Method 608.3 - Organochlorine Pesticides and 

PCBs by GC/HSD

December 2014
U.S. Environmental Protection Agency

Office of Water

Office of Science and Technology

Engineering and Analysis Division (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA-821-R-14-016

Method 608.3 i December 2014
METHOD 608.3 - ORGANOCHLORINE PESTICIDES AND PCBS BY GC/HSD

1. Scope and Application

1.1 This method is for determination of organochlorine pesticides and polychlorinated biphenyls 

(PCBs) in industrial discharges and other environmental samples by gas chromatography (GC) 

combined with a halogen-specific detector (HSD; e.g., electron capture, electrolytic conductivity), 

as provided under 40 CFR 136.1. This revision is based on a previous protocol (Reference 1), on 

the revision promulgated October 26, 1984 (49 FR 43234), on an inter-laboratory method 

validation study (Reference 2), and on EPA Method 1656 (Reference 16). The analytes that may 

be qualitatively and quantitatively determined using this method and their CAS Registry numbers 

are listed in Table 1.

1.2 This method may be extended to determine the analytes listed in Table 2. However, extraction or 

gas chromatography challenges for some of these analytes may make quantitative determination 

difficult.

1.3 When this method is used to analyze unfamiliar samples for an analyte listed in Table 1 or Table 2, 

analyte identification must be supported by at least one additional qualitative technique. This 

method gives analytical conditions for a second GC column that can be used to confirm and 

quantify measurements.

Additionally, Method 625 provides gas chromatograph/mass spectrometer (GC/MS) conditions 

appropriate for the qualitative confirmation of results for the analytes listed in Tables 1 and 2 using 

the extract produced by this method, and Method 1699 (Reference 18) provides high resolution 

GC/MS conditions for qualitative confirmation of results using the original sample. When such 

methods are used to confirm the identifications of the target analytes, the quantitative results should 

be derived from the procedure with the calibration range and sensitivity that are most appropriate 

for the intended application.

1.4 The large number of analytes in Tables 1 and 2 makes testing difficult if all analytes are determined 

simultaneously. Therefore, it is necessary to determine and perform quality control (QC) tests for 

the “analytes of interest” only. The analytes of interest are those required to be determined by a 

regulatory/control authority or in a permit, or by a client. If a list of analytes is not specified, the 

analytes in Table 1 must be determined, at a minimum, and QC testing must be performed for these 

analytes. The analytes in Table 1 and some of the analytes in Table 2 have been identified as Toxic 

Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants (40 CFR 423, Appendix A). 

1.5 In this revision to Method 608, Chlordane has been listed as the alpha- and gamma- isomers in 

Table 1. Reporting may be by the individual isomers, or as the sum of the concentrations of these

isomers, as requested or required by a regulatory/control authority or in a permit. Technical

Chlordane is listed in Table 2 and may be used in cases where historical reporting has only been

the Technical Chlordane. Toxaphene and the PCBs have been moved from Table 1 to Table 2

(Additional Analytes) to distinguish these analytes from the analytes required in quality control 

tests (Table 1). QC acceptance criteria for Toxaphene and the PCBs have been retained in Table 4

and may continue to be applied if desired, or if these analytes are requested or required by a 

regulatory/control authority or in a permit. Method 1668C (Reference 17) may be useful for 

determination of PCBs as individual chlorinated biphenyl congeners, and Method 1699

(Reference 18) may be useful for determination of the pesticides listed in this method. However, 

at the time of writing of this revision, Methods 1668C and 1699 had not been approved for use at 

40 CFR part 136. 

Method 608.3 1 December 2014
1.6 Method detection limits (MDLs; Reference 3) for the analytes in Tables 1 and some of the analytes 

in Table 2 are listed in those tables. These MDLs were determined in reagent water (Reference 3). 

Advances in analytical technology, particularly the use of capillary (open-tubular) columns, 

allowed laboratories to routinely achieve MDLs for the analytes in this method that are 2 - 10 times 

lower than those in the version promulgated in 1984 (40 FR 43234). The MDL for an analyte in a 

specific wastewater may differ from those listed, depending upon the nature of interferences in the 

sample matrix.

1.6.1 EPA has promulgated this method at 40 CFR Part 136 for use in wastewater compliance 

monitoring under the National Pollutant Discharge Elimination System (NPDES). The data 

reporting practices described in Section 15.2 are focused on such monitoring needs and 

may not be relevant to other uses of the method. 

1.6.2 This method includes “reporting limits” based on EPA’s “minimum level” (ML) concept 

(see the glossary in Section 23). Tables 1 and 2 contain MDL values and ML values for 

many of the analytes. The MDL for an analyte in a specific wastewater may differ from 

those listed in Tables 1 or 2, depending upon the nature of interferences in the sample 

matrix.

1.7 The separatory funnel and continuous liquid-liquid sample extraction and concentration steps in 

this method are essentially the same as those steps in Methods 606, 609, 611, and 612. Thus, a 

single sample may be extracted to measure the analytes included in the scope of each of these 

methods. Samples may also be extracted using a disk-based solid-phase extraction (SPE) 

procedure developed by the 3M Corporation and approved by EPA as an Alternate Test Procedure 

(ATP) for wastewater analyses in 1995 (Reference 20).

1.8 This method is performance-based. It may be modified to improve performance (e.g., to overcome 

interferences or improve the accuracy of results) provided all performance requirements are met.

1.8.1 Examples of allowed method modifications are described at 40 CFR 136.6. Other 

examples of allowed modifications specific to this method are described in Section 8.1.2. 

1.8.2 Any modification beyond those expressly permitted at 40 CFR 136.6 or in Section 8.1.2 of 

this method shall be considered a major modification subject to application and approval of 

an alternate test procedure under 40 CFR 136.4 and 136.5. 

1.8.3 For regulatory compliance, any modification must be demonstrated to produce results 

equivalent or superior to results produced by this method when applied to relevant 

wastewaters (Section 8.1.2).

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

GC/HSD. The laboratory must demonstrate the ability to generate acceptable results with this 

method using the procedure in Section 8.2. 

1.10 Terms and units of measure used in this method are given in the glossary at the end of the method. 

2. Summary of Method

2.1 A measured volume of sample, the amount required to meet an MDL or reporting limit (nominally 

1-L), is extracted with methylene chloride using a separatory funnel, a continuous liquid/liquid 

extractor, or disk-based solid-phase extraction equipment. The extract is dried and concentrated for 

Method 608.3 2 December 2014
cleanup, if required. After cleanup, or if cleanup is not required, the extract is exchanged into an 

appropriate solvent and concentrated to the volume necessary to meet the required compliance or 

detection limit, and analyzed by GC/HSD. 

2.2 Qualitative identification of an analyte in the extract is performed using the retention times on 

dissimilar GC columns. Quantitative analysis is performed using the peak areas or peak heights for 

the analyte on the dissimilar columns with either the external or internal standard technique.

2.3 Florisil®, alumina, a C18 solid-phase cleanup, and an elemental sulfur cleanup procedure are 

provided to aid in elimination of interferences that may be encountered. Other cleanup procedures 

may be used if demonstrated to be effective for the analytes in a wastewater matrix.

3. Contamination and Interferences

3.1 Solvents, reagents, glassware, and other sample processing lab ware may yield artifacts, elevated 

baselines, or matrix interferences causing misinterpretation of chromatograms. All materials used 

in the analysis must be demonstrated free from contamination and interferences by running blanks 

initially and with each extraction batch (samples started through the extraction process in a given 

24-hour period, to a maximum of 20 samples). Specific selection of reagents and purification of 

solvents by distillation in all-glass systems may be required. Where possible, lab ware is cleaned 

by extraction or solvent rinse, or baking in a kiln or oven. All materials used must be routinely 

demonstrated to be free from interferences under the conditions of the analysis by running blanks 

as described in Section 8.5. 

3.2 Glassware must be scrupulously cleaned (Reference 4). Clean all glassware as soon as possible 

after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent 

washing with hot water, and rinses with tap water and reagent water. The glassware should then be 

drained dry, and heated at 400 °C for 15-30 minutes. Some thermally stable materials, such as 

PCBs, may require higher temperatures and longer baking times for removal. Solvent rinses with 

pesticide quality acetone, hexane, or other solvents may be substituted for heating. Volumetric lab 

ware should not be heated excessively or for long periods of time. After drying and cooling, 

glassware should be sealed and stored in a clean environment to prevent accumulation of dust or 

other contaminants. Store inverted or capped with aluminum foil. 

3.3 Interferences by phthalate esters can pose a major problem in pesticide analysis when using the 

electron capture detector. The phthalate esters generally appear in the chromatogram as large late 

eluting peaks, especially in the 15 and 50% fractions from Florisil®. Common flexible plastics 

contain varying amounts of phthalates that may be extracted or leached from such materials during 

laboratory operations. Cross contamination of clean glassware routinely occurs when plastics are 

handled during extraction steps, especially when solvent-wetted surfaces are handled. 

Interferences from phthalates can best be minimized by avoiding use of non-fluoropolymer plastics 

in the laboratory. Exhaustive cleanup of reagents and glassware may be required to eliminate 

background phthalate contamination (References 5 and 6). Interferences from phthalate esters can 

be avoided by using a microcoulometric or electrolytic conductivity detector. 

3.4 Matrix interferences may be caused by contaminants co-extracted from the sample. The extent of 

matrix interferences will vary considerably from source to source, depending upon the nature and 

diversity of the industrial complex or municipality being sampled. Interferences extracted from 

samples high in total organic carbon (TOC) may result in elevated baselines, or by enhancing or 

suppressing a signal at or near the retention time of an analyte of interest. Analyses of the matrix 

spike and duplicate (Section 8.3) may be useful in identifying matrix interferences, and the cleanup 

Method 608.3 3 December 2014
procedures in Section 11 may aid in eliminating these interferences. EPA has provided guidance 

that may aid in overcoming matrix interferences (Reference 7); however, unique samples may 

require additional cleanup approaches to achieve the MDLs listed in Table 3. 

4. Safety

4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined; 

however, each chemical compound should be treated as a potential health hazard. From this 

viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever 

means available. The laboratory is responsible for maintaining a current awareness file of OSHA 

regulations regarding the safe handling of the chemicals specified in this method. A reference file 

of safety data sheets (SDSs, OSHA, 29 CFR 1910.1200[g] ) should also be made available to all 

personnel involved in sample handling and chemical analysis. Additional references to laboratory 

safety are available and have been identified (References 8 and 9) for the information of the 

analyst.

4.2 The following analytes covered by this method have been tentatively classified as known or 

suspected human or mammalian carcinogens: 4,4'-DDT, 4,4'-DDD, the BHCs, and the PCBs. 

Primary standards of these toxic analytes should be prepared in a chemical fume hood, and a 

NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are 

handled. 

4.3 This method allows the use of hydrogen as a carrier gas in place of helium (Section 5.8.2). The 

laboratory should take the necessary precautions in dealing with hydrogen, and should limit 

hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air. 

5. Apparatus and Materials

Note: Brand names and suppliers are for illustration purposes only. No endorsement is implied. 

Equivalent performance may be achieved using equipment and materials other than those 

specified here. Demonstrating that the equipment and supplies used in the laboratory 

achieve the required performance is the responsibility of the laboratory. Suppliers for 

equipment and materials in this method may be found through an on-line search. Please 

do not contact EPA for supplier information. 

5.1 Sampling equipment, for discrete or composite sampling 

5.1.1 Grab sample bottle—amber glass bottle large enough to contain the necessary sample

volume (nominally 1 L), fitted with a fluoropolymer-lined screw cap. Foil may be 

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

available, protect samples from light. Unless pre-cleaned, the bottle and cap liner must be 

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

contamination. 

5.1.2 Automatic sampler (optional)—the sampler must use a glass or fluoropolymer container 

and tubing for sample collection. If the sampler uses a peristaltic pump, a minimum length 

of compressible silicone rubber tubing may be used. Before use, however, the 

compressible tubing should be thoroughly rinsed with methanol, followed by repeated 

rinsing with reagent water to minimize the potential for sample contamination. An 

Method 608.3 4 December 2014
integrating flow meter is required to collect flow proportional composites. The sample 

container must be kept refrigerated at <6 °C and protected from light during compositing. 

5.2. Lab ware

5.2.1 Extraction 

5.2.1.1 pH measurement 

5.2.1.1.1 pH meter, with combination glass electrode 

5.2.1.1.2 pH paper, wide range (Hydrion Papers, or equivalent) 

5.2.1.2 Separatory funnel—Size appropriate to hold the sample and extraction solvent 

volumes, equipped with fluoropolymer stopcock. 

5.2.1.3 Continuous liquid-liquid extractor—Equipped with fluoropolymer or glass 

connecting joints and stopcocks requiring no lubrication. (Hershberg-Wolf 

Extractor, Ace Glass Company, Vineland, NJ, or equivalent.) 

5.2.1.3.1 Round-bottom flask, 500-mL, with heating mantle

5.2.1.3.2 Condenser, Graham, to fit extractor 

5.2.1.4 Solid-phase extractor—90-mm filter apparatus (Figure 2) or multi-position 

manifold 

5.2.1.4.1 Vacuum system—Capable of achieving 0.1 bar (25 inch) Hg (house 

vacuum, vacuum pump, or water aspirator), equipped with shutoff 

valve and vacuum gauge

5.2.1.4.2 Vacuum trap—Made from 500-mL sidearm flask fitted with singlehole rubber stopper and glass tubing 

Note: The approved ATP for solid-phase extraction is limited to disk-based extraction

media and associated peripheral equipment. 

5.2.2 Filtration

5.2.2.1 Glass powder funnel, 125- to 250-mL

5.2.2.2 Filter paper for above, Whatman 41, or equivalent 

5.2.2.3 Prefiltering aids—90-mm 1-μm glass fiber filter or Empore® Filter Aid 400 

5.2.3 Drying column 

5.2.3.1 Chromatographic column—approximately 400 mm long x 15 mm ID, with 

fluoropolymer stopcock and coarse frit filter disc (Kontes or equivalent).

5.2.3.2 Glass wool—Pyrex, extracted with methylene chloride or baked at 450 °C for 1 

hour minimum 

Method 608.3 5 December 2014
5.2.4 Column for Florisil® or alumina cleanup—approximately 300 mm long x 10 mm ID, with 

fluoropolymer stopcock. (This column is not required if cartridges containing Florisil® are 

used.) 

5.2.5 Concentration/evaporation 

Note: Use of a solvent recovery system with the K-D or other solvent evaporation 

apparatus is strongly recommended. 

5.2.5.1 Kuderna-Danish concentrator

5.2.5.1.1 Concentrator tube, Kuderna-Danish—10-mL, graduated (Kontes or 

equivalent). Calibration must be checked at the volumes employed 

for extract volume measurement. A ground-glass stopper is used to 

prevent evaporation of extracts.

5.2.5.1.2 Evaporative flask, Kuderna-Danish—500-mL (Kontes or 

equivalent). Attach to concentrator tube with connectors. 

5.2.5.1.3 Snyder column, Kuderna/Danish—Three-ball macro (Kontes or 

equivalent) 

5.2.5.1.4 Snyder column—Two-ball micro (Kontes or equivalent)

5.2.5.1.5 Water bath—Heated, with concentric ring cover, capable of 

temperature control (± 2 °C), installed in a hood using appropriate 

engineering controls to limit exposure to solvent vapors.

5.2.5.2 Nitrogen evaporation device—Equipped with heated bath that can be maintained 

at an appropriate temperature for the solvent and analytes. 

(N-Evap, Organomation Associates, Inc., or equivalent)

5.2.5.3 Rotary evaporator—Buchi/Brinkman-American Scientific or equivalent, equipped 

with a variable temperature water bath, vacuum source with shutoff valve at the 

evaporator, and vacuum gauge.

5.2.5.2.1 A recirculating water pump and chiller are recommended, as use of 

tap water for cooling the evaporator wastes large volumes of water and 

can lead to inconsistent performance as water temperatures and 

pressures vary.

5.2.5.2.2 Round-bottom flask – 100-mL and 500-mL or larger, with groundglass fitting compatible with the rotary evaporator

Note: This equipment is used to prepare copper foil or copper powder for removing 

sulfur from sample extracts (see Section 6.7.4).

5.2.5.4 Automated concentrator—Equipped with glassware sufficient to concentrate 3-

400 mL extract to a final volume of 1-10 mL under controlled conditions of 

temperature and nitrogen flow (Turbovap, or equivalent). Follow manufacturer’s 

directions and requirements.

Method 608.3 6 December 2014
5.2.5.5 Boiling chips—Glass, silicon carbide, or equivalent, approximately 10/40 mesh. 

Heat at 400 °C for 30 minutes, or solvent rinse or Soxhlet extract with methylene 

chloride.

