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METHOD 641: ANALYSIS OF THIABENDAZOLE
IN WASTEWATER BY LIQUID CHROMATOGRAPHY
1. SCOPE AND APPLICATION
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1.1 This method covers the determination of thiabendazole in municipal
and industrial wastewater.
Parameter CAS No.
Thiabendazole 148-79-8
1.2 The estimated detection limit (EDL) for thiabendazole is listed in
Table 1. The EDL was calculated from the minimum detectable
response being equal to 5 times the background noise using a 100-uL
injection. The EDL for a specific wastewater may be different
depending on the nature of interferences in the sample matrix.
1.3 This is a liquid chromatographic method applicable to the
determination of thiabendazole in municipal and industrial
discharges. When this method is used to analyze unfamiliar samples
for thiabendazole, compound identification should be supported by
at least one additional qualitative technique.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the operation of liquid chromatographs and
in the interpretation of chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this
method using the procedure described in Sections 9.2 and 9.3.
2. SUMMARY OF METHOD
2.1 Thiabendazole is analyzed in the sample matrix after solubilization
with acid and filtration to remove particulate matter.
Chromatographic conditions are described which permit the
separation and accurate measurement of thiabendazole by direct
aqueous injectionHPLC with fluorescence detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation in liquid chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running laboratory reagent blanks as
described in Section 9.1.
3.1.1 The use of high-purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
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U.S. l&vtrotTmental Protection Agency
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3.2
3.1.2 Glassware must be scrupulously cleaned (1). Clean all
glassware as soon a$ possible after use by rinsing with the
last solvent used in it. This should be followed by
detergent washing with hot water and rinses with tap water
and reagent water. It 'should then be drained dry and heated
in a muffle furnace at 400*C for 15 to 30 minutes. Solvent
rinses with acetone and pesticide-quality hexahe may be
substituted for the muffle furnace heating. Volumetric ware
should not be heated in a muffle furnace. After drying and
cooling, glassware should be sealed and stored in a clean
environment to prevent any accumulation of dust or other
contaminants. Store the glassware inverted or capped with
aluminum foil.
Matrix interferences may be caused by fluorescing contaminants that
coelute with thiabendazole. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
being sampled. Matrix interferences caused by the presence of
particulate matter are removed by filtration. Unique samples may
require additional cleanup approaches to achieve the detection
limit listed in Table 1.
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the
lowest possible level by whatever means available. The laboratory
is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
available and have been identified (2-4) for the information of the
analyst.
5. APPARATUS AND EQUIPMENT
5.1 SAMPLING EQUIPMENT FOR DISCRETE SAMPLING
5.1.1 Vial - 25 mL capacity or larger, equipped with a screw cap
with hole in center (Pierce #13074 or equivalent).
Detergent wash, rinse with tap and distilled water, and dry
at 105* before use.
5.1.2 Vial - 3.5 ml, equipped with a screw cap with hole in center
(Pierce #13019 or equivalent).
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5.1.3 Septum - Teflon^ - faced silicone (Pierce #12722 or
equivalent). Detergent wash and dry at 105*C for one hour
before use.
5.1.4 Septum - TeflonR - Faced silicone (Pierce #12712 or
equivalent). Detergent wash and dry at 105 for 1 hour
before use.
5.2 Syringe Glass, 5 ml with Leur tip.
5.3 Syringe filter holder Stainless steel with Leur connection
(Rainin 38-101 or equivalent).
5.4 Filters 13 mm, Nylon 66, 0.45-y pore (Rainin 38-112 or
equivalent).
5.5 Balance Analytical, capable of accurately weighing to the
nearest 0.0001 gram.
5.6 High performance liquid chromatography (HPLC) apparatus
Analytical system complete with liquid chromatograph and all
required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified
detectors and strip-chart recorder. A data system is recommended
for measuring peak areas.
5.6.1 Isocratic pumping system, constant flow.
5.6.2 Injector valve (Rheodyne 7125 or equivalent) with 100-uL
loop.
5.6.3 Column 250 mm by 4.6 mm ID, stainless steel, packed with
reverse-phase Ultrasphere ODS, 10u.
5.6.4 Fluorescence detector, for excitation at 300 nm and emission
at 360 nm. (Perkin Elmer 650-10S or equivalent).
Fluorometer should have dispersive optics for excitation and
utilize either filter or dispersive optics at the emission
detector.
5.6.5 Strip-chart recorder compatible with detector, 250-mm. (A
data system for measuring peak areas is recommended.)
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagent water Reagent water is defined as a water in which an
interferent is not observed at the EDL of each parameter of
interest.
6.2 Sodium hydroxide solution (ION) Dissolve 40 grams of NaOH in
reagent water and dilute to 100 mL.
6.3 Sodium thiosulfate (ACS) Granular.
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6.4 Sulfuric acid solution (1+1) Slowly add 50 ml of
(specific gravity 1.84) to 50 mL of reagent water.
