Environmental Technology Verification Report Strategic Diagnostics Inc. Microtox Rapid Toxicity Testing System


November 2003
Environmental Technology
Verification Report
Strategic Diagnostics Inc.
Microtox®
Rapid Toxicity Testing System
Prepared by
Battel le
1#
Batteiie
Putting Technology To Work
Under a cooperative agreement with
SEPA U.S. Environmental Protection Agency
ETV ElV ElV

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November 2003
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
Strategic Diagnostics Inc.
Microtox®
Rapid Toxicity Testing System
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 seven environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at
http ://www. ep a. go v/et v/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. Many thanks go to Battelle's Medical
Research and Evaluation Facility for providing the facilities for and personnel capable of
working with chemical warfare agents and biotoxins. We would also like to thank
Karen Bradham, U.S. EPA National Exposure Research Laboratory; Steve Allgeier, U.S. EPA
Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern California; Yves
Mikol, New York City Department of Environmental Protection; and Stanley States, Pittsburgh
Water and Sewer Authority, for their careful review of the test/QA plan and this verification
report.
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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 	4
3.1	Introduction	4
3.2	Test Design 	5
3.3	Test Samples 	6
3.3.1	Quality Control Samples 	6
3.3.2	Drinking Water Fortified with Contaminants	7
3.3.3	Drinking Water Fortified with Potential Interferences	7
3.4	Test Procedure	7
3.4.1	Test Sample Preparation and Storage	7
3.4.2	Test Sample Analysis Procedure 	8
3.4.3	Stock Solution Confirmation Analysis	9
4	Quality Assurance/Quality Control	 12
4.1	Quality Control of Stock Solution Confirmation Methods 	 12
4.2	Quality Control of Drinking Water Samples 	 12
4.3	Audits 	 13
4.3.1	Performance Evaluation Audit		13
4.3.2	Technical Systems Audit 		14
4.3.3	Audit of Data Quality		14
4.4	QA/QC Reporting 		14
4.5	Data Review 		15
5	Statistical Methods and Reported Parameters	 16
5.1	Endpoints and Precision 		16
5.2	Toxicity Threshold		17
5.3	False Positive/Negative Responses 		17
5.4	Other Performance Factors 		18
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6	Test Results	 19
6.1	Endpoints and Precision 	 19
6.1.1	Contaminants	 19
6.1.2	Potential Interferences 	24
6.1.3	Precision 	25
6.2	Toxicity Threshold	26
6.3	False Positive/Negative Responses 	26
6.4	Other Performance Factors 	27
7	Performance Summary	28
8	References 	29
Figures
Figure 2-1. Microtox® Rapid Toxicity Testing System	2
Tables
Table 3-1. Contaminants and Potential Interferences 	5
Table 3-2. Summary of Quality Control Contaminant Test Samples	8
Table 3-3. Dose Confirmation Results 	 10
Table 3-4. Water Quality Parameters	 11
Table 4-1. Summary of Performance Evaluation Audit 	 13
Table 4-2. Summary of Data Recording Process	 15
Table 6-la. Aldicarb Percent Inhibition Results	20
Table 6-lb. Colchicine Percent Inhibition Results 	20
Table 6-lc. Cyanide Percent Inhibition Results 	21
Table 6-Id. Dicrotophos Percent Inhibition Results	21
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Table 6-le.	Thallium Sulfate Percent Inhibition Results	22
Table 6-If.	Botulinum Toxin Percent Inhibition Results 	22
Table 6-lg.	Ricin Percent Inhibition Results 	23
Table 6-lh.	Soman Percent Inhibition Results 	23
Table 6-li.	VX Percent Inhibition Results	24
Table 6-2.	Potential Interferences Results	25
Table 6-3.	Toxicity Thresholds	26
Table 6-4.	False Negative Responses 	27
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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 water
DDW
dechlorinated drinking water from Columbus, Ohio
ec50
effective concentration causing 50% inhibition
EPA
U.S. Environmental Protection Agency
ETV
Environmental Technology Verification
HDPE
high-density polyethylene
ID
identification
LD
lethal dose
[iL
microliter
mL
milliliter
NSDWR
National Secondary Drinking Water Regulations
%D
percent difference
PE
performance evaluation
QA
quality assurance
QC
quality control
QMP
quality management plan
SOP
standard operating procedure
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 accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative 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 Strategic Diagnostics Inc. Microtox® rapid toxicity
testing system. Rapid toxicity testing systems 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 environ-
mental monitoring technologies for air, water, and soil. This verification report provides results
for the verification testing of Microtox® Following is a description of Microtox®, based on
information provided by the vendor. The information provided below was not subjected to
verification in this test.
Microtox® is an in vitro testing system that uses bioluminescent bacteria to detect toxins in air,
water, soil, and sediment. Microtox® is a metabolic inhibition test that provides both acute
toxicity and genotoxic analyses. Microtox® uses a strain of naturally occurring luminescent
bacteria. Vibrio fischeri. Vibrio fischeri are non-pathogenic, marine, luminescent bacteria that
are sensitive to a wide range of toxicants. When properly grown, luminescent bacteria produce
light as a by-product of their cellular respiration. Cell respiration is fundamental to cellular
metabolism and all associated life processes. Bacterial bioluminescence is tied directly to cell
respiration, and any inhibition of cellular activity (toxicity) results in a decreased rate of
respiration and a corresponding decrease in the rate of luminescence.
Microtox® Model 500 Analyzer was tested as a stand-alone instrument along with the Microtox®
reagent. The Vibrio fischeri are supplied in a standard freeze-dried (lyophilized) state and, to
analyze water samples, are reconstituted in a salt solution, 2.5 millili ters (mL) of the water
sample are diluted with 250 microliters
