METHODS FOR OR6ANOCHLORINE PESTICIDES AND CHLOROPHENOXY
ACID HERBICIDES IN DRINKING WATER AND RAW SOURCE WATER
INTERIM
Pending Issuance of
>^ Methods for Organic Analysis
\ of Water and Wastes
U. S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 45268
July 1978
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FOREWORD
The National Interim Primary Drinking Mater Regulations
promulgated on December 24, 1975, in accordance with the provisions of
the Safe Drinking Water Act (Public Law 93-523), have set maximum
contaminant levels for a variety of pollutants. The methods contained
herein are provided to determine compliance with para. 141.12 (a)
chlorinated hydrocarbon insecticides and 141.12 (b) chlorophenoxy
herbicides. Endrin, Undane, methoxychlor and toxphene may be determined
by the chlorinated hydrocarbon method while 2,4-0 and 2,4,5-TP (311vex)
may be determined by the chlorophenoxy method.
These methods have been assembled by the staff of the
Environmental Monitoring and Support Laboratory - Cincinnati (EMSL-CIN)
and are provided only for the interim period until the manual, "Methods
for Organic Anaysis of Water and Wastes," becomes available.
Dwight G. Ballinger, Director
Environmental Monitoring and Support Laboratory - Cincinnati
11
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DISCLAIMER
The mention of trade names or commercial products 1n this manual
Is for Illustration purposes, and does not constitute endorsement or
recommendation for use by the U. S. Environmental Protection Agency.
ill
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METHOD FOR ORGANOCHLORINE PESTICIDES IN DRINKING WATER
1. Scope and Application
1.1 This method covers the determination of organochlorine
pesticides in drinking water and raw source water. Lindane,
endrin, methoxychlor and toxaphene are determined by this
procedure.
1.2 The method sensitivity is 0.001 to 0.010 ug/1 for single
component pesticides and 0.050 to 1.0 ug/1 for
muHi-component pesticides when analyzing a 1 liter sample
with the electron capture detector.
1.3 Other organochlorine pesticides, such as BHCr~heptachlor,
aldrin, heptachlor epoxide, dieldrin, Captan, DOE, ODD, DDT,
endosulfan, dichloran, mi rex, pentachloronitrobenzene, tri-
fluralin, Strobane, chlordane (tech.) and others may also be
determined by this method.
2. Summary
2.1 The method offers several analytical alternatives, dependent
on the analyst's assessment of the nature and extent of
interferences and/or the complexity of the pesticide mix-
tures found. Specifically, the procedure describes the use
of an effective co-solvent for efficient sample extraction;
provides, through use of column chromatography and liquid-
liquid partition, methods for elimination of non-pesticide
interferences and the pre-separation of pesticide mixtures.
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Identification is made by selective gas chromatographic
separations and may be corroborated through the use of two
or more unlike columns. Detection and measurement is
accomplished by electron capture, microcoulometric or
electrolytic conductivity gas chromatography. Results are
reported 1n micrograms per liter.
2.2 Confirmation of the identity of the compounds should be made
by GC-MS when a new or undefined sample type is being
analyzed and the concentration is adequate for such
determination.
2.3 This method 1s recommended for use only by experienced
pesticide analysts or under the close supervision of such
qualified persons.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing
hardware may yield discrete artifacts and/or elevated base-
lines, causing misinterpretation of gas chromatograms.
All of these materials must be demonstrated to be free from
Interferences under the conditions of the analysis. Specific
selection of reagents and purification of solvents by dis-
tillation in all-glass systems may be required. Refer to
Part I, Sections 1.4 and 1.5 (1).
3.2 The interferences in drinking water should not pose any
difficulty in obtaining accurate and precise measurement of
organochlorine pesticides.
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3.3 Polychlorinated Biphenyls (PCBs) - Special attention is
called to industrial plasticizers and hydraulic fluids such
as the PCBs, which are a potential source of interference in
pesticide analysis. The presence of PCBs is indicated by a
large number of partially resolved or unresolved peaks which
may occur throughout the entire chromatogram. Par-
ticularly severe PCB interference will require special
separation procedures (2,3).
3.4 Phthalate Esters - These compounds, widely used as plasti-
cizers, respond to the electron capture detector and are a
source of interference in the determination of organo-
chlorine pesticides using this detector. Water leaches
these materials from plastics, such as polyethylene bottles
and tygon tubing. The presence of phthaiate esters is
implicated in samples that respond to electron capture but
not to the mlcrocoulometric or electrolytic conductivity
halogen detectors.
3.5 Organophosphorus Pesticides - A number of organophosphorus
pesticides, such as those containing a nitro group, e.g.,
parathlon, respond to the electron capture detector and may
interfere with the determination of the organochlorine
pesticides. Such compounds can be identified by their
response to the flame photometric detector (4).
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4. Apparatus and Materials
4.1 Gas Chromatograph - Equipped with glass lined injection port.
4.2 Detector Options:
4.2.1 Electron Capture - Radioactive (tritium or nickel-63)
4.2.2 Microcoulometric Titration
4.2.3 Electrolytic Conductivity
4.3 Recorder - Potentiometric strip chart (10 in.) compatible
with the detector.
