METHODS FOR NONCONVENTIONAL
PESTICIDE ANALYSIS OF INDUSTRIAL
AND MUNICIPAL WASTEWATER
U.S. ENVIRONMENTAL PROTECTION AGENCY
George M. Jett
Project Officer
January 31, 1983
fL"\ ~'^™r«rt?i Fraction Agency
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Disclaimer
This report has been reviewed by the Effluent Guidelines Division, U.S.
Environmental Protection Agency, and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
15,3. Livl.^.-.r^r.b1, Protection Agency
U.S. Environmental Protection Agency
Effluent Guidelines Division
Waterside Mall (WH-552)
401 M. Street, S.W.
Washington, D.C. 20460
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Foreword
The Environmental Protection Agency proposes to establish test procedures
for the analysis of 66 nonconventional pesticide pollutants for which effluent
limitations guidelines and standards were proposed on November 30, 1982, in
40 CFR Part 455 (47 FR 53994). These procedures will be used in supporting
the effluent limitations guidelines and standards proposed in 47 FR 53994 and
would also be used for filing applications for the National Pollutant
Discharge Elimination System (NPDES) permits, for state certification, and for
compliance monitoring under the Clean Water Act.
Under the authority of Sections 304(h) and 501(a) of the Clean Water Act, 33
U.S.C. 1251 et seq (the Federal Water Pollution Control Act Amendments of
1972, as amended by the Clean Water Act of 1977) the Agency is required to
promulgate guidelines establishing test procedures for the analysis of
pollutants and is authorized to prescribe such regulations as are necessary to
carry out its functions under this Act.
The test procedures in this document are a compilation of three sets of
analytical methods: those developed by the Pesticide Industry found in
Part A, those developed by the Agency contractors found in Part B, and those
developed/approved by the Agency's Environmental Monitoring and Support
Laboratory—Cincinnati (EMSL—Ci) found in Part C. All methods contained
herein follow an Agency specified format while still maintaining the integrity
of the original method.
A fourth section of this document (Part D) contains Agency requirements for
sample collection, preservation, and handling; quality control; and safety.
These requirements were designed for universal use and can be applied to each
nethod found in Parts A, B, and C of this document.
Jeffeizy D. Denit, Director
Effluent Guidelines Division
111
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Acknowledgements
This document was developed under the supervision and guidance of Mr. George
M. Jett, who served as project officer for the development of the November 30,
1982 pesticide technicaj regulations and the January 1983 pesticide analytical
regulation. Other Agency key personnel who assisted on this document were Mr.
Devereaux Barnes, Ms. Nancy Parkinson, Mr. S. Scott Saavedra and Ms Susan Schmedes,
Additional technical assistance was provided by Dr. James Longbottom, Dr.
James Lichtenberg, Dr. Robert Booth, and Mr Edward Kerns of the Environmental
Monitoring and Support Laboratory in Cincinnati, Ohio.
This document was prepared by Environmental Science and Engineering, Inc.,
(ESE) under contract number 68-01-6701. The project was managed by Ms.
Barbara Brown, under the direction of Mr. James B. Cowart. Technical guidance
and direction were provided by Dr. John Mousa and Mr. Stuart Whitlock. Key
personnel included Dr. Dilna Victor, Ms. Karen Hatfield, Mr. Peter Beck, and
Ms. Paula Anderson. Ms. Suzanne Albrecht coordinated the production of the
document, which was edited by Ms. Patricia McGhee and typed in it entirety by
Ms. Carolyn Granger. Analytical methods were provided by ESE and two addi-
tional contractors, whose efforts are acknowledged as follows: Dr. Herbert
C. Miller and Ms. Ruby James of Southern Research Institute: Mr. John Clausen,
Mr. Bob Beimer, Mr. Mike O'Rell and Mr. Bill Coleman of TRW Systems, Inc.
