## 6640 ACIDIC HERBICIDE COMPOUNDS*

6640 A. Introduction

1. Sources and Significance

Agricultural chemicals used for weed control can be found as contaminants in various aquatic systems. 1 Although formerly only two chlorinated phenoxy acid herbicides have been among the substances regulated by the U.S. Environmental Protection Agency in drinking water, and thus were routinely measured, several other types of carboxylic acid compounds used for their toxic effects now are under regulatory control. 2

2. Selection of Method

The micro liquid-liquid extraction gas chromatographic method presented here is applicable to the determination of salts and esters of analyte acids. The form of each acid is not routinely distinguished by this method. Results are calculated and reported for each listed compound as the total free acid. Since only the free acid is quantified, the herbicide Lactofen will be quantified as Acifluorfen because their structures represent different esters of the same acid herbicide. Additionally, this method is unable to quantify the degradation products of Dacthal separately from the parent compound unless the optional wash step is performed. If this wash step is not performed, all forms of Dacthal (the parent compound and the mono- and di-acid degradation products) will be quantified as Dacthal. 3. Sampling and Storage

Collect grab samples in accordance with conventional sampling practices (see Section 1060B), using amber glass containers with polytetrafluoroethylene (PFTE) lined screw caps and capacities of at least 40 mL. Add sodium sulfite crystals (approximately 2 mg/40 mL sample) if residual chlorine is present in the sample. Fill sample bottles but take care not to flush out the sodium sulfite. Because the target analytes of this method are not volatile, it is not necessary to ensure that the sample bottles are completely headspace-free. Ice samples during shipment so that the temperature does not exceed 10°C during the first 48 h after collection. Confirm that samples are at or below 10°C when they are received at the laboratory. Store samples in the laboratory at or below 6°C and protect from light until extraction. Do not freeze samples.

Because of the several pH adjustments made to the samples in the course of this method, the addition of organic or inorganic biocides, including hydrochloric acid, has been omitted. Theanalyst should be aware of the potential for the biological degradation of the analytes. Extract samples as soon as possible and within no more than 14 d.

4. References

1. U.S. ENVIRONMENTAL PROTECTION A GENCY . 1990. National Pesticide Survey: Summary Results of EPA’s National Survey of Pesticides inDrinking Water Wells. EPA 57019-90-015, U.S. Environmental Protection Agency, Cincinnati, Ohio. 2. U.S. ENVIRONMENTAL PROTECTION A GENCY . 1992. National PrimaryDrinking Water Regulations; Synthetic Organic Chemicals and Inorganic Chemicals; Final Rule. 40 CFR Parts 141 and 142, Part 111; Federal Register 57, No.138.



## 6640 B. Micro Liquid-Liquid Extraction Gas Chromatographic Method

1. General Discussion

a. Application: This is a gas chromatography (GC) method for the determination of chlorinated acids in drinking water. It can be used to measure various acidic organic compounds and their corresponding acid salts, although the form of each acid is not differentiated and the calculated amount of each is expressed as free acid. Accuracy, precision, and detection level data have been generated in reagent water and finished ground and surface waters for compounds listed in Table 6640:I. Also see 6640A.2.

b. Principle: A 40-mL sample is adjusted to pH  12 with 4N sodium hydroxide and is kept for 1 h at room temperature to hydrolyze derivatives. N OTE : Because the chlorphenoxy acid herbicides are formulated as a variety of esters and salts, thehydrolysis step is required and may not be skipped. After hydrolysis, an optional wash step using a 90:10 hexane:methyl

tert-butyl ether (MtBE) mixture may be performed as a sample cleanup and to remove Dacthal. It is only necessary to perform this wash if the quantitation of the Dacthal degradation products is desired. The aqueous sample then is acidified with sulfuric acid to pH 1 and extracted with 4 mL of MtBE that contains the internal standard. The chlorinated acids, which have been partitioned into the MtBE, then are converted to methyl esters by derivatization with diazomethane. The target esters are separated and detected by capillary column gas chromatography using an electron capture detector (GC/ECD). Analytes are quantified using an internal-standard-based calibration curve.

c. Interferences: Clean all glassware meticulously by washing with detergent and tap water, rinsing with tap water, and rinsing with reagent water. A final rinse with solvents may be needed. In * Approved by Standard Methods Committee, 2006. Joint Task Group: 21st Edition—Russell Chinn (chair), Wenta Liao, David J. Munch, Theresa A. Prato.

1place of a solvent rinse, heat nonvolumetric glassware in amuffle furnace at 400°C for 2 h. Do not heat volumetric glassware in an oven above 120°C. Store glassware inverted or capped with aluminum foil. Method interferences may be caused by contaminants in solvents, reagents (including reagent water), sample bottles and caps, and other sample-processing hardware that lead to discrete artifacts and/or elevated base lines in the chromatograms. Interferences by phthalate esters can pose a major problem in pesticide analysis when an electron capture detector (ECD) is used. These compounds generally appear in the chromatogram as large peaks. Common flexible plastics contain varying amounts of phthalates that are easily extracted or leached during laboratory operations. Cross-contamination ofclean glassware routinely occurs when plastics are handled during extraction steps, especially when solvent-wetted surfaces are present. Interferences from phthalates can best be minimized byavoiding the use of plastics in the laboratory. Exhaustive purification of reagents and glassware may be required to eliminate background phthalate contamination. 1,2 Routinely demonstrate that all sources of reagents are free from interferences, i.e., less than one-third the method reporting limit (MRL) for each targetanalyte, under the conditions of the analysis, by analyzing laboratory reagent blanks. Do not subtract blank values from sample results.