5.2.5 Solid-phase extraction disks—90-mm extraction disks containing 2 g of 8-μm octadecyl 

(C18) bonded silica uniformly enmeshed in a matrix of inert PTFE fibrils (3M Empore® or 

equivalent). The disks should not contain any organic compounds, either from the PTFE or 

the bonded silica, which will leach into the methylene chloride eluant. One liter of reagent 

water should pass through the disks in 2-5 minutes, using a vacuum of at least 25 inches of 

mercury.

Note: Extraction disks from other manufacturers may be used in this procedure, provided 

that they use the same solid phase materials (i.e., octadecyl bonded silica). Disks of 

other diameters also may be used, but may adversely affect the flow rate of the 

sample through the disk. 

5.3 Vials

5.3.1 Extract storage—10- to 15-mL, amber glass, with fluoropolymer-lined screw cap

5.3.2 GC autosampler—1- to 5-mL, amber glass, with fluoropolymer-lined screw- or crimp-cap, 

to fit GC autosampler

5.4 Balances

5.4.1 Analytical—capable of accurately weighing 0.1 mg

5.4.2 Top loading—capable of weighing 10 mg 

5.5 Sample cleanup

5.5.1 Oven—For baking and storage of adsorbents, capable of maintaining a constant 

temperature (± 5 °C) in the range of 105-250 °C. 

5.5.2 Muffle furnace—Capable of cleaning glassware or baking sodium sulfate in the range of 

400-450 °C. 

5.5.3 Vacuum system and cartridges for solid-phase cleanup (see Section 11.2)

5.5.3.1 Vacuum system—Capable of achieving 0.1 bar (25 in.) Hg (house vacuum, 

vacuum pump, or water aspirator), equipped with shutoff valve and vacuum 

gauge

5.5.3.2 VacElute Manifold (Analytichem International, or equivalent)

5.5.3.3 Vacuum trap—Made from 500-mL sidearm flask fitted with single-hole rubber 

stopper and glass tubing 

5.5.3.4 Rack for holding 50-mL volumetric flasks in the manifold

Method 608.3 7 December 2014
5.5.3.5 Cartridge—Mega Bond Elute, Non-polar, C18 Octadecyl, 10 g/60 mL 

(Analytichem International or equivalent), used for solid-phase cleanup of 

sample extracts (see Section 11.2)

5.5.3.5.1 Cartridge certification—Each cartridge lot must be certified to 

ensure recovery of the analytes of interest and removal of 2,4,6-

trichlorophenol. To make the test mixture, add the trichlorophenol 

solution (Section 6.7.2.1) to the same standard used to prepare the 

Quality Control Check Sample (Section 6.8.3). Transfer the mixture 

to the column and dry the column. Pre-elute with three 10-mL 

portions of elution solvent, drying the column between elutions. 

Elute the cartridge with 10 mL each of methanol and water, as in 

Section 11.2.3.3. 

5.5.3.5.2 Concentrate the eluant to per Section 10.3.3, exchange to isooctane 

or hexane per Section 10.3.3, and inject 1.0 μL of the concentrated 

eluant into the GC using the procedure in Section 12. The recovery 

of all analytes (including the unresolved GC peaks) shall be within 

the ranges for calibration verification (Section 13.6 and 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.

5.5.4 Sulfur removal tube—40- to 50-mL bottle, test tube, or Erlenmeyer flask with 

fluoropolymer-lined screw cap

5.6 Centrifuge apparatus 

5.6.1 Centrifuge—Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes at 

5,000 rpm minimum 

5.6.2 Centrifuge bottle—500-mL, with screw cap, to fit centrifuge 

5.6.3 Centrifuge tube—15-mL, with screw cap, to fit centrifuge 

5.7 Miscellaneous lab ware—graduated cylinders, pipettes, beakers, volumetric flasks, vials, syringes, 

and other lab ware necessary to support the operations in this method 

5.8 Gas chromatograph—Dual-column with simultaneous split/splitless, temperature programmable 

split/splitless (PTV), or on-column injection; temperature program with isothermal holds, and all 

required accessories including syringes, analytical columns, gases, and detectors. An autosampler 

is highly recommended because it injects volumes more reproducibly than manual injection 

techniques. Alternatively, two separate single-column gas chromatographic systems may be 

employed. 

5.8.1 Example columns and operating conditions 

5.8.1.1 DB-608 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 0.83-μm 

film thickness.

5.8.1.2 DB-1701 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 1.0-μm 

film thickness. 

Method 608.3 8 December 2014
5.8.1.3 Suggested operating conditions used to meet the retention times shown in Table 3 

are:

Carrier gas flow rate: approximately 7 mL/min

Initial temperature: 150 °C for 0.5 minute, 

 Temperature program: 150-270 °C at 5 °C/min, and 

 Final temperature: 270 °C, until trans-Permethrin elutes 

Note: Other columns, internal diameters, film thicknesses, and operating conditions 

may be used, provided that the performance requirements in this method are met. 

However, the column pair chosen must have dissimilar phases/chemical 

properties in order to separate the compounds of interest in different retention 

time order. Columns that only differ in the length, ID, or film thickness, but use 

the same stationary phase do not qualify as “dissimilar.” 

5.8.2 Carrier gas—Helium or hydrogen. Data in the tables in this method were obtained using 

helium carrier gas. If hydrogen is used, analytical conditions may need to be adjusted for 

optimum performance, and calibration and all QC tests must be performed with hydrogen 

carrier gas. See Section 4.3 for precautions regarding the use of hydrogen as a carrier gas.

5.8.3 Detector—Halogen-specific detector (electron capture detector (ECD), electrolytic 

conductivity detector (ELCD), or equivalent). The ECD has proven effective in the 

analysis of wastewaters for the analytes listed in Tables 1 and 2, and was used to develop 

the method performance data in Section 17 and Tables 4 and 5. 

5.8.4 Data system—A computer system must be interfaced to the GC that allows continuous 

acquisition and storage of data from the detectors throughout the chromatographic program. 

The computer must have software that allows searching GC data for specific analytes, and 

for plotting responses versus time. Software must also be available that allows integrating 

peak areas or peak heights in selected retention time windows and calculating 

concentrations of the analytes.

6. Reagents and Standards

6.1 pH adjustment

6.1.1 Sodium hydroxide solutions

6.1.1.1 Concentrated (10 M)—Dissolve 40 g of NaOH (ACS) in reagent water and dilute 

to 100 mL. 

6.1.1.2 Dilute (1 M)—Dissolve 40 g NaOH in 1 L of reagent water.

6.1.2 Sulfuric acid (1+1)—Slowly add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent 

water.

6.1.3 Hydrochloric acid—Reagent grade, 6 N 

6.2 Sodium thiosulfate—(ACS) granular.

Method 608.3 9 December 2014
6.3 Sodium sulfate—Sodium sulfate, reagent grade, granular anhydrous (Baker or equivalent), rinsed 

with methylene chloride (20 mL/g), baked in a shallow tray at 450 °C for 1 hour minimum, cooled 

in a desiccator, and stored in a pre-cleaned glass bottle with screw cap which prevents moisture 

from entering. If, after heating, the sodium sulfate develops a noticeable grayish cast (due to the 

presence of carbon in the crystal matrix), that batch of reagent is not suitable for use and should be 

discarded. Extraction with methylene chloride (as opposed to simple rinsing) and baking at a lower 

temperature may produce sodium sulfate suitable for use. 

6.4 Reagent water—Reagent water is defined as water in which the analytes of interest and interfering 

compounds are not observed at the MDLs of the analytes in this method. 

6.5 Solvents—methylene chloride, acetone, methanol, hexane, acetonitrile, and isooctane, high purity 

pesticide quality, or equivalent, demonstrated to be free of the analytes and interferences (Section 

3). Purification of solvents by distillation in all-glass systems may be required.

Note: The standards and final sample extracts must be prepared in the same final solvent.

6.6 Ethyl ether—Nanograde, redistilled in glass if necessary

 Ethyl ether must be shown to be free of peroxides before use, as indicated by EM Laboratories 

Quant test strips (available from Scientific Products Co. and other suppliers). Procedures 

recommended for removal of peroxides are provided with the test strips. After removal of 

peroxides, add 20 mL of ethyl alcohol preservative to each liter of ether.

6.7 Materials for sample cleanup

6.7.1 Florisil®—PR grade (60/100 mesh), activated at 650 - 700 °C, stored in the dark in a glass 

container with fluoropolymer-lined screw cap. Activate each batch immediately prior to 

use for 16 hours minimum at 130 °C in a foil-covered glass container and allow to cool. 

Alternatively, 500 mg cartridges (J.T. Baker, or equivalent) may be used. 

6.7.2 Solutions for solid-phase cleanup

6.7.2.1 SPE cartridge calibration solution—2,4,6-trichlorophenol, 0.1 μg/mL in acetone.

6.7.2.2 SPE elution solvent—methylene chloride:acetonitrile:hexane (50:3:47).

6.7.3 Alumina, neutral, Brockman Activity I, 80-200 mesh (Fisher Scientific certified, or 

equivalent). Heat in a glass bottle for 16 hours at 400 to 450 °C. Seal and cool to room 

temperature. Add 7% (w/w) reagent water and mix for 10 to 12 hours. Keep bottle tightly 

sealed.

6.7.4 Sulfur removal

6.7.4.1 Copper foil or powder—Fisher, Alfa Aesar, or equivalent. Cut copper foil into 

approximately 1-cm squares. Copper must be activated on each day it will be 

used, as described below. 

6.7.4.1.1 Place the quantity of copper needed for sulfur removal (Section 

11.5.1.3) in a ground-glass-stoppered Erlenmeyer flask or bottle. 

Cover the foil or powder with methanol. 

Method 608.3 10 December 2014
6.7.4.1.2 Add HCl dropwise (0.5 - 1.0 mL) while swirling, until the copper 

brightens.

6.7.4.1.3 Pour off the methanol/HCl and rinse 3 times with reagent water to 

remove all traces of acid, then 3 times with acetone, then 3 times 

with hexane.

6.7.4.1.4 For copper foil, cover with hexane after the final rinse. Store in a 

stoppered flask under nitrogen until used. For the powder, dry on a 

rotary evaporator. Store in a stoppered flask under nitrogen until 

used. 

6.7.4.2 Tetrabutylammonium sulfite (TBA sulfite)

6.7.4.2.1 Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHSO4

6.7.4.2.2 Sodium sulfite, Na2SO3

6.7.4.2.3 Dissolve approximately 3 g tetrabutylammonium hydrogen sulfate in 

100 mL of reagent water in an amber bottle with fluoropolymer-lined 

screw cap. Extract with three 20-mL portions of hexane and discard 

the hexane extracts.

6.7.4.2.4 Add 25 g sodium sulfite to produce a saturated solution. Store at 

room temperature. Replace after 1 month.

6.8 Standard solutions—Purchase as solutions or mixtures with certification to their purity, 

concentration, and authenticity, or prepare from materials of known purity and composition. If 

compound purity is 96% or greater, the weight may be used without correction to compute the 

concentration of the standard. Store neat standards or single analyte standards in the dark at -20 to 

-10 °C in screw-cap vials with fluoropolymer-lined caps. Store multi-analyte standards at 4°C or 

per manufacturer’s recommendations. Place a mark on the vial at the level of the solution so that 

solvent evaporation loss can be detected. Bring the vial to room temperature prior to use to redissolve any precipitate.

6.8.1 Stock standard solutions—Standard solutions may be prepared from pure standard 

materials or purchased as certified solutions. Traceability must be to a national standard, 

when available. Except as noted below for solutions spiked into samples, prepare stock 

standards in isooctane or hexane. Observe the safety precautions in Section 4. The 

following procedure may be used to prepare standards from neat materials.

6.8.1.1 Dissolve an appropriate amount of assayed reference material in solvent. For 

example, weigh 10 mg of aldrin in a 10-mL ground-glass-stoppered volumetric 

flask and fill to the mark with isooctane or hexane. Larger volumes may be used 

at the convenience of the laboratory. After the aldrin is completely dissolved, 

transfer the solution to a 15-mL vial with fluoropolymer-lined cap. 

6.8.1.2 Check for signs of degradation prior to preparation of calibration or performancetest standards.

6.8.1.3 Replace stock solutions after 12 months, or sooner if comparison with quality 

control check standards indicates a change in concentration.

Method 608.3 11 December 2014
6.8.2 Calibration solutions—It is necessary to prepare calibration solutions for the analytes of 

interest (Section 1.4) only using an appropriate solvent (isooctane or hexane may be used). 

Whatever solvent is used, both the calibration standards and the final sample extracts must 

use the same solvent. Other analytes may be included as desired. 

6.8.2.1 Prepare calibration standards for the single-component analytes of interest and 

surrogates at a minimum of three concentration levels (five are suggested) by 

adding appropriate volumes of one or more stock standards to volumetric flasks. 

One of the calibration standards should be at a concentration of the analyte near 

the ML in Table 1 or 2. The ML value may be rounded to a whole number that is 

more convenient for preparing the standard, but must not exceed the ML values 

listed in Tables 1 or 2 for those analytes which list ML values. Alternatively, the 

laboratory may establish the ML for each analyte based on the concentration of 

the lowest calibration standard in a series of standards obtained from a 

commercial vendor, again, provided that the ML values does not exceed the MLs 

in Table 1 and 2, and provided that the resulting calibration meets the acceptance 

criteria in Section 7.5.2. based on the RSD, RSE, or R2

. 

The other concentrations should correspond to the expected range of 

concentrations found in real samples or should define the working range of the 

GC system. A minimum of six concentration levels is required for a second 

order, non-linear (e.g., quadratic; ax2

 + bx + c) calibration. Calibrations higher 

than second order are not allowed. 

Given the number of analytes included in this method, it is highly likely that 

some will coelute on one or both of the GC columns used for the analysis. 

Therefore, divide the analytes two or more groups and prepare separate 

calibration standards for each group, at multiple concentrations (e.g., a five-point 

calibration will require ten solutions to cover two groups of analytes).

Note: Many commercially available standards are divided into separate mixtures to 

address this issue.

The other concentrations should correspond to the expected range of 

concentrations found in real samples or should define the working range of the 

GC system. A separate standard near the MDL may be analyzed as a check on 

sensitivity, but should not be included in the linearity assessment. A minimum of 

six concentration levels is required for a non-linear (e.g., quadratic) calibration 

(Section 7.5.2 or 7.6.2). The solvent for the standards must match the final 

solvent for the sample extracts (e.g., isooctane or hexane). 

Note: The option for non-linear calibration may be necessary to address specific 

instrumental techniques. However, it is not EPA's intent to allow non-linear 

calibration to be used to compensate for detector saturation or to avoid proper 

instrument maintenance.

6.8.2.2 Multi-component analytes (e.g., PCBs as Aroclors, and Toxaphene) 

6.8.2.2.1 A standard containing a mixture of Aroclor 1016 and Aroclor 1260 

will include many of the peaks represented in the other Aroclor 

mixtures. As a result, a multi-point initial calibration employing a 

mixture of Aroclors 1016 and 1260 at three to five concentrations 

Method 608.3 12 December 2014
should be sufficient to demonstrate the linearity of the detector 

response without the necessity of performing multi-point initial 

calibrations for each of the seven Aroclors. In addition, such a 

mixture can be used as a standard to demonstrate that a sample does 

not contain peaks that represent any one of the Aroclors. This 

standard can also be used to determine the concentrations of either 

Aroclor 1016 or Aroclor 1260, should they be present in a sample. 

 Therefore, prepare a minimum of three calibration standards 

containing equal concentrations of both Aroclor 1016 and Aroclor 

1260 by dilution of the stock standard with isooctane or hexane. The 

concentrations should correspond to the expected range of 

concentrations found in real samples and should bracket the linear 

range of the detector.

6.8.2.2.2 Single standards of each of the other five Aroclors are required to aid 

the analyst in pattern recognition. Assuming that the Aroclor 

1016/1260 standards described in Section 6.8.2.2.1 have been used to 

demonstrate the linearity of the detector, these single standards of the 

remaining five Aroclors also may be used to determine the 

calibration factor for each Aroclor. Prepare a standard for each of 

the other Aroclors. The concentrations should generally correspond 

to the mid-point of the linear range of the detector, but lower 

concentrations may be employed at the discretion of the analyst 

based on project requirements.

6.8.2.2.3 For Toxaphene, prepare a minimum of three calibration standards 

containing Toxaphene by dilution of the stock standard with 

isooctane or hexane. The concentrations should correspond to the 

expected range of concentrations found in real samples and should 

bracket the linear range of the detector.

6.8.3 Quality Control (QC) Check Sample —Also known as the Laboratory Control Sample 

(LCS). Prepare a mid-level standard mixture in acetone (or water miscible solvent) from a 

stock solution from the same source as the calibration standards. This standard will be used 

to generate extracts to evaluate the capability of the laboratory. 

6.8.4 Second Source Standard—Obtain standards from a second source (different manufacturer 

or different certified lot), and prepare a mid-level standard mixture in isooctane or hexane. 

This standard will be analyzed with the calibration curve to verify the accuracy of the 

calibration. 

