EPA-821-R-00-018
September 2000
METHOD 1657
ORGANO-PHOSPHORUS PESTICIDES IN WASTEWATER,
SOIL, SLUDGE, SEDIMENT, AND TISSUE BY GC/FPD
Revision A
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Method 1657, Revision A
Acknowledgments
This method was developed under the direction of William A. Telliard of the Engineering and Analysis
Division (BAD) within the U.S. Environmental Protection Agency's (EPA's) Office of Science and
Technology (OST).
Disclaimers
This Method has been reviewed and approved for publication by the Analytical Methods Staff within
EPA's Engineering and Analysis Division. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
Introduction
Method 1657 was developed by the U.S. Environmental Protection Agency's (EPA's) Office of Science and
Technology for measurement of organo-phosphorus (O-P) pesticides in wastewater, soil, sludge, and
sediment. EPA Method 1657 has been expanded through revision A to include measurement of O-P
pesticides in fish to support EPA's National Fish Survey.
Further Information
Questions concerning this Method or its application should be addressed to:
W.A. Telliard, Director
Analytical Methods Staff (4303)
Office of Science and Technology
U.S. Environmental Protection Agency
Ariel Rios Building
1200 Pennsylvania Avenue, N.W.
Washington, D.C. 20460
Phone: 202/260-7134
Fax: 202/260-7185
Requests for copies of this Method should be addressed to:
EPA Sample Control Center
c/o DynCorp Environmental
6101 Stevenson Avenue
Alexandria, VA 22314
Phone: 703-461-2100
Fax: 703-461-8056
E-mail: SCC@,DYNCORP.COM
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Method 1657, Revision A
Note: This method is performance based. The laboratory is permitted to omit any step or modify any
procedure provided that all performance requirements in this Method are met. The laboratory may not
omit any quality control analyses. The terms "shall," "must," and "may not" define procedures required
for producing reliable results. The terms "should" and "may" indicate optional steps that may be
modified or omitted if the laboratory can demonstrate that the modified method produces results
equivalent or superior to results produced by this Method.
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Method 1657, Revision A
Method 1657, Revision A
Organo-Phosphorus Pesticides in Wastewater, Soil, Sludge,
Sediment, and Tissue by GC/FPD
1.0 Scope and Application
1.1 Method 1657, Revision A (Method 1657A, the "Method") is for determination of organo-phosphorus
pesticides and other analytes amenable to extraction and analysis by wide-bore capillary column gas
chromatography combined with a flame photometric detector (GC/FPD). The Method is for use in EPA's
data gathering and monitoring programs associated with the Clean Water Act, the Resource Conservation
and Recovery Act, the Comprehensive Environmental Response, Compensation and Liability Act, and
the Safe Drinking Water Act.
1.2 The analytes listed in Table 1 may be determined in wastewater, soil, sediment, sludge, and tissue by this
Method. In addition, Method 1657A may be applicable to other phosphorus containing pesticides. The
quality control (QC) requirements in this Method give the steps necessary to determine this applicability.
Not all analytes listed in Table 1 have corresponding calibration data in Table 3 or QC acceptance criteria
in Table 4. Calibration data for such analytes may be found in other EPA methods (References 1 and 2).
1.3 This Method is applicable to a large number of analytes. Calibrating the GC systems for all analytes is
time-consuming. If only a single analyte or small number of analytes are to be tested for, it is only
necessary to calibrate the GC systems and meet the performance specifications detailed in this Method
for the analyte(s) of interest. In addition, the GC conditions can be optimized for these analytes provided
that all performance specifications in this Method are met.
1.4 When this Method is applied to analysis of unfamiliar samples, analyte identity must be supported by at
least one additional qualitative technique. This Method describes analytical conditions for a second gas
chromatographic column that can be used to confirm measurements made with the primary column. Gas
chromatography/mass spectrometry (GC/MS) can be used to confirm analytes in extracts produced by
this Method when analyte levels are sufficient.
1.5 The detection limits of this Method are usually dependent on the level of interferences rather than
instrumental limitations. The method detection limits (MDLs; 40 CFR 136, Appendix B) in Table 2
typify the minimum quantities that can be detected with no interferences present.
1.6 This Method is for use by or under the supervision of analysts experienced in the use of a gas
chromatography and in the interpretation of gas chromatographic data. Each laboratory that uses this
Method must demonstrate the ability to generate acceptable results using the procedure in Section 9.2.
2.0 Summary of Method
2.1 Extraction—The extraction technique depends on the nature and solids content of the sample.
2.1.1 Aqueous samples containing less than 1% solids.
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Method 1657, Revision A
2.1.1.1 Samples containing water-insoluble analytes—A 1-L sample is extracted with methylene
chloride using continuous extraction techniques.
2.1.1.2 Samples containing highly water-soluble analytes such as methamidophos—Salt is added
to a 1-L sample and the sample is extracted with an azeotropic mixture of
chloroform:acetone using continuous extraction techniques.
2.1.2 Samples containing greater than 1% solids.
2.1.2.1 Non-sludge samples—If the solids content is 1 to 30%, the sample is diluted to 1% solids
with reagent water, homogenized ultrasonically, and extracted as an aqueous sample. If the
solids content is greater than 30%, the sample is extracted with methylene chloride:acetone
using ultrasonic techniques.
2.1.2.2 Sewage sludge samples and other intractable sample types—If the solids content is less than
30%, the sample is diluted to 1% solids and extracted as an aqueous sample. If the solids
content is greater than 30%, the sample is extracted with acetonitrile and then methylene
chloride using ultrasonic techniques. The extract is back-extracted with 2% (w/v) sodium
sulfate in reagent water to remove water-soluble interferences and residual acetonitrile.
2.1.3 Fish and other tissue samples are homogenized, dried with sodium sulfate, and extracted with 1:1
methylene chloride:hexane using Soxhlet extraction (Section 12.3).
2.2 Concentration—Each extract is dried over sodium sulfate and concentrated using a Kuderna-Danish
evaporator.
2.3 Cleanup—Cleanup procedures include gel permeation chromatography (GPC) and/or solid-phase
extraction. After cleanup, the extract is concentrated (Section 13).
2.4 Gas chromatography—A 1-//L aliquot of the concentrated extract is injected into the gas
chromatography (GC). The analytes are separated on a wide-bore, fused-silica capillary column and
detected using a flame photometric detector. For analyte confirmation, a 1-jwL aliquot of the extract
is injected into a column containing a dissimilar stationary phase.
2.5 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention times of
the analyte on two dissimilar columns with the respective retention times of an authentic standard.
Analyte identity is confirmed when the retention times agree within their respective windows.
2.6 Quantitative analysis is performed by using an authentic standard to produce a calibration factor or
calibration curve, and using the calibration data to determine the concentration of a pollutant in the
extract. The concentration in the sample is calculated using the sample weight or volume and the
extract volume.
2.7 Quality is assured through reproducible calibration and testing of the extraction and GC systems.
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3.0 Definitions
Definitions are given in the glossary at the end of this Method.
4.0 Contamination and Interferences
4.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or elevated
baselines causing misinterpretation of chromatograms. All materials used in the analysis shall be
demonstrated to be free from interferences under the conditions of analysis by running Method blanks
as described in Section 9.5.
4.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at 450 °C for
a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials, such as PCBs, may
not be eliminated by this treatment and thorough rinsing with acetone and pesticide-quality hexane may
be required.
4.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems may be
required.
4.4 Interferences co-extracted from samples will vary considerably from source to source, depending on
the diversity of the site being sampled. The cleanup procedures given in this Method can be used to
overcome many of these interferences, but unique samples may require additional cleanup to achieve
the minimum levels given in Table 2.
4.5 Cleanup of tissue—The natural lipid content of tissue can interfere in the analysis of tissue samples.
The lipid contents of different species and portions of tissue can vary widely. Lipids are soluble to
varying degrees in various organic solvents and may be present in sufficient quantity to overwhelm the
column chromatographic purification procedures used for sample extracts. Lipids are removed by the
cleanup procedures in Section 13.
5.0 Safety
5.1 The toxicity or carcinogenicity of each analyte or reagent used in this Method has not been precisely
determined; however, each analyte or reagent should be treated as a potential health hazard. Exposure
to these analytes should be reduced to the lowest possible level.
5.2 This Method does not address all safety issues associated with its use. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations for the safe handling of the chemicals
specified in this Method. OSHA rules require that a reference file of material safety data sheets
(MSDSs) must be made available to all personnel involved in these analyses (29 CFR 1917.28,
appendix E). It is also suggested that the laboratory perform personal hygiene monitoring of each
analyst who uses this Method and that the results of this monitoring be made available to the analyst.
Personnel hygiene monitoring should be performed using OSHA or NIOSH approved personal hygiene
monitoring methods. Additional information on laboratory safety can be found in References 3-5.
5.3 Unknown samples may contain high concentrations of volatile toxic analytes. Sample containers should
be opened in a hood and handled with gloves that will prevent exposure. The oven used for sample
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Method 1657, Revision A
drying to determine percent moisture should be located in a hood so that vapors from samples do not
create a health hazard in the laboratory.
6.0 Apparatus and Materials
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is
implied. Equivalent performance may be achieved using apparatus and materials other than those specified
here, but demonstration of equivalent performance meeting the requirements of this Method is the
responsibility of the laboratory.
6.1 Sampling equipment for discrete or composite sampling.
6.1.1 Sample bottles and caps.
6.1.1.1 Liquid samples (waters, sludges and similar materials that contain less than 5%
solids)—Sample bottle, amber glass, 1-L or 1-quart, with screw-cap.
6.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar materials that
contain greater than 5% solids)—Sample bottle, wide-mouth, amber glass, 500-mL
minimum.
6.1.1.3 If amber bottles are not available, samples shall be protected from light.
6.1.1.4 Bottle caps—Threaded to fit sample bottles. Caps shall be lined with fluoropolymer.
6.1.1.5 Cleaning.
6.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent or baked at 450 °C for a
minimum of 1 hour before use.
6.1.1.5.2 Liners are detergent water washed, then rinsed with reagent water and solvent, and baked
at approximately 200 °C for a minimum of 1 hour prior to use.
6.1.2 Compositing equipment—Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at 0 to 4 °C
during sampling. Glass or fluoropolymer tubing only shall be used. If the sampler uses a
peristaltic pump, a minimum length of compressible silicone rubber tubing may be used in the
pump only. Before use, the tubing shall be thoroughly rinsed with methanol, followed by repeated
rinsings with reagent water to minimize sample contamination. An integrating flow meter is used
to collect proportional composite samples.
6.2 Equipment for glassware cleaning - Laboratory sink with overhead fume hood.
6.3 Equipment for sample preparation.
6.3.1 Laboratory fume hood of sufficient size to contain the sample preparation equipment listed below.
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6.3.2 Glove box (optional).
6.3.3 Equipment for tissue grinding and homogenization
6.3.3.1 Tissue homogenizer—VirTis Model 45 Macro homogenizer (American Scientific Products
H-3515, or equivalent) with stainless steel Macro-shaft and Turbo-shear blade.
6.3.3.2 Meat grinder—Hobart, or equivalent, with 3-5 mm holes in inner plate.
6.3.4 Equipment for determining percent moisture.
6.3.4.1 Oven, capable of maintaining a temperature of 110° C (±5°C).
6.3.4.2 Desiccator.
6.3.4.3 Crucibles, porcelain.
6.3.4.4 Weighing pans, aluminum.
6.3.5 Balances.
6.3.5.1 Analytical—Capable of weighing 0.1 mg.
6.3.5.2 Top loading—Capable of weighing 10 mg.
6.4 Extraction equipment.
6.4.1 Equipment for ultrasonic extraction.
6.4.1.1 Sonic disrupter—3 75-watt with pulsing capability and 1A" or %" disrupter horn
(Ultrasonics, Inc, Model 375C, or equivalent).
6.4.1.2 Sonabox (or equivalent)—For use with disrupter.
6.4.2 Equipment for liquid-liquid extraction.
6.4.2.1 Continuous liquid-liquid extractor—Fluoropolymer or glass connecting joints and stopcocks
without lubrication, 1.5- to 2-L capacity (Hershberg-Wolf Extractor, Cal-Glass, Costa
Mesa, California, 1000- to 2000-mL continuous extractor, or equivalent).
