Method 615

The Determination of

Chlorinated Herbicides in

Municipal and Industrial

Wastewater
Method 615

The Determination of Chlorinated Herbicides in Municipal and

Industrial Wastewater

1. SCOPE AND APPLICATION

1.1 This method covers the determination of certain chlorinated herbicides. The following

parent acids can be determined by this method:

Parameter STORET No. CAS No.

2,4-D 39736 94-75-7

Dalapon - 75-99-0

2,4-DB - 94-82-6

Dicamba - 1918-00-9

Dichlorprop - 120-36-5

Dinoseb - 88-85-7

MCPA - 94-74-6

MCPP - 7085-19-0

2,4,5-T 39740 93-76-5

2,4,5-TP 39760 93-72-1

1.2 This method is also applicable to the determination of salts and esters of these

compounds. These include, but are not limited to: the isobutyl and isooctyl esters of

2,4-D; the isobutyl and isooctyl esters of 2,4-DB; the isooctyl ester of MCPA; and the

isooctyl ester of 2,4,5-TP. The actual form of each acid is not distinguished by this

method. Results are calculated and reported for each listed parameter as total free acid.

1.3 This is a gas chromatographic (GC) method applicable to the determination of the

compounds listed above in industrial and municipal discharges as provided under

40 CFR 136.1. Any modification of this method beyond those expressly permitted shall

be considered a major modification subject to application and approval of alternative test

procedures under 40 CFR 136.4 and 136.5.

1.4 The method detection limit (MDL, defined in Section 15) for each parameter is listed in

Table 1. The MDL for a specific wastewater may differ from those listed, depending

upon the nature of interferences in the sample matrix.

1.5 This method is restricted to use by or under the supervision of analysts experienced in

the use of gas chromatography and in the interpretation of gas chromatograms. Each

analyst must demonstrate the ability to generate acceptable results with this method

using the procedure described in Section 8.2.

1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds

above, compound identifications should be supported by at least one additional

qualitative technique. This method describes analytical conditions for alternative gas

chromatographic columns that can be used to confirm measurements made with the
Method 615

primary column. Section 15 provides gas chromatograph/ mass spectrometer (GC/MS)

criteria appropriate for the qualitative confirmation of compound identifications.

2. SUMMARY OF METHOD

2.1 A measured volume of sample, approximately 1 L, is acidified. The acid herbicides and

their esters and salts are extracted with ethyl ether using a separatory funnel. The

derivatives are hydrolyzed with potassium hydroxide and extraneous organic material

is removed by a solvent wash. After acidification, the acids are extracted and converted

to their methyl esters using diazomethane as the derivatizing agent. Excess reagent is

removed, and the esters are determined by electron capture (EC) gas chromatography.1

3. INTERFERENCES

3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware,

and other sample-processing apparatus that lead to discrete artifacts or elevated baselines

in gas chromatograms. All reagents and apparatus must be routinely demonstrated to

be free from interferences under the conditions of the analysis by running laboratory

reagent blanks as described in Section 8.5.

3.1.1 Glassware must be scrupulously cleaned. Clean all glassware as soon as possible 2

after use by thoroughly rinsing with the last solvent used in it. Follow by

washing with hot water and detergent and thorough rinsing with dilute acid, tap

and reagent water. Drain dry, and heat in an oven or muffle furnace at 400°C for

15 to 30 minutes. Do not heat volumetric ware. Thermally stable materials, such

as PCBs, may not be eliminated by this treatment. Thorough rinsing with acetone

and pesticide-quality hexane may be substituted for the heating. After drying

and cooling, seal and store glassware in a clean environment to prevent any

accumulation of dust or other contaminants. Store inverted or capped with

aluminum foil.

3.1.2 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.

3.2 The acid forms of the herbicides are strong organic acids, which react readily with

alkaline substances and can be lost during analysis. Glassware and glass wool must be

acid-rinsed with (1+9) hydrochloric acid and the sodium sulfate must be acidified with

sulfuric acid prior to use to avoid this possibility.