Use a grade of sodium sulfate suitable for pesticide residue analysis. If the suitability of the available sodium sulfate is in question, extract and analyze a laboratory reagent blank to test for interferences before sample processing. Matrix interferences may be caused by contaminants that are extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the water sampled. Also, contaminants in local ground water and surface water may interfere with the analysis of Dalapon on the primary column. A slow initial temperature program can ensure separation of the interferent from Dalapon.Confirm analyte identifications with the confirmation column specified, another column that is dissimilar to the primary column, or by GC/MS if the concentrations are high enough.

d. Method detection level (MDL): Method detection levels are compound-, instrument-, and matrix-dependent. The MDL is defined as the statistically calculated minimum amount that can be measured with 99% confidence that the reported value is greater than zero. 3 Experimentally determined MDLs for the ana

lytes are provided in Table 6640:I. The MDL differs from, and is usually lower than (but never above), the minimum reporting level (MRL). The concentration range for target analytes in this method was evaluated between 0.5 and 20  g/L for a 40-mL sample. e. Safety: See Section 1090. The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined. Regard each chemical compound as a potential health hazard andminimize exposure to these chemicals. Maintain in the laboratory a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of MSDSs also should be made available to allpersonnel involved in the chemical analysis. Additional references to laboratory safety are available. 4–6

The toxicity of the extraction solvent methyl t-butyl ether (MtBE) has not been well defined. Susceptible individuals may experience adverse affects upon skin contact or inhalation of vapors. Therefore, use protective clothing and gloves, and use MtBE only in a chemical fume hood or glove box. The same precaution applies to pure standard materials.

Diazomethane is a toxic carcinogen that can explode under certain conditions. Use the diazomethane generator behind a safety shield in a well-ventilated fume hood. Never allow thegenerator to be heated above 90°C and avoid all grinding surfaces, such as ground-glass joints, sleeve bearings, and glass stirrers. To minimize safety hazards, the diazomethane generatorapparatus used in the esterification procedure produces micromolar amounts of diazomethane in solution. If the procedure is followed carefully, no possibility for explosion exists.

Although hydrogen can be safely used as a carrier gas, the potential for fire or explosion does exist if the gas system is mishandled. If unsure of the safety guidelines for using hydrogen as a carrier gas, seek advice from the instrument manufacturer.

2. Apparatus

a. Sample containers, amber glass bottles with a minimum volume of 40 mL, fitted with PTFE-lined screw caps.

b. Extraction vials, 60-mL clear glass vials with PTFE-lined
screw caps.

c. Autosampler vials, 2.0-mL vials with screw or crimp cap and a PTFE-faced seal.

d. Standard solution storage containers, 10- to 20-mL amber glass vials with PTFE-lined screw caps.

e. Clear vials, 7-mL glass, disposable, with PTFE-lined screw caps for extract drying and derivatization.

f. Pasteur pipets, glass, disposable. g. Pipets, Class A, 2.0-mL and 4.0-mL glass, or adjustable volume dispensers.

h. Volumetric flasks, Class A, suggested sizes 5 mL, 10 mL, and 100 mL. T ABLE 6640:I. S INGLE -LABORATORY M ETHOD D ETECTION LEVELS IN REAGENT WATER

Compound* Fortification Level


g/L Primary Column MDL


g/L Secondary Column MDL


g/L
Dalapon 0.100 0.05 0.07 3,5-Dichlorobenzoic acid 0.050 0.21 0.05 Dicamba 0.050 0.03 0.04 Dichlorprop 0.100 0.43 0.12 2,4-D 0.100 0.06 0.07 Pentachlorophenol 0.010 0.01 0.08 Silvex 0.025 0.03 0.02 2,4,5-T 0.025 0.02 0.03 2,4-DB 0.100 0.25 0.18 Chloramben 0.050 0.06 0.08 Dinoseb 0.100 0.17 0.08 Bentazon 0.100 0.07 0.19 Dacthal 0.050 0.11 0.11 Quinclorac 0.050 0.08 0.11 Picloram 0.050 0.08 0.05 Acifluorfen 0.250 0.31 0.09

* To assure exact identification of analytes, CAS numbers are listed as follows: Acifluorfen, 50594-66-6; Bentazon, 25057-89-0; Chloramben, 133-90-4; 2,4-D, 94-75-7; Dacthal, 1861-32-1; Dalapon, 75-99-0; 2,4-DB, 94-82-6; Dicamba, 1918-00-9; 3,5-Dichlorobenzoic acid, 51-36-5; Dichloroprop, 120-36-5; Dinoseb, 88-85-7; pentachlorophenol 87-86-5; Picloram, 1918-02-1; Quinclorac, 84087- 01-4; Silvex, 93-72-1; 2,4,5-T, 93-76-5. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

2i. Microsyringes, various sizes. j. Balance, analytical, capable of weighing to the nearest 0.0001 g.

k. Diazomethane generator: See Figure 6251:3 for a diagram of an all-glass system custom-made for these validation studies. Micromolar generators also are available from commercial sources.* If a generator from a commercial source is used, follow manufacturer’s instructions.

l. Gas chromatograph, capillary GC.† Modifications shouldinclude a high-pressure (50 psi) split/splitless injector, fast temperature ramp oven (50°C/min) and a low-volume (150-  L) micro ECD detector. A data system capable of fast sampling (20 points/peak) also is required.

m. Primary GC column, ‡ 180-  m ID, 40 m long, 0.2-  m film thickness, fused silica capillary with chemically bonded (14% cyanopropylphenymethylpolysiloxane), or equivalent bonded, fused silica column.

n. Confirmation GC column, § 180-  m ID, 40 m long, 0.2-  m film thickness, fused silica capillary with chemically bonded (5% phenylmethylpolysiloxane), or equivalent bonded, fused silica column. Columns of other dimensions may be used as long as the QC criteria of the method are met.

3. Reagents

Use reagent-grade or better chemicals in all analyses. For reagents b through f , use high-purity HPLC-grade materials demonstrated to be free from analytes and interferences. Unlessotherwise indicated, use reagents conforming to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first determined that the reagent is of sufficiently high purity to permit its use without lessening the quality of the determination.

