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METHOD 505
ANALYSIS OF ORGANOHALIDE PESTICIDES AND
COMMERCIAL POLYCHLORINATED BIPHENYL (PCB) PRODUCTS
IN WATER BY MICROEXTRACTION AND GAS CHROMATOGRAPHY
Revision 2.1
Edited by J.W. Munch (1995)
T. W. Winfield - Method 505, Revision 1.0 (1986)
T. W. Winfield - Method 505, Revision 2.0 (1989)
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 505
ANALYSIS OF ORGANOHALIDE PESTICIDES AND COMMERCIAL
POLYCHLORINATED BIPHENYL (PCB) PRODUCTS IN WATER BY
MICROEXTRACTION AND GAS CHROMATOGRAPHY
1.0 SCOPE AND APPLICATION
1.1 This method is applicable to the determination of the following analytes in1,2,3
finished drinking water, drinking water during intermediate stages of treatment,
and the raw source water:
Analyte Registry Number
Chemical Abstract Services
Alachlor 15972-60-8
Aldrin 309-00-2
Atrazine 1912-24-9
Chlordane 57-74-9
alpha-Chlorodane 5103-71-9
gamma-Chlorodane 5103-74-2
Dieldrin 60-57-1
Endrin 72-20-8
Heptachlor 76-44-8
Heptachlor Epoxide 1024-57-3
Hexachlorobenzene 118-74-1
Hexachlorocyclopentadiene 77-74-4
Lindane 58-89-9
Methoxychlor 72-43-5
cis-Nonachlor 5103-73-1
trans-Nonachlor 39765-80-5
Simazine 122-34-9
Toxaphene 8001-35-2
Aroclor 1016 12674-11-2
Aroclor 1221 11104-28-2
Aroclor 1232 11141-16-5
Aroclor 1242 53469-21-9
Aroclor 1248 12672-29-6
Aroclor 1254 11097-69-1
Aroclor 1260 11096-82-5
1.2 The analyst must demonstrate the applicability of the method by collecting
precision and accuracy data on fortified samples (i.e., groundwater, tap water)4
and provide qualitative confirmation of results by Gas Chromatography/Mass
Spectrometry (GC/MS) , or by GC analysis using dissimilar columns.5
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1.3 Method detection limits (MDL) for the above organohalides and Aroclors have6
been experimentally determined (Section 13.2). Actual detection limits are highly
dependent upon the characteristics of the gas chromatographic system used
(e.g., column type, age, and proper conditioning; detector condition; and injector
mode and condition).
1.4 This method is restricted to use by or under the supervision of analysts
experienced in the use of GC and in the interpretation of gas chromatograms.
Each analyst must demonstrate the ability to generate acceptable results with this
method using the procedure described in Section 11.0.
2.0 SUMMARY OF METHOD
2.1 Thirty-five mL of sample are extracted with 2 mL of hexane. One to 2 µL of the
extract are then injected into a gas chromatograph equipped with a linearized
electron capture detector for separation and analysis. Analytes are quantitated
using procedural standard calibration (Section 3.12).
2.2 The extraction and analysis time is 40-70 minutes per sample depending upon the
analytes and the analytical conditions chosen. (See Section 6.9).
3.0 DEFINITIONS
3.1 Laboratory Duplicates (LD1 and LD2) -- Two sample aliquots taken in the
analytical laboratory and analyzed separately with identical procedures. Analyses
of LD1 and LD2 give a measure of the precision associated with laboratory
procedures, but not with sample collection, preservation, or storage procedures.
3.2 Field Duplicates (FD1 and FD2) -- Two separate samples collected at the same
time and place under identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of FD1 and FD2 give a
measure of the precision associated with sample collection, preservation and
storage, as well as with laboratory procedures.
3.3 Laboratory Reagent Blank (LRB) -- 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 other samples.
The LRB is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the apparatus.
3.4 Field Reagent Blank (FRB) -- Reagent water placed in a sample container in the
laboratory and treated as a sample in all respects, including exposure to sampling
site conditions, storage, preservation and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other interferences are present
in the field environment.
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3.5 Laboratory Performance Check Solution (LPC) -- A solution of method analytes,
surrogate compounds, and internal standards used to evaluate the performance
of the instrument system with respect to a defined set of method criteria.