6.4.2.2 Round-bottom flask—500-mL, with heating mantle.
6.4.2.3 Condenser—Graham, to fit extractor.
6.4.2.4 pH meter—With combination glass electrode.
6.4.2.5 pH paper—Wide-range (Hydrion Papers, or equivalent).
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Method 1657, Revision A
6.4.3 Separatory runnels—250-, 500-, 1000-, and 2000-mL, with fluoropolymer stopcocks.
6.4.4 Filtration apparatus.
6.4.4.1 Glass powder runnels—125- to 250-mL.
6.4.4.2 Filter paper for above (Whatman 41, or equivalent).
6.4.5 Beakers.
6.4.5.1 1.5- to 2-L, calibrated to 1 L.
6.4.5.2 400- to 500-mL.
6.4.6 Spatulas—Stainless steel or fluoropolymer.
6.4.7 Drying column—400 mm long x 15 to 20 mm ID Pyrex chromatographic column equipped with
coarse glass frit or glass wool plug.
6.4.7.1 Pyrex glass wool—Extracted with solvent or baked at 450°C for a minimum of 1 hour.
6.4.8 Evaporation/concentration apparatus.
6.4.8.1 Kuderna-Danish (K-D) apparatus.
6.4.8.1.1 Evaporation flask—500-mL (Kontes K-570001-0500, or equivalent), attached to
concentrator tube with springs (Kontes K-662750-0012).
6.4.8.1.2 Concentrator tube—10-mL, graduated (Kontes K-570050-1025, or equivalent) with
calibration verified. Ground-glass stopper (size 19/22 joint) is used to prevent
evaporation of extracts.
6.4.8.1.3 Snyder column—Three-ball macro (Kontes K-503000-0232, or equivalent).
6.4.8.1.4 Snyder column—Two-ball micro (Kontes K-469002-0219, or equivalent).
6.4.8.1.5 Boiling chips.
6.4.8.1.5.1 Glass or silicon carbide—Approximately 10/40 mesh, extracted with methylene
chloride and baked at 450 °C for a minimum of 1 hour.
6.4.8.1.5.2 Fluoropolymer (optional)—Extracted with methylene chloride.
6.4.8.2 Water bath—Heated, with concentric ring cover, capable of temperature control (±2°C),
installed in a fume hood.
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Method 1657, Revision A
6.4.8.3 Nitrogen-evaporation device—Equipped with heated bath that can be maintained at 35 to
60°C (N-Evap, Organomation Associates, Inc., or equivalent).
6.4.8.4 Sample vials—Amber glass, 1- to 5-mL with fluoropolymer-lined screw- or crimp-cap, to
fit GC auto sampler.
6.4.9 Equipment for Soxhlet extraction.
6.4.9.1 Soxhlet—5 0-mm ID, 200-mL capacity with 500-mL flask (Cal-Glass LG-6900, or
equivalent, except substitute 500-mL round-bottom flask for 300-mL flat-bottom flask).
6.4.9.2 Thimble—43 x 123 to fit Soxhlet (Cal-Glass LG-6901-122, or equivalent).
6.4.9.3 Moisture trap—Dean Stark or Barret with fluoropolymer stopcock, to fit Soxhlet.
6.4.9.4 Heating mantle—Hemispherical, to fit 500-mL round-bottom flask (Cal-Glass LG-8801-
112, or equivalent).
6.4.9.5 Variable transformer—Powerstat (or equivalent), 110-volt, 10-amp.
6.5 Apparatus for extract cleanup.
6.5.1 Automated gel permeation chromatography (Analytical Biochemical Labs, Inc, Columbia, MO,
Model GPC Autoprep 1002, or equivalent).
6.5.1.1 Column—600 to 700 mm long x 25 mm ID, packed with 70 g of SX-3 Bio-beads (Bio-Rad
Laboratories, Richmond, CA, or equivalent).
6.5.1.2 Syringe—10-mL, with Luer fitting.
6.5.1.3 Syringe-filter holder—Stainless steel with glass fiber or fluoropolymer filters (Gelman
Acrodisc-CR, 1 to 5 \a, or equivalent).
6.5.1.4 UV detectors—254 nm, preparative or semi-prep flow cell (Isco, Inc., Type 6; Schmadzu,
5 mm path length; Beckman-Altex 152W, 8 \\L micro-prep flow cell, 2 mm path; Pharmacia
UV-1, 3 mm flow cell; LDC Milton-Roy UV-3, monitor #1203; or equivalent).
6.5.2 Oven-For baking and storage of absorbents, capable of maintaining a constant temperature (±5
°C) in the range of 105-250 °C.
6.5.3 Vacuum system and cartridges for solid-phase extraction (SPE).
6.5.3.1 Vacuum system—Capable of achieving 0.1 bar (house vacuum, vacuum pump, or water
aspirator), with vacuum gauge.
6.5.3.2 VacElute Manifold (Analytichem International, or equivalent).
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Method 1657, Revision A
6.5.3.3 Vacuum trap—Made from 500-mL sidearm flask fitted with single-hole rubber stopper and
glass tubing.
6.5.3.4 Rack—For holding 50-mL volumetric flasks in the manifold.
6.5.3.5 Column—Mega Bond Elut, Non-polar, CIS Octadecyl, 10 g/60 mL (Analytichem
International Cat. No. 607H060, or equivalent).
6.5.4 Chromatographic column—400 mm long x 22 mm ID, with fluoropolymer stopcock and coarse
frit (Kontes K-42054, or equivalent).
6.6 Centrifuge apparatus.
6.6.1 Centrifuge—Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at 5,000
rpm minimum.
6.6.2 Centrifuge bottles—500-mL, with screw-caps, to fit centrifuge.
6.6.3 Centrifuge tubes—12- to 15-mL, with screw-caps, to fit centrifuge.
6.6.4 Funnel—Buchner, 15 cm.
6.6.4.1 Flask—Filter, for use with Buchner funnel.
6.6.4.2 Filter paper—15 cm (Whatman #41, or equivalent).
6.7 Miscellaneous glassware.
6.7.1 Pipettes—Glass, volumetric, 1-, 5-, and 10-mL.
6.7.2 Syringes—Glass, with Luerlok tip, 0.1-, 1- and 5-mL. Needles for syringes, 2", 22-gauge.
6.7.3 Volumetric flasks—10-, 25-, and 50-mL.
6.7.4 Scintillation vials—Glass, 20- to 50-mL, with fluoropolymer-lined screw-caps.
6.7.5 Glass funnel-125-250mL.
6.7.6 Glass-fiber filter paper-Whatman GF/D (or equivalent), to fit glass funnel in Section 6.7.5.
6.8 Gas chromatography —Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a flame photometric detector at the end of each column, temperature
program with isothermal holds, data system capable of recording simultaneous signals from the two
detectors, and shall meet all of the performance specifications in Section 14.
6.8.1 GC columns—Bonded-phase fused-silica capillary.
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6.8.1.1 Primary—30 m (±3 m) long x 0.5 mm (±0.05 mm) ID DB-1, or equivalent.
6.8.1.2 Confirmatory—DB-1701, or equivalent, with same dimensions as primary column.
6.8.2 Data system—Shall collect and record GC data, store GC runs on magnetic disk or tape, process
GC data, compute peak areas, store calibration data including retention times and calibration
factors, identify GC peaks through retention times, compute concentrations, and generate reports.
6.8.2.1 Data acquisition—GC data shall be collected continuously throughout the analysis and
stored on a mass storage device.
6.8.2.2 Calibration factors and calibration curves—The data system shall be used to record and
maintain lists of calibration factors and multipoint calibration curves (Section 10).
Computations of relative standard deviation (RSD; coefficient of variation) are used for
testing calibration linearity. Statistics on initial (Section 9.2) and ongoing (Section 15.5)
performance shall be computed and maintained.
6.8.2.3 Data processing—The data system shall be used to search, locate, identify, and quantify the
analytes of interest in each GC analysis. Software routines shall be employed to compute
and record retention times and peak areas. Displays of chromatograms and library
comparisons are required to verify results.
6.8.2.4 Flame photometric detector—Capable of detecting 11 pg of malathion under the analysis
conditions given in Table 2.
7.0 Reagents and Standards
7.1 Sample preservation—Sodium thiosulfate (ACS), granular.
7.2 pH adjustment.
7.2.1 Sodium hydroxide—Reagent grade.
7.2.1.1 Concentrated solution (ION)—Dissolve 40 g NaOH in 100-mL reagent water.
7.2.1.2 Dilute solution (0.1M)—Dissolve 4 g NaOH in 1 L of reagent water.
7.2.2 Sulfuric acid (1+1)—Reagent grade, 6N in reagent water. Slowly add 50 mL H2SO4 (specific
gravity 1.84) to 50 mL reagent water.
7.2.3 Potassium hydroxide—37% (w/v); dissolve 37 g KOH in 100 mL reagent water.
7.3 Solution drying and back-extraction.
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7.3.1 Sodium sulfate—Reagent grade, granular anhydrous (Baker 3375, or equivalent), rinsed with
methylene chloride (20 mL/g), baked at 450 °C for a minimum of 1 hour, cooled in a desiccator,
and stored in a pre-cleaned glass bottle with screw-cap which prevents moisture from entering.
7.3.2 Sodium sulfate solution—2% (w/v) in reagent water, pH adjusted to 8.5 to 9.0 with KOH or
H2SO4.
7.3.3 Sodium sulfate, reagent grade, powdered, treated as in Section 7.3.1- Used for drying solids and
tissue samples.
7.3.4 Activated silica gel—100-200 mesh, Supelco 1-3651 (or equivalent), rinsed with methylene
chloride, baked at 180° C for a minimum of 1 hour, cooled in a desiccator, and stored in a pre-
cleaned glass bottle with screw-cap that will prevent moisture from entering.
7.4 Solvents—Methylene chloride, hexane, acetone, acetonitrile, isooctane, and methanol; pesticide-quality;
lot-certified to be free of interferences.
7.5 GPC calibration solution—Solution containing 300 mg/mL corn oil, 15 mg/mL bis(2-
ethylhexyl)phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
7.6 Sample cleanup.
7.6.1 Solid-phase extraction.
7.6.1.1 SPE cartridge calibration solution—2,4,6-trichlorophenol, 0.1 (jg/mL in acetone.
7.6.1.2 SPE elution solvent—methylene chloride:acetonitrile:hexane (50:3:47).
7.6.2 GPC column packing-Bio-beads, SX-3, (Bio-Rad Laboratories, Richmond, CA, or equivalent).
7.7 Reference matrices.
7.7.1 Reagent water—Water in which the analytes of interest and interfering analytes are not detected
by this Method.
7.7.2 High-solids reference matrix—Playground sand or similar material in which the analytes of
interest and interfering analytes are not detected by this Method. Maybe prepared by extraction
with methylene chloride and/or baking at 450 °C for 4 hours minimum.
7.7.3 Tissue reference matrix—Corn or other vegetable oil, prepared by extraction with methylene
chloride.
7.8 Standard solutions—Purchased as solutions or mixtures with certification to their purity, concentration,
and authenticity, or prepared from materials of known purity and composition. If analyte purity is 96%
or greater, the weight may be used without correction to compute the concentration of the standard.
When not being used, standards are stored in the dark at -20 to -10 °C in screw-capped vials with
fluoropolymer-lined lids. A mark is placed on the vial at the level of the solution so that solvent
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Method 1657, Revision A
evaporation loss can be detected. The vials are brought to room temperature prior to use. Any
precipitate is redissolved and solvent is added if solvent loss has occurred.
7.9 Preparation of stock solutions—Prepare in isooctane per the steps below. Observe the safety
precautions in Section 5.
7.9.1 Dissolve an appropriate amount of assayed reference material in solvent. For example, weigh 10
mg of malathion in a 10-mL ground-glass stoppered volumetric flask and fill to the mark with
isooctane. After the malathion is completely dissolved, transfer the solution to a 15-mL vial with
fluoropolymer-lined cap.
7.9.2 Stock solutions should be checked for signs of degradation prior to the preparation of calibration
or performance test standards.
7.9.3 Stock standard solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
7.10 Secondary mixtures—Using stock solutions (Section 7.9), prepare mixtures atthe levels shown in Table
3 for calibration and calibration verification (Sections 10.3 and 15.4), for initial and ongoing precision
and recovery (Sections 9.2 and 15.5), and for spiking into the sample matrix (Section 9.4).