Observe safety precautions; see 6640B.1 e

. a. Reagent water: Use purified water that does not contain anymeasurable quantities of any target analytes or interfering compounds greater than 1/3 the MRL for each compound of interest.

b. Methyl tert- butyl ether (MtBE). c. Acetone

. d. Carbitol (diethylene glycol monoethyl ether). e. Hexane:MtBE wash solvent, 90:10 (v/v), unpreserved. f. Hexane

. g. Sodium sulfate, Na2 SO 4 , pesticide-grade, granular, anhy

drous. If interferences are observed (see 6640B.1 c ), it may be necessary to heat the sodium sulfate in a shallow tray at 400°C for up to 4 h to remove phthalates and other interfering organic substances. Alternatively, extract it with methylene chloride in a Soxhlet apparatus for 48 h. Store in a capped glass bottle rather than a plastic container.

h. Acidified sodium sulfate: Acidify by slurrying 100 g of muffled sodium sulfate with enough ethyl ether to just cover the solid. Add 0.5 mL conc sulfuric acid dropwise while mixing thoroughly. Remove ether under vacuum. Mi x 1 g of resulting solid with 5 mL reagent water and measure pH of the mixture. The pH must be below 4. Store in a desiccator or at 100°C to keep reagent dry.

i. Copper II sulfate pentahydrate , CuSO 4  5H 2 O. j. Sodium hydroxide solution, NaOH, 4N: Dissolve 16 g NaOH pellets in reagent water and dilute to 100 mL.

k. Potassium hydroxide solution, KOH, 37% (w/v): Dissolve 37 g KOH pellets in reagent water and dilute to 100 mL.

l. Sodium sulfite, Na 2 SO 3 , for use as a dechlorinating agent.

m. N- methyl -N- nitroso -p- toluenesulfonamide (MNTS) solution: Prepare a solution containin g 5 g MNTS in 50 mL of a 50:50 (v/v) mixture of MtBE and carbitol. This solution is stable for 1 month or longer when stored at 4°C in an amber bottle with a PTFE-lined screw cap.

n. Sulfuric acid, H 2 SO 4 , conc. o. Silica gel, 35 to 60 mesh. p. Hydrogen, 99.999% pure or better, GC carrier gas. q. Nitrogen, 99.999% pure or better, ECD make-up gas. r. Internal standard solutions: 4,4  -Dibromooctafluorobiphenyl (99 %) is used as an internal standard for the method. This compound has been shown to be an effective internal standard for the method analytes, but other compounds may be used if the QC requirements are met. The solutions listed in ¶s 1) and 2) below are stable for at least 30 d. Replace solution if ongoing QC indicates a problem.

1) Internal standard stock solution, 2.0 mg/mL: Prepare aninternal standard stock solution by accurately weighing approximately 0.0200 g of neat 4,4  -dibromooctafluorobiphenyl. Dissolve the neat material in MtBE and dilute to volume in a 10-mL volumetric flask. Transfer solution to an amber glass vial with a PTFE-lined screw cap and store at 0°C. The resulting concentration of the stock internal standard solution will be approximately 2.0 mg/mL.

2) Internal standard primary dilution standard, 2.5  g/mL:Prepare an internal standard fortification solution at approximately 2.5  g/mL by adding 12.5  L of the stock standard to 10 mL MtBE. Transfer primary dilution to an amber glass vial with a PTFE-lined screw cap and store at 0°C.

3) MtBE extraction solvent with internal standard, 2.5  g/mL: The internal standard 4,4  -dibromooctafluorobiphenyl is added to the extraction solvent before analyte extraction to compensate for any volumetric differences encountered during sample processing. Make this solution just before extraction. The addition of 1 mL of primary dilution standard (2.5  g/mL) to 99 mL MtBE results in a final internal standard concentration of 25 ng/mL.

s. Surrogate (SUR) analyte standard solution: 2,4-dichlorophenylacetic acid (99 %) is used as a surrogate compound toevaluate extraction and derivatization procedures. This compound is an effective surrogate for the method analytes, but other compounds may be used if QC requirements are met. The solutions listed in 1) and 2) below are stable for at least 30 d. Replace solution if ongoing QC indicates a problem.

1) Surrogate stock solution, 1.0 mg/mL: Prepare a surrogate stock standard solution of 2,4-dichlorophenylacetic acid by weighing approximately 0.0100 g of neat material. Dissolve theneat material in acetone and dilute to volume in a 10-mL volumetric flask. Transfer solution to an amber glass vial with a * Aldrich Catalog, No. Z10, 889-8, or equivalent. † Hewlett Packard Model 6890, or equivalent. ‡ DB-1701, or equivalent. § DB-5, or equivalent. Diazald ® , Aldrich Chemical Co., or equivalent. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

3PTFE-lined screw cap and store at 0°C. The resulting concentration will be 1.0 mg/mL.

2) Surrogate primary dilution standard/sample fortification solution, 100  g/mL: Prepare a primary dilution standard at approximately 100 g/mL by the addition of 1 mL stock standard to 10 mL acetone. Transfer primary dilution to an amber glass vial with a PTFE-lined screw cap and store at 0°C. The addition of 10  L of primary dilution standard to the 40-mL aqueous sample results in a surrogate concentration of 25 ng/mL.

t. Analyte standard solutions: Obtain analytes as neat or solidfree acid standards or as commercially prepared ampulized solutions from a reputable standard manufacturer. Do not use premethylated standards for preparing analyte standards. Prepare analyte stock and primary dilution standards as described below. The solutions listed in ¶s 1) and 2) below are stable for at least 30 d. Replace solution if ongoing QC indicates a problem.

1) Analyte stock standard solution: Prepare separate stock standard solutions for each analyte of interest at a concentration of 1 to 5 mg/mL in acetone. Method analytes may be obtained as neat materials or ampulized solutions (  99% purity) from a number of commercial suppliers. Store stock standard solutions at 0°C.

For analytes that are solids in their pure form, prepare stock standard solutions by accurately weighing approximately 0.01 to 0.05 g pure material in a 10-mL volumetric flask. Dilute to volume with acetone.

For analytes that are liquid in their pure form at room temperature, place approximately 9.8 mL acetone in a 10-mL volumetric flask. Let flask stand, unstoppered, for about 10 min to allow solvent film to evaporate from the inner walls of flask, and weigh to the nearest 0.1 mg. Use a 10- L syringe and immediately add 10.0  L standard material to the flask by keeping syringe needle just above the surface of the acetone. Be sure that the standard material falls dropwise directly into the acetone without contacting inner wall of flask. Calculate concentration in milligrams per milliliter from net gain in weight. Dilute to volume, stopper, then mix by inverting flask several times.