6.8.5 Internal standard solution—If the internal standard calibration technique is to be used, 

prepare pentachloronitrobenzene (PCNB) at a concentration of 10 μg/mL in ethyl acetate. 

Alternative and multiple internal standards; e.g., tetrachloro-m-xylene, 4,4'-

dibromobiphenyl, and/or decachlorobiphenyl may be used provided that the laboratory 

performs all QC tests and meets all QC acceptance criteria with the alternate or additional 

internal standard(s) as an integral part of this method.

6.8.6 Surrogate solution—Prepare a solution containing one or more surrogates at a 

concentration of 2 μg/mL in acetone. Potential surrogates include: dibutyl chlorendate 

(DBC), tetrachloro-m-xylene (TCMX), 4,4'-dibromobiphenyl, or decachlorobiphenyl

Method 608.3 13 December 2014
provided that the laboratory performs all QC tests and meets all QC acceptance criteria 

with the alternative surrogate(s) as an integral part of this method. If the internal standard 

calibration technique is used, do not use the internal standard as a surrogate.

6.8.7 DDT and endrin decomposition (breakdown) solution—Prepare a solution containing 

endrin at a concentration of 1 μg/mL and 4,4'-DDT at a concentration of 2 μg/mL, in 

isooctane or hexane. 

6.8.8 Quality control check sample (laboratory control sample; LCS) concentrate—See Sections 

8.2.1 and 8.4.

6.8.9 Stability of solutions—Analyze all standard solutions (Sections 6.8.1 through 6.8.8) within 

48 hours of preparation. Replace purchased certified stock standard solutions per the 

expiration date. Replace stock standard solutions prepared by the laboratory or mixed with 

purchased solutions after one year, or sooner if comparison with QC check samples 

indicates a problem.

7. Calibration 

7.1 Establish gas chromatographic operating conditions equivalent to those in Section 5.8.1 and 

Footnote 2 to Table 3. Alternative temperature program and flow rate conditions may be used. 

The system may be calibrated using the external standard technique (Section 7.5) or the internal 

standard technique (Section 7.6). It is necessary to calibrate the system for the analytes of interest 

(Section 1.4) only. 

7.2 Separately inject the mid-level calibration standard for each calibration mixture. Store the 

retention time on each GC column. 

7.3 Demonstrate that each column/detector system meets the MDLs in Table 3 or demonstrates 

sufficient sensitivity for the intended application and passes the DDT/endrin decomposition test 

(Section 13.5).

7.4 Injection of calibration solutions—Inject a constant volume in the range of 0.5 to 2.0 μL of each 

calibration solution into the GC column/detector pairs. Beginning with the lowest level mixture 

and proceeding to the highest level mixture may limit the risk of carryover from one standard to the 

next, but other sequences may be used. A blank sample should be analyzed after the highest 

standard to demonstrate that there is no carry-over within the system for this calibration range. For 

each analyte, compute, record, and store, as a function of the concentration injected, the retention 

time and peak area on each column/detector system. If multi-component analytes are to be 

analyzed, store the retention time and peak area for the three to five exclusive (unique large) peaks

for each PCB or technical chlordane. Use four to six peaks for toxaphene.

7.5 External standard calibration 

7.5.1 From the calibration data (Section 7.4), calculate the calibration factor (CF) for each 

analyte at each concentration according to the following equation:

CF = 

As

Cs

Method 608.3 14 December 2014
where: 

Cs = Concentration of the analyte in the standard (ng/mL) 

As = Peak height or area

For multi-component analytes, choose a series of characteristic peaks for each analyte (3 to 

5 for each Aroclor, 4 to 6 for toxaphene) and calculate individual calibration factors for 

each peak. Alternatively, for toxaphene, sum the areas of all of the peaks in the standard 

chromatogram and use the summed area to determine the calibration factor. (If this 

alternative is used, the same approach must be used to quantitate the analyte in the 

samples.)

7.5.2 Calculate the mean (average) and relative standard deviation (RSD) of the calibration 

factors. If the RSD is less than 20%, linearity through the origin can be assumed and the 

average CF can be used for calculations. Alternatively, the results can be used to fit a 

linear or quadratic regression of response ratios, As/Ais, vs. concentration ratios Cs/Cis. If 

used, the regression must be weighted inversely proportional to concentration. The 

coefficient of determination (R2

) of the weighted regression must be greater than 0.99. 

Alternatively, the relative standard error (Reference 10) may be used as an acceptance 

criterion. As with the RSD, the RSE must be less than 20%. If an RSE less than 20% 

cannot be achieved for a quadratic regression, system performance is unacceptable and the 

system must be adjusted and re-calibrated. 

Note: Regression calculations are not included in this method because the calculations 

are cumbersome and because many GC/ECD data systems allow selection of 

weighted regression for calibration and calculation of analyte concentrations.

7.6 Internal standard calibration

7.6.1 From the calibration data (Section 7.4), calculate the response factor (RF) for each analyte 

at each concentration according to the following equation: 

RF = (As x Cis)

(Ais x Cs)

where:

As = Response for the analyte to be measured.

Ais = Response for the internal standard. 

Cis = Concentration of the internal standard (ng/mL) 

Cs = Concentration of the analyte to be measured (ng/mL). 

7.6.2 Calculate the mean (average) and relative standard deviation (RSD) of the response factors. 

If the RSD is less than 15%, linearity through the origin can be assumed and the average 

RF can be used for calculations. Alternatively, the results can be used to prepare a 

calibration curve of response ratios, As/Ais, vs. concentration ratios, Cs/Cis, for the analyte. 

A minimum of six concentration levels is required for a non-linear (e.g., quadratic) 

regression. If used, the regression must be weighted inversely proportional to 

concentration, and the correlation coefficient of the weighted regression must be greater 

than 0.99. The relative standard error (Reference 11) may also be used as an acceptance 

criterion. As with the RSD, the RSE must be less than 15%. If an RSE less than 15% 

cannot be achieved for a quadratic regression, system performance is unacceptable and the 

system must be adjusted and re-calibrated.

Method 608.3 15 December 2014
7.7 Second source standard—After the calibration curves are analyzed, analyze a second source 

standard at the mid-level concentration. This standard confirms the accuracy of the calibration 

curve. The concentrations must be within 20% difference of the true value. If the observed 

concentration exceeds this criteria, a third source may be analyzed to determine which standard 

was not accurate, and subsequent corrective actions taken. 

7.8 The working calibration curve, CF, or RF must be verified at the beginning and end of each 24-

hour shift by the analysis of a mid-level calibration standard or the combined QC standard (Section 

6.8.2.1.3). Requirements for calibration verification are given in Section 13.6 and Table 4. 

Alternatively, calibration verification may be performed after a set number of injections (e.g., every 

20 injections), to include injection of extracts of field samples, QC samples, instrument blanks, etc. 

(i.e., it is based on the number of injections performed, not sample extracts). 

Note: The 24-hour shift begins after analysis of the combined QC standard (calibration 

verification) and ends 24 hours later. The ending calibration verification standard is run 

immediately after the last sample run during the 24-hour shift, so the beginning and ending 

calibration verifications are outside of the 24-hour shift. If calibration verification is based 

on the number of injections instead of time, then the ending verification standard for one 

group of 20 injections may be used as the beginning verification for the next group of 20 

injections.

7.9 Florisil® calibration—The column cleanup procedure in Section 11.3 utilizes Florisil column 

chromatography. Florisil® from different batches or sources may vary in adsorptive capacity. To 

standardize the amount of Florisil® which is used, use of the lauric acid value (Reference 11) is 

suggested. The referenced procedure determines the adsorption from a hexane solution of lauric 

acid (mg) per g of Florisil®. The amount of Florisil® to be used for each column is calculated by 

dividing 110 by this ratio and multiplying by 20 g. If cartridges containing Florisil® are used, then 

this step is not necessary.

8. Quality Control 

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. 

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

capability and ongoing analysis of spiked samples and blanks to evaluate and document data 

quality. The laboratory must maintain records to document the quality of data generated. Ongoing 

data quality checks are compared with established performance criteria to determine if the results 

of analyses meet performance requirements of this method. A quality control check standard 

(LCS, Section 8.4) must be prepared and analyzed with each batch of samples to confirm that the 

measurements were performed in an in-control mode of operation. A laboratory may develop its 

own performance criteria (as QC acceptance criteria), provided such criteria are as or more 

restrictive than the criteria in this method.

8.1.1 The laboratory must make an initial demonstration of the capability (IDC) to generate 

acceptable precision and recovery with this method. This demonstration is detailed in 

Section 8.2. On a continuing basis, the laboratory should repeat demonstration of 

capability (DOC) annually.

8.1.2 In recognition of advances that are occurring in analytical technology, and to overcome 

matrix interferences, the laboratory is permitted certain options (Section 1.8 and 40 CFR 

136.6(b) [Reference 12]) to improve separations or lower the costs of measurements. These 

options may include alternative extraction (e.g., other solid-phase extraction materials and 

Method 608.3 16 December 2014
formats), concentration, and cleanup procedures, and changes in GC columns (Reference 12). 

Alternative determinative techniques, such as the substitution of spectroscopic or 

immunoassay techniques, and changes that degrade method performance, are not allowed. If 

an analytical technique other than the techniques specified in this method is used, that 

technique must have a specificity equal to or greater than the specificity of the techniques in 

this method for the analytes of interest. The laboratory is also encouraged to participate in 

performance evaluation studies (see Section 8.8).

8.1.2.1 Each time a modification listed above is made to this method, the laboratory is 

required to repeat the procedure in Section 8.2. If the detection limit of the method 

will be affected by the change, the laboratory is required to demonstrate that the 

MDLs (40 CFR Part 136, Appendix B) are lower than one-third the regulatory 

compliance limit or as low as the MDLs in this method, whichever are greater. If 

calibration will be affected by the change, the instrument must be recalibrated per 

Section 7. Once the modification is demonstrated to produce results equivalent or 

superior to results produced by this method as written, that modification may be 

used routinely thereafter, so long as the other requirements in this method are met 

(e.g., matrix spike/matrix spike duplicate recovery and relative percent difference).

8.1.2.1.1 If an allowed method modification, is to be applied to a specific 

discharge, the laboratory must prepare and analyze matrix 

spike/matrix spike duplicate (MS/MSD) samples (Section 8.3) and 

LCS samples (Section 8.4). The laboratory must include surrogates 

(Section 8.7) in each of the samples. The MS/MSD and LCS 

samples must be fortified with the analytes of interest (Section 1.4). 

If the modification is for nationwide use, MS/MSD samples must be 

prepared from a minimum of nine different discharges (See Section 

8.1.2.1.2), and all QC acceptance criteria in this method must be 

met. This evaluation only needs to be performed once other than for 

the routine QC required by this method (for example it could be 

performed by the vendor of an alternate material) but any laboratory 

using that specific material must have the results of the study 

available. This includes a full data package with the raw data that 

will allow an independent reviewer to verify each determination and 

calculation performed by the laboratory (see Section 8.1.2.2.5, items 

a-q). 

8.1.2.1.2 Sample matrices on which MS/MSD tests must be performed for 

nationwide use of an allowed modification: 

(a) Effluent from a POTW

(b) ASTM D5905 Standard Specification for Substitute Wastewater

(c) Sewage sludge, if sewage sludge will be in the permit

(d) ASTM D1141 Standard Specification for Substitute Ocean 

Water, if ocean water will be in the permit

(e) Untreated and treated wastewaters up to a total of nine matrix 

types (see http:water.epa.gov/scitech/wastetech

/guide/industry.cfm) for a list of industrial categories with 

existing effluent guidelines).

At least one of the above wastewater matrix types must have at 

least one of the following characteristics:

Method 608.3 17 December 2014
(i) Total suspended solids greater than 40 mg/L 

(ii) Total dissolved solids greater than 100 mg/L 

(iii) Oil and grease greater than 20 mg/L

(iv) NaCl greater than 120 mg/L

(v) CaCO3 greater than 140 mg/L 

The interim acceptance criteria for MS, MSD recoveries that do not 

have recovery limits specified in Table 5, and recoveries for 

surrogates that do not have recovery limits specified in Table 8, must 

be no wider than 60 -140 %, and the relative percent difference 

(RPD) of the concentrations in the MS and MSD that do not have 

RPD limits specified in Table 5 must be less than 30%. 

Alternatively, the laboratory may use the laboratory’s in-house limits 

if they are tighter.

(f) A proficiency testing (PT) sample from a recognized provider, in 

addition to tests of the nine matrices (Section 8.1.2.1.1). 

8.1.2.2 The laboratory must maintain records of modifications made to this method. 

These records include the following, at a minimum:

8.1.2.2.1 The names, titles, street addresses, telephone numbers, and 

e-mail addresses of the analyst(s) that performed the analyses and 

modification, and of the quality control officer that witnessed and will 

verify the analyses and modifications.

8.1.2.2.2 A list of analytes, by name and CAS Registry number.

8.1.2.2.3 A narrative stating reason(s) for the modifications.

8.1.2.2.4 Results from all quality control (QC) tests comparing the modified

method to this method, including:

a) Calibration (Section 7).

b) Calibration verification (Section 13.6).

c) Initial demonstration of capability (Section 8.2).

d) Analysis of blanks (Section 8.5).

e) Matrix spike/matrix spike duplicate analysis (Section 8.3).

f) Laboratory control sample analysis (Section 8.4).

8.1.2.2.5 Data that will allow an independent reviewer to validate each 

determination by tracing the instrument output (peak height, area, or 

other signal) to the final result. These data are to include:

a) Sample numbers and other identifiers.

b) Extraction dates.

c) Analysis dates and times.

d) Analysis sequence/run chronology.

e) Sample weight or volume (Section 10).

f) Extract volume prior to each cleanup step (Sections 10 and 11).

g) Extract volume after each cleanup step (Section 11).

h) Final extract volume prior to injection (Sections 10 and 12).

i) Injection volume (Sections 12.3 and 13.2).

j) Sample or extract dilution (Section 15.4).

k) Instrument and operating conditions.

Method 608.3 18 December 2014
l) Column (dimensions, material, etc).

m) Operating conditions (temperatures, flow rates, etc).

n) Detector (type, operating conditions, etc).

o) Chromatograms and other recordings of raw data.

p) Quantitation reports, data system outputs, and other data to link 

the raw data to the results reported.

q) A written Standard Operating Procedure (SOP)

8.1.2.2.6 Each individual laboratory wishing to use a given modification must 

perform the start-up tests in Section 8.1.2 (e.g., DOC, MDL), with 

the modification as an integral part of this method prior to applying 

the modification to specific discharges. Results of the DOC must 

meet the QC acceptance criteria in Table 5 for the analytes of interest 

(Section 1.4), and the MDLs must be equal to or lower than the 

MDLs in Table 3 for the analytes of interest.

8.1.3 Before analyzing samples, the laboratory must analyze a blank to demonstrate that 

interferences from the analytical system, lab ware, and reagents, are under control. Each 

time a batch of samples is extracted or reagents are changed, a blank must be extracted and 

analyzed as a safeguard against laboratory contamination. Requirements for the blank are 

given in Section 8.5. 

8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of 5% of all 

samples in a batch (Section 22.2) or from a given site or discharge, in duplicate, to monitor 

and evaluate method and laboratory performance on the sample matrix. This procedure is 

described in Section 8.3. 

8.1.5 The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control 

check sample (laboratory control sample, LCS; on-going precision and recovery sample, 

OPR) that the measurement system is in control. This procedure is described in Section 

8.4. 

8.1.6 The laboratory should maintain performance records to document the quality of data that is 

generated. This procedure is given in Section 8.7. 

8.1.7 The large number of analytes tested in performance tests in this method present a 

substantial probability that one or more will fail acceptance criteria when all analytes are 

tested simultaneously, and a re-test (reanalysis) is allowed if this situation should occur. If, 

however, continued re-testing results in further repeated failures, the laboratory should 

document the failures and either avoid reporting results for the analytes that failed or report 

the problem and failures with the data. A QC failure does not relieve a discharger or 

permittee of reporting timely results.

8.2 Demonstration of capability (DOC)—To establish the ability to generate acceptable recovery and 

precision, the laboratory must perform the DOC in Sections 8.2.1 through 8.2.6 for the analytes of 

interest initially and in an on-going manner at least annually. The laboratory must also establish 

MDLs for the analytes of interest using the MDL procedure at 40 CFR 136, Appendix B. The 

laboratory's MDLs must be equal to or lower than those listed in Table 3 or lower than one-third 

the regulatory compliance limit, whichever is greater. For MDLs not listed in Tables 1 or 2, the 

laboratory must determine the MDLs using the MDL procedure at 40 CFR 136, Appendix B under 

the same conditions used to determine the MDLs for the analytes listed in Tables 1 and 2. All 

procedures used in the analysis, including cleanup procedures, must be included in the DOC. 