(fiL) of a Microtox® reagent, then
approximately 1 mL of water sample is
added to 100 [iL of the reconstituted
bacteria. Luminescence readings are
taken prior to adding the drinking water
and then at 5 and 15 minutes after the
addition. When analyzing unknown
samples, it is recommended that
inhibition data be collected at both time
intervals to determine the most
appropriate data collection time since the
rates can vary depending on how the
toxicant affects the bacteria. Results are
displayed as absolute light units.
Figure 2-1. Microtox® Rapid Toxicity Testing
System
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The temperature-controlled Microtox® maintains the test organisms and samples at a standard
temperature of 15°C. As such, the Microtox® must be operated in a laboratory setting at ambient
temperatures of between 15 and 30°C. It detects light intensity at 490 nanometers, the
wavelength emitted by the bacteria. Microtox® can be used with Microtox®Omni™ software and
a personal computer to collect, analyze, track, and store test data. Microtox® weighs 21 pounds,
measures 7-1/8 inches x 15-3/8 inches x 16-1/8 inches, and runs on 120/240 volts alternating
current. The Microtox® Model 500 costs $17,895, and the reagents cost $360 for approximately
200 samples.
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Chapter 3
Test Design and Procedures
3.1 Introduction
The objective of this verification test of rapid toxicity technologies was to evaluate their ability
to detect certain toxins and to determine their susceptibility to interfering chemicals in a
controlled experimental matrix. Rapid toxicity technologies do not identify or determine the
concentration of specific contaminants, but serve as a screening tool to quickly determine
whether water is potentially toxic. Rapid toxicity technologies use bacteria (e.g., Vibrio
fischeri), enzymes (e.g., luciferase), or small crustaceans (e.g., Daphnia magna) that either
directly, or in combination with reagents, produce a background level of light or use dissolved
oxygen at a steady rate in the absence of toxic contaminants. Toxic contaminants in water are
indicated by a change in the color or intensity of light produced or by a decrease in the dissolved
oxygen uptake rate in the presence of the contaminants.
As part of this verification test, Microtox® was subjected to various concentrations of
contaminants such as industrial chemicals, pesticides, rodenticides, pharmaceuticals, nerve
agents, and biological toxins. Each contaminant was added to separate drinking water samples
and analyzed. In addition to determining whether Microtox® can detect the toxicity caused by
each contaminant, its response to interfering compounds in clean drinking water, such as water
treatment chemicals and by-products, was evaluated. Table 3-1 shows the contaminants and
potential interferences that were evaluated during this verification test.
This verification test was conducted according to procedures specified in the Test/QA Plan for
Verification of Rapid Toxicity Technologies.(1) Microtox® was verified by analyzing a
dechlorinated drinking water (DDW) sample from Columbus, Ohio, fortified with various
concentrations of the contaminants and interferences shown in Table 3-1. Hereafter in this
report, DDW will refer to dechlorinated drinking water from Columbus, Ohio. Where possible,
the concentration of each contaminant or potential interference was confirmed independently by
Aqua Tech Environmental Laboratories (ATEL), Marion, Ohio, or by Battelle, depending on the
analyte.
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Table 3-1. Contaminants and Potential Interferences
Category
Contaminant
Carbamate pesticide
aldicarb
Pharmaceutical
colchicine
Industrial chemical
cyanide
Organophosphate pesticide
dicrotophos
Rodenticide
thallium sulfate
Biological toxins
botulinum toxin, ricin
Nerve agents
soman, VX
Potential interferences
aluminum, copper, iron, manganese, zinc,
chloramination by-products, and chlorination
by-products
Microtox® was evaluated by
•	Endpoint and precision—percent inhibition for all concentration levels of contaminants and
potential interfering compounds and precision of replicate analyses
•	Toxicity threshold for each contaminant
•	False negative responses—contaminants that were reported as producing inhibition results
similar to the negative control when the contaminant was present at lethal concentrations
•	False positive responses—occurrence of inhibition significantly greater than the inhibition
reported for unspiked American Society for Testing and Materials (ASTM) Type II deionized
(DI) water samples (zero inhibition)
•	Ease of use
•	Throughput.
3.2 Test Design
Microtox® was used to analyze the DDW sample fortified with contaminants at concentrations
ranging from lethal levels to concentrations 1,000 times less than the lethal dose. The lethal dose
of each contaminant was determined by calculating the concentration at which 250 mL of water
would probably cause the death of a 154-pound person. These calculations were based on
toxicological data available for each contaminant. For soman, the stock solution confirmation
showed degradation in the water; therefore, the concentrations analyzed were less than
anticipated. Whether the concentration is still a lethal dose, as is the case for all contaminants,
depends on the characteristics of the individual person and the amount of contaminant ingested.
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Inhibition results (endpoints) from four replicates of each contaminant at each concentration
level were evaluated to assess the ability of Microtox® to detect toxicity at various
concentrations of contaminants, as well as to measure the precision of Microtox® results.
The response of Microtox® to compounds used during the water treatment process (identified as
potential interferences in Table 3-1) was evaluated by analyzing separate aliquots of DDW
fortified with each potential interference at approximately one-half of the concentration limit
