April 2005
Environmental Technology
Verification Report
Industrial Test Systems, Inc.
Cyanide ReagentStrip™ Test Kit
Prepared by
Battelle
Batteiie
The B us mess of Innovation
Under a cooperative agreement with
A U.S. Environmental Protection Agency
ETV ElV ElV
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April 2005
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Industrial Test Systems, Inc.
Cyanide ReagentStrip™ Test Kit
by
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
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Foreword
The U.S. Environmental Protection Agency (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 verification 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/center 1. html.
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We would like to thank Billy Potter,
U.S. Environmental Protection Agency, National Exposure Research Laboratory; Ric DeLeon,
Metropolitan Water District of Southern California; William Burrows, U.S. Army Center for
Environmental Health Research; and Kenneth Wood, Du Pont Corporate Environmental
Engineering Group, for their technical review of the test/quality assurance plan and for their
careful review of this verification report. We also would like to thank Jeff Wilson, City of
Montpelier, VT; Christopher Jones, Des Moines, IA, Water Works; Wylie Harper, City of
Seattle, WA; Jamie Shakar, City of Tallahassee, FL; and Tom Burkhart, City of Flagstaff, AZ.
These water distribution facilities provided post-treatment water samples for evaluation.
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations viii
1 Background 1
2 Technology Description 2
3 Test Design and Procedures 3
3.1 Introduction 3
3.2 Reference Method 4
3.3 Test Design 4
3.4 Test Samples 5
3.4.1 Quality Control Samples 5
3.4.2 Performance Test Samples 6
3.4.3 Lethal/Near-Lethal Concentrations of Cyanide in Water 7
3.4.4 Surface Water; Drinking Water from Around the U.S.;
and Columbus, OH, Drinking Water 7
3.5 Test Procedure 9
3.5.1 Sample Preparation 9
3.5.2 Sample Identification 9
3.5.3 Sample Analysis 9
4 Quality Assurance/Quality Control 12
4.1 Reference Method Quality Control Results 12
4.2 Audits 14
4.2.1 Performance Evaluation Audit 14
4.2.2 Technical Systems Audit 14
4.2.3 Audit of Data Quality 15
4.3 Quality Assurance/Quality Control Reporting 15
4.4 Data Review 15
5 Statistical Methods and Reported Parameters 17
5.1 Accuracy 17
5.2 Precision 17
5.3 Linearity 18
5.4 Method Detection Limit 18
5.5 Inter-Unit Reproducibility 18
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5.6 Lethal or Near-Lethal Dose Response 19
5.7 Operator Bias 19
5.8 Field Portability 19
5.9 Ease of Use 19
5.10 Sample Throughput 19
6 Test Results 20
6.1 Accuracy 20
6.2 Precision 22
6.3 Linearity 29
6.4 Method Detection Limit 32
6.5 Inter-Unit Reproducibility 32
6.6 Lethal or Near-Lethal Dose Response 34
6.7 Operator Bias 35
6.8 Field Portability 36
6.9 Ease of Use 37
6.10 Sample Throughput 38
7 Performance Summary 39
8 References 42
Figures
Figure 2-1. Industrial Test Systems, Inc., Cyanide ReagentStrip™ Test Kit 2
Figure 3-1. Sample Preparation and Analysis of Surface and Drinking Water Samples 8
Figure 3-2. Cyanide ReagentStrip™ Test Kit Color Charts 11
Figure 6-1. Non-technical Operator Linearity Results (0.03 to 25 mg/L) 30
Figure 6-2. Technical Operator Linearity Results (0.03 to 25 mg/L) 30
Figure 6-3. Non-technical Operator Linearity Results (0.03 to 1 mg/L) 31
Figure 6-4. Technical Operator Linearity Results (0.03 to 1 mg/L) 31
Figure 6-5. Inter-Unit Reproducibility Results 33
Figure 6-6. Operator Bias Results 36
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Tables
Table 3-1. Test Samples 6
Table 4-1. Reference Method Quality Control Standard Results 13
Table 4-2. Reference Method Laboratory-Fortified Matrix Analysis Results 13
Table 4-3. Summary of Performance Evaluation Audit 14
Table 4-4. Summary of Data Recording Process 16
Table 6-la. Cyanide Results from Performance Test Samples 21
Table 6-lb. Cyanide Results from Surface Water 22
Table 6-lc. Cyanide Results from U.S. Drinking Water 23
Table 6-Id. Cyanide Results from Columbus, OH, Drinking Water 24
Table 6-2a. Percent Accuracy of Performance Test Sample Measurements 25
Table 6-2b. Percent Accuracy of Surface Water Measurements 26
Table 6-2c. Percent Accuracy of U.S. Drinking Water Measurements 26
Table 6-2d. Percent Accuracy of Columbus, OH, Drinking Water Measurements 26
Table 6-3. Semi-Quantitative Accuracy Evaluation 27
Table 6-4a. Precision of Performance Test Measurements 27
Table 6-4b. Precision of Surface Water Measurements 27
Table 6-4c. Precision of U.S. Drinking Water Measurements 28
Table 6-4d. Precision of Columbus, OH, Drinking
Water Measurements 28
Table 6-5. Results of Method Detection Limit Assessment 33
Table 6-6. Lethal/Near-Lethal Concentration Sample Results 34
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List of Abbreviations
AMS Advanced Monitoring Systems
ASTM American Society for Testing and Materials
ATEL Aqua Tech Environmental Laboratories
DI deionized
DPD N,N-di ethyl -/^-phenyl enedi ami ne
DW drinking water
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
ID identification
KCN potassium cyanide
L liter
LFM laboratory-fortified matrix
MDL method detection limit
mg milligram
mL milliliter
NaOH sodium hydroxide
NIST National Institute of Standards and Technology
PE performance evaluation
PT performance test
QA quality assurance
QC quality control
QCS quality control standard
QMP quality management plan
RB reagent blank
RPD relative percent difference
RSD relative standard deviation
TSA technical systems audit
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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 tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by substantially accelerating the acceptance
and use of improved and cost-effective technologies. ETV seeks to achieve this goal by provid-
ing 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 tech-
nologies 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 EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the Industrial Test Systems, Inc., Cyanide ReagentStrip™
test kit in detecting the presence of cyanide in water. Portable cyanide analyzers were identified
as a priority technology verification category through the AMS Center stakeholder process.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of the Cyanide ReagentStrip™ test kit. Following is a
description of the Cyanide ReagentStrip™ test kit, based on information provided by the vendor.
The information provided below was not verified in this test.
The Industrial Test Systems, Inc., Cyanide ReagentStrip™ test kit is designed to detect free
cyanide in water. This is done by converting cyanide in water to cyanogen chloride, which, in the
presence of isonicotinic and barbituric acids, produces a color change that can be detected
visually or with a colorimeter. Results can be determined by three methods, and the method
selected is dependent upon the data needs of the user: (1) A semi-quantitative result in increments
ranging from <0.1 milligram per liter (mg/L) to >10 mg/L can be obtained in approximately 1
minute by comparing the color change on ReagentStrip™ #2 to a color chart; (2) a
semi-quantitative result for an expanded range of 0 mg/L to >200 mg/L can be obtained in
10 minutes by visually comparing the color of the water sample in a microcuvette with a separate
color chart designed for use with microcuvettes; and
(3) a quantitative determination can be obtained in
10 minutes when the microcuvette is inserted into
the optional ReagentStrip™ C07500 colorimeter
(also identified as the ReagentStrip™ Reader), and
the intensity of the color is measured quantitatively.
The ReagentStrip™ Reader generates a result in
absorbance units that are converted to concentration
units using the reference table provided by
Industrial Test Systems, Inc. The absorbance units
on the reference table convert to concentrations
ranging from <0.01 mg/L to >60 mg/L.
The Cyanide ReagentStrip™ test kit includes one bottle each of Cyanide ReagentStrip™ #1 and
#2, one graduated pipette, 20 microcuvettes, one microcuvette holder, one ReagentStrip™
Reader, two semi-quantitative visual color charts, one colorimeter absorbance reference chart,
one instruction sheet, and a material safety data sheet. The list price of the Cyanide
ReagentStrip™ test kit, including the optional ReagentStrip™ Reader, is $559.99 for 50 tests.
Reagent Strips™ #1 and #2 for additional tests can be purchased separately at an approximate
cost of $40 for 50 additional tests.
p m m m m w ¦ * as«
" OP
cjEDcncn
Figure 2-1. Industrial Test Systems, Inc.,
Cyanide ReagentStrip™ Test Kit
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Chapter 3
Test Design and Procedures
3.1 Introduction
Cyanide can be present in various forms in water. This verification test focused on detecting the
free cyanide ion prepared using potassium cyanide (KCN) and is referred to as simply "cyanide"
in this report. At high doses, this form of cyanide inhibits cellular respiration and, in some cases,
can result in death. Because of the toxicity of cyanide to humans, the EPA has set 0.2 mg/L as
the maximum concentration of cyanide that can be present in drinking water (DW). In DW and
surface water under ambient conditions, cyanide evolves from aqueous hydrogen cyanide,
sodium cyanide, potassium cyanide, and other metal or ionic salts where cyanide is released
when dissolved in water. Heavier cyanide complexes (e.g., iron) are bound tightly, requiring an
acid distillation to liberate the toxic free cyanide ion, a step not verified as part of this test since
the distillation step would prevent these analyzers from being field portable. Because disassocia-
tion of the free cyanide ion is unlikely under ambient conditions, the heavier salts are considered
much less toxic than simple cyanide salts such as potassium and sodium cyanide.
This verification test was conducted according to procedures specified in the Test/QA Plan for
Verification of Portable Analyzers for Detection of Cyanide in Water.(1) The verification was
based on comparing the cyanide concentrations of water samples analyzed using the Cyanide
ReagentStrip™ test kit with cyanide concentrations analyzed using a laboratory-based reference
method. The Cyanide ReagentStrip™ test kit was verified by analyzing performance test (PT),
lethal/near-lethal concentration, surface, and DW samples. A comparison of the analytical
results from the Cyanide ReagentStrip™ test kit and the reference method provided the basis for
the quantitative results presented in this report.
The Cyanide ReagentStrip™ test kit performance was evaluated in terms of
¦ Accuracy
¦ Precision
¦ Matrix effects
¦ Linearity
¦ Method detection limit
¦ Inter-unit reproducibility
¦ Lethal or near-lethal dose response
¦ Operator bias
¦ Field portability
¦ Ease of use
¦ Sample throughput.
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3.2 Reference Method
Aqua Tech Environmental Laboratories (ATEL) in Marion, OH, performed the reference
analyses of all test samples. ATEL received the samples from Battelle labeled with an
identification (ID) number meaningful only to Battelle, performed the analyses, and submitted to
Battelle the results of the analyses without knowledge of the prepared or fortified concentration
of the samples.
