United States
Environmental Protection
^1 Agency
Office of Water EPA 821-R-16-009
www.epa.gov December 2016
Method 608.3: Organochlorine
Pesticides and
PCBs by GC/HSD
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This document contains the text of Method 608.3 as published in 40 CFR part 136, Appendix A; but
formatted as a more user friendly stand-alone document.
Please address questions or comments to:
CWA Methods Team
Engineering and Analytical Support Branch/EAD (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460
www.epa.gov/cwa-methods/forms/contact-us-about-cwa-analytical-methods
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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 625.1 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 part 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
Method 608.3
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writing of this revision, Methods 1668C and 1699 had not been approved for use at 40 CFR part
136.
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 (49 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 15.6 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.
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.
Method 608.3
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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
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 - see Glossary for detailed definition), as described in
Section 8.5. Specific selection of reagents and purification of solvents by distillation in all-glass
systems may be required. Where possible, labware is cleaned by extraction or solvent rinse, or
baking in a kiln or oven.
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. Do not heat
volumetric labware above 90 °C. After drying and cooling, store inverted or capped with solvent-
rinsed or baked aluminum foil in a clean environment to prevent accumulation of dust or other
contaminants.
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
Method 608.3
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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 matrix spike duplicate (Section 8.3) may be useful in identifying matrix interferences,
and the cleanup 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 Tables 1 and 2.
4. Safety
4.1 Hazards associated with each reagent used in this method have 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 1910.12009(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
Method 608.3
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of compressible silicone rubber tubing may be used. Before use, rinse the compressible
tubing thoroughly with methanol, followed by repeated rinsing with reagent water to
minimize the potential for sample contamination. An 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
Note: The approved ATP for solid-phase extraction is limited to disk-based extraction
media and associated peripheral equipment.
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 single-
hole rubber stopper and glass tubing
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 l-(j,m 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).
Method 608.3
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5.2.3.2 Glass wool - Pyrex, extracted with methylene chloride or baked at 450 °C for 1
hour minimum
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.
5.2.5.3.1 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.
5.2.5.3.2 Round-bottom flask - 100-mL and 500-mL or larger, with ground-
glass 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).
Method 608.3
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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.
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.
5.2.6 Solid-phase extraction disks - 90-mm extraction disks containing 2 g of 8-|im octadecyl
(CI 8) 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
Method 608.3
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5.5.3.4 Rack for holding 50-mL volumetric flasks in the manifold
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)
5.5.4 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-|a,m
film thickness
5.8.1.2 DB-1701 (or equivalent), 30-m long x 0.53-mm ID fused-silica capillary, 1.0-|a,m
film thickness
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
Method 608.3
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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 H2S04 (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.
6.3 Sodium sulfate - Sodium sulfate, reagent grade, granular anhydrous (Baker or equivalent), rinsed
with methylene chloride, 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.
Method 608.3
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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.
6.7.1.1 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 6.7.1.3) 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.
6.7.1.2 Concentrate the eluantto per Section 10.3.3, exchange to isooctane or hexane per
Section 10.3.3, and inject 1.0 |o,L 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), the recovery of trichlorophenol shall be less than 5%, and no peaks
interfering with the target analytes shall be detected. Otherwise the Florisil®
cartridge is not performing properly and the cartridge lot shall be rejected.
6.7.1.3 Florisil® cartridge calibration solution - 2,4,6-Trichlorophenol, 0.1 |a,g/mL in
acetone.
6.7.2 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 before it may 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.
6.7.4.1.2 Add HC1 dropwise (0.5-1.0 mL) while swirling, until the copper
brightens.
Method 608.3
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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.
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.
Inspect the copper foil or powder before each use. It must have a
bright, non-oxidized appearance to be effective. Copper foil or
powder that has oxidized may be reactivated using the procedure
described above.
6.7.4.2 Tetrabutylammonium sulfite (TBA sulfite) - Prepare as described below.
6.7.4.2.1 Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHS04
6.7.4.2.2 Sodium sulfite, Na2S03
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.
