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Table 6-2. Lethal Dose Level Preservative Blank Percent Inhibition Results
Preservative
Blank
Inhibition
(%)
Average
(%)
Standard
Deviation
(%)
Negative
Control
-l
0
3
-2
(b)
3
Positive Control
8a
Ricin
-14
-5
9
-7
-8
-8
Soman/VX00
-3
-4
4
-8
-6
1
Botulinum
Toxin Complex
B
-12
-5
9
-6
7
-9
ta) Positive control percent inhibition less than suggested by Lab_Bell.
(b) Removed -45% because result was an obvious outlier.
Table 3-2 details the concentrations of preservatives in the lethal dose samples of each
contaminant. These data could be evaluated in two ways to determine the sensitivity of the
LuminoTox SAPS to contaminants stored in preservatives. The first approach would be to
determine the inhibition of the test samples containing preservatives with respect to the back-
ground negative control, as was the case for the contaminants that were not stored in
preservatives. This technique, however, could indicate that the LuminoTox SAPS Test Kit was
sensitive to the contaminant when, in fact, it was sensitive to one of the preservatives. Since
these contaminants are only available (either commercially or from the government) in aqueous
formulations with the preservatives, this may be appropriate. The second approach would be to
fortify negative control samples with the same concentrations of preservative contained in all the
samples so that the inhibition resulting from the preservatives could be subtracted from the
inhibition caused by the contaminant. This approach would greatly increase the number of
samples required for analysis. Therefore, for this test, aspects of both approaches were
incorporated without substantially increasing the number of samples. Negative control samples
fortified with a concentration of each preservative equivalent to the concentration in the lethal
dose test samples (preservative blanks) were analyzed prior to and with every set of test samples.
For those sets of test samples for which it was especially difficult to determine whether
inhibitory effects were from the contaminant or the preservative, the preservative blank was
diluted identically to all the contaminant samples and analyzed so a background subtraction
could take place if necessary.
During the initial analysis of the preservative blanks (Table 6-2), none of the preservative blank
samples generated an inhibition significantly greater than the DDW negative control. Because
the preservatives apparently do not have toxic effects at the lethal dose concentration, no
additional preservative blanks were analyzed to determine whether there were toxic effects from
each individual concentration level. Each concentration level was evaluated and compared with
27
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the negative control to determine any toxic effects. The lethal dose preservative blank was
determined with each contaminant sample set and is shown with each contaminant inhibition
regardless of the result of the initial preservative blank analysis. Neither the contaminant test
samples nor the preservative blank differed significantly from the negative control for botulinum
toxin complex B, ricin, soman, or VX. Therefore, none of these contaminants caused detectable
inhibition.
A positive control sample was analyzed with every set of analyses and, overall, the positive
control inhibition was somewhat inconsistent throughout the test of the LuminoTox SAPS Test
Kit. Prior to the verification test, Battelle had not been informed of a defined performance
criterion for the positive control; so, if an inhibition greater than the negative control was
generated, the testing staff considered the LuminoTox SAPS Test Kit to be operating properly.
The average inhibition across all of the positive control samples was 28% ± 12%, with each
positive control exhibiting more inhibition than its associated negative control. After the
completion of the test, Lab_Bell expressed concern that the positive control samples did not
always generate a percent inhibition as high as expected. Battelle was then provided a detailed
protocol that stated that the 0.01-mg/L solution of atrazine was expected to have an inhibition of
approximately 43% ± 5%. Had this information been available at the time of testing, the sample
sets not meeting the required positive control inhibition would have been reanalyzed once to try
to bring the control into the acceptable range as defined by Lab_Bell. However, because this was
not available to Battelle until after testing, the data are being reported as collected, and the tables
containing data from sample sets with positive control data less than 38% are noted as such in a
footnote. Three of the positive controls had an inhibition of 40% and above, four were between
30% and 40%, and seven were less than 30%. Three sample sets contained positive controls that
generated an inhibition less than 10% (aldicarb—8%, colchicine—9%, and the preservative
blanks—8%). Despite the low positive control inhibition, the three highest concentration levels
of aldicarb generated detectable inhibition; therefore, in that case, a positive control inhibition of
8% seemed to indicate the adequate functioning of the LuminoTox SAPS. Additionally, nicotine,
with a positive control response of 28% had detectable inhibition at the top two concentrations.
