EPA 600/4-81-053
Environmental Protection r- f) s : \ t
Agency X * ' ' ' '
&EPA Research and
Development
Methods For Organochlorlne Pesticides and Chlorophenoxy
Acid Herbicides 1n Drinking Water and Raw Source Water
Prepared for
Joseph A. Cotruvo
Dtrector
Crtterta and Standards Division
Office of Drinking Water
Prepared by
Janes J. Llchtenberg
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
U.S. Environ- -n-i P," --tion Agency.
Region V, I/ '."•.'.-'
230 Soutii [;e~r •:•-•.-: '--^
Chicago, Illinois 60604
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METHODS FOR ORGANOCHLORINE PESTICIDES AND. CHLOROPHENOXY
ACID HERBICIDES IN DRINKING WATER AND RAH 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
U.S. Enviro/irri~ri*nS Protection Agency
Region V, IJ-r-v-y
230 South Dearbo-n Street
Chicago, Illinois 60604
July 1978
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FOREWORD
The National Interim Primary Drinking Water Regulations
promulgated on December 24, 1975,' 1n 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. Endrln, Undane, methoxychlor and toxphene may be determined
by the chlorinated hydrocarbon method while 2,4-0 and 2,4,5-TP (Sllvex)
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 Anaysls of Water and Wastes," becomes available.
Dwlght 6. Sallinger, Director
Environmental Monitoring and Support Laboratory - Cincinnati
Protection Agency
11
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DISCLAIMER
The mention of trade names or commercial products 1n this manual
1s for Illustration purposes, and does not constitute endorsement or
recommendation for use by the U. S. Environmental Protection Agency.
111
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As water bath for the distilling flask, set up a 2-11tar beaker on a
stlrplate (hot plate and stlrrer}, maintaining temperature at 7Q°C.
Dissolve 6 g KCH 1n 10 ml water 1n the distilling flask (no heat).
Add 35 ml Carbltol (dlethylene glycol monoethyl ether), stirring bar, and
another 10 ml ether. Connect the distilling flask to the condenser and
lonerse distilling flask 1n water bath. By,means of the dropping funnel,
add a solution of 21.5 g Olazald in 140 nl ether over a period of 20
nrinutes. After distillation 1s apparently complete, add another 20 ml
ether and continue distilling until distillate 1s colorless. Combine the
contents of the two receivers 1n a glass bottle (WITHOUT ground glass
neck), stopper with cork, and freeze overnight. Decant the dlazomethane
from the 1ce 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 Olazald reacted 1n this manner produce about 3 g of
Olazooethane.
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METHOD FOR OR6ANOCTLORINE PESTICIDES IN PRIMING HATER
1. Scope and Application
1.1 This wethod covers the determination of organochlorlne
pesticides 1n drinking water and raw source water. Undane,
endrln, nethoxychlor and toxaphene are determined by this
procedure.
1.2 The Method sensitivity 1s 0.001 to 0.010 ug/1 for single
component pesticides and O.OSO to 1.0 ug/1 for
rnultl-component pesticides when analyzing a 1 liter sample
with the electron capture detector.
1.3 Other organochlorlne pesticides, such as BHC,—heptachlor,
aldrfn, heptachlor epoxlde, dleldrln, Captan, DOE, 000, DOT,
endosulfan, dlchloran, mlrex, pentachloronltrobenzene, trf-
fluralln, 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 I1qu1d-
I1qu1d partition, methods for elimination of non-pesticide
Interferences and the pre-separatlon of pesticide mixtures.
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Identification 1s node by selective gas chromatographic
separations and may be corroborated through the use of two
or more unlike columns. Detection and measurement 1s
accomplished by electron capture, mlcrocou Tome trie or
electrolytic conductivity gas chromatography. Results are
reported In mlcrograms per liter.
2.2 Confirmation of the Identity of the compounds should be made
by SC-MS when a new or undefined sample type 1s being
analyzed and the concentration 1s 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 nrisInterpretation 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 1n all-glass systems may be required. Refer to
Part I. Sections 1.4 and 1.5 (1).
3.2 The Interferences 1n drinking water should not pose any
difficulty 1n obtaining accurate and precise measurement of
organochlorlne pesticides.