recommended by the EPA's National Secondary Drinking Water Regulations (NSDWR)(2)
guidance. For analysis of by-products of the chlorination process, the unspiked DDW was
analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the
analysis of by-products of the chloramination process, a separate drinking water sample from
St. Petersburg, Florida, which uses chloramination as its disinfection process, was obtained. The
samples were analyzed after residual chlorine was removed using sodium thiosulfate.
Sample throughput was measured based on the number of samples analyzed per hour. Ease of
use and reliability were determined based on documented observations of the operators and the
verification test coordinator.
3.3 Test Samples
Test samples used in the verification test included drinking water and quality control (QC)
samples. Table 3-2 shows the number and type of samples analyzed. QC samples included
method blanks and positive and negative control samples. The fortified drinking water samples
were prepared from a single drinking water sample collected from the Columbus, Ohio, system.
The water was dechlorinated using sodium thiosulfate and then fortified with various concen-
trations of contaminants and interferences. Using this DDW (Columbus, Ohio, dechlorinated
drinking water), individual solutions containing each contaminant and potential interference
were prepared and analyzed. The DDW containing the potential interferences was analyzed at a
single concentration level, while four dilutions (made using the DDW) were analyzed for each
contaminant using Microtox®. Mixtures of contaminants and interfering compounds were not
analyzed.
3.3.1 Quality Control Samples
QC samples included method blank samples, which consisted of ASTM Type IIDI water;
positive control samples, which consisted of ASTM Type II DI water or DDW (depending on
vendor preference) fortified with a contaminant and concentration selected by the vendor; and
negative control samples, which consisted of the unspiked DDW. The method blank samples
were used to help ensure that no sources of contamination were introduced in the sample
handling and analysis procedures. Either zinc sulfate or phenol were suggested by the vendor for
use as positive control samples, and both were used at times throughout the verification test.
While performance limits were not placed on the results, significant inhibition for either of these
contaminants indicated to the operator that Microtox® was functioning properly. The negative
control sample was used to set a background inhibition of the DDW, the matrix in which each
test sample was prepared.
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3.3.2 Drinking Water Fortified with Contaminants
Approximately 150 liters of Columbus, Ohio, tap water were collected in a high-density
polyethylene (HDPE) container. The sample was dechlorinated with 0.5 mL of 0.4 M sodium
thiosulfate for every liter of water. All subsequent test samples were prepared from this DDW
and stored in glass containers to avoid chlorine leaching from HDPE containers.
A stock solution of each contaminant was prepared in ASTM Type IIDI water at concentrations
above the lethal dose level. The stock solution was diluted in DDW to obtain one sample con-
taining the lethal dose concentration for each contaminant and three additional samples with
concentrations 10, 100, and 1,000 times less than the lethal dose. Table 3-2 lists each concentra-
tion level and the number of samples analyzed at each level.
3.3.3 Drinking Water Fortified with Potential Interferences
Individual aliquots of the DDW were fortified with one-half the concentration specified by the
EPA's NSDWR for each potential interference. Table 3-2 lists the interferences, along with the
concentrations at which they were tested. Four replicates of each of these samples were
analyzed. To test the sensitivity of Microtox® to by-products of the chlorination process as
potential interferences, the unspiked DDW (same as the negative control) was used since the
water sample originated from a utility that uses chlorination as its disinfectant procedure. In a
similar test involving the by-products of the chloramination process, an additional water sample
was obtained from St. Petersburg, Florida, a city that uses chloramination as its disinfectant
procedure. The residual chlorine in both of these samples was removed using sodium thiosulfate,
and then the samples were analyzed in replicate with no additional fortification of contaminants.
3.4 Test Procedure
3.4.1 Test Sample Preparation and Storage
A drinking water sample was collected as described in Section 3.3.2 and, because free chlorine
kills the bacteria within the Microtox® reagent and can degrade the contaminants during storage,
was immediately dechlorinated with sodium thiosulfate. Prior to preparing each stock solution,
dechlorination of the water sample was qualitatively confirmed by adding an n,n-diethyl-p-
phenylenediamine tablet to a 25-mL aliquot of the DDW. Once dechlorination was confirmed,
all the contaminant samples, potential interference samples, and negative control QC samples
were made from this DDW, while the method blank sample was prepared from ASTM Type II
DI water. The positive control samples were made using ASTM Type II DI water in Class A
volumetric glassware. All QC samples were prepared prior to the start of the testing and stored at
room temperature for a maximum of 60 days. The aliquots of DDW containing the contaminants
were prepared within seven days of testing and stored in the dark at room temperature without
chemical preservation. Aliquots to be analyzed by each technology were placed in uniquely
labeled sample containers. The sample containers were assigned an identification (ID) number.
A master log of the samples and sample ID numbers for each technology was kept by Battelle.
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Table 3-2. Summary of Quality Control Contaminant Test Samples
Type of Sample
Sample Characteristics
Concentration
Levels (mg/L)
No. of Sample Analyses