3.3 Organic acids and phenols, especially chlorinated compounds, cause the most direct

interference with the determination. Alkaline hydrolysis and subsequent extraction of

the basic solution remove many chlorinated hydrocarbons and phthalate esters that might

otherwise interfere with the electron capture analysis.

3.4 Matrix interferences may be caused by contaminants that are coextracted from the

sample. The extent of matrix interferences will vary considerably from source to source,

depending upon the nature and diversity of the industrial complex or municipality

sampled. The cleanup procedure in Section 11 can be used to overcome many of these
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interferences, but unique samples may require additional cleanup approaches to achieve

the MDL 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 must 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 for the 3-5

information of the analyst.

4.2 Diazomethane is a toxic carcinogen and can explode under certain conditions. The

following precautions must be followed:

4.2.1 Use only a well-ventilated hood; do not breath vapors.

4.2.2 Use a safety screen.

4.2.3 Use mechanical pipetting aides.

4.2.4 Do not heat above 90°C: EXPLOSION may result.

4.2.5 Avoid grinding surfaces, and avoid the use of ground-glass joints, sleeve bearings,

and glass stirrers: EXPLOSION may result.

4.2.6 Do not store near alkali metals: EXPLOSION may result.

4.2.7 Solutions of diazomethane decompose rapidly in the presence of solid materials

such as copper powder, calcium chloride, and boiling chips.

5. APPARATUS AND MATERIALS

5.1 Sampling equipment, for discrete or composite sampling.

5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume,

fitted with screw-caps lined with TFE-fluorocarbon. Aluminum foil may be

substituted for TFE if the sample is not corrosive. If amber bottles are not

available, protect samples from light. The container and cap liner must be

washed, rinsed with acetone or methylene chloride, and dried before use to

minimize contamination.

5.1.2 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
Method 615

be used. Before use, however, the compressible tubing must 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 Glassware. (All specifications are suggested. Catalog numbers are included for

illustration only.)

5.2.1 Separatory funnels: 60-mL and 2000-mL, with TFE-fluorocarbon stopcocks,

ground- glass or TFE stoppers.

5.2.2 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or

equivalent). Calibration must be checked at the volumes employed in the test.

Ground-glass stopper is used to prevent evaporation of extracts.

5.2.3 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or

equivalent). Attach to concentrator tube with springs.

5.2.4 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or

equivalent).

5.2.5 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or

equivalent).

5.2.6 Erlenmeyer flask: Pyrex, 250-mL with 24/40 ground-glass joint.

5.2.7 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.

5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a

Soxhlet extraction with methylene chloride.

5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C).

The bath should be used in a hood.

5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.

5.6 Diazomethane generator: assemble from two test tubes 150 mm long by 20 mm ID, two

Neoprene rubber stoppers, and a source of nitrogen. The generator assembly is shown

in Figure 1.

5.7 Glass wool: Acid-washed (Supelco 2-0383 or equivalent).

5.8 Gas chromatograph: Analytical system complete with gas chromatograph suitable for

on-column injection and all required accessories including syringes, analytical columns,

gases, detector, and strip-chart recorder. A data system is recommended for measuring

peak areas.
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5.8.1 Column 1: 180 cm long by 4 mm ID glass, packed with 1.5% SP-2250/1.95%

SP-2401 on Supelcoport (100/120 mesh) or equivalent. This column was used to

develop the method performance statements in Section 16. Alternative columns

may be used in accordance with the provisions described in Section 13.1.

5.8.2 Column 2: 180 cm long by 4 mm ID glass, packed with 5% OV-210 on Gas

Chrom Q (100/120 mesh) or equivalent.

5.8.3 Column 3: 180 cm long by 2 mm ID glass, packed with 0.1% SP-1000 on

Carbopak C (80/100 mesh) or equivalent.