6.7.5 Sodium chloride - Reagent grade, prepare at 5% (w/v) solution in reagent water.
6.8 Stock standard solutions - Stock standard solutions may be prepared from pure materials, or
purchased as certified solutions. Traceability must be to the National Institute of Standards and
Technology (NIST) or other national or international standard, when available. Stock solution
concentrations alternative to those below may be used. Because of the toxicity of some of the
compounds, primary dilutions should be prepared in a hood, and a NIOSH/MESA approved toxic
gas respirator should be worn when high concentrations of neat materials are handled. The
following procedure may be used to prepare standards from neat materials.
6.8.1 Accurately weigh about 0.0100 g of pure material in a 10-mL volumetric flask. Dilute to
volume in pesticide quality hexane, isooctane, or other suitable solvent. Larger volumes
may be used at the convenience of the laboratory. When compound purity is assayed to be
96% or greater, the weight may be used without correction to calculate the concentration of
the stock standard. Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or by an independent source.
6.8.1.1 Unless stated otherwise in this method, store non-aqueous standards in
fluoropolymer-lined screw-cap, or heat-sealed, glass containers, in the dark at
-20 to -10 °C. Store aqueous standards; e.g., the aqueous LCS (Section 8.4), in the
dark at < 6 °C, but do not freeze.
6.8.1.2 Standards prepared by the laboratory may be stored for up to one year, except
when comparison with QC check standards indicates that a standard has degraded
or become more concentrated due to evaporation, or unless the laboratory has data
on file to prove stability for a longer period. Commercially prepared standards
6.7.4.1.3
6.7.4.1.4
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may be stored until the expiration date provided by the vendor, except when
comparison with QC check standards indicates that a standard has degraded or
become more concentrated due to evaporation, or unless the laboratory has data
from the vendor on file to prove stability for a longer period.
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 at or below the ML
specified in Table 1, or 2, or as specified by a regulatory/control authority or in a
permit. The ML value may be rounded to a whole number that is more
convenient for preparing the standard, but must not exceed the ML value listed in
Tables 1 or 2 for those analytes which list ML values. Alternatively, the
laboratory may establish an ML for each analyte based on the concentration of
the lowest calibration standard in a series of standards produced by the laboratory
or obtained from a commercial vendor, again, provided that the ML does not
exceed the ML 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 = 0) calibration (Section 7.5.2 or
7.6.2). Calibrations higher than second order are not allowed. A separate
standard near the MDL may be analyzed as a check on sensitivity, but should not
be included in the linearity assessment. 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.
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.
Divide the analytes into 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). Table 7 provides
information on dividing the target analytes into separate calibration mixtures that
should minimize or eliminate co-elutions. This table is provided solely as
guidance, based on the GC columns suggested in this method. If an analyte
listed in Table 7 is not an analyte of interest in a given laboratory setting, then it
need not be included in a calibration mixture.
Note: Many commercially available standards are divided into separate
mixtures to address this issue.
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If co-elutions occur in analysis if a sample, a co-elution on one column is
acceptable so long as effective separation of the co-eluting compounds can be
achieved on the second column.
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
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 Concentrate - Prepare one or more mid-level
standard mixtures (concentrates) in acetone (or other water miscible solvent). The
concentrate is used as the spiking solution with which to prepare the Demonstration of
Capabilities (DOC) samples, the Laboratory Control Sample (LCS), and Matrix Spike (MS)
and Matrix Spike Duplicate (MSD) samples described in Section 8. If prepared by the
laboratory (as opposed the purchasing it from a commercial supplier), the concentrate must
be prepared independently from the standards used for calibration, but may be prepared
from the same source as the second-source standard used for calibration verification
(Section 7.7). Regardless of the source, the concentrate must be in a water-miscible
solvent, as noted above.