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3.3 Polychlorlnated Blphenyls (PC8s) - Special attention 1s
called to Industrial plastlclzers and hydraulic fluids such
as the PC8s, which are a potential source of Interference 1n
pesticide analysis. The presence of PCSs 1s Indicated by a
large number of partially resolved or unresolved peaks which
nay occur throughout the entire chromatogram. Par-
ticularly severe PCS Interference will require special
separation procedures (2,3).
3.4 Phthalate Esters - These compounds, widely used as plastl-
clzers, respond to the electron capture detector and are a
source of Interference 1n the determination of organo-
chlorlne pesticides using this detector. Water leaches
these materials from plastics, such as polyethylene bottles
and tygon tubing. The presence of phthalate esters 1s
Implicated In samples that respond to electron capture but
not to the nrfcrocoulometrlc or electrolytic conductivity
halogen detectors.
3.5 Organophosphorus Pesticides - A number of organophosphorus
pesticides, such as those containing a nltro group, e.g.,
parathlon, respond to the electron capture detector and may
Interfere with the determination of the organochlorlne
pesticides. Such compounds can be Identified by their
response to the flame photometric detector (4).
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Apparatus and Materials
4.1 Sas Chromatograph - Equipped with glass lined Injection port.
4.2 Detector Options:
4.2.1 Electron Capture - Radioactive (tritium or n1cke!-63)
4.2.2 M1crocoulometr1c T1trat1on
4.2.3 Electrolytic Conductivity
4.3 Recorder - Potent1ometr1c strip chart (10 1n.) compatible
with the detector.
4.4 Gas Chromatograph1c Column Materials:
4.4.1 Tubing - Pyrex (180 on long X 4 nui ID)
4.4.2 Glass Wool - SHanized
4.4.3 Solid Support - Gas-Chroro-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, 5X
4.4.4.3 OV-17, 1.5* plus QF-1 or OV-210, 1.9SX
4.4.4.4 QF-1, 6X plus SE-30, 4«
4.5 Kuderna-Oanlsh (K-0) 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 Chromatographlc Column - Chroma/lex (400 nw long x 19 m 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 Chromatographlc Column - pyrex (approximately 400 nm long x
20 IM ID) with coarse fritted plate on bottom.
4.8 Micro Syringes - 10, 25, 50 and 100 ul.
4.9 Separator/ 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 Sulfuric Add - (ACS) Mix equal volumes of cone. HjSO^
with distilled water.
5.4 Florlsll - PR Grade (60-100 mesh); purchase activated at
12SO°F and store 1n the dark 1n glass containers with
glass stoppers or foil-lined screw caps. Before use,
activate each batch overnight at 130°C 1n foil-covered
glass container. Determine 1 auric-acid value (See Section
13).
5.5 Dlethyl Ether - Nanograde, redistilled 1n glass, 1f
necessary.
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5.5.1 Must b« 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 1n glass 1f
necessary.
5.7 Pesticide Standards - Reference grade.
6. Calibration
6.1 6as chronatographlc operating conditions are considered
acceptable 1f the response to dlcapthon 1s at least 501 of
•
full scale when "<0.06 ng 1s Injected for electron capture
detection and <100 ng 1s Injected for n1crocoulometr1c or
electrolytic conductivity detection. For all quantitative
Measurements, the detector oust be operated within Its
linear response range and the detector noise level should be
less than 2X of full scale.
6.2 Standards are Injected frequently as a check on the sta-
bility of operating conditions. Sas chronatograns of
several standard pesticides are shown 1n Figures 1, 2, 3 and
4 and provide reference operating conditions for the four
recomnended columns.
6.3 The elutlon order and retention ratios of various organo-
chlorlne pesticides are provided 1n Table 1, as a guide.
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s
21
II II
IflfNINN TIME IN MIMIffS
Fifun 1. CalHMi Pickiif 1.5% IV-U * 1.15% OF 1, Cirriu Gat: Arm/Mitiiii at II •)/•!•.
Tmiiiilin: 211 C, Piticlir: flietni Capturi.
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15 10 SO
KTENT10N TIME III MIN0TES
Figure 2. Column Packing: 5% OV-210, Carrier Sas: Argon/Methane
at 70 ml/nia, Column Temperature: 180 C, Detector:
Electron Capture.
8
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IS
II
li II
IfKNTIIN mi m MOUTH
Fi|«n 3. CilNMi Packiif: 1% Of 1 + 4% SE 31, Cirrllf fiat: Ar|ii/Mitiin ai SO •l/«ii.