5.8.4 Detector: Electron capture. This detector has proven effective in the analysis of

wastewaters for the parameters listed in the scope and was used to develop the

method performance statements in Section 15. Alternative detectors, including

a mass spectrometer, may be used in accordance with the provisions described

in Section 13.1.

6. REAGENTS

6.1 Reagent water: Reagent water is defined as a water in which an interferant is not

observed at the method detection limit of each parameter of interest.

6.2 Acetone, hexane, methanol: Pesticide-quality or equivalent.

6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as

indicated by EM Quant test strips (available from Scientific Products Co., Cat.

No. P1126-8, and other suppliers). Procedures recommended for removal of peroxides

are provided with the test strips. After cleanup, 20 mL ethyl alcohol preservative must

be added to each liter of ether.

6.4 Sodium sulfate: ACS, granular, acidified, anhydrous. Condition heating in a shallow

tray at 400°C for a minimum of 4 hours to remove phthalates and other interfering

organic substances. Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or

perform a Soxhlet extraction with methylene chloride for 48 hours. Acidify by slurrying

100 g sodium sulfate with enough ethyl ether to just cover the solid. Add 0.1 mL

concentrated sulfuric acid and mix thoroughly. Remove the ether under vacuum. Mix

1 g of the resulting solid with 5 mL of reagent water and measure the pH of the mixture.

It must be below pH 4. Store at 130°C.

6.5 Hydrochloric acid (1+9): Add one volume of concentrated acid (ACS) to 9 volumes

reagent water.

6.6 Potassium hydroxide solution: 37% aqueous solution (w/v). Dissolve 37 g ACS-grade

potassium hydroxide pellets in reagent water and dilute to 100 mL.

6.7 Sulfuric acid solution (1+1): Slowly add 50 mL H SO (sp. gr. 1.84) to 50 mL of reagent 2 4

water.
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6.8 Sulfuric acid solution (1+3): Slowly add 25 mL H SO (sp. gr. 1.84) to 75 mL of reagent 2 4

water. Maintain at 4°C.

6.9 Carbitol: Diethylene glycol monoethyl ether, ACS. Available from Aldrich Chemical Co.

6.10 Diazald: N-methyl-N-nitroso-p-toluenesulfonamide, ACS. Available from Aldrich

Chemical Co.

6.11 Silicic acid: Chromatographic grade, nominal 100 mesh. Store at 130°C.

6.12 Stock standard solutions (1.00 μg/μL): Stock standard solutions can be prepared from

pure standard materials or purchased as certified solutions.

6.12.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure

acids. Dissolve the material in pesticide-quality ethyl ether and dilute to volume

in a 10-mL volumetric flask. Larger volumes can be used at the convenience of

the analyst. If 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.

6.12.2 Transfer the stock standard solutions into PTFE-sealed screw-cap vials. Store at

4°C and protect from light. Stock standard solutions should be checked

frequently for signs of degradation or evaporation, especially just prior to

preparing calibration standards from them.

6.12.3 Stock standard solutions must be replaced after 1 week, or sooner if comparison

with check standards indicates a problem.

7. CALIBRATION

7.1 Establish gas chromatographic operating parameters equivalent to those indicated in

Table 1. The gas chromatographic system must be calibrated using the external standard

technique.

7.2 External standard calibration procedure.

7.2.1 For each parameter of interest, prepare working standards of the free acids at a

minimum of three concentration levels by adding accurately measured volumes

of one or more stock standards to a 10-mL volumetric flask containing 1.0 mL

methanol and diluting to volume with ethyl ether. One of the external standards

should be representative of a concentration near, but above, the method detection

limit. The other concentrations should correspond to the range of concentrations

expected in the sample concentrates or should define the working range of the

detector.