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The concentrate is used to prepare the DOC and LCS (Sections 8.2.1 and 8.4) and
MS/MSD samples (Section 8.3). Depending on the analytes of interest for a given sample
(see Section 1.4), multiple solutions and multiple LCS or MS/MSD samples may be
required to account for co-eluting analytes. However, a co-elution on one column is
acceptable so long as effective separation of the co-eluting compounds can be achieved on
the second column. In addition, the concentrations of the MS/MSD samples should reflect
any relevant compliance limits for the analytes of interest, as described in Section 8.3.1. If
a custom spiking solution is required for a specific discharge (Section 8.3.1), prepare it
separately from the DOC and LCS solution.
Note: Some commercially available standards are divided into separate mixtures to
address the co-elution issue.
6.8.4 Calibration Verification Standards - In order to verify the results of the initial calibration
standards, prepare one or more mid-level standard mixtures in isooctane or hexane, using
standards obtained from a second source (different manufacturer or different certified lot
from the calibration standards). These standards will be analyzed to verify the accuracy of
the calibration (Sections 7.7 and 13.6.2). As with the QC sample concentrate in Section
6.8.3, multiple solutions may be required to address co-elutions among all of the analytes.
6.8.5 Internal standard solution - If the internal standard calibration technique is to be used,
prepare pentachloronitrobenzene (PCNB) at a concentration of 10 |a,g/mL in ethyl acetate.
Alternative and multiple internal standards; e.g., tetrachloro-m-xylene, 4,4'-dibromo-
biphenyl, and/or decachlorobiphenyl may be used provided that the laboratory performs all
QC tests and meets all QC acceptance criteria with the alternative 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 |a,g/mL in acetone. Potential surrogates include: dibutyl chlorendate
(DBC), tetrachloro-m-xylene (TCMX), 4,4'-dibromobiphenyl, or decachlorobiphenyl.
Alternative surrogates and concentrations may be used, provided 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 50 ng/mL and 4,4'-DDT at a concentration of 100 ng/mL, in
isooctane or hexane. A 1-fj.L injection of this standard will contain 50 picograms (pg) of
endrin and 100 pg of DDT. The concentration of the solution may be adjusted by the
laboratory to accommodate other injection volumes such that the same masses of the two
analytes are introduced into the instrument.
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.
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7.2 Separately inject the mid-level calibration standard for each calibration mixture. Store the
retention time on each GC column.
7.3 Injection of calibration solutions - Inject a constant volume in the range of 0.5 to 2.0 |a,L of each
calibration solution into the GC column/detector pairs. An alternative volume (see Section 12.3)
may be used provided all requirements in this method are met. 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. An instrument blank should be analyzed
after the highest standard to demonstrate that there is no carry-over within the system for this
calibration range.
7.4 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:
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, As, vs. concentration Cs. If used, the regression
must be weighted inversely proportional to concentration. The coefficient of determination
(R2) of the weighted regression must be greater than 0.920. 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.
Method 608.3
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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 = (A1xQ!)
(Ais x Cs)
where:
As = Response for the analyte to be measured.
Ajs = Response for the internal standard.
C1S = 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/A1S, vs. concentration ratios, CS/C1S, 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 coefficient of determination of the weighted regression must be
greater than 0.920. Alternatively, the relative standard error (Reference 10) may 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.
7.7 The working calibration curve, CF, or RF must be verified immediately after calibration and at the
beginning and end of each 24-hour shift by the analysis of a mid-level calibration standard. The
calibration verification standard(s) must be obtained from a second manufacturer or a
manufacturer's batch prepared independently from the batch used for calibration (Section 6.8.4).
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). The time for the
injections may not exceed 24 hours.
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 injections may be used as the beginning verification for the next group of
injections.
7.8 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.
Method 608.3
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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 must repeat demonstration of capability
(DOC) at least 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 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
Method 608.3
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routine QC required by this method (for example it could be
performed by the vendor of an alternative 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 publicly owned treatment works (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 www.epa.gov/eg/industrial-effluent-guidelines 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:
(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) CaC03 greater than 140 mg/L
The interim acceptance criteria for MS, MSD recoveries that do
not have recovery limits in Table 4 or developed in Section 8.3.3,
and for surrogates that do not have recovery limits developed in
Section 8.6, 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 in Table 4 or developed in
Section 8.3.3, 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, and business 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.