The analytical results for the Cyanide ReagentStrip™ test kit were compared with the results
obtained from analysis using semi-automated colorimetry according to EPA Method 335.1,
Cyanides Amenable to Chlorination.{2) This method was selected because it measures the
concentration of the cyanide ion in water samples under ambient conditions, which is the same
form of cyanide that the participating technologies are designed to measure. For the reference
method analyses, the concentration of free cyanide was determined by the difference of two
measurements of total cyanide. One colorimetric determination was made after the free cyanide
in the sample had been chlorinated to cyanogen chloride, which degrades quickly, and a second
was made without chlorination. Typically, samples were sent to the reference laboratory for
analysis each testing day. The reference analysis was performed within 14 days of sample
collection.
3.3 Test Design
The verification test was conducted between September 22, 2004, and October 5, 2004. All
analyses were performed according to the manufacturer's recommended procedures. The
verification test involved challenging the Cyanide ReagentStrips™ test kit with a variety of test
samples, including sets of DW and surface water samples representative of those likely to be
analyzed by buyers and users of the Cyanide ReagentStrips™ test kit. The results from the
Cyanide ReagentStrips™ test kits were compared with the reference method to qualitatively and
quantitatively assess performance. Multiple aliquots of each test sample were analyzed
separately to assess the precision of the Cyanide ReagentStrips™ test kit and the reference
method.
Results were generated using the Cyanide ReagentStrips™ test kit by a technical and a
non-technical operator to assess operator bias. The non-technical operator had no previous
laboratory experience. Both operators watched a brief training video provided by the vendor to
become acquainted with the basic operation of the test kit. Both operators analyzed all of the test
samples. Each operator manipulated separate water samples and reagents to generate a solution
in which cyanide could be detected photometrically. Then, the operators analyzed their
respective solutions using two ReagentStrip™ Readers to evaluate inter-unit reproducibility.
Sample throughput was estimated based on the time required to prepare and analyze a sample.
Ease of use was based on documented observations by the operator and the Battelle Verification
Test Coordinator. The Cyanide ReagentStrip™ test kit was used in a field environment as well
as in a laboratory setting to assess the impact of field conditions on performance.
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3.4 Test Samples
Test samples used in the verification test are shown in Table 3-1 and include quality control
(QC) samples, PT samples, lethal/near-lethal concentration samples, DW samples, and surface
water samples. The QC, PT, and lethal/near-lethal samples were prepared from National Institute
of Standards and Technology (NIST) traceable standards. The PT and QC sample concentrations
were targeted to the EPA maximum contaminant level in DW, which for cyanide is 0.2 mg/L.(3)
The PT samples ranged from 0.03 mg/L to 25 mg/L. The performance of the Cyanide
ReagentStrip™ test kit also was evaluated quantitatively with samples prepared with cyanide
concentrations up to 250 mg/L that could be lethal if ingested. Two surface water sources
(Olentangy River and Alum Creek Reservoir) were sampled and analyzed. In addition, five
sources of DW from around the United States and two sources of Columbus, OH, DW were
evaluated.
3.4.1 Quality Control Samples
QC samples included laboratory reagent blanks (RBs), quality control standards (QCSs), and
laboratory-fortified matrix (LFM) samples (Table 3-1). The RBs consisted of American Society
for Testing and Materials (ASTM) Type II deionized (DI) water and were exposed to handling
and analysis procedures identical to other prepared samples, including the addition of all
reagents. These samples were used to help ensure that no sources of contamination were
introduced in the sample handling and analysis procedures. One RB sample was analyzed for
every batch of about 10 water samples. In several instances, the RB samples produced results
slightly above <0.01 mg/L. When that was the case, the RB result (background) was subtracted
from each result in the sample batch. QCSs of 0.2 mg/L cyanide were prepared in ASTM Type II
DI water and analyzed (without defined performance expectations) after approximately every
10th sample by the Cyanide ReagentStrip™ test kit to demonstrate proper functioning to the
operator. The LFM samples were prepared as aliquots of DW and surface water samples spiked
with KCN as free cyanide to make the cyanide concentration also 0.2 mg/L. Four LFM samples
were analyzed for each source of DW and surface water. These samples were used to determine
whether matrix effects had an influence on the analytical results from both the Cyanide
ReagentStrip™ test kits and the reference method.
QCSs were analyzed approximately every 10th sample to ensure the proper calibration of the
reference instrument. According to its standard operating procedure for this reference method,
the reference laboratory prepared the QCSs for its use at 0.2 or 0.15 mg/L from a stock solution
independent of the one used to prepare the QCS analyzed using the Cyanide ReagentStrip™ test
kit. The reference method required that the concentration of each QCS be within 25% of the
known concentration. If at any point the difference was larger than 25%, the data collected since
the most recent QCS would have been flagged; and proper maintenance would have been
performed to regain accurate cyanide measurement, according to ATEL protocols. Section 4.1
describes these samples in more detail.
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Table 3-1. Test Samples'3'
Type of Sample
Sample Characteristics
Cyanide
Concentration (mg/L)
Number of Samples
QC
RB
0 mg/L
every 10th sample
QCS
0.2 mg/L®
every 10th sample
PT
Detection limit determination
0.05 mg/L
7
Spiked DI water
0.03 mg/L
4
Spiked DI water
0.1 mg/L
4
Spiked DI water
0.2 mg/L
4
Spiked DI water
0.4 mg/L
4
Spiked DI water
1 mg/L
4
Spiked DI water
5 mg/L
4
Spiked DI water
15 mg/L
4
Spiked DI water
25 mg/L
4
Lethal/
Near-Lethal
Samples
Spiked DI water
50 mg/L
4
Spiked DI water
100 mg/L
4
Spiked DI water
250 mg/L
4
Surface Water and
DW
Alum Creek Reservoir
Unspiked background
4
0.2 mg/L LFM
4
Olentangy River
Unspiked background
4
0.2 mg/L LFM
4
Des Moines, IA
Unspiked background
4
0.2 mg/L LFM
4
Flagstaff, AZ
Unspiked background
4
0.2 mg/L LFM
4
Montpelier, VT
Unspiked background
4
0.2 mg/L LFM
4
Seattle, WA
Unspiked background
4
0.2 mg/L LFM
4
Tallahassee, FL
Unspiked background
4
0.2 mg/L LFM
4
Columbus, OH, city water
Unspiked background
12
0.2 mg/L LFM
12
Columbus, OH, well water
Unspiked background
12
0.2 mg/L LFM
12
(^ Samples were analyzed in random order.
(b:i Maximum contaminant level for cyanide.
3.4.2 Performance Test Samples
The PT samples (Table 3-1) were prepared in the laboratory using ASTM Type IIDI water. The
samples were used to determine the accuracy, precision, linearity, and detection limit of the
Cyanide ReagentStrip™ test kit. Seven non-consecutive replicate analyses of a 0.05-mg/L
solution were made to obtain precision data with which to determine the method detection limit
(MDL).(4) Eight other solutions were prepared to assess the linearity over a 0.03- to 25-mg/L
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range of cyanide concentrations. Four aliquots of each of these solutions were analyzed
separately to assess the precision of the Cyanide ReagentStrip™ test kit. The concentrations of
the PT samples are listed in Table 3-1. The operator analyzed the PT samples blindly and in
random order to minimize bias.
3.4.3 Lethal/Near-Lethal Concentrations of Cyanide in Water
To assess the response of the Cyanide ReagentStrip™ test kit when cyanide is present in DW at
lethal and near-lethal concentrations, samples were prepared in ASTM Type IIDI water at
concentrations of 50, 100, and 250 mg/L. The quantitative and semi-quantitative results
generated by the Cyanide ReagentStrip™ test kit were compared to results from the reference
method.
3.4.4 Surface Water; Drinking Water from Around the U.S.; and
Columbus, OH, Drinking Water
Water samples, including fresh surface water and DW (well and local distribution sources) were
collected from a variety of sources and used to evaluate technology performance. Surface water
was collected near the shoreline by submerging 10-L high-density polyethylene containers no
more than one inch below the surface of the water. In a similar container, representatives of five
city water treatment facilities provided Battelle with a sample of water that had completed the
treatment process, but had not yet entered the water distribution system. Two Columbus, OH,
water samples were collected from local residential homes, one from a home with city water and
one from a home with well water. Surface water samples were collected from
¦ Alum Creek Reservoir (OH)
¦ Olentangy River (OH).
DW samples were collected from
¦ Local distribution source water (post-treatment) from five cities (Des Moines, IA; Flagstaff,
AZ; Montpelier, VT; Seattle, WA; and Tallahassee, FL).
¦ Columbus, OH, city water
¦ Columbus, OH, well water.
The water samples collected as part of this verification test were not characterized in any way
(i.e., hardness, alkalinity, etc.) other than for cyanide concentration. Each sample was tested for
the presence of chlorine, dechlorinated if necessary, and split into two subsamples. Figure 3-1 is
a diagram of the process leading from sampling to aliquot analysis. One subsample was spiked
with 0.2 mg/L of cyanide to provide LFM aliquots, and the other subsample remained unspiked
(background). Four aliquots were taken from each subsample and analyzed separately using the
Cyanide ReagentStrip™ test kit. Also, eight aliquots were taken from the background subsample
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Figure 3-1. Sample Preparation and Analysis of Surface and
Drinking Water Samples
and used for analysis by the reference method. Four of the aliquots were left unspiked and
analyzed by the reference method, and four of the ali quots were fortified with 0.2 mg/L of KCN
as free cyanide at the reference laboratory just before the reference analyses took place. This was
done to closely mimic the time elapsed between when the LFM samples were fortified with
0.2 mg/L KCN as free cyanide and when they were analyzed during the testing of the Cyanide
ReagentStrip™ test kit.
Columbus, OH, city and well water samples were used to verify the field portabili ty of the
Cyanide ReagentStrip™ test kit. Approximately 20 L of water were collected from an outside
spigot at two participating residences, one with well water and one with Columbus, OH, city
water. The sample aliquots prepared for analysis by the Cyanide ReagentStrip™ test kit were
first analyzed in a Battelle laboratory. Then the samples were transported to the indoor field
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location and analyzed there. Finally, the samples were taken to the outdoor field location for
analysis. In both the laboratory and indoor field locations, the sample temperature was
approximately 21°C at the time of analysis, while the outdoor temperature decreased the sample
temperature at the time of analysis to approximately 17°C. Because the same sample aliquots
were analyzed at the different locations on the same day, only one set of reference samples was
sent to the reference laboratory for analysis. Each of the samples was treated as described above
and as shown in Figure 3-1.
3.5 Test Procedure
3.5.1 Sample Preparation
QC and PT samples were prepared from a commercially available and NIST-traceable standard.