2) Primary dilution standard (PDS): Prepare primary dilutionstandard solution by combining and diluting stock standard solutions with acetone. Store primary dilution standard solution at

0°C. As a guideline to the analyst, the analyte concentrations, in micrograms per milliliter, used in the primary dilution standard solution during method development are listed as follows.

Acifluorfen 5.0 Bentazon 10 Chloramben 5.0 2,4-D 10 Dalapon 10 2,4-DB 10 Dacthal acid degradation 5.0 Dicamba 5.0 3,5-Dichlorobenzoic acid 5.0 Dichlorprop 10 Dinoseb 10 Pentachlorophenol 1.0 Picloram 5.0 2,4,5-T 2.5 2,4,5-TP (Silvex) 2.5 Quinclorac 5.0 u. Calibration standards: Prepare a five-point calibration curve by fortifying reagent water with the primary dilution standard. Let the standard reach room temperature, then add a designated amount of each calibration standard in acetone intoseparate, 40-mL portions of reagent water to produce a calibration curve ranging from below or at the MRL to approximately 10 to 20 times the lowest calibration level. Treat these aqueouscalibration standards like samples and therefore add all preservatives and other reagents. Extract by the procedure described below. Store calibration standard solutions in acetone at 0°C.

4. Procedure

a. Sample preparation: Remove samples from storage and let them equilibrate to room temperature. Place 40 mL water sample in a pre-cleaned, 60-mL glass vial with a PTFE-lined screw cap, using a graduated cylinder. Add 10 L room temperature surrogate standard (100  g/mL 2,4-dichlorophenylacetic acid in acetone) to the aqueous sample.

b. Hydrolysis : Because many of the herbicides included in this method are applied as a variety of esters and salts, hydrolyze them to the parent acid before extraction. Include this step in analysis of all extracted field samples, laboratory reagent blanks (LRBs), laboratory-fortified matrices (LFMs) and calibration standards. Failure to perform this step may result in data that are biased low for some targets in field samples. 7 Add 1 mL 4N NaOH solution to each glass vial, then cap and shake for several seconds. Check sample pH with pH paper or a pH meter. If sample does not have a pH  12, adjust pH by adding more 4N NaOH solution. Let sample remain at room temperature for 1 h, shaking contents periodically.

c. Hexane:MtBE wash (optional): Use this wash step to quantify the Dacthal degradation products from the parent Dacthal. This wash aids in sample cleanup and removes any Dacthal from the sample that would interfere with the quantitation of the Dacthal degradation products. After hydrolysis, add 5 mL 90:10 (v:v) hexane:MtBE and shake vigorously for 3 min. Let phases separate for approximately 5 min, then remove and discard the top hexane/MtBE layer. If differentiation between Dacthal and its degradation products is not required, this step may be omitted.

d. Microextraction: Adjust pH to 1 by adding approximately 1 mL conc sulfuric acid. Cap, shake, and then check pH with a pH meter or narrow-range pH paper. Add more sulfuric acid as needed to properly adjust pH.

Quickly add approximately 2 g copper II sulfate pentahydrate and shake until dissolved. This colors the aqueous phase blue and allows the analyst to better distinguish between the aqueous phase and the organic phase.

Quickly add approximately 16 g muffled sodium sulfate and shake until almost all is dissolved. Sodium sulfate addition increases the ionic strength of the aqueous phase, further drivingthe chlorophenoxy acids into the organic phase, and also decreases MtBE solubility in the aqueous phase, allowing greater volumetric recovery. Add this salt and the copper II sulfate pentahydrate quickly so the heat generated from addition of the acid will help dissolve the salts.

Add exactly 4.0 mL MtBE extraction solvent with internal standard to each vial and shake vigorously for 3 min. Let phases separate for approximately 5 min. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

4e. Preparation of diazomethane: Assemble diazomethane generator (Figure 6251:3) in a hood. See 6640B.2 k above. The collection vessel is a 10- or 15-mL glass vial equipped with a PTFE-lined screw cap and maintained at 0 to 5°C.

Add a sufficient amount of MtBE (approximately 7 mL) to first tube to cover the impinger. Add 10 mL MtBE to collection vial. Set nitrogen flow at 5 to 10 mL/min. Add 4 mL MNTS solution (6640B.3 m ) and 3 mL 37% KOH solution to the second impinger. Connect tubing as shown in Figure 6251:3 and let nitrogen flow purge diazomethane from reaction vessel into the collection vial for 30 min. Cap vial when collection is completeand maintain at 0 to 5°C. When stored at 0 to 5°C, this diazomethane solution may be used over a period of 72 h.

Using a Pasteur pipet, transfer sample extract (upper MtBE layer) to a 7-mL glass vial. Add 0.6 g acidified sodium sulfate and shake. This step dries the MtBE extract.

Using a Pasteur pipet, transfer extract to a second, 7-mL glass vial. Add 250  L diazomethane solution to each vial. The contents of the vial should remain slightly yellow, indicating an excess of diazomethane. More diazomethane may be added if necessary. Let the esterification reaction proceed for 30 min.

Remove any unreacted diazomethane by adding 0.1 g silica gel. Effervescence (evolution of nitrogen) is an indication that excess diazomethane was present. Let extracts stand for 0.5 h.

Transfer extract to an autosampler vial. A duplicate vial may be filled with excess extract.

Analyze sample extracts as soon as possible. The sample extract may be stored up to 21 d if kept at 0°C or less. Keep extracts away from light in amber glass vials with PTFE-lined caps.

f. Gas chromatography: Use apparatus specified in 6640B.2 l,

m, n, and hydrogen carrier gas, 6640B.3 p

. C AUTION : Observe safety guidelines when using hydrogen as a carrier gas . If further safety information is required, seek advice from the instrument manufacturer regarding hydrogen use.

Pay strict attention to established column installation guidelines for proper cutting and placement of capillary columns within the instrument. If a loss of response is noted for an analyte, trimming approximately 1 m from the head of the column may restore response. A guard column is recommendedif conditions in the laboratory necessitate frequent column trimming.