Method 608.3 19 December 2014
8.2.1 For the DOC, a QC check sample concentrate containing each analyte of interest (Section 

1.4) is prepared in a water-miscible solvent using the solution in Section 6.8.3. The QC 

check sample concentrate must be prepared independently from those used for calibration, 

but should be from the same source and prepared in a water-miscible solvent. The 

concentrate should produce concentrations of the analytes of interest in water at or below 

the mid-point of the calibration range. Multiple solutions may be required. 

Note: QC check sample concentrates are no longer available from EPA.

8.2.2 Using a pipet or syringe, prepare four QC check samples by adding an appropriate volume 

of the concentrate and of the surrogate(s) to each of four 1-L aliquots of reagent water. 

Swirl or stir to mix.

8.2.3 Extract and analyze the well-mixed QC check samples according to the method beginning 

in Section 10. 

8.2.4 Calculate the average percent recovery ( X ) and the standard deviation (s) of the percent 

recovery for each analyte using the four results.

8.2.5 For each analyte, compare s and X with the corresponding acceptance criteria for precision 

and recovery in Table 4. For analytes in Table 2 that are not listed in Table 4, QC 

acceptance criteria must be developed by the laboratory. EPA has provided guidance for 

development of QC acceptance criteria (References 12 and 13). If s and X for all analytes 

of interest meet the acceptance criteria, system performance is acceptable and analysis of 

blanks and samples can begin. If any individual s exceeds the precision limit or any 

individual X falls outside the range for recovery, system performance is unacceptable for 

that analyte.

Note: The large number of analytes in Tables 1 and 2 present a substantial probability 

that one or more will fail at least one of the acceptance criteria when many or all 

analytes are determined simultaneously. 

8.2.6 When one or more of the analytes tested fail at least one of the acceptance criteria, repeat 

the test for only the analytes that failed. If results for these analytes pass, system 

performance is acceptable and analysis of samples and blanks may proceed. If one or more

of the analytes again fail, system performance is unacceptable for the analytes that failed 

the acceptance criteria. Correct the problem and repeat the test (Section 8.2). See Section 

8.1.7 for disposition of repeated failures. 

Note: To maintain the validity of the test and re-test, system maintenance and/or 

adjustment is not permitted between this pair of tests.

8.3 Matrix spike and matrix spike duplicate (MS/MSD)—The laboratory must, on an ongoing basis, 

spike at least 5% of the samples in duplicate from each sample site being monitored to assess 

accuracy (recovery and precision). The data user should identify the sample and the analytes of 

interest (Section 1.4) to be spiked. If direction cannot be obtained, the laboratory must spike at 

least one sample in duplicate per extraction batch of up to 20 samples (Section 22.2) with the 

analytes in Table 1. Spiked sample results should be reported only to the data user whose sample 

was spiked, or as requested or required by a regulatory/control authority. 

8.3.1. If, as in compliance monitoring, the concentration of a specific analyte will be checked 

against a regulatory concentration limit, the concentration of the spike should be at that 

Method 608.3 20 December 2014
limit; otherwise, the concentration of the spike should be one to five times higher than the 

background concentration determined in Section 8.3.2, at or near the midpoint of the 

calibration range, or at the concentration in the LCS (Section 8.4) whichever concentration 

would be larger. When no information is available, the mid-point of the calibration may be 

used, as long as it is the same or less than the regulatory limit.

 

8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the each 

analyte of interest. If necessary to meet the requirement in Section 8.3.1, prepare a new 

check sample concentrate (Section 8.2.1) appropriate for the background concentration. 

Spike and analyze two additional sample aliquots of the same volume as the original 

sample, and determine the concentrations after spiking (A1 and A2) of each analyte. 

Calculate the percent recoveries (P1 and P2) as: 

Px= Ax - B

T x 100 

where T is the known true value of the spike.

Also calculate the relative percent difference (RPD) between the concentrations (A1 and 

A2): 

RPD = 

|A1 − A2|

A1 + A2

2

x 100

8.3.3 Compare the percent recoveries (P1 and P2) and the RPD for each analyte in the MS/MSD 

aliquots with the corresponding QC acceptance criteria for recovery (P) and RPD in Table 

4. 

If any individual P falls outside the designated range for recovery in either aliquot, or the 

RPD limit is exceeded, the result for the analyte in the unspiked sample is suspect and may 

not be reported or used for permitting or regulatory compliance. See Section 8.1.7 for 

disposition of failures.

For analytes in Table 2 not listed in Table 5, QC acceptance criteria must be developed by 

the laboratory. EPA has provided guidance for development of QC acceptance criteria 

(References 12 and 13).

8.3.4 After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate, 

the laboratory must calculate and apply in-house QC limits for recovery and RPD of future 

MS/MSD samples (Section 8.3). The QC limits for recovery are calculated as the mean 

observed recovery ± 3 standard deviations, and the upper QC limit for RPD is calculated as 

the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be 

updated at least every two years and re-established after any major change in the analytical 

instrumentation or process. At least 80% of the analytes tested in the MS/MSD must have 

in-house QC acceptance criteria that are tighter than those in Table 4. If an in-house QC 

limit for the RPD is greater than the limit in Table 4, then the limit in Table 4 must be used. 

Similarly, if an in-house lower limit for recovery is below the lower limit in Table 4, then 

the lower limit in Table 4 must be used, and if an in-house upper limit for recovery is above 

the upper limit in Table 4, then the upper limit in Table 4 must be used. The laboratory 

must evaluate surrogate recovery data in each sample against its in-house surrogate 

recovery limits. The laboratory may use 60 -140% as interim acceptance criteria for 

surrogate recoveries until in-house limits are developed. 

Method 608.3 21 December 2014
8.4 Laboratory control sample (LCS)—A QC check sample (laboratory control sample, LCS; on-going 

precision and recovery sample, OPR) containing each single-component analyte of interest 

(Section 1.4) must be extracted, concentrated, and analyzed with each extraction batch of up to 20 

samples (Section 3.1) to demonstrate acceptable recovery of the analytes of interest from a clean 

sample matrix. If multi-peak analytes are required, extract and prepare at least one as an LCS for 

each batch. Alternatively, the laboratory may set up a program where multi-peak LCS is rotated 

with a single-peak LCS.

8.4.1 Prepare the LCS by adding QC check sample concentrate (Section 8.2.1) to reagent water. 

Include all analytes of interest (Section 1.4) in the LCS. The volume of reagent water must 

be the same as the nominal volume used for the sample, the DOC (Section 8.2), the blank 

(Section 8.5), and the MS/MSD (Section 8.3). Also add a volume of the surrogate solution 

(Section 6.8.6).

8.4.2 Analyze the LCS prior to analysis of samples in the extraction batch (Section 3.1). 

Determine the concentration (A) of each analyte. Calculate the percent recovery as:

Ps = 

A

T x 100

where T is the true value of the concentration in the LCS.

8.4.3 For each analyte, compare the percent recovery (P) with its corresponding QC acceptance 

criterion in Table 4. For analytes of interest in Table 2 not listed in Table 4, use the QC 

acceptance criteria developed for the MS/MSD (Section 8.3.3.2). If the recoveries for all 

analytes of interest fall within the designated ranges, analysis of blanks and field samples 

may proceed. If any individual recovery falls outside the range, proceed according to 

Section 8.4.4. 

Note: The large number of analytes in Tables 1 and 2 present a substantial probability 

that one or more will fail the acceptance criteria when all analytes are tested 

simultaneously. Because a re-test is allowed in event of failure (Sections 8.1.7 and 

8.4.4), it may be prudent to extract and analyze two LCSs together and evaluate 

results of the second analysis against the QC acceptance criteria only if an analyte 

fails the first test.

8.4.4 Repeat the test only for those analytes that failed to meet the acceptance criteria (P). If 

these analytes now pass, system performance is acceptable and analysis of blanks and 

samples may proceed. Repeated failure, however, will confirm a general problem with the 

measurement system. If this occurs, repeat the test using a fresh LCS (Section 8.2.1) or an 

LCS prepared with a fresh QC check sample concentrate (Section 8.2.1), or perform and 

document system repair. Subsequent to repair, repeat the LCS test (Section 8.4). See 

Section 8.1.7 for disposition of repeated failures. 

8.4.5 After analysis of 20 LCS samples, the laboratory must calculate and apply in-house QC 

limits for recovery to future LCS samples (Section 8.4). Limits for recovery in the LCS are 

calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the 

analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table 

4. As noted in Section 8.6, each laboratory must develop QC acceptance criteria for the 

surrogates they employ. The laboratory should use 60 -140% as interim acceptance criteria 

for recoveries of spiked analytes and surrogates until in-house LCS and surrogate limits are 

developed. If an in-house lower limit for LCS recovery is lower than the lower limit in 

Method 608.3 22 December 2014
Table 4, the lower limit in Table 4 must be used, and if an in-house upper limit for recovery 

is higher than the upper limit in Table 4, the upper limit in Table 4 must be used. 

8.5 Blank—Extract and analyze a blank with each extraction batch (Section 22.2) to demonstrate that 

the reagents and equipment used for preparation and analysis are free from contamination.

8.5.1 Prepare the blank from reagent water and spike it with the surrogates. The volume of 

reagent water must be the same as the volume used for samples, the DOC (Section 8.2), the 

LCS (Section 8.4), and the MS/MSD (Section 8.3). Extract, concentrate, and analyze the 

blank using the same procedures and reagents used for the samples, LCS, and MS/MSD in 

the batch. Analyze the blank immediately after analysis of the LCS (Section 8.4) and prior 

to analysis of the MS/MSD and samples to demonstrate freedom from contamination. 

8.5.2 If any analyte of interest is found in the blank at a concentration greater than the MDL for 

the analyte, at a concentration greater than one-third the regulatory compliance limit, or at a 

concentration greater than one-tenth the concentration in a sample in the batch (Section 

3.1), whichever is greatest, analysis of samples must be halted and samples in the batch 

must be re-extracted and the extracts reanalyzed. Samples in a batch must be associated 

with an uncontaminated blank before the results for those samples may be reported or used 

for permitting or regulatory compliance purposes. If re-testing of blanks results in repeated 

failures, the laboratory should document the failures and report the problem and failures 

with the data.

8.6 Surrogate recovery—As a quality control check, the laboratory must spike all samples with the 

surrogate standard spiking solution (Section 6.8.6) per Section 10.2.2 or 10.4.2, analyze the 

samples, and calculate the percent recovery of each surrogate. QC acceptance criteria for 

surrogates must be developed by the laboratory. EPA has provided guidance for development of 

QC acceptance criteria (References 12 and 13). If any recovery fails its criterion, attempt to find 

and correct the cause of the failure, and if sufficient volume is available, re-extract another aliquot 

of the affected sample. Surrogate recoveries from the blank and LCS may be used as pass/fail 

criteria by the laboratory or as required by a regulatory authority, or may be used to diagnose 

problems with the analytical system.

8.7 As part of the QC program for the laboratory, it is suggested but not required that method accuracy 

for wastewater samples be assessed and records maintained. After analysis of five or more spiked 

wastewater samples as in Section 8.4, calculate the average percent recovery ( X ) and the standard 

deviation of the percent recovery (sp). Express the accuracy assessment as a percent interval from 

X -2sp to X +2sp. For example, if X = 90% and sp = 10%, the accuracy interval is expressed as 70 

- 110%. Update the accuracy assessment for each analyte on a regular basis to ensure process 

control (e.g., after each 5 - 10 new accuracy measurements).

8.8 It is recommended that the laboratory adopt additional quality assurance practices for use with this 

method. The specific practices that are most productive depend upon the needs of the laboratory 

and the nature of the samples. Field duplicates may be analyzed to assess the precision of 

environmental measurements. When doubt exists over the identification of a peak on the 

chromatogram, confirmatory techniques such as gas chromatography with another dissimilar 

column, specific element detector, or mass spectrometer must be used. Whenever possible, the 

laboratory should analyze standard reference materials and participate in relevant performance 

evaluation studies.

Method 608.3 23 December 2014
9. Sample Collection, Preservation, and Handling

9.1 Collect samples as grab samples in glass bottles, or in refrigerated bottles using automatic sampling 

equipment. Collect 1-L of ambient waters, effluents, and other aqueous samples. If high 

concentrations of the analytes of interest are expected (e.g., for untreated effluents or in-process 

waters), collect a smaller volume (e.g., 250 mL), but not less than 100 mL, in addition to the 1-L 

sample. Follow conventional sampling practices, except do not pre-rinse the bottle with sample 

before collection. Automatic sampling equipment must be as free as possible of polyvinyl chloride 

or other tubing or other potential sources of contamination. If needed, collect additional sample(s) 

for the MS/MSD (Section 8.3).

9.2 Ice or refrigerate the sample at <6°C from the time of collection until extraction, but do not freeze. 

If aldrin is to be determined and residual chlorine is present, add 80 mg/L of sodium thiosulfate but 

do not add excess. Any method suitable for field use may be employed to test for residual chlorine 

(Reference 14). If sodium thiosulfate interferes in the determination of the analytes, an alternative 

preservative (e.g., ascorbic acid or sodium sulfite) may be used. 

9.3 Extract all samples within seven days of collection and completely analyze within 40 days of 

extraction (Reference 1). If the sample will not be extracted within 72 hours of collection, adjust 

the sample pH to range of 5.0 - 9.0 with sodium hydroxide solution or sulfuric acid. Record the 

volume of acid or base used. 

10. Sample Extraction 

10.1 This section contains procedures for separatory funnel liquid-liquid extraction (SFLLE, Section 

10.2), continuous liquid-liquid extraction (CLLE, Section 10.4), and disk-based solid-phase 

extraction (SPE, Section 10.5). SFLLE is faster, but may not be as effective as CLLE for 

extracting polar analytes. SFLLE is labor intensive and may result in formation of emulsions that 

are difficult to break. CLLE is less labor intensive, avoids emulsion formation, but requires more 

time (18-24 hours), more hood space, and may require more solvent. SPE can be faster, unless the 

particulate load in an aqueous sample is so high that it slows the filtration process. If an alternative 

extraction scheme to those detailed in this method is used, all QC tests must be performed and all 

QC acceptance criteria must be met with that extraction scheme as an integral part of this method. 

10.2 Separatory funnel liquid-liquid extraction (SFLLE)

10.2.1 The SFLLE procedure below assumes a sample volume of 1 L. When a different sample 

volume is extracted, adjust the volume of methylene chloride accordingly.

10.2.2 Mark the water meniscus on the side of the sample bottle for later determination of sample 

volume. Pour the entire sample into the separatory funnel. Pipet the surrogate standard 

spiking solution (Section 6.8.6) into the separatory funnel. If the sample will be used for 

the LCS or MS or MSD, pipet the appropriate QC check sample concentrate (Section 8.2.1) 

into the separatory funnel. Mix well. If the sample arrives in a larger sample bottle, 1 L 

may be measured in a graduated cylinder, then added to the separatory funnel. 

Note: Instances in which the sample is collected in an oversized bottle should be reported 

by the laboratory to the data user. Of particular concern is that fact that this 

practice precludes rinsing the empty bottle with solvent as described below, which 

could leave hydrophobic pesticides on the wall of the bottle, and underestimate the 

actual sample concentrations.

Method 608.3 24 December 2014
10.2.3 Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 seconds to 

rinse the inner surface. Transfer the solvent to the separatory funnel and extract the sample 

by shaking the funnel for two minutes with periodic venting to release excess pressure. 

Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If 

an emulsion forms and the emulsion interface between the layers is more than one-third the 

volume of the solvent layer, employ mechanical techniques to complete the phase 

separation. The optimum technique depends upon the sample, but may include stirring, 

filtration of the emulsion through glass wool, centrifugation, freezing, or other physical 

methods. Collect the methylene chloride extract in a flask. If the emulsion cannot be 

broken (recovery of less than 80% of the methylene chloride, corrected for the water 

solubility of methylene chloride), transfer the sample, solvent, and emulsion into the 

extraction chamber of a continuous extractor and proceed as described in Section 10.4. 

10.2.4 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the 

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

extraction in the same manner. Proceed to macro-concentration (Section 10.3.1).

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

transferring the liquid to an appropriately sized graduated cylinder. Record the sample 

volume to the nearest 5 mL. Sample volumes may also be determined by weighing the 

container before and after extraction or filling to the mark with water.

10.3 Concentration 

10.3.1 Macro concentration

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

concentrator tube to a 500-mL evaporative flask. Other concentration devices or 

techniques may be used in place of the K-D concentrator so long as the 

requirements of Section 8.2 are met.

10.3.1.2 Pour the extract through a solvent-rinsed drying column containing about 10 cm 

of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. 

Rinse the flask and column with 20-30 mL of methylene chloride to complete the 

quantitative transfer.

10.3.1.3 If no cleanup is to be performed on the sample, add 500 μL (0.5 mL) of isooctane 

to the extract to act as a keeper during concentration.