The standard was dissolved and diluted to appropriate concentrations using ASTM Type EDI
water in Class A volumetric glassware. The QC and PT samples were prepared within one day of
testing. Samples sent to the reference laboratory were preserved with sodium hydroxide (NaOH)
at a pH >12, and stored at 4°C until analysis.
Surface and DW samples were collected from the sources indicated in Section 3.4.4 and were
stored in high-density polyethylene containers. Because free chlorine degrades cyanide during
storage, at the time of sample receipt, before NaOH preservation, all of the samples were tested
for free chlorine by adding one N,N-diethyl-/;-phenylenediamine (DPD) chlorine indicator tablet
(Orbeco Analytical Systems, Inc.) to 25 milliliters (mL) of the water sample and crushed with a
glass stirring rod. If the water turned pink, the presence of chlorine was indicated. All the DW
samples were tested in this manner; and, if the presence of chlorine was indicated,
approximately 60 mg of ascorbic acid were added per L of bulk sample to dechlorinate the
sample. A separate DPD indicator test (as described above) was done to confirm adequate
dechlorination of the sample (indicated by no color change).
3.5.2 Sample Identification
Aliquots to be analyzed were drawn from the standard solutions or from source and DW samples
and placed in uniquely identified sample containers for subsequent analysis. The sample
containers were identified by a unique ID number. A master log of the samples and sample ID
numbers for each unit being verified was kept by Battelle. The ID number, date, person
collecting, sample location, and time of collection were recorded on a chain-of-custody form for
all field samples.
3.5.3 Sample Analysis
Each day, test samples were prepared from the cyanide standard in either DI water, surface
water, or DW matrix. Each sample was prepared in its own container and labeled only with a
sample ID number that also was recorded in a laboratory record book, along with details of the
sample preparation. Prior to the analysis of each sample, the verification staff recorded the
sample ID number on a sample data sheet; then, after the analysis was complete, the result was
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recorded on the sample data sheet. Four replicates of each test sample were analyzed. Method
blank and QC standards were allowed to come to the same temperature as the samples prior to
analysis.
The Cyanide ReagentStrip™ test kit testing procedure included the following steps for analyzing
water samples for the presence of cyanide: (1)2 mL of water sample (with pH adjusted to
between 5 and 11) were added to a new disposable microcuvette using a disposable graduated
pipette supplied in the kit. (2) ReagentStrip™ #1 was dipped repeatedly into the sample for
30 seconds (using a timer) with a constant up-and-down motion at a rate of about one up-and-
down motion per second. The motion allowed the ReagentStrip™ to gently touch the bottom of
the microcuvette. After 30 seconds, ReagentStrip™ #1 was removed and discarded.
(3) ReagentStrip™ #2 was dipped into the sample for 30 seconds, with a constant up-and-down
motion as in Step 2. After 30 seconds, ReagentStrip™ #2 was removed, shaken once to remove
excess liquid, and immediately matched to the closest color on the color chart labeled
"ReagentStrip™ Colors" (shown in Figure 3-2) to obtain a semi-quantitative cyanide result.
Color matching was completed within 2 minutes, before the strip dried. If the color was between
two blocks, the concentration was estimated to a concentration half-way between the two blocks.
(4) The color of the solution in the microcuvette was then allowed to develop for a reaction time
according to the sample temperature. The wait time guidelines were as follows: 10 minutes (but
not more than 13 minutes) if the sample was 21°C to 28°C (all samples analyzed in the
laboratory and at the indoor field location fit into this category), 20 minutes (but not more than
26 minutes) if the sample was 15°C to 19°C, and 40 minutes (but not more than 50 minutes) if
the sample was 5°C to 14°C. (5) At the end of the microcuvette wait time, the microcuvette was
placed on the color chart labeled "Microcuvette Colors" (as shown in Figure 3-2). Looking from
above and down from the top, the microcuvette was moved within the various color boxes until
the closest match to the dark band in the center of the microcuvette was found. Matching was
completed within 1 to 2 minutes. If the color was between two blocks, the concentration was
estimated to a concentration half-way between the two blocks. (6) Immediately after determining
the semi-quantitative visual microcuvette result, 2 mL of unreacted water sample were added to
a clean microcuvette marked "reference sample" and inserted into the ReagentStrip™ Reader so
that the window faced front to back. It was pushed down fully so the microcuvette was locked
into place. The ReagentStrip™ Reader was zeroed by pressing the gray button marked "R," and
the orange button marked "T" was pressed to verify that the blank read 0.00 absorbance units.
The "reference sample" microcuvette was then set aside. (7) In a similar manner, the fully
reacted sample microcuvette was inserted into the ReagentStrip™ Reader, and the orange button
marked "T" was pressed to obtain an absorbance reading instantaneously. The absorbance value
was converted to cyanide concentration by the operator, using the reference table provided with
the kit. (8) The absorbance reading was obtained using both ReagentStrip™ Readers to obtain
inter-unit comparability data.
10
-------
ReagentStrip™ Colors
ppm (mg/L) - Free Cyanide
mr
wvrw.SENSAFE.com
<0.1 0.5 1 2 3 5 >10
III
MICROCUVETTE Colors ClED
ppm (mg/L) - Free Cyanide
0.05 0.1 0.2
0.5 >3 >20 >200
~ ~~~
MFfcCMMM - MT1
Figure 3-2. Cyanide ReagentStrip™ Test Kit Color
Charts
Results were recorded manually on appropriate data sheets. In addition to the analytical results,
the data sheets and corresponding laboratory notebooks included records of the time required for
sample analysis and operator observations concerning the use of the Cyanide ReagentStrip™
test kit (i.e., ease of use, maintenance, etc.).
While the Cyanide ReagentStrip™ test kit was being tested, a replicate sample set was being
analyzed by the reference laboratory. The reference instrument was operated according to the
recommended procedures in the instruction manual, and samples were analyzed according to
EPA Method 335.1<2) and ATEL standard operating procedures. Results from the reference
analyses were recorded electronically and compiled by ATEL into a report, including the sample
ED and the analyte concentration for each sample.
11
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Chapter 4
Quality Assurance/Quality Control
QA/QC procedures were performed in accordance with the quality management plan (QMP) for
the AMS Center(3) and the test/QA plan for this verification test.(1)
4.1 Reference Method Quality Control Results
Analyses of QC samples were used to document the performance of the reference method. To
ensure that no sources of contamination were present, RB samples were analyzed. The test/QA
plan stated that if the analysis of an RB sample indicated a concentration above the reporting
limit for the reference method, the contamination source was to be corrected and proper blank
reading achieved before proceeding with the verification test. Fourteen RB samples were
analyzed, and all of them were reported as below the 0.005-mg/L MDL for the reference method.
The reference instrument was calibrated initially according to the procedures specified in the
reference method. The accuracy of the reference method was verified with QCS samples
analyzed with each sample batch. One of two QCS samples, one with a concentration of 0.15
mg/L and the other with a concentration of 0.2 mg/L, were analyzed with each analytical batch
(approximately every 10 water samples). The test/QA plan(1) required the QCS results to always
be within the percent recovery range of 75 to 125%. As shown in Table 4-1, the percent
recoveries were always between 95 and 107%.
Reference LFM samples were analyzed to confirm the proper functioning of the reference
method and to assess whether matrix effects influenced the results of the reference method. The
LFM percent recovery of the spiked solution was calculated from the following equation:
c - c
% Recovery = — X 100 ^ '
s
where Cxis the reference concentration of the spiked sample, C is the reference concentration of
the background sample which, in this case, was always zero (results were below the MDL for the
reference method), and 5 is the fortified concentration of the cyanide spike. If the percent
recovery of an LFM fell outside the range of 75 to 125%, a matrix effect or some other analytical
problem was suspected. As shown in Table 4-2, there were no such instances during this
verification test. To mimic the elapsed time between fortification and analysis by the
ReagentStrip™ test kit, the reference LFM samples were spiked just minutes prior to analysis
12
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Table 4-1. Reference Method Quality Control Standard Results
Date
Reference Method
Result (mg/L)
Known QCS
Concentration (mg/L)
% Recovery
9/27/2004
0.145
0.150
97
9/27/2004
0.189
0.200
95
9/29/2004
0.144
0.150
96
9/29/2004
0.199
0.200
100
9/30/2004
0.159
0.150
106
9/30/2004
0.207
0.200
104
10/1/2004
0.156
0.150
104
10/1/2004
0.213
0.200
107
10/4/2004
0.153
0.150
102
10/4/2004
0.206
0.200
103
10/4/2004
0.154
0.150
103
10/5/2004
0.156
0.150
104
10/5/2004
0.212
0.200
106
10/5/2004
0.150
0.150
100
10/5/2004
0.206
0.200
103
using the reference method. The precision of the reference method was evaluated for each set of
samples analyzed by the reference method by calculating the relative standard deviation (RSD)
(formula shown in Section 5.2). These results also are shown in Table 4-2. All sample sets
resulted in RSDs <10%, indicating very reproducible results.
Table 4-2. Reference Method Laboratory-Fortified Matrix Analysis Results
Fortified Average Reference
Concentration
Concentration
%
%
Sample Description
(mg/L)
(mg/L)
Recovery
RSD
Alum Creek LFM
0.200
0.219
110
2
Olentangy River LFM
0.200
0.203
102
5
Des Moines, IA, LFM
0.200
0.206
103
1
Flagstaff, AZ, LFM
0.200
0.206
103
7
Montpelier, VT, LFM
0.200
0.189
95
4
Seattle, WA, LFM
0.200
0.190
95
7
Tallahassee, FL, LFM
0.200
0.227
113
10
Columbus, OH, City Water LFM
0.200
0.196
98
4
Columbus, OH, Well Water LFM
0.200
0.190
95
6
13
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4.2 Audits
4.2.1 Performance Evaluation Audit
A performance evaluation (PE) audit was conducted to assess the quality of the reference
measurements in this verification test. For the PE audit, an independent standard was obtained
from a different vendor than the one that supplied the QCSs. The relative percent difference
(RPD) of the measured concentration and the known concentration was calculated using the
following equation:
M
RPD=—x 100 (2)
A
where Mis the absolute difference between the measured and known concentrations, and A is
the mean of the same two concentrations. An RPD of <25% was required for the reference
measurements to be considered acceptable. Failure to achieve this agreement would have
triggered a repeat of the PE comparison. As shown in Table 4-3, all of the PE sample results
ranged from 0.5 to 2%, well below the required range.