3.6 Laboratory Fortified Blank (LFB) -- An aliquot of reagent water to which known
quantities of the method analytes are added in the laboratory. The LFB is
analyzed exactly like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of making
accurate and precise measurements at the required method detection limit.
3.7 Laboratory Fortified Sample Matrix (LFM) -- An aliquot of an environmental
sample to which known quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample, and its purpose is to
determine whether the sample matrix contributes bias to the analytical results.
The background concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.8 Stock Standard Solution -- A concentrated solution containing a single certified
standard that is a method analyte, or a concentrated solution of a single analyte
prepared in the laboratory with an assayed reference compound. Stock standard
solutions are used to prepare primary dilution standards.
3.9 Primary Dilution Standard Solution (PDS) -- A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted as needed
to prepare calibration solutions and other needed analyte solutions.
3.10 Calibration Standard (CAL) -- A solution prepared from the primary dilution
standard solution and stock standard solutions of the internal standards and
surrogate analytes. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.11 Quality Control Sample (QCS) -- A sample matrix containing method analytes or
a solution of method analytes in a water miscible solvent which is used to fortify
reagent water or environmental samples. The QCS is obtained from a source
external to the laboratory, and is used to check laboratory performance with
externally prepared test materials.
3.12 Procedural Standard Calibration -- A calibration method where aqueous
calibration standards are prepared and processed (e.g., purged, extracted, and/or
derivatized) in exactly the same manner as a sample. All steps in the process
from addition of sampling preservatives through instrumental analyses are
included in the calibration. Using procedural standard calibration compensates
for any inefficiencies in the processing procedure.
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4.0 INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents,
glassware and other sample processing apparatus that lead to discrete artifacts or
elevated baselines in gas chromatograms. All reagents and apparatus must be
routinely demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.2.
4.1.1 Glassware must be scrupulously cleaned . Clean all glass- ware as soon2
as possible after use by thoroughly rinsing with the last solvent used in
it. Follow by washing with hot water and detergent and thorough rinsing
with tap and reagent water. Drain dry, and heat in an oven or muffle
furnace at 400°C for one hour. Do not heat volumetric ware. Thermally
stable materials, such as PCBs, might not be eliminated by this treatment.
Thorough rinsing with acetone may be substituted for the heating. After
drying and cooling, seal and store glassware in a clean environment to
prevent any accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by distilla- tion in all-glass
systems may be required.
Warning: When a solvent is purified, stabilizers put into the solvent by
the manufacturer are removed thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives put into the
solvent by the manufacturer are removed thus potentially reducing the
shelf-life.
4.2 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a sample containing
relatively high concentrations of analytes. Between-sample rinsing of the sample
syringe and associated equipment with hexane can minimize sample cross
contamination. After analysis of a sample containing high concentrations of
analytes, one or more injections of hexane should be made to ensure that accurate
values are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are coextract- ed from
the sample. Also, note that all the analytes listed in the scope and application
section are not resolved from each other on any one column, i.e., one analyte of
interest may be an interferant for another analyte of interest. The extent of matrix
interferences will vary considerably from source to source, depending upon the
water sampled. Cleanup of sample extracts may be necessary. Analyte
identifications should be confirmed (Section 11.4).
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4.4 It is important that samples and working standards be contained in the same
solvent. The solvent for working standards must be the same as the final solvent
used in sample preparation. If this is not the case, chromatographic comparability
of standards to sample may be affected.
4.5 Caution must be taken in the determination of endrin since it has been reported
that the splitless injector may cause endrin degradation . The analyst should be7
alerted to this possible interference resulting in an erratic response for endrin.
4.6 Variable amounts of pesticides and commercial PCB products from aqueous
solutions adhere to glass surfaces. It is recommended that sample transfers and
glass surface contacts be minimized, and that adequate rinsing of glass surfaces
be performed.
4.7 Aldrin, hexachlorocyclopentadiene, and methoxychlor are rapidly oxidized by
chlorine. Dechlorination with sodium thiosulfate at time of collection will stop
further oxidation of these compounds.
Warning: An interfering, erratic peak has been observed within the retention
window of heptachlor during many analyses of reagent, tap, and groundwater.