Method blank
NS(a)
9
Quality control
Positive control
115 (Phenol)
25 (Zinc sulfate)
9
17

Negative control (unspiked
DDW)
NS
51

Aldicarb
280; 28; 2.8; 0.28
4 per concentration level

Colchicine
240; 24; 2.4; 0.24
4 per concentration level

Cyanide
250; 25; 2.5; 0.25
4 per concentration level

Dicrotophos
1,400; 140; 14; 1.4
4 per concentration level
DDW fortified
with contaminants
Thallium sulfate
Botulinum toxin03'
2,400; 240; 24; 2.4
0.30; 0.030;
0.0030; 0.00030
4 per concentration level
4 per concentration level

Ricin(c)
15; 1.5; 0.15; 0.015
4 per concentration level

Soman(d)
0.068; 0.0068;
0.00068; 0.000068
4 per concentration level

VX
0.22; 0.022;
0.0022; 0.00022
4 per concentration level

Aluminum
0.36
4
DDW fortified
Copper
Iron
Manganese
0.65
0.069
0.26
4
A
with potential
interferences
4

Zinc
3.5
4
Disinfectant
Chloramination by-products
NS
4
by-products
Chlorination by-products
NS
4
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time since the rates can vary depending on how the toxicant affects the bacteria. Since these
samples were all treated as unknowns, both data collection times were used.
For each contaminant, Microtox® analyzed the lethal dose concentration and three additional
concentration levels four times. Only one concentration of potential interference was analyzed.
The absolute light units were recorded and the percent inhibition was calculated for each
sample. When Microtox® produced percent inhibitions greater than 50% for a contaminant, EC50
(effective concentration causing 50% inhibition) values were also calculated and reported. Two
operators performed all the analyses using Microtox®. Both held bachelor's degrees in the
sciences and spent approximately four hours with the vendor to become familiar with using
Microtox®.
3.4.3 Stock Solution Confirmation Analysis
The concentrations of the contaminant and interfering compound stock solutions were verified
with standard analytical methods, with the exception of colchicine, ricin, and botulinum
toxin—contaminants without standard analytical methods. Aliquots to be analyzed by standard
methods were preserved as prescribed by the method. In addition, the same standard methods
were used to measure the concentrations of each contaminant/potential interference in the
unspiked DDW so that background concentrations of contaminants or potential interferences
were accounted for within the displayed concentration of each contaminant/potential
interference sample. Table 3-3 lists the standard methods used to measure each analyte; the
results from the stock solution confirmation analyses (obtained by reporting the lethal dose
concentration for the contaminants and the single concentration that was analyzed for the
potential interferences); and the background levels of the contaminants and potential inter-
ferences measured in the DDW sample, which were all non-detect or negligible.
Standard methods were also used to characterize several water quality parameters such as the
concentration of trihalomethanes, haloacetic acids, and total organic halides; turbidity; dissolved
organic carbon content; pH; alkalinity; specific conductivity; and hardness. Table 3-4 lists these
measured water quality parameters for both the water sample collected in Columbus, Ohio,
representing a water system using chlorination as the disinfecting process, and the water sample
collected in St. Petersburg, Florida, representing a water system using chloramination as the
disinfecting process.
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Table 3-3. Dose Confirmation Results

Method
Average Concentration ±
Standard Deviation N = 4
(mg/L)
Background
in DDW
(mg/L)
Contaminant

Aldicarb
EPA 531.1(3)
280 ± 28
<0.0007
Colchicine
(a)
NA®
NA
Cyanide
EPA 335.1(4)
250 ± 15
0.008
Dicrotophos
EPA SW846 (8141A)®
1,400 ± 140
<0.002
Thallium sulfate
EPA 200.8(6)
2,400 ± 24
<0.001
Botulinum toxin
(a)
NA
NA
Ricin
(a)
NA
NA
Soman
(<0
0.068(d) ± 0.001
<0.05
VX
(<0
0.20 ± 0.02
<0.05
Potential
Interference

Aluminum
EPA 200.8
0.36 ±0.01
<0.10
Copper
EPA 200.8
0.65 ±0.01
0.011
Iron
EPA 200.8
0.069 ± 0.008
<0.04
Manganese
EPA 200.8
0.26 ±0.01
<0.01
Zinc
EPA 200.8
3.5 ±0.35
0.3
(a) No standard method available. QA audits and balance calibration assured accurately prepared solutions.
(b) NA = Not applicable.
(c) Purity analyses performed on chemical and biological agent materials using Battelle standard operating procedures.
(d) The result of the dose confirmation analysis for soman was 23% of the expected concentration of 0.30 mg/L.
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Table 3-4. Water Quality Parameters
Parameter
Method
Dechlorinated Columbus,
Ohio, Tap Water (disinfected
by chlorination)
Dechlorinated
St. Petersburg, Florida,
Tap Water (disinfected by
chloramination)
Turbidity
EPA 180.1(7)
0.1 NTU(a)
0.3 NTU
Organic carbon
SM 5310(8)
2.5 mg/L
2.9 mg/L
Specific conductivity
SM 2510(S)
364 |imho
460 |imho
Alkalinity
SM 2320(S)
42 mg/L
97 mg/L
pH
EPA 150.1(9)
7.65
7.95
Hardness
EPA 130.2(9)
112 mg/L
160 mg/L
Total organic halides
SM 5320B(8)
190 [ig/L
83 jig/L
Total trihalomethanes
EPA 524.2(10)
52.8 jig/L
2.4 |ig/L
Total haloacetic acids
EPA 552.2(11)
75.7 fig/L
13.5 fig/L
(a)NTU = nephelometric turbidity unit
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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(12) and the test/QA plan for this verification test.(1)
4.1 Quality Control of Stock Solution Confirmation Methods
The stock solutions for aldicarb, cyanide, dicrotophos, and thallium sulfate were analyzed using
a standard reference method at ATEL. As part of ATEL's standard operating procedures (SOPs)
various QC samples were analyzed with each sample set. These included matrix spike,
laboratory control spike, and method blank samples. According to the standard methods used for
the analyses, recoveries of the QC spike samples analyzed with samples from this verification
test were within acceptable limits of 75% to 125%, and the method blank samples were below
the detectable levels for each analyte. For VX and soman, the confirmation analyses were
performed at Battelle using a Battelle SOP. Calibration standard recoveries of VX and soman
were always between 69% and 130%, and most of the time were between 90% and 100%.
Standard analytical methods for colchicine, ricin, and botulinum toxin were not available and,
therefore, not performed. QA audits and balance calibrations assured that solutions for these
compounds were accurately prepared.
4.2 Quality Control of Drinking Water Samples
A method blank sample consisting of ASTM Type IIDI water was analyzed once by Microtox®
for approximately every 20 drinking water samples that were analyzed. No detectable inhibition
was observed in any of these samples. A positive control sample also was analyzed once for
approximately every 20 drinking water samples. While performance limits were not placed on
the results of the positive control sample, the vendor informed Battelle that, if the positive
control samples did not cause greater than approximately 50% inhibition, it would indicate to
the operator that Microtox® was operating incorrectly. For nine positive control samples of
phenol, inhibitions of 86% ±1% and 87% ± 2% were measured at 5 and 15 minutes,
respectively. For 17 positive control samples of zinc sulfate, inhibitions of 77% ± 1% and 96%
± 2% were measured at 5 and 15 minutes. These inhibition values indicated the proper
functioning of Microtox®. A negative control sample (unspiked DDW) was analyzed with
approximately every four samples. The percent inhibition calculation incorporated the inhibition
of the negative control; therefore, by definition, the negative control samples had 0% inhibition.
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4.3 Audits
4.3.1 Performance Evaluation Audit
The concentration of the standards used to prepare stock solutions of the contaminants and
potential interferences was confirmed by analyzing solutions of each analyte prepared in ASTM
Type IIDI water from two separate commercial vendors using the confirmation methods. The
standards from one source were used to prepare the stock solutions during the verification test,
while the standards from a second source were used exclusively to confirm the accuracy of the
measured concentration of the first source. The percent difference (%D) between the measured
concentration of the performance evaluation (PE) sample, and the prepared concentration of that
sample was calculated using the following equation:
%D=—Xl00%
A (1)
where M is the absolute value of the difference between the measured and the prepared concen-
tration, and A is the prepared concentration. The %D between the measured concentration of the
PE standard and the prepared concentration had to be less than 25% for the measurements to be
considered acceptable. Table 4-1 shows the results of the PE audit for each compound. All %D
values were less than 25.
Table 4-1. Summary of Performance Evaluation Audit