10.3.1.4 Add one or two clean boiling chips and attach a three-ball Snyder 

column to the K-D evaporative flask. Pre-wet the Snyder column by 

adding about 1 mL of methylene chloride to the top. Place the K-D 

apparatus on a hot water bath (60 - 65 °C) so that the concentrator tube is 

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

of the flask is bathed with hot vapor. Adjust the vertical position of the 

apparatus and the water temperature as required to complete the 

concentration in 15 - 20 minutes. At the proper rate of evaporation the 

balls of the column will actively chatter but the chambers will not flood 

with condensed solvent. When the apparent volume of liquid reaches 1 

mL or other determined amount, remove the K-D apparatus from the 

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

Method 608.3 25 December 2014
10.3.1.5 If the extract is to be cleaned up by a procedure for sulfur removal, 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 in methylene chloride and proceed 

to sulfur removal (Section 11.5). If the extract is to cleaned up using one of the 

other cleanup procedures or is to be injected into the GC, proceed to KudernaDanish micro-concentration (Section 10.3.2) or nitrogen evaporation and solvent 

exchange (Section 10.3.3). 

10.3.2 Kuderna-Danish micro concentration

10.3.2.1 Add another one or two clean boiling chips to the concentrator tube and attach a 

two-ball micro-Snyder column. Pre-wet the Snyder column by adding about 0.5 

mL of methylene chloride to the top. Place the K-D apparatus on a hot water 

bath (60 – 65 °C) so that the concentrator tube is partially immersed in hot water. 

Adjust the vertical position of the apparatus and the water temperature as 

required to complete the concentration in 5 - 10 minutes. At the proper rate of 

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

flood with condensed solvent. When the apparent volume of liquid reaches 

approximately 1 mL or other required amount, remove the K-D apparatus from 

the water bath and allow it to drain and cool for at least 10 minutes. Remove the 

Snyder column and rinse the flask and its lower joint into the concentrator tube 

with approximately 0.2 mL of methylene chloride, and proceed to Section 10.3.3 

for nitrogen evaporation and solvent exchange. 

10.3.3 Nitrogen evaporation and solvent exchange—Extracts to be subjected to solid-phase 

cleanup (SPE) are exchanged into 1.0 mL of the SPE elution solvent (Section 6.7.2.2). 

Extracts to be subjected to Florisil® or alumina cleanups are exchanged into hexane. 

Extracts that have been cleaned up and are ready for analysis are exchanged into isooctane 

or hexane, to match the solvent used for the calibration standards. 

10.3.3.1 Transfer the vial containing the sample extract to the nitrogen evaporation 

(blowdown) device (Section 5.2.5.2). Lower the vial into a 50-55 °C water bath 

and begin concentrating. During the solvent evaporation process, do not allow 

the extract to become dry. 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.

10.3.3.2 Solvent exchange 

10.3.3.2.1 When the volume of the liquid is approximately 500 μL, add 2 to 3 

mL of the desired solvent (SPE elution solvent for SPE cleanup, 

hexane for Florisil or alumina, or isooctane for final injection into 

the GC) and continue concentrating to approximately 500 μL. 

Repeat the addition of solvent and concentrate once more.

10.3.3.3.2 Adjust the volume of an extract to be cleaned up by SPE, Florisil®, 

or alumina to 1.0 mL. Proceed to extract cleanup (Section 11). 

10.3.3.3 Extracts that have been cleaned up and are ready for analysis—Adjust the final 

extract volume to be consistent with the volume extracted and the sensitivity 

Method 608.3 26 December 2014
desired. The goal is for a full-volume sample (e.g., 1-L) to have a final extract 

volume of 10 mL, but other volumes may be used. 

10.3.4 Transfer the concentrated extract to 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 

4 °C. Analyze the extract by GC per the procedure in Section 12. 

10.4 Continuous liquid/liquid extraction (CLLE)

10.4.1 Use CLLE when experience with a sample from a given source indicates an emulsion 

problem, or when an emulsion is encountered using SFLLE. CLLE may be used for all 

samples, if desired.

10.4.2 Mark the water meniscus on the side of the sample bottle for later determination of sample 

volume. Transfer the sample to the continuous extractor and, using a pipet, add surrogate 

standard spiking solution. If the sample will be used for the LCS, MS, or MSD, pipet the 

appropriate check sample concentrate (Section 8.2.1 or 8.3.2) into the separatory funnel. 

Mix well. Add 60 mL of methylene chloride to the sample bottle, seal, and shake for 30 

seconds to rinse the inner surface. Transfer the solvent to the extractor.

10.4.3 Repeat the sample bottle rinse with two additional 50-100 mL portions of methylene 

chloride and add the rinses to the extractor.

10.4.4 Add a suitable volume of methylene chloride to the distilling flask (generally 

200 - 500 mL) and sufficient reagent water to ensure proper operation of the extractor, and 

extract the sample for 18 - 24 hours. A shorter or longer extraction time may be used if all 

QC acceptance criteria are met. Test and, if necessary, adjust the pH of the water during 

the second or third hour of the extraction. After extraction, allow the apparatus to cool, 

then detach the distilling flask. Dry, concentrate, solvent exchange, and transfer the extract 

to a vial with fluoropolymer-lined cap, per Section 10.3. 

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

transferring the liquid to an appropriately sized graduated cylinder. Record the sample 

volume to the nearest 5 mL. Sample volumes may also be determined by weighing the 

container before and after extraction or filling to the mark with water.

10.5 Solid-phase extraction of aqueous samples

The steps in this section address the extraction of aqueous field samples using disk-based solidphase extraction (SPE) media, based on an ATP approved by EPA in 1995 (Reference 20). This 

application of SPE is distinct from that used in this method for the cleanup of sample extracts in 

Section 11.2. Analysts must be careful not to confuse the equipment, supplies, or the procedural 

steps from these two different uses of SPE.

Note: Changes to the extraction conditions described below may be made by the laboratory under 

the allowance for method flexibility described in Section 8.1, provided that the performance 

requirements in Section 8.2 are met. However, changes in SPE materials, formats, and 

solvents must meet the requirements in Section 8.1.2 and its subsections. 

Method 608.3 27 December 2014
10.5.1 Mark the water meniscus on the side of the sample bottle for later determination of sample 

volume. If the sample contains particulates, let stand to settle out the particulates before 

extraction.

10.5.2 Extract the sample as follows:

10.5.2.1 Place a 90-mm standard filter apparatus on a vacuum filtration flask or manifold 

and attach to a vacuum source. The vacuum gauge should read at least 25 in. of 

mercury when all valves are closed. Position a 90-mm C18 extraction disk onto 

the filter screen. Wet the entire disk with methanol. To aid in filtering samples 

with particulates, a 1-μm glass fiber filter or Empore® Filter Aid 400 can be 

placed on the top of the disk and wetted with methanol. Install the reservoir and 

clamp. Resume vacuum to dry the disk. Interrupt the vacuum. Wash the disk 

and reservoir with 20 mL of methylene chloride. Resume the vacuum briefly to 

pull methylene chloride through the disk. Interrupt the vacuum and allow the 

disk to soak for about a minute. Resume vacuum and completely dry the disk. 

10.5.2.2 Condition the disk with 20 mL of methanol. Apply vacuum until nearly all the 

solvent has passed through the disk, interrupting it while solvent remains on the 

disk. Allow the disk to soak for about a minute. Resume vacuum to pull most of 

the methanol through, but interrupting it to leave a layer of methanol on the 

surface of the disk. Do not allow disk to dry.

 For uniform flow and good recovery, it is critical the disk not be allowed to dry 

from now until the end of the extraction. Discard waste solvent. Rinse the disk 

with 20 mL of deionized water. Resume vacuum to pull most of the water 

through, but interrupt it to leave a layer of water on the surface of the disk. Do 

not allow the disk to dry. If disk does dry, recondition with methanol as above. 

10.5.2.3 Add the water sample to the reservoir and immediately apply the vacuum. If 

particulates have settled in the sample, gently decant the clear layer into the 

apparatus until most of the sample has been processed. Then pour the remainder 

including the particulates into the reservoir. Empty the sample bottle completely. 

When the filtration is complete, dry the disk for three minutes. Turn off the 

vacuum. 

10.5.3 Discard sample filtrate. Insert tube to collect the eluant. The tube should fit around the 

drip tip of the base. Reassemble the apparatus. Add 5.0 mL of acetone to the center of the 

disk, allowing it to spread evenly over the disk. Turn the vacuum on and quickly off when 

the filter surface nears dryness but still remains wet. Allow to soak for 15 seconds. Add 20 

mL of methylene chloride to the sample bottle, seal and shake to rinse the inside of the 

bottle. Transfer the methylene chloride from the bottle to the filter. Resume the vacuum 

slowly so as to avoid splashing. 

Interrupt the vacuum when the filter surface nears dryness but still remains wet. Allow 

disk to soak in solvent for 20 seconds. Rinse the reservoir glass and disk with 10 mL of 

methylene chloride. Resume vacuum slowly. Interrupt vacuum when disk is covered with 

solvent. Allow to soak for 20 seconds. Resume vacuum to dry the disk. Remove the 

sample tube.

10.5.4 Dry, concentrate, solvent exchange, and transfer the extract to a vial with fluoropolymerlined cap, per Section 10.3. 

Method 608.3 28 December 2014
10.5.5 Determine the original sample volume by refilling the sample bottle to the mark and 

transferring the liquid to an appropriately sized graduated cylinder. Record the sample 

volume to the nearest 5 mL. Sample volumes may also be determined by weighing the 

container before and after extraction or filling to the mark with water.

11. Extract Cleanup

11.1 Cleanup may not be necessary for a relatively clean sample matrix. If particular circumstances 

require the use of a cleanup procedure, the laboratory may use any or all of the procedures below or 

any other appropriate procedure (e.g., gel permeation chromatography). However, the laboratory 

must first repeat the tests in Sections 8.2, 8.3, and 8.4 to demonstrate that the requirements of those 

sections can be met using the cleanup procedure(s) as an integral part of this method. This is 

particularly important when the target analytes for the analysis include any of the single component 

pesticides in Table 2, because some cleanups have not been optimized for all of those analytes.

11.1.1 The solid-phase cartridge (Section 11.2) removes polar organic compounds such as 

phenols. 

11.1.2 The Florisil® column (Section 11.3) allows for selected fractionation of the organochlorine 

analytes and will also eliminate polar interferences.

11.1.3 Alumina column cleanup (Section 11.4) also removes polar materials.

11.1.4 Elemental sulfur, which interferes with the electron capture gas chromatography of some of 

the pesticides, may be removed using activated copper, or TBA sulfite. Sulfur removal 

(Section 11.5) is required when sulfur is known or suspected to be present. Some 

chlorinated pesticides which also contain sulfur may be removed by this cleanup. 

11.2 Solid-phase extraction (SPE) as a cleanup

 In order to use the C18 SPE cartridge in Section 5.5.3.5 as a cleanup procedure, the sample extract 

must be exchanged from methylene chloride to methylene chloride: acetonitrile:hexane. Follow 

the solvent exchange steps in Section 10.3.3.2 prior to attempting solid-phase cleanup. 

Note: This application of SPE is distinct from that used in this method for the extraction of 

aqueous samples in Section 10.5. Analysts must be careful not to confuse the equipment, 

supplies, or procedural steps from these two different uses of SPE.

11.2.1 Setup

11.2.1.1 Attach the VacElute Manifold (Section 5.5.3.2) to a water aspirator or vacuum 

pump with the trap and gauge installed between the manifold and vacuum 

source. 

11.2.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and adjust 

the vacuum to 5 to 10 psi. 

11.2.2 Cartridge washing—Pre-elute each cartridge prior to use sequentially with 10-mL portions 

each of hexane, methanol, and water using vacuum for 30 seconds after each eluting 

solvent. Follow this pre-elution with 1 mL methylene chloride and three 10-mL portions of 

the elution solvent (Section 6.7.2.2) using vacuum for 5 minutes after each eluting solvent. 

Method 608.3 29 December 2014
Tap the cartridge lightly while under vacuum to dry between solvent rinses. The three 

portions of elution solvent may be collected and used as a cartridge blank, if desired. 

Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum for 

30 seconds after each eluant.

11.2.3 Extract cleanup

11.2.3.1 After cartridge washing (Section 11.2.2), release the vacuum and place the rack 

containing the 50-mL volumetric flasks (Section 5.5.3.4) in the vacuum 

manifold. Re-establish the vacuum at 5 to 10 psi. 

11.2.3.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE 

cartridge. Apply vacuum for five minutes to dry the cartridge. Tap gently to aid 

in drying. 

11.2.3.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL 

portions of the methylene chloride:acetonitrile:hexane (50:3:47) elution solvent 

(Section 6.7.2.2), using vacuum for five minutes after each portion. Collect the 

eluants in the 50-mL volumetric flasks.

11.2.3.4 Release the vacuum and remove the 50-mL volumetric flasks.

11.2.3.5 Concentrate the eluted extracts per Section 10.3.

11.3 Florisil®

 

 In order to use Florisil cleanup, the sample extract must be exchanged from methylene chloride to 

hexane. Follow the solvent exchange steps in Section 10.3.3.2 prior to attempting Florisil®

cleanup. 

Note: Alternative formats for this cleanup may be used by the laboratory, including cartridges 

containing Florisil®. If an alternative format is used, consult the manufacturer’s 

instructions and develop a formal documented procedure to replace the steps in Section 11.3 

of this method and demonstrate that the alternative meets the relevant quality control 

requirements of this method. 

11.3.1 If the chromatographic column does not contain a frit at the bottom, place a small plug of 

pre-cleaned glass wool in the column (Section 5.2.4) to retain the Florisil®. Place the mass

of Florisil® (nominally 20 g) predetermined by calibration (Section 7.9 and Table 6) in a 

chromatographic column. Tap the column to settle the Florisil® and add 1 to 2 cm of 

granular anhydrous sodium sulfate to the top. 

11.3.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil®. Just prior to 

exposure of the sodium sulfate layer to the air, stop the elution of the hexane by closing the 

stopcock on the chromatographic column. Discard the eluant. 

11.3.3 Transfer the concentrated extract (Section 10.3.3) onto the column. Complete the transfer 

with two 1-mL hexane rinses, drawing the extract and rinses down to the level of the 

sodium sulfate. 

11.3.4 Place a clean 500-mL K-D flask and concentrator tube under the column. Elute Fraction 1 

with 200 mL of 6% (v/v) ethyl ether in hexane at a rate of approximately 5 mL/min. 

Method 608.3 30 December 2014
Remove the K-D flask and set it aside for later concentration. Elute Fraction 2 with 200 

mL of 15% (v/v) ethyl ether in hexane into a second K-D flask. Elute Fraction 3 with 200 

mL of 50% (v/v) ethyl ether in hexane into a third K-D flask. The elution patterns for the 

pesticides and PCBs are shown in Table 6. 

11.3.5 Concentrate the fractions as in Section 10.3, except use hexane to prewet the column and 

set the water bath at about 85 °C. When the apparatus is cool, remove the Snyder column 

and rinse the flask and its lower joint into the concentrator tube with hexane. Adjust the 

volume of Fraction 1 to approximately 10 mL for sulfur removal (Section 11.5), if required; 

otherwise, adjust the volume of the fractions to 10 mL, 1.0 mL, or other volume needed for 

the sensitivity desired. Analyze the concentrated extract by gas chromatography (Section 

12). 

11.4 Alumina 

The sample extract must be exchanged from methylene chloride to hexane. Follow the solvent 

exchange steps in Section 10.3.3.2 prior to attempting alumina cleanup. 

11.4.1 If the chromatographic column does not contain a frit at the bottom, place a small plug of 

pre-cleaned glass wool in the chromatographic column (Section 5.2.4) to retain the 

alumina. Add 10 g of alumina (Section 6.7.3) on top of the plug. Tap the column to settle 

the alumina. Place 1 - 2 g of anhydrous sodium sulfate on top of the alumina. 

11.4.2 Close the stopcock and fill the column to just above the sodium sulfate with hexane. Add 

25 mL of hexane. Open the stopcock and adjust the flow rate of hexane to approximately 2 

mL/min. Do not allow the column to go dry throughout the elutions. 

11.4.3 When the level of the hexane is at the top of the column, quantitatively transfer the extract 

to the column. When the level of the extract is at the top of the column, slowly add 25 mL 

of hexane and elute the column to the level of the sodium sulfate. Discard the hexane. 

11.4.4 Place a K-D flask (Section 5.2.5.1.2) under the column and elute the pesticides with 

approximately 150 mL of hexane:ethyl ether (80:20 v/v). It may be necessary to adjust the 

volume of elution solvent for slightly different alumina activities.

11.4.5 Concentrate the extract per Section 10.3.

11.5 Sulfur removal—Elemental sulfur will usually elute in Fraction 1 of the Florisil® column cleanup. 

If Florisil® cleanup is not used, or to remove sulfur from any of the Florisil® fractions, use one of 

the sulfur removal procedures below. These procedures may be applied to extracts in hexane, ethyl 

ether, or methylene chloride.

Note: Separate procedures using copper or TBA sulfite are provided in this section for sulfur 

removal. They may be used separately or in combination, if desired.

