EPA/600/R-14/307
February 2014
THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
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ETV Joint Verification Statement
TECHNOLOGY TYPE: COLIFORM DETECTION
APPLICATION: ANALYSIS OF E. C0L/IN DRINKING WATER
TECHNOLOGY NAME: ENDETEC TECTA™ B-16
COMPANY: Pathogen Detection Systems, Inc.
ADDRESS: Suite 4697, Biosciences Complex, 116 Barrie Street
Kingston, Ontario, Canada K7L 3N6
PHONE: 866-362-0993
WEB SITE: www.endetec.com
E-MAIL: peter.gallant@veoliawater.com
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 a system for coliform detection in drinking water (DW). This verification
statement provides a summary of the test results for Pathogen Detection System, Inc.'s ENDETEC
TECTA™ B-16.
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VERIFICATION TEST DESCRIPTION
In February 2013, EPA revised the 1989 Total Coliform Rule (TCR), a national primary drinking water
regulation (NPDWR). The revised rule establishes a maximum contaminant level goal (MCLG) of zero for
E. coli (EC), a more specific indicator of fecal contamination and potential harmful pathogens than total
coliform. EPA has removed the 1989 MCLG and maximum contaminant level for total coliform. In the
revised TCR, total coliforms serve as an indicator of a potential pathway of contamination into the
distribution system. A PWS that exceeds a specified frequency of total coliform occurrence must conduct an
assessment to determine if any sanitary defects exist and, if found, correct them. In order to comply with
the revised TCR (RTCR), water utilities need coliform detection technologies that are able to detect EC at
concentrations of one colony forming unit (CPU) per 100 milliliters (mL). While it is difficult to determine
if a single target organism is present in 100 mL of water, when approximately half of the analyzed replicates
are positive and half are negative, the density of the organism has become adequately low so that a positive
result can be considered single organism detection. Therefore, for the purpose of this verification, the
objective was to prepare spiked DW dilution sets that provided 50 ±25% positive results for EC with the
Colilert-18 reference method and then confirm the results from the TECTA™ B-16 (through analysis of the
spent media from the initial samples) with the Colilert-18 reference method.
The verification test of the TECTA™ B-16 was conducted from August 21 through August 23, 2013 at the
City of Columbus Division of Water (CDW) laboratory in Columbus, Ohio with the reference method
analyses being performed at Superior Laboratories in Galloway, Ohio (which is a 15 minute drive from the
CDW laboratory). Technology operation and sample handling and analysis were performed according to
the operating documentation and method description provided by the vendor. Both reference method and
TECTA B-16 sample analysis results were reported in presence/absence format, consistent with the
requirements of the RTCR.
Sample analysis results from the TECTA B-16 were evaluated by calculating the true positive (and true
negative) results through confirmation analyses using Colilert-18 as described above. These calculations
include the comparison of false positive rate (or specificity) and false negative rate (or sensitivity). In
addition, statistical testing was performed on the initial reference method and TECTA B-16 results.
Sustainable operational factors such as ease of use, required reagents, analysis time, and laboratory space
and utilities required are reported.
QA oversight of verification testing was provided by Battelle and EPA. Battelle and EPA QA staff
conducted technical systems audits of the testing and Battelle QA staff conducted a data quality audit of at
least 25% 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 all available at www.epa.gov/etv/centers/center 1 .html.
TECHNOLOGY DESCRIPTION
The TECTA B-16 is a bench top detection and data logging system for the analysis of TC and EC in water
samples. It utilizes an enzyme substrate test to simultaneously detect the presence of TC ((3-galactosidase
enzyme) and EC ((3-glucuronidase enzyme). The system consists of single-use cartridges that contain pre-
measured reagents and an embedded optical sensor. A 100 mL water sample is added to the cartridge and
then up to 16 of the cartridges are incubated in and analyzed by the TECTA B-16.
The enzymes produced by TC and EC bacteria cleave the fluorogenic substrates in the growth media,
resulting in the release of fluorescent products. The fluorescent product molecules rapidly accumulate in an
optical sensor formed from a polymeric material embedded at the center of the test cartridge base, which is
continuously illuminated by an ultraviolet light source in the bottom of each TECTA B-16 sample chamber.
The light emitted by the polymer when fluorescent indicator products are present is detected at wavelengths
specific to each fluorescent product that are in turn specific to detection of TC and EC bacteria. Optical
detection is performed automatically by a charge-coupled device. Test management software within the
TECTA B-16 interprets these optical signals continuously throughout the test cycle, and provides an alert of
a positive sample detection for both EC and TC as soon as a threshold level of fluorescence is detected.
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Samples not displaying detectable fluorescence at the wavelengths of interest are determined to be absent of
bacteria after 18 hours. Due to its continuous monitoring capability, positive sample results can be detected
in less than 18 hours.
In continous mode, the TECTA B-16 can analyze up to 16 samples in 18 hours (h). The vendor provided
three units for testing, providing simultaneous sample analysis capacity of 48 samples. The TECTA B-16
can also be operated in read mode where the detector in the sample chamber can be used to make an
instantaneous measurement of light emission from the polymer. Read mode can be used to confirm results
obtained in continuous mode or if a sample cartridge has been incubated under appropriate conditions
outside of the TECTA B-16. In read mode, the results display on the screen in the same way as for the
continuous measurements, except that a "time-to-result" indication is not available for samples processed in
read mode.
VERIFICATION RESULTS
Positive Results. The positive TC and EC test results for the TECTA B-16 and reference method (Colilert-18)
are presented in Table 1. One of the two dilutions (0.5 CFU/100 mL) yielded the target 50 ± 25% split in
responses from the reference method. The other dilution generated results that were 100% positive.