Method 608.3
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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.
1) 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
Tables 1 and 2 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 samples to monitor and
evaluate method and laboratory performance on the sample matrix. The procedure for
spiking and analysis is given 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,
Method 608.3
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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 part 136, appendix B. The
laboratory's MDLs must be equal to or lower than those listed in Tables 1 or 2, 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 part 136, appendix
B under the same conditions used to determine the MDLs for the analytes listed in Tables 1 and 2.
When analyzing the PCBs as Aroclors, it is only necessary to establish an MDL for one of the
multi-component analytes (e.g., PCB 1254), or the mixture of Aroclors 1016 and 1260 may be used
to establish MDLs for all of the Aroclors. Similarly, MDLs for other multi-component analytes
(e.g., Chlordanes) may be determined using only one of the major components. All procedures
used in the analysis, including cleanup procedures, must be included in the DOC.
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.
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.
Method 608.3
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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 purpose of the MS/MSD requirement is
to provide data that demonstrate the effectiveness of the method as applied to the samples in
question by a given laboratory, and both the data user (discharger, permittee, regulated entity,
regulatory/control authority, customer, other) and the laboratory share responsibility for provision
of such data. The data user should identify the sample and the analytes of interest (Section 1.4) to
be spiked and provide sufficient sample volume to perform MS/MSD analyses. The laboratory
must, on an ongoing basis, spike at least 5% of the samples in duplicate from each discharge being
monitored to assess accuracy (recovery and precision). If direction cannot be obtained from the
data user, the laboratory must spike at least one sample in duplicate per extraction batch of up to 20
samples 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, or in a
permit.
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
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.
.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 anew
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 (Ai and A2) of each analyte.
Calculate the percent recoveries (Pi and P2) as:
Ax-B
Px= - x 100
where T is the known true value of the spike.
Also calculate the relative percent difference (RPD) between the concentrations (Ai and
A2):
| A^ A21
RPD = ~a7T~a7" x 100
Method 608.3
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8.3.3 Compare the percent recoveries (Pi 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 4, 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,
and if the laboratory chooses to develop and apply optional in-house QC limits, the
laboratory should calculate and apply the optional in-house QC limits for recovery and
RPD of future MS/MSD samples (Section 8.3). The optional in-house 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 and the remaining analytes (those not included in the 80%) must meet
the acceptance criteria 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. Alternatively, surrogate recovery limits may be developed from laboratory
control charts. In-house QC acceptance criteria must be updated at least every two years.
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 (Sections 6.8.3 and 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).
Method 608.3
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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:
A
Ps = - 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), or limits based on
laboratory control charts. 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 analysis of the LCS prepared with a fresh sample
concentrate, or to system repair, repeat the LCS test (Section 8.4). If failure of the LCS
indicates a systemic problem with samples in the batch, re-extract and re-analyze the
samples in the batch. See Section 8.1.7 for disposition of repeated failures.
8.4.5 After analysis of 20 LCS samples, and if the laboratory chooses to develop and apply
optional in-house QC limits, the laboratory should calculate and apply the optional in-house
QC limits for recovery of future LCS samples (Section 8.4). Limits for recovery in the
LCS should be 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, and the remaining analytes (those not included in the 80%) must meet the
acceptance criteria in Table 4. If an in-house lower limit for recovery is lower than the
lower limit in 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. Many of the analytes and surrogates do not contain acceptance criteria. The
laboratory should use 60 -140% as interim acceptance criteria for recoveries of spiked
analytes and surrogates that do not have recovery limits specified in Table 4, and at least
80% of the surrogates must meet the 60 - 140% interim criteria until in-house LCS and
surrogate limits are developed. Alternatively, acceptance criteria for analytes that do not
have recovery limits in Table 4 may be based on laboratory control charts. In-house QC
acceptance criteria must be updated at least every two years.
8.5 Blank - Extract and analyze a blank with each extraction batch (Section 3.1) to demonstrate that
the reagents and equipment used for preparation and analysis are free from contamination.