7.2.2 Prepare calibration standards by esterification of 1.00-mL volumes of the working

standards as described in Section 11. Using injections of 2 to 5 μL of each

calibration standard, tabulate peak height or area responses against the mass of
Method 615

free acid represented by the injection. The results can be used to prepare a

calibration curve for each parameter. Alternatively, the ratio of the response to

the mass injected, defined as the calibration factor (CF), can be calculated for each

parameter at each standard concentration. If the relative standard deviation of

the calibration factor is less than 10% over the working range, the average

calibration factor can be used in place of a calibration curve.

7.2.3 The working calibration curve or calibration factor must be verified on each

working shift by the preparation of one or more calibration standards. If the

response for any parameter 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 calibration factor must be prepared for that parameter.

7.3 Before using any cleanup procedure, the analyst must process a series of calibration

standards through the procedure to validate elution patterns and the absence of

interference from the reagents.

8. QUALITY CONTROL

8.1 Each laboratory using this method is required to operate a formal quality control

program. The minimum requirements of this program consist of an initial demonstration

of laboratory capability and the analysis of spiked samples as a continuing check on

performance. The laboratory is required to maintain performance records to define the

quality of data that is generated.

8.1.1 Before performing any analyses, the analyst must demonstrate the ability to

generate acceptable accuracy and precision with this method. This ability is

established as described in Section 8.2.

8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is

permitted certain options to improve the separations or lower the cost of

measurements. Each time such modifications to the method are made, the analyst

is required to repeat the procedure in Section 8.2.

8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to

monitor continuing laboratory performance. This procedure is described in

Section 8.4.

8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must

perform the following operations.

8.2.1 Select a representative spike concentration for each compound (acid or ester) to

be measured. Using stock standards, prepare a quality control check sample

concentrate in acetone, 1000 times more concentrated than the selected

concentrations.

8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a

minimum of four 1000-mL aliquots of reagent water. A representative

wastewater may be used in place of the reagent water, but one or more additional
Method 615

aliquots must be analyzed to determine background levels, and the spike level

must exceed twice the background level for the test to be valid. Analyze the

aliquots according to the method beginning in Section 10.

8.2.3 Calculate the average percent recovery (R), and the standard deviation of the

percent recovery (s), for the results. Wastewater background corrections must be

made before R and s calculations are performed.

8.2.4 Using the appropriate data from Table 2, determine the recovery and single

operator precision expected for the method, and compare these results to the

values calculated in Section 8.2.3. If the data are not comparable, review potential

problem areas and repeat the test.

8.3 The analyst must calculate method performance criteria and define the performance of

the laboratory for each spike concentration and parameter being measured.

8.3.1 Calculate upper and lower control limits for method performance as follows:

Upper Control Limit (UCL) = R + 3s

Lower Control Limit (LCL) = R − 3s

where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used

to construct control charts that are useful in observing trends in performance. 6

8.3.2 The laboratory must develop and maintain separate accuracy statements of

laboratory performance for wastewater samples. An accuracy statement for the

method is defined as R ± s. The accuracy statement should be developed by the

analysis of four aliquots of wastewater as described in Section 8.2.2, followed by

the calculation of R and s. Alternatively, the analyst may use four wastewater

data points gathered through the requirement for continuing quality control in

Section 8.4. The accuracy statements should be updated regularly.6

8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor

spike recoveries. The frequency of spiked sample analysis must be at least 10% of all

samples or one spiked sample per month, whichever is greater. One aliquot of the

sample must be spiked and analyzed as described in Section 8.2. If the recovery for a

particular parameter does not fall within the control limits for method performance, the

results reported for that parameter in all samples processed as part of the same set must

be qualified as described in Section 14.3. The laboratory should monitor the frequency

of data so qualified to ensure that it remains at or below 5%.

8.5 Before processing any samples, the analyst must demonstrate through the analysis of a

1-L aliquot of reagent water that all glassware and reagent interferences are under

control. Each time a set of samples is extracted or there is a change in reagents, a

laboratory reagent blank must be processed as a safeguard against laboratory

contamination.