Acknowledgement is also made of the cooperation of personnel in many plants
in the pesticide chemicals industry who provided valuable data and information
relating to nonconventional pesticide analytical methods developed and used by
their companies.
iv
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Conbents
Introduction vii
Proposed Test Procedures for Nonconventional Pesticides ix
Part A. Industry Analytical Methods
Alachlor, Butachlor, and Propachlor Method 101
Alachlor, Butachlor, and Propachlor Method 102
AOP, ZAC, Zineb, and Ziram Method 103
Benfluralin, Ethalf luralin, and Isopropalin Method 104
Benomyl and Carbendazim Method 105
Benomyl and Carbendazim Method 106
Bentazon (BAS 351-H) Method 107
Bolstar Method 108
Bromacil, Hexazinone, Oxamyl, Terbacil, and Methomyl..Method 109
Busan 40, Busan 85, and KM- Methyl Method 110
Carbofuran Method 111
Chlorobenzilate Method 112
Chlorpyrifos and Chlorpyrifos methyl Method 113
Coumaphos Method 114
Cyanazine Method 115
Cyanazine [BLADEX(TM)] and Stirofos ,.,,.., Method 116
2,4-DB Method 117
Deet Method 118
Mevinphos [PHOSDRIN(TM)], Dichlorvos [VAPONA(TM)],
Naled [Dibrom(TM)], and Stirofos [RABON(TM)] Method 119
Mevinphos [PHOSDRIN(TM)], Dichlorvos [VAPONA(TM)],
Naled [DIBROM(TM)], and Stirofos [GARDONA(TM)] Method 120
Dinoseb (2-Sec-Butyl-4,6-Dinitrophenol) Method 121
Dinoseb Method 122
EthiontTM] Method 123
Etridiazole [TERRAZOLE(TM)] Method 124
Fensulfothion [DASANIT(TM)] Method 125
Fenthion Method 126
Glyphosate Method 127
Mancozeb [DITHANE M-45(TM)] Method 128
Maneb Method 129
Mephosfolan [CYTROLANE(TM)], Phorate [THIMET(TM)],
and Terbufos [COONTER(TM)] Method 130
Metham [VAPAM(TM)] „ Method 131
Methomyl [NUDRIN(TM)] Method 132
Methomyl Method 133
Mevinphos Method 134
Prof luralin Method 135
Simetryn Method 136
Triadimefon [BAYLETON(TM)] Method 137
Trichloronate [AGRITOX(TM)] Method 138
Tricyclazole Method 139
Glyphosate Method 140
Hexazinone, Terbacil, and Bromacil Method 141
Ziram Method 142
Propachlor Method 143
Fluometuron Method 144
Metribuzin [SENCOR(TM)] Method 145
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Part B. Contractor Analytical Methods
AOF, Ferbam, Niacide, ZAC, Zineb, and Ziram Method 401
Benomyl and CarbendazLm Method 402
Carbofuran Method 403
Chlorobenzilate, Terbutryn, and Profluralin Method 404
2,4-DB, 2,4-DB Isobutyl Ester (2,4-DB IBE), and
2,4-DB Isooctyl Ester (2,4-DB IOE) Method 405
Dinoseb Method 406
Dinoseb Method 407
Methomyl Method 408
Cyanazine Method 409
Part C. EMSL Analytical Methods
Pentachlorophenol sal t Method 604
Chlorobenzilate, Etridiazole, Propachlor, and
Dibromochloropropane (DBCP) Method 608.1
Ethion Method 614
2,4-DB, 2,4-DB IBE, 2,4-DB IOE, and Dinoseb Method 615
Carbophenothion Method 617
Simetryn and Terbutryn Method 619
Bolstar, Chlorpyrifos, Chlorpyrifos methyl, Coumaphos,
Dichlorvos, Fensulfothion, Fenthion, Mevinphos,
Naled, Phorate, Rocinel, Stirofos, and
Trichloronate Method 622
Pentachlorophenol salt Method 625 »
Benfluralin, Ethalfluralin, Isopropalin, and
Profluralin Method 627
Cyanazine Method 629
AOP, Busan 40, Busan 85, Carbam-S, Ferbam,
KN Methyl, Mancozeb, Maneb, Metham, Nabam,
Niacide, ZAC, Zineb, and Ziram Method 630
Benomyl and Carbendazim Method 631
Carbofuran, Fluometuron, Methomyl, and Oxamyl Method 632
Bromacil, Deet, Hexazinone, Metribuzin, Terbacil,
Triadimefon, Tricyclazole ..Method 633
Dichlofenthion, Dioxathion and Carbophenothion........Method 701
Part D. Quality Assurance Requirements
Procedure A — Requirements for Sample Collection, Preservation,
and Handling
Procedure B — Quality Control Requirements
Procedure C — Safety Requirements
Appendix A. Definition aad Procedure for the Determination of the Method
Detection Limit
Appendix B. Abbreviations, Acronyms, and Other Terms Used in this Document
VI
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Introduction
Under the Clean Water Act, the Agency is required to regulate three broad
categories of pollutants. These categories are toxic pollutants, conventional
pollutants, and nonconventional pollutants. Toxic po-llutants are a defined
list of 126 pollutants derived from the NRDC Consent Decree, 8 ERG 2120
(D.D.C. 1976), modified 12 ERC 1833 (D.D.C. 1979). Conventional pollutants
are biological oxygen demand (BOD), total suspended solids (TSS), oil and
grease, fecal coliform, and pH. A nonconventional pollutant is any pollutant
not identified as a toxic pollutant (Section 307(a)(l) of the Act) or as a
conventional pollutant (Section 304(a)(4) of the Act).