Chromatographic conditions and analyte retention times for the primary column are given in Table 6640:II A chromatogram from this column is provided in Figure 6640:1. Chromatographic conditions and analyte retention times for the secondary column are given in Table 6640:III. A chromatogram from this column is provided in Figure 6640:2.

g. Calibration: Establish GC operating parameters equivalent to the suggested specifications in Table 6640:II. Calibrate GC system using the internal standard (IS) technique. Other columns or conditions may be used if equivalent or better performance can be demonstrated. Prepare a set of at least five calibration standards as described in 6640B.3 u . The lowest concentration of calibration standard must be at or below the MRL; preferably use a minimum of four calibration standards between the MRL and the upper quantitative range.

Use GC data system software to generate a linear regression or quadratic calibration curve using the internal standard. This curve may be generated by plotting A
x /A is vs. Q
x /Q is , where A
x and A is are integrated peak areas of the analyte and internal standard, respectively, and Q
x and Q is are quantity of analyte and internal standard injected in concentration units, respectively. T ABLE 6640:II. C HROMATOGRAPHIC C ONDITIONS AND A VERAGE RETENTION T IME D ATA FOR PRIMARY C OLUMN

Peak Number (Figure 6640:1) Compound Average RT* min RSD %
1 Dalapon 7.06 0.017 2 3,5 Dichlorobenzoic acid 11.32 0.002 3 2,4-Dichlorophenylacetic acid (SUR) 11.80 0.002 4 Dicamba 11.91 0.002 5 Dichlorprop 12.21 0.002 6 4,4  -Dibromooctafluorobiphenyl (IS) 12.25 0.002 7 2,4-D 12.37 0.002 8 Pentachlorophenol 12.49 0.002 9 Silvex 12.70 0.002 10 2,4,5-T 12.90 0.002 11 2,4-DB 13.10 0.003 12 Chloramben 13.15 0.003 13 Dinoseb 13.44 0.003 14 Bentazon 13.60 0.003 15 Dacthal 13.68 0.002 16 Quinclorac 13.85 0.004 17 Picloram 14.02 0.003 18 Acifluorfen 15.20 0.004

* Average of seven runs. Primary column: DB-1701, 40 m  0.180 mm ID, 0.20-mm film thickness, injector temperature 200°C, liner 2 mm straight Siltek deactivated, injection volume 1  L of 200 ng/mL (highest level component) standard, splitless injection. Hold to 1 min then purge @ 50 mL/min, detector temperature 360°C, detector make -up gas nitrogen at 20 mL/min. Temperature program: 45°C initial, program at 5°C/min to 80°C, then 50°C/min to 220°C, then 20°C/min to 280°C and hold for 3 min. Data coll ection via HP GC Chemstation at a rate of 20 Hz. Carrier gas: Hydrogen (UHP). Detector make-up gas: Nitrogen (UHP). ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

5Figure 6640:1. Chromatogram of chlorphenoxy herbicides on a primary column. Peak identifications and chromatographic conditions are given in Table 6640:II. Peak has been attenuated for illustration.

T ABLE 6640:III. C HROMATOGRAPHIC C ONDITIONS AND A VERAGE RETENTION T IME D ATA FOR C ONFIRMATION C OLUMN

Peak Number (Figure 6640:2) Compound Average RT* min RSD %
1 Dalapon 5.04 0.057 2 3,5-Dichlorobenzoic acid 10.33 0.007 3 2,4-Dichlorophenylacetic acid (SUR) 11.31 0.004 4 Dicamba 11.46 0.006 5 Dichlorprop 12.19 0.006 6 4,4  -Dibromooctafluorobiphenyl (IS) 12.71 0.004 7 2,4-D 12.34 0.005 8 Pentachlorophenol 13.16 0.006 9 Silvex 13.29 0.004 10 2,4,5-T 13.48 0.005 11 2,4-DB 13.94 0.005 12 Chloramben 13.34 0.004 13 Dinoseb 14.01 0.004 14 Bentazon 14.28 0.006 15 Dacthal 15.00 0.004 16 Quinclorac 14.88 0.004 17 Picloram 14.59 0.004 18 Acifluorfen 16.38 0.003

* Average of seven runs. Confirmation column: DB-5, 40 m  0.180 mm ID, 0.20-mm film thickness, injector temperature 200°C, liner 2 mm straight Siltek® deactivated, injection volume 1  L of 100 ng/mL (highest level component) standard, splitless injection. Hold to 1 min then purge @ 50 mL/min, detector temperature 360°C, detector make-up gas nitrogen at 20 mL/min. Temperature program: 45°C initial, program at 4°C/min to 80°C, then program at 30°C/min to 280°C and hold for 2 min. Data collection via HP GC Chemstation at a rate of 20 Hz. Carrier gas: Hydrogen (UHP). Detector make-up gas: Nitrogen (UHP). ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

65. Quality Control

The quality control practices considered to be an integral part of each method are summarized in Table 6020:I.

Quality control (QC) requirements include the initial demonstration of capability (IDC), the determination of the method detection level (MDL), and subsequent analysis in each analysis batch of a laboratory reagent blank (LRB), continuing calibration check (CCC) standards, a laboratory-fortified sample matrix (LFM), and either a laboratory-fortified sample matrix duplicate (LFMD) or a field duplicate (FD) sample. This section details thespecific requirements for each QC parameter. The criteria discussed below are considered the minimum acceptable QC criteria. Laboratories are encouraged to institute additional QC practices to meet their specific needs.

Process all QC samples through all steps of the procedure (6640B.4 a

– f

), including hydrolysis and methylation. Add sample preservatives as described in 6640A.3 before extracting and analyzing the QC samples.

a. Initial demonstration of capability IDC: Requirements for this demonstration are described below.

1) Initial demonstration of low system background—Before any samples are analyzed, demonstrate that a LRB is reasonably free of contamination and that the criteria in ¶ c below are met. Process all QC samples through all steps of the method.

2) Initial demonstration of precision—Prepare, extract, and analyze four to seven replicate LFBs fortified at 5  g/L, or near the mid-range of the initial calibration curve. Process all QC samples through all steps of the analysis procedure. The relative standard deviation (RSD) of the results of the replicate analyses should be less than 20%.