8.6 It is recommended that the laboratory adopt additional quality assurance practices for

use with this method. The specific practices that are most productive depend upon the

needs of the laboratory and the nature of the samples. Field duplicates may be analyzed
Method 615

to monitor the precision of the sampling technique. When doubt exists over the

identification of a peak on the chromatogram, confirmatory techniques such as gas

chromatography with a dissimilar column, specific element detector, or mass

spectrometer must be used. Whenever possible, the laboratory should perform analysis

of quality control materials and participate in relevant performance evaluation studies.

9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING

9.1 Grab samples must be collected in glass containers. Conventional sampling practices7

should be followed; however, the bottle must not be prerinsed with sample before

collection. Composite samples should be collected in refrigerated glass containers in

accordance with the requirements of the program. Automatic sampling equipment must

be as free as possible of plastic and other potential sources of contamination.

9.2 The samples must be iced or refrigerated at 4°C from the time of collection until

extraction.

9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of

extraction.

10. SAMPLE EXTRACTION

10.1 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 with

wide-range pH paper and adjust to pH less than 2 with sulfuric acid (1+1).

10.2 Add 150 mL ethyl ether to the sample bottle, cap the bottle, and shake 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 excess 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 upon the sample, but may include stirring, filtration of the

emulsion through glass wool, centrifugation, or other physical means. Drain the aqueous

phase into a 1000-mL Erlenmeyer flask and collect the extract in a 250-mL ground-glass

Erlenmeyer flask containing 2 mL of 37% potassium hydroxide solution. Approximately

80 mL of the ethyl ether will remain dissolved in the aqueous phase.

10.3 Add a 50-mL volume of ethyl ether to the sample bottle and repeat the extraction a

second time, combining the extracts in the Erlenmeyer flask. Perform a third extraction

in the same manner.

10.4 Add 15 mL reagent water and one or two clean boiling chips to the 250-mL flask and

attach a three-ball Snyder column. Prewet the Snyder column by adding 1 mL ethyl

ether to the top. Place the apparatus on a hot water bath (60 to 65°C), such that the

bottom of the flask is bathed in the water vapor. Although the ethyl ether will evaporate

in about 15 minutes, continue heating for a total of 60 minutes, beginning from the time

the flask is placed on the water bath. Remove the apparatus and let stand at room

temperature for at least 10 minutes.
Method 615

10.5 Transfer the solution to a 60-mL separatory funnel using 5 to 10 mL of reagent water.

Wash the basic solution twice by shaking for one minute with 20-mL portions of ethyl

ether. Discard the organic phase. The free acids remain in the aqueous phase.

10.6 Acidify the contents of the separatory funnel to pH 2 by adding 2 mL of cold (4°C)

sulfuric acid (1+3). Test with pH indicator paper. Add 20 mL ethyl ether and shake

vigorously for 2 minutes. Drain the aqueous layer into the 250-mL Erlenmeyer flask,

then pour the organic layer into a 125-mL Erlenmeyer flask containing about 0.5 g of

acidified anhydrous sodium sulfate. Repeat the extraction twice more with 10-mL

aliquots of ethyl ether, combining all solvent in the 125-mL flask. Allow the extract to

remain in contact with the sodium sulfate for approximately 2 hours.

10.7 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube

to a 500- mL evaporative flask. Other concentration devices or techniques may be used

in place of the K-D if the requirements of Section 8.2 are met.

10.8 Pour the combined extract through a funnel plugged with acid-washed glass wool, and

collect the extract in the K-D in concentrator. Use a glass rod to crush any caked sodium

sulfate during the transfer. Rinse the Erlenmeyer flask and column with 20 to 30 mL of

ethyl ether to complete the quantitative transfer.