11.5.1 Removal with copper (Reference 15) 

Note: Some of the analytes in Table 2 are not amenable to sulfur removal with copper 

(e.g., atrazine and diazinon). Therefore, before using copper to remove sulfur from 

an extract that will be analyzed for any of the non-PCB analytes in Table 2, the 

laboratory must demonstrate that the analytes can be extracted from an aqueous 

sample matrix that contains sulfur and recovered from an extract treated with 

Method 608.3 31 December 2014
copper. Acceptable performance can be demonstrated through the preparation 

and analysis of a matrix spike sample that meets the QC requirements for recovery. 

11.5.1.1 Quantitatively transfer the extract to a 40- to 50-mL flask or bottle. If there is 

evidence of water in the K-D or round-bottom flask after the transfer, rinse the 

flask with small portions of hexane:acetone (40:60) and add to the flask or bottle. 

Mark and set aside the concentration flask for future use.

11.5.1.2 Add 10 - 20 g of granular anhydrous sodium sulfate to the flask. Swirl to dry the 

extract.

11.5.1.3 Add activated copper (Section 6.7.4.1.4) and allow to stand for 30 - 60 minutes, 

swirling occasionally. If the copper does not remain bright, add more and swirl 

occasionally for another 30 - 60 minutes.

11.5.1.4 After drying and sulfur removal, quantitatively transfer the extract to a nitrogenevaporation vial or tube and proceed to Section 10.3.3 for nitrogen evaporation 

and solvent exchange, taking care to leave the sodium sulfate and copper foil in 

the flask.

11.5.2 Removal with TBA sulfite

11.5.2.1 Using small volumes of hexane, quantitatively transfer the extract to a 40- to 50-

mL centrifuge tube with fluoropolymer-lined screw cap.

11.5.2.2 Add 1 - 2 mL of TBA sulfite reagent (Section 6.7.4.2.4), 2 - 3 mL of 2-propanol, 

and approximately 0.7 g of sodium sulfite (Section 6.7.4.2.2) crystals to the tube. 

Cap and shake for 1 - 2 minutes. If the sample is colorless or if the initial color is 

unchanged, and if clear crystals (precipitated sodium sulfite) are observed, 

sufficient sodium sulfite is present. If the precipitated sodium sulfite disappears, 

add more crystalline sodium sulfite in approximately 0.5-g portions until a solid 

residue remains after repeated shaking.

11.5.2.3 Add 5 - 10 mL of reagent water and shake for 1 - 2 minutes. Centrifuge to settle 

the solids. 

11.5.2.4 Quantitatively transfer the hexane (top) layer through a small funnel containing a 

few grams of granular anhydrous sodium sulfate to a nitrogen-evaporation vial or 

tube and proceed to Section 10.3.3 for micro-concentration and solvent 

exchange.

12. Gas Chromatography 

12.1 Establish the same operating conditions used in Section 7.1 for instrument calibration. 

12.2 If the internal standard calibration procedure is used, add the internal standard solution (Section 

6.9.3) to the extract as close as possible to the time of injection to minimize the possibility of loss 

by evaporation, adsorption, or reaction. For example, add 1 μL of 10 μg/mL internal standard 

solution into the extract, assuming no dilutions. Mix thoroughly.

Method 608.3 32 December 2014
12.3 Simultaneously inject an appropriate volume of the sample extract or standard solution onto both 

columns, using split, splitless, solvent purge, large-volume, or on-column injection. Alternatively, 

if using a single-column GC configuration, inject an appropriate volume of the sample extract or 

standard solution onto each GC column independently. If the sample is injected manually, the 

solvent-flush technique should be used. The injection volume depends upon the technique used 

and the sensitivity needed to meet MDLs or reporting limits for regulatory compliance. Injected 

volumes must be the same for all standards and sample extracts. Record the volume injected to the 

nearest 0.05 μL.

12.4 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.

12.5 Perform all qualitative and quantitative measurements as described in Sections 14 and 15. When 

standards and extracts are not being used for analyses, store them refrigerated at <6°C, protected 

from light, in screw-cap vials equipped with un-pierced fluoropolymer-lined septa. 

13. System and Laboratory Performance

13.1 At the beginning of each shift during which standards or extracts are analyzed, GC system 

performance and calibration must be verified for all analytes and surrogates on both 

column/detector systems. Adjustment and/or recalibration (per Section 7) are performed until all 

performance criteria are met. Only after all performance criteria are met may samples, blanks and 

other QC samples, and standards be analyzed. 

13.2 Inject an aliquot of the combined QC standard (Section 6.8.4) on both columns. Inject an aliquot 

of each of the multi-component standards. 

13.3 Retention times—The absolute retention times of the peak maxima shall be within ±2 seconds of 

the retention times in the calibration verification (Section 7.8).

13.4 GC resolution—Resolution is acceptable if the valley height between two peaks (as measured from 

the baseline) is less than 40% of the shorter of the two peaks.

13.4.1 DB-608 column —DDT and endrin aldehyde 

13.4.2 DB-1701 column—alpha and gamma chlordane 

Note: If using other GC columns or stationary phases, these resolution criteria apply to these 

four target analytes and any other closely eluting analytes on those other GC columns. 

13.5 Decomposition of DDT and endrin—If DDT, endrin, or their breakdown products are to be 

determined, this test must be performed prior to calibration verification (Section 13.6). DDT 

decomposes to DDE and DDD. Endrin decomposes to endrin aldehyde and endrin ketone. 

13.5.1 Inject 1 μL of the DDT and endrin decomposition solution (Section 6.9.5). 

13.5.2 Measure the areas of the peaks for DDT, DDE, DDD, Endrin, Endrin aldehyde, and Endrin 

ketone in the chromatogram and calculate the percent breakdown as shown in the equations 

below: 

Method 608.3 33 December 2014
% breakdown of DDT = sum of degradation peak areas (DDD + DDE)

sum of all peak areas (DDT + DDE + DDD) x 100

% breakdown of Endrin = sum of degradation peak areas (Endrin aldehyde + Endrin ketone)

sum of all peak areas (Endrin + Endrin aldehyde + Endrin ketone)

 x 100

13.5.3 Both the % breakdown of DDT and of Endrin must be less than 20%, otherwise the system 

is not performing acceptably for DDT and endrin. In this case, repair the GC column 

system that failed and repeat the performance tests (Sections 13.2 to 13.6) until the 

specification is met.

Note: DDT and endrin decomposition are usually caused by accumulations of particulates 

in the injector and in the front end of the column. Cleaning and silanizing the 

injection port liner, and breaking off a short section of the front end of the column 

will usually eliminate the decomposition problem. Either of these corrective actions 

may affect retention times, GC resolution, and calibration linearity. 

13.6 Calibration verification 

13.6.1 Compute the percent recovery of each analyte and of the coeluting analytes, based on the 

initial calibration data (Section 7.5 or 7.6).

13.6.2 For each analyte or for coeluting analytes, compare the concentration with the limits for 

calibration verification in Table 4. For coeluting analytes, use the coeluting analyte with 

the least restrictive specification (the widest range). For analytes in Table 2 not listed in 

Table 4, QC acceptance criteria must be developed by the laboratory. EPA has provided 

guidance for development of QC acceptance criteria (References 13 and 14). If the 

recoveries for all analytes meet the acceptance criteria, system performance is acceptable 

and analysis of blanks and samples may continue. If, however, any recovery falls outside 

the calibration verification range, system performance is unacceptable for that analyte. If 

this occurs, repair the system and repeat the test (Section 13.6), or prepare a fresh 

calibration standard and repeat the test, or recalibrate (Section 7). See Section 8.1.7 for 

information on repeated test failures. 

13.7 Laboratory control sample

13.7.1 Analyze the extract of the combined QC standard (a.k.a. LCS) (Section 6.8.3) extracted 

with each sample batch (Section 8.4). 

13.7.2 Compute the percent recovery of each analyte and of the coeluting analytes.

13.7.3 For each analyte or coeluting analytes, compare the percent recovery with the limits for “P” 

in Table 4. For coeluting analytes, use the coeluting 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, reextract the sample batch, and repeat the ongoing precision and recovery test.

13.7.4 It is suggested, but not required, that the laboratory update statements of data quality. Add 

results that pass the specifications in Section 13.7.3 to initial (Section 8.7) 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 the average percent recovery (R) and the standard deviation of percent recovery, 

Method 608.3 34 December 2014
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%. 

13.8 Internal standard response—If internal standard calibration is used, verify that detector sensitivity 

has not changed by comparing the response (area or height) of each internal standard in the sample, 

blank, LCS, MS, and MSD to the response in the combined QC standard (Section 6.8.3). The peak 

area or height of the internal standard should be within 50% to 200% (1/2 to 2x) of its respective 

peak area or height in the verification standard. If the area or height is not within this range, 

compute the concentration of the analytes using the external standard method (Section 7.5). 

14. Qualitative Identification

14.1 Identification is accomplished by comparison of data from analysis of a sample, blank, or other QC 

sample with data from calibration verification (Section 7.7.1 or 13.5), and with data stored in the 

retention-time and calibration libraries (Section 7.7). The retention time window is determined as 

described in Section 14.2. Identification is confirmed when retention time agrees on both GC 

columns, as described below.

14.2 Establishing retention time windows 

14.2.1 Using the data from the multi-point initial calibration (Section 7.4), determine the retention 

time in decimal minutes (not minutes:seconds) of each peak representing a singlecomponent target analyte on each column/detector system. For the multi-component 

analytes, use the retention times of the five largest peaks in the chromatograms on each 

column/detector system.

14.2.2 Calculate the standard deviation of the retention times for each single-component analyte 

on each column/detector system and for the three to five exclusive (unique large) peaks for 

each multi-component analyte.

14.2.3 Define the width of the retention time window as three times that standard deviation. 

Establish the center of the retention time window for each analyte by using the absolute 

retention time for each analyte from the calibration verification standard at the beginning of 

the analytical shift. For samples run during the same shift as an initial calibration, use the 

retention time of the mid-point standard of the initial calibration. If the calculated RT 

window is less than 0.02 minutes, then use 0.02 minutes as the window. 

Note: Procedures for establishing retention time windows from other sources may be employed 

provided that they are clearly documented and provide acceptable performance. Such 

performance may be evaluated using the results for the spiked QC samples described in 

this method, such as laboratory control samples and matrix spike samples.

14.2.4 New retention time windows must be established when a new GC column is installed or if a 

GC column has been shortened during maintenance to a degree that the retention times of 

analytes in the calibration verification standard have shifted close to the lower limits of the 

established retention time windows. 

14.2.5 RT windows should be checked periodically by examining the peaks in spiked samples 

such as the LCS or MS/MSD to confirm that peaks for known analytes are properly 

identified.

Method 608.3 35 December 2014
14.2.6 If the retention time of an analyte in the initial calibration data has been evaluated as 

described in Section 7.4.1 and it varied by more than 5 seconds across the calibration range 

as a function of the concentration of the standard (see Section 7.4.2), then using the 

standard deviation of the retention times to set the width of the retention time window may 

not adequately serve to identify the analyte in question under routine conditions. In such 

cases, data from additional analyses of standards may be required to adequately model the 

chromatographic behavior of the analyte.

14.3 Identifying the analyte in a sample

14.3.1 In order to identify a single-component analyte from analysis of a sample, blank, or other 

QC sample, the peak representing the analyst must fall within its respective retention time 

windows on both column/detector systems (as defined in Section 14.2). That identification 

is further supported by the comparison of the numerical results on both columns, as 

described in Section 15.7. 

14.3.2 In order to identify a multi-component analyte, pattern matching (fingerprinting) may be 

used, or the three to five exclusive (unique, baseline resolved, and largest) peaks for that 

analyte must fall within their respective retention time windows on both column/detector 

systems (as defined in Section 14.2). That identification is further supported by the 

comparison of the numerical results on both columns, as described in Section 15.7. 

14.4 GC/MS confirmation

When the concentration of an analyte is sufficient, or if the presence or identity is suspect, its 

presence should be confirmed by GC/MS. In order to match the sensitivity of the GC/ECD, 

confirmation will have to be by SIM-GC/MS, or estimated the concentration would have to be 100 

times higher than the GC/ECD calibration range. 

14.5 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 may use the cleanup procedures in this method (Section 11) on a new 

sample aliquot to attempt to remove the interferent. After 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.

14.5.1 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 calibration, 

calibration verification, blank, LCS, and MS/MSD, the RT for the analyte of interest in the 

sample should be within this variation regardless of the window established in Section 14.2. 

If the analyte is not within this variation on both columns, it is likely not present. 

14.5.2 The possibility exists that the RT for the analyte in a sample could shift if extraneous 

materials are present. This possibility may be able to be confirmed or refuted by the 

behavior of the surrogates in the sample. If multiple surrogates are used that span the 

length of the chromatographic run, the RTs for the surrogates on both columns are 

consistent with their RTs in calibration, calibration verification, blank, LCS, and MS/MSD, 

it is unlikely that the RT for the analyte of interest has shifted. 

Method 608.3 36 December 2014
14.5.3 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 analyte is present, because 

shifts nearly always occur in the same direction on both columns. 

15. Quantitative Determination

15.1 External standard quantitation—Calculate the concentration of the analyte in the extract using the 

calibration curve or average calibration factor determined in calibration (Section 7.5.2) and the 

following equation: 

Cex = 

As

CF

where: 

Cex = Concentration of the analyte in the extract (ng/mL) 

As = Peak height or area for the analyte in the standard or sample

CF = Calibration factor, as defined in Section 7.5.1 

15.2 Internal standard quantitation—Calculate the concentration of the analyte in the extract using the 

calibration curve or average response factor determined in calibration (Section 7.6.2) and the 

following equation: 

Cex = 

As x Cis

Ais x RF

where: 

Cex = Concentration of the analyte in the extract (ng/mL) 

As = Peak height or area for the analyte in the standard or sample

Cis = Concentration of the internal standard (ng/mL) 

Ais = Area of the internal standard

RF = Response factor, as defined in Section 7.6.1 

15.3 Calculate the concentration of the analyte in the sample using the concentration in the extract, the 

extract volume, the sample volume, and the dilution factor, per the following equation:

Cs = 

Cex x Vex x DF

Vs x 1000

where: 

Cs = Concentration of the analyte in the sample (μg/L)

Vex = Final extract volume (mL)

Cex = Concentration in the extract (ng/mL) 

Vs = Volume of sample (L) 

DF = Dilution factor

and the factor of 1,000 in the denominator converts the final units from ng/L to μg/L 

15.4 If the concentration of any target analyte exceeds the calibration range, either extract and analyze a 

smaller sample volume, or dilute and analyze the diluted extract.

Method 608.3 37 December 2014
15.5 Quantitation of multi-component analytes

15.5.1 PCBs as Aroclors

Quantify an Aroclor by comparing the sample chromatogram to that of the most similar 

Aroclor standard as indicated in Section 14.3.2. Compare the responses of 3 to 5 major 

peaks in the calibration standard for that Aroclor with the peaks observed in the sample 

extract. The amount of Aroclor is calculated using the individual calibration factor for each 

of the 3 to 5 characteristic peaks chosen in Sec. 7.5.1. Determine the concentration of each 

of the characteristic peaks, using the average calibration factor calculated for that peak in 

Sec. 7.5.2, and then those 3 to 5 concentrations are averaged to determine the concentration 

of that Aroclor. 

15.5.2 Other multi-component analytes 

Quantify any other multi-component analytes (technical chlordane or toxaphene) using the 

same peaks used to develop the average calibration factors in Section 7.5.2. Determine the 

concentration of each of the characteristic peaks, and then the concentrations represented

by those characteristic peaks are averaged to determine the concentration of the analyte. 

Alternatively, for toxaphene, the analyst may determine the calibration factor in Section 

7.5.2 by summing the areas of all of the peaks for the analyte and using the summed of the 

peak areas in the sample chromatogram to determine the concentration. However, the 

approach used for toxaphene must be the same for the calibration and the sample analyses.

15.6 Reporting of results

As noted in Section 1.6.1, EPA has promulgated this method at 40 CFR Part 136 for use in 

wastewater compliance monitoring under the National Pollutant Discharge Elimination System 

(NPDES). The data reporting practices described here are focused on such monitoring needs and 

may not be relevant to other uses of the method. 

15.6.1 Report results for wastewater samples in μg/L without correction for recovery. (Other units 

may be used if required by in a permit.) Report all QC data with the sample results.

15.6.2 Reporting level

Unless otherwise specified in by a regulatory authority or in a discharge permit, results for 

analytes that meet the identification criteria are reported down to the concentration of the 

ML established by the laboratory through calibration of the instrument (see Section 7.5 or 

7.6 and the glossary for the derivation of the ML). EPA considers the terms “reporting 

limit,” “quantitation limit,” and “minimum level” to be synonymous. 