Average Measured
Concentration ±
Standard Deviation
(mg/L)
Actual Concentration
(mg/L)
Percent
Difference

Aldicarb
0.00448 ± 0.000320
0.00500
11
Contaminant
Cyanide
Dicrotophos
0.207 ± 0.026
0.00728 ± 0.000699
0.200
0.00748
4
3

Thallium sulfate
0.090 ± 0.004
0.100
10
Potential
interference
Aluminum
Copper
0.512 ±0.013
0.106 ±0.002
0.500
0.100
2
6

Iron
0.399 ± 0.004
0.400
0.30

Manganese
0.079 ± 0.003
0.100
21

Zinc
0.106 ±0.016
0.100
6
Given the lack of confirmation methodology for some of the contaminants in this verification
test, PE audits were not performed for all of the contaminants. PE audits were performed when
13
-------
more than one source of the contaminant or potential interference was commercially available
and when methods were available to perform the confirmation. To assure the purity of the other
standards, documentation, such as certificates of analysis, was obtained for colchicine,
botulinum toxin, and ricin. In the cases of VX and soman, which were obtained from the U.S.
Army, the reputation of the source, combined with the confirmation analysis data, provided
assurance of the concentration analyzed.
4.3.2 Technical Systems Audit
The Battelle Quality Manager 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.(12) As part of the audit, the Battelle Quality Manager reviewed the contaminant standard
and stock solution confirmation methods, compared actual test procedures with 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 significant action. The
records concerning the TSA are permanently stored with the Battelle Quality Manager.
The EPA Quality Manager also conducted a TSA to ensure that the verification test was
performed in accordance with the test/QA plan(1) and the AMS Center QMP.(12) As part of the
audit, the EPA Quality Manager compared actual test procedures with those specified in the
test/QA plan and reviewed data acquisition and sample preparation records and procedures. No
significant findings were observed during the EPA TSA. The records concerning the TSA are
permanently stored with the EPA Quality Manager.
4.3.3 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.
4.4 QA/QC Reporting
Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5
of the QMP for the ETV AMS Center.(12) 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.
14
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4.5 Data Review
Records generated in the verification test were reviewed before these records were used to
calculate, evaluate, or report verification results. Table 4-2 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.
Table 4-2. 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
Battelle
Laboratory
record books
Start/end of test, and
at each change of a
test parameter
Used to organize/check
test results; manually
incorporated in data
spreadsheets as
necessary
Sample preparation
(dates, procedures,
concentrations)
Battelle
Laboratory
record books
When each sample
was prepared
Used to confirm the
concentration and
integrity of the samples
analyzed
Procedures entered into
laboratory record books
Test parameters
(contaminant
concentrations,
location, etc.)
Battelle
Laboratory
record books
When set or
changed
Used to organize/check
test results, manually
incorporated in data
spreadsheets as
necessary
Stock solution
confirmation
analysis, sample
analysis, chain of
custody, and
results
Battelle or
contracted
laboratory
Laboratory
record books,
data sheets, or
data acquisition
system, as
appropriate
Throughout sample
handling and
analysis process
Transferred to
spreadsheets/agreed
upon report
(a) All activities subsequent to data recording were carried out by Battelle.
15
-------
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 Endpoints and Precision
Microtox® reports absolute light units as a measure of light intensity (/). Each sample was com-
pared with a reference sample that, for this verification test, was unspiked DDW. This
comparison was made by calculating gamma (Gr), the ratio of the light lost at time t to the light
remaining at time t, using the two following equations:
R, -
hi (2)
r Rt 'hi i
Gt — 1
1 St
(3)
where Rt corrects for any inhibition induced by the negative control sample and Ict and Ici are the
absolute light intensities produced by the negative control at time t and at the initial time, /,
respectively. In addition, Isi and Ist are the light intensities produced by the water sample at the
initial time, i, and time t, respectively. Gt is converted to percent inhibition using the following
equation:
% inhibition = , xlOO ^
1+Gt
Percent inhibition data were calculated and are presented with respect to each test sample
analyzed as a part of this verification test. For contaminants that induced inhibition of greater
than 50%, the concentration of contaminant that affects 50% of the bacteria in the Microtox®
reagent (EC50) was estimated from the linear regression of the log of each concentration level of
the contaminant versus the percent inhibition. For contaminants that did not induce inhibition of
greater than 50%, this calculation was not appropriate.
16
-------
The standard deviation (S) of the results for the replicate samples was calculated, as follows, and
used as a measure of technology precision at each concentration.
n / -s2
X('* -')
1/2
(5)
where n is the number of replicate samples, Ik is the percent inhibition measured for the klh
sample, and I is the average percent inhibition of the replicate samples. Because the average
inhibitions were frequently near zero for this data set, relative standard deviations would often
have greatly exceeded 100%, making the results difficult to interpret. Therefore, the precision
results were left in the form of standard deviations so the reader could easily view the
uncertainty around the average for results that were both near zero and significantly larger than
zero.
5.2 Toxicity Threshold
The toxicity threshold was defined as the lowest concentration of contaminant to exhibit a
percent inhibition significantly greater than the negative control. Also, the inhibition of the
toxicity threshold had to be significantly different than the inhibition of the other concentrations
analyzed. Since the inhibition of the test samples was calculated with respect to the inhibition of
each negative control sample, the percent inhibition of the negative control was always zero. An
inhibition was significantly greater than the negative control if the average inhibition plus or
minus the standard deviation did not include zero.
5.3 False Positive/Negative Responses
A response would be considered false positive if an unspiked drinking water sample produced
an inhibition significantly greater than zero when determined with respect to ASTM Type IIDI
water. Depending on the degree of inhibition in the sample, toxicity due to subsequent contam-
ination of that sample may not be detectable or could be exaggerated as a result of the baseline
inhibition. To test for this possibility, the percent inhibition of the unspiked drinking water was
determined with respect to ASTM Type II DI water. Drinking water samples collected from
water systems using chlorination and chloramination as the disinfecting process were analyzed
in this manner. An inhibition was considered significantly different from zero if the average
inhibition, plus or minus the standard deviation, did not include zero.
A response was considered false negative when Microtox® was subjected to a lethal concentra-
tion of some contaminant in the DDW and did not indicate inhibition significantly greater than
the negative control (zero inhibition) and the other concentration levels analyzed. Requiring the
inhibition of the lethal dose sample to be significantly greater than zero and the other concentra-
tion levels more thoroughly incorporated the uncertainty of all the measurements made by
17
-------
Microtox® in determining a false negative result. A difference was considered significant if the
average inhibition plus or minus the standard deviation did not encompass the value or range of
values that were being compared.
5.4 Other Performance Factors
Ease of use (including clarity of the instruction manual, user-friendliness of software, and
overall convenience) was qualitatively assessed throughout the verification test through
observations of the operators and verification test coordinator. Sample throughput was evaluated
quantitatively based on the number of samples that could be analyzed per hour.
18
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Chapter 6
Test Results
6.1 Endpoints and Precision
Tables 6-la-i present the percent inhibition data for nine contaminants, and Table 6-2 presents
data for five potential interferences and the drinking water samples disinfected by both chlorina-
tion and chloramination. Given in each table are the concentrations analyzed, the percent
inhibition results for each replicate at each concentration, and the average and standard
deviation of the inhibition of the four replicates at each concentration. Results are given for the
samples analyzed at five minutes and then again at 15 minutes. For the most part, the results at
both time intervals were consistent, but according to the vendor protocol for unknown samples,
both data sets were collected and reported. EC50 values also are given when applicable. Samples
that produced negative percent inhibition values indicated an increase in light production by the
bacteria and were considered non-toxic.
6.1.1 Contaminants
The contaminants that were analyzed by Microtox® during this verification test produced one of
two trends apparent from Tables 6-la-i. Contaminants caused percent inhibitions that, starting
from the lowest concentration that produced inhibitions near zero, either increased in proportion
to the concentration in the sample so the two highest concentrations had significantly higher
inhibition, or did not change considerably, regardless of what concentration was analyzed.
Aldicarb, dicrotophos, and thallium sulfate (15 minutes only) fall into the former category, while
botulinum toxin, ricin, VX, and soman fall into the latter category. The only exceptions to these
trends were colchicine, for which the lethal dose (highest concentration) exhibited a higher
percent inhibition than for the rest of the concentration levels, and cyanide, for which the two
highest concentrations exhibited a higher percent inhibition than for the other two concentration
levels at 5 minutes. At 15 minutes, all four concentration levels of cyanide had distinct
inhibitions. For aldicarb, cyanide, and dicrotophos, whose inhibitions increased with
concentration and spanned the range from approximately no inhibition to greater than 50%
inhibition, EC50 values were calculated and reported in Tables 6-la, 6-lc, and 6-Id. Because
inhibitions did not reach 50% for the other contaminants, EC50 values could not be calculated.
19
-------
Table 6-la. Aldicarb Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation EC50
(%) (%) (%) (mg/L)
-3
0.28 2 3 4
6
45
-2
2 3 5
10