7.11 Surrogate spiking solution—Prepare tributyl phosphate and triphenyl phosphate each at a concentration
of 10 (jg/mL in acetone.
7.12 Stability of solutions—All standard solutions (Section 7.8) shall be analyzed within 48 hours of
preparation and on a monthly basis thereafter for signs of degradation. Standards will remain
acceptable if the peak area remains within ±15% of the area obtained in the initial analysis of the
standard.
7.13 Combined QC standard—Mix equal volumes of the high concentration calibration solutions (Table 3)
to produce this standard. It is used for calibration (Section 10.4), calibration verification (Section
15.4), and for initial (Section 9.2.) and ongoing (Section 15.5) demonstrations of method performance.
8.0 Sample Preservation
8.1 Collect aqueous samples in glass containers following conventional sampling practices (Reference 7),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples that flow
freely are collected in refrigerated bottles using automatic sampling equipment. Solid samples are
collected as grab samples using wide-mouth jars. Tissue samples may be collected in jars or wrapped
in aluminum foil.
8.2 Storage Temperature
8.2.1 Aqueous samples—Maintain at 0-4 °C from the time of collection until extraction.
8.2.2 Solid samples—Maintain at 0-4 ° C from time of collection until extraction, or freeze at <-10 ° C.
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8.2.3 Tissue samples—Freeze at <-10 °C upon collection unless the sample will be extracted within
12 hours, in which case maintain at 0-4 °C from the time of collection until extraction.
8.3 Aqueous sample preservation
8.3.1 If the sample will not be extracted within 72 hours of collection, adjust the sample to a pH of 5.0
to 9.0 using sodium hydroxide or sulfuric acid solution. Record the volume of acid or base used.
8.3.2 If residual chlorine is present in an aqueous sample, add 80 mg sodium thiosulfate per liter of
water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 8).
8.4 Holding times
8.4.1 Aqueous and unfrozen solid samples—Begin sample extraction within 7 days of collection
8.4.2 Frozen solid and tissue samples—If stored in the dark at <-10°C, solid, semi-solid, multi-phase,
and tissue samples may be stored for up to one year prior to extraction.
8.4.3 Analyze all extracts within 40 days of extraction.
9.0 Quality Control
9.1 Each laboratory that uses this Method is required to operate a formal quality control (QC) program
(Reference 6). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, ongoing analyses of standards and blanks as tests of continued performance, and
analysis of matrix spike samples to assess recovery and precision. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of this Method. For the performance tests, a reference matrix is used that
most closely simulates the matrix of interest. For aqueous, solid, and tissue samples, reference matrices
of reagent water (Section 7.7.1), playground sand (Section 7.7.2), or corn oil (Section 7.7.3),
respectively, are suggested.
9.1.1 The laboratory shall make an initial demonstration of the ability to generate acceptable precision
and recovery with this Method. This ability is established as described in Section 9.2.
9.1.2 The laboratory is permitted to modify this Method to improve separations or lower the costs of
measurements, provided all performance requirements are met. These options include use of
alternate extraction techniques such as solid-phase extraction and accelerated solvent extraction,
alternate concentration techniques such as rotary evaporation, and alternate cleanup techniques
such as high-performance liquid chromatography. Changes in the principle of the determinative
technique, such as the use of colorimetry, are not allowed.
9.1.2.1 Each time a modification is made to this Method, the laboratory is required to repeat the
procedure in Section 9.2 to demonstrate performance. If a detection limit will be affected
by the modification, the laboratory is required to demonstrate that the MDL (40 CFR Part
136, Appendix B) is less than or equal to the MDL in this Method for the analyte(s) of
interest. If this Method will be used for compliance monitoring, the MDL for the modified
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Method 1657, Revision A
method must be less than or equal to the MDL of this Method or one-third the regulatory
compliance limit, whichever is greater. If the change will affect calibration, the instrument
must be recalibrated according to Section 10.
9.1.2.2 The laboratory is required to maintain records of modifications made to this Method. These
records include the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone numbers of the analyst(s) who performed the
analyses and modification, and the quality control officer who witnessed and will verify
the analyses and modification
9.1.2.2.2 A narrative stating the reason(s) for the modification(s)
9.1.2.2.3 Results from all quality control (QC) tests comparing the modified method to this
Method, including the following:
(a) Calibration (Section 10)
(b) Initial precision and recovery (Section 9.2)
(c) Analysis of blanks (Section 9.5)
(d) Matrix spike analysis (Section 9.4)
(e) Ongoing precision and recovery (Section 15.5)
(f) Surrogate recovery (Section 9.3)
(g) Method detection limit (Section 9.1.2.1)
9.1.2.2.4 Data that will allow an independent reviewer to validate each determination by tracking
the instrument output to the final result. These data are to include the following:
(a) Sample numbers and other identifiers
(b) Processing dates
(c) Analysis dates
(d) Analysis sequence/run chronology
(e) Sample weight or volume
(f) Copies of logbooks, chart recorder, or other raw data output
(g) Calculations linking raw data to the results reported
9.1.3 The laboratory shall spike all samples with at least one surrogate compound to monitor method
performance. This test is described in Section 9.3. When results of these spikes indicate atypical
method performance for samples, the samples are diluted to bring method performance within
acceptable limits (Section 18).
9.1.4 Analyses of matrix spike (MS) samples are required to demonstrate the recovery and to monitor
matrix interferences. Section 9.4 describes the procedure and QC criteria for spiking.
9.1.5 The laboratory shall, on an ongoing basis, demonstrate through calibration verification and the
analysis of the combined QC standard (Section 7.13) that the analysis system is in control. These
procedures are described in Sections 15.1, 15.4, and 15.5.
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Method 1657, Revision A
9.1.6 The laboratory should maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 9.4.4 and 15.5.4.
9.1.7 Analyses of blanks are required to demonstrate freedom from contamination. The procedures and
criteria for analysis of a blank are described in Section 9.5.
9.1.8 Other analytes may be determined by this Method. The procedure for establishing a QC
acceptance criteria for a new analyte is given in Section 9.6.
9.1.9 QC using this Method is controlled by an analytical batch. An analytical batch is a set of
samples started through the extraction process during a given 12-hour shift, to a maximum of 20.
A batch may be from 1 to as many as 20 samples. Each batch must be accompanied by a blank,
an OPR, and MS.
9.2 Initial demonstration of laboratory capability
9.2.1 Method detection limit—To establish the ability to detect the analytes of interest in this Method,
the laboratory shall achieve MDLs that are less than or equal to the MDLs listed in Table 2 or
one-third the regulatory compliance limit, whichever is greater. The MDLs shall be determined
according to the procedure at 40 CFR 136, Appendix B using the apparatus, reagents, and
standards that will be used in the practice of this Method. These MDLs shall be used for
determination of laboratory capability only, and should be determined when a new operator
begins work or whenever, in the judgment of the laboratory, a change in instrument hardware or
operating conditions would dictate reevaluation of capability.
9.2.2 Initial precision and recovery—To establish the ability to generate acceptable precision and
recovery, the laboratory shall perform the following operations.
9.2.2.1 For aqueous samples, extract, concentrate, and analyze one set of four 1-L aliquots of
reagent water spiked with the combined QC standard (Section 7.13) according to the
procedure in Section 11. Alternatively, sets of four replicates of the individual calibration
groups (Table 3) may be used. For samples containing high solids, a set of four 30-g
aliquots of the high-solids reference matrix (Section 7.7.2) are used. For tissue samples, a
set of four 10-g aliquots of the tissue reference matrix (Section 7.7.3) are used.
9.2.2.2 Using results of the set of four analyses, compute the average percent recovery (X) and the
relative standard deviation (RSD) of percent recovery for each analyte.
9.2.2.3 For each analyte, compare X and the RSD and with the corresponding limits for initial
precision and recovery in Table 4. For coeluting analytes, use the coeluted compound with
the least restrictive specification (largest RSD and widest range). If X and RSD for all
analytes meet the acceptance criteria, system performance is acceptable and analysis of
blanks and samples may begin. If, however, any individual RSD exceeds the precision limit
or any individual X falls outside the range for recovery, system performance is unacceptable
for that analyte. In this case, correct the problem and repeat the test.
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Method 1657, Revision A
9.3 Surrogates - The laboratory shall spike all samples with surrogates (Section 7.11) to assess method
performance on the sample matrix.
9.3.1 Analyze each sample according to this Method beginning in Section 11.
9.3.2 Compute the percent recovery (P) of the surrogates.
9.3.3 The recovery of the surrogates shall be in the range of 40 - 120%. If the recovery of a surrogate
falls outside of these limits, method performance is unacceptable for that sample. Water samples
are diluted, and smaller amounts of soils, sludges, sediments, and tissues are reanalyzed per
Section 18.
9.4 Matrix spike—The laboratory shall spike (matrix spike) at least 5% of the samples from a given matrix
type (e.g., influent to treatment, treated effluent, produced water, river sediment, soil, tissue). If only
one sample from a given matrix type is analyzed, a separate aliquot of that sample shall be spiked.
9.4.1 The concentration of the matrix spike shall be determined as follows:
9.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in the sample is being
checked against a regulatory concentration limit, the MS shall be at that limit or at 1 to 5
times higher than the background concentration determined in Section 9.4.2, whichever
concentration is greater.
9.4.1.2 If the concentration of an analyte in the sample is not being checked against a limit specific
to the analyte, the MS shall be at the concentration of the combined QC standard (Section
7.13) or at 1 to 5 times higher than the background concentration, whichever concentration
is greater.
9.4.1.3 If it is impractical to determine the background concentration before spiking (e.g., maximum
holding times will be exceeded), the matrix spike concentration shall be the regulatory
concentration limit, if any; otherwise, the greater of either 5 times the expected background
concentration or at the concentration of the combined QC standard (Section 7.13).
9.4.2 Analyze one sample aliquot to determine the background concentration (B) of each analyte. If
necessary, prepare a standard solution appropriate to produce a level in the sample 1 to 5 times
the background concentration. Spike a second sample aliquot with the standard solution and
analyze it to determine the concentration after spiking (A) of each analyte. Calculate the percent
recovery (P) of each analyte:
Equation 1
P = (100(A-B)/T)
where
T = True value of the spike
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Method 1657, Revision A
9.4.3 The matrix spike recovery in sample should be 25 - 150%. For the OPR, compare the percent
recovery for each analyte with the corresponding QC acceptance criteria in Table 4.
9.4.3.1 If the results of both the MS and the OPR fall outside the acceptance criteria, the analytical
system is judged to be not in control. The laboratory must identify and correct the problem
and reanalyze all samples in the sample batch.
9.4.3.2 If results for the MS fail the acceptance criteria, and recovery for the OPR standard (Section
15.5) for the analytical batch is within the acceptance criteria in Table 4, an interference is
present and the results may not be reported or otherwise used for permitting or regulatory
compliance purposes. If the interference can be attributed to sampling, the site or discharge
should be resampled. If the interference can be attributed to a method deficiency, the
laboratory must modify this Method, repeat the test required in Section 9.1.2, and repeat
analysis of the sample and MS.
9.4.3.3 If the interference cannot be overcome after this Method has been modified, the sample is
complex and must be diluted and reanalyzed per Section 18.
9.4.4 As part of the QC program for the laboratory, it is suggested but not required that method
precision and recovery be assessed and records be maintained. After the analysis of five spiked
samples of a given matrix type (water, soil, sludge, sediment, tissue) in which the analytes pass
the tests in Section 9.4.3, compute the average percent recovery (P) and the standard deviation
of the percent recovery for each analyte (or coeluting analyte group). Express the accuracy
assessment as a percent recovery interval from P - 2sp to P + 2sp for each matrix. For example,
if P=90% and sp=10% for five analyses of compost, the accuracy interval is expressed as 70 to
110%. Update the accuracy assessment for each analyte in each matrix on a regular basis (e.g.,
after each 5 to 10 new accuracy measurements).
9.5 Blanks—Reference matrix blanks are analyzed to demonstrate freedom from contamination.
9.5.1 Extract and concentrate a 1-L reagent water blank, a 30-g high-solids reference matrix blank, or
a 10-g tissue matrix blank (whichever is appropriate) with each sample batch (Section 9.1.9).