4.4 Gas Chromatographic Column Materials:
4.4.1 Tubing - Pyrex (180 cm long X 4 mm ID)
4.4.2 Glass Wool - Silanized
4.4.3 Solid Support - Gas-Chrom-Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
4.4.4.1 OV-1, 3*
4.4.4.2 OV-210, 535
4.4.4.3 OV-17, 1.5% plus QF-1 or OV-210, 1.95%
4.4.4.4 QF-1, 6% plus SE-30, 4%
4.5 Kuderna-Danish (K-D) Glassware
4.5.1 Snyder Column - three ball (macro) and two ball
(micro)
4.5.2 Evaporative Flasks - 500 ml
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers
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4.6 Chromatographic Column - Chromaflex (400 run long x 19 mm ID)
with coarse fritted plate on bottom and Teflon stopcock; 250
ml reservoir bulb at top of column with flared out funnel
shape at top of bulb - a special order (Kontes K-420540-
9011).
4.7 Chromatographic Column - pyrex (approximately 400 mm long x
20 mm ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and 100 ul.
4.9 Separatory funnels • 125 ml, 1000 ml and 2000 ml with Teflon
stopcock.
4.10 Graduated cylinders - 100 and 250 ml.
5. Reagents, Solvents, and Standards
5.1 Sodium Hydroxide - (ACS) 10 N in distilled water.
5.2 Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at
400 C for 4 hrs.).
5.3 SuIfuric Acid - (ACS) Mix equal volumes of cone. H2S04
with distilled water.
5.4 Florisll - PR Grade (60-100 mesh); purchase activated at
1250°F and store in the dark in glass containers with
glass stoppers or foil-lined screw caps. Before use,
activate each batch overnight at 130°C in foil-covered
.glass container. Determine 1auric-acid value (See Section
13).
5.5 Diethyl Ether - Nanograde, redistilled in glass, if
necessary.
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5.5.1 Must be free of peroxides as Indicated by EM Quant
test strips. (Test strips are available from EM
Laboratories, Inc., 500 Executive Blvd., Elmsford, NY
10523.)
5.5.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.6 Hexane, Methanol, Methylene Chloride, Petroleum Ether
(boiling range 30-60 C) - nanograde, redistill in glass if
necessary.
5.7 Pesticide Standards - Reference grade.
6. Calibration
6.1 Gas chromatographic operating conditions are considered
acceptable if the response to dicapthon 1s at least 502 of
full scale when ~<0.06 ng is injected for electron capture
detection and *100 ng 1s Injected for microcoulometric or
electrolytic conductivity detection. For all quantitative
measurements, the detector must be operated within its
linear response range and the detector noise level should be
less than 22 of full scale.
6.2 Standards are injected frequently as a check on the sta-
bility of operating conditions. Gas chromatograms of
several standard pesticides are shown in Figures 1, 2, 3 and
4 and provide reference operating conditions for the four
recommended columns.
6.3 The elution order and retention ratios of various organo-
chlorine pesticides are provided in Table 1, as a guide.
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IS 10
RETENTION TIME IN MINUTES
Figure 1. Column Packing: 1.5% OV-17 + 1.95% QF-1, Carrier Gas: Argon/Methane at 60 ml/rain,
Column Temperature: 200 C, Detector: Electron Capture.
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IS
0
10 5
RETENTION TIME IN MINUTES
Figure 2. Column Packing: 5% OY-210, Carrier Gas: Argon/Methane
at 70 ml/rain. Column Temperature: 180 C, Detector:
Electron Capture.
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25
IS 10
RETENTION TIME IN MINUTES
Figure 3. Column Packing: 6% QF-1 t 4% SE-30, Carrier Gas: Argon/Methane at 60 ml/min,
Column Temperature: 200 C, Detector: Electron Capture.
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a:
CJ
25
21
5
IS II
IETENTIOH TIME IN MINUTES
Figure 4. Column Packing: 3% 0V 1, Carrier Gas: Argon/Methane at 70 ml/mm,
Column Temperature: 180 C, Detector: Electron Capture.
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Table 1
RETENTION RATIOS OF VARIOUS ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Liquid
Phase' 1
Column Temp.