It appears to be related to dibutyl phthalate; however, the specific source has not
yet been definitively determined. The observed magnitude and character of this
peak randomly varies in numerical value from successive injections made from
the same vial.
5.0 SAFETY
5.1 The toxicity and carcinogenicity of chemicals used in this method have not been
precisely defined; each chemical should be treated as a potential health hazard,
and exposure to these chemicals should be minimized. Each laboratory is
responsible for maintaining awareness of OSHA regulations regarding safe
handling of chemicals used in this method. Additional references to laboratory
safety are available for the information of the analyst.8-10
5.2 The following organohalides have been tentatively classified as known or
suspected human or mammalian carcinogens: aldrin, commercial PCB products,
chlordane, dieldrin, heptachlor, hexachlorobenzene, and toxaphene. Pure
standard materials and stock standard solutions of these compounds should be
handled in a hood or glovebox.
Warning: When a solvent is purified, stabilizers put into the solvent by the
manufacturer are removed thus potentially making the solvent hazardous.
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6.0 EQUIPMENT AND SUPPLIES (All specifications are suggested. Catalog numbers are
included for illustration only.)
6.1 Sample Containers -- 40 mL screw cap vials (Pierce #13075 or equivalent) each
equipped with a size 24 cap with a flat, disc-like TFE facing backed with a
polyethylene film/foam extrusion (Fisher #02-883-3F or equivalent). Prior to use,
wash vials and septa with detergent and rinse with tap and distilled water.
Allow the vials and septa to air dry at room temperature, place the vials in a
400°C oven for one hour, then remove and allow to cool in an area known to be
free of organics.
6.2 Vials -- auto sampler, screw cap with septa, 1.8 mL, Varian #96-000099-00 or
equivalent or any other autosampler vials not requiring more than 1.8 mL sample
volumes.
6.3 Auto Sampler -- Hewlett-Packard 7671A, or equivalent.
6.4 Micro Syringes -- 10 µL and 100 µL.
6.5 Micro Syringe -- 25 µL with a 2 inch by 0.006 inch needle - Hamilton 702N or
equivalent.
6.6 Pipettes -- 2.0 mL and 5.0 mL transfer.
6.7 Volumetric Flasks -- 10 mL and 100 mL, glass stoppered.
6.8 Standard Solution Storage Containers -- 15 mL bottles with PTFE-lined screw
caps.
6.9 Gas Chromatograph -- Analytical system complete with temperature
programmable GC and split/splitless injector suitable for use with capillary
columns and all required accessories including syringes, analytical columns, gases,
a linearized electron capture detector and stripchart recorder. A data system is
recommended for measuring peak areas. Table 1 lists retention times observed
for method analytes using the columns and analytical conditions described below.
6.9.1 Three gas chromatographic columns are recommended. Column 1
(Section 6.9.2) should be used as the primary analytical column unless
routinely occurring analytes are not adequately resolved. Validation data
presented in this method were obtained using this column. Columns 2
and 3 are recommended for use as confirmatory columns when GC/MS
confirmation is not available. Alternative columns may be used in
accordance with the provisions described in Section 9.4.
6.9.2 Column 1 (primary column) - 0.32 mm ID x 30 M long fused silica
capillary with chemically bonded methyl polysiloxane phase (DB-1, 1.0 µm
film, or equivalent). Helium carrier gas flow is about 25 cm/sec. linear
velocity, measured at 180° with 9 psi column head pressure. The oven
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temperature is programmed from 180-260°C at 4°C/min. and held at
260°C until all expected compounds have eluted. Injector temperature:
200°C. Splitless Mode: 0.5 minute. Detector temperature: 290°C. Sample
chromatograms for selected pesticides are presented in Figures 1 and 2.
Chromatograms of the Aroclors, toxaphene, and technical chlordane are
presented in Figures 3 through 11.
6.9.3 Column 2 (alternative Column 1) - 0.32mm ID x 30 M long fused silica
capillary with a 1:1 mixed phase of dimethyl silicone and polyethylene
glycol (Durawax-DX3, 0.25 µm film, or equivalent). Helium carrier gas
flow is about 25 cm/sec. linear velocity and oven temperature is
programmed from 100-210°C at 8°C/min., and held at 210°C until all
expected compounds have eluted. Then the post temperature is
programmed to 240°C at 8°C/min. for five minutes.