: 200 C, Ditictir: Elictrn Cailnri.
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01
5
. -
815
MKEX
ICIJIII
KPT. EPBXBE
—— ALMl
TIIFLIULl
SDIVEMT
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Table 1
RETENTION RATIOS OF VARIOUS ORSANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Liquid
Phase' 1
Col urn Temp.
Argon/Methane
Carrier Flow
Pesticide
THfluralln
-8HC
PCN8
Llndane
01 chl or an
Heptachlor
Aldrln
Heptachlor Epoxlde
Endosulfan I
P.P'-OOE
01e1dr1n
Cap tan
Endrln
o.p'-QOT
p,p'-000
Endosulfan II
P,P'-OOT
Hlrex
Methoxychlor
Aldrln
(M1n. absolute)
1.5X OV-17
.95X QF-1*
200 C
60 m1/m1n
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 n1/m1n
RR
i.n
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
3X
OV-1
180 C .-
70 nl/n1n
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
6S QF-1
+
4X SE-30
200 C
60 m1/m1n
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
4.7*
4.60
5.6
Ull columns glass, 180 on x 4 mm ID, solid support Gas-Chrom Q (100/120
mesh)
ZOV-210 also may be used
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7. Sample Collection and Handling
7.1 Wash all bottles and cap liners 1n 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 1n a dust-free
environment.
7.5 Collect samples 1n 1 quart narrow-mouth bottles with a
Teflon lined screw cap (option 1 quart wldemouth screw-cap
bottles with Teflon I1d Hner). Collect all samples 1n
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 l/m1n. Fill the bottle about 90X 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 15X
methylene chloride 1n 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 1n the separatory funnel vigorously for two
nrfnutas. 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 lOO-ml beaker and then
pass 1t through a column containing 3 to 4 Inches of
anhydrous sodium sulfate, and collect 1t 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
Erlemneyer 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 1n the sane manner.
8.3 Combine the extracts and concentrate 1n 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 1f greater sensitivity 1s required.
9. Clean-up and Separation Procedures
9.1 Interferences 1n the form of distinct peaks and/or high
background 1n the Initial gas chromatograph-fc analysis,- as
well as the physical characteristics of the extract (color,
cloudiness, viscosity) and background knowledge of the
sample will Indicate whether clean-up 1s 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 ml 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 155 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 - 8y using an equivalent quantity of any
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batch of F1or1s11, as determined by Its Uurlc add
value,the pesticides will be separated into the eluates
Indicated below:
6X Eluate
Aldrln DOT Pentachloronltrobenzene
8HC Heptachlor Strobane
Chlordane Heptachlor Epoxlde Toxaphene
000 Undane Tr1f1ural1n
OOE Methoxychlor PCS's
M1rex
15X Eluate SOX Eluate
Endosulfan I Cndosulfan II
Endrln Captan
01eldr1n
01chloran
Phthaiate esters
Certain thlophosphate pesticides can occur 1n each of the
above fractions as well as the 100X 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 1s extracted, a method blank 1s
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
15
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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/liter - l£LJL|L_iVt)
(Vj) (Vs)
A » nq standard
Standard area
8 » Sample aliquot area
V^» 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 laurlc add
13.1 A rapid method for determining adsorptive capacity of
F1or1s1l is based on adsorption of lauric acid from hexane
solution (6) (3). An excess of laurlc acid is used and the
amount not adsorbed is measured by alkali titration. The
weight of laurlc add adsorbed is used to calculate, by
simple proportion, equivalent quantities of Florisil for
batches having different adsorptive capacities.
13.2 Apparatus
13.2.1 Suret - 25 ml with 1/70 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
phenolphthaleln.
13.3.2 Hexane - Distilled from all glass apparatus.
13.3.3 Laurie add - Purified, CP.
13.3.4 Laurie acid solution - Transfer 10.000 g laurlc
acid to 500 ml volumetric flask, dissolve 1n
hexane, and dilute to 500 ml (1 ml » 20 mg).
13.3.5 Phenolphthaleln Indicator - Dissolve 1 g 1n
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
(1JI). Dilute 25 ml IN. NaOH to 500 ml with water
(0.05N). Standardize as follows: Weigh 100-200
mg laurlc acid Into T25 ml Erlenmeyer flask. Add
50 ml neutralized ethyl alcohol and 3 drops
phenolphthaleln Indicator; titrate to permanent
end point. Calculate mg laurlc acid/ml 0.05K NaOH
(about 10 mg/nl).