Argon/Methane
Carrier Flow
Pesticide
Tr1flural1n
-BHC
PCNB
Lindane
Oichloran
Heptachlor
Aldrln
Heptachlor Epoxide
Endosulfan I
p,p'-OOE
Dieldrln
Captan
Endrln
o.p'-DDT
p.p'-DDO
Endosulfan II
p,p'-ODT
Mi rex
Methoxychlor
Aldrin
(Win. absolute)
1.556 OV-17
*
.95% QF-l2
200 C
60 ml/min
RR
0.39
0.54
0.68
0.69
0.77
0.82
1.00
1.54
1.95
2.23
2.40
2.59
2.93
3.16
3.48
3.59
4.18
6.1
7.6
3.5
5%
OV-210
180 C
70 ml/min
RR
1.11
0.64
0.85
0.81
1.29
0.87
1.00
1.93
2.48
2.10
3.00
4.09
3.56
2.70
3.75
4.59
4.07
3.78
6.5
2.6
3%
OV-1
180 C
70 ml/min
RR
0.33
0.35
0.49
0.44
0.49
0.78
1.00
1.28
1.62
2.00
1.93
1.22
2.18
2.69
2.61
2.25
3.50
6.6
5.7
4.0
6% QF-1
+
4* SE-30
200 C
60 ml/min
RR
0.57
0.49
0.63
0.60
0.70
0.83
1.00
1.43
1.79
1.82
2.12
1.94
2.42
2.39
2.55
2.72
3.12
4.79
4.60
5.6
columns glass, 180 cm x 4 mm ID, solid support Gas-Chrom Q (100/120
mesh)
'OV-210 also may be used
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7. Sample Collection and Handling
7.1 Wash all bottles and cap liners in detergent water. Rinse
with tap water and finally distilled water.
7.2 Allow bottles and cap liners to air-dry. Muffle the sample
bottles at 400°C for 1 hour.
7.3 Rinse the cap liners with pesticide-grade hexane.
7.4 When cool, seal the bottles and store in a dust-free
environment.
7.5 Collect samples in 1 quart narrow-mouth bottles with a
Teflon lined screw cap (option 1 quart widemouth screw-cap
bottles with Teflon lid liner). Collect all samples in
duplicate.
7.6 Sampling from a water tap: Turn on water and allow system
to flush. When the temperature has stabilized, adjust the
flow to about 1 1/min. Fill the bottle about 90* full and
seal.
7.7 The sample should be maintained near 4°C until analysis
and should be extracted as soon as possible after collection.
8. Sample Procedure
8.1 Quantitatively transfer the entire sample into a two-liter
separatory funnel. Rinse the container with 60 ml of 15%
methylene chloride in hexane (v:v) and add to the separatory
funnel. Also rinse the sample bottle with each succeeding
volume of extracting solvent.
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8.2 Shake the sample in the separatory funnel vigorously for two
minutes. Allow the mixed solvent to separate from the
sample, then draw the water into a one-liter Erlenmeyer
flask. Pour the organic layer into a 100-ml beaker and then
pass it through a column containing 3 to 4 inches of
anhydrous sodium sulfate, and collect it in a 500-ml K-0
flask equipped with a 10 ml ampul. Return the water phase
to the separatory funnel. Rinse the sample bottle and the
Erlenmeyer flask with a second 60 ml volume of solvent; add
the solvent to the separatory funnel and complete the
extraction procedure a second time. Perform a third
extraction in the same manner.
8.3 Combine the extracts and concentrate in the K-0 evaporator
on a hot water bath.
8.4 Adjust the ampul volume to 10.0 ml with hexane.
8.5 Analyze by gas chromatography. If interferences are noted,
proceed to Section 9.
8.6 The sample extract can be further concentrated using a micro
Snider column if greater sensitivity is required.
9. Clean-up and Separation Procedures
9.1 Interferences in the form of distinct peaks and/or high
background in the initial gas chromatographic analysis, as
well as the physical characteristics of the extract (color,
cloudiness, viscosity) and background knowledge of the
sample will indicate whether clean-up is required. When
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these interfere with measurement of the pesticides, or
affect column life or detector sensitivity, proceed as
directed below.
9.2 Florisil Column Adsorption Chromatography
9.2.1 Adjust the sample extract-volume to 10 ml.
9.2.2 Place a charge of activated Florisil (weight deter-
mined by lauric-acid value, see Section 13) in a
Chromaflex column. After settling the Florisil by
tapping the column, add about one-half inch layer of
anhydrous granular sodium sulfate to the top.
9.2.3 Pre-elute the column, after cooling, with 50-60 mT of
petroleum ether. Discard the eluate and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by
decantation and subsequent petroleum ether washings.
Adjust the elution rate to about 5 ml per minute and,
separately, collect the two eluates in 500 ml K-0
flasks equipped with 10 ml ampuls (see Eluate
Composition 10.3.). Perform the first elution with
200 ml of 6% ethyl ether in petroleum ether, and the
second elution with 200 ml of 15S ethyl ether in
petroleum ether.
9.2.4 Concentrate the eluates to 6-10 ml in the K-0
evaporator in a hot water bath.
9.2.5 Analyze by gas Chromatography.
9.3 Eluate Composition - By using an equivalent quantity of any
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batch of Florisil, as determined by its lauric acid
value,the pesticides will be separated into the eluates
indicated below:
6% Eluate
Aldrin DOT Pentachloronitrobenzene
BHC Heptachlor Strobane
Chlordane Heptachlor Epoxide Toxaphene
ODD Lindane Trifluralin
DDE Methoxychlor PCB's
Mi rex
15% Eluate 50% Eluate
Endosulfan I Endosulfan II
Endrin Captan
Dieldrin
Dichloran
Phthalate esters
Certain thiophosphate pesticides can occur in each of the
above fractions as well as the 100* fraction. For
additional information regarding eluate composition, refer
to the FDA Pesticide Analytical Manual (6).
