5549
905R80112
METHOD 643: ANALYSIS OF BENTAZON
IN WASTEWATER BY LIQUID CHROMATOGRAPHY
1. SCOPE AND APPLICATION
V .f
1.1 This method covers the determination of bentazon in municipal and
industrial wastewater.
Parameter CAS No.
Bentazon (Basagran) 25057-89-0
1.2 The estimated detection limit (EDL) for bentazon is listed in
Table 1. The EOL was calculated from the minimum detectable
response being equal to 5 times the background noise using a 5-mL
final extract volume of a 1-liter sample and an injection volume of
100 yL. The EDL for a specific wastewater may be different
depending on the nature of interferences in the sample matrix.
1.3 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of bentazon in municipal and
industrial discharges. When this method is used to analyze
unfamiliar samples for bentazon, compound identification should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second HPLC column
that can be used to confirm measurements made with the primary
column.
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 liquid chromatograms.
2. SUMMARY OF METHOD
2.1 Bentazon is removed from an acidified sample matrix by extraction
with methylene chloride. The extract is discarded after back
extraction with aqueous base. HPLC conditions are described which
permit the separation and measurement of bentazon in the aqueous
extract.
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.
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230 South Dearborn Street
Chicago, Illinois 60604
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Environmental Protection Agency
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3.1.2 Glassware must be scrupulously cleaned (1). Clean all
glassware as soon as 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 hexane 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.
3.2 Matrix interferences may be caused by UV-active contaminants that
are co-extracted from the samples. 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. 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 SAMPLE CONTAINERS - Narrow-mouth glass bottles, 1-liter or 1-quart
volume, equipped with polytetrafluoroethylene (PTFE)-lined screw
caps. Wide-mouth glass bottles, 1-quart volume, equipped with
PTFE-lined screw caps may also be used. Prior to use, wash bottles
and cap liners with detergent and rinse with tap and reagent
water. Allow the bottles and cap liners to air dry, then muffle
the bottles at 400*C for 1 hour. After cooling, rinse the bottle
and cap liners-with hexane, seal the bottles, and store in a
dust-free environment.
5.1.1 Automatic sampler (optional)—Must incorporate glass sample
containers for the collection of a minimum of 250 mL.
Sample containers must be kept refrigerated at 4*C and
protected from light during compositing. If the sampler
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uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing should be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect
flow-proportional composites.
5.2 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.
5.2.1 Gradient pumping system, constant flow.
5.2.2 Injector valve (Rheodyne 7125 or equivalent) with 100-nL
loop.
5.2.3 Column 1 — 250 mm by 4.6 mm ID, stainless steel, packed
with reverse-phase Ultrasphere OOS, 10 u, or equivalent.
5.2.4 Column 2 — 300 mm by 4.0 mm ID, packed with reverse phase
Bondapak CIS, 10u, (Waters Associates), or equivalent.
5.2.5 Ultraviolet detector, variable wavelength, capable of
monitoring at 340 nm.
5.2.6 Strip-chart recorder compatible with detector, 250-mm. (A
data system for measuring peak areas is recommended.)
5.3 MISCELLANEOUS
5.3.1 Balance—analytical, capable of accurately weighing to the
nearest 0.0001 g.
5.3.2 Separatory funnels—2-liter, and 250 mL, equipped with PTFE
stopcocks.
5.3.3 Standard solution storage containers—15-mL bottles with
PTFE-lined screw caps.
5.3.4 Pasteur pipets with bulbs.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 REAGENTS
6.1.1 Acetone, hexane, methanol, and methylene
chloride—Demonstrated to be free of analytes and
interferences.
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5.1.2 Reagent water—Reagent water is defined as a water in which
an interferent is not observed at the method detection limit
of each parameter of interest.
6.1.3 Sodium hydroxide solution (0.1N)—Dissolve 0.4 gram of NaOH
in reagent water and dilute to 100 ml.
6.1.4 Sodium chloride—(ACS) Crystals.
6.1.5 Sodium thiosulfate—(ACS) Granular.
6.1.6 Sulfuric acid solution (1+1)—Slowly add 50 ml of H2S04
(specific gravity 1.84) to 50 mL of reagent water.
6.1.7 Sodium hydroxide solution (6N)—Dissolve 24 g of NaOH in
reagent water and dilute to 100 mL.
6.1.8 Acetate buffer solution—Dissolve 0.41 g of anhydrous sodium
acetate (ACS) and 1.5 ml of glacial acetic acid (ACS) in 100
mi of reagent water.
6.1.9 Glacial acetic acid—(ACS).
6.1.10 HPLC mobile phase buffer (pH 4.7, 0.062 M acetate)—
Dissolve 0.87 g of anhydrous sodium acetate (ACS) and 3.0 mL
of glacial acetic acid (ACS) in 1 L of reagent water.
6.2 STANDARD STOCK SOLUTION (1.00 wg/uL)—This solution may be
purchased as a certified solution or prepared from a pure standard
material using the following procedures.
6.2.1 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 10-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.
6.2.2 Transfer the stock standards in TeflonR-sealed screw-cap
bottles. Store at 4*C and protect from light. Stock
standards should be checked frequently for signs of
degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.2.3 Stock standards must be replaced after 6 months, or when
comparison with quality control check samples indicates a
problem.
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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 extraction. If the samples will not be extracted
within 48 hours of collection, the sample should be adjusted to a
pH range of 6.0 to 8.0 with sodium hydroxide or sulfuric acid and
35 mg of sodium thiosulfate per ppm of free chlorine per liter must
be added.
7.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of extraction. (6)
8. CALIBRATION
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 bentazon by adding volumes of the stock standard to a
volumetric flask and diluting to volume with HPLC mobile phase
(35-percent methanol in HPLC mobile phase buffer or
40-percentmethanol in HPLC mobile phase buffer). 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 the analytes.
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 bentazon 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 pattern and the absence of interferences from the reagents.
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9. QUALITY CONTROL
9.1 MONITORING FOR INTERFERENCES
Analyze a laboratory reagent blank 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
bentazon, 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.
9.2.1.1 Laboratory Control Standard Concentrate (100 ug/L)—
From stock standards prepared as described in
Section 6.2, prepare a laboratory control standard
concentrate that contains bentazon at a
concentration of 2 ug/uL in methanol.(7)
9.2.1.2 Laboratory Control Standard — Using a pipet or
micro liter syringe, add 50.0 uL of the laboratory
control standard concentrate to a 1.0 L aliquot of
reagent water contained in a 1-L volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described
in Section 10. Calculate the percent recovery
(Pi) with the equation:
P. , . Si
Ti
where S-j - the analytical results from the
laboratory control standard, in ug/L; and
T-j m the known concentration of the spike,
in ug/L.
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.(7)
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
samples for at least 10% of all samples. To the extent
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practical, the samples for duplication should contain
reportable levels of bentazon.
9.3.2 Calculate the relative range (7) (RR-j) with the equation:
100 R,
RR
i
Xi
where R-j - the absolute difference between the
duplicate measurements X^ and Xg, in
ug/L; and
XT m the average concentration found ([X^ +
X2]/2), in yg/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. PROCEDURE
10.1 SAMPLE EXTRACTION
10.1.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume. Pour the entire
sample (approximately one liter) into a 2-liter separatory
funnel. Add 35 mg/L of sodium thiosulfate per ppm of free
chlorine. Check the pH of the sample with wide-range pH
paper and adjust to within the range of 2.5 to 3.5 with
sulfuric acid. Add 200 grams of sodium chloride and mix to
dissolve.
10.1.2 Add 60 ml of methylene chloride to the sample bottle and
shake for 30 seconds to rinse the walls. Transfer the
solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 minutes with periodic venting to
release vapor pressure. Allow the organic layer to separate
from the water phase for a minimum of 10 minutes. If the
emulsion interface between layers is more than one-third the
volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends on the sample, but may include
stirring, filtration of the emulsion through glass wool, or
centrifugation. Collect the extract in a 250-mL separatory
funnel.
10.1.3 Add an additional 60-mL volume of methylene chloride to the
sample bottle and complete the extraction procedure a second
time, combining the extracts in the 250-mL separatory funnel.