The low positive control inhibition may indicate a lower sensitivity of the LuminoTox SAPS
Test Kit than when a positive control inhibition of greater than 40% is obtained, but at least for
aldicarb and nicotine, the sensitivity seemed to be adequate.
The preservative blank inhibition also seems to suggest that the positive control inhibition was
adequate for confirming the function of the technology. The lethal dose preservative blank was
analyzed once prior to and once with the analysis of the contaminant samples. During the first
analysis prior to contaminant testing, the positive control inhibition was 8%. During subsequent
contaminant testing, every applicable positive control inhibition was higher (botulinum toxin
complex B—41%, ricin—22%, soman—33%, and VX—22%) and all of the lethal dose
preservative blanks generated an inhibition that was either the same as or extremely similar to
what was determined during the first analysis, therefore confirming the inhibition results from
the initial analysis that may have otherwise been in question because of the low positive control
inhibition. Additionally, the repeatability of results across all of the contaminants was very
good. Eighty-six percent of the time the standard deviation was less than 5% inhibition. This
suggests that even an inhibition of 8 or 9% is likely a significant inhibition with respect to the
negative control. All positive control results are reported along with their respective contaminant
set in Tables 6-la through 6-lj.
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6.1.2 Potential Interferences
All of the potential interference samples were prepared in DDW and compared with the negative
control to determine the level of inhibition. This determination is crucial because the ability of
the LuminoTox SAPS Test Kit to detect toxicity is dependent on the background fluorescence
production in whatever drinking water matrix is being used. If the background drinking water
sample completely inhibits background fluorescence, inhibition caused by contaminants could
not be detected. Table 6-3 presents the results from the samples that were analyzed to test the
effect of potential interferences on the LuminoTox SAPS Test Kit. Of the five metal solutions
that were evaluated as possible interferences, none exhibited an inhibition that was significantly
different from the DDW negative control. Therefore, it seems that there is little risk of
interference for these metals because enough fluorescence is produced for inhibition as a result
of contamination.
To investigate whether the LuminoTox SAPS Test Kit is sensitive to by-products of disinfecting
processes, DDW samples from water systems that use chlorination and chloramination were
analyzed and compared with ASTM Type IIDI water as the control sample. In the absence of a
background water sample, it seems likely that DI water may be used as a "clean water" control;
therefore, it would be helpful to know what the results would be if this is done. The sample from
the water supply disinfected by chlorination (N=56) exhibited an average inhibition of -8% ±
23%, while the sample from the water supply disinfected by chloramination exhibited an
inhibition of 0% ± 5% on four replicates. The difference in the number of replicates is because
the dechlorinated water was used as the negative control with each sample set; therefore, much
more data were collected on that water. These inhibition data suggest that samples disinfected by
either process are not likely to interfere with the LuminoTox SAPS Test Kit results.
6.1.3 Precision
Across all the contaminants and potential interferences, the standard deviation (not relative
standard deviation) was measured and reported for each set of four replicates to evaluate the
LuminoTox SAPS Test Kit precision. Out of 78 opportunities, the standard deviation of the four
replicate inhibition measurements was less than 5% inhibition 67 times (86% of the time),
between 5% and 10% inhibition 10 times (13% of the time), and greater than 10% inhibition just
1 time (1%). As described in Section 3.2.2, the analysis procedure required that each replicate
undergo the entire analysis process; therefore, the measurement of precision represents the
precision of the analysis method performed on a single water sample on a given day. The
precision does not reflect the repeatability of the method across more than one day or more than
one preparation of reagents or more than one operator.