3) Initial demonstration of accuracy—Using the same set ofreplicate data generated for ¶ 2) above, calculate average recovery. The average recovery of the replicate values should be within  20% of the true value.

4) Method detection level (MDL)—Prepare, extract, and analyze at least seven replicate LFBs at a concentration estimated to be near the MDL, over a period of at least 3 d (both extraction and analysis should be conducted over at least 3 d) using the analysis procedure of 6640B.4a

– f

. Process all QC samples through all steps of the method. Select a fortification level with a signal of two to five timesthe noise level and at or below the laboratory’s MRL. The appropriate concentration will depend on the sensitivity of the GC/ECD system. Add sample preservatives as described in 6640A.3 to these samples. Calculate the MDL as follows:

MDL  St ( n
 1, 1

   0.99)

where:

t ( n
 1, 1

   0.99)  Student’s t value for the 99% confidence level with n
 1 degrees of freedom,

n  number of replicates, and S  standard deviation of replicate analyses. Figure 6640:2. Chromatogram of the chlorphenoxy herbicides on confirmation column. Peak identifications and chromatographic conditions are given in Table 6640:III. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

7N OTE : Calculated MDLs need only be less than 1/3 of the laboratory’s MRL to be considered acceptable. Do not subtract blank values when performing MDL calculations. The MDL is a statistical determination of precision only. 1 If the MDL repli

cates are fortified at a low enough concentration, it is likely that they will not meet precision and accuracy criteria, and may result in a calculated MDL that is higher than the fortified concentration.

b. Minimum reporting level (MRL): The MRL is the threshold concentration of an analyte that a laboratory can expect to quantitate accurately in an unknown sample. The MRL should not be established at an analyte concentration that is less thaneither three times the MDL or a concentration yielding a signalto-noise (S/N) ratio of less than five. Depending upon the study’s data quality objectives, it may be set at a higher concentration. Although the lowest calibration standard should be at or below the MRL, the MRL cannot be established at a concentration lower than the lowest calibration standard.

c. Laboratory reagent blank (LRB): An LRB is required with each extraction batch of samples to determine the background system contamination. If the LRB produces a peak within the retention-time window of any analyte that would prevent thedetermination of that analyte, determine the source of contamination, and eliminate the interference before processing samples. Reduce background contamination to an acceptable level before proceeding. Background from method analytes or contaminants that interfere with the measurement of method analytes should be below 1/3 of the MRL. If target analytes are detected in the LRB at concentrations equal to, or greater than, this level, then all data for the problem analyte(s) may be considered invalid for all samples in the extraction batch.

d. Continuing calibration check (CCC): A CCC is a standard, prepared in the same extraction batch as the samples of interest, that contains all compounds of interest and is extracted in the same manner as the procedural standards used to prepare the initial calibration curve. Prepare an appropriate number of CCCswith each extraction batch. The CCC verifies the initial calibration at the beginning and end of each group of analyses, and after every tenth sample. The beginning CCC for each analysis batch should be at or below the MRL in order to verify instrumentsensitivity before any analyses. Subsequent CCCs should alternate between a medium and high concentration. Calculate theconcentration of each analyte and surrogate in the check standard. The calculated amount for each analyte for medium and high level CCCs should be within  30% of the true value. Thecalculated amount for the lowest calibration level for each analyte should be within  50% of the true value. If these conditions do not exist, consider all data for the problem analyte invalid,and take remedial action, which may require recalibration. Reanalyze any field sample extracts analyzed since the last acceptable calibration verification after adequate calibration has beenrestored, with the following exception. If the continuing calibration fails because the calculated concentration is greater than 130% (150% for the low-level CCC) for a particular target compound, and field sample extracts show no detection for thattarget compound, non-detects may be reported without re-analysis. Prepare calibration checks, with the samples being analyzed, at the beginning of each day that samples are analyzed, after every ten samples, and at the end of any group of sample analyses. e. Laboratory fortified blank (LFB): Because this method utilizes procedural calibration standards, which are fortified reagent waters extracted with each analytical batch, there is no difference between the LFB and the continuing calibration check standard. Consequently, the analysis of an LFB is not required; however the acronym LFB is used for clarity in the IDC.

f. Internal standard (IS): Monitor the peak area of each internal standard in all injections during each analysis day. The IS response (as indicated by peak area) for any chromatographic run should not deviate by more than  50% from the average area measured during the initial calibration for that IS. A poor injection could cause the IS area to exceed these criteria. Inject a second portion of the suspect extract to determine whether the failure is due to poor injection or instrument response drift. If thereinjected portion produces an acceptable internal standard response, report the results. If the internal standard area for thereinjected extract deviates more than 50% from the initial calibration average, check the CCC standards that ran before and after the sample. If the CCC fails the criteria of ¶ d above, recalibrate according to 6640B.4g . If the CCC is acceptable, repeat sample extraction provided that the sample is still withinholding time. Otherwise, report results obtained from the reinjected extract, but indicate as suspect.

g. Surrogate recovery: The surrogate standard is fortified into the aqueous portion of all samples, LRBs, and LFMs and LFMDs before extraction. It is also added to the calibration curve and CCC standards. The surrogate is a means of assessing method performance from extraction to final chromatographic measurement. When surrogate recovery from a sample, blank, or CCC is less than 70% or greater than 130%, check calculations to locate possible errors, standard solutions for degradation, contamination, and instrument performance. If those steps do not reveal the cause of the problem, re-analyze extract. If extract re-analysis meets the surrogate recovery criterion, report data only for re-analyzed extract. If extract re-analysis fails the 70 to 130% recovery criterion, check calibration by injecting the last calibration standard that passed. If the calibration standard fails the criteria of ¶ d above, recalibrate according to 6640B.4g . Ifcalibration standard is acceptable, repeat sample extraction provided that the sample is still within the holding time. If sample re-extract also fails the recovery criterion, report all data for that sample as a quantitative estimate because of surrogate recovery.

h. Laboratory-fortified sample matrix and duplicate (LFM and LFMD): Include LFMs and LFMDs in each extraction batch to determine whether the sample matrix adversely affects method accuracy. If the occurrence of target analytes in the samples is infrequent, or if historical trends are unavailable, include a second LFM or LFMD from a duplicate field sample to assess method precision. Extraction batches that contain LFMDs will not require the analysis of a FD. If a variety of different sample matrices are analyzed regularly, for example, drinking water from groundwater and surface water sources, establish method performance for each. Over time, document LFM data for all routine sample sources for the laboratory. Within each extraction batch, fortify a minimum of one field sample as an LFM for every 20 samples extracted. Prepare the LFM by adding to a sample an appropriate amount of analyte as given in ¶ 3 t2). Select a fortifying concentration that is at least twice the matrix background concentration, if known. Use historical data and ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

8rotate through the designated concentrations when selecting a fortifying concentration.