6.9.4 Column 3 (alternative Column 2) - 0.32mm ID x 25 M long fused silica
capillary with chemically bonded 50:50 Methyl-Phenyl silicone (OV-17,
1.5 µm film thickness, or equivalent). Helium carrier gas flow is about
40 cm/sec. linear velocity and oven temperature is programmed from
100-260°C at 4°C/min. and held at 260°C until all expected compounds
have eluted.
7.0 REAGENTS AND STANDARDS
Warning: When a solvent is purified, stabilizers put into the solvent by the
manufacturer are removed thus potentially making the solvent hazardous. Also, when
a solvent is purified, preservatives put into the solvent by the manufacturer are removed
thus potentially making the shelf-life short.
7.1 Reagents
7.1.1 Hexane Extraction Solvent -- UV Grade, Burdick and Jackson #216 or
equivalent.
7.1.2 Methyl Alcohol, ACS Reagent Grade -- Demonstrated to be free of
analytes.
7.1.3 Sodium Chloride, NaCl, ACS Reagent Grade -- For pretreatment before
use, pulverize a batch of NaCl and place in a muffle furnace at room
temperature. Increase the temperature to 400°C and hold for 30 minutes.
Store in a glass (not plastic) bottle to avoid phthalate contamination..
7.1.4 Sodium thiosulfate, Na S O , ACS Reagent Grade -- For preparation of2 2 3
solution (0.04 g/mL), mix 1 g of Na S O with reagent water and bring to2 2 3
25 mL volume in a volumetric flask. Verify the stability of this solution
and replace as necessary.
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7.2 Reagent Water -- Reagent water is defined as water free of interference when
employed in the procedure described herein.
7.2.1 A Millipore Super-Q Water System or its equivalent may be used to
generate deionized reagent water.
7.2.2 Test reagent water each day it is used by analyzing it according to
Section 9.2.
7.3 Stock Standard Solutions (SSS) -- These solutions may be obtained as certified
solutions or prepared from pure standard materials using the following
procedures:
7.3.1 Prepare stock standard solutions (5000 µg/mL) by accurately weighing
about 0.0500 g of pure material. Dissolve the material in methanol and
dilute to volume in a 10 mL volumetric flask. Larger volumes can be used
at the convenience of the analyst. When compound purity is assayed to
be 96% or greater, the weight can be used without correction to calculate
the concentration of the stock standard. Commercially prepared stock
standards can be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.3.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles.
Store at 4°C and protect from light. Stock standard solutions should be
checked frequently for signs of degradation or evaporation, especially just
prior to preparing calibration standards from them.
7.3.3 Stock standard solutions must be replaced after six months, or sooner if
comparison with check standards indicates a problem.
7.4 Primary Dilution Standard Solutions (PDS) -- Use stock standard solutions to
prepare primary dilution standard solutions that contain the analytes in methanol.
The primary dilution standards should be prepared at concentrations that can be
easily diluted to prepare aqueous calibration standards (Section 10.2.1) that will
bracket the working concentra- tion range. Store the primary dilution standard
solutions with minimal headspace and check frequently for signs of deterioration
or evaporation, especially just before preparing calibration standards. The storage
time described for stock standard solutions in Section 7.3.3 also applies to primary
dilution standard solutions.
Note: Primary dilution standards for toxaphene, chlordane and each of the
Aroclors must be prepared individually.
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8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 Sample Collection
8.1.1 Collect all samples in 40 mL bottles into which 3 mg of sodium thiosulfate
crystals have been added to the empty bottles just prior to shipping to the
sampling site. Alternately, 75 µL of freshly prepared sodium thiosulfate
solution (0.04 g/mL) may be added to empty 40 mL bottles just prior to
sample collection.
8.1.2 When sampling from a water tap, open the tap and allow the system to
flush until the water temperature has stabilized (usually about
10 minutes). Adjust the flow to about 500 mL/min. and collect samples
from the flowing stream.
8.1.3 When sampling from a well, fill a wide-mouth bottle or beaker with
sample, and carefully fill 40 mL sample bottles.
8.2 Sample Preservation
8.2.1 The samples must be chilled to 4°C at the time of collection and
maintained at that temperature until the analyst is prepared for the
extraction process. Field samples that will not be received at the