6.2 Toxicity Threshold
Table 6-4 gives the toxicity thresholds, as defined in Section 5.2, for each contaminant. Note the
difference between detectability with respect to the negative control and the toxicity threshold
with respect to the other concentration levels analyzed. A contaminant concentration level can
have an inhibition significantly different from the negative control (thus detectable), but if its
inhibition is not
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Table 6-3. Potential Interferences Results
Potential
Interferences
Concentration
(mg/L)
Inhibition
(%)
Average
(%)
Standard Deviation
(%)
Negative control
(Metals)
Positive Control
(Metals)
Aluminum
NA
NA
0.5
-3
0
1
3
45
-3
2
0
5
Copper
0.6
Iron
0.15
2
2
-1
-1
Manganese
0.25
3
4
-3
-2
Zinc
2.5
-4
-1
5
-2
Negative control
(By-products)
Positive control
(By-products)
Chlorination
by-products
NA
NA
NA
1
0
-3
2
34'
«
(b)
23
Chloramination
by-products
NA
-4
0
-3
7
NA = Not applicable.
/a\
Positive control percent inhibition less than suggested by Lab_Bell.
(b) Average inhibition across all DDW negative control samples (N=56).
30
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Table 6-4. Toxicity Thresholds
Contaminant
Concentration (mg/L)
Aldicarb
26
Botulinum toxin complex B
ND
Colchicine
ND
Cyanide
250
Dicrotophos
ND
Nicotine
280
Ricin
ND
Soman
ND
Thallium sulfate
ND
VX
ND
ND = Significant inhibition was not detected.
significantly different from the concentration levels below it, it would not be considered the
toxicity threshold because in the context of this test, its inhibition would not be distinguishable
from that of the lower concentrations. The lowest toxicity threshold concentration was for
aldicarb at 26 mg/L.
6.3 False Positive/Negative Responses
None of the LuminoTox SAPS Test Kit results would be considered false positive because
neither the chlorination nor chloramination by-product samples were inhibitory and, therefore,
fluorescence production was adequate to allow inhibition to occur if a contaminant was present
that produced a detectable toxic effect. Since the background inhibition is not complete, it can be
accounted for by using negative control samples that are very similar to the water being
analyzed. If samples are analyzed daily, a good practice would be to archive a negative control
sample each day in case of contamination the next day.
Table 6-5 shows the LuminoTox SAPS Test Kit false negative responses, which are described in
Section 5.3. Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, thallium sulfate,
and VX did not exhibit a detectable inhibition at the lethal concentration.
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Table 6-5. False Negative Responses
Contaminant
Lethal Dose
Concentration (Mg/L)
False Negative
Aldicarb
260
no
Botulinum toxin
complex B
0.30
yes
Colchicine
240
yes
Cyanide
250
no
Dicrotophos
1,400
yes
Nicotine
2,800
no
Ricin
15
yes
Soman
1.4
yes
Thallium sulfate
2,800
yes
VX
2.0
yes
6.4 Other Performance Factors
6.4.1 Ease of Use
The LuminoTox SAPS Test Kit contained detailed instructions and clear illustrations. The
contents of the LuminoTox SAPS Test Kit were well identified with labels on the vials. Storage
requirements were stated in the instructions and on the reagent vials. Overall, the test was easy to
perform; but additional practice helped the operators become accustomed to the timing involved
with running a large number of test samples.
Preparation of the test samples for analysis was straightforward. The analyzer, including a piece
of foil covering the cuvette opening, was easy to use, but the necessity to record four numbers as
raw data was somewhat burdensome. Lab Bell has indicated that this is undergoing modification.
After testing, the fluorometer was easily wiped clean and required no routine maintenance other
than selecting the SAPS mode prior to the start of sample analysis.
No formal scientific education would be required to use the LuminoTox SAPS Test Kit.
However, good laboratory skills, especially pipetting technique, would be beneficial.
Verification testing staff were able to operate the LuminoTox SAPS Test Kit after a 4-hour
training session with the vendor. With every sample, approximately 2 mL of liquid waste were
generated, along with leftover SAPS and a 3-mL disposable syringe.
6.4.2 Field Portability
The LuminoTox SAPS Test Kit was transported from a laboratory setting to a storage room for
the field portability evaluation. The storage room contained several tables and light and power
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sources, but no other laboratory facilities. No carrying case was provided with the LuminoTox
SAPS Test Kit; however, all materials were transported by one person in a small cardboard box.