10. Quality Control
10.1 Duplicate and spiked sample analyses are recommended as
quality control checks. Quality control charts (5) should
be developed and used as a check on the analytical system.
Quality control check samples and performance evaluation
samples should be analyzed on a regular basis.
10.2 'Each time a set of samples is extracted, a method blank is
determined on a volume of distilled water equivalent to that
used to dilute the sample.
11. Calculation of Results
11.1 Determine the pesticide concentration by using the absolute
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calibration procedure described below or the relative cali-
bration procedure described in Part I, Section 3.4.2 (1).
( 1 ) Micrograms/1 i ter =
(Vs
A = ng standard
Standard area
8 = Sample aliquot area
VT = Volume of extract injected (ul)
Vt= Volume of total extract (ul)
Vs= Volume of water extracted (ml)
12. Reporting Results
12.1 Report results in micrograms per liter without correction
for recovery data. When duplicate and spiked samples are
analyzed, all data obtained should be reported.
13. Standardization of Florisil column by weight adjustment based on
adsorption of lauric acid
13.1 A rapid method for determining adsorptive capacity of
Florisil is based on adsorption of lauric acid from hexane
solution (6) (8). An excess of lauric acid is used and the
amount not adsorbed is measured by alkali titration. The
weight of lauric acid adsorbed is used to calculate, by
simple proportion, equivalent quantities of Florisil for
batches having different adsorptive capacities.
13.2 Apparatus
13.2.1 Buret - 25 ml with 1/10 ml graduations.
13.2.2 Erlenmeyer flasks - 125 ml narrow mouth and 25 ml
glass stoppered.
13.2.3 Pipet - 10 and 20 ml transfer.
13.2.4 Volumetric flasks - 500 ml.
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13.3 Reagents and Solvents
13.3.1 Alcohol, ethyl - USP or absolute, neutralized to
phenolphthalein.
13.3.2 Hexane - Distilled from all glass apparatus.
13.3.3 Laurie acid - Purified, CP.
13.3.4 Laurie acid solution - Transfer 10.000 g lauric
acid to 500 ml volumetric flask, dissolve in
hexane, and dilute to 500 ml (1 ml - 20 mg).
13.3.5 Phenolphthalein Indicator - Dissolve 1 g in
alcohol and dilute to 100 ml.
13.3.6 Sodium hydroxide - Dissolve 20 g NaOH (pellets,
reagent grade) in water and dilute to 500 ml
(IN). Dilute 25 ml IN NaOH to 500 ml with water
(0.05N0. Standardize as follows: Weigh 100-200
mg lauric acid into 125 ml Erlenmeyer flask. Add
50 ml neutralized ethyl alcohol and 3 drops
phenolphthalein indicator; titrate to permanent
end point. Calculate mg lauric acid/ml O.OSjY NaOH
i.
(about 10 mg/ml).
13.4 Procedure
13.4.1 Transfer 2.000 g F1or1s1l to 25-ml glass-stoppered
Erlenmeyer flasks. Cover loosely with aluminum
•
foil and heat overnight at 130°C. Stopper, cool
to room temperature, add 20.0 ml lauric acid
solution (400 mg), stopper, and shake occasionally
for .15 minutes. Let the adsorbent settle and
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pipet 10.0 ml of supernatant into 125 ml
Erlenmeyer flask. Avoid inclusion of any Florisil.
13.4.2 Add 50 ml of neutral alcohol and 3 drops of
indicator solution; titrate with 0.05f< to a
permanent end point.
13.5 Calculation of lauric acid value and adjustment of column
weight
13.5.1 Calculate amount of lauric acid adsorbed on
Florisil as follows:
Lauric add value a mg lauric acid/g Florisil 3
200 - (ml required for titration x mg lauric
add/ml 0.05N NaOH).
13.5.2 To obtain an equivalent quantity of any batch of
Florisil, divide 110 by lauric acid value for that
batch and multiply by 20 g. Verify proper elution
of pesticides by 13.6.
13.6 Test for proper elution pattern and recovery of pesticides
13.6.1 Prepare a test mixture containing aldrln,
heptachlor epoxide, p,p'-ODE, dieldrin, Parathion
and malathion. Oieldrin and Parathion should
elute in the 15% eluate; all but a trace of
malathion in the 50% eluate and the others in the
6% eluate.
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REFERENCES
1. "Method for Organic Pesticides in Water and Wastewater",
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio, 45268, 1971.
2. Monsanto Methodology for Aroclors - Analysis of Environmental
Materials for Biphenyls, Analytical Chemistry Method 71-35,
Monsanto Company, St. Louis, Missouri, 63166, 1970.
3. "Method for Polychlorinated Biphenyls in Industrial Effluents,"
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio, 45268, 1973.
4. "Method for Organophosphorus Pesticides in Industrial Effluents,"
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio, 45268, 1973.
5. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," Chapter 6, Section 6.4, U.S. Environmental Pro-
tection Agency, National Environmental Research Center, Analytical
Quality Control Laboratory, Cincinnati, Ohio, 45268, 1973.
6. "Pesticide Analytical Manual," U.S. Dept. of Health, Education and
Welfare, Food and Drug Administration, Washington, O.C.
7. "Analysis of Pesticide Residues in Human and Environmental
Samples," U.S. Environmental Protection Agency, Perrine Primate
Research Laboratories, Perrine, Florida, 33157, 1971.
8. Mills, P.A., "Variation of Florisil Activity: Simple Method for
Measuring Adsorbent Capacity and Its Use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical
Chemists. 5J_, 29 (1968).
9. Goerlitz, O.F. and Brown, E., "Methods for Analysis of Organic
Substances in Water," Techniques of Water Resources Investigations
of the'United States Geological Survey, Book 5, Chapter A3, U.S.
Department of the Interior, Geological Survey, Washington, O.C.
20242, 1972, pp. 24-40.
10. Steere, N.V., editor, "Handbook of Laboratory Safety," Chemical
Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio, 44128,
1971, pp. 250-254.
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METHOD FOR CHLOROPHENOXY ACID HERBICIDES IN DRINKING WATER
1. Scope and Application
1.1 This method covers the determination of chlorinated phenoxy acid
herbides in drinking water and raw source water. The compounds
2,4-dichlorophenoxy-acetic acid (2,4-D) and 2-(2,4,5-trichloro-
phenoxy) propionic acid (silvex) are determined by this
procedure.
1.2 The detection limits are 20 ng/1 for 2,4-0 and 5 ng/1 for Silvex
and 2,4,5-T.
1.3 Since these compounds may occur in water in various forms (i.e.,
acid, salt, ester, etc.} a hydrolysis step is included to permit
the determination of the active part of the herbicide. The
method may be applied to additional phenoxy acids such as
2,3-dichloro-o-anisic acid (dicamba) and 2,4,5-trichlorophenoxy-
acetic acid (2,4,5-T) and certain phenols.
2. Summary
2.1 Chlorinated phenoxy acids and their esters are extracted from
the acidified water sample with ethyl ether. The esters are
hydrolyzed to adds and extraneous organic material is removed
by a solvent wash. The acids are converted to methyl esters
which are extracted from the aqueous phase. Identification of
•
the esters is made by selective gas chromatographic separations
and may be corroborated through the use of two or more unlike
columns. Detection and measurement is accomplished by electron
20
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capture, microcoulometric or electrolytic conductivity gas
chromatography (1). Results are reported in micrograms per
liter.
2.2 This method is recommended for use only by experienced pesticide
analysts or under the close supervision of such qualified
persons.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing
hardware may yield discrete artifacts and/or elevated baselines
causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to be free from interference
under the conditions of the analysis. Specific selection of
reagents and purification of solvents by distillation in
a11-glass systems may be required. Refer to Part 1, Section 1.4
and 1.5, (2).
3.2 The interferences encountered in drinking water should not pose
great difficulty in obtaining accurate and precise measurement
of chlorinated phenoxy acid herbicides.
3.3 Organic acids, expecially chlorinated acids, cause the most
direct interference with the determination. Phenols including
chlorophenols will also interfere with this procedure.
3.4 Alkaline hydrolysis and subsequent extraction eliminates many of
the predominant chlorinated insecticides which might otherwise
•
interfere with the test.
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3.5 The herbicides, being strong organic acids, react readily with
alkaline substances and may be lost during analysis. Glassware
and glass wool should be acid-rinsed and sodium sulfate should
be acidified with sulfuric acid to avoid this possibility.
4. Apparatus and Materials
4.1 Gas Chromatograph - Equipped with glass lined injection port.
4.2 Detector Options:
4.2.1 Electron Capture - Radioactive (tritium or nickel-63)
4.2.2 Microcoulometric Titration
4.2.3 Electrolytic Conductivity
4.3 Recorder - Potentiometric strip chart (10 in.) compatible with
the detector.
4.4 Gas Chromatograph1c Column Materials:
4.4.1 Tubing - Pyrex (180 cm long X 4 mm ID)
4.4.2 Glass Wool - Silanizsd
4.4.3 Solid Support - Gas-Chrom-Q (100-120 mesh)
4.4.4 Liquid Phases - Expressed as weight percent coated on
solid support.
4.4.4.1 OV-210, 5X
4.4.4 2 OV-17, 1.5X plus QF-1, 1.95X
4.5 Kuderna-Danlsh (K-0) Glassware
•
4.5.1 Snyder Column - three ball (macro) and two ball (micro)
4.5.2 Evaporative Flasks - 250 ml
4.5.3 Receiver Ampuls - 10 ml, graduated
4.5.4 Ampul Stoppers
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4.6 Graduated cylinders - 100 and 250 ml.
4.7 Erlenmeyer flasks - 125 ml, 250 ml ground glass T 24/40 with
stopper
4.8 Micro Syringes - 10, 25, 50 and 100 ul.
4.9 Pipets - Pasteur, glass disposable (140 mm long X 5 mm ID).
4.10 Separatory Funnels - 60 ml and 2000 ml with Teflon stopcock.
4.11 Glass wool - Filtering grade, acid washed.
4.12 Diazald Kit - Recommended for the generation of diazomethane
(available from Aldrich Chemical Co., Cat. #210,025-2)
4.13 Florisil - PR grade (60-100 mesh) purchased activated at 1250F
and stored at 130 C.