Section 304(h) of the Clean Water Act directs the Agency to approve
analytical methods for the analysis of pollutants. These methods are
necessary for filing applications for the National Pollutant Discharge
Elimination System (NPDES) permits, for state certifications, and for
compliance monitoring under the Clean Water Act.
On November 30, 1982, the Agency proposed effluent limitations guidelines
and standards for 34 specific toxic pollutants, BOD, COD, TSS, pH, and 137
nonconventional pesticide pollutants in the Pesticide Chemicals Industry
(47 FR 53994). An integral part of the effluent limitations guidelines and
standards for the Pesticides Industry is the regulation proposing approved
test procedures. To date, EPA has established analytical methods for only
49 of the nonconventional pollutants regulated by the proposed pesticide
effluent limitations guidelines and standards. EPA projected that additional
nonconventional pesticide analytical methods would be proposed by January
1983. In support of these regulations the Agency has compiled test procedures
for an additional 66 nonconventional pesticide pollutants.
The Agency is proposing nonconventional pesticide analytical methods for 66
of the 137 nonconventional pesticide pollutants for which Agency-approved
procedures do not currently exist. The remaining 71 nonconventional
pesticide pollutants regulated in the November 30, 1982 document proposing
effluent limitations guidelines and standards for the Pesticide Industry have
either 304(h) approved methods or require no method since EPA proposed to
establish "no discharge of process wastewater" as the applicable regulation.
The January 1983 proposed rulemaking (Guidelines Establishing Test
Procedures for the Analysis of Nonconventional Pesticide Pollutants in the
Pesticide Industry) includes a compilation of three sets of methods: those
developed/used by the industry, those developed/used by Agency contractors,
and those developed/approved by the Environmental Monitoring and Support
Laboratory in Cincinnati (EMSL-Ci). The Agency is proposing to incorporate by
reference these test procedures for nonconventional pesticide pollutants in 40
CFR Part 455 Section 170 pursuant to Subpart 401.13.
This document includes the abovementioned methods and is divided into four
parts. Part A includes 45 industry-developed/used methods involving such
instrumental techniques as gas chromatography (GC), spectrophotometry, high
pressure liquid chromatography (HPLC), thin layer chromatography (TLC), gas
chromatography/mass spectrometry (GC/MS), and titration and are numbered 101
through 145. Part B includes nine contractor-developed/used methods including
GC, spectrophotometric, HPLC, and GC/MS methods and are numbered 401 through
409. Part C includes 15 EMSL-developed/approved methods such as GC, GC/MS,
HPLC, and spectrophotometric methods and are numbered 604, 608.1, 614, 615,
617, 619, 622, 625, 627, 629 to 633 and 701. In addition to analytical
VII
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methodology, Section D contains quality assurance (QA) requirements for
collection, preservation, and handling of samples; quality control (QC); and
safety. These requirements are applicable to all methods enclosed in this
document. The EMSL procedure for determining the method detection limit is
presented in Appendix A. Appendix B lists abbreviations, acronyms, and other
terms used in this document.
The industry and contractor methods contained herein have been assembled
into a format consistent with the EMSL-developed methods. This effort was
completed for ease of reading and consistency; however, the integrity of the
original method is still maintained. The methods in their original form are
referenced at the end of each procedure.
Storet and CAS numbers were not available for all nonconventional pesticides
identified in this document (see Scope and Application section of each
method). Therefore, