Method 632.1
The Determination of
Carbamate and Amide
Pesticides in Municipal and
Industrial Wastewater
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Method 632.1
The Determination of Carbamate and Amide Pesticides in Municipal
and Industrial Wastewater
1. Scope and Application
1.1 This method covers the determination of certain carbamate/amide pesticides in municipal
and industrial wastewater. The following parameters may be determined by this
1.2 The estimated detection limits (EDLs) for the parameters above are listed in Table 1. The
EDL was calculated from the minimum detectable response being equal to five times the
background noise using a 10-mL final extract volume of a 1-L sample and an injection
volume of 100 pL. 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 the compounds listed above in municipal and industrial discharges.
When this method is used to analyze unfamiliar samples for any or all of the compounds
above, 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 The carbamate/amide pesticides are removed from the sample matrix by extraction with
methylene chloride. The extract is dried, exchanged to HPLC mobile phase and analyzed
by liquid chromatography with ultraviolet (UV) detection.
3. Interferences
3.1 Solvent, reagents, glassware, and other sample processing hardware may yield discrete
artifacts and/or elevated baselines causing misinterpretation of liquid chromatograms.
All of these materials must be demonstrated to be free from interferences under the
method.
Parameter
Napropamide
Propanil
Vacor
CAS No.
15299-99-1
709-98-8
53558-25-1
conditions of the analysis by running laboratory reagent blanks as described in Section
9.1.
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Method 632.1
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.
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 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 coextracted 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 cleanup approaches to achieve
the detection limits 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 identified2 4 for the
information of the analyst.
5. Apparatus and Equipment
5.1 Sample Containers: Narrow-mouth glass bottles, 1-L 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 distilled 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 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
3.1.1
3.1.2
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Method 632.1
minimize the potential for contamination of the sample. An integrating flow
meter is required to collect flow-proportional composites.
5.2 Rotary evaporator: With 24/40 joints and associated water bath and vacuum for
operation at reduced pressure (Servo Instruments VE-1000-B or equivalent).
5.3 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.
5.3.1 Gradient pumping system.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with IOO41L loop.
5.3.3 Column 1: 250 mm long by 4.0 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 5 |i, or equivalent.
5.3.4 Column 2: 250 mm long by 4.6 mm ID, packed with reverse phase Dupont
Zorbax ODS, 10 |i, or equivalent.
5.3.5 Ultraviolet detector, variable wavelength, capable of monitoring at 254 nm.
5.3.6 Strip-chart recorder compatible with detector, 250 mm. (A data system for
measuring peak areas is recommended.)
5.4 Boiling flask: 250-mL, flat-bottom, 24/40 joint.
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped
with coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or
perform a Soxhlet extraction with methylene chloride for 2 hours.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Volumetric flasks: 5-mL and 10-mL, Class A.
5.6.6
Pasteur pipettes with bulbs.
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Method 632.1
6. Reagents and Consumable Materials
6.1 Reagents.
6.1.1 Acetone, acetonitrile, hexane, and methylene chloride: Demonstrated to be free
of analytes and interferences.
6.1.2 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 sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours
in a shallow tray.
6.1.4 HPLC mobile phase, Column 1: Add 400 mL of acetonitrile to a 1-L volumetric
flask and dilute to volume with reagent water.
6.1.5 HPLC mobile phase, Column 2: Add 550 mL of acetonitrile to a 1-L volumetric
flask and dilute to volume with reagent water.
6.1.6 Sodium hydroxide solution (1.0N): Dissolve 40 g of NaOH in reagent water and
dilute to 1000 mL.
6.1.7 Sodium chloride: ACS, crystals.
6.1.8 Sodium thiosulfate: ACS, granular.
6.1.9 Sulfuric acid solution (1+1): Slowly add 50 mL of H2S04 (specific gravity 1.84) to
50 mL of reagent water.
6.2 Standard stock solutions (1.00 |ig/|_iL): These solutions may be purchased as certified
solutions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality (9:1) acetonitrile/acetone and
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% 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 to PTFE-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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Method 632.1
7. Sample Collection, Preservation, and Storage
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers.
Conventional sampling practices5 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. 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 extracted
within 48 hours of collection, the sample should be adjusted to a pH of 2.0 to 4.0 with
sulfuric acid, and add 35 mg of sodium thiosulfate per liter of sample for each part per
million of free chlorine.
7.3 All samples must be extracted within 7 days and completely analyzed within 30 days of
extraction.
8. Calibration
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in
Table 1. The chromatographic system can be calibrated using the external standard
technique (Section 8.2).
8.2 External standard calibration procedure.
8.2.1 Prepare calibration standards at a minimum of three concentration levels of the
analytes by adding volumes of the stock standard to a volumetric flask and
diluting to volume with HPLC mobile phase. One of the 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.
8.2.2 Using injections of 100 |iL of each calibration standard, tabulate peak height or
area response 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%
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.
8.2.3 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 any analyte varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration standard. Alternatively, a new
calibration curve or factor must be prepared.
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Method 632.1
9. Quality Control
9.1 Monitoring for interferences.
9.1.1 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 the analytes, 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 standard may not be used for calibration
of the analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.2, prepare a laboratory control standard
concentrate that contains the analytes at a concentration of 10 |ig/mL in
acetonitrile.6
9.2.1.2 Laboratory control standard: Using a pipette, add 1.0 mL of the
laboratory control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10.
Calculate the percent recovery (P;) with the equation:
Equation 1
100 s,
where
S; = Analytical results from the laboratory control standard, in p.g/L
Ti = Known concentration of the spike, in jig/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.