5. Reagents. Solvents and Standards
5.1 Boron Trifluoride-Methanol-esterification-reagent, 14 percent
boron trifluorlde by weight.
5.2 N-methyl-N-nitroso-p-toluenesulfonamide (Diazald) - High purity,
melting point range 60-62 C. Precursor for the generation of
diazomethane (see Appendix I).
5.3 Potassium Hydroxide Solution - A 37 percent (w:v) aqueous
solution prepared from reagent grade potassium hydroxide pellets
and reagent water.
5.4 Sodium Sulfate, Acidified - (ACS) granular sodium sulfate,
treated as follows: Add 0.1 ml of cone, sulfuric acid to lOOg
of sodium sulfate slurried with enough ethyl ether to just cover
the solid. Remove the ether with the vacuum. Mix 1 g of the
resulting solid with 5 ml of reagent water and ensure the
mixture to have a pH below 4. Store at 130 C.
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5.5 Su If uric acid - (ACS) concentrated, Sp. Gr. 1.84.
5.5.1 Sulfuric Acid (1:1) - Carefully mix equal volumes of
HgSt^ (5.5) with distilled water. Cool while
adding acid.
5.5.2 Sulfuric Acid (1+3) - Carefully mix 1 volume of
H2S04 (5.5) with 3 volumes of distilled water.
Cool while adding acid.
5.6 Carbitol (diethylene glycol monoethyl ether).
5.7 Oiethyl Ether - Nanograde, redistilled in glass, if necessary.
5.7.1 Must be free of peroxides as indicated by EM Quant
test strips (available from EM Laboratories, Inc., 500
Executive Blvd., Elmsford, N.Y., 10523).
5.7.2 Procedures recommended for removal of peroxides are
provided with the test strips.
5.8 Benzene Hexane - Nanograde, redistilled in glass, if necessary.
5.9 Pesticide Standards - Acids and Methyl Esters, reference grade.
5.9.1 Stock standard solutions - Dissolve 100 mg of each
herbicide in 50 ml ethyl ether; then make to 100 ml
with redistilled hexane. Solution contains 1 rag/ml.
5.9.2 Working standard - Pipet 1.0 ml of each stock solution
into a single 100 ml volumetric flask. Make to volume
•
with a mixture of ethyl ether and hexane (1:1).
Solution contains 10 ug/ml of each standard.
5.9.3 Standard for Chromatography (Oiazomethane Procedure) -
Pipet 1.0 ml of the working standard into a glass
stoppered test tube and evaporate off the solvent
24
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using steam bath. Add 2 ml diazomethane to the
residue. Let stand 10 minutes with occasional
shaking, then allow the solvent to evaporate
spontaneously. Dissolve the residue in 200 ul of
hexane for gas chromatography.
5.9.4 Standard for Chromatgraphy (Boron Trifluoride
Procedure) - Pipet 1.0 ml of the working standard into
a glass stoppered test tube. Add 0.5 ml of benzene
and evaporate to 0.4 ml using a two-ball Snyder
microcolumn and a steam bath. Proceed as in 10.3.1.
Esters are then ready for gas chromatography.
6. Calibration
6.1 Gas chromatographic operating conditions are considered
acceptable if the response to dicapthon 1s at least 50% of full
scale when 0.06 ng is injected for electron capture detection
and 100 ng is injected for microcoulometric or electrolytic
conductivity detection. For all quantitative measurements, the
detector must be operated within its linear response range and
the detector noise level should be less than 2% of full scale.
6.2 Standards, prepared from methyl esters of phenoxy acid
herbicides calculated as the acid equivalent, are injected
frequently as a check on the stability of operating conditions.
Gas chromatograms of several chlorophenoxys are shown in Figure
1.
6.3 The elution order and retention ratios of methyl esters of
25
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64
RETENTION
3
IN
0
MINUTES
TIME
Ftq. I Column: 1.5 % 0V -17 + 1.93 % OF- I ,
Carrier Gas : Argon (5%) / Methane : 70ml/min.,
Column Temp. 185 C, Detector: Electron Capture .
26
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Table 1
RETENTION RATIOS FOR METHYL ESTERS OF SOME CHLORINATED
PHENOXY ACID HERBICIDES RELATIVE TO 2,4-D
Liquid Phase1
Column Temp.
Argon /Methane
Carrier FTow
Herbicide
dlcamba
2,4-0
sllvex
2,4,5-T
2,4-0
(minutes absolute)
1.5* OV-17
1.95* QF-1
185 C
70 ml/m1n
RR
0.60
1.00
1.34
1.72
2.00
5* OV-210
185 C
70 ml/min
RR
0.61
1.00
1.22
1.51
1.62
Ull columns alas:., 180 cm x 4 mm ID, solid support
Gas Chrom Q (100/120 mesh)
27
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chlorinated phenoxy acid herbicides are provided in Table 1, as a
guide.
