THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
xvEPA
ntal Protection Agra
Baireiie
Business of Innovation
TECHNOLOGY TYPE: MICROCYSTIN TEST KIT
APPLICATION:
RECREATIONAL WATER MICROCYSTIN
DETECTION
TECHNOLOGY NAME: Microcystin Strip Test Kit
COMPANY: Abraxis
ADDRESS:
WEB SITE:
54 Steamwhistle Drive
Warminster, PA 18974
http://www.abraxiskits.com/
PHONE: 215-357-3911
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 microcystin test kits for water monitoring. This verification statement provides a
summary of the test results for the Abraxis Microcystin Strip Test Kit.
VERIFICATION TEST DESCRIPTION
This verification test of the Abraxis Microcystin Strip Test Kit was conducted from July 26 through August 12,
2010 at Battelle laboratories in Columbus, OH. Reference analyses by liquid chromatography tandem mass
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spectrometry (LC-MS/MS) were performed the week of August 16, 2010 by the University of Nebraska Water
Sciences Laboratory.
The objective of this verification test was to evaluate the microcystin test kit performance in analyzing known
concentrations of microcystin in ASTM International Type II deionized (DI) water and in natural recreational
water (RW) samples. The technology was used to analyze a variety of water samples for the variants
microcystin-LR, microcystin-LA, and microcystin-RR. Because the technology cannot specify between the more
than 80 microcystin variants, the samples prepared for this test were spiked with three individual variants. The
Microcystin Strip Test Kit provided a quantitative determination of microcystins and was evaluated in terms of:
• Accuracy - comparison of test kit results (samples prepared in DI) to results from a reference method;
• Precision - repeatability of test kit results from three sample replicates analyzed in DI water, matrix
interference, and RW samples;
• Linearity - determination of whether or not the test kit response increases in direct proportion to the
known concentration of microcystin;
• 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 - general operation, data acquisition, setup, consumables, etc.
Each microcystin test kit was operated according to the vendor's instructions by a vendor-trained Battelle
technician. Samples and calibration standards were analyzed in duplicate and positive and negative controls were
analyzed at the vendor-specified frequency. The ability of the Abraxis Microcystin Strip Test Kit to determine
the concentration of microcystin was challenged using quality control (QC) samples, performance test (PT)
samples and RW samples. 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. 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.
Unlike many contaminants, certified microcystin standards are not commercially available. In planning this
verification test, multiple sources of standards were investigated. With agreement from the stakeholders, all
vendors and the EPA project officer, the standards used for this verification were purchased from the most
reputable sources (LR and RR from Canadian National Research Council and LA from Abraxis), based on a
Performance Evaluation Audit, and used for both the testing solutions and the reference method calibration.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted technical
systems audits of the both the laboratory and field testing, and Battelle QA staff 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 www.epa.gov/etv/centers/center 1 .html.
TECHNOLOGY DESCRIPTION
Following is a description of the Abraxis Microcystin StripTest Kit, based on information provided by the vendor.
The information provided below was not verified in this test.
The Microcystin Strip Test Kit is a rapid immunochromatographic test, designed solely for use in the qualitative
screening of microcystins and nodularins in RW. A rapid cell lysis step (QuikLyse™) performed prior to testing
is required to measure total microcystins (dissolved or free, plus cell bound). The Microcystin Strip Test Kit
provides only preliminary qualitative test results. If necessary, positive samples can be confirmed by ELISA,
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high-pressure liquid chromatography (HPLC) or other conventional methods. The test is designed for field use,
requiring no instrumentation or other equipment, no power sources, and no refrigerated storage.
The test is based on the recognition of microcystins, nodularins and their variants by specific antibodies. The
toxin conjugate competes for antibody binding sites with microcystins/nodularins that may be present in the water
sample. The test device consists of a conical flip-top vial with specific antibodies for microcystins and nodularins
labeled with a gold colloid and a membrane strip to which a conjugate of the toxin is attached. A control line,
produced by a non-microcystin antibody/antigen reaction, is also present on the membrane strip. The control line
is not influenced by the presence or absence of microcystins in the water sample, and therefore, it should be
present in all reactions. In the absence of toxin in the water sample, the colloidal gold labeled antibody complex
moves with the water sample by capillary action to contact the immobilized microcystins conjugate producing a
visible line in the test line region.
If microcystins are present in the water sample, they compete with the immobilized toxin conjugate in the test
area for the antibody binding sites on the colloidal gold labeled complex. If a sufficient amount of toxin is
present, it will fill all of the available binding sites, thus preventing attachment of the labeled antibody to the toxin
conjugate and preventing the development of a second line in the test line region. If a second line is not visible in
the test line region, or if the test line is lighter than the negative control line, microcystin is present at levels of
concern (>10 ppb). Semi-quantitative results in the range of 0 to 10 ppb can be obtained by comparing the test
line intensity to those produced by solutions of known microcystins concentrations (control solutions). During
this verification, the results were considered 0 to 10 ppb, or greater than 10 ppb.