10.9 Add one to two clean boiling chips to the evaporative flask and attach a three-ball Snyder

column. Prewet the Snyder column by adding about 1-mL ethyl ether to the top. Place

the K-D apparatus on a hot water bath, 60 to 65°C, so that the concentrator tube is

partially immersed in the hot water, and the entire lower rounded surface of the flask is

bathed with hot vapor. Adjust the vertical position of the apparatus and the water

temperature as required to complete the concentration in 15 to 20 minutes. At the proper

rate of distillation the balls of the column will actively chatter but the chambers will not

flood. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus and

allow it to drain and cool for at least 10 minutes.

10.10 Remove the Snyder column and rinse the flask and its lower joint into the concentrator

tube with 1 to 2 mL of ethyl ether. A 5-mL syringe is recommended for this operation.

Add a fresh boiling chip. Attach a micro-Snyder column to the concentrator tube and

prewet the column by adding about 0.5 mL of ethyl ether to the top. Place the micro

K-D apparatus on the water bath so that the concentrator tube is partially immersed in

the hot water. Adjust the vertical position of the apparatus and the water temperature

as required to complete concentration in 5 to 10 minutes. When the apparent volume

of liquid reaches 0.5 mL, remove the micro K-D from the bath and allow it to drain and

cool. Remove the micro Snyder column and add 0.1 mL of methanol. Rinse the walls

of the concentrator tube while adjusting the volume to 1.0 mL with ethyl ether.

11. ESTERIFICATION OF ACIDS

11.1 Assemble the diazomethane generator (see Figure 1) in a hood using two test tubes 150

mm long by 20 mm ID. Use neoprene rubber stoppers with holes drilled in them to

accommodate glass delivery tubes. The exit tube must be drawn to a point to bubble

diazomethane through the sample extract.
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11.2 Add 5 mL of ethyl ether to the first test tube. Add 1 mL of ethyl ether, 1 mL of carbitol,

1.5 mL of 37% aqueous KOH, and 0.1-0.2 g Diazald to the second test tube. Immediately

place the exit tube into the concentrator tube containing the sample extract. Apply

nitrogen flow (10 mL/min.) to bubble diazomethane through the extract for 10 minutes

or until the yellow color of diazomethane persists.

11.3 Remove the concentrator tube and seal it with a neoprene or PTFE stopper. Store at

room temperature in a hood for 20 minutes.

11.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 g silicic acid to the

concentrator tube. Allow to stand until the evolution of nitrogen gas has stopped.

Adjust the sample volume to 10.0 mL with hexane. Stopper the concentrator tube and

store refrigerated if further processing will not be performed immediately. It is

recommended that the methylated extracts be analyzed immediately to minimize any

transesterification and other potential reactions that may occur. Analyze by gas

chromatography.

11.5 Determine the original sample volume by refilling the sample bottle to the mark and

transferring the water to a 1000-mL graduated cylinder. Record the sample volume to

the nearest 5 mL.

12. CLEANUP AND SEPARATION

12.1 No cleanup procedures were required to analyze the wastewaters described in Section 16.

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 for the cleanup procedure is no less than 85%.

13. GAS CHROMATOGRAPHY

13.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph.

Included in this table are estimated retention times and method detection limits that can

be achieved by this method. Examples of the separations achieved for the methyl esters

are shown in Figures 2 to 3. Other packed columns, chromatographic conditions, or

detectors may be used if the requirements of Section 8.2 are met. Capillary

(open-tubular) columns may also be used if the relative standard deviations of responses

for replicate injections are demonstrated to be less than 6% and the requirements of

Section 8.2 are met.

13.2 Calibrate the system daily as described in Section 7.

13.3 Inject 1 to 5 μL of the sample extract using the solvent-flush technique. Record the 8

volume injected to the nearest 0.05 μL, and the resulting peak size in area or peak height

units. An automated system that consistently injects a constant volume of extract may

also be used.