5
2.8 ^ 5 2
2
6
2 4 3
8
3

29
30
28 30 1
30
30
29
31 2
33 31
32
41
280 11
(Lethal ' 79 2
Dose) ^
79
83 81 2
80

Table 6-lb. Colchicine Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation EC50
(%) (%) (%) (mg/L)
8
0.24 ^ 5 3
6

5
2 3 2
1 3
3
NA(a)
7
2.4 ^ 6 1
6
6
KT A (a)
3
6 5 1
6
7
24 5 5 2
2

5
3
2 3
1 3
-1
240 !o
(Lethal " 11 1
Dose) j j

11
11 12 1
13
13
(a) NA = Not applicable.
20
-------
Table 6-lc. Cyanide Percent Inhibition Results

Results after 5 minutes

Results after 15 minutes
Concen-

Standard

tration
Inhibition Average
Deviation
EC50
Inhibition
Average
Deviation
EC-50
(mg/L)
(%)
(%)
(%)
(mg/L)
(%)
(%)
(%)
(mg/L)

0.25
5
5
11
7
3

5
7
11
8
3

2.5
5
7
9
4

41
44
46
4

8
48

25
83
83
85
83
1

96
96
95
96
1

250
(Lethal
Dose)
100

100

100
100
100
100
0

Table 6-Id. Dicrotophos Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation EC50
(%) (%) (%) (mg/L)
1
13
I"4 5 7 5
9
160
0
4 2 3
0
6
200
11
14 ® 8 3
8
10
2 6 4
4
41
39
140 35 37 3
34
39
35
34 3
32
33
1-400 f
(Lethal ° 82 1
Dose) 81
80
TO 80 2
79
21
-------
Table 6-le. Thallium Sulfate Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation EC50
(%) (%) (%) (mg/L)
3
14
2.4 5 7 5
4
NA(a)
2
8 4 3
3
2
NA
3
24 I 4 2
6
3
2
5 6 3
9
6
240 8 6 1
4
16
18 17 1
16
17
27
2,400
(Lethal ° 17 7
Dose) j j
41
21 32 6
28
(a) NA = Not applicable.
Table 6-If. Botulinum Toxin Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation EC50
(%) (%) (%) (mg/L)
-6
0.00030 A -4 2
-2
NA(a)
-2
i -2 1
0
NA
3
0.0030 ~2 -1 3
-3
3
~-l -1 2
-2
-1
0.030 ^-2 3
-6
-4
7
;2 o 5
-1
4
0.30 ^
(Lethal 2 0 3
Dose) j
-2
-5
(a) NA = Not applicable.
22
-------
Table 6-lg. Ricin Percent Inhibition Results

Results after 5 minutes

Results after 15 minutes
Concen-

Standard

tration
Inhibition Average
Deviation
EC-50
Inhibition Average
Deviation
ec50
(mg/L)
(%)
(%)
(%)
(mg/L)
(%)
(%)
(%)
(mg/L)

-2

-5

0.015
-1
2
1
0
2

-2
-1
0
-2
2

0.15
-3
2
0
3

-7
1
0
4

-1

NA(a)
1

-1

1.5
-2
0
-1
-1
2

-4
-2
-7
-4
3

15
-1

-2
1
-2
0

(Lethal
Dose)
D
-1
2
0
2

-1
2

(a) NA = Not applicable.
Table 6-lh. Soman Percent Inhibition Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
tration Inhibition Average Deviation EC50
(mg/L) (%) (%) (%) (mg/L)
Standard
Inhibition Average Deviation
(%) (%) (%)
EC-50
(mg/L)
1
0.000068 "J -4 5
-9
NA(a)
-9
NA
5
0.00068 1 1 3
1
-4 0 4
-2
-2
0.0068 ^-2 2
0
-1
^-2 2
-3
-3
0.068(b) 3
(Lethal 7. -1 4
Dose) ^
2
3
-2 ° 3
-2
(a) NA = Not applicable.
-------
Table 6-li. VX Percent Inhibition Results

Results after 5 minutes

Results after 15 minutes
Concen-

Standard

tration
Inhibition Average
Deviation
EC-50
Inhibition
Average
Deviation
EC-50
(mg/L)
(%)
(%)
(%)
(mg/L)
(%)
(%)
(%)
(mg/L)

-1

0.00022
5
2
1
2
2

4
2
1
3
2

-2

0.0022
11
20
14
8

6
16
9
9

NA(a)
18

-2

-3

0.022
0
1
12
3
7

-3
1
-1
-2
2

0.22
32

(Lethal
Dose)
-4
-2
0
7
17

-4
-1
6
18

(a) NA = Not applicable.
6.1.2 Potential Interferences
Table 6-2 presents the results from the samples that were analyzed to test the effect of potential
interferences on Microtox®. Aluminum and iron exhibited percent inhibitions near zero,
indicating little or no response to those compounds, while manganese exhibited inhibitions of
about 9% and zinc at 5 minutes had an inhibition at 6%. Exhibiting higher inhibitions were
copper, 40% and 61% at 5 and 15 minutes, respectively, and zinc, 28% at 15 minutes only.
All of the contaminant and potential interference samples were prepared in the DDW and
compared with unspiked DDW. Therefore, any background inhibition in the DDW was
mathematically corrected. To investigate whether Microtox® is sensitive to by-products of
disinfecting processes, DDW from water systems that use chlorination and chloramination were
analyzed and compared with ASTM Type IIDI water as the control sample. This determination