Method 608.3
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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 - 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 (Section 8.4). 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;
otherwise, see Section 8.1.7 for disposition of repeated failures.
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 8.3, 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). If desired, statements of accuracy for
laboratory performance, independent of performance on samples, may be developed using LCSs.
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.
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
Method 608.3
24
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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 a 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. Pipetthe 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 8.3 or
8.4) 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. Ofparticular 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.
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,
Method 608.3
25
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filtration of the emulsion through glass wool, use of phase-separation paper, centrifugation,
salting, 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 |_iL (0.5 mL) ofisooctane
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.
10.3.1.5 If the extract is to be cleaned up by sulfur removal or acid back extraction,
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) or acid back extraction
(Section 11.6). If the extract is to cleaned up using one of the other cleanup
procedures or is to be injected into the GC, proceed to Kuderna-Danish micro-
Method 608.3
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concentration (Section 10.3.2) or nitrogen evaporation and solvent exchange
(Section 10.3.3).
10.3.2 Kuderna-Danish micro concentration - 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 (J.L. 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 (J.L. 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
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
< 6 C. Analyze the extract by GC per the procedure in Section 12.
Method 608.3
27
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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 to a range
of 5.0 - 9.0 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
transferthe 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 solid-
phase 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.
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 must 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
Method 608.3
28
December 2016
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with particulates, a 1 -|im 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 fluoropolymer-
lined cap, per Section 10.3.
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.
Method 608.3
29
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11. Extract Cleanup
11.1 Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater,
drinking water). 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.
11.1.5 Acid back extraction (Section 11.6) may be useful for cleanup of PCBs and other
compounds not adversely affected by sulfuric acid.
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 (50:3:47).
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.
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.
Method 608.3
30
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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 7.8 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.
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
Method 608.3
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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 prewetthe 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 copper. Acceptable
performance can be demonstrated through the preparation and analysis of a matrix spike
sample that meets the QC requirements for recovery.
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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 nitrogen-
evaporation 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 ofhexane, quantitatively transfer the extract to a40-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.
11.6 Acid back extraction (Section 6.1.2)
11.6.1 Quantitatively transfer the extract (Section 10.3.1.5) to a 250-mL separatory funnel.
11.6.2 Partition the extract against 50 mL of sulfuric acid solution (Section 6.1.2). Discard the
aqueous layer. Repeat the acid washing until no color is visible in the aqueous layer, to a
maximum of four washings.
11.6.3 Partition the extract against 50 mL of sodium chloride solution (Section 6.7.5). Discard the
aqueous layer.
11.6.4 Proceed to Section 10.3.3 for micro-concentration and solvent exchange.
Method 608.3
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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 (iL of 10 (ig/mL internal standard
solution into the extract, assuming no dilutions. Mix thoroughly.
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. Injection
volumes must be the same for all extracts. Record the volume injected to the nearest 0.05 (.iL.
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 calibration verification 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.
Method 608.3
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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 |a,L of the DDT and endrin decomposition solution (Section 6.8.7). As noted in
Section 6.8.7, other injection volumes may be used as long as the concentrations of DDT
and endrin in the solution are adjusted to introduce the masses of the two analytes into the
instrument that are listed in Section 6.8.7.
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:
sum of degradation peak areas (DDD + DDE)
% breakdown of DDT = ; — x 100
sum of all peak areas (DDT + DDE + DDD)
sum of degradation peak areas (Endrin aldehyde + Endrin ketone)
% breakdown of Endrin = —— - . .———— .—- x 100
sum of all peak areas (Endrin + Endrin aldehyde + Endrin ketone)
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 LCS (Section 6.8.3) extracted with each sample batch (Section
8.4). See Section 8.4 for criteria acceptance of the LCS.
Method 608.3
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13.7.2 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,
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 calibration verification (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). If the
analytes are affected, re-prepare and reanalyze the sample, blank, LCS, MS, or MSD, and repeat
the pertinent test.