15.6.2.1 Report the lower result from the two columns (see Section 15.7 below) for each 

analyte in each sample, blank, or standard at or above the ML to 3 significant 

figures. Report a result for each analyte found in each sample below the ML as 

“<ML,” or as required by the regulatory authority or permit. Results are reported 

without blank subtraction unless requested or required by a regulatory authority 

or in a permit. In this case, both the sample result and the blank results must be 

reported together. 

15.6.2.2 In addition to reporting results for samples and blank(s) separately, the 

concentration of each analyte in a blank or field blank associated with that 

Method 608.3 38 December 2014
sample may be subtracted from the result for that sample, but only if requested or 

required by a regulatory authority or in a permit. In this case, both the sample 

result and the blank results must be reported together. 

15.6.2.3 Report the result for an analyte in a sample or extract that has been diluted at the 

least dilute level at which the peak area is within the calibration range (i.e., above 

the ML for the analyte) and the MS/MSD recovery and RPD are within their 

respective QC acceptance criteria (Table 4). This may require reporting results 

for some analytes from different analyses.

The results for each analyte in the MS/MSD samples should be reported from the 

same GC column as used to report the results for that analyte in the unspiked 

sample. If the MS/MSD recoveries and RPDs calculated in this manner do not 

meet the acceptance criteria in Table 4, then the analyst may use the results from 

the other GC column to determine if the MS/MSD results meet the acceptance 

criteria. If such a situation occurs, the results for the sample should be 

recalculated using the same GC column data as used for the MS/MSD samples, 

and reported with appropriate annotations that alert the data user of the issue.

15.6.2.4 Results from tests performed with an analytical system that is not in control (i.e., 

that does not meet acceptance criteria for all of QC tests in this method) must not 

be reported or otherwise used for permitting or regulatory compliance purposes, 

but do not relieve a discharger or permittee of reporting timely results. If the 

holding time would be exceeded for a re-analysis of the sample, the 

regulatory/control authority should be consulted for disposition. 

15.6.3 Analyze the sample by GC/MS or on a third column when analytes have co-eluted or 

interfere with determination on both columns. 

Note: Dichlone and kepone do not elute from the DB-1701 column and must be 

confirmed on a DB-5 column, or by GC/MS. 

15.7 Quantitative information that may aid in the confirmation of the presence of an analyte 

15.7.1 As noted in Section 14.3, the relative agreement between the numerical results from the 

two GC columns may be used to support the identification of the target analyte by 

providing evidence that that co-eluting interferences are not present at the retention time of 

the target analyte. Calculate the percent difference (%D) between the results for the analyte 

from both columns, as follows: 

%D = 

Higher result − Lower result

Higher result x 100

In general, if the %D of the two results is less than 50% (e.g., a factor of 2), then the 

pesticide is present. This %D is generous and allows for the pesticide that has the largest 

measurement error.

Note: Laboratories may employ metrics less than 50% for this comparison, including 

those specified in other analytical methods for these pesticides (e.g., CLP or 

SW-846). 

Method 608.3 39 December 2014
15.7.2 If the amounts do not agree, and the RT data indicate the presence of the analyte (per 

Section 14), it is likely that a positive interference is present on the column that yielded the 

higher result. That interferent may be represented by a separate peak on the other column

that does not coincide with the retention time of any of the target analytes. If the 

interfering peak is evident on the other column, report the result from that column and 

advise the data user that the interference resulted in a %D value greater than 50%.

 If an interferent is not identifiable on the second column, then the results must be reported 

as “not detected” at the lower concentration. In this event, the pesticide is not confirmed 

and the reporting limit is elevated.

Note: The resulting elevation of the reporting limit may not meet the requirements for 

compliance monitoring and the use of additional cleanup procedures may be 

required.

16. Analysis of Complex Samples

16.1 Some samples may contain high levels (greater than 1 μg/L) of the analytes of interest, interfering 

analytes, and/or polymeric materials. Some samples may not concentrate to 1.0 mL (Section 

10.3.3.3.2); others may overload the GC column and/or detector. 

16.2 When an interference is known or suspected to be present, the laboratory should attempt to clean 

up the sample extract using the SPE cartridge (Section 11.2), by Florisil® (Section 11.3), Alumina 

(Section 11.4), sulfur removal (Section 11.5), or another clean up procedure appropriate to the 

analytes of interest. If these techniques do not remove the interference, the extract is diluted by a 

known factor and reanalyzed (Section 12). Dilution until the extract is lightly colored is preferable. 

Typical dilution factors are 2, 5, and 10. 

16.3 Recovery of surrogate(s)—In most samples, surrogate recoveries will be similar to those from 

reagent water. If surrogate recovery is outside the range developed in Section 8.6, the sample is reextracted and reanalyzed if there is sufficient sample and if it is within the 7-day extraction holding 

time. If the surrogate recovery is still outside this range, extract and analyze one-tenth the volume 

of sample to overcome any matrix interference problems. If a sample is highly colored or 

suspected to be high in concentration, a 1-L sample aliquot and a 100-mL sample aliquot could be 

extracted simultaneously and still meet the holding time criteria, while providing information about 

a complex matrix. 

16.4 Recovery of the matrix spike and matrix spike duplicate (MS/MSD)—In most samples, MS/MSD 

recoveries will be similar to those from reagent water. If either the MS or MSD recovery is outside 

the range specified in Section 8.3.3, one-tenth the volume of sample is spiked and analyzed. If the 

matrix spike recovery is still outside the range, the result for the unspiked sample may not be 

reported or used for permitting or regulatory compliance purposes. Poor matrix spike recovery 

does not relieve a discharger or permittee of reporting timely results.

17. Method Performance

17.1 This method was tested for linearity of spike recovery from reagent water and has been 

demonstrated to be applicable over the concentration range from 4x MDL to 1000x MDL with the 

following exceptions: Chlordane recovery at 4x MDL was low (60%); Toxaphene recovery was 

demonstrated linear over the range of 10x MDL to 1000x MDL (Reference 3).

Method 608.3 40 December 2014
17.2 The 1984 version of this method was tested by 20 laboratories using reagent water, drinking water, 

surface water, and three industrial wastewaters spiked at six concentrations (Reference 2). 

Concentrations used in the study ranged from 0.5 to 30 μg/L for single-component pesticides and 

from 8.5 to 400 μg/L for multi-component analytes. These data are for a subset of analytes 

described in the current version of the method. 

17.3 During the development of Method 1656, a similar EPA procedure for the organochlorine 

pesticides, single-operator precision, overall precision, and method accuracy were found to be 

directly related to the concentration of the analyte and essentially independent of the sample 

matrix. Linear equations to describe these relationships are presented in Table 5. 

18. Pollution Prevention

18.1 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, the 

laboratory should use pollution prevention techniques to address waste generation. When wastes 

cannot be reduced at the source, the Agency recommends recycling as the next best option.

18.2 The analytes in this method 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. This method 

utilizes significant quantities of methylene chloride. Laboratories are encouraged to recover and 

recycle this and other solvents during extract concentration.

18.3 For information about pollution prevention that may be applied to laboratories and research 

institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction

(Reference 19).

19. Waste Management 

19.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing 

waste management, particularly the hazardous waste identification rules and land disposal 

restrictions, and to protect the air, water, and land by minimizing and controlling all releases from 

fume hoods and bench operations. Compliance is also required with any sewage discharge permits 

and regulations. An overview of requirements can be found in Environmental Management Guide 

for Small Laboratories (EPA 233-B-98-001). 

19.2 Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured down a 

drain, or must be handled as hazardous waste. 

19.3 Many analytes in this method decompose above 500 oC. Low-level waste such as absorbent paper, 

tissues, animal remains, and plastic gloves may be burned in an appropriate incinerator. Gross 

quantities of neat or highly concentrated solutions of toxic or hazardous chemicals should be 

packaged securely and disposed of through commercial or governmental channels that are capable 

of handling toxic wastes. 

Method 608.3 41 December 2014
20. References

1. “Determination of Pesticides and PCBs in Industrial and Municipal Wastewaters,” EPA 600/4-82-

023, National Technical Information Service, PB82-214222, Springfield, Virginia 22161, April 

1982. 

2. “EPA Method Study 18 Method 608-Organochlorine Pesticides and PCBs,” EPA 600/4-84-061, 

National Technical Information Service, PB84-211358, Springfield, Virginia 22161, June 1984. 

3. “Method Detection Limit and Analytical Curve Studies, EPA Methods 606, 607, and 608,” Special 

letter report for EPA Contract 68-03-2606, U.S. Environmental Protection Agency, Environmental 

Monitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980. 

4. ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practice for Preparation of 

Sample Containers and for Preservation of Organic Constituents,” American Society for Testing 

and Materials, Philadelphia.

5. Giam, C.S., Chan, H.S., and Nef, G.S. “Sensitive Method for Determination of Phthalate Ester 

Plasticizers in Open-Ocean Biota Samples,” Analytical Chemistry, 47, 2225 (1975). 

6. Giam, C.S. and Chan, H.S. “Control of Blanks in the Analysis of Phthalates in Air and Ocean 

Biota Samples,” U.S. National Bureau of Standards, Special Publication 442, pp. 701-708, 1976. 

7. Solutions to Analytical Chemistry Problems with Clean Water Act Methods, EPA 821-R-07-002, 

March 2007. 

8. “Carcinogens-Working With Carcinogens,” Department of Health, Education, and Welfare, Public 

Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, 

Publication No. 77-206, August 1977. 

9. “Occupational Exposure to Hazardous Chemicals in Laboratories,” (29 CFR Part 1910, Subpart 

1450), Occupational Safety and Health Administration, OSHA . 

10. 40 CFR 136.6(b)(4)(j) 

11. Mills, P.A. “Variation of Florisil Activity: Simple Method for Measuring Absorbent Capacity and 

Its Use in Standardizing Florisil Columns,” Journal of the Association of Official Analytical 

Chemists, 51, 29, (1968). 

12. 40 CFR 136.6(b)(2)(i) 

13. Protocol for EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater 

and Drinking Water (EPA-821-B-98-003) March 1999 

14. Methods 4500 Cl F and 4500 Cl G, Standard Methods for the Examination of Water and 

Wastewater, published jointly by the American Public Health Association, American Water Works 

Association, and Water Environment Federation, 1015 Fifteenth St., Washington, DC 20005, 20th 

Edition, 2000. 

15. “Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental 

Samples,” EPA-600/8-80-038, U.S. Environmental Protection Agency, Health Effects Research 

Laboratory, Research Triangle Park, North Carolina.

Method 608.3 42 December 2014
16. USEPA, 2000, Method 1656 Organo-Halide Pesticides In Wastewater, Soil, Sludge, Sediment, and

Tissue by GC/HSD, EPA-821-R-00-017, September 2000.

17. USEPA, 2010, Method 1668C Chlorinated Biphenyl Congeners in Water, Soil, Sediment,

Biosolids, and Tissue by HRGC/HRMS, EPA-820-R-10-005, April 2010.

18. USEPA, 2007, Method 1699: Pesticides in Water, Soil, Sediment, Biosolids, and Tissue by

HRGC/HRMS, EPA-821-R-08-001, December 2007.

19. “Less is Better,” American Chemical Society on-line publication,

http://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafety/publications/

less-is-better.pdf

20. EPA Method 608 ATP 3M0222, An alternative test procedure for the measurement of

organochlorine pesticides and polychlorinated biphenyls in waste water. Federal Register/Vol. 60,

No. 148 August 2, 1995

Method 608.3 43 December 2014
21. Tables

Table 1 – Pesticides 1

Analyte CAS Number

MDL2

(ng/L)

ML3

(ng/L

Aldrin 309-00-2 8 24

alpha-BHC 319-84-6 6 18

beta-BHC 319-85-7 7 21

delta-BHC 319-86-8 5 15

gamma-BHC (Lindane) 58-89-9 1 33

alpha-Chlordane 5103-71-9 9 27

gamma-Chlordane 5103-74-2 8 24

4,4'-DDD 72-54-8 5 15

4,4'-DDE 72-55-9 10 30

4,4'-DDT 50-29-3 12 36

Dieldrin 60-57-1 6 18

Endosulfan I 959-98-8 11 33

Endosulfan II 33213-65-9 8 24

Endosulfan sulfate 1031-07-8 7 21

Endrin 72-20-8 4 12

Endrin aldehyde 7421-93-4 11 33

Heptachlor 76-44-8 5 15

Heptachlor epoxide 1024-57-3 12 36

1 All analytes in this table are Priority Pollutants (40 CFR 423, Appendix A) 2 40 CFR 136, Appendix B. MDLs were obtained by a single laboratory 

with an electrolytic conductivity detector, and are estimates of what can be 

achieved using an electron capture detector. 3 ML = Minimum Level – see Glossary for definition and derivation

Method 608.3 44 December 2014
Table 2 – Additional Analytes

Analyte CAS Number

MDL3

(ng/L)

ML4

(ng/L

Acephate 30560-19-1 2,000 6,000

Alachlor 15972-60-8 20 60

Atrazine 1912-24-9 500 1,500

Benfluralin (Benefin) 1861-40-1 20 60

Bromacil 314-40-9 70 210

Bromoxynil octanoate 1689-99-2 30 90

Butachlor 23184-66-9 30 90

Captafol 2425-06-1 100 300

Captan 133-06-2 100 300

Carbophenothion (Trithion) 786-19-6 50 150

Chlorobenzilate 510-15-6 25 75

Chloroneb (Terraneb) 2675-77-6

Chloropropylate (Acaralate) 5836-10-2

Chlorothalonil 1897-45-6 15 45

Cyanazine 21725-46-2

DCPA (Dacthal) 1861-32-1 3 9

2,4'-DDD 53-19-0

2,4'-DDE 3424-82-6

2,4'-DDT 789-02-6

Diallate (Avadex) 2303-16-4 45 135

1,2-Dibromo-3-chloropropane (DBCP) 96-12-8

Dichlone 117-80-6

Dichloran 99-30-9

Dicofol 115-32-2

Endrin ketone 53494-70-5 8 24

Ethalfluralin (Sonalan) 55283-68-6 5 15

Etridiazole 2593-15-9

Fenarimol (Rubigan) 60168-88-9 20 30

Hexachlorobenzene 1 118-74-1

Hexachlorocyclopentadiene 1 77-47-4

Isodrin 465-73-6 13 39

Isopropalin (Paarlan) 33820-53-0 20 60

Kepone 143-50-0 100 300

Methoxychlor 72-43-5 30 90

Metolachlor 51218-45-2

Metribuzin 21087-64-9 5 15

Mirex 2385-85-5 4 12

Nitrofen (TOK) 1836-75-5 13 39

cis-Nonachlor 5103-73-1

trans-Nonachlor 39765-80-5

Norfluorazon 27314-13-2 50 150

Octachlorostyrene 29082-74-4

Oxychlordane 27304-13-8

PCNB (Pentachloronitrobenzene) 82-68-8 6 18

Pendamethalin (Prowl) 40487-42-1

cis-Permethrin 61949-76-6 200 600

trans-Permethrin 61949-77-7 200 600

Method 608.3 45 December 2014
Table 2 – Additional Analytes

Analyte CAS Number

MDL3

(ng/L)

ML4

(ng/L

Perthane (Ethylan) 72-56-0

Propachlor 1918-16-7

Propanil 709-98-8

Propazine 139-40-2

Quintozene 82-68-8

Simazine 122-34-9 400 1,200

Strobane 8001-50-1

Technazene 117-18-0

Technical Chlordane2

Terbacil 5902-51-2 200 600

Terbuthylazine 5915-41-3 300 900

Toxaphene 1 8001-35-2 910 2,730

Trifluralin 1582-09-8 50 150

PCB-1016 1 12674-11-2 150 450

PCB-1221 1 11104-28-2 150 450

PCB-1232 1 11141-16-5 150 450

PCB-1242 1 53469-21-9 150 450

PCB-1248 1 12672-29-6 150 450

PCB-1254 1 11097-69-1 150 450

PCB-1260 1 11096-82-5 140 420

1 Priority Pollutants (40 CFR 423, Appendix A) 2 Technical Chlordane may be used in cases where historical reporting has only been for this 

form of Chlordane. 3 40 CFR 136, Appendix B. MDLs were obtained by a single laboratory with an electrolytic 

conductivity detector, and are estimates of what can be achieved using an electron capture 

detector. 4 ML = Minimum Level – see Glossary for definition and derivation

Method 608.3 46 December 2014
Table 3 – Example Retention Times1

Analyte

Retention time (min)2

DB-608 DB-1701

Acephate 5.03 – 3

Trifluralin 5.16 6.79

Ethalfluralin 5.28 6.49

Benfluralin 5.53 6.87

Diallate-A 7.15 6.23

Diallate-B 7.42 6.77

alpha-BHC 8.14 7.44

PCNB 9.03 7.58

Simazine 9.06 9.29

Atrazine 9.12 9.12

Terbuthylazine 9.17 9.46

gamma-BHC (Lindane) 9.52 9.91

beta-BHC 9.86 11.90

Heptachlor 10.66 10.55

Chlorothalonil 10.66 10.96

Dichlone 10.80 – 4

Terbacil 11.11 12.63

delta-BHC 11.20 12.98

Alachlor 11.57 11.06

Propanil 11.60 14.10

Aldrin 11.84 11.46

DCPA 12.18 12.09

Metribuzin 12.80 11.68

Triadimefon 12.99 13.57

Isopropalin 13.06 13.37

Isodrin 13.47 11.12

Heptachlor epoxide 13.97 12.56

Pendamethalin 14.21 13.46

Bromacil 14.39 – 3

alpha-Chlordane 14.63 14.20

Butachlor 15.03 15.69

gamma-Chlordane 15.24 14.36

Endosulfan I 15.25 13.87

4,4’-DDE 16.34 14.84

Dieldrin 16.41 15.25

Captan 16.83 15.43

Chlorobenzilate 17.58 17.28

Endrin 17.80 15.86

Nitrofen (TOK) 17.86 17.47

Kepone 17.92 – 3, 5

4,4’-DDD 18.43 17.77

Endosulfan II 18.45 18.57

Bromoxynil octanoate 18.85 18.57

Method 608.3 47 December 2014
Table 3 – Example Retention Times1

Analyte

Retention time (min)2

DB-608 DB-1701

4,4’-DDT 19.48 18.32

Carbophenothion 19.65 18.21

Endrin aldehyde 19.72 19.18

Endosulfan sulfate 20.21 20.37

Captafol 22.51 21.22

Norfluorazon 20.68 22.01

Mirex 22.75 19.79

Methoxychlor 22.80 20.68

Endrin ketone 23.00 21.79

Fenarimol 24.53 23.79

cis-Permethrin 25.00 23.59

trans-Permethrin 25.62 23.92

PCB-1242

PCB-1232

PCB-1016

PCB-1221

PCB-1248

PCB-1254

PCB-1260 (5 peaks) 15.44 14.64

15.73 15.36

16.94 16.53

17.28 18.70

19.17 19.92

Toxaphene (5 peaks) 16.60 16.60

17.37 17.52

18.11 17.92

19.46 18.73

19.69 19.00

1 Data from EPA Method 1656 (Reference 16) 2 Columns: 30-m long x 0.53-mm ID fused-silica capillary; 

DB-608, 0.83 μm; and DB-1701, 1.0 μm. 