13.4 The width of 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 can be used to calculate
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a suggested window size for a compound. However, the experience of the analyst

should weigh heavily in the interpretation of chromatograms.

13.5 If the response for the peak exceeds the working range of the system, dilute the extract

and reanalyze.

13.6 If the measurement of the peak response is prevented by the presence of interferences,

further cleanup is required.

14. CALCULATIONS

14.1 Determine the concentration of individual compounds in the sample. Calculate the

amount of free acid injected from the peak response using the calibration curve or

calibration factor in Section 7.2.2. The concentration in the sample can be calculated as

follows:

Equation 1

where

A = Amount of material injected, in ng

V = Volume of extract injected, in μL i

V = Volume of total extract, in μL t

V = Volume of water extracted, in mL s

14.2 Report results in micrograms per liter as acid equivalent without correction for recovery

data. When duplicate and spiked samples are analyzed, report all data obtained with the

sample results.

14.3 For samples processed as part of a set where the laboratory spiked sample recovery falls

outside of the control limits in Section 8.3, data for the affected parameters must be

labeled as suspect.
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15. GC/MS CONFIRMATION

15.1 It is recommended that GC/MS techniques be judiciously employed to support

qualitative compound identifications made with this method. The mass spectrometer

should be capable of scanning the mass range from 35 amu to a mass 50 amu above the

molecular weight of the methyl ester of the acid herbicide. The instrument must be

capable of scanning the mass range at a rate to produce at least 5 scans per peak but not

to exceed 7 seconds per scan utilizing a 70 V (nominal) electron energy in the electron

impact ionization mode. A GC-to-MS interface constructed of all glass or glass-lined

materials is recommended. A computer system should be interfaced to the mass

spectrometer that allows the continuous acquisition and storage on machine-readable

media of all mass spectra obtained throughout the duration of the chromatographic

program.

15.2 Gas chromatographic columns and conditions should be selected for optimum separation

and performance. The conditions selected must be compatible with standard GC/MS

operating practices. Chromatographic tailing factors of less than 5.0 must be achieved.9

15.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS

system must be checked to see that all decafluorotriphenyl phosphine (DFTPP)

performance criteria are achieved.10

15.4 To confirm an identification of a compound, the background-corrected mass spectrum

of the methyl ester must be obtained from the sample extract and compared with a mass

spectrum from a stock or calibration standard analyzed under the same chromatographic

conditions. It is recommended that at least 25 ng of material be injected into the GC/MS.

The criteria below must be met for qualitative confirmation.

15.4.1 All ions that are present above 10% relative abundance in the mass spectrum of

the standard must be present in the mass spectrum of the sample with agreement

to ±10%. For example, if the relative abundance of an ion is 30% in the mass

spectrum of the standard, the allowable limits for the relative abundance of that

ion in the mass spectrum for the sample would be 20 to 40%.

15.4.2 The retention time of the compound in the sample must be within 6 seconds of

the same compound in the standard solution.

15.4.3 Compounds that have very similar mass spectra can be explicitly identified by

GC/MS only on the basis of retention time data.

15.5 Where available, chemical ionization mass spectra may be employed to aid in the

qualitative identification process.

15.6 Should these MS procedures fail to provide satisfactory results, additional steps may be

taken before reanalysis. These may include the use of alternate packed or capillary GC

columns or additional cleanup.
Method 615

16. METHOD PERFORMANCE

16.1 The method detection limit (MDL) is defined as the minimum concentration of a

substance that can be measured and reported with 99% confidence that the value is above

zero. The MDL concentrations listed in Table 1 were obtained from reagent water with 11

an electron capture detector.1

16.2 In a single laboratory (West Coast Technical Services, Inc.), using reagent water and

effluents from publicly owned treatment works (POTW), the average recoveries presented

in Table 2 were obtained. The standard deviations of the percent recoveries of these 1

measurements are also included in Table 2.
Method 615

References

1. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No. 68-03-2697.