Analyze the blank immediately after analysis of the combined QC standard (Section 7.13) to
demonstrate freedom from contamination.
9.5.2 If any of the analytes of interest (Table 1) or any potentially interfering substance is found in a
blank at a concentration greater than the concentration of the low concentration calibration
solution in Table 3 (assuming the same calibration factor as for triphenyl phosphate for analytes
not listed in Table 1), analysis of samples is halted until the source of contamination is eliminated
and a blank shows no evidence of contamination at this level.
9.6 Application of this Method to other analytes.
9.6.1 To establish QC acceptance criteria for an analyte, determine the precision and recovery by
analyzing four replicates of the analyte along with the combined QC standard per the procedure
in Section 9.2. If the analyte coelutes with an analyte in the QC standard, prepare a new QC
standard without the coeluting component(s).
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Method 1657, Revision A
9.6.2 Compute the average percent recovery (A) and the RSD of percent recovery (RSDn) for the new
analyte, and measure the recovery and RSD for the other analytes. The data for the new analyte
are valid if the precision and recovery specifications for the other analytes are met; otherwise, the
analytical problem is corrected and the test is repeated. Establish a preliminary QC limit of
2RSDn for precision and of A ± 2RSDn for recovery for the new analyte and add to the
acceptance criteria in Table 4.
9.7 The specifications contained in this Method can be met if the apparatus used is calibrated properly, then
maintained in a calibrated state. The standards used for calibration (Sections 7.8 - 7.13), calibration
verification (Section 15.4), and for initial (Section 9.2) and ongoing (Section 15.5) precision and
recovery should be identical, so that the most precise results will be obtained. The GC instruments will
provide the most reproducible results if dedicated to the settings and conditions required for the analyses
of the analytes given in this Method.
9.8 Depending on specific program requirements, field replicates and field spikes of the analytes of interest
into samples may be required to assess the accuracy of the sampling and sample transporting
techniques.
10.0 Setup and Calibration
10.1 Configure the GC system as given in Section 6.8 and establish the operating conditions in Table 2.
10.2 Attainment of MDL—Determine that each column/detector system meets the MDLs in Table 2.
10.3 Calibration.
10.3.1 Injection of calibration solutions.
10.3.1.1 Analytes with calibration data in Table 3—The analytes in each calibration group in Table
3 were chosen so that each analyte would be separated from the others by approximately
1 minute on the primary column. The concentrations were chosen to bracket the working
range of the FPD. However, because the response of some models of FPD are greater than
others, it may be necessary to inject a larger volume of calibration solution for these
detectors.
10.3.1.2 Analytes without calibration data in Table 3—Prepare calibration standards at a minimum
of three concentration levels. One of these concentrations should be near, but above, the
MDL (Table 2) and the other concentrations should define the working range of the
detectors.
10.3.1.3 Set the automatic injector to inject a constant volume in the range of 0.5 to 5.0 yL of each
calibration solution into the GC column/detector pairs, beginning with the lowest level
mixture and proceeding to the highest. For each analyte, compute and store, as a function
of the concentration injected, the retention time and peak area on each column/detector
system (primary and confirmatory).
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Method 1657, Revision A
10.3.2 Retention time—The polar nature of some analytes causes the retention time to decrease as the
quantity injected increases. To compensate this effect, the retention time for analyte
identification is correlated with the analyte level.
10.3.2.1 If the difference between the maximum and minimum retention times for any analyte is less
than 5 seconds over the calibration range, the retention time for that analyte can be
considered constant and an average retention time may be used for analyte identification.
10.3.2.2 Retention-time calibration curve (retention time vs. amount)—If the retention time for an
analyte in the lowest level standard is more than 5 seconds greater than the retention time
for the analyte in the highest level standard, a retention-time calibration curve shall be used
for identification of that analyte.
10.3.3 Calibration factor (ratio of area to amount injected).
10.3.3.1 Compute the RSD of the calibration factor over the calibration range for each analyte on
each column/detector system.
10.3.3.2 Linearity—If the calibration factor for any analyte is constant (RSD <20%) over the
calibration range, an average calibration factor may be used for that analyte; otherwise,
the complete calibration curve (area vs. amount) for that analyte shall be used.
10.4 Combined QC standards—To preclude periodic analysis of all of the individual calibration groups of
analytes (Table 3), calibration of the GC systems are verified with combined solutions as a final step.
Not all of the analytes in these standards will be separated by the GC columns used in this Method.
Retention times and calibration factors are verified for the analytes that are resolved, and calibration
factors are obtained for the unresolved peaks. These combined QC standards are prepared at the level
the mid-range calibration standard (Table 3).
10.4.1 Analyze the combined QC standard on each column/detector pair.
10.4.1.1 For those analytes that exhibit a single, resolved GC peak, the retention time shall be
within ±5 seconds of the retention time of the peak in the medium level calibration standard
(Table 3), and the calibration factor using the primary column shall be within ±20% of the
calibration factor in the medium level standard (Table 3). If the retention times are outside
of these windows, readjust the analytical conditions until this test is met, or recalibrate and
repeat this test.
10.4.1.2 For coeluted compounds, compute and store the retention times at the peak maxima on
both columns (primary and confirmatory), and also compute and store the calibration
factors on both columns. These results will be used for calibration verification (Section
15.2 and 15.4) and for precision and recovery studies (Sections 9.2 and 15.5).
11.0 Sample Preparation
11.1 Determination of percent solids (for non-tissue samples expected or known to contain >1% solids).
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Method 1657, Revision A
11.1.1 Weigh 5 to 10 g of sample into a tarred beaker. Record the weight to three significant figures.
11.1.2 Dry overnight (12 hours minimum) at 110°C (±5 °C), and cool in a desiccator.
11.1.3 Determine percent solids as follows:
Equation 2
,., might of dry sample
% sokds = — — ±— x 100
weight of wet sample
11.2 Preparation of samples for extraction.
11.2.1 Samples containing 0-30% solids.
11.2.1.1 Aqueous samples containing 1% solids or less—Extract the sample directly using
continuous liquid-liquid extraction techniques.
11.2.1.1.1 Measure 1 L (±0.01 L) of sample into a clean 1.5- to 2-L beaker.
11.2.1.1.2 Spike 1.0 mL of the surrogate spiking solution (Section 7.11) into the sample aliquot.
Proceed to preparation of the QC aliquots for low-solids samples (Section 11.2.1.3).
11.2.1.2 Samples containing 1 to 30% solids.
11.2.1.2.1 Mix sample thoroughly.
11.2.1.2.2 Using the percent solids found in Section 11.1.3, determine the weight of sample
required to produce 1-L of solution containing 1% solids as follows:
Equation 3
1000 p
sample weight =
% solids
11.2.1.2.3 Place the weight determined in Section 11.2.1.2.2 in a clean 1.5- to 2.0-L beaker.
Discard all sticks, rocks, leaves, and other foreign material prior to weighing.
11.2.1.2.4 Bring the volume of the sample aliquot(s) to 100- to 200-mL with reagent water.
11.2.1.2.5 Spike 1.0 mL of the appropriate surrogate spiking solution (Section 7.11) into each
sample aliquot.
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Method 1657, Revision A
11.2.1.2.6 Using a clean metal spatula, break any solid portions of the sample into small pieces.
11.2.1.2.7 Place the %" horn on the ultrasonic probe approximately 1A" below the surface of each
sample aliquot and pulse at 50% for 3 minutes at full power. If necessary, remove the
probe from the solution and break any large pieces using the metal spatula or a stirring
rod and repeat the sonication. Clean the probe with methylene chloride:acetone (1:1)
between samples to preclude cross-contamination.
11.2.1.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
11.2.1.3 Preparation of QC aliquots for samples containing low solids (<30%).
11.2.1.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at the same
time, place two 1.0-L (±0.1 L) aliquots of reagent water in clean 1.5- to 2.0-L beakers.
11.2.1.3.2 Blank—Spike 1.0 mL of the pesticide surrogate spiking solution (Section 7.11) into
one reagent water aliquot.
11.2.1.3.3 OPR—Spike 1.0 mL of the pesticide surrogate spiking solution (Section 7.11) and the
combined QC standard (Section 7.13) into the second reagent water aliquot.
11.2.1.3.4 Matrix Spike—Prepare an aliquot at the concentrations specified in Section 9.4.
11.2.1.3.5 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Extract the samples
and QC aliquots per Section 12.1.
11.2.2 Samples containing 30% solids or greater.
11.2.2.1 Mix the sample thoroughly.
11.2.2.2 Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker. Discard all sticks, rocks, leaves,
and other foreign material prior to weighing.
11.2.2.3 Spike 1.0 mL of the surrogate spiking solution (Section 7.11) into the sample aliquot.
11.2.2.4 QC aliquots—For each sample or sample batch (to a maximum of 20) to be extracted at
the same time, place two 30-g (±0.3 g) aliquots of the high-solids reference matrix in clean
400- to 500-mL beakers.
11.2.2.4.1 Blank—Spike 1.0 mL of the surrogate spiking solution (Section 7.11) into one aliquot
of the high-solids reference matrix.
11.2.2.4.2 OPR—Spike 1.0 mL of the surrogate spiking solution (Section 7.11) and the combined
QC standard (Section 7.13) into the second high-solids reference matrix aliquot.
11.2.2.4.3 Matrix spike—Prepare an aliquot at the concentration specified in Section 9.4
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Method 1657, Revision A
11.2.2.5 Extract the high-solids samples per Section 12.0.
11.2.3 Tissue samples
11.2.3.1 Prior to processing tissue samples, the laboratory must determine the exact tissue to be
analyzed. Common requests for analysis offish tissue include whole fish-skin on, whole
fish-skin removed, edible fish fillets (filleted in the field or by the laboratory), specific
organs, and other portions. Once the appropriate tissue has been determined, the sample
must be homogenized.
11.2.3.2 Homogenization
11.2.3.2.1 Samples are homogenized while still frozen, where practical. If the laboratory must
dissect the whole fish to obtain the appropriate tissue for analysis, the unused tissues
may be rapidly refrozen and stored in a clean glass jar for subsequent use.
11.2.3.2.2 Each analysis requires 10 g of tissue (wet weight). Therefore, homogenize at least 30
g of tissue to allow for the MS and for re-extraction of a second aliquot of the same
homogenized sample, if re-analysis is required. When whole fish analysis is required,
the entire fish is homogenized.
11.2.3.2.3 Homogenize the sample in a tissue homogenizer (Section 6.3.3.1) or grind in a meat
grinder (Section 6.3.3.2). Cut tissue too large to feed into the grinder into smaller
pieces. To assure homogeneity, grind three times.
11.2.3.2.4 Transfer approximately 10 g (wet weight) of homogenized tissue to a clean, tared, 400-
to 500-mL beaker. Record the weight to 3 significant figures.
11.2.3.2.5 Spike 1.0 mL of the surrogate spiking solution (Section 7.11) into the sample.
11.2.3.2.6 Transfer the remaining homogenized tissue to a clean jar with a fluoropolymer-lined
lid. Seal the jar and store the tissue at <-10 °C. Return any tissue that was not
homogenized to its original container and store at <-10 °C.
11.2.3.3 Preparation of QC aliquots.
11.2.3.3.1 For each sample or sample batch (Section 9.1.9), weigh two 10-g (± 1 g) aliquots of
the tissue reference matrix (Section 7.7.3) into clean 400- to 500-mL beakers. One
aliquot will serve as the blank and the other as the OPR.
11.2.3.3.2 Spike 1.0 mL of the surrogate spiking solution (Section 7.11) into each aliquot.
11.2.3.3.3 OPR—Spike the combined QC standard (Section 7.13) into one of the aliquots.
11.2.3.3.4 MS—Prepare an aliquot at the concentrations specified in Section 9.4.
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11.2.4 Samples containing methamidophos and other highly water-soluble analytes—Prepare samples
containing less than 30% solids per Section 11.2.1; prepare samples containing greater than
30% solids per Section 11.2.2; and prepare tissue samples per Section 11.2.3.
11.2.4.1 Interferences—If interferences are expected, aqueous samples can be pre-extracted with
methylene chloride to remove these interferences. This extract can be used for
determination of insoluble or slightly soluble analytes and the surrogates. Methamidophos
is only sightly soluble in methylene chloride and will not be in this extract unless carried
by polar species in the sample matrix. If analytes other than methamidophos are not to be
determined, the methylene chloride extract can be discarded.