7. Sample Collection and Handling
7.T Mash all bottles and cap liners in detergent water. Rinse with
tap water and finally distilled water.
7.2 Allow bottles and cap liners to air-dry. Muffle the sample
bottles at 400°C for 1 hour.
7.3 Rinse the cap liners with pesticide-grade hexane.
7.4 When cool, seal the bottles and store in a dust-free environment.
7.5 Collect samples in 1 quart narrow-mouth bottles with a Teflon
lined screw cap (option 1 quart widemouth screw-cap bottles with
Teflon lid liner). Collect all samples in duplicate.
7.6 Sampling from a water tap: Turn on water and allow system to
flush. When the temperature has stabilized, adjust the flow to
about 1 1/min. Fill the bottle about 90% full and seal.
7.7 The sample should be maintained near 4°C until analysis and
should be extracted as soon as possible after collection.
8. Sample Procedure
8.1 Quantitatively transfer 1 liter of sample into a two-liter
separatory funnel, and acidify to approximately pH 2 with
concentrated suIfuric acid. Check pH with indicator paper.
8.2 Add 150 ml of ether to the sample in the separatory funnel and
shake vigorously for one minute. Allow the contents to separate
for at least ten minutes. After the layers have separated,
drain the water phase into a one-liter Erlenmeyer flask. Then
28
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collect the extract in a 250 ml ground-glass Erlenmeyer flask
containing 2 ml of 37 percent aqueous potassium hydroxide.
8.3 Extract the sample two more times using 50 ml of ether each
time, and combine the extracts in the Erlenmeyer flask. (Rinse
the one-liter flask with each additional aliquot of extracting
solvent.)
9. Hydrolysis
9.1 Add 15 ml of distilled water and a small boiling stone to the
flask containing the ether extract, and fit the flask with a
3-ball Snyder column. Evaporate the ether on a steam bath and
continue heating for a total of 60 minutes.
9.2 Transfer the concentrate to a 60 ml separatory funnel. Extract
the basic solution two times with 20 ml of ether and discard the
ether layers. The herbicides remain in the aqueous phase.
9.3 Acidify the contents of the separatory funnel by adding 2 ml of
cold (4°C) sulfuric acid (1+3). Extract the herbicides once
with 20 ml of ether and then two more times with 10 ml of
ether. Collect the extracts in a 125 ml Erlenmeyer flask
containing about 0.5 g of acidified anhydrous sodium sulfate
(5.4). Allow the extract to remain in contact with the sodium
sulfate for approximately two hours.
10. Esterification (4,5)
10.1 Transfer the ether extract into a Kuderna-Oanish flask equipped
with a 10-ml graduated ampul. Use liberal washings of ether.
Using a glass rod, crush any caked sodium sulfate during the
washing.
29
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TO.1.1 If ester ification is to be done with diazomethane,
evaporate to approximately 4 ml on a steam bath (do
not immerse the ampul in water) and proceed as
directed in Section 10.2.
10.1.2 If esterification is to be done with boron
trifluoride, add 0.5 ml benzene and evaporate to about
5 ml on a steam bath. Remove the ampul from the flask
and further concentrate the extract to 0.4 ml using a
two-ball Snyder microcolumn and proceed as in 10.3.
10.2 Oiazomethane Esterification
10.2.1 Disconnect the ampul from the K-D flask and place in a
hood away from steam bath. Adjust volume to 4 ml with
ether, add 2 ml diazomethane, and let stand 10 minutes
with occasional swirling.
10.2.2 Rinse inside wall of ampul with several hundred
micro liters of ethyl ether. Take sample to
approximately 2 ml to remove excess diazomethane by
allowing solvent to evaporate spontaneously (room
temperature).
10.2.3 'Dissolve residue in 5 ml of hexane. Analyze by gas
chromatography.
10.2.4 If further clean-up of the sample is required, proceed
as in 10.3.4.
10.3 Boron Trifluoride Esterification
10.3.1 After the benzene solution in the ampul has cooled.
30
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add 0.5 ml of boron trifluoride-methanol reagent. Use
the two-ball Snyder microcolumn as an air-cooled
condenser and hold the contents of the ampul at 50°C
for 30 minutes on the steam bath.
10.3.2 Cool and add about 4.5 ml of a neutral 5 percent
aqueous sodium sulfate solution. Seal the flask with
a ground glass stopper and shake vigorously for about
one minute. Allow to stand for three minutes for
phase separation. Using a pipet, withdraw the bottom
water phase and discard.
10.3.4 Pipet the solvent layer from the ampul to the top of a
small column prepared by plugging a disposable Pasteur
pipet with glass wool and packing with 2.0 cm of
sodium sulfate over 1.5 cm of Florisil adsorbent.
Collect the eluate in a graduated ampul. Complete the
transfer by repeatedly rinsing the ampul with small
quantities of benzene and passing the rinses through
the column until a final volume of 5.0 ml of eluate is
obtained. Analyze by gas chromatography.