Available from U.S. Environmental Protection Agency, Environmental Monitoring and

Support Laboratory, Cincinnati, Ohio.

2. 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, PA, p. 679, 1980.

3. "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.

4. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational

Safety and Health Administration, OSHA 2206 (Revised, January 1976).

5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,

Committee on Chemical Safety, 3rd Edition, 1979.

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 45268, March 1979.

7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling

Water," American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.

8. Burke, J. A. "Gas Chromatography for Pesticide Residue Analysis; Some Practical

Aspects," Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).

9. McNair, H.M. and Bonelli, E. J. "Basic Chromatography," Consolidated Printing,

Berkeley, California, p. 52, 1969.

10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to Calibrate Ion

Abundance Measurement in Gas Chromatography-Mass Spectrometry," Analytical

Chemistry, 47, 995 (1975).

11. Glaser, J.A. et al. "Trace Analysis for Wastewaters," Environmental Science & Technology,

15, 1426 (1981).
Method 615

Table 1. Chromatographic Conditions and Method Detection Limits

Parameter Method Detection

(as methyl ester) Limit (μg/L) Column 1 Column 2 Column 3

Retention Time

Dicamba 1.2 1.0 ) 0.27

2,4-D 2.0 1.6 ) 1.20

2,4,5-TP 2.7 2.0 ) 0.17

2,4,5-T 3.4 2.4 ) 0.20

2,4-DB 4.1 ) ) 0.91

Dalapon ) ) 5.0 5.80

MCPP 3.4 ) ) 192.00

MCPA 4.1 ) ) 249.00

Dichlorprop 4.8 ) ) 0.65

Dinoseb 11.2 ) ) 0.07

Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/1.95% SP-2401

packed in a glass column 1.8 m long by 4 mm ID with 95% argon/5% methane carrier gas at a

flow rate of 70 mL/min. Column temperature: isothermal at 185°C, except for MCPP, MCPA,

dichlorprop and dinoseb, where the column temperature was held at 140°C for 6 minutes and

then programmed to 200°C at 10°/min. An electron capture detector was used to measure MDL.

Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 5% OV-210 packed in a glass

column 1.8 m long by 4 mm ID with 95% argon/5% methane carrier gas at a flow rate of

70 mL/min. Column temperature: isothermal at 185°C.

Column 3 conditions: Carbopak C (80/100 mesh) coated with 0.1% SP-1000 packed in a glass

column 1.8 m long by 2 mm ID with nitrogen carrier gas at a flow rate of 25 mL/min. Column

temperature: programmed at injection from 100 to 150°C at 10°/min.
Method 615

Table 2. Single-Operator Accuracy and Precision*

Parameter Type (μg/L) (%) (%)

Sample Spike Mean Recovery Deviation

Standard

2,4-D DW 10.9 75 4

MW 10.1 77 4

MW 200.0 65 5

Dalapon DW 23.4 66 8

MW 23.4 96 13

MW 468.0 81 9

2,4-DB DW 10.3 93 3

MW 10.4 93 3

MW 208.0 77 6

Dicamba DW 1.2 79 7

MW 1.1 86 9

MW 22.2 82 6

Dichlorprop DW 10.7 97 2

MW 10.7 72 3

MW 213.0 100 2

Dinoseb MW 0.5 86 4

MW 102.0 81 3

MCPA DW 2020.0 98 4

MW 2020.0 73 3

MW 21400.0 97 2

MCPP DW 2080.0 94 4

MW 2100.0 97 3

MW 20440.0 95 2

2,4,5-T DW 1.1 85 6

MW 1.3 83 4

MW 25.5 78 5

2,4,5-TP DW 1.0 88 5

MW 1.3 88 4

MW 25.0 72 5

*All results based upon seven replicate analyses.

DW = Reagent water

MW = Municipal water
Method 615
Method 615
Method 615