6.5 HPLC buffer (pH 8.2) Add 8 ml of triethanolamine (Eastman 1599)
and 1 mi of glacial acetic acid (ACS) to 1 liter of reagent water.
6.6 High-purity methanol HPLC quality, distilled in glass.
6.7 Stock standard solution (1.0 ug/uL) Stock standard solutions are
prepared from pure standard material or purchased as a certified
solution.
6.7.1 Prepare the stock standard solution by accurately weighing
about 0.0100 gram of pure material. Dissolve the material
in pesticide quality methanol, dilute to volume in a IQ-mL
volumetric flask. Larger volumes can be used at the
convenience of the analyst. When compound purity is
certified at 96 percent or greater, the weight can be used
without correction to calculate the concentration of the
stock standard. Commercially prepared stock standards can
be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard in Teflon^-sealed screw-cap
bottle. Store at 4*C and protect from light. The stock
standard should be checked frequently for signs of
degradation or evaporation, especially just prior to
preparing calibration standards.
6.7.3 The stock standard must be replaced after 6 months, or when
comparison with quality control check samples indicates a
problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected
in glass containers. Conventional sampling practices (5) should be
followed, except that the bottle must not be prewashed with sample
before collection.
7.2 The samples must be iced or refrigerated at 4*C from the time of
collection until analysis. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be analyzed
within 48 hours of collection, the sample should be adjusted to a
pH range of 1.0 to 3.0 with sodium hydroxide or sulfuric acid, and
35 mg of sodium thiosulfate per ppm of free chlorine should be
added.
7.3 All samples must be analyzed within 30 days of collection. (6)
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8. CALIBRATION AND STANPARIZATION .
8.1 Establish liquid chromatographic operating parameters equivalent to
those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration
levels of thiabendazole by adding volumes of the stock standard to
a volumetric flask and diluting to volume with HPLC mobile phase.
One of the external standards should be at a concentration near,
but greater than, the EDL, and the other concentrations should
correspond to the expected range of concentrations found in real
samples or should define the working range of the detector.8.3.
Using injections of 100 uL of each calibration standard, tabulate
peak height or area responses against the mass injected. The
results are used to prepare a calibration curve for thiabendazole.
Alternatively, if the ratio of response to amount injected
(calibration factor) is a constant over the working range (<10
percent relative standard deviation, RSD), linearity of the
calibration curve can be assumed and the average ratio or
calibration factor can be used in place of a calibration curve.
8.4 The working calibration curve or calibration factor must be
verified on each working day by the measurement of one or more
calibration standards. If the response for thiabendazole varies
from the predicted response by ±10 percent, the test must be
repeated using a fresh calibration standard. Alternatively, a new
calibration curve or factor must be prepared.
8.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the reagents.
9. QUALITY CONTROL
9.1 MONITORING FOR INTERFERENCES
Analyze a laboratory reagent blank as described in section 10 each time
a set of samples is extracted. A laboratory reagent blank is an aliquot
of reagent water. If the reagent blank contains a reportable level of
thiabendazole, immediately check the entire analytical system to locate
and correct for possible interferences and repeat the test.
9.2 ASSESSING ACCURACY
9.2.1 After every 10 samples, and preferably in the middle of each
day, analyze a laboratory control standard. Calibration
standards may not be used for accuracy assessments and the
laboratory control standrd may not be used for calibration
of the analytical system.
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9.2.1.1 Laboratory Control Standard Concentrate - from the
stock standard prepared as described in Section 6.7,
prepare a laboratory control standard concentrate
that contains thiabendazole at a concentration of 2
ug/mL in methanol or other suitable sol vent.(7)
9.2.1.2 Laboratory Control Standard - using a pipet or
microliter syringe, add 50.0 uL of the laboratory
control standard concentrate to a 10 mL aliquot of
reagent water contained in a 10-mL volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described
in Section 10. Calculate the percent recovery
with the equation:
P.. 100Si
I ^^^^^^^^H^
where S-j = the analytical results from the
laboratory control standard, in ug/L; and
T-J = the known concentration of the spike,
in
9.2.2 At least annually, the laboratory should participate in
formal performance evaluation studies, where solutions of
unknown concentrations are analyzed and the performance of
all participants is compared.
9.3 ASSESSING PRECISION
9.3.1 Precision assessments for this method are based upon the
analysis of field duplicates (Sect. 7.1). Analyze both
sample vials for at least 10% of all samples. To the extent
practical, the samples for duplication should contain
reportable levels of thiabendazole.
9.3.2 Calculate the relative range (RRi) with the equation:
100 R.
1 T
where Ri = 1 the absolute difference between the
duplicate measurements X] and X2, in
ug/L; and
XT = the average concentration found ([X] +
X23/2), in ug/L.