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Method 632.1
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field
duplicates (Section 7.1). Analyze both aliquots for at least 10% of all samples. To
the extent practical, the samples for duplication should contain reportable levels
of the analytes.
9.3.2 Calculate the relative range6 (RR j) with the equation:
Equation 2
100/?,
RR;
X:
where
Rt = Absolute difference between the duplicate measurements X, and X2, in jig/L
X; = Average concentration found
in |ig/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 into a 2-L separatory funnel. Check
the pH of the sample with wide-range pH paper and adjust to within the range
of 6.5 to 7.5 with sodium hydroxide or sulfuric acid by slow addition and
thorough mixing. Add 200 g 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 Erlenmeyer flask.
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
Erlenmeyer flask.
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Method 632.1
10.1.4 Perform a third extraction in the same manner. Pour the combined extract
through a drying column containing about 10 cm of anhydrous sodium sulfate,
collecting the extract in a 250-mL flat-bottom boiling flask. Rinse the Erlenmeyer
flask and column with about 30 mL of methylene chloride to complete the
transfer.
10.1.5 Concentrate the combined methylene chloride extracts to about 1 mL on a rotary
evaporator with bath temperature between 35 and 40°C. Add 15 mL of
acetonitrile, and reconcentrate to about 1 mL. Transfer the extract to a 10-mL
volumetric flask. Rinse the boiling flask with about 1 mL of acetonitrile, and
transfer to the volumetric flask. A 5-mL syringe is recommended for this
operation. Rinse the boiling flask further with a 1-mL portion of acetonitrile, and
transfer to the volumetric flask.
10.1.6 Add exactly 5.0 mL of HPLC-grade water to the flask, and dilute to 10 mL with
acetonitrile. If the extracts will be stored longer than 2 days, they should be
transferred to PTFE-sealed screw-cap bottles. If the sample extract requires no
cleanup, proceed with chromatographic analysis. If the sample requires cleanup,
proceed to Section 10.2.
10.1.7 Determine the original sample volume by refilling the sample bottle to the mark
and transferring the liquid to a 1000-mL graduated cylinder. Record the sample
volume to the nearest 5 mL.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix.
If particular circumstances demand the use of a cleanup procedure, the analyst
must determine the elution profile and demonstrate that the recovery of each
compound of interest is no less than 85%.
10.2.2 Proceed with liquid chromatography as described in Section 10.3.
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 limits that can be achieved by this method. An example of
the separation achieved by the primary column of the analytes is shown in
Figures 1 and 2. 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.
10.3.3 Inject 100 |iL of the sample extract. Monitor the column eluent at 254 nm.
Record the resulting peak size in area or peak height units.
10.3.4 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
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Method 632.1
be used to calculate a suggested window size; however, the experience of the
analyst should weigh heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the
sample with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of
interferences, cleanup is required.
11. Calculations
11.1 Determine the concentration of analytes in the sample.
11.1.1 Calculate the amount of analytes 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 equation:
Equation 3
(A) (V)
Concentration, ug/L =
(V) (V)
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in {iL
Vt = Volume of total extract, in {iL
Vs = Volume of water extracted, in mL
11.2 Report results in milligrams per liter 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 EDLs and associated chromatographic conditions for the analytes are listed in Table
l.7 The EDL is defined as the minimum response being equal to five times the
background noise, assuming a 10-mL final extract volume of a 1-L sample and an HPLC
injection volume of 100 |iL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science
and Engineering, Inc., in the designated matrix. The results of these studies are
presented in Table 2.
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Method 632.1
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials,
Philadelphia, Pennsylvania, p. 679, 1986.
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, August 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, Pennsylvania, p. 76,
1986.
6. "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, March 1979.
7. "Evaluation of Ten Pesticides," U.S. Environmental Protection Agency, Contract
68-03-1760, Task No. 11, U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio (in preparation.).
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Method 632.1
Table 1. Chromatographic Conditions and Estimated Detection Limits
Retention Time (min) Estimated Detection
Parameter Column 1 | Column 2 Limit (ptg/L)
Vacor (RH 787) 6.0 3.8 0.20
Propanil 12.4 6.9 0.85
Napropamide 15.2 9.5 0.31
Column 1: 25 cm long by 4 mm ID, stainless steel, packed with Ultrasphere ODS (particle size
5 p); mobile phase: 40% acetonitrile/HPLC water programmed to 65% acetonitrile/HPLC water
over 10 minutes at a flow rate of 1.0 mL/min at ambient temperature.
Column 2: 25 cm long by 4.6 mm ID, stainless steel, packed with Zorbax ODS (DuPont); mobile
phase: Isocratic elution with 55% acetonitrile/HPLC water at a flow rate of 1.0 mL/min for 6
minutes then linear flow gradient to 1.5 mL/min over 3 minutes at ambient temperature.
Table 2. Single-Laboratory Accuracy and Precision
Spike
Number
Average
Standard
Matrix
Range
of
Percent
Deviation
Parameter
Type*
(m/L)
Replicates
Recovery
(%)
Napropamide
1
11.5
7
113.8
15.7
1
597.0
7
104.0
16.0
Propanil
1
14.0
7
99.8
12.4
1
676.0
7
96.4
7.6
Vacor (RH787)
1
12.9
7
98.2
17.5
1
655.0
7
111.2
5.2
*1 = Spiked municipal wastewater
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Method 632.1
Retention Time (minutes)
A52-002-64A
Figure 1. HPLC Chromatogram of Carbamates/Amides on Column 1
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