is crucial because the ability of Microtox® to detect toxicity is dependent on the light production
of the reagents in a clean drinking water matrix. If clean drinking water produces 100%
inhibition of light, the detection of subsequently added contaminants would not be possible. On
average, the chlorinated sample exhibited no detectable inhibition, indicating no toxicity, while
the chloraminated sample exhibited nearly complete inhibition (87% and 98% at 5 and
15 minutes, respectively. This suggests that samples that have been disinfected by using a
chloramination process are likely to interfere with the Microtox® results because the background
water sample would completely inhibit the Microtox® reagent.
24
-------
Table 6-2. Potential Interferences Results
Results after 5 minutes
Results after 15 minutes
Concen- Standard
Potential tration Inhibition Average Deviation
Interferences (mg/L) (%) (%) (%)
Standard
Inhibition Average Deviation
(%) (%) (%)
3
2
Aluminum 0.36 ^ 2 1
2
8
~3 1 5
0
0
37
Copper 0.65 ^ 40 3
38
62
62 ai 1
60
59
-2
Iron 0.069 2 ^
-1
-4
i -5 2
-4
8
Manganese 0.26 9 2
11
9
8 9 3
5
13
5
Zinc 3.5 ^ 6 1
6
29
g -
26
-11
Chlorination (a) -11
by-products 2
1
-15
- »
-10
Chloramin- ^
ation by- NA ^ 87 1
products QO
OO
O
OO
00 00 00 00
o\ o\ o\ G\
(a) NA = Not applicable.
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation was measured
and reported for each set of four replicates to evaluate the Microtox® precision. The standard
deviation of the four replicate measurements was greater than 10% for only one sample and, in
most cases, it was less than 5%.
25
-------
6.2 Toxicity Threshold
Table 6-3 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. The
lowest toxicity threshold concentration was for cyanide at 0.25 mg/L after the 15-minute
reaction time, indicating that Microtox® was most sensitive to cyanide. For botulinum toxin,
ricin, soman, and VX, no inhibition greater than the negative control was detected, regardless of
the concentration level, indicating that the technology was not highly responsive to these
contaminants.
Table 6-3. Toxicity Thresholds
Contaminant
Concentration (mg/L)
(a)
Aldicarb
Colchicine
Cyanide
Dicrotophos
Thallium sulfate
Botulinum toxin
Ricin
Soman
VX
28
240
25 (5 minutes)
0.25 (15 minutes)
140
ND(b) (5 minutes)
240
ND
ND
ND
ND
(a) Unless otherwise noted, toxicity thresholds were the same at 5 and 15 minutes.
(b) ND = Significant inhibition was not detected.
6.3 False Positive/Negative Responses
False positive responses were observed for unspiked chloraminated tap water. As described in
Section 6.1.2, for a clean tap water sample that had been disinfected using a chloramination
process, Microtox® reported almost complete inhibition (87% and 98%). By-products of the
chloramination process apparently inhibited the Microtox® reagent. The water sample treated by
chlorination and then subsequently dechlorinated caused no detectable inhibition. Similarly, the
method blank samples caused no significant decrease of absolute light units and, therefore, were
not the cause for any false positive responses.
A false negative response is when a lethal dose of contaminant is present in the water sample
and no inhibition is detected. Table 6-4 gives each contaminant's lethal dose concentration and
shows whether or not each contaminant exhibited a false negative response at that concentration
level, Microtox® detected an inhibition induced by lethal doses of aldicarb, colchicine, cyanide,
dicrotophos, and thallium sulfate (15 minutes only), while botulinum toxin, ricin, soman, and
VX inhibitions were not detected at the lethal dose, indicating false negative responses.
26
-------
Table 6-4. False Negative Responses
Contaminant
Lethal Dose
Concentration
(mg/L)
False Negative
Response
Aldicarb
280
no
Colchicine
240
no
Cyanide
250
no
Dicrotophos
1,400
no
Thallium sulfate
2,400
yes (5 minutes)