Table 4-3. Summary of Performance Evaluation Audit
Measured
Known
Concentration
Concentration
RPD
Sample
Date of Analysis
(mg/L)
(mg/L)
(%)
PE-A
9/17/2004
0.199
0.200
0.50
PE-B
9/17/2004
0.199
0.200
0.50
PE-C
9/17/2004
0.196
0.200
2.02
PE-D
9/17/2004
0.199
0.200
0.50
4.2.2 Technical Systems Audit
Prior to using ATEL as the reference laboratory, the Battelle Quality Manager performed an audit
to ensure that ATEL was proficient in the reference analyses. This audit entailed a review of the
appropriate training records, state certification data, and the laboratory QMP. The Battelle
Quality Manager also conducted a technical systems audit (TSA) to ensure that the verification
test was performed in accordance with the test/QA plan(1) and the AMS Center QMP.(3) As part of
the audit, the Battelle Quality Manager compared the reference method used to the ATEL
standard operating procedures, compared actual test procedures to those specified in the test/QA
plan, and reviewed data acquisition and handling procedures. Observations and findings from
this audit were documented and submitted to the Battelle Verification Test Coordinator for
response. No findings were documented that required any corrective action. The records
concerning the TSA are stored for at least seven years with the Battelle Quality Manager.
14
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4.2.3 Audit of Data Quality
At least 10% of the data acquired during the verification test were audited. Battelle's Quality
Manager traced the data from the initial acquisition, 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.
4.3 Quality Assurance/Quality Control Reporting
Each assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5 of the
QMP for the ETV AMS Center.(3) Once the assessment report was prepared, the Battelle
Verification Test Coordinator ensured that a response was provided for each adverse finding or
potential problem and implemented any necessary follow-up corrective action. The Battelle
Quality Manager ensured that follow-up corrective action was taken. The results of the TSA were
sent to the EPA.
4.4 Data Review
Records generated in the verification test were reviewed before these records were used to
calculate, evaluate, or report verification results. Table 4-4 summarizes the types of data
recorded. The review was performed by a technical staff member involved in the verification test,
but not the staff member who originally generated the record. The person performing the review
added his/her initials and the date to a hard copy of the record being reviewed.
15
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Table 4-4. Summary of Data Recording Process
Data to be
Recorded
Responsible
Party
Where Recorded
How Often
Recorded
Disposition of Data(a)
Dates, times of test
events
Reference method
sample analysis,
chain of custody,
results
Battelle
Test parameters Battelle
(sample
temperature, analyte
concentrations,
location, etc.)
Water sampling data Battelle
ATEL
Laboratory record
books
Laboratory record
books
Laboratory record
books
Laboratory record
book/data sheets or
data acquisition
system, as
appropriate
Start/end of test; at
each change of a
test parameter
When set or
changed, or as
needed to
document stability
At least at the time
of sampling
Throughout sample
handling and
analysis process
Used to organize/
check test results;
manually incorporated
data into spreadsheets
as necessary
Used to organize/
check test results;
manually incorporated
data into spreadsheets
as necessary
Used to organize/
check test results;
manually incorporated
data into spreadsheets
as necessary
Excel spreadsheets
(fl> All activities subsequent to data recording were carried out by Battelle.
16
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Chapter 5
Statistical Methods and Reported Parameters
The statistical methods presented in this chapter were used to verify the performance parameters
listed in Section 3.1.
5.1 Accuracy
Accuracy was assessed relative to the results obtained from the reference analyses. Samples were
analyzed by both the reference method and the Cyanide ReagentStrip™ test kit. The results for
each set of analyses were averaged, and the accuracy was expressed in terms of a relative average
bias (B) as calculated from the following equation:
where d is the average difference between the Cyanide ReagentStrip™ test kit results and the
result from the reference method, and CR is the average of the reference measurements. The
semi-quantitative results were not evaluated using a bias calculation because of their subjective
nature. Because the color charts have only discrete colors from which to choose, the bias is
influenced by the number of possible results between one color and the next. To better summarize
the semi-quantitative results, each test sample was assigned the color on each test strip
representing the concentration closest to the concentration determined by the reference method.
The frequency with which the test results matched that color exactly was evaluated, as well as
how often the test results were within one color of the color closest to the reference concentration
color.
5.2 Precision
The standard deviation (S) of the results for the replicate samples was calculated and used as a
measure of Cyanide ReagentStrip™ test kit precision at each concentration.
d
B = = x 100
Cr
(3)
1/2
(c'-c)2
(4)
k=1
17
-------
where n is the number of replicate samples, Ck is the concentration measured for the kth sample,
and C is the average concentration of the replicate samples. The Cyanide ReagentStrip™ test kit
precision at each concentration was reported in terms of the RSD, e.g.,
RSD
S
c
x 100% (5)
For the semi-quantitative strip and microcuvette color chart results, the precision was evaluated
by showing whether or not the same result was determined for each replicate analysis. A "yes"
indicates that all four results were reported as the same color, and a "no" indicates that they were
not.
5.3 Linearity
Linearity was assessed by linear regression, with the analyte concentration measured by the
reference method as independent variable and the ReagentStrip™ Reader result from the Cyanide
ReagentStrip™ test kit as the dependent variable. Linearity is expressed in terms of the slope,
intercept, and the coefficient of determination (r2). The semi-quantitative results were not
conducive to an evaluation of linearity because of the relatively large concentration ranges
encompassed by each individual color.
5.4 Method Detection Limit
The MDL(4) for the Cyanide ReagentStrip™ test kit was assessed from the seven replicate
analyses of a fortified sample with a cyanide concentration of approximately five times the
manufacturer's reported detection limit. The MDL was calculated from the following equation:
MDL = txS (6)
where t is the Student's value for a 99% confidence level, and S is the standard deviation of the
replicate samples. The MDL for each ReagentStrip™ Reader was reported separately. Again, the
semi-quantitative results were not conducive to a statistical evaluation of MDL. A qualitative
evaluation of the concentration levels that produced detectable semi-quantitative results was
reported.
5.5 Inter-Unit Reproducibility
The quantitative results obtained from two ReagentStrip™ Readers were compiled and compared
to assess inter-unit reproducibility. The results were interpreted using a linear regression of the
results for one ReagentStrip™ Reader plotted against the results produced by the other
ReagentStrip™ Reader. If the ReagentStrip™ Readers function identically, the slope of such a
regression will not differ significantly from unity.
18
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5.6 Lethal or Near-Lethal Dose Response
The Cyanide ReagentStrip™ test kit was not designed to quantitatively measure near-lethal or
lethal concentrations of cyanide in water. However, the Cyanide ReagentStrip™ test kit
semi-quantitative strip and microcuvette analysis options give semi-quantitative information in
addition to the indication that a sample has reached the top of the quantitative range.
Additionally, the operators and the Battelle Verification Test Coordinator made qualitative
observations of the Cyanide ReagentStrip™ test kit operation while analyzing such samples.
Observations of unusual operational characteristics (rate of color change, unusually intense color,
unique digital readout, etc.) were documented and reported.
5.7 Operator Bias
To assess operator bias for the Cyanide ReagentStrip™ test kit, the results obtained from a
technical and non-technical operator were compiled independently and subsequently compared.
The results were interpreted using a linear regression of the non-technical operator's results
plotted against the results produced by the technical operator. If the operators obtained identical
results, the slope of such a regression would not differ significantly from unity. The
semi-quantitative strip and microcuvette results were evaluated in this manner by determining the
frequency by which both operators produced results within one color gradient of one another.
5.8 Field Portability
The results obtained from the measurements made on DW samples in the laboratory and indoor
and outdoor field settings were compared to assess the accuracy of the measurements under the
different analysis conditions. The results were interpreted qualitatively because of the small
number of samples.
5.9 Ease of Use
Ease of use was a qualitative measure of the user friendliness of the Cyanide ReagentStrip™ test
kit, including how easy or hard the instruction manual was to use.
5.10 Sample Throughput
Sample throughput indicated the amount of time required to analyze a set of samples, including
both sample preparation and analysis.
19
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Chapter 6
Test Results
The results of the verification test of the Cyanide ReagentStrip™ test kit are presented in this
section.
6.1 Accuracy
Tables 6-1 a-d present the cyanide results from analysis of the PT samples; surface water; DW
from various regions of the United States; and DW from Columbus, OH, respectively, for both
the reference analyses and the Cyanide ReagentStrip™ test kit. The Cyanide ReagentStrip™ test
kit results include the quantitative results from the ReagentStrip™ Reader and semi-quantitative
results from the strips and microcuvettes. Results are shown for both ReagentStrip™ Readers that
were tested (labeled as Unit #1 and #2), as well as for the technical and non-technical operators.
On Tables 6-1 a-d, the individual results are shaded to indicate how the color matched with
respect to the color that represented the reference concentration.
Tables 6-2a-d present the percent accuracy of the quantitative Cyanide ReagentStrip™ test kit
results. The bias values were determined according to Equation (3), Section 5.1. Bias was not
calculated for background samples with non-detectable concentrations of cyanide. The bias
values shown in Tables 6-2a-d can be summarized by the range of bias observed with different
sample sets. For example, for the quantitative results, biases ranged from -47 to 25% for the PT
samples, -27 to 28% for the surface water samples, -41 to 3% for the DW samples from around
the United States, and -91 to 30% for the Columbus, OH, DW samples. However, if the outdoor
samples are removed, the range of biases changes to -42 to 30%.
The impact of the various surface water and DW sample matrices was not clear when evaluating
the accuracy results. No particular source of water (DI water included) produced results with
consistently low biases. In general, the bias results of the surface water and DW samples were in
a range similar to the PT samples that were prepared in DI water; therefore, it seems that the
matrix effect on these results was minimal.
The semi-quantitative results were not evaluated this way because of their subjective nature.
Since the color charts have only discrete colors from which to choose, the bias could be greatly
influenced because of the number of possible results between one color and the next. For
example, for the strips, if the test concentration was 0.200 mg/L, the closest detectable result on
20
-------
Table 6-la. Cyanide Results from Performance Test Samples
Non-technical Operator Technical Operator
Prepared Cone.
Ref. Cone.