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. Alternatively, GC/MS identification may be used to provide another
means of identification.
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 single-
component 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.
Method 608.3
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14.2.4 The retention time windows must be recentered 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.
14.2.6 If the retention time of an analyte in the calibration (Section 7.4) varies by more than 5
seconds across the calibration range as a function of the concentration of the standard,
using the standard deviation of the retention times (Section 14.2.3) 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 analyte 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 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. Alternatively, GC/MS
identification may be used. Differentiation among some of the Aroclors may require
evaluation of more than five peaks to ensure correct identification.
14.4 GC/MS confirmation
When the concentration of an analyte is sufficient and 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 would need to be by GC/MS-SIM, or the estimated concentration would need to be
100 times higher than the GC/ECD calibration range. The extract may be concentrated by an
additional amount to allow a further attempt at GC/MS confirmation.
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
Method 608.3
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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.
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:
As
r = —
ex 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:
_ As x C;s
ex ~ 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
C1S = 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:
Cex X Vex X DF
s Vs x1000
where:
Cs = Concentration of the analyte in the sample ((.ig/L)
Vex = Final extract volume (mL)
Cex = Concentration in the extract (ng/mL)
Method 608.3
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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 (ig/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.
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 Section 7.5.1. Determine the concentration of
each of the characteristic peaks, using the average calibration factor calculated for that peak
in Section 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 (ig/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 specified otherwise 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 15.7below) for each
analyte in each sample or QC standard at or above the ML to 3 significant
Method 608.3
39
December 2016
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figures. Report a result for each analyte in each sample or QC standard below
the ML as "
-------
Higher result — Lower result
%D = — — - x 100
Higher result
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).
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. See Section 8.1.7 for disposition of problem results.
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 j^ig/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 limits developed in Section 8.6, re-extract and
reanalyze the sample if there is sufficient sample and if it is within the 7-day extraction holding
time. If 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
Method 608.3 41 December 2016
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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. See Section 8.1.7 for
dispositions of failures. 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 lOOOx MDL with the
following exceptions: Chlordane recovery at 4x MDL was low (60%); Toxaphene recovery was
demonstrated linear over the range of lOx MDL to lOOOx MDL (Reference 3).
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 |a,g/L for single-component pesticides and
from 8.5 to 400 |a,g/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), available from the American Chemical Society's Department of Governmental
Relations and Science Policy, 1155 16th Street NW, Washington DC 20036, 202-872-4477.
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
Method 608.3
42
December 2016
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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 handled and disposed of as hazardous
waste, or neutralized and disposed of in accordance with all federal, state, and local regulations. It
is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions. The laboratory using this method has the responsibility 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. For further
information on waste management, see "The Waste Management Manual for Laboratory
Personnel," also available from the American Chemical Society at the address in Section 18.3.
19.3 Many analytes in this method decompose above 500 °C. 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.
19.4 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction,
available from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street NW, Washington, DC 20036, 202-872-4477.
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, D3 694-78. "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.
Method 608.3
43
December 2016
-------
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 CI F and 4500 CI G, Standard Methods for the Examination of Water and Wastewater,
published jointly by the American Public Health Association, American Waterworks 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.
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 (2002),
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
44
December 2016
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21. Tables
Table 1 - Pesticides 1
Analyte
CAS Number
MDL2 (ng/L)
ML3 (ng/L)
Aldrin
309-00-2
4
12
alpha-BHC
319-84-6
3
9
beta-BHC
319-85-7
6
18
delta-BHC
319-86-8
9
27
gamma-EHC (Lindane)
58-89-9
4
12
alpha-Chlordme 4
5103-71-9
14
42
gamma-Chlordane 4
5103-74-2
14
42
4,4'-DDD
72-54-8
11
33
4,4'-DDE
72-55-9
4
12
4,4'-DDT
50-29-3
12
36
Dieldrin
60-57-1
2
6
Endosulfan I
959-98-8
14
42
Endosulfan II
33213-65-9
4
12
Endosulfan sulfate
1031-07-8
66
198
Endrin
72-20-8
6
18
Endrin aldehyde
7421-93-4
23
70
Heptachlor
76-44-8
3
9
Heptachlor epoxide
1024-57-3
83
249
1 All analytes in this table are Priority Pollutants (40 CFR part 423, appendix A)
2 40 CFR part 136, appendix B, June 30, 1986.