no (15 minutes)
Botulinum toxin
0.30
yes
Ricin
15
yes
Soman
0.068(a)
yes
VX
0.22
yes
-------
Chapter 7
Performance Summary
Parameter
Compound
Lethal
Dose (LD)
Cone.
(mg/L)
Average Inhibitions at Concentrations
Relative to the LD Concentration
(% after 15 minutes) %
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)'1"
LD
LD/10
LD/100
LD/1,000
Contaminants in
DDW
Aldicarb
280
81
31
4
3
2-5
28
Colchicine
240
12
2
5
3
1-3
240
Cyanide
250
100
96
46
8
0^1
0.25
Dicrotophos
1,400
80
34
6
2
2-4
140
Thallium
sulfate
2,400
32
17
6
4
1-6
240
Botulinum
toxin(b)
0.30
-4
0
-1
-2
1-5
ND(C)
Ricin(d)
15.0
-1
-4
0
-2
2-4
ND
Soman
0.068(e)
0
-2
0
-4
2-4
ND
VX
0.22
6
-2
9
3
2-18
ND
Potential
interferences in
DDW
Interference
Cone.
(mg/L)
Average Inhibitions at a
Single Concentration
(% after 15 minutes)
Standard
Deviation
(%)

Aluminum
0.36
1
5
Copper
0.65
61
1
Iron
0.069
-5
2
Manganese
0.26
9
3
Zinc
3.5
28
1
False positive
response
Nearly complete (87% and 98%) inhibition in water from a system disinfected by chloramination.
Inhibition due to contamination may not be detectable due to background inhibition. Inhibition
from water disinfected by chlorination was not significantly greater than zero.
False negative
response
No inhibition was detected for lethal doses of botulinum toxin, ricin, soman, and VX.
Other
performance
factors
The pictorial manual was useful, initial light measurements served as a good check of bacterial
health and instrument operation, sample handling was easy, and sample throughput was
15 samples per hour. Although the operators had scientific backgrounds, operators with little
technical training would probably be able to analyze samples successfully using only the
instructions as a guide. Microtox® was not tested in a non-laboratory setting because it is designed
to be only a laboratory benchtop instrument.
See Tables 6-la-i in the report for the precision around each individual inhibition result.
Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.
ND = Not detectable.
Lethal dose solution also contained 3 mg/L phosphate, 26 mg/L sodium chloride, and 2 mg/L sodium azide.
Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the concentration of the
lethal dose was 23% of the expected concentration of 0.30 mg/L.
28
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Chapter 8
References
1. Test/QA Plan for Verification of Rapid Toxicity Technologies, Battelle, Columbus, Ohio,
June 2003.
2. United States Environmental Protection Agency, National Secondary Drinking Water
Regulations: Guidance for Nuisance Chemicals, EPA/810/K-92/001, July 1992.
3. U.S. EPA Method 531.1, "Measurement of n-Methylcarbamoyloximes and
n-methylcarbamates in Water by Direct Aqueous Injection HPLC with Post Column
Derivatization," in Methods for the Determination of Organic Compounds in Drinking
Water—Supplement 111, EPA/600/R-95/131, 1995.
4. U.S. EPA Method 335.1, "Cyanides, Amenable to Chlorination," in Methods for the
Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
5. SW846 Method 8141 A, "Organophosphorous Compounds by Gas Chromatography:
Capillary Column Technique," Revision 1, September 1994.
6. U.S. EPA Method 200.8, "Determination of Trace Elements in Waters and Wastes by
Inductively-Coupled Plasma Mass Spectrometry," in Methods for the Determination of
Metals in Environmental Samples, Supplement I, EPA/600/R-94/111, 1994.
7. U.S. EPA Method 180.1, "Turbidity (Nephelometric)," Methods for the Determination of
Inorganic Substances in Environmental Samples, EPA/600/R-93/100, 1993.
8. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
9. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
10. U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking
Water—Supplement 111, EPA/600/R-95/131.
11. U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector," Methods for the Determination of
Organic Compounds in Drinking Water—Supplement III EPA/600/R-95/131.
29
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12. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 4.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, December 2002.
30
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