Unit #1
Unit #2
Strip
Microcuvette
Unit #1
Unit #2
Strip
Microcuvette
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
0.03
0.038
0.03
0.03
<0.1
0.05
0.03
0.04
<0.1
0.05
0.03
0.035
0.04
0.02
<0.1
0.05
0.01
0.01
<0.1
0.05
0.03
0.024
0.03
0.04
<0.1
0.05
0.02
0.01
<0.1
0.05
0.03
0.035
0.02
0.03
<0.1
0.05
0.01
0.01
<0.1
0.05
0.1
0.108
0.08
0.08
<0.1
0.05
0.06
0.08
0.5
0.1
0.1
0.071
0.07
0.07
0.5
0.1
0.05
0.06
<0.1
0.075
0.1
0.093
0.06
0.08
<0.1
0.05
0.05
0.05
<0.1
0.075
0.1
0.091
0.05
0.05
<0.1
0.05
0.04
0.05
<0.1
0.075
0.2
0.211
0.16
0.17
0.5
0.2
0.14
0.14
0.25
0.1
0.2
0.195
0.12
0.14
0.5
0.2
0.14
0.14
0.25
0.2
0.2
0.209
0.11
0.13
0.5
0.2
0.13
0.13
0.25
0.15
0.2
0.174
0.10
0.10
0.5
0.2
0.13
0.14
0.25
0.15
0.4
0.392
0.43
0.36
0.5
>3
0.28
0.28
0.75
0.5
0.4
0.416
0.46
0.58
0.5
>3
0.30
0.31
0.75
0.5
0.4
0.366
0.32
0.35
1
>3
0.28
0.28
0.75
0.5
0.4
0.406
0.33
0.36
0.5
>3
0.24
0.26
0.75
0.2
1
0.955
1.1
1.2
2
>3
0.97
1.16
2.5
0.5
1
0.905
0.95
1.0
2
>3
0.85
0.92
2.5
0.5
1
1.01
0.96
1.1
2
>3
0.69
0.74
2.5
0.5
1
0.965
0.87
0.96
2
>3
0.67
0.72
1.5
0.5
5
4.90
5.0
4.9
5
>3
5
5
7.5
>3
5
4.88
6.9
6.9
5
>3
5
5.4
5
>3
5
4.79
5.8
5.4
5
>3
5.4
5.0
5
>3
5
4.60
6.2
6.2
5
>3
4.5
4.9
5
>3
15
14.0
17
16
>10
>20
14
14
10
>20
15
15.8
26
21
>10
>20
16
14
10
>20
15
13.2
15
14
>10
>20
17
16
10
>20
15
15.5
14
13
>10
>3
18
17
10
>20
25
22.8
21
19
>10
>3
20
19
10
>20
25
23.8
22
20
>10
>20
28
22
10
>20
25
23.1
31
24
>10
>3
19
16
10
>20
25
25.4
29
22
>10
>3
29
24
10
>20
exact match to the color that should I 1 within one color of the color that should
represent the reference laboratory result represent the reference laboratory result
the strip color chart would be 0.5 mg/L, which would result in a bias of approximately 150%. The
colors representing discrete concentration ranges cause a similar problem in evaluating the
accuracy of the microcuvette result. To better summarize the semi-quantitative results, each test
sample was assigned the color on each test strip representing the concentration closest to the
concentration determined by the reference method. Then the frequency with which the test results
(1) matched that color exactly, (2) were within one color of the "correct" color with respect to the
reference concentration, or (3) were within two colors of the reference concentration was
evaluated. These categories are shown with colored shading on the data tables.
21
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Table 6-lb. Cyanide Results from Surface Water
Non-technical Operator Technical Operator
Cone. Unit #1
Unit #2
Strip
Microcuvette
Unit #1
Unit #2
Strip
Microcuvette
(mg/L) (mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Alum Creek Background
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Alum Creek LFM
0.216 0.19
0.19
0.5
0.5
0.18
0.18
0.25
0.2
0.215 0.21
0.19
0.5
0.5
0.18
0.18
0.5
0.5
0.221 0.12
0.09
0.5
0.5
0.15
0.16
0.5
0.5
0.224 0.16
0.17
0.5
0.5
0.2
0.22
0.5
0.5
Olentangy River Background
<0.005 <0.01
<0.01
<0.1
0
0.03
0.03
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Olentangy River LFM
0.197 ' 0.26
0.27
0.5
0.5
0.25
0.26
0.5
0.5
0.191 0.25
0.23
0.5
0.5
0.30
0.31
0.5
0.5
0.214 0.24
0.22
0.5
0.5
0.20
0.21
0.5
0.2
0.21 0.20
0.21
0.5
0.5
0.25
0.26
0.5
0.5
exact match to the color that should I 1 within one color of the color that should
represent the reference laboratory result represent the reference laboratory result
Table 6-3 summarizes this semi-quantitative accuracy evaluation for each type of test sample. For
all strip results, 84% of the PT sample results matched the exact color and 16% were within one
color on the color chart; 100% of the surface water and U.S. DW samples matched the exact color;
and 83% of the Columbus, OH, DW samples matched exactly, with the remaining 17% within one
color. For all microcuvette results, 64% of the PT samples matched exactly and 36% were within
one color; 56% of the surface water samples matched exactly, with 44% being within one color.
For U.S. DW, these numbers were 88% and 12%, respectively. For the Columbus, OH, DW
samples, 66% matched exactly, 17% were within one color, and 17% were not within one color on
the color chart.
6.2 Precision
Tables 6-4a-d show the RSD of the cyanide analysis results from PT samples; surface water; DW
from around the U.S.; and DW from Columbus, OH, respectively, for the Cyanide ReagentStrip™
test kit and the reference method. Results are shown for both units that were tested. RSDs were not
calculated for results reported as less than the reporting limit for the Cyanide ReagentStrip™ test
kit. The RSD values shown in Tables 6-4a-d can be summarized by the range of RSDs observed
with different sample sets. For example, the RSDs ranged from 4 to 86% for the PT samples
22
-------
Table 6-lc. Cyanide Results from U.S. Drinking Water
Non-technical Operator Technical Operator
Cone.
Unit #1
Unit #2
Strip
Microcuvette
Unit #1
Unit #2
Strip
Microcuvette
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Des Moines, IA, Background
<0.005
<0.01
<0.01
<0.1
0
0.01
<0.01
<0.1
0
<0.005
0.02
0.02
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Des Moines, IA, LFM
0.208
0.14
0.14
0.5
0.5
0.16
0.15
0.5
0.15
0.207
0.15
0.15
0.5
0.5
0.15
0.14
0.5
0.15
0.203
0.14
0.14
0.5
0.2
0.15
0.16
0.5
0.2
0.206
0.12
0.13
0.5
0.2
0.18
0.18
0.5
0.15
Flagstaff, AZ, Background
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Flagstaff, AZ, LFM
0.22
0.11
0.11
0.5
0.5
0.12
0.12
0.5
0.2
0.193
0.12
0.14
0.5
0.5
0.11
0.12
0.5
0.2
0.206
0.11
0.11
0.5
0.5
0.14
0.14
0.5
0.2
0.206
0.16
0.18
0.5
0.5
0.12
0.12
0.5
0.15
Montpelier, VT, Background
<0.005
0.01
0.01
<0.1
0
<0.01
<0.01
<0.1
0
0.018
0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
0.33
<0.01
<0.01
<0.1
0
0.01
<0.01
<0.1
0
<0.005
<0.01
0.01
<0.1
0
0.01
<0.01
<0.1
0
Montpelier, VT, Background
0.187
0.15
0.15
0.5
0.2
0.18
0.18
0.5
0.2
0.194
0.17
0.17
0.5
0.2
0.21
0.21
0.5
0.2
0.197
0.15
0.15
0.5
0.2
0.18
0.18
0.5
0.2
0.198
0.17
0.18
0.5
0.2
0.21
0.21
0.5
0.2
Seattle, WA, Background
<0.005
<0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005
0.02
0.02
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
0.01
0.02
<0.1
0
0.01
<0.01
<0.1
0
Seattle, WA, LFM
0.199
0.15
0.16
0.5
0.2
0.17
0.18
0.5
0.2
0.171
0.14
0.14
0.5
0.2
0.19
0.20
0.5
0.2
0.202
0.15
0.15
0.5
0.2
0.20
0.21
0.5
0.2
0.188
0.15
0.17
0.5
0.2
0.15
0.16
0.5
0.2
Tallahassee, FL, Background
<0.005
<0.01
<0.01
<0.1
0
0.02
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Tallahassee, FL Background
0.208
0.15
0.16
0.5
0.5
0.17
0.16
0.5
0.2
0.21
0.16
0.16
0.5
0.5
0.16
0.14
0.5
0.15
0.231
0.16
0.17
0.5
0.5
0.16
0.17
0.5
0.15
0.257
0.15
0.15
0.5
0.5
0.18
0.17
0.5
0.2
exact match to the color that should within one color of the color that should
represent the reference laboratory result represent the reference laboratory result
23
-------
Table 6-ld. Cyanide Results from Columbus, OH, Drinking Water
Non-technical Operator'3'
Technical Operator
Ref. Cone. Unit #1
Unit #2
Strip Microcuvette
Unit #1
Unit #2
Strip
Microcuvette
(mg/L) (mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
City Water Background
- Outdoor Field Site
<0.005 0.01
0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
City Water Background
- Indoor Field Site
<0.005 <0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.01
0.02
<0.1
0
City Water Background
- Lab
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
City LFM - Outdoor Field Site
0.203 0.05
0.05
<0.1
0.05
0.04
0.06
<0.1
0.05
0.191 0.03
0.03
<0.1
0.05
<0.01
0.01
<0.1
0.05
0.187 0.02
0.03
<0.1
0.05
0.03
0.03
<0.1
0.05
0.201 0.05
0.06
<0.1
0.05
<0.01
<0.01
<0.1
0
City LFM - Indoor Field Site
0.203 0.23
0.25
0.5
0.5
0.25
0.26
0.5
0.5
0.191 0.16
0.18
0.5
0.5
0.24
0.26
0.5
0.5
0.187 0.22
0.24
0.5
0.5
0.25
0.26
0.5
0.5
0.201 0.27
0.28
0.5
0.5
0.24
0.24
0.5
0.5
City LFM - Lab
0.203 0.19
0.21
0.5
0.5
0.24
0.24
0.5
0.5
0.191 0.20
0.23
0.5
0.2
0.23
0.24
0.5
0.5
0.187 0.16
0.16
0.5
0.2
0.25
0.26
0.5
0.5
0.201 0.21
0.20
0.5
0.5
0.19
0.21
0.5
0.5
Well Water Background
- Outdoor Field Site
<0.005 <0.01
<0.01
<0.1
0
0.06
0.05
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.04
0.05
<0.1
0
<0.005 0.05
0.05
<0.1
0
<0.01
<0.01
<0.1
0
Well Water Background
- Indoor Field Site
<0.005 <0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.05
0.05
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.02
0.02
<0.1
0
<0.005 <0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
24
-------
Table 6-ld. Cyanide Results from Columbus, OH, Drinking Water (continued)
Non-technical Operator1
Ja)
Technical Operator
Ref. Cone.