Calculate the percent recovery (R) for each analyte by using the equation:

R  A

 B
C  100

where:

A  measured concentration in fortified sample, B  measured concentration in unfortified sample, and C  fortification concentration.
Analyte recoveries may exhibit matrix bias. For samples fortified at or above their native concentration, recoveries should range between 70 and 130%, except for low-level fortification near or at the MRL, where 50 to 150% recoveries may be acceptable. If the accuracy of any analyte falls outside the designated range, and the laboratory performance for that analyte is shown to be in control in the LFB, the recovery may be judgedto be matrix-biased. Label result for that analyte in the unfortified sample as suspect because of matrix effects.

If an LFMD is analyzed instead of a FD, calculate relative percent difference (RPD) for values in duplicate LFMs (LFM and LFMD) by using the equation:

RPD  LFM

 LFMD  LFM  LFMD /2  100

RPDs for duplicate LFMs should fall in the range of  30% for samples fortified at or above their native concentration. Greater variability may be observed when LFMs are spiked near the MRL. At the MRL, RPDs should fall in the range of  50% for samples fortified at or above their native concentration. If the accuracy of any analyte falls outside the designated range and the laboratory performance for that analyte is shown to be incontrol in the LFB, the recovery may be judged to be matrixbiased. Label result for that analyte in the unfortified sample as suspect because of matrix effects.

i. Field duplicates (FD1 and FD2): Within each extraction batch, include a minimum of one field duplicate (FD) or LFMD (¶ 5 h above). FDs check the precision associated with sample collection, preservation, storage, and laboratory procedures. Iftarget analytes are not routinely observed in field samples, analyze a LFMD to substitute for this requirement. Extraction batches that contain LFMDs may not require the analysis of a FD. Calculate the relative percent difference (RPD) for duplicate measurements (FD1 and FD2) using the equation:

RPD  FD 1

 FD 2  FD 1  FD 2 /2  100 RPDs for duplicates should be in the range of  30%. Greater variability may be observed when analyte concentrations are near the MRL. At the MRL, RPDs should fall in the range of
 50%. If the accuracy of any analyte falls outside the designated range, and the laboratory performance for that analyte is shown to be in control in the LFB, the recovery may be judged to be matrix-biased. Label result for that analyte in the unfortified sample as suspect because of matrix effects.

j. Quality control (QC) sample: Analyze a QC sample each time that new primary dilution standards, ¶ 3 t2), are prepared. Ideally, obtain QC sample from second vendor. If a second vendor is not available, use a different lot of the standard. Although use of pre-methylated standards is prohibited forpreparing analyte standard solutions, pre-methylated standards may be used to prepare the QC sample. The QC sample may be injected as a calibration standard or fortified into reagent water and analyzed as a LFB. If the QC sample is analyzed as a continuing calibration, acceptance criteria are the same as for the CCC. If the QC sample is analyzed as aLFB, acceptance criteria are the same as for a LFB. If measured analyte concentrations are not of acceptable accuracy,check entire analytical procedure to locate and correct problem source.

6. Precision and Bias

Precision and bias data for this method are presented in Table 6640:IV. The effect of sample holding time on recovery from a chlorinated surface water is shown in Table 6640:V and the effect of extract holding time in Table 6640:VI.

7. References

1. GIAM , C.S., H.S. C HAN & G.S. NEF . 1975. Sensitive method for

determination of phthalate ester plasticizers in open ocean biota samples. Anal. Chem. 47:2225. 2. GIAM , C.S. & H.S. C HAN . 1976. Control of blanks in the analysis ofphthalates in air and ocean biota samples. U.S. National Bur. Standards, Special Publ. 442, p. 701. 3. GLASER , J.A., D.L. F OERST , G.D. M C KEE , S.A. Q UAVE & W.L. B UDDE

. 1981. Trace analyses for wastewaters. Environ. Sci. Technol. 15: 1426. 4. O CCUPATIONAL S AFETY AND H EALTH A DMINISTRATION . 1976. OSHA Safety and Health Standards, General Industry. 29 CRF 1910; OSHA 2206 (rev.), Occupational Safety & Health Admin., Washington, D.C. 5. U.S. PUBLIC H EALTH S ERVICE . 1977. Carcinogens—Working with Carcinogens. Publ. No. 77-206, U.S. Dep. Health, Education, and Welfare, Public Health Serv., Center for Disease Control, National Inst. Occupational Safety & Health, Atlanta, Ga. 6. C OMMITTEE ON C HEMICAL S AFETY , AMERICAN C HEMICAL S OCIETY . 1979. Safety in Academic Chemistry Laboratories, 3rd ed. American Chemical Soc., Washington, D.C. 7. B ASSETT , M.V., B.V. PEPICH & D.M. M UNCH . 2000. The role playedby basic hydrolysis in the validity of acid herbicide data — Recommendations for future preparation of herbicide performance evaluation standards. Environ. Sci. Technol. 34:1117. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