11.2.4.2 Determine the percent solids and prepare a 1-L sample aliquot and the QC aliquots per
Sections 11.2.1.3 and 11.2.2.4 and 11.2.3.3, except do not spike the surrogate into the
sample aliquot if the methylene chloride extract will be discarded (Section 11.2.4.1).
11.2.4.3 Extract the aliquots per Section 12.0 using methylene chloride to remove interferences.
11.2.4.4 After extraction, remove the water and methylene chloride from the extractor. Decant the
aqueous portion into a beaker and combine the remaining methylene chloride with the
extract in the distilling flask. If the methylene chloride extract is to be used for
determination of other analytes and the surrogate, proceed to Section 12.4 with that
extract.
11.2.4.5 Saturate the water sample with sodium chloride. Approximately 350 g will be required.
NOTE: As a result of the increased density of the water caused by saturation with salt, the sample may sink
to where the water enters the siphon tube of the continuous extractor. To prevent this from occurring, use
a smaller volume of water (e.g., 800 mL) in the extractor. Correct for this adjustment in the calculation of
the concentration of the analytes in the extract (Section 15).
11.2.4.6 If the methylene chloride extract was discarded, spike the surrogates into the sample
aliquot.
11.2.4.7 Extract the sample per Section 12.0 except use a chloroform:acetone azeotrope (2:1 v/v
or 4:1 w/w) for the extraction.
12.0 Extraction and Concentration
12.1 Continuous extraction of low-solids (aqueous) samples—Place 100 to 150 mL solvent (methylene
chloride or 2:1 v/v chloroform:acetone, as appropriate) in each continuous extractor and 200 to 300 mL
in each distilling flask.
12.1.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass containers
with 50 to 100 mL methylene chloride and add to the respective extractors. Include all solids
in the extraction process.
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Method 1657, Revision A
12.1.2 Adjust the pH of the waters in the extractors to 5- 9 with NaOH or H2SO4 while monitoring
with a pH meter.
NOTE: Warning—Some samples require acidification in a hood because of the potential for generating
hydrogen sulfide.
12.1.3 Begin the extraction by heating the flask until the solvent is boiling. When properly adjusted,
one to two drops of solvent per second will fall from the condenser tip into the water. Test and
adjust the pH of the waters after the first 1 to 2 hours of extraction. Extract for 18 to 24 hours.
12.1.4 Remove the distilling flask, estimate and record the volume of extract (to the nearest 100 mL),
and pour the contents through a prerinsed drying column containing 7 to 10 cm of granular
anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of methylene chloride and
pour through the drying column. For extracts to be cleaned up using GPC, collect the solution
in a 500-mL K-D evaporator flask equipped with a 10-mL concentrator tube. Seal, label, and
concentrate per Sections 12.4 to 12.5.
12.2 Ultrasonic extraction of high-solids samples—Procedures are provided for extraction of non-sewage
sludge (Section 12.2.1) and sewage sludge samples (Section 12.2.2).
12.2.1 Ultrasonic extraction of non-sewage sludge high-solids aliquots.
12.2.1.1 Add 60 to 70 g of powdered anhydrous sodium sulfate to the sample and QC aliquots.
Mix each aliquot thoroughly. Some wet sludge samples may require more than 70 g for
complete removal of water. All water must be removed prior to addition of organic solvent
so that the extraction process is efficient.
12.2.1.2 Add 100 mL (±10 mL) of acetone methylene chloride (1:1) to each of the aliquots and mix
thoroughly.
12.2.1.3 Place the %" horn on the ultrasonic probe approximately 1A" below the surface of the
solvent but above the solids layer and pulse at 50% for 3 minutes at full power. If
necessary, remove the probe from the solution and break any large pieces using a metal
spatula or a stirring rod and repeat the sonication.
12.2.1.4 Decant the extract through a prerinsed drying column containing 7 to 10 cm of granular
anhydrous sodium sulfate into 500- to 1000-mL graduated cylinder.
12.2.1.5 Repeat the extraction steps (Sections 12.2.1.2 to 12.2.1.4) twice more for each sample and
QC aliquot. On the final extraction, swirl the sample or QC aliquot, pour into its
respective drying column, and rinse with acetone:methylene chloride. Record the total
extract volume. If necessary, transfer the extract to a centrifuge tube and centrifuge for
10 minutes to settle fine particles. Concentrate the extract per Section 12.4 to 12.5.
12.2.2 Ultrasonic extraction of high solids sewage sludge aliquots.
12.2.2.1 Add 100 mL (±10 mL) of acetonitrile to each of the aliquots and mix thoroughly.
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Method 1657, Revision A
12.2.2.2 Place the %" horn on the ultrasonic probe approximately 1A" below the surface of the
solvent but above the solids layer and pulse at 50% for 3 minutes at full power. If
necessary, remove the probe from the solution and break any large pieces using a metal
spatula or a stirring rod and repeat the sonication.
12.2.2.3 Decant the extract through filter paper into a 1000- to 2000-mL separatory funnel.
12.2.2.4 Repeat the extraction and filtration steps (Sections 12.2.2.2 to 12.2.2.3) using a second
100 mL (±10 mL) of acetonitrile.
12.2.2.5 Repeat the extraction step (Sections 12.2.2.2 and 12.2.2.2) using 100 mL (±10 mL) of
methylene chloride. On this final extraction, swirl the sample or QC aliquot, pour into its
respective filter paper, and rinse with methylene chloride. Record the total extract volume.
12.2.2.6 For each extract, prepare 1.5 to 2 L of reagent water containing 2% sodium sulfate.
Adjust the pH of the water to 6.0 to 9.0 with NaOH or H2SO4 (Section 7.3.2).
12.2.2.7 Back-extract each extract three times sequentially with 500 mL of the aqueous sodium
sulfate solution, returning the bottom (organic) layer to the separatory funnel the first two
times while discarding the top (aqueous) layer. On the final back-extraction, filter each
pesticide extract through a prerinsed drying column containing 7 to 10 cm of granular
anhydrous sodium sulfate into a 500- to 1000-mL graduated cylinder. Record the final
extract volume.
12.2.3 For extracts to be cleaned up using GPC, filter these extracts through Whatman #41 paper into
a 5 00-mL K-D evaporator flask equipped with a 10-mL concentrator tube. Rinse the graduated
cylinder or centrifuge tube with 30 to 50 mL of methylene chloride and pour through filter to
complete the transfer. Seal and label the K-D flask. Concentrate these fractions per Section
12.4 and 12.5.
12.3 Tissue extraction and determination of lipid content.
12.3.1 Pre-extraction of the Soxhlet apparatus.
12.3.1.1 Charge a clean extraction thimble (Section 6.4.9.2) with 5.0 g of silica (Section 7.6.2).
12.3.1.2 Place the thimble in a clean extractor. Place 30 to 40 mL of methylene chloride :hexane
(1:1) mixture in the receiver and 200 to 250 mL of methylene chloride:hexane (1:1)
mixture in the flask.
12.3.1.3 Pre-extract by heating the flask until the methylene chloride :hexane (1:1) mixture is
boiling. When properly adjusted, one to two drops of methylene chloride:hexane (1:1)
mixture will fall per second from the condenser tip into the receiver. Extract the apparatus
for a minimum of three hours.
12.3.1.4 After pre-extraction, cool and disassemble the apparatus. Rinse the thimble with
methylene chloride :hexane (1:1) mixture and allow to air dry.
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Method 1657, Revision A
12.3.2 Sample extraction.
12.3.2.1 Add 30 to 40 g of powdered anhydrous sodium sulfate (Section 7.3.2) to each sample and
QC aliquot (Sections 11.2.3.2 and 11.2.3.3) and mix thoroughly. Cover the beakers with
aluminum foil and allow to equilibrate for 12-24 hours. Remix prior to extraction to
prevent clumping. If necessary, add more sodium sulfate and re-mix. The sample must
be dehydrated completely for efficient extraction. Complete dehydration is indicated by
a free-flowing sample/sodium sulfate mixture.
12.3.2.2 Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of methylene
chloride :hexane to the reflux flask.
12.3.2.3 Transferthe sample/sodium sulfate mixture (Section 12.3.2.1) to the Soxhlet thimble, and
install the thimble in the Soxhlet apparatus.
12.3.2.4 Rinse the beaker with several portions of solvent mixture and add to the thimble. Fill the
thimble/receiver with solvent. Extract for 18 to 24 hours.
12.3.2.5 After extraction, cool and disassemble the apparatus. Filter each extract through
Whatman #41 paper into a 500-mL K-D evaporator flask equipped with a 10-mL
concentrator tube. Rinse the extraction apparatus with 30 to 50 mL of methylene chloride
and add to the K-D flask.
12.3.3 Percent lipid determination—The lipid content is determined by extraction of tissue with the
same solvent system (methylene chloride :hexane) that was used in EPA's National Dioxin Study
so that lipid contents are consistent with that study.
12.3.3.1 Concentrate the tissue extract to near dryness using the macro concentration procedure in
Section 12.4.
12.3.3.2 Complete the removal of the solvent—Transfer the vial containing the sample extract to
the nitrogen blowdown device (Section 6.4.8.3). Adjust the flow of nitrogen so that the
surface of the solvent is just visibly disturbed. Lower the vial into a water bath
temperature of approximately 60 °C. Weigh the receiver, record the weight, and return
the receiver to the blowdown apparatus, concentrating the residue until a constant weight
is obtained. Record the weight of the receiver, boiling chips, and residue.
12.3.3.3 Redissolve the residue in methylene chloride and quantitatively transfer to a vial for GPC
cleanup (Section 13.2), retaining the boiling chips in the receiver.
12.3.3.4 Allow the receiver and chips to dry and weigh the receiver (including the boiling chips).
12.3.3.5 Calculate the lipid content to the nearest three significant figures as follows:
September 2000 25
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Method 1657, Revision A
Equation 4
WPJOnt Ol TP$1u1/IP \0\
% lipid = g J w x 100
weight of tissue (g)
12.3.3.6 It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.
12.4 Macro concentration.
12.4.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL concentrator
tubes. Add one to two clean boiling chips to the flask and attach a three-ball macro Snyder
column. Prewet the column by adding approximately 1 mL of methylene chloride through the
top. Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of
the flask is bathed with steam. 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.
12.4.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from the
bath and allow the solvent to drain and cool for at least 10 minutes.
12.4.3 If the extract is to be cleaned up using GPC, remove the Snyder column and rinse the flask and
its lower joint into the concentrator tube with 1 to 2 mL of methylene chloride. A 5-mL syringe
is recommended for this operation. Adjust the final volume to 10 mL and proceed to GPC
cleanup in Section 13. If the extract is to be cleaned up by SPE or injected into the GC, proceed
per Section 12.5.
12.5 Solvent exchange—Extracts to be cleaned up by SPE are exchanged into the SPE elution solvent
(Section 7.6.1.2); extracts to be injected into the GC are exchanged in to toluene:acetone (1:10).
12.5.1 Remove the Snyder column, add approximately 10 mL of the appropriate solvent and a clean
boiling chip, and reattach the Snyder column. Concentrate the extract per Section 12 except
use the appropriate solvent to pre-wet the column. The elapsed time for concentration to
approximately 2 mL should be 5 to 10 minutes.
12.5.2 Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with
the respective solvent. Proceed to micro concentration and solvent exchange (Section 12.6).
12.6 Micro-concentration and solvent exchange—Extracts to be cleaned up using SPE are exchanged into
1.0 mL of SPE elution solvent (Section 7.6.1.2). Extracts that have been cleaned up and are ready for
GC analysis are exchanged into toluene:acetone.
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Method 1657, Revision A
12.6.1 Transfer the vial containing the sample extract to the nitrogen blowdown device (Section
6.4.8.3). Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed.
A large vortex in the solvent may cause analyte loss.
12.6.2 Lower the vial into a 45 °C water bath and continue concentrating.
12.6.2.1 If the extract is to be concentrated to dryness for weight determination (Section 12.3.3.2),
blow dry at 60 °C until a constant weight is obtained.
12.6.2.2 If the extract is to be concentrated for injection into the GC or the solvent is to be
exchanged for extract cleanup, proceed as follows:
12.6.3 When the volume of the liquid is approximately 0.5 mL, add 2 to 3 mL of the desired solvent
and continue concentrating to approximately 0.5 mL. Repeat the addition of solvent and
concentrate once more.
12.6.4 Adjustthe volume of an extractto be cleaned up to l.OmL. Proceed to extract cleanup (Section
13.3).
12.6.5 For an extract that was cleaned up and is ready for GC analysis, adjust the volume as follows:
12.6.5.1 Aqueous samples containing <1% solids—Adjustthe final extract volume to be consistent
with the volume extracted.
12.6.5.1.1 For extracts that have been cleaned up using GPC, adjust the final extract volume to
1/200 of the volume extracted. For example, if the volume extracted is 950 mL, adjust
the final extract volume to 4.8 mL.
12.6.5.1.2 For extracts that have not been cleaned up using GPC, adjust the final extract volume
to 1/100 of the volume extracted. For example, if the volume extracted is 1000 mL,
adjust the final extract volume to 10 mL.
NOTE: The difference in volume fractions (1/200 or 1/100) accounts for the 50% loss in the GPC cleanup.
Also, by preserving the ratio between the volume extracted and the final extract volume, the concentrations,
detection limits, and minimum levels ofquantitation do not need to be adjusted for differences in the volume
extracted.
12.6.5.2 For aqueous samples containing >1% solids, soil, sludge, and tissue samples, adjust the
final extract volume to 5.0 mL if the extract was cleaned up by GPC; otherwise, adjust the
volume to 10 mL (the difference accounts for the 50% loss in the GPC cleanup).
12.6.6 Transfer the concentrated extractto a vial with fluoropolymer-lined cap. Seal the vial and label
with the sample number. Store in the dark at room temperature until ready for GC analysis.
If GC analysis will not be performed on the same day, store the vial in the dark at <-10 °C.
Analyze the extract by GC per the procedure in Section 14.
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Method 1657, Revision A
13.0 Extract Cleanup
13.1 Except for tissues, soils, and sediments cleanup procedures may not be necessary. If particular
circumstances require the use of a cleanup procedure, the analyst may use any or all of the procedures
below or any other appropriate procedure. However, the analyst shall first repeat the tests in Section
9.2 to demonstrate that the requirements of Section 9.2 can be met using the cleanup procedure(s) as
an integral part of this Method. Figure 1 outlines the cleanup steps.
13.1.1 Gel permeation chromatography (Section 13.2) removes many high molecular weight
interferences that cause GC column performance to degrade. It is used for all soil, sediment,
and tissue extracts and may be used for water extracts that are expected to contain high
molecular weight organic analytes (e.g., polymeric materials, humic acids).
13.1.2 The solid-phase extraction cartridge (Section 13.3) removes polar organic analytes such as
phenols.
13.2 Gel permeation chromatography (GPC).
13.2.1 Column packing.
13.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
13.2.1.2 Coverthe beads with methylene chloride and allow to swell overnight (12 hours minimum).
13.2.1.3 Transfer the swelled beads to the column and pump solvent through the column, from
bottom to top, at 4.5 to 5.5 mL/min prior to connecting the column to the detector.
13.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column head pressure
to 7 to 10 psig, and purge for 4 to 5 hours to remove air. Maintain a head pressure of 7
to 10 psig. Connect the column to the detector.
13.2.2 Column calibration.
13.2.2.1 Load 5 mL of the calibration solution (Section 7.5) into the sample loop.
13.2.2.2 Inject the calibration solution and record the signal from the detector. The elution pattern
will be corn oil, bis(2-ethylhexyl) phthalate, pentachlorophenol, perylene, and sulfur.
13.2.2.3 Set the "dump time" to allow >85% removal of the corn oil and >85% collection of the
phthalate.
13.2.2.4 Set the "collect time" to the peak minimum between perylene and sulfur.
13.2.2.5 Verify the calibration with the calibration solution after every 20 extracts. Calibration is
verified if the recovery of the pentachlorophenol is greater than 85%. If calibration is not
verified, the system shall be recalibrated using the calibration solution, and the previous
28 September 2000
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Method 1657, Revision A
batch of samples (those affected by the calibration) shall be re-extracted and cleaned up
using a calibrated GPC system.
13.2.3 Extract cleanup—GPC requires that the column not be overloaded. The column specified in
this Method is designed to handle a maximum of 0.5 g of high molecular weight material in a
5-mL extract. If the extract is known or expected to contain more than 0.5 g, the extract is split
into fractions for GPC and the fractions are combined after elution from the column. The solids
content of the extract may be obtained gravimetrically by evaporating the solvent from a 5Q-yL
aliquot.
13.2.3.1 Filter the extract or load through the filter holder to remove particulate. Load the 5.0 mL
extract onto the column.
13.2.3.2 Elute the extract using the calibration data determined in Section 13.2.2. Collect the eluate
in a clean 400- to 500-mL beaker.
13.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between extracts to
prepare for the next sample.
13.2.3.4 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride blank shall be
run through the system to check for carry-over.
13.2.3.5 Concentrate the extract and exchange into hexane per Sections 12.4 and 12.5. Adjust the
final volume to 5.0 mL.
13.3 Solid-phase extraction (SPE).
13.3.1 Setup.
13.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with the trap and
gauge installed between the manifold and vacuum source.
13.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and adjust the
vacuum to 5 to 10 psig.
13.3.2 Cartridge washing—Pre-elute each cartridge prior to use sequentially with 10-mL portion each
of hexane, methanol, and water using vacuum for 30 seconds after each eluant. Follow this
pre-elution with 1 mL methylene chloride and three 10-mL portions of the elution solvent
(Section 7.6.1.2) using vacuum for 5 minutes after each eluant. Tap the cartridge lightly while
under vacuum to dry between eluants. The three portions of elution solvent may be collected
and used as a blank if desired. Finally, elute the cartridge with 10 mL each of methanol and
water, using the vacuum for 30 seconds after each eluant.
13.3.3 Cartridge certification—Each cartridge lot must be certified to ensure recovery of the analytes
of interest and removal of 2,4,6-trichlorophenol.
September 2000 29
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Method 1657, Revision A
13.3.3.1 To make the test mixture, add the trichlorophenol solution (Section 7.6.1.1) to the
combined calibration standard (Section 7.5). Elute the mixture using the procedure in
Section 13.3.4.
13.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 yL of the concentrated eluant into the GC
using the procedure in Section 13. The recovery of all analytes (including the unresolved
GC peaks) shall be within the ranges for recovery specified in Table 4, and the peak for
trichlorophenol shall not be detectable; otherwise the SPE cartridge is not performing
properly and the cartridge lot shall be rejected.
13.3.4 Extract cleanup.
13.3.4.1 After cartridge washing (Section 13.3.2), release the vacuum and place the rack containing
the 50 mL volumetric flasks (Section 6.7.3) in the vacuum manifold. Reestablish the
vacuum at 5 to 10 psig.
13.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract (Section 12.5.4) to the SPE
cartridge. Apply vacuum for five minutes to dry the cartridge. Tap gently to aid in drying.
13.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL portions of the
elution solvent (Section 7.6.1.2), using vacuum for 5 minutes after each portion. Collect
the eluants in the 50-mL volumetric flasks.
13.3.4.4 Release the vacuum and remove the 50-mL volumetric flasks.
13.3.4.5 Concentrate the eluted extracts per Sections 12.4 and 12.5.
14.0 Gas Chromatography
Table 2 summarizes the recommended operating conditions for the gas chromatography. Included in
this table are the retention times and estimated detection limits that can be achieved under these
conditions. Examples of the separations achieved by the primary and confirmatory columns are shown
in Figure 2.
14.1 Calibrate the system as described in Section 10.
14.2 Set the auto sampler to inject the same volume that was chosen for calibration (Section 10.3) for all
standards and extracts of blanks and samples.
14.3 Set the data system or GC control to start the temperature program upon sample injection, and begin
data collection after the solvent peak elutes. Set the data system to stop data collection after the last
analyte is expected to elute and to return the column to the initial temperature.
15.0 System and Laboratory Performance
15.1 At the beginning of each 12-hour shift during which analyses are performed, GC system performance
and calibration are verified for all pollutants and surrogates on both column/detector systems. Forthese
30 September 2000
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Method 1657, Revision A
tests, analysis of the combined QC standard (Section 10.4) shall be used to verify all performance
criteria. Adjustment and/or recalibration (per Section 10) shall be performed until all performance
criteria are met. Only after all performance criteria are met may samples, blanks, and precision and
recovery standards be analyzed.
15.2 Retention times—The absolute retention times of the peak maxima shall be within ±10 seconds of the
retention times in the initial calibration (Section 10.4.1).
15.3 GC resolution—Resolution is acceptable if the valley height between two peaks (as measured from the
baseline) is less than 10% of the taller of the two peaks.
15.3.1 Primary column (DB-1)—Malathion and ethyl parathion.
15.3.2 Confirmatory column (DB-1701)—Terbufos and diazinon.
15.4 Calibration verification—Calibration is verified for the combined QC standard only.
15.4.1 Inject the combined QC standard (Section 7.13).
15.4.2 Compute the percent recovery of each analyte or coeluting analytes, based on the calibration
data (Section 10.4).
15.4.3 For each analyte or coeluted analytes, compare this calibration verification recovery with the
corresponding limits for ongoing recovery in Table 4. For coeluting analytes, use the coeluted
analyte with the least restrictive specification (the widest range). If the recoveries for all
analytes meet the acceptance criteria, system performance is acceptable and analysis of blanks
and samples may begin. If, however, any recovery falls outside the calibration verification
range, system performance is unacceptable for that analyte. In this case, correct the problem
and repeat the test, or recalibrate (Section 10).
15.5 Ongoing precision and recovery.
15.5.1 Analyze the extract of the OPR extracted with each sample batch (Sections 11.2.1.3.3,
11.2.2.4.3, 11.2.3.3.3).
15.5.2 Compute the percent recovery of each analyte and coeluting analytes.
15.5.3 For each analyte or coeluted analytes, compare the percent recovery with the limits for ongoing
recovery in Table 4. For coeluted analytes, use the coeluted analyte with the least restrictive
specification (widest range). If all analytes pass, the extraction, concentration, and cleanup
processes are in control and analysis of blanks and samples may proceed. If, however, any of
the analytes fail, these processes are not in control. In this event, correct the problem, re-extract
the sample batch, and repeat the ongoing precision and recovery test.
15.5.4 If desired, add results that pass the specifications in Section 15.5.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by calculating
September 2000 31
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Method 1657, Revision A
the average percent recovery (R) and the standard deviation of percent recovery sr. Express the
accuracy as a recovery interval from R - 2sr to R + 2sr. For example, if R = 95% and sr = 5%,
the accuracy is 85 to 105%.
16.0 Qualitative Determination
16.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or blank
with data from analysis of the shift standard (Section 15.1), and with data stored in the retention-time
and calibration libraries (Section 10.4.3). Identification is confirmed when retention time and amounts
agree per the criteria below.
16.2 For each analyte on each column/detector system, establish a retention-time window ±20 seconds on
either side of the retention time in the calibration data (Section 10.4). For analytes that have a
retention-time curve (Section 10.3.2.2), establish this window as the minimum -20 seconds and
maximum +20 seconds.
16.2.1 Analytes not requiring a retention-time calibration curve—If a peak from the analysis of a
sample or blank is within a window (as defined in Section 16.2) on the primary column/detector
system, it is considered tentatively identified. A tentatively identified analyte is confirmed when
(1) the retention time for the analyte on the confirmatory column/detector system is within the
retention-time window on that system, and (2) the computed amounts (Section 17) on each
system (primary and confirmatory) agree within a factor of 2.
16.2.2 Analytes requiring a retention-time calibration curve—If a peak from the analysis of a sample
or blank is within a window (as defined in Section 16.2) on the primary column/detector system,
it is considered tentatively identified. A tentatively identified analyte is confirmed when (1) the
retention times on both systems (primary and confirmatory) are within ±30 seconds of the
retention times for the computed amounts (Section 17), as determined by the retention-time
calibration curve (Section 10.3.3.2), and (2) the computed amounts (Section 17) on each system
(primary and confirmatory) agree within a factor of 2.
16.2.3 When the concentration of an analyte is sufficient, it should be further identified by gas
chromatography/mass spectrometry (GC/MS).
16.2.4 Additional information that may aid the laboratory in the identification of an analyte—The
occurrence of peaks eluting near the retention time of an analyte of interest increases the
probability of a false positive for the analyte. If the concentration is insufficient for
confirmation by GC/MS, the laboratory must use all of the cleanup procedures in this Method
(Section 18.2) to attempt to remove the interferences. After all attempts at cleanup are
exhausted, the following steps may be helpful to assure that the substance that appears in the
RT windows on both columns is the analyte of interest.
16.2.4.1 Determine the consistency of the RT data for the analyte on each column. For example,
if the RT is very stable (i.e., varies by no more than a few seconds) for the VER, BLK,
OPR, and MS, the RT for the analyte of interest in the sample should be within this
variation regardless of the window established in Section 16.2. If the analyte is not within
this variation on both columns, it is likely not present.
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Method 1657, Revision A
16.2.4.2 The possibility exists that the RT for the analyte in a sample could shift if extraneous
materials are present. This possibility can be confirmed or refuted by the behavior of the
surrogates in the sample. If the RTs for the surrogates on both columns are consistent
with their RTs in the VER, BLK, OPR and MS data, the RT for the analyte of interest
cannot shift.
16.2.4.3 If the RT for the analyte is shifted slightly later on one column and earlier on the other, and
the surrogates have not shifted, it is highly unlikely that the pesticide is present because
shifts nearly always occur in the same direction on both columns.
17.0 Quantitative Determination
17.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in //g/niL)
using the calibration factor or calibration curve (Section 10.3.2.2).
17.2 Liquid samples
17.2.1 Compute the concentration in the sample using the following equation:
Equation 5
c .,„£=>
where Cs = Concentration in the sample, in jUg/L
Vex = Final extract total volume, in mL
Cex = Concentration in the extract, in jUg/mL
Vs = Sample extracted, in L
17.2.2. Samples containing <1% solids - If the final extract volume was adjusted to be consistent
with the sample volume extracted (Section 12.6.5.1), adjust the concentrations of the
surrogate and matrix spike compounds for the sample volume (Section 12.6.5.1). For
example, if the sample volume was 950mL, adjust the concentration of the surrogates and
matrix spike compounds by multiplying the concentration by 1000/950.
17.3 Solid samples—Compute the concentration in the solid phase of the sample using the following
equation:
September 2000 33
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Method 1657, Revision A
Equation 6
C =,0 _ _
1000 (W) (solids)
where
Cs = Concentration in the sample, in (Jg/kg
Vex= Final extract total volume, in mL
Cex= Concentration in the extract, in (jg/mL
1000 = Conversion factor, g to kg
Ws = Sample weight, in g
_ solids = Percent solids in Section 12 divided by 100
17.4 Tissue samples.
17.4 .1 Total sample—calculate the concentration in the total tissue sample using the following
equation:
Equation 7
ws
where
Cs = Concentration in the sample, in
Ve = Extract volume, in mL
Cex = Concentration in the extract, in
Ws = Sample weight, in g
17.4 .2 Concentration in lipid fraction—Calculate the concentration in the lipid fraction using the
following equation:
Equation 8
100C
c i
where
Cl = concentration in lipid fraction, in /u,g/g
Cs = total concentration (from Equation 6)
%lipid = the percent lipid content
34 September 2000
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Method 1657, Revision A
17.5 If the concentration of any analyte exceeds the calibration range of the system, the extract is diluted
by a factor of 10, and an aliquot of the diluted extract is analyzed.
17.6 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at which the
concentration is in the calibration range.
17.7 The laboratory must report the lower value from the two columns. The laboratory must analyze the
sample by GC/MS or on a third column when analytes have co-eluted or interfere with
determination on one of the two columns used in the analysis.
17.8 Quantitative information that may aid in the confirmation of the presence of an analyte
17.8.1 Sections 16.2.1 and 16.2.2 state that if the amounts on both column/detector systems agree
within a factor of 2, the pesticide is present. This factor of 2 is generous and allows for the
pesticide that has the largest measurement error. A better indication of the actual
measurement error can be gained from the IPR and VER data. Using these data, establish a
measurement window ±3 standard deviations (or ±3 RSD) around the mean on one column.
If the concentration for the pesticide determined on the other column/detector system agrees
within this window, and the RT data agree (from Section 16), the pesticide is definitely
confirmed.
17.8.2 If the amounts do not agree, and the RT data indicate the presence of the analyte (per
Section 16), it is likely that an interference is present on the column that yielded the highest
result. Look for a separate peak for this interferent on the other column. It should be near
the peak for the analyte of interest, and the area of the peak, when added to the area of the
peak for the analyte of interest, should provide an estimate of what the concentration would
be if the peaks coincided. This concentration should roughly agree with the concentration
determined for the analyte on the column on which the suspected interference occurred. In
this event, the pesticide is confirmed and lowest concentration should be reported.
18.0 Analysis of Complex Sam pies
18.1 Some samples may contain high levels (>1000 ng/L) of the analytes of interest, interfering analytes,
and/or polymeric materials. Some samples may not concentrate to 10 mL (Section 12.5); others
may overload the GC column and/or detector.
18.2 The analyst shall attempt to clean up all complex samples using GPC (Section 13.2), and the SPE
cartridge (Section 13.3). If these techniques do not remove the interfering analytes, the extract is
diluted by a factor of 10 and reanalyzed (Section 17.4).
18.3 Recovery of surrogates—In most samples, surrogate recoveries will be similar to those from reagent
water or from the high solids reference matrix. If the surrogate recovery is outside the range
September 2000 35
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Method 1657, Revision A
specified in Section 9.3.3, the sample shall be re-extracted and reanalyzed. If the surrogate recovery
is still outside this range, the sample is diluted by a factor of 10 and reanalyzed (Section 17.4).
18.4 Recovery of matrix spikes—In most samples, matrix spike recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the matrix spike recovery is outside the
range specified in Table 4, the sample shall be diluted by a factor of 10, respiked, and reanalyzed.
If the matrix spike recovery is still outside the range, this Method may not apply to the sample being
analyzed and the result may not be reported or used for permitting or regulatory compliance
purposes. However, a failure to meet the performance criteria in this Method does not relieve a
discharger from timely reporting.
19.0 Method Performance
19.1 Development of this Method is detailed in References 9 and 10.
20.0 Pollution Prevention
20.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity
of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory
operations. EPA has established a preferred hierarchy of environmental management techniques
that places pollution prevention as the management option of first choice. Whenever feasible,
laboratory personnel should use pollution prevention techniques to address waste generation. When
wastes cannot be reduced feasiblely at the source, the Agency recommends recycling as the next
best option. The solvents used in this Method should be reused as practicable by purifying by
distillation. The only other chemicals used in this Method are the neat materials used in preparing
standards. These standards are used in extremely small amounts and pose little threat to the
environment when managed properly. Standards should be prepared in volumes consistent with
laboratory use to minimize the disposal of excess volumes of expired standards.
20.2 For information about pollution prevention that may be applied to laboratories and research
institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction,
available from the American Chemical Society's Department of Governmental Relations and
Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-4477.
21.0 Waste Management
21.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly hazardous waste identification rules and land disposal restrictions,
and for protecting the air, water, and land by minimizing and controlling all releases from fume
hoods and bench operations. Compliance with all sewage discharge permits and regulations is also
required. An overview of requirements can be found in Environmental Management Guide for
Small Laboratories (EPA 233-B-98-001).
21.2 Acids, bases, and samples at pH <2 must be neutralized before being disposed of, or must be
handled as hazardous waste.
36 September 2000
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Method 1657, Revision A
21.3 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste
Reduction, both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
22.0 References
1. "Guideline Establishing Test Procedures for the Analysis of Pollutants under the Clean Water Act;
Final Rule and Interim Final Rule and Proposed Rule," 40 CFR Part 136.
2. "Methods for the Determination of Organic Analytes in Drinking Water," U.S. Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio: EPA-600/4-
88/039, December 1988.
3. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare; Public
Health Service; Center for Disease Control; National Institute for Occupational Health and Safety:
Publication 77-206, August 1977.
4. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety and
Health Administration: January 1976.
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on Chemical
Safety: 1979.
6. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-79-019,
March 1979.
7. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society for
Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
8. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/4-70-020, March
1979.
9. "Consolidated GC Method for the Determination of ITD/RCRA Pesticides using Selective GC
Detectors," S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref 32145-01,
Document R70, September 1986.
10. "Method Development and Validation, EPA Method 1618," Pesticide Center, Department of
Environmental Health, Colorado State University: November 1988, January 1989, and March
1992.
September 2000 37
-------
Method 1657, Revision A
23.0 Tables
Table 1 . Organo-Phosphorus Pesticides Determined by Large-Bore,
Chromatography
EPA EGD
468
453
461
469
443
479
471
460
450
455
449
452
458
467
463
446
454
447
464
474
475
456
444
470
459
448
457
465
473
477
476
with Flame Photometric Detector
Analyte
Acephate
Azinphos ethyl
Azinphos methyl
Chlorfevinphos
Chlorpyrifos
Coumaphos
Crotoxyphos
DBF
Demeton
Diazinon
Dichlofenthion
Dichlorvos
Dicrotophos
Dimethoate
Dioxathion
Disulfoton
EPN
Ethion
Ethoprop
Famphur
Fensulfothion
Fenthion
Hexamethylphosphoramide
Leptophos
Malathion
Merphos *
Methamidophos
Methyl chlorpyrifos
Methyl parathion
Methyl trithion
Mevinphos
Monocrotophos
Naled
Parathion (ethyl)
Phorate
Phosmet
Phosphamidon
Ronnel
Sulfotepp
Sulprofos (Bolstar)
TEPP
Fused-Silica Capillary Column Gas
CAS Registry
30560-19-1
2642-71-9
86-50-0
470-90-6
2921-88-2
56-72-4
7700-17-6
78-48-8
8065-48-3
333-41-5
97-17-6
62-73-7
141-66-2
60-51-5
78-34-2
298-04-4
2104-64-5
563-12-2
13194-48-4
52-85-7
115-90-2
55-38-9
680-31-9
21609-90-5
121-75-5
150-50-5
10265-92-6
5598-13-0
298-00-0
953-17-3
7786-34-7
6923-22-4
300-76-5
56-38-2
298-02-2
732-11-6
13171-21-6
299-84-3
3689-24-5
35400-43-2
107-49-3
38
September 2000
-------
Method 1657, Revision A
Table 1. Organo-Phosphorus Pesticides Determined by Large-Bore, Fused-Silica Capillary Column Gas
Chromatography with Flame Photometric Detector
EPA EGD Analyte CAS Registry
472 Terbufos 13071-79-9
466 Tetrachlorvinphos 22248-79-9
Tokuthion 34643-46-4
445 Trichlorfon 52-68-6
Trichloronate 327-98-0
451 Tricresylphosphate 78-30-8
462 Trimethylphosphate 512-56-1
This analyte will convert to DBF in the very hot GC injection port.
September 2000 39
-------
Method 1657, Revision A
Table 2.
EPA
EGD
450
444
445
471
459
455
470
477
457
449
452
472
473
458
460
456
475
447
448
469
461
479
466
454
Gas Chromatography of Organo-Phosphorus Pesticides
Retention Time 1 (min)
Analyte
DB-1
Dichlorvos 6.56
Mevinphos 11.85
Acephate 12.60
Trichlorofon 12.69
Methamidophos 15.10
Demeton-A 17.70
Ethoprop 18.49
Naled 18.92
Dicrotophos 19.33
Monocrotophos 19.62
Sulfotepp 20.04
Phorate 20.12
Dimethoate 20.59
Demeton-B 21.40
Dioxathion 22.24
Terbufos 22.97
Phosphamidon-E 23.70
Disulfoton 23.89
Diazinon 24.03
Tributyl phosphate (surr) 24.50
Phosphamidon-Z 25.88
Methyl parathion 25.98
Dichlorofenthion 26.11
Methyl chlorpyrifos 26.29
Ronnel 27.33
Malathion 28.87
Fenthion 29.14
Parathion 29.29
Chlorpyrifos 29.48
Trichloronate 30.44
Chlorfevinphos 32.05
Crotoxyphos 32.65
Tokuthion 33.30
Tetrachlorvinphos 33.40
DEF 34.05
Merphos-B 35.16
Fensulfothion 36.58
Methyl trithion 36.62
MDL "' J
DB-1 701 (ng/L)
9.22 4
16.20 74
17.40 500
18.85 1503
19.20 100
20.57 19
21.43 7
23.00 18
26.30 81
29.24 85
23.68 6
23.08 10
29.29 27
25.52 21
26.70 121
24.55 26
29.89 28
27.01 32
26.10 38
17.20
32.62 116
32.12 18
28.66 6
29.53 13
30.09 11
33.49 11
32.16 22
34.61 10
32.15 4
32.12 14
36.08 2
37.58 81
37.17 2
37.85 12
37.50 50
37.37 18
43.86 104
40.52 10
40
September 2000
-------
Method 1657, Revision A
Table 2.