10.1.4 Perform a third extraction in the same manner. Add 2 mL of
0.1 M NaOH in reagent water to the 250-mL separatory funnel,
and extract by shaking the funnel for 2 minutes with
periodic venting to release vapor pressure. Allow the
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organic layer to separate from the water phase for a minimum
of 10 minutes. Drain the methylene chloride into a 250-mL
Erlenmeyer flask. Transfer the aqueous layer with a Pasteur
pipet to a 5-mL volumetric flask.
10.1.5 Add the methylene chloride back to the 250-mL separatory
funnel, and extract with an additional 2 ml of 0.1 M NaOH.
Combine the extracts in the 5-mL volumetric flask.
10.1.6 Add two drops of glacial acetic acid to the volumetric
flask, and dilute to volume with acetate buffer solution
(Section 6.1.7).
10.1.7 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1,000-mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
10.2 CLEANUP AND SEPARATION
10.2.1 The cleanup procedure recommended in this method involves
the back extraction of a methylene chloride extract with
aqueous base, and has been used for the analysis of various
clean waters and industrial effluents. If additional
cleanup is required, a one liter sample is adjusted to pH 12
with 6N NaOH and extracted with three, 60 mL aliquots of
methylene chloride in a 2L seporatary funnel. The methylene
chloride extracts are discarded and the aqueous sample
adjusted to pH range of 2.5 to 3.5 with 1:1 sulphuric acid
solution for re-extraction as per sect. 10.1.1. If
additional cleanup is required, or if particular
circumstances demand the use of an alternate cleanup
procedure, the analyst must determine the elution profile
and demonstrate that the recovery for each compound of
interest is no less than 85 percent.
10.3 LIQUID CHROMATOGRAPHY ANALYSIS
10.3.1 Table 1 summarizes the recommended operating conditions for
the liquid chromatograph. Included in this table are the
estimated retention times and estimated detection limit that
can be achieved by this method. An example of the
separation achieved by Column 2 is shown in Figure 1. 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 \il of the sample extract. Monitor the column eluent at
340 nm. 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, additional cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration of bentazon in the sample.
11.1.1 Calculate the amount of bentazon injected from the peak
response from the calibration curve. The concentration in
the sample can be calculated from the equation:
(A) (Vt)
Concentration, ug/L » (V ) (V )
where:
A - Amount of bentazon injected (nanograms),
V^ « Volume of extract injected (uL),
V^ « Volume of total extract (uL), and
Vs * Volume of water extracted (mL).
11.2 Report results in vg/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 bentazon are
listed in Table 1(8). The EDL is defined as the minimum response
being equal to 5 times the background noise, assuming a 5-mL final
extract volume of a 1-liter sample and an HPLC injection volume of
100 UL.
12.2 Single operator accuracy and precision studies were conducted by
Environmental Science and Engineering, Incorporated (6), in the
designated matrices. The results of these studies are presented in
Table 2.
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I
REFERENCES " " ;
t
f
1. ASTM Annual Book of Standards, Part 31, 03694, "Standara 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.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice
for Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report
No. 68-03-2897 (In Preparation). Unpublished report available from
U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 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 Pesticide Methods," U.S: Environmental
Protection Agency Contract No. 68-03-1760, Task No. 11, U.S.
Environmenal Monitoring and Support Laboratory, Cincinnati, Ohio
45268.
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TABLE Z. SINGLE LABORATORY ACCURACY AND PRECISION
Parameter
Bentazon
Matrix
Type*
1
2
Range
ug/L
125
20,400
No. of
Replicates
7
7
Average
Percent
Recovery
85.1
88.4
Standard
Deviation
(%)
4.8
8.4
*1 » 50-percent industrial effluent + 50-percent POTW effluent.
2 » 100-percent industrial effluent.
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DUE
U.S. Environmental Protection Agency
Region V, Library
230 South Dearborn Street
Chicago, Illinois 60604
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u
4)
J
Q 1 2 3 4 5
Retention Time (Min.)
FIGURE 1. HPLC OF BENTAZON COLUMN 2
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