9.3.3 Individual relative range measurements are pooled to
determine average relative range or to develop an expression
of relative range as a function of concentration.10.
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10. PROCEDURE
10.1 SAMPLE PREPARATION
10.1.1 Adjust the pH of the sample to pH 1-3 with sulfuric acid
solution.
10.1.2 Assemble the syringe-filtration assembly by attaching the
filter holder (with filter) to a 5-mL glass syringe equipped
with a Leur tip.
10.1.3 Remove the barrel from the syringe and pour a 4 to 5 ml
aliquot of the acified sample into the syringe, allowing room
for reinsertion of the syringe barrel.
10.1.4 Filter a portion of the sample through 0.45-p filter using a
syringe filter holder. The first few milliliters should be
discarded. Collect the filtrate in a 4-mL vial equipped with
a TeflonR-sealed screw cap.
10.1.5 The syringe and filter holder should be rinsed with acetone
or methanol and then HPLC water between samples.
10.2 CLEANUP AND SEPARATION
10.2.1 Cleanup procedures may not be necessary for a relatively
clean sample matrix. Use of fluorescent detectors, however,
often obviates the necessity for cleanup of relatively clean
sample matrices. If particular circumstances demand the use
of an alternative cleanup procedure, the analyst must
determine the elution profile and demonstrate that recovery
is no less than 85 percent.
10.3 LIQUID CHROMATOGRAPHIC ANALYSIS
10.3.1 Table 1 summarizes the recommended operating conditions for
the liquid chromatograph. Included in this table are the
estimated retention time and estimated detection limit that
can be achieved by this method. An example chromatogram
achieved by this column is shown in Figure 1. Figure 2 is a
chromatogram of thiabendazole in a POTW wastewater sample.
Other columns, chromatographic conditions, or detectors may
be used if data quality comparable to table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.4 Inject 100 yL of the filtered aqueous sample. Monitor the column
eluent at excitation wavelength 300 nm (5-nm slit width) and
emission wavelength 360 nm (10-nm slit width). Record the resulting
peak size in area or peak height units.
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10.5 The retention time window used to make identifications should be
based upon measurements of actual retention time variations of
standards over the course of a day. Three times the standard
deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the
analyst should weigh heavily in the interpretation of chromatograms.
10.6 If the response for the peak exceeds the working range of the
system, dilute the sample with mobile phase and reanalyze.
10.7 If the measurement of the peak response is prevented by the presence
of interferences, further cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration of thiabendazole in the sample.
11.1.1 Calculate the amount of thiabendazole injected from the peak
response using the calibration curve or calibration factor in
Section 8.2.2. The concentration in the sample can be
calculated from the following equation:
Concentration, ug/L = (A) (1000)
where:
A = Amount of thiabendazole injected (nanograms) and
V-j « Volume of sample injected (yL).
11.2 Report results in wg/L without correction for recovery data. When
duplicate and spiked samples are analyzed, report all data obtained
with the sample results.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for thiabendazole
are listed in Table 1(8). The EDL is defined as the minimum
response being equal to 5 times the background noise, using a
100-uL injection.
12.2 Single operator accuracy and precision studies were conducted by
Environmental Science and Engineering, Incorporated (6), using a
spiked POTW sample. The results of these studies are presented in
Table 2.
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REFERENCES
1. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice
for Preparation of Sample Containers and for Preservation,"
American Society for Testing and Materials, Philadelphia, PA, p.
679, 1980.
2. , "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised,
January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report
#68-03-2897, unpublished report available from U.S. Environmental
Protection Agency, Environdmental Monitoring and Support
Laboratory, Cincinnati, Ohiio 45268.
7. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1979.
8. "Evaluation of Ten Pesticides Methods," U.S. Environmental
Protection Agency, Contract No. 68-03-1760, Task No. 11, U.S.
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio
45268.
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TABLE 1
LIQUID CHROMATOGRAPHY OF THIABENDAZOLE*
Compound
Retention Time
(minutes)
Estimated Detection
Limit (ug/L)
Thiabendazole
4.3
1.7
* HPLC conditions: 10 y reverse-phase Ultrasphere ODS, 4.6 mm by 250 mm
column; isocratic 70 percent methaneI/30 percent buffer; flow rate 1
mL/min.
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Thiabendazole
Spike
Concentration
(ug/L)
12.5
625
Number of
Replicates
7
7
Average
Percent
Recovery
100
92.8
Standard
Deviation
(%)
9.5
4.5
*POTW effluent.
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0123456
Retention Time (Win.)
FIGURE 1. HPLC OF THIABENDAZOLE
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~»ATE DUE
US Environmental Protection Agency
Chicago,
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0 '1 2 3 4 5 6
Retention Time (Min.)
FIGURE 2. CHROMATOGRAM OF THIABENDAZOLE
IN WASTEWATER SAMPLE.
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