11. Quality Control
11.1 Duplicate and spiked sample analyses are recommended as quality
control checks. Quality control charts (3) should be developed
*
and used as a check on the analytical system. Quality control
check samples and performance evaluation samples should be
analyzed on a regular basis.
31
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11.2 Each time a set of samples is extracted, a method blank 1s
determined on a volume of distilled water equivalent to that
used to dilute the sample.
12. Calculation of Results
12.1 Determine the methyl ester concentration by using the absolute
calibration procedure described below or the relative
calibration procedure described in Part I, Section 3.4.2 (2).
(1) Mlcrograms/liter - (A) (B).JYt)
\»1J l»s'
A * ng standard
standard area
B » Sample aliquot area
VT Volume of extract Injected (ul)
Vt" Volume of totaT extract (ul)
Vsa Volume of water extracted (ml)
12.2 Molecular weights for the calculation of methyl esters as the
acid equivalents.
2,4-0 222.0 Olcamba 221.0
2,4-0 methyl ester 236.0 Dicamba methyl ester 236.1
.SHvex 269.5 2,4,5-T 255.5
Sllvex methyl ester 283.5 2,4,5-T methyl ester 269.5
13. Reporting Results
13.1 Report results in micrograms per liter as the acid equivalent
without correction for recovery data. When duplicate and spiked
samples are analyzed all data obtained should be reported.
32
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REFERENCES
(1) Goerlitz, D. G., and Lamar, W. L., "Determination of Phenoxy and
Herbicides in Water by Electron-Capture and Mlcrocoulometric Gas
Chromatography", U. S. Geol. Survey Water-Supply Paper 1817-C (1967).
(2) "Methods for Organic Pesticides in Water and Wastewater", (1971),
U. S. Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio, 45268.
(3) "Handbook for Analytical Quality Control 1n Water and Wastewater
Laboratories" (1972), U. S. Environmental Protection Agency, National
Environmental Research Center, Analytical Quality Control Laboratory,
Cincinnati, Ohio, 45268.
(4) Metcalf, L. 0., and Schnrttz, A. A., "The Rapid Preparation of Fatty
«1d Jft?CL!?r Sas Chromatograpnlc Analysis", Analytical Chemistry.
33, 363 (1961).
(5) Schlenk, H. and Gellerman, J. L., "Esterlficatlon of Fatty Acids with
Dlazomethane on a Small Scale", Analytical Chemistry. 32, 1412 (1960).
(6) "Pesticide Analytical Manual", U. S. Department of Health, Education
and Welfare, Food and Drug Administration, Washington, 0. C.
(7) Steere, N. V., editor, "Handbook of Laboratory Safety", Chemical
Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio, 44128, 1971,
pp. 250-254.
33
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APPENDIX I
Dlazomethane 1n ether (6)
1. CAUTIONS:
Diazomethane 1s very toxic. It can explode under certain conditions.
The following precautions should be observed.
Use only 1n we 11-ventHated hood.
Use safety screen.
Do not pipette solution of dlazomethane by mouth.
For pouring solutions of dlazomethane, use of gloves 1s optional.
Do not heat solutions to 100°C (EXPLOSIONS).
Store solutions of gas at low temperatures (freezer compartment of
explosion-proof refrigerators).
Avoid ground glass apparatus, glass stlrrers and sleeve bearings
where grinding may occur (EXPLOSIONS).
Keep solutions away from alkali metals (EXPLOSIONS).
Solutions of dlazomethane decompose rapidly In presence of solid
material such as copper powder, calcium chloride, boiling stones,
etc. These solid materials cause polymethylene and nitrogen
gas to form.
2. PREPARATION:
Use a we 11-ventilated hood and cork stoppers for all connections.
«
Fit a 125 ml long-neck distilling flask with a dropping funnel and an
efficient condenser set downward for distillation. Connect the condenser
to two receiving flasks in a series - a 500 ml Erlenmeyer followed by a
125 ml Erlenmeyer containing 30 ml ether. The inlet to the 125 ml
Erlenmeyer should dip below the ether. Cool both receivers to 0°C.
34
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As water bath for the distilling flask, set up a 2-liter beaker on a
stirplate (hot plate and stinrer), maintaining temperature at 70°C.
Dissolve 6 g KOH in 10 ml water in the distilling flask (no heat).
Add 35 ml Carbitol (diethylene glycol monoethyl ether), stirring bar, and
another 10 ml ether. Connect the distilling flask to the condenser and
immerse distilling flask in water bath. By means of the dropping funnel,
add a solution of 21.5 g Diazald in 140 ml ether over a period of 20
minutes. After distillation is apparently complete, add another 20 ml
ether and continue distilling until distillate is colorless. Combine the
contents of the two receivers in a glass bottle (WITHOUT ground glass
neck), stopper with cork, and freeze overnight. Decant the diazomethane
from the ice crystals Into a glass bottle, stopper with cork, and store
In freezer until ready for use. The final solution may be stored up to
six months without marked deterioration.
The 21.5 g of Diazald reacted in this manner produce about 3 g of
Diazomethane.
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