VERIFICATION RESULTS
The verification of the Abraxis Microcystin Strip Test Kit is summarized in Table 1 and by the parameters
described below.
Table 1. Abraxis Microcystin Strip Test Kit Performance Summary
Sample
Description
Spiked Cone.
0.10 ppb
0.50 ppb
1.0 ppb
2.0 ppb
4.0 ppb
7.0 ppb
15 ppb
LR Results
(Observation and
Interpretation)
Dark Line
Dark Line
Dark Line
Line
Line
Line
Line
Line
Line
Line
Line
Line
Faint line
Faint line
Faint line
NA
NA
NA
No line
No line
No line
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
NA
NA
NA
> 10 ppb
> 10 ppb
> 10 ppb
LA Results
(Observation and
Interpretation)
Dark Line
Dark Line
Dark Line
Line
Line
Line
Line
Line
Line
Line
Line
Line
Faint line
Faint line
Faint line
Very Faint line
Very Faint line
Very Faint line
No line
No line
No line
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
> 10 ppb
> 10 ppb
> 10 ppb
RR Results
(Observation and
Interpretation)
Dark Line
Dark Line
Dark Line
Line
Line
Line
Line
Line
Line
Line
Line
Line
Faint line
Faint line
Faint line
No line
No line
No line
No line
No line
No line
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
0-10 ppb
> 10 ppb
> 10 ppb
> 10 ppb
> 10 ppb
> 10 ppb
> 10 ppb
NA = not applicable; 7.0 ppb level was not performed for LR samples.
Accuracy. The DI samples (concentrations between 0.10 and 15 ppb) for the three variants of microcystin were
used during this verification test. All of the samples were analyzed in triplicate and each of them produced a
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control line on the Microcystin Strip Test Kit to indicate that the test was functioning properly. As the
concentrations of the various microcystin variants increased, the line generated in the test area region was
observed to change as expected. The lowest concentration samples generated dark lines, the mid-level
concentrations generated lighter lines, and the highest concentrations were either very faint lines or generated no
line at all. The Microcystin Strip Test Kit correctly detected each of the 15 ppb samples as being greater than 10
ppb. The Microcystin Strip Test Kit results were inconsistent with the reference method in only one sample set
out of 18. The 7.0 ppb RR DI water sample did not generate a line, indicating a concentration of greater than 10
ppb.
Precision. The line colors were consistent within the three replicates at each concentration, with one exception.
With the exception of RW 5, the triplicate measurements of the samples generated lines that were very similar in
intensity. In two replicates, RW 5 generated faint lines indicating a 0 to 10 ppb concentration and one replicate
that had no line, indicating a concentration of greater than 10 ppb.
Matrix Interference. The RW matrix interferent and chlorophyll-a interferent sample results for the Microcystin
Strip Test Kit all agreed and the interpretation of the results was consistent with the spiked amount of
microcystins in the samples. There was no indication that different matrices affected the test kit performance.
Recreational Water (RW). RW sample results for the Microcystin Strip Test Kit were also consistent with the
reference method results. While the total measured microcystin result may have other variants present that were
not measured by the reference method, with the exception of RW 5, the sample results were all consistent with the
reference laboratory results of the RW samples. That is, when the reference concentrations were greater than 10
ppb, there was no line generated; when the reference concentration was between 2.0 and 4.0 ppb, the lines were
medium dark or faint, and when the reference concentrations were lower than 2.0 ppb, the lines generated were
dark.
Operational Factors. The test kit operator reported that the Microcystin Strip Test Kit was very easy to use and
needs no technical skills to operate. The brochure and flow charts with illustrations were clear and easy to follow.
There is no solution or sample preparation needed. The entire procedure takes approximately 40 minutes,
including the QuikLyse™ procedure and the microcystins analysis. The QuikLyse™ process uses 1 mL of
sample through two, 8-minute incubation periods. The sample is then transferred into the microcystins reagent
conical tube. The sample is incubated for 10 minutes and then the test strip is added to the conical tube. The test
strip is interpreted according to the figure in the brochure after 5 minutes of exposure to the sample. No
consumables were required for this technology. Once the analysis was complete, the remaining solutions were
disposed in the trash in accordance with local regulations. The listed price for the Microcystin Strip Test Kit at
the time of the verification test was $480 for a 20-strip kit and $150 for a five-strip kit. The kit has a 12-month
shelf life when received, and should be stored at room temperature.
Signed by Tracy Stenner 9/22/10 Signed by Sally Gutierrez 10/25/10
Tracy Stenner Date Sally Gutierrez Date
Manager Director
Environmental Product Line National Risk Management Research Laboratory
National Security Global Business Office of Research and Development
Battelle 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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