3 ML = Minimum Level - see Glossary for definition and derivation, calculated as
3 times the MDL.
4 MDL based on the MDL for Chlordane
Method 608.3
45
December 2016
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Table 2 - Additional Analytes
Analyte
CAS Number
MDL3 (ng/L)
ML4 (ng/L
Acephate
30560-19-1
Alachlor
15972-60-8
Atrazine
1912-24-9
Benfluralin (Benefin)
1861-40-1
Bromacil
314-40-9
Bromoxynil octanoate
1689-99-2
Butachlor
23184-66-9
Captafol
2425-06-1
Captan
133-06-2
Carbophenothion (Trithion)
786-19-6
Chlorobenzilate
510-15-6
Chloroneb (Terraneb)
2675-77-6
Chloropropylate (Acaralate)
5836-10-2
Chlorothalonil
1897-45-6
Cyanazine
21725-46-2
DCPA (Dacthal)
1861-32-1
2,4'-DDD
53-19-0
2,4'-DDE
3424-82-6
2,4'-DDT
789-02-6
Diallate (Avadex)
2303-16-4
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
Ethalfluralin (Sonalan)
55283-68-6
Etridiazole
2593-15-9
Fenarimol (Rubigan)
60168-88-9
Hexachlorobenzene1
118-74-1
Hexachlorocyclopentadiene1
77-47-4
Isodrin
465-73-6
Isopropalin (Paarlan)
33820-53-0
Kepone
143-50-0
Methoxychlor
72-43-5
Metolachlor
51218-45-2
Metribuzin
21087-64-9
Mirex
2385-85-5
Nitrofen (TOK)
1836-75-5
cis-Nonachlor
5103-73-1
trans-Nonachlor
39765-80-5
Norfluorazon
27314-13-2
Octachlorostyrene
29082-74-4
Oxychlordane
27304-13-8
PCNB (Pentachloronitrobenzene)
82-68-8
Pendamethalin (Prowl)
40487-42-1
cy.v-Pcrmcthrin
61949-76-6
/ram-Pcrmcthrin
61949-77-7
Perthane (Ethylan)
72-56-0
Method 608.3
46
December 2016
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Table 2 - Additional Analytes
Analyte
CAS Number
MDL3 (ng/L)
ML4 (ng/L
Propachlor
1918-16-7
Propanil
709-98-8
Propazine
139-40-2
Quintozene
82-68-8
Simazine
122-34-9
Strobane
8001-50-1
Technazene
117-18-0
Technical Chlordane2
Terbacil
5902-51-2
Terbuthylazine
5915-41-3
Toxaphene1
8001-35-2
240
720
Trifluralin
1582-09-8
PCB-1016 1
12674-11-2
PCB-12211
11104-28-2
PCB-1232 1
11141-16-5
PCB-1242 1
53469-21-9
65
95
PCB-1248 1
12672-29-6
PCB-1254 1
11097-69-1
PCB-1260 1
11096-82-5
PCB-1268
11100-14-4
Priority Pollutants (40 CFR part 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 part 136, appendix B, June 30, 1986.
4 ML = Minimum Level - see Glossary for definition and derivation, calculated as 3 times the
MDL.
Method 608.3
47
December 2016
-------
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-EHC
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-EHC (Lindane)
9.52
9.91
beta-SHC
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
«//;/?a-Chlordanc
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
Method 608.3
48
December 2016
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Table 3 - Example Retention Times1
Analyte
Retention time (min)2
DB-608
DB-1701
Bromoxynil octanoate
18.85
18.57
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
c/.v-Pcrmcthrin
25.00
23.59
/ram-Pcrmc thrin
25.62
23.92
PCB-1016
PCB-1221
PCB-1232
PCB-1242
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 |am; andDB-1701, 1.0 |am.