Table 1. Results Summary for Positive TECTA B-16 Results for TC and EC
Dilution
(target concentration)
A
(5CFU/100mL)
B
(0.5 CFU/100 mL)
TECTA B-16
TC and EC
N
20
6
% of total
samples
100%
30%
Colilert-18
N
20
11
% of total
samples
100%
55%
Because the reference method results were between 25% and 75% positive, the reference method results
suggested that the 0.5 CFU/100 mL solutions prepared for the evaluation were at the single organism per 100
mL concentration level. Therefore the TECTA B-16 results were able to be used along with the reference
method confirmation data to determine the effectiveness of the TECTA B-16 in detecting such low
concentrations. Specifically, following analysis using the TECTA B-16, 1 mL of the resulting suspension
was inoculated into 99 mL of sterilized water and analyzed by the reference method. The result of these
analyses provided confirmation of the presence/absence results for each replicate sample. Table 2
summarizes the confirmed true positive and true negative TC and EC results for the TECTA B-16.
Table 2. TECTA B-16 True Positive and Negative Results Summary
Dilution
(target concentration)
A
(5CFU/100mL)
B
(0.5 CFU/100 mL)
N
20
20
Confirmed
(True Positive)
20
6
Confirmed
(True Negative)
0
14
False Results
0
0
N= Number of replicates
Sensitivity, Specificity, False Positive (FP) Rate, False Negative (FN) Rate. Table 3 summarizes the
specificity, sensitivity, FP rate, and FN rate for TC and EC for 18 and 24 h incubations. Sensitivity is defined
as the percent of positive samples correctly identified as positive and specificity is defined as the percent of
negative samples correctly identified as negative. For the 0.5 CFU/100 mL sample, the sensitivity and
specificity of the TECTA B-16 was 100% with no false positives or false negatives. For the 5 CFU/100 mL
samples, the sensitivity was also 100% (with no false negatives. For both concentrations, the results were all
confirmed by the reference method.
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Table 3. Results Summary of TECTA B-16
Incubation Time (h)
Sensitivity
Specificity
False Positive
False Negative
0.5 CFU/100 mL
100%
100%
0%
0%
5 CFU/100 mL
100%
NA
NA
0%
NA - not applicable because zero in denominator of calculation
Comparability. In another approach of comparison, a chi-square test for independence was performed to
compare the TECTA B-16 against the reference method (Colilert-18). Because only the 0.5 CFU/100 mL
dilution had both positive and negative results, the chi-squared analysis was only performed for that solution.
This analysis generated a p-value that was greater than 0.05 indicating that the TECTA B-16 results were not
significantly different from the initial Colilert-18 results (at the 95% confidence level), a result that is
consistent with the confirmatory analyses described above (which indicated identical results between the
TECTA B-16 and Colilert-18 confirmatory analysis of each TECTA B-16 replicate).
Additional Concentrations in Continuous Operation. The objective of this component of the testing was to
verify the TECTA B-16 capability of reporting analysis results as soon as determined by the TECTA B-16
rather than waiting for the end of an incubation time period such as 18 or 24 h. Four concentrations of EC
ATCC 25922 (8, 100, 1,000, and 8,600 CFU/100 mL) were analyzed four times each. The TECTA B-16
generated positive TC and EC responses for all of the samples. The required analysis time for TC ranged
from 10 to 14 h and for EC ranged from 9 to 13 h. The amount of time until detection for the TC and EC
samples decreased with each increasing concentration level and generally the EC took about 30 to 50 minutes
less time for detection.
Operational Factors. The TECTA B-16 was operated in continuous measurement mode for the simultaneous
measurement of TC and EC in up to 16 different samples. To initiate analysis, 100 mL of each individual
water sample were dispensed into each cartridge and the cartridge was snapped firmly shut, then swirled to
dissolve the contents. The cartridges were loaded into the TECTA B-16 in the same manner and the 18 h
incubation/analysis was started by closing the lid. The samples (16 at a time) were incubated within the
TECTA B-16 at 35 °C and results were reported on the screen (and available to the operator as electronic
alerts) as soon as the TECTA B-16 was able to make a conclusive positive determination of TC and/or EC
based on the fluorescence measurement. The result of each measurement was displayed on the screen and the
operator recorded the result on a sample data sheet. Each result could also be downloaded for review and
viewed on a computer containing the TECTA B-16 software or a standard web browser. The CDW operator
noted that the technology was very user friendly and eliminated the need for a technician to be present outside
of working hours to read the results.
The TECTA B-16 has dimensions of 48 cm wide * 62 cm deep * 34 cm high (18.8 inches wide * 24.5 inches
deep x 13.5 inches high) and weighs approximately 28 kilograms (61.7 pounds). The TECTA B-16 is
completely self-contained and does not require any additional equipment or materials to perform analyses.
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Signed by Spencer Pugh 2/28/14 Signed by Cynthia Sonich-Mullin 3/3/14
Spencer Pugh Date Cynthia Sonich-Mullin Date
General Manager Director
Energy and Environment Business Unit National Risk Management Research Laboratory
Battelle Office of Research and Development
U.S. Environmental Protection Agency
NOTICE: ETV verifications are based on an evaluation of technology performance under specific,
predetermined criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or
implied warranties as to the performance of the technology and do not certify that a technology will always
operate as verified. The end user is solely responsible for complying with any and all applicable federal, state,
and local requirements. Mention of commercial product names does not imply endorsement.
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