13.4 Procedure
13.4.1 Transfer 2.000 g FloHsIl to 25-ml glass-stoppered
Erlenmeyer flasks. Cover loosely with alunrinura
foil and heat overnight at 130°C. Stopper, cool
to room temperature, add 20.0 ml laurlc acid
solution (400 mg), stopper, and shake occasionally
for 15 minutes. Let the adsorbent settle and
17
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plpet 10.0 ml of supernatant into 125 ml
Erlemneyer flask. Avoid Inclusion of any Florlsil.
13.4.2 Add 50 ml of neutral alcohol and 3 drops of
Indicator solution; titrate with 0.05£ to a
permanent end point.
13.5 Calculation of laurlc add value and adjustment of column
weight
13.5.1 Calculate amount of laurlc add adsorbed on
F1or1s11 as follows:
Laurie add value » mg laurlc add/g F1or1s11 *
200 - (ml required for tltratlon x mg laurlc
acid/ml 0.05N NaOH).
13.5.2 To obtain an equivalent quantity of any batch of
F1or1s11, divide 110 by laurlc add value for that
batch and multiply by 20 g. Verify proper elutlon
of pesticides by 13.6.
13.6 Test for proper elutlon pattern and recovery of pesticides
13.6.1 Prepare a test mixture containing aldrln,
heptachlor epoxlde, p,p'-OOE, dleldrln, Parathlon
and malathion. 01e1dr1n and Parathlon should
elute in the 15X eluate; all but a trace of
malathion in the SOX 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 or Environmental
Materials for Blphenyls, Analytical Chemistry Method 71-35,
Monsanto Company, St. Louis, Missouri, 63166, 1970.
3. 'Method for PolychloHnated Blphenyls 1n Industrial Effluents,"
Environmental Protection Agency, National Environmental Research
Center, Cfnc1rmat1, 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 1n 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. Oept. of Health, Education and
Welfare, Food and Drug Administration, Washington, O.C.
7. "Analysis of Pesticide Residues 1n Human and Environmental
Samples," U.S. Environmental Protection Agency, Perrfne Primate
Research Laboratories, PerHne, Florida, 33157, 1971.
8. Mills, P.A., "Variation of Flor1s11 Activity: Simple Method for
Measuring Adsorbent Capacity and Its Use 1n Standardizing F1or1s1l
Columns," Journal of the Association of Official Analytical
Chemists, bl^ 29 (195871
9. Goer11tz, D.F. and Brown, E,, "Methods for Analysis of Organic
Substances 1n 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
herbldes 1n drinking water and raw source water. The compounds
2,4-d1chlorophenoxy-acet1c acid (2,4-0) and 2-(2,4,5-tr1ch1oro-
phenoxy) pro(5Ionic acid (sllvex) are determined by this
procedure.
1.2 The detection limits are 20 ng/1 for 2,4-0 and 5 ng/1 for Sllvex
and 2,4,5-T.
1.3 Since these compounds may occur 1n water 1n various forms (I.e.,
acid, salt, ester, etc.) a hydrolysis step 1s 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-d1chloro-o-an1s1c add (dlcamba) and 2,4,5-trlchlorophenoxy-
acetlc 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 acids and extraneous organic material 1s removed
by a solvent wash. The acids are converted to methyl esters
which are extracted from the aqueous phase. Identification of
the esters 1s made by selective gas chromatographic separations
and may be corroborated through the use of two or more unlike
columns. Detection and measurement 1s accomplished by electron
20
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capture, m1crocoulometr1c or electrolytic conductivity gas
chromatography (1). Results are reported 1n mlcrograms per
liter.
2.2 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 baselines
causing misinterpretation of gas chroraatograms. 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 1n
all-glass systems may be required. Refer to Part 1, Section 1.4
and 1.5, (2).
3.2 The Interferences encountered 1n drinking water should not pose
great difficulty 1n obtaining accurate and precise measurement
of chlorinated phenoxy add herbicides.