Unit #1
Unit #2
Strip
Microcuvette
Unit #1
Unit #2
Microcuvette
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
Strip (mg/L)
(mg/L)
Well Water Background
-Lab
<0.005
<0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005
<0.01
<0.01
<0.1
0
0.01
0.01
<0.1
0
<0.005
0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
<0.005
0.01
<0.01
<0.1
0
<0.01
<0.01
<0.1
0
Well Water LFM - Outdoor Field Site
0.188
0.01
0.01
<0.1
0
0.07
0.08
<0.1
0.05
0.186
0.05
0.06
<0.1
0.05
0.02
0.01
<0.1
0.05
0.205
0.02
0.03
<0.1
0
0.04
0.05
<0.1
0.05
0.18
0.02
0.02
<0.1
0
<0.01
<0.01
<0.1
0
Well Water LFM - Indoor Field Site
0.188
0.12
0.13
0.5
0.5
0.20
0.21
0.5
0.15
0.186
0.08
0.09
0.5
0.2
0.21
0.21
0.5
0.2
0.205
0.12
0.12
0.5
0.5
0.18
0.19
0.5
0.15
0.18
0.12
0.12
0.5
0.2
0.15
0.16
0.5
0.2
Well Water LFM - Lab
0.188
0.14
0.15
0.5
0.2
0.14
0.14
0.5
0.2
0.186
0.11
0.13
0.5
0.2
0.14
0.15
0.5
0.2
0.205
0.10
0.11
0.5
0.2
0.16
0.16
0.5
0.2
0.18
0.11
0.11
0.5
0.2
0.13
0.14
0.5
0.2
131 The non-technical operator's outdoor field results were recorded after a 40-minute reaction time.
| 1 exact match to the color that should ~
represent the reference laboratory
result
within one color of the color
that should represent the
reference laboratory result
within two colors of the color
that should represent the
reference laboratory result
Table 6-2a. Percent Accuracy of Performance Test Sample Measurements
Sample Non-technical Operator Technical Operator
Concentration
Unit #1 (%
Unit #2
Unit #1
Unit #2
(mg/L)
bias)
(% bias)
(% bias)
(% bias)
0.03
-9
-9
-47
-47
0.1
-28
-23
-45
-34
0.2
-38
-32
-32
-30
0.4
-3
4
-30
-28
1
1
11
-17
-8
5
25
22
4
6
15
23
10
11
4
25
8
-11
1
-15
25
-------
Table 6-2b. Percent Accuracy of Surface Water Measurements
Non-technical Operator
Technical Operator
Unit #1 Unit #2
Unit #1
Unit #2
(% bias) (% bias)
(% bias)
(% bias)
Alum Creek LFM
-22 -27
-19
-16
Olentangy River LFM
17 15
23
28
Table 6-2c. Percent Accuracy of U.S. Drinking Water Measurements
Non-technical Operator
Technical Operator
Unit #1 Unit #2
Unit #1
Unit #2 (%
(% bias) (% bias)
(% bias)
bias)
Des Moines, IA, LFM
-33 -32
-22
-24
Flagstaff, AZ, LFM
-39 -35
-41
-39
Montpelier, VT, LFM
-15 -14
3
3
Seattle, WA, LFM
-22 -18
-7
-1
Tallahassee, FL, LFM
-32 -29
-26
-29
Table 6-2d. Percent Accuracy of Columbus, OH, Drinking Water Measurements
Non-technical Operator'3' Technical Operator
Unit #1 (% Unit #2 Unit #1 (%
Unit #2 (%
bias) (% bias) bias)
bias)
City Water LFM - Outdoor Field Site
I
00
1
00
1
-87
City Water LFM - Indoor Field Site
13 21 25
30
City Water LFM - Lab
-3 2 16
21
Well Water LFM - Outdoor Field Site
-87 -84 -83
-82
Well Water LFM - Indoor Field Site
-42 -39 -3
1
Well Water LFM - Lab
-39 -34 -25
-22
(a) The non-technical operator's outdoor field results were recorded after a 40-minute reaction time.
26
-------
Table 6-3. Semi-Quantitative Accuracy Evaluation
Strip Results Microcuvette Results
(number of
samples)
Qualitative Criteria
Non-
technical
Technical
Overall
Non-
technical
Technical
Overall
Exact Color Match
81% (26)
88% (28)
84% (54)
47% (15)
81% (26)
64% (41)
PT (32)
Within One Color
19% (6)
13% (4)
16% (6)
53% (17)
19% (6)
36% (23)
Not Within One Color
0% (0)
0% (0)
0% (0)
0% (0)
0% (0)
0% (0)
Exact Color Match
100% (16)
100% (16)
100% (32)
50% (8)
62% (10)
56% (18)
Surface (16)
Within One Color
0% (0)
0% (0)
0% (0)
50% (8)
38% (6)
44% (14)
Not Within One Color
0% (0)
0% (0)
0% (0)
0% (0)
0% (0)
0% (0)
U.S. DW
(40)
Exact Color Match
100% (40)
100% (40)
100% (80)
75% (30)
100% (40)
88% (70)
Within One Color
0%
0%
0% (0)
25% (10)
0% (0)
12% (10)
Not Within One Color
0%
0%
0% (0)
0% (0)
0% (0)
0% (0)
Columbus,
Exact Color Match
83% (40)
83% (40)
83% (80)
66% (32)
66% (32)
66% (64)
OH, DW
Within One Color
17% (8)
17% (8)
17% (16)
17% (8)
17% (8)
17% (16)
(32)
Not Within One Color
0% (0)
0% (0)
0% (0)
17% (8)
17% (8)
17% (16)
Table 6-4a. Precision of Performance Test Measurements
Sample Ref. Non-technical Operator Technical Operator
Cone.
Method
Unit #1
Unit #2
Unit #1
Unit #2
(mg/L)
(% RSD)
(% RSD)
(% RSD)
Strip
Microcuvette
(% RSD)
(% RSD)
Strip
Microcuvette
0.03
19
27
27
Yes
Yes
55
86
Yes
Yes
0.1
17
20
20
No
No
16
24
No
Yes
0.2
9
21
21
Yes
Yes
4
4
Yes
No
0.4
5
18
27
No
Yes
9
7
Yes
No
1
4
10
10
Yes
Yes
18
23
No
Yes
5
3
14
15
Yes
Yes
7
4
No
Yes
15
8
30
22
Yes
No
11
10
Yes
Yes
25
5
19
10
Yes
No
22
17
Yes
Yes
"Yes" or "no" indicates whether or not the four replicate samples generated the same results.
Table 6-4b. Precision of Surface Water Measurements
Non-technical Operator Technical Operator
Ref.
Method Unit #1
Unit #2
Unit #1
Unit #2
(% RSD) (% RSD)
(% RSD)
Strip
Microcuvette
(% RSD)
(% RSD)
Strip
Microcuvette
Alum Creek LFM
2 23
30
Yes
Yes
12
14
Yes
No
Olentangy River LFM
5 11
11
Yes
Yes
16
16
Yes
No
"Yes" or "no" indicates whether or not the four replicate samples generated the same results.
27
-------
Table 6-4c. Precision of U.S. Drinking Water Measurements
R
-------
evaluation, if a result was extrapolated between two colors, the result was rounded up to the
nearest color. For the strip results, the same result was obtained for all four replicates in 11 out of
16 PT sample sets, all four of the surface water sample sets, all 10 U.S. DW sample sets, and 11
out of 12 sample sets of Columbus, OH, DW. For the microcuvettes, consistent results were
obtained for 11 of 16 PT sample sets, 2 out of 4 surface water sample sets, 9 out of 10 U.S. DW
sample sets, and 7 out of 12 sample sets of Columbus, OH, DW.
6.3 Linearity
The linearity of the Cyanide ReagentStrip™ test kit was assessed by using a linear regression of
the quantitative PT results (0.03 to 25 mg/L) against the reference method results (Table 6-la).
Figures 6-1 and 6-2 show scatter plots of the results from the non-technical and technical
operators, respectively, versus the reference results. A dotted regression line with a slope of unity
and intercept of zero also is shown in Figures 6-1 and 6-2. A linear regression of the data in
Figure 6-1 for the non-technical operator gives the following regression equation.
y (non-technical operator results in mg/L) = 1.03 (± 0.06)x (reference result in mg/L)
+ 0.19 (± 0.55) mg/L with r2 = 0.947 and N = 72 (64 PT and 8 MDL).
where the values in parentheses represent the 95% confidence interval (two times the standard
error) of the slope and intercept, r2 is the coefficient of determination, and N is the total number
of results with corresponding reference analyses.
A linear regression of the data in Figure 6-2 for the technical operator gives the following
regression equation:
y (technical operator results in mg/L) = 0.97 (± 0.04)x (reference result in mg/L)
+ 0.07 (± 0.42) mg/L with r2 = 0.965 and N = 72 (64 PT and 8 MDL).
The slopes of these regressions are not significantly different from unity, neither intercept is
significantly different from zero, and the r2 values are both above 0.94.
Because of the 0.2-mg/L EPA maximum contaminant level for cyanide, this type of cyanide
detection technology is often used to measure concentrations near the low end of the 0.03 to
25 mg/L concentration range. The linearity of the performance test samples ranging in
concentration from 0.03 to 1 mg/L is shown in Figures 6-3 and 6-4. A linear regression of the
data in Figure 6-3 for the non-technical operator gives the following regression equation:
y (non-technical operator results in mg/L) = 1.08 (± 0.05)x (reference result in mg/L)
- 0.03 (± 0.02) mg/L with r2 = 0.973 and N = 48 (40 PT and 8 MDL).
29
-------
Reference Method Results (mg/L)
Figure 6-1. Non-technical Operator Linearity Results (0.03 to 25 mg/L)
Reference Method Results (mg/L)
Figure 6-2. Technical Operator Linearity Results (0.03 to 25 mg/L)
30
-------
Reference Method Results (mg/L)
Figure 6-3. Non-technical Operator Linearity Results (0.03 to 1 mg/L)
Reference Method Results (mg/L)
Figure 6-4. Technical Operator Linearity Results (0.03 to 1 mg/L)
31
-------
A linear regression of the data in Figure 6-4 for the technical operator gives the following
regression equation:
y (technical operator results in mg/L) = 0.88 (± 0.07)x (reference result in mg/L)
- 0.03 (±0.03) mg/L with r2 = 0.934 and N = 48 (40 PT and 8 MDL).
Within this smaller concentration range, both slopes were significantly different from unity, while
the intercepts were either not significantly different from zero or very close to it. This suggests
that the non-technical operator's results have a slightly high bias, and the technical operator's
results have a slightly low bias. This effect was not apparent with the wider concentration range.
Linearity was not evaluated for the semi-quantitative results because the colors on the color
charts encompassed discrete concentration ranges.