Gas Chromatography of Organo-Phosphorus Pesticides
Retention Time1 (min)
EPA Analyte
EGD
463 Ethion
Sulprofos
446 Famphur
465 Phosmet
467 EPN
453 Azinphos methyl
474 Leptophos
468 Azinphos ethyl
Triphenyl phosphate (surr)
443 Coumaphos
DB-1
37.61
38.10
38.24
41.24
41.94
43.33
44.32
45.55
47.68
48.02
\DB-1 701
41.67
41.74
46.37
48.22
47.52
50.26
47.36
51.88
40.43
56.44
MDL23
(ng/L)
13
6
27
14
9
9
14
22
24
Notes:
1. Columns: 30 m long x 0.53 mm ID; DB-1: 1.5 \a; DB-1701: 1.0 \i. Conditions suggested to meet
retention times shown: 110°C for 0.5 min, 110 to 250° at 3 °C/min, 250°C until coumaphos elutes.
Carrier gas flow rate approximately 7 mL/min.
2. 40 CFR Part 136, Appendix B.
3. Estimated. Detection limits for soils (in ng/kg) are estimated to be 30 to 100 times this level.
September 2000
41
-------
Method 1657, Revision A
Table 3. Concentrations of Calibration Solutions
Concentration ([ig/mL)
EPA EGD
Calibration
453
450
458
447
Calibration
461
469
471
449
446
474
456
445
451
Calibration
468
479
454
444
459
448
465
Analyte
Group 1
Azinphos methyl
Dichlorvos
Dicrotophose
Disulfoton
Fenthion
Hexamethyl phosphoramide
Merphos-A
Merphos-B
Methyl trithion
Ronnel
Sulprofos
Trimethyl phosphate
Group 2
Chlorfevinphos
Chlorpyrifos
Demeton-A
Demeton-B
Dichlofenthion
Dimethoate
Famphur
Leptophos
Methyl parathion
Trichlorofon
Tricre sylphosphate
Tokothion
Group 3
Azinphos ethyl
Crotoxyphos
DBF
Fensulfothion
Methyl chlorpyrifos
Mevinphos
Naled
Parathion
Phosmet
Low
0.1
0.5
0.5
0.2
0.2
0.2
0.2
0.2
0.5
0.2
0.2
1.0
0.2
0.2
0.2
0.2
0.2
0.1
0.5
0.2
0.2
0.5
1.0
0.2
0.2
0.5
0.2
0.5
0.2
0.5
0.5
0.2
0.5
Medium
0.5
2.5
2.5
1.0
1.0
1.0
1.0
1.0
2.5
1.0
1.0
5.0
1.0
1.0
1.0
1.0
1.0
0.5
2.5
1.0
1.0
2.5
5
1.0
1.0
2.5
1.0
2.5
1.0
2.5
2.5
1.0
2.5
High
2.0
10.0
10.0
4.0
4.0
4.0
4.0
4.0
10.0
4.0
4.0
20.0
4.0
4.0
4.0
4.0
4.0
2.0
10.0
4.0
4.0
10.0
20.0
4.0
4.0
10.0
4.0
10.0
4.0
10.0
10.0
4.0
10.0
42
September 2000
-------
Method 1657, Revision A
Table 3. Concentrations of Calibration Solutions
Concentration ([ig/mL)
EPA EGD
473
477
472
Calibration
443
460
467
463
475
457
466
Analyte
Phosphamidon-E
Phosphamidon-Z
Sulfotepp
TEPP
Terbufos
Group 4
Coumaphos
Diazinon
Dioxathion
EPN
Ethion
Ethoprop
Malathion
Methamidophos
Monocrotophos
Phorate
Tetrachlorvinphos
Trichloronate
Low
0.5
0.5
0.2
2.5
0.2
0.5
0.2
0.5
0.2
0.2
0.2
0.2
0.5
1.0
0.2
0.2
0.2
Medium
2.5
2.5
1.0
12.5
1.0
2.5
1.0
2.5
1.0
1.0
1.0
1.0
2.5
5.0
1.0
1.0
1.0
High
10.0
10.0
4.0
50.0
4.0
10.0
4.0
10.0
4.0
4.0
4.0
4.0
10.0
40.0
4.0
4.0
4.0
For analytes listed in Table 2 that are not listed in this table, determine appropriate ranges for
calibration standards.
September 2000
43
-------
Method 1657, Revision A
Table 4.
Acceptance Criteria for Performance Tests for Organo-Phosphorus Analytes
Acceptance Criteria
EGD No. Analyte
468
453
461
469
443
479
471
460
450
455
449
452
458
467
463
446
454
447
464
474
475
456
444
470
459
44
Acephate
Azinphos ethyl
Azinphos methyl
Chlorfevinphos
Chlorpyrifos
Coumaphos
Crotoxyphos
DBF
Demeton
Diazinon
Dichlofenthion
Dichlorvos
Dicrotophos
Dimethoate
Dioxathion
Disulfoton
EPN
Ethion
Ethoprop
Famphur
Fensulfothion
Fenthion
Hexamethylphos-
phoramide
Leptophos
Malathion
Merphos-B
Methamidophos :
Methyl chlorpyrifos
Methyl parathion
Methyl trithion
Mevinphos
Monocrotophos
Naled
Initial Precision
Spike and Accuracy
Level in (%)
the extract
(ng/mL) s
50000 40
10 40
5 40
10 40
10 40
25 40
25 46
10 40
10 40
10 40
10 40
25 40
X
32-140
60-140
52-140
56-140
60-140
60-140
28-140
45-140
33-140
60-140
60-140
52-140
not recovered
5 89
40
10 40
10 40
10 40
10 40
25 40
25 65
10 40
27-140
59-140
46-140
60-140
60-140
60-140
60-140
13-140
60-140
not recovered
10 40
10 40
10 40
10000 40
10 40
10 40
25 40
25 40
60-140
60-140
60-140
60-140
60-140
60-140
21-140
24-140
not recovered
25 40
0-148
Calibration
Verification
(%)
68-132
70-130
70-130
70-130
70-130
70-130
68-136
68-132
64-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
42-139
70-137
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-135
19-206
70-130
Recovery/
Ongoing
Accuracy,
R(%)
28-150
50-150
37-150
37-150
48-150
50-150
6-150
42-150
16-150
50-150
50-150
39-150
22-150
49-150
33-150
50-150
47-150
50-150
50-150
0-150
50-150
50-150
50-150
50-150
50-150
50-150
50-150
0-166
7-150
0-176
September 2000
-------
Method 1657, Revision A
Table 4. Acceptance Criteria for Performance
Spike
Level in
the extrac
EGD No. Analyte (ng/mL)
448
457
465
473
477
476
472
466
445
451
462
Parathion
Phorate
Phosmet
Phosphamidon-Z
Ronnel
Sulfotepp
Sulprofos
TEPP
Terbufos
Tetrachlorvinphos
Tokuthion
Trichlorofon 2
Trichloronate
Tricre sylphosphate
Trimethylphosphate
10
10
25
25
10
10
10
10
10
100
25
10
50
Tests for Organo-Phosphorus Analytes
Acceptance Criteria
Initial Precision
and Accuracy
(%)
t
s
40
40
40
45
40
X
60-140
54-140
44-140
0-140
60-140
40 60-140
40 60-140
not recovered
40
40
40
42
40
60-140
48-140
60-140
43-195
60-140
40 60-140
not recovered
Calibration
Verification
(%)
70-130
70-130
61-159
70-130
70-130
70-130
70-130
70-130
70-130
70-130
70-130
58-142
70-130
70-130
70-130
Recovery/
Ongoing
Accuracy,
R(%)
50-150
43-150
25-150
0-150
50-150
50-150
50-150
47-150
32-150
50-150
37-201
50-150
50-150
Notes:
1. With salt and azeotropic extraction
2. With salt
September 2000
45
-------
Method 1657, Revision A
Percent Solids
< 30% Solids
> 30% Solids
Oil. To 1% Solids
ACN&CH2C12Sonic
CH2C!2 Liq./Liq. Ext.
H2O Back Extract
Methamidophos
"Normal" Compounds
Add NaCI
CHCI3:(CH3)2CO L/L Ext
Concentrate
To Cleanup
Extraction and Concentration Steps
From Extraction
I
Gel Permeation
Solid-Phase Ext.
1
GC/FPD
Cleanup and Analysis Steps
AS2-O02-B3J
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
46
September 2000
-------
Method 1657, Revision A
(B)4
(A) I
* 1
ffl 9
Is
8- |
3 t
ts
. . 1
I f I
Retention Time (minutes)
s i
I I
Figure 2. Gas Chromatogram of Selected Organo-Phosphorus Compounds
September 2000
47
-------
Method 1657, Revision A
24.0 Glossary
The definitions and purposes below are specific to this Method, but have been conformed to common usage
as much as possible.
24.1 Ambient Water—Waters in the natural environment (e.g., rivers, lakes, streams, and other
receiving waters), as opposed to effluent discharges.
24.2 Analytical Batch—A batch of up to 20 samples that are oxidized with the same batch of reagents
and analyzed during the same 12-hour shift. Each analytical batch must also include an OPR and a
QCS. In addition, MS/MSD samples must be prepared at a frequency of 10% per analytical batch
(one MS/MSD for every 10 samples).
24.3 Intercomparison Study—An exercise in which samples are prepared and split by a reference
laboratory, then analyzed by one or more testing laboratories and the reference laboratory. The
intercomparison, with a reputable laboratory as the reference laboratory, serves as the best test of
the precision and accuracy of the analyses at natural environmental levels.
24.4 Matrix Spike (MS) and Matrix Spike Duplicate (MSD)—Aliquots of an environmental sample to
which known quantities of the analyte(s) of interest is added in the laboratory. The MS and MSD
are analyzed exactly like a sample. Their purpose is to quantify the bias and precision caused by
the sample matrix. The background concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the MS and MSD corrected for these
background concentrations.
24.5 May—This action, activity, or procedural step is allowed but not required.
24.6 May not—This action, activity, or procedural step is prohibited.
24.7 Minimum Level (ML)—The lowest level at which the entire analytical system must give a
recognizable signal and acceptable calibration point for the analyte. It is equivalent to the
concentration of the lowest calibration standard, assuming that all method-specified sample weights,
volumes, and cleanup procedures have been employed. The ML is calculated by multiplying the
MDL by 3.18 and rounding the result to the number nearest to (1, 2, or 5) x 10", where n is an
integer.
24.8 Must—This action, activity, or procedural step is required.
24.9 Quality Control Sample (QCS)—A sample containing analytes at known concentrations. The
QCS is obtained from a source external to the laboratory, or is prepared from a source of standards
different from the source of calibration standards. It is used as an independent check of instrument
calibration.
24.10 Reagent Water—Water demonstrated to be free from analytes at the MDLs of this Method. It is
prepared from 18 MQ ultrapure deionized water starting from a prepurified source. Reagent water
is used to wash bottles, as trip and field blanks, and in the preparation of standards and reagents.
48 September 2000
-------
Method 1657, Revision A
24.11 Regulatory Compliance Limit—A limit on the concentration or amount of a pollutant or
contaminant specified in a nationwide standard, in a permit, or otherwise established by a regulatory
authority.
24.12 Shall—This action, activity, or procedure is required.
24.13 Should—This action, activity, or procedure is suggested, but not required.
24.14 Stock Solution—A solution containing an analyte that is prepared from a reference material
traceable to EPA, NIST, or a source that will attest to the purity and authenticity of the reference
material.
September 2000 49
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