3^3 Organic acids, expeclally 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 ac1d-r1nsed and sodium sulfate should
be acidified with sulfurlc 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 n1ckel-63)
4.2.2 Mlcrocouloraetrlc Tltratlon
4.2.3 Electrolytic Conductivity
4.3 Recorder - Potent1ometr1c strip chart (10 1n.) compatible with
the detector.
4.4 Gas Chromatograph1c Column Materials:
4.4.1 Tubing - Pyrex (180 on long X 4 mm ID)•
4.4.2 Glass Wool - Sllanlzed
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, 1.9SX
4.5 Kuderna-Oanlsh (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
22
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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 Plpets - Pasteur, glass disposable (140 mm long X 5 an ID).
4.10 Separatory Funnels - 50 ml and 2000 ml with Teflon stopcock.
m
4.11 Glass wool • Filtering grade, add washed.
4.12 Olazald Kit • Recommended for the generation of dlazomethane
(available from Aldrlch Chemical Co., Cat. #210,025-2)
4.13 F1or1s11 - PR grade (60-100 mesh) purchased activated at 1250F
and stored at 130 C.
5. Reagents, Solvents and Standards
5.1 Boron Tr1fluor1de-Methanol-ester1f1cat1on-reagent, 14 percent
•
boron trlfluorlde by weight.
5.2 N-methyl-N-«1troso-p-toluenesulfonam1de (Olazald) - High purity,
melting point range 60-62 C. Precursor for the generation of
dlazomethane (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, sulfurlc 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.
23
-------
5.5 Sulfurfc acid - (ACS) concentrated, Sp. Sr. 1.84.
5.5.1 SulfuHc Acid (1:1) - Carefully mix equal volumes of
HjSOa (5.5) with distilled water. Cool while •
adding acid.
5.5.2 SulfuHc Add (1+3) - Carefully mix 1 volume of
HjSO^ (5.5) with 3 volumes of distilled water.
Cool while adding acid.
5.6 Carbltol (diethylene glycol monoethyl ether).
5.7 Dlethyl Ether - Nanograde, redistilled 1n glass, 1f 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 1n glass, 1f necessary.
5.9 Pesticide Standards - Acids and Methyl Esters, reference grade.
5.9.1 Stock standard solutions - Dissolve 100 mg of each
herbicide 1n 60 ml ethyl ether; then make to 100 ml
with redistilled hexane. Solution contains 1 mg/ml.
5.9.2 Working standard - P1pet 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 (01azomethane Procedure) -
P1pet 1.0 ml of the working standard into a glass
stoppered test tube and evaporate off the solvent
-------
using steam bath. Add 2 ml dlazomethane to the
residue. Let stand 10 minutes with occasional
shaking, then allow the solvent to evaporate
spontaneously. Dissolve the residue in 2(Xf ul of
hexane for gas chromatography.
5.9.4 Standard for Chromatgraphy (Boron Trlfluorlde
. . Procedure) - P1pet 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
nrlcrocolumn and a steam bath. Proceed as 1n 10.3.1.
Esters are then ready for gas chromatography.
6. Calibration
6.1 Gas chromatographlc operating conditions are considered
*
acceptable ff the response to dlcapthon Is at least SOX of full
scale when 0.06 ng 1s Injected for electron capture detection
• and 100 ng 1s Injected for ra1crocoulometr1c 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 2X of full scale.
6.2 Standards, prepared from methyl esters of phenoxy add
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 1n Figure
1.
6.3 The elutlon order and retention ratios of methyl esters of
25
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X3A1IS -
f
Is
» -
4
CM? fc
W > ~ *
a o j< «
c"=5
us
5
*i
sl
y o
«OK -
-------
Table 1
RETENTION RATIOS FOR METHYL ESTERS OF SOME CHLORINATED
PHENOXY ACID HERBICIDES RELATIVE TO 2,4-0
Liquid Phase1
Colunm Tenp.
Argon/Methane
Carrier Flow
Herbicide
dlcanfca
2,4-0
si 1 vex
2,4,5-T
2,4-0
( »1nutes absolute)
1.5X OV-17
1.95X QF-1
185 C
70 nl/nrin
RR
0.60
1.00
1.34
1.72
2.00
5% OV-210
185 C
70 nl/nrin
RR*
0.61
1.00
1.22
1.51
1.62
columns glass, 180 en x 4 on ID, solid support
6as Chroa Q (100/120 mesh)
27
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chlorinated phenoxy acid herbicides are provided 1n Table 1, as a
guide.