6.4 Method Detection Limit
The manufacturer's estimated detection limit for the Cyanide ReagentStrip™ test kit is 0.01
mg/L cyanide. The MDL(4) was determined by analyzing seven replicate samples at a
concentration of 0.05 mg/L. Table 6-5 shows the results of the MDL assessment. The MDLs
determined as described in Equation (6) of Section 5.4 were 0.04 and 0.03 mg/L when used by
the non-technical operator and 0.02 for both units when used by the technical operator. A
quantitative evaluation of MDL was not performed for the semi-quantitative strips and
microcuvettes. However, the results for the 0.05-mg/L samples are shown in Table 6-5. In all
cases, the strips had non-detectable (i.e.,<0.1 mg/L) results, and all of the microcuvette results
were 0.05 mg/L. In both cases, the results were as expected for this concentration. An evaluation
of the PT sample results reveals that the strips generated detectable results at all concentration
levels 0.2 mg/L and above and the microcuvette at all concentration levels tested (0.03 mg/L and
above).
6.5 Inter-Unit Reproducibility
The inter-unit reproducibility of the ReagentStrip™ Reader was assessed by using a linear
regression of the results produced by one ReagentStrip™ Reader plotted against the results
produced by the other ReagentStrip™ Reader. The results from all of the samples that had
detectable amounts of cyanide (including the PT, surface, and DW samples) were included in this
regression. Figure 6-5 shows a scatter plot of the results from both Cyanide ReagentStrip™ test
kits. A dotted regression line with a slope of unity and intercept of zero also is shown in
Figure 6-5.
32
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Table 6-5. Results of Method Detection Limit Assessment
Sample Non-technical Operator Technical Operator
Cone.
Unit #1
Unit #2
Strip
Microcuvette
Unit #1
Unit #2
Strip
Microcuvette
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
(mg/L)
PT-0.05
0.05
0.05
<0.1
0.05
0.03
0.03
<0.1
0.05
PT-0.05
0.02
0.02
<0.1
0.05
0.02
0.03
<0.1
0.05
PT-0.05
0.03
0.03
<0.1
0.05
0.02
0.02
<0.1
0.05
PT-0.05
0.02
0.02
<0.1
0.05
0.02
0.02
<0.1
0.05
PT-0.05
0.03
0.03
<0.1
0.05
0.03
0.03
<0.1
0.05
PT-0.05
0.04
0.03
<0.1
0.05
0.02
0.02
<0.1
0.05
PT-0.05
0.04
0.03
<0.1
0.05
0.03
0.02
<0.1
0.05
Std. Dev.
1.11x1 0"2
l.OOxlO"2
NA(a)
NA
5.34xl0"3
5.35xl0"3
NA
NA
t (n=7)
3.14
3.14
NA
NA
3.14
3.14
NA
NA
MDL
0.04
0.03
NA
NA
0.02
0.02
NA
NA
(a) NA = Not applicable because a statistical evaluation of MDL for the semi-quantitative results was not appropriate.
exact match to the color that should represent the
reference laboratory results
Figure 6-5. Inter-Unit Reproducibility Results
33
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A linear regression of the data in Figure 6-5 for the inter-unit reproducibility assessment gives the
following regression equation:
y (Unit #1 result in mg/L) = 1.17 (± 0.02)x (Unit #2 in mg/L) - 0.08 (± 0.08) mg/L
with r2 = 0.991 and N = 213 (includes all pairs of detectable results).
The intercept was not significantly different from zero; however, the slope was significantly
larger than unity, suggesting that, in general, Unit #1 gave higher responses than Unit #2.
6.6 Lethal or Near-Lethal Dose Response
Samples at 50-, 100-, and 250-mg/L concentrations (close to what may be lethal if a volume the
size of a typical glass of water were ingested) were analyzed by the Cyanide ReagentStrip™ test
kit. Table 6-6 presents the measured cyanide results from analysis of the lethal/near-lethal
concentration samples for both the reference analyses and the Cyanide ReagentStrip™ test kit.
Results are shown in Table 6-6 for both ReagentStrip™ Readers. The vendor states in the
instructions that cyanide concentrations above 40 mg/L exceed the linear range of the
ReagentStrip™ Reader and need to be diluted to obtain quantitative results. Because of the
various lower concentrations that were tested as PT samples, additional dilutions were not
performed. The ReagentStrip™ Reader results for these concentrations ranged from 36 to
>60 mg/L (the response to indicate that the top of the linear range of the ReagentStrip™ Reader
has been reached). For the 50-mg/L samples, 11 out of 16 results produced a >60-mg/L result.
For the 100- and 200-mg/L samples, 23 out of 32 results correctly produced a >60-mg/L result.
Table 6-6. Lethal/Near-Lethal Concentration Sample Results
Non-technical Operator Technical Operator
Prepared
Cone.
(mg/L)
Ref. Cone.
(mg/L)
Unit #1
(mg/L)
Unit #2
(mg/L)
Strip
(mg/L)
Microcuvette
(mg/L)
Unit #1
(mg/L)
Unit #2
(mg/L)
Strip
(mg/L)
Microcuvette
(mg/L)
50
44.5
>60
>60
>10
>3
>60
>60
>10
>20
50
45.8
>60
>60
>10
>20
>60
41
>10
>20
50
45.5
>60
46
>10
>3
>60
38
>10
>20
50
49.8
>60
50
>10
>3
>60
55
>10
>20
100
102
>60
55
>10
>20
>60
>60
>10
>20
100
95.5
>60
>60
>10
>20
>60
>60
>10
>20
100
102
>60
60
>10
>3
>60
>60
>10
>20
100
94
>60
>60
>10
>3
>60
>60
>10
>20
250
265
>60
>60
>10
>20
>60
50
>10
>20
250
245
55
36
>10
>20
>60
36
>10
>20
250
239
>60
60
>10
>3
>60
41
>10
>20
250
253
>60
>60
>10
>3
>60
55
>10
>20
exact match to the color that ~ within one color ot the color within two colors of the color
should represent the reference that should represent the sjlouy represent the
laboratory result reference laboratory result reference laboratory result
34
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For the semi-quantitative strips, all 12 samples for both operators correctly resulted in a
>10-mg/L (the highest concentration color) result. For the microcuvettes, all 12 samples tested by
the technical operator resulted in >20-mg/L results. Eight of these (50 and 100 mg/L) were an
exact color match and four were within one color. The microcuvettes analyzed by the non-
technical operator generated results that included 3 exact color matches, 7 within one color, and
2 that were more than one color from the chart color that should have matched the reference
result. For both operators, the 250-mg/L samples did not change to the color representing the
>200-mg/L concentration. Considering only a visual observation of the lethal dose sample
analysis process, the color changes on the strips and of the solution in the microcuvettes were
quick and pronounced with respect to the much lower concentrations. In this manner, the
presence of a high concentration of cyanide was easily ascertained through the use of all three
detection mechanisms.
6.7 Operator Bias
The possible difference in results produced by the non-technical and technical operators was
assessed by using a linear regression of the results produced by the non-technical operator plotted
against the results produced by the technical operator. The results from all of the samples that had
detectable amounts of cyanide (including the PT, surface, and DW samples) from both
technologies were included in this regression. The total number of results with corresponding
reference analyses (N) is smaller here than for the inter-unit comparability because of the large
number of samples for which one unit or the other was non-detectable. Figure 6-6 shows a scatter
plot of the results from both technologies. A dotted regression line with a slope of unity and
intercept of zero also is shown in Figure 6-6. A linear regression of the data in Figure 6-6 for the
inter-unit comparability assessment gives the following regression equation:
y (non-technical operator result in mg/L) = 1.04 (± 0.04)x (technical operator result in
mg/L) + 0.08 (± 0.26) mg/L with r2 = 0.936 and N = 167 (includes all pairs of detectable
results).
This regression, with a slope and intercept that are not different from unity and zero, respectively,
suggests that the results generated by both operators were not different from one another. This is
consistent with the wider concentration range linearity results. However, the linearity results over
the concentration range of 0.03 to 1 mg/L suggested that the non-technical operator's results were
biased slightly high, while the technical operator's results were biased slightly low. In this
instance, because neither operator's results are necessarily closer to the reference method result
(as the linearity data show), it is unlikely that the difference between the two operators was
related to differences in training or experience. It is apparently the result of the normal variability
of two different people performing the analyses at these lower concentrations.
For the semi-quantitative strip and microcuvette results, the operator-specific data were evaluated
by evaluating the number of results in which one operator's results were more than one color
chart color different from the other operator's result. For example, three of the colors on the
microcuvette color chart represent, in order, 0.2, 0.5, and >3 mg/L; therefore, if one operator
determined that the color of the microcuvette was most similar to the >3-mg/L color and the other
35
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0 5 10 15 20 25 30 35
Technical Operator Results (mg/L)
Figure 6-6. Operator Bias Results
operator determined that it was most similar to the 0.2-mg/L color, that would be considered
more than one color different. For the strips, all of the results were within one color. For the
microcuvettes, one out of 108 results was more than one color different from one another. This
outlier had one result as the >3-mg/L color and one as 0.2 mg/L. The color closest to representing
the reference concentration (0.4 mg/L) in this case would have been the 0.5 mg/L color. For this
comparison, all extrapolations between two colors were rounded up.
For the lethal/near-lethal samples, the non-technical operator's result was a >3-mg/L result in 7
out of 12 instances when the seemingly correct result (according to the reference concentration)
and the result of the technical operator was >20 mg/L. These colors are adjacent to one another
on the color chart, but the results stood out from the rest because the concentrations are so
different. Similarly, for the 0.4-mg/L and 1-mg/LPT samples, the non-technical operator's result
was >3 mg/L, while the technical operator's was (seemingly more correctly) 0.5 mg/L. Overall, it
seems that the non-technical operator had some difficulty distinguishing between the colors on
the microcuvette color chart, especially between the 0.2 and 0.5, 0.5 and >3 mg/L, and >3 mg/L
and >20 mg/L colors. The discrepancy between the results generated by the two operators may
have more to do with the vision of the operators than their level of education and experience.
While the non-technical operator was not previously aware of any color vision deficiency, this
may have had some impact on the results.
6.8 Field Portability
The Cyanide ReagentStrip™ test kit was operated in laboratory and field settings during this
verification test. Tables 6-1 d, 6-2d, and 6-3d show the results of these measurements. From an
operational standpoint, the Cyanide ReagentStrip™ test kit was easily transported to the field
36
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setting, and the samples were analyzed in the same fashion as they were in the laboratory. No
functional aspects of the Cyanide ReagentStrip™ test kit were compromised by performing the
analyses in the field setting. However, performing analyses under cool conditions negatively
affected the performance of the reagents. The low temperatures severely inhibited the chemical
reaction rates, which decreased the color formation in the LFM samples spiked with cyanide.