Conditions suggested to meet retention times shown:
150 °C for 0.5 minute, 150-270 °C at 5 °C/min, and 270 °C
until /nmv-Pcrmcthrin 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
49
December 2016
-------
Table 4 - QC Acceptance Criteria
Analyte
Calibration
verification
(%)
Test
concen-
tration
(Hg/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-EHC
75 - 125
2.0
38
39-130
17 - 147
44
delta- BHC
75 - 125
2.0
43
51 - 130
19 - 140
52
gamma-SRC
75 - 125
2.0
29
43 - 130
32 - 140
39
a//;/?a-Chlordanc
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 for the DOC (Section 8.2.4).
X = Average of four recovery measurements for the DOC (Section 8.2.4)
P = Recovery for the LCS (Section 8.4.3)
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
50
December 2016
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Table 5 - Precision and Recovery as Functions of Concentration
Analyte
Recovery, X'
(M'g/L)
Single analyst
precision, sr' (ng/L)
Overall precision,
S' (M'g/L)
Aldrin
0.81C + 0.04
0.16(X) -0.04
0.20(X) -0.01
alpha-EHC
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 |a,g/L.
Method 608.3
51
December 2016
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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-EHC
97
delta-BHC
98
gamma-EHC (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
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
52
December 2016
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Table 7 - Suggested Calibration Groups1
Analyte
Calibration Group 1
Calibration Group 4
Acephate
Benfluralin
Alachlor
Chlorobenzilate
Atrazine
Dieldrin
betaSWC
Endosulfan I
Bromoxynil octanoate
Mirex
Captafol
Terbacil
Diallate
Terbuthylazine
Endosulfan sulfate
Triadimefon
Endrin
Calibration Group 5
Isodrin
alpha-Chlordane
Pendimethalin (Prowl)
Captan
/ram-Pcrmc thrin
Chlorothalonil
Calibration Group 2
4,4'-DDD
alpha-BHC
Norfluorazon
DCPA
Simazine
4,4'-DDE
Calibration Group 6
4,4'-DDT
Aldrin
Dichlone
delta-BHC
Ethalfluralin
Bromacil
Fenarimol
Butachlor
Methoxychlor
Endosulfan II
Metribuzin
Heptachlor
Calibration Group 3
Kepone
gamma-BHC (Lindane)
Calibration Group 7
gamma-Chlordane
Carbophenothion
Endrin ketone
Chloroneb
Heptachlor epoxide
Chloropropylate
Isopropalin
DBCP
Nitrofen (TOK)
Dicofol
PCNB
Endrin aldehyde
c/.v-Pcrmcthrin
Etridiazole
Trifluralin
Perthane
Propachlor
Propanil
Propazine
1 The analytes may be organized in other calibration groups, provided that there are no coelution
problems and that all QC requirements are met.
Method 608.3
53
December 2016
-------
22. Figures
(B)
i
f I
* *
I &
I *
Jj U
/LjLjU
J
|»TM^TT|TTii>i»n|ii»rm^fwniumTniTTi^rnmri}inimn|i!mim|'mmni|nnHm|»niMif)iT>rMlTf^
¦¦]'nunii|Tini^n|iiiuiiU|mMint|iifmTrr|TTiTmTrpTTmut|
-------
1000
— 750
500
— 250
90-mm GMF150 Filter
90-mm SPE Disk
o
1-Liter Suction Flask
Figure 2 Disk-based solid-phase extraction apparatus
Method 608.3
55
December 2016
-------
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
°c
degrees Celsius
Mg
microgram
(jL
microliter
<
less than
<
less than or equal to
>
greater than
0/
/o
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.
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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 standard (Section 6.8.4) used to verify calibration (Sections
7.8 and 13.6).
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
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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 part 136,
appendix B. The MDLs determined by EPA are listed in Tables 1 and 2. As noted in Section 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
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
RPD - Relative percent difference
RSD - See relative standard deviation
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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 to §1910.1200. 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.
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