The LuminoTox SAPS Test Kit was set up easily in less than 10 minutes, and a source of
electricity was not required since the fluorometer ran on batteries. Minimum space requirements
in the field would be a mostly flat surface of approximately 45 by 60 centimeters. The only items
needed for field use not provided in the LuminoTox SAPS Test Kit was a timer and a waste
reservoir. Overall, the LuminoTox SAPS Test Kit was easy to transport to the field and was
deployed in a matter of minutes. The limiting factor for testing in the field would be the
approximately 90 minutes required to expose the SAPS to light prior to testing. After the light
exposure, results were obtained within 10 minutes of starting the test. The LuminoTox SAPS
Test Kit was tested with one contaminant, cyanide, at the lethal dose concentration. The results
of the test (see Table 6-ld) were very similar to the laboratory results. Inhibition in the
laboratory was 17% ± 2%, and in the non-laboratory location, 16% ± 4%, suggesting that
location did not impact the performance of the LuminoTox SAPS Test Kit.
6.4.3 Throughput
Once the SAPS were prepared, approximately 20 analyses were completed per hour. The
20 analyses included method blanks and positive and negative controls, as well as test samples.
Approximately 50 samples could be analyzed with the supplies contained in one LuminoTox
SAPS Test Kit.
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Chapter 7
Performance Summary
Parameter
Compound
Lethal
Dose (LD)
Cone.
(mg/L)
Average Inhibition at Concentrations
Relative to the LD Concentration
(%)
LD
LD/10
LD/100
LD/1,000
Range of
Standard
Deviations
(%)
Toxicity
Thresh.
(mg/L)
Aldicarb
260
50
14
0
1-3
26
Botulinum toxin
complex B
0.3
-10
-6
-5
1-8
ND
Colchicine
240
1-5
ND
Cyanide
250
17
10
2-3
250
Contaminants in
DDW
Dicrotophos
1,400
-11
-12
-10
1-2
ND
Nicotine
2,800
34
10
1-4
280
Ricin
15
2-6
ND
Soman
1.4
2-3
ND
Thallium
sulfate
2,800
-3
-4
2-3
ND
VX
3
-1
Interference
Cone.
(mg/L)
Average Inhibition
(%)
Potential
interferences in
DDW
Aluminum
0.5
1
Copper
0.6
Iron
0.15
Manganese
0.25
Zinc
2.5
-1
Standard
Deviation (%)
False positive
response
None of the LuminoTox SAPS Test Kit responses were considered false positive. All disinfection by-
product test samples left enough fluorescence for inhibition due to contamination.
False negative
response
Botulinum toxin complex B, colchicine, dicrotophos, ricin, soman, thallium sulfate, and VX exhibited
non-detectable responses at the lethal dose concentration.
Ease of use
The LuminoTox SAPS Test Kit contained detailed instructions and clear illustrations. The contents
were well identified with labels on the vials. Storage requirements were stated in the instructions and
on the reagent vials. Preparation of the test samples for analysis was straightforward. The necessity to
record four numbers as raw data was somewhat burdensome; however, this feature is being modified
according to Lab_Bell. No formal scientific education would be required to use the LuminoTox SAPS
Test Kit.
Field portability
The LuminoTox SAPS Test Kit was transported from a laboratory setting to a storage room for the
field portability evaluation. The limiting factor for testing in the field would be the approximately
90 minutes required to allow the SAPS to be exposed to light prior to testing. The LuminoTox SAPS
Test Kit was tested with one contaminant, cyanide, at the lethal dose concentration. The results of the
test were very similar to the laboratory results. Inhibition in the laboratory was 17% ± 2%, and in the
non-laboratory location, 16% ±4%.
Throughput
Approximately 20 analyses were completed per hour, and 50 samples could be analyzed with the
supplies contained in one LuminoTox SAPS Test Kit.
ND = Significant inhibition was not detected.
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Chapter 8
References
1. Test/QA Plan for Verification of Rapid Toxicity Technologies, Battelle, Columbus, Ohio,
June 2003; Amendment 1: June 9, 2005; Amendment 2: August 19, 2005.
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 335.3, "Cyanide, Total—Colorimetric, Automated UV," in Methods for
the Chemical Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
4. 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.
5. U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.
6. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, 1997. Washington, DC.
7. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020.
8. 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 I iPA/600/R-95/131.
9. 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 III, EPA/600/R-95/131.
10. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater, 20th edition, 1998, Washington, DC.
11. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, December 2004.
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