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 1n a dust-free environment.
7.5 Collect samples 1n 1 quart narrow-mouth bottles with a Teflon
lined screw cap (option 1 quart wldemouth screw-cap bottles with
Teflon I1d liner). Collect all samples 1n 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 l/m1n. Fill the bottle about 90X 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
3.1 Quantitatively transfer 1 liter of sample Into a two-liter
separatory funnel, and acidify to approximately pH 2 with
concentrated sulfuHc add. Check pH with Indicator paper.
3.2 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 1n 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
coW (4°C) sulfurlc 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 1n contact with the sodium
sulfate for approximately two hours.
10. Ester1f1cat1on (4,5)
10.1 Transfer the ether extract Into a Kudema-Oanlsh 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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10.1.1 If ester1f1cation 1s to be done with dlazomethane,
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 ester1f1cat1on 1s to be done with boron
trlfluorlde-, 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 nricrocolumn and proceed as 1n 10.3.
10.2 Dlazomethane Ester1f1cat1on
10.2.1 Disconnect the ampul from the K-0 flask and place 1n a
hood away from steam bath. Adjust volume to 4 ml with
ether, add 2 ml dlazomethane, 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 dlazomethane by
allowing solvent to evaporate spontaneously (room
temperature).
10.2.3 Dissolve residue 1n 5 nl of hexane. Analyze by gas
chromatography.
10.2.4 If further clean-up of the sample 1s required, proceed
as In 10.3.4.
10.3 Boron Trlfluorlde Esterif1cat ion
10.3.1 After the benzene solution 1n the ampul has cooled,
30
-------
add 0.5 ml of boron trlfluoMde-methanol reagent. Use
the two-ball Snyder mlcrocolumn 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 plpet, withdraw the bottom
water phase and discard.
10.3.4 Plpet the solvent layer from the ampul to the top of a
small column prepared by plugging a disposable Pasteur
plpet with glass wool and packing with 2.0 cm of
*
sodium sulfate over 1.5 cm of Flor1s11 adsorbent.
Collect the eluate 1n 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 1s
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 fs extracted, a method blank is
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) M1crograms/I1ter - (*)„ ffiiuW
V»1/ l»sJ
A • nq standard
standard area
8 « Sample aliquot area
V-j- Volume of extract Injected (ul)
Yt" Volume of total extract (ul)
Vs» 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 Olcamba methyl ester 236.1
SHvex 269.5 2,4,5-T 2S5.S
SHvex methyl ester 283.5 2,4,5-T methyl ester 269.5
13. Reporting Results
13.1 Report results in mlcrograms per liter as the add equivalent
without correction for recovery data. When duplicate and spiked
samples are analyzed all data obtained should be reported.
32
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(1) 6oerl1tz, 0. 6., and Lamar, W. L., "Determination of Phenoxy and
Herbicides In Water by Electron-Capture and M1crocou1ometr1c Sas
Chromatography", U. S. Geol. Survey Watar-Supply Paper 1817-C (1967).
(2) "Methods for Organic Pesticides In Water and Wastewater*, (1977),
U. S. Environmental Protection Agency, National Environmental
Research Center, Cincinnati, Ohio, 45268.
(3) "Handbook for Analytical Quality Control In 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 Scharltz, A. A., "The Rapid Preparation of Fatty
Acid Esters for Sas Chromatographlc Analysis*, Analytical Chemistry.
33, 363 (1961). l ^
(5) Schlenk, H. and Seller-man, J. U, "Ester1f1cat1on of Fatty Acids with
Olazomethane on a Small Scale*, Analytical Chemistry. 32, 1412 (I960).
(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
Olazomethane In ether (6)
1. CAUTIONS:
Olazonethane Is very toxic. It can explode under certain conditions.
The following precautions should be observed.
Use only In Me11-ventilated hood.
Use safety screen.
Oo 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 stlnrers and sleeve bearings
where grinding may occur (EXPLOSIONS).
Keep solutions away from alkali netals (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 we11-ventilated hood and cork stoppers for all connections.
Fit a 125 nl long-neck distilling flask with a dropping funnel and an
efficient condenser set downward for distillation. Connect the condenser
to two receiving flasks 1n a series - a 500 ml Erlenroeyer 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.
U S. Environmental Protection Agency
Region V, 1.^7
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