Table 6-2d shows the bias of the samples analyzed in the field settings (indoors with sample
temperatures of approximately 21°C and outdoors with sample temperatures of 17°C) and of the
identical samples analyzed in the laboratory at approximately 21°C. The samples analyzed
outdoors had extremely large negative biases ranging from -78 to -91%. According to the
Cyanide ReagentStrip™ test kit instructions, a 20-minute reaction time was to be used because of
the sample temperature of 17°C. Because of the lack of color development after 20 minutes,
several samples were analyzed after a 40-minute reaction time to see if additional time would
improve the results. When little or no additional color development took place, data again were
collected after the 20-minute reaction time. The non-technical operator did not revert back to the
20-minute reaction time; therefore, the non-technical results collected outdoors represent a 40-
minute reaction time.
No trend in the accuracy of samples analyzed in the laboratory and the indoor field location was
apparent. For both operators, bias results from the laboratory ranged from -39 to +21% and from
the indoor field location from -42 to +30%. Both operators had examples of very low biases for
these samples. For example, at the laboratory using the city water sample, the non-technical
operator had biases of -3 and +2%. Similarly, at the indoor field location using well water, the
technical operator produced biases of -3 and 1%. However, as the above ranges indicate, there
were examples with larger biases above and below the target concentration. With the exception of
the low-temperature analyses, the Cyanide ReagentStrip™ test kit performance at the field
location was similar to that in the laboratory.
6.9 Ease of Use
The Cyanide ReagentStrip™ test kit was easy to operate. The written instructions provided were
clear, and the accompanying instructional video (lasting less than five minutes) was detailed and
easy to understand. Because the required reagents were transferred into the test sample entirely by
the repeated dipping of the two types of ReagentStrips, there was no measuring or mixing. The
operators only had to hold strictly to the appropriate color development time as given in the
instructions. The ReagentStrip™ Reader also was easy to use; the sample microcuvette was
inserted and one button pushed to read the absorbance value. That absorbance value was
compared to a table of absorbances to determine the corresponding cyanide concentration. Water
samples within a pH range of 5 and 11 could be analyzed directly without pH adjustment.
Samples with pHs outside that range required adjustment using NaOH or hydrochloric acid. This
step required the availability of acid and/or base, pH paper or meter, and some knowledge of pH
adjustment. Instructions for pH adjustment were not provided. Cleanup was simple and free of
mess.
37
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The semi-quantitative ReagentStrips and microcuvettes were also easy to use. For the strips, the
color at the end of a paper strip was compared to a color chart immediately after dipping the strip
into the test sample. For the microcuvettes, the color of the test solution after the color develop-
ment time was compared to another color chart. Both color charts were conveniently designed to
compare colors (see Figure 3-2). The color bar on the ReagentStrip color chart was in the shape
of the end of a ReagentStrip so the strip could be held directly between two colors and compared.
Similarity, the shape of the colors on the microcuvette color chart were outlines of squares of a
size that allowed the microcuvettes to fit within the outline. The operators could look down at the
sample in the microcuvette and compare the color of the solution to the color of the outlined
squares.
6.10 Sample Throughput
Sample preparation, including accurate volume measurement and the addition of reagents (i.e.,
dipping the ReagentStrips) took only one to two minutes per sample. After preparing the sample,
typically a 10-minute period of color development was required before sample analysis (because
the sample temperature in the laboratory was approximately 21°C). Therefore, if only one sample
is analyzed, it would take approximately 12 minutes. However, both operators were able to
stagger the start of the color development period every two minutes for subsequent samples, so a
typical sample set of 10 analyses took 30 to 40 minutes. The strip results were available within
1 minute of measuring the samples into the sample microcuvette.
38
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Chapter 7
Performance Summary
The quantitative accuracy of the ReagentStrip™ Reader-derived results were evaluated by
calculating biases with respect to the reference concentrations. Biases for the Cyanide
ReagentStrip™ test kit ranged from -47 to 25% for the PT samples; -27 to 28% for the surface
water samples; -41 to 3% for the DW samples from around the United States; and, with the
exception of the samples analyzed outdoors, -42 to 30% for the Columbus, OH, DW samples.
The matrix effect on these results was apparently minimal because the range of the bias for the
surface water and DW results was similar to that of the PT samples that were prepared in DI
water. For the semi-quantitative accuracy results, 84% of the PT sample results matched the exact
color that should represent the reference concentration, and 16% were within one color on the
color chart; 100% of the surface water and U.S. DW samples matched the exact color; and 83%
of the Columbus, OH, DW samples matched exactly, with the remaining 17% within one color.
For the microcuvette results, 64% of the PT samples matched exactly and 36% were within one
color; 56% of the surface water samples matched exactly, with 44% being within one color; 88%
of the U.S. DW samples matched exactly, and 12% were within one color; and 66% of the
Columbus, OH, DW samples matched exactly, 17% were within one color, and 17% were within
two colors on the color chart.
The quantitative precision of the Cyanide ReagentStrip™ test kit using the ReagentStrip™
Reader was evaluated by calculating RSDs for each sample set. The RSDs ranged from 4 to 86%
for the PT samples (if the 0.03 mg/L samples are removed, the upper end of that range was 27%);
11 to 30%) for the surface water samples; 3 to 25% for the DW samples from around the United
States; and, except for the outdoor field site, 2 to 21% for the Columbus, OH, DW samples. The
RSDs were similar regardless of the water matrix. For the semi-quantitative strip evaluation, the
precision was evaluated by determining the frequency by which the qualitative result (color) was
produced for each sample set. The same result was obtained for four replicates in 11 out of 16 PT
sample sets, all of the surface water sample sets, all 10 U.S. DW sample sets, and 11 out of 12
sample sets of Columbus, OH, DW. For the microcuvettes, consistent results were obtained for
11 of 16 PT sample sets, 2 out of 4 surface water sample sets, 9 out of 10 U.S. DW sample sets,
and 7 out of 12 sample sets of Columbus, OH, DW.
A linear regression of the PT sample results, ranging in concentration from 0.03 to 25 mg/L, gave
the following regression equations:
y (non-technical operator results in mg/L) = 1.03 (± 0.06)x (reference result in mg/L)
+ 0.19 (± 0.55) mg/L with r2 = 0.947 and N = 72.
39
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y (technical operator results in mg/L) = 0.97 (± 0.04)x (reference result in mg/L)
+ 0.08 (± 0.42) mg/L with r2 = 0.965 and N = 72.
where the values in parentheses represent the 95% confidence interval (two times the standard
error) of the slope and intercept, r2 is the coefficient of determination, and N is the total number
of results with corresponding reference analyses. The slopes of these regressions are not
significantly different from unity, neither intercept is significantly different from zero, and the r2
values are both above 0.94. Linear regressions were also generated for a concentration range of
0.03 to 1 mg/L. Within this smaller concentration range, both slopes were significantly different
from unity, while the intercepts were either not significantly different from zero or very close to
it. This suggests that the non-technical operator's results have a slightly high bias, and the
technical operator's results have a slightly low bias. This effect was not apparent with the wider
concentration range.
The quantitative MDL of the Cyanide ReagentStrip™ test kit was determined by analyzing seven
replicate samples at a concentration of 0.05 mg/L. The MDLs were 0.04 and 0.03 mg/L for the
non-technical operator and 0.02 for both units when used by the technical operator. The strips
generated detectable results at all concentration levels 0.2 mg/L and above, and the microcuvettes
generated detectable results at all concentrations 0.03 mg/L and above.
A linear regression of the data for the inter-unit reproducibility assessment gave the following
regression equation:
y (Unit #1 result in mg/L) = 1.17 (± 0.02)x (Unit #2 in mg/L) - 0.08 (± 0.08) mg/L
with r2 = 0.991 and N = 213.
The intercept was not significantly different from zero; however, the slope was significantly
larger than unity, suggesting that, on average, Unit #1 gave slightly higher responses than Unit
#2.
The ReagentStrip™ Reader results for lethal to near-lethal concentrations ranged from 36 to
>60 mg/L. According to the vendor, concentrations >40 mg/L exceed the linear range of the
ReagentStrip™ Reader so these results would need to be clarified through dilution and reanalysis
of samples. For the semi-quantitative strips, all 12 samples for both operators correctly resulted in
a >10-mg/L (the highest concentration color) result. For the microcuvettes, all 12 samples tested
by the technical operator resulted in >20-mg/L results, but the 250-mg/L samples did not appear
to change to the >200 mg/L color. If the sample analysis procedure was only observed visually,
the color changes on the strips and of the solution (for the lethal dose concentrations) in the
microcuvettes were quick and pronounced with respect to the much lower concentrations. In this
manner, the presence of a high concentration of cyanide was easily ascertained through use of all
three detection mechanisms.
A linear regression of the data for the operator bias assessment gave the following regression
equation:
y (non-technical operator result in mg/L) = 1.04 (± 0.04)x (technical operator result in
mg/L) + 0.08 (± 0.26) mg/L with r2 = 0.936 and N = 167.
40
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This regression, with a slope and intercept that are not different from unity and zero, respectively,
suggests that the results generated by both operators were not different from one another. This is
consistent with the wider concentration range linearity results. However, the linearity results over
the concentration range of 0.03 to 1 mg/L suggested that the non-technical operator's results were
biased slightly high, while the technical operator's results were biased slightly low. In this
instance, because neither operator's results are necessarily closer to the reference method result
(as the linearity data show), it is unlikely that the difference between the two operators was
related to differences in training or experience. It is apparently the result of the normal variability
of two different people performing the analyses at these lower concentrations. For the semi-
quantitative microcuvette results, in only one out of 108 results was one operator's result more
than one color different from the other operator's result. For the strips, the results from both
operators were always within one color of one another.
No functional aspects of the Cyanide ReagentStrip™ test kit were compromised by performing
the analyses in the field setting. However, performing analyses under cool conditions negatively
affected the performance of the reagents as was evidenced by the large negative biases for the
samples analyzed outdoors.
The Cyanide ReagentStrip™ test kit was easy to operate. The written instructions provided were
clear, and the accompanying instructional video (lasting less than five minutes) was detailed and
easy to understand. Because the required reagents were transferred into the test sample entirely by
the repeated dipping of the two types of ReagentStrips™, there was no measuring or mixing. The
strips, microvettes, and ReagentStrip™ Reader were easy to use; and cleanup was minimal.
The analysis of a set of approximately 10 samples, including sample preparation and reaction
time, took 30 to 40 minutes.
41
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Chapter 8
References
Test/QA Plan for Verification of Portable Analyzers for Detection of Cyanide in Water,
Battelle, Columbus, Ohio, January 2003.
U.S. EPAMethod 335.1, Cyanides Amenable to Chlorination, 1974, in "Methods for
Chemical Analysis of Water and Wastes," EPA/600/4-79/020, March 1983.
Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, March 2004.
Code of Federal Regulations, Title 40, Part 136, Appendix B, "Definition and Procedure
for the Determination of the Method Detection Limit-Revision 1.11."
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