Conditions suggested to meet retention times shown: 

150 °C for 0.5 minute, 150-270 °C at 5°C/min, and 270 °C 

until trans-Permethrin elutes.

Carrier gas flow rates approximately 7 mL/min. 3 Does not elute from DB-1701 column at level tested. 4 Not recovered from water at the levels tested. 5 Dichlone and Kepone do not elute from the DB-1701 

column and should be confirmed on DB-5.

Method 608.3 48 December 2014
Table 4 – QC Acceptance Criteria

Analyte

Calibration 

verification 

(%)

Test 

concentration 

(μg/L)

Limit for 

s (% SD)

Range for 

X (%)

Range 

for P 

(%)

Maximum 

MS/MSD 

RPD (%)

Aldrin 75 - 125 2.0 25 54 - 130 42 - 140 35

alpha-BHC 69 - 125 2.0 28 49 - 130 37 - 140 36

beta-BHC 75 - 125 2.0 38 39 - 130 17 - 147 44

delta-BHC 75 - 125 2.0 43 51 - 130 19 - 140 52

gamma-BHC 75 - 125 2.0 29 43 - 130 32 - 140 39

alpha-Chlordane 73 - 125 50.0 24 55 - 130 45 - 140 35

gamma-Chlordane 75 - 125 50.0 24 55 - 130 45 - 140 35

4,4'-DDD 75 - 125 10.0 32 48 - 130 31 - 141 39

4,4'-DDE 75 - 125 2.0 30 54 - 130 30 - 145 35

4,4'-DDT 75 - 125 10.0 39 46 - 137 25 - 160 42

Dieldrin 48 - 125 2.0 42 58 - 130 36 - 146 49

Endosulfan I 75 - 125 2.0 25 57 - 141 45 - 153 28

Endosulfan II 75 - 125 10.0 63 22 - 171 D - 202 53

Endosulfan sulfate 70 - 125 10.0 32 38 - 132 26 - 144 38

Endrin 5 - 125 10.0 42 51 - 130 30 - 147 48

Heptachlor 75 - 125 2.0 28 43 - 130 34 - 140 43

Heptachlor epoxide 75 - 125 2.0 22 57 - 132 37 - 142 26

Toxaphene 68 - 134 50.0 30 56 - 130 41 - 140 41

PCB-1016 75 - 125 50.0 24 61 - 103 50 - 140 36

PCB-1221 75 - 125 50.0 50 44 - 150 15 - 178 48

PCB-1232 75 - 125 50.0 32 28 - 197 10 - 215 25

PCB-1242 75 - 125 50.0 26 50 - 139 39 - 150 29

PCB-1248 75 - 125 50.0 32 58 - 140 38 - 158 35

PCB-1254 75 - 125 50.0 34 44 - 130 29 - 140 45

PCB-1260 75 - 125 50.0 28 37 - 130 8 - 140 38

S = Standard deviation of four recovery measurements (Section 8.2.4).

Note: These criteria were developed from data in Table 5 (Reference 2). Where necessary, limits for recovery have 

been broadened to assure applicability to concentrations below those in Table 5. 

Method 608.3 49 December 2014
Table 5 – Precision and Recovery as Functions of Concentration

Analyte

Recovery, X'

(μg/L)

Single analyst 

precision, sr' (μg/L)

Overall precision, 

S' (μg/L)

Aldrin 0.81C + 0.04 0.16( X ) - 0.04 0.20( X ) - 0.01

alpha-BHC 0.84C + 0.03 0.13( X ) + 0.04 0.23( X ) - 0.00

beta-BHC 0.81C + 0.07 0.22( X ) - 0.02 0.33( X ) - 0.05

delta-BHC 0.81C + 0.07 0.18( X ) + 0.09 0.25( X ) + 0.03

gamma-BHC (Lindane) 0.82C - 0.05 0.12( X ) + 0.06 0.22( X ) + 0.04

Chlordane 0.82C - 0.04 0.13 ( X ) + 0.13 0.18( X ) + 0.18

4,4'-DDD 0.84C + 0.30 0.20( X ) - 0.18 0.27( X ) - 0.14

4,4'-DDE 0.85C + 0.14 0.13( X ) + 0.06 0.28( X ) - 0.09

4,4'-DDT 0.93C - 0.13 0.17( X ) + 0.39 0.31( X ) - 0.21

Dieldrin 0.90C + 0.02 0.12( X ) + 0.19 0.16( X ) + 0.16

Endosulfan I 0.97C + 0.04 0.10( X ) + 0.07 0.18( X ) + 0.08

Endosulfan II 0.93C + 0.34 0.41( X ) - 0.65 0.47( X ) - 0.20

Endosulfan sulfate 0.89C - 0.37 0.13( X ) + 0.33 0.24( X ) + 0.35

Endrin 0.89C - 0.04 0.20( X ) + 0.25 0.24( X ) + 0.25

Heptachlor 0.69C + 0.04 0.06( X ) + 0.13 0.16( X ) + 0.08

Heptachlor epoxide 0.89C + 0.10 0.18( X ) - 0.11 0.25( X ) - 0.08

Toxaphene 0.80C + 1.74 0.09( X ) + 3.20 0.20( X ) + 0.22

PCB-1016 0.81C + 0.50 0.13( X ) + 0.15 0.15( X ) + 0.45

PCB-1221 0.96C + 0.65 0.29( X ) - 0.76 0.35( X ) - 0.62

PCB-1232 0.91C + 10.8 0.21( X ) - 1.93 0.31( X ) + 3.50

PCB-1242 0.93C + 0.70 0.11( X ) + 1.40 0.21( X ) + 1.52

PCB-1248 0.97C + 1.06 0.17( X ) + 0.41 0.25( X ) - 0.37

PCB-1254 0.76C + 2.07 0.15( X ) + 1.66 0.17( X ) + 3.62

PCB-1260 0.66C + 3.76 0.22( X ) - 2.37 0.39( X ) - 4.86

X' = Expected recovery for one or more measurements of a sample containing a concentration of C, in μg/L.

Method 608.3 50 December 2014
Table 6 – Distribution of Chlorinated Pesticides and PCBs into Florisil® Column Fractions

Analyte

Percent Recovery by Fraction1

1 2 3

Aldrin 100

alpha-BHC 100

beta-BHC 97

delta-BHC 98

gamma-BHC (Lindane) 100

Chlordane 100

4,4'-DDD 99

4,4'-DDE 98

4,4'-DDT 100

Dieldrin 0 100

Endosulfan I 37 64

Endosulfan II 0 7 91

Endosulfan sulfate 0 0 106

Endrin 4 96

Endrin aldehyde 0 68 26

Heptachlor 100

Heptachlor epoxide 100

Toxaphene 96

PCB-1016 97

PCB-1221 97

PCB-1232 95 4

PCB-1242 97

PCB-1248 103

PCB-1254 90

PCB-1260 95

1 Eluant composition:

Fraction 1 - 6% ethyl ether in hexane

Fraction 2 - 15% ethyl ether in hexane

Fraction 3 - 50% ethyl ether in hexane

Method 608.3 51 December 2014
22. Figures

Figure 1 Example Chromatogram of Selected Organochlorine Pesticides 

Method 608.3 52 December 2014
Figure 2 Disk-based solid-phase extraction apparatus 

Method 608.3 53 December 2014
23. Glossary

These definitions and purposes are specific to this method but have been conformed to common 

usage to the extent possible.

23.1 Units of weight and measure and their abbreviations

23.1.1 Symbols

oC degrees Celsius

μg microgram

μL microliter

< less than

≤ less than or equal to

> greater than

% percent

23.1.2 Abbreviations (in alphabetical order)

cm centimeter

g gram

hr hour

ID inside diameter

in. inch

L liter

M molar solution - one mole or gram molecular weight of solute in one liter of solution

mg milligram

min minute

mL milliliter

mm millimeter

N Normality - one equivalent of solute in one liter of solution

ng nanogram

psia pounds-per-square inch absolute

psig pounds-per-square inch gauge

v/v volume per unit volume

w/v weight per unit volume

23.2 Definitions and acronyms (in alphabetical order)

Analyte – A compound or mixture of compounds (e.g., PCBs) tested for by this method. The 

analytes are listed in Tables 1 and 2.

Analytical batch – The set of samples analyzed on a given instrument during a 24-hour period that 

begins and ends with calibration verification (Sections 7.8 and 13). See also “Extraction batch.”

Blank (method blank; laboratory blank) – An aliquot of reagent water that is treated exactly as a 

sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and 

surrogates that are used with samples. The blank is used to determine if analytes or interferences 

are present in the laboratory environment, the reagents, or the apparatus.

Calibration factor (CF) – See Section 7.5.1.

Method 608.3 54 December 2014
Calibration standard – A solution prepared from stock solutions and/or a secondary standards and 

containing the analytes of interest, surrogates, and internal standards. This standard is used to 

model the response of the GC instrument against analyte concentration.

Calibration verification – The process of confirming that the response of the analytical system 

remains within specified limits of the calibration.

Calibration verification standard – The combined QC standard (Section 7.7) used to verify 

calibration (Section 13.5) and for LCS tests (Section 8.4).

Extraction Batch – A set of up to 20 field samples (not including QC samples) started through the 

extraction process in a given 24-hour shift. Each extraction batch of 20 or fewer samples must be 

accompanied by a blank (Section 8.5), a laboratory control sample (LCS, Section 8.4), a matrix 

spike and duplicate (MS/MSD; Section 8.3), resulting in a minimum of five samples (1 field 

sample, 1 blank, 1 LCS, 1 MS, and 1 MSD) and a maximum of 24 samples (20 field samples, 1 

blank, 1 LCS, 1 MS, and 1 MSD) for the batch. If greater than 20 samples are to be extracted in a 

24-hour shift, the samples must be separated into extraction batches of 20 or fewer samples.

Field Duplicates – Two samples collected at the same time and place under identical conditions, and 

treated identically throughout field and laboratory procedures. Results of analyses the field 

duplicates provide an estimate of the precision associated with sample collection, preservation, and 

storage, as well as with laboratory procedures.

Field blank – An aliquot of reagent water or other reference matrix that is placed in a sample 

container in the field, and treated as a sample in all respects, including exposure to sampling site 

conditions, storage, preservation, and all analytical procedures. The purpose of the field blank is to 

determine if the field or sample transporting procedures and environments have contaminated the 

sample. See also “Blank.”

GC – Gas chromatograph or gas chromatography

Gel-permeation chromatography (GPC) – A form of liquid chromatography in which the analytes 

are separated based on exclusion from the solid phase by size.

Internal standard – A compound added to an extract or standard solution in a known amount and 

used as a reference for quantitation of the analytes of interest and surrogates. Also see Internal 

standard quantitation.

Internal standard quantitation – A means of determining the concentration of an analyte of interest 

(Tables 1 and 2) by reference to a compound not expected to be found in a sample.

IDC – Initial Demonstration of Capability (Section 8.2); four aliquots of a reference matrix spiked 

with the analytes of interest and analyzed to establish the ability of the laboratory to generate 

acceptable precision and recovery. An IDC is performed prior to the first time this method is used 

and any time the method or instrumentation is modified.

Laboratory Control Sample (LCS; laboratory fortified blank; Section 8.4) – An aliquot of reagent 

water spiked with known quantities of the analytes of interest and surrogates. The LCS is analyzed 

exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain 

within the limits specified in this method for precision and recovery.

Laboratory Fortified Sample Matrix – See Matrix spike

Method 608.3 55 December 2014
Laboratory reagent blank – See blank

Matrix spike (MS) and matrix spike duplicate (MSD) (laboratory fortified sample matrix and 

duplicate) – Two aliquots of an environmental sample to which known quantities of the analytes of 

interest and surrogates are added in the laboratory. The MS/MSD are prepared and analyzed 

exactly like a field sample. Their purpose is to quantify any additional bias and imprecision 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/MSD corrected for background 

concentrations.

May – This action, activity, or procedural step is neither required nor prohibited.

May not – This action, activity, or procedural step is prohibited.

Method detection limit (MDL) – A detection limit determined by the procedure at 40 CFR 136, 

Appendix B. The MDLs determined by EPA are listed in Tables 1 and 2. As noted in Sec. 1.6, use 

the MDLs in Tables 1 and 2 in conjunction with current MDL data from the laboratory actually 

analyzing samples to assess the sensitivity of this procedure relative to project objectives and 

regulatory requirements (where applicable)

Minimum level (ML) – The term “minimum level” refers to either the sample concentration 

equivalent to the lowest calibration point in a method or a multiple of the method detection limit 

(MDL), whichever is higher. Minimum levels may be obtained in several ways: They may be 

published in a method; they may be based on the lowest acceptable calibration point used by a 

laboratory; or they may be calculated by multiplying the MDL in a method, or the MDL determined 

by a laboratory, by a factor of 3. For the purposes of NPDES compliance monitoring, EPA

considers the following terms to be synonymous: “quantitation limit,” “reporting limit,” and 

“minimum level.” 

MS – Mass spectrometer or mass spectrometry

Must – This action, activity, or procedural step is required.

Preparation blank – See blank

Quality control sample (QCS) – A sample containing analytes of interest at known concentrations. 

The QCS is obtained from a source external to the laboratory or is prepared from standards obtained 

from a different source than the calibration standards. The purpose is to check laboratory 

performance using test materials that have been prepared independent of the normal preparation 

process.

Reagent water – Water demonstrated to be free from the analytes of interest and potentially 

interfering substances at the MDLs for the analytes in this method.

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/control 

authority.

Relative standard deviation (RSD) – The standard deviation times 100 divided by the mean. Also 

termed “coefficient of variation.”

RF – Response factor. See Section 7.6.2

Method 608.3 56 December 2014
RPD – Relative percent difference

RSD – See relative standard deviation

Safety Data Sheet (SDS) – Written information on a chemical’s toxicity, health hazards, physical 

properties, fire, and reactivity, including storage, spill, and handling precautions that meet the 

requirements of OSHA, 29 CFR 1910.1200(g) and Appendix D. United Nations Globally 

Harmonized System of Classification and Labelling of Chemicals (GHS), third revised edition, 

United Nations, 2009.

Should – This action, activity, or procedural step is suggested but not required.

SPE – Solid-phase extraction; a sample extraction or extract cleanup technique in which an analyte 

is selectively removed from a sample or extract by passage over or through a material capable of 

reversibly adsorbing the analyte.

Stock solution – A solution containing an analyte that is prepared using a reference material 

traceable to EPA, the National Institute of Science and Technology (NIST), or a source that will 

attest to the purity and authenticity of the reference material.

Surrogate – A compound unlikely to be found in a sample, which is spiked into the sample in a 

known amount before extraction, and which is quantified with the same procedures used to 

quantify other sample components. The purpose of the surrogate is to monitor method performance 

with each sample.

Method 608.3 57 December 2014