9T ABLE 6640:IV. M ETHOD PRECISION AND B IAS IN S ELECTED M ATRICES

Low-Level Fortification Mid-Level Fortification

Matrix Compound Fortification Concentration


g/L Mean Recovery % RSD* % S/N† Fortification Concentration


g/L Mean Recovery % RSD* %
Reagent water Dalapon 1.0 108 2.2 578 10 107 2.3 3,5-Dichlorobenzoic acid 0.50 117 16 9 5.0 96 2.3 Dicamba 0.50 97 3.7 35 5.0 102 1.5 Dichlorprop 1.0 95 5.7 19 10 107 0.6 2,4-D 1.0 98 5.2 23 10 106 1.4 Pentachlorophenol 0.10 76 7.4 21 1.0 103 1.4 Silvex 0.25 84 20 9 2.5 107 4.4 2,4,5-T 0.25 96 7.3 16 2.5 105 1.3 2,4-DB 1.0 97 8.4 6 10 93 1.6 Chloramben 0.50 90 9.0 113 5.0 105 2.9 Dinoseb 1.0 103 6.7 8 10 119 4.6 Bentazon 1.0 100 2.1 41 10 98 0.7 Dacthal 0.50 92 6.5 100 5.0 100 1.9 Quinclorac 0.10 106 9.3 20 1.0 101 2.9 Picloram 1.0 92 2.6 23 10 99 3.1 Acifluorfen 0.50 77 5.9 80 5.0 107 4.5 2,4-Dichlorophenyl-acetic

acid (SUR) 25 107 8.0 0 25 112 1.3 Chlorinated surface water Dalapon 1.0 104 2.1 — 10 100 2.5 3,5-Dichlorobenzoic acid 0.50 110 3.0 — 5.0 104 2.6 Dicamba 0.50 93 4.4 — 5.0 102 2.9 Dichlorprop 1.0 93 11 — 10 101 1.2 2,4-D 1.0 97 5.2 — 10 101 1.2 Pentachlorophenol 0.10 86 3.2 — 1.0 100 1.0 Silvex 0.25 103 4.3 — 2.5 101 1.0 2,4,5-T 0.25 100 3.5 — 2.5 100 1.6 2,4-DB 1.0 104 5.9 — 10 101 2.3 Chloramben 0.50 93 2.0 — 5.0 102 2.6 Dinoseb 1.0 99 1.2 — 10 101 1.6 Bentazon 1.0 91 2.2 — 10 98 1.8 Dacthal 0.50 92 4.6 — 5.0 99 2.0 Quinclorac 0.10 96 15 — 1.0 105 2.5 Picloram 1.0 102 2.4 — 10 107 3.2 Acifluorfen 0.50 98 4.1 — 5.0 92 2.4 2,4-Dichlorophenyl-acetic

acid (SUR) 25 107 1.5 — 25 106 1.4 Chlorinated ground water Dalapon 1.0 105 1.5 — 10 97 5.5 3,5-Dichlorobenzoic acid 0.50 104 6.0 — 5.0 95 2.1 Dicamba 0.50 100 2.2 — 5.0 98 1.6 Dichlorprop 1.0 109 3.5 — 10 79 0.7 2,4-D 1.0 108 10 — 10 99 1.6 Pentachlorophenol 0.10 103 17 — 1.0 100 4.2 Silvex 0.25 96 3.0 — 2.5 97 1.6 2,4,5-T 0.25 91 3.4 — 2.5 97 1.5 2,4-DB 1.0 94 3.1 — 10 92 1.1 Chloramben 0.50 88 2.7 — 5.0 102 2.6 Dinoseb 1.0 100 4.0 — 10 105 2.6 Bentazon 1.0 101 2.8 — 10 101 1.8 Dacthal 0.50 62‡ 9.9 — 5.0 99 1.2 Quinclorac 0.10 107 5.7 — 1.0 94 1.5 Picloram 1.0 97 2.8 — 10 103 2.5 Acifluorfen 0.50 92 4.2 — 5.0 84 2.5 2,4-Dichlorophenyl acetic

acid (SUR) 25 95 3.1 — 25 106 1.1

* n  7 † Signal-to-noise ratios were calculated for each target compound peak by dividing peak height for each compound by peak-to-peak noise, which was determined for each component from method blank over a period of time equal to full peak width in target analyte’s retention time window. ‡ Dacthal was present in the chlorinated surface water at about 1/5th fortification level. This was taken into account in the determination of the mean recovery. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

6-10T ABLE 6640:V. EFFECT OF S AMPLE H OLDING T IME ON RECOVERY FOR S AMPLES FROM A C HLORINATED S URFACE WATER F ORTIFIED WITH M ETHOD ANALYTES *
Recovery % Compound Day 0 Day 2 Day 7 Day 14
Dalapon 91 90 91 108 3,5-Dichlorobenzoic acid 98 97 85 91 Dicamba 92 92 96 99 Dichlorprop 89 88 91 95 2,4-D 88 85 87 92 Pentachlorophenol 92 85 86 93 Silvex 91 88 86 94 2,4,5-T 89 89 85 91 2,4-DB 87 84 86 93 Chloramben 86 86 88 100 Dinoseb 105 95 99 111 Bentazon 91 87 95 99 Dacthal 92 91 86 94 Quinclorac 90 80 84 91 Picloram 94 85 85 99 Acifluorfen 109 96 103 123

* Sample storage stability is expressed as a percent recovery value calculated as described in 6640B.5 h

. T ABLE 6640:VI. EFFECT OF EXTRACT H OLDING T IME ON RECOVERY FOR S AMPLES FROM A C HLORINATED S URFACE WATER F ORTIFIED WITH M ETHOD ANALYTES *†
Recovery %

Compound Initial Injection Day 21 Reinjection
Dalapon 100 89 3,5-Dichlorobenzoic acid 104 113 Dicamba 102 104 Dichlorprop 100 101 2,4-D 101 102 Pentachlorophenol 100 102 Silvex 101 103 2,4,5-T 99 102 2,4-DB 101 102 Chloramben 102 105 Dinoseb 99 81 Bentazon 98 100 Dacthal 99 103 Quinclorac 105 127 Picloram 107 98 Acifluorfen 92 104 2,4-Dichlorophenylacetic acid 106 105

* Sample storage stability is expressed as a percent recovery value calculated as described in 6640B.5 h

. † All samples fortified at the same level used to collect the mid-level precision and bias. ACIDIC HERBICIDE COMPOUNDS (6640)/Micro Liquid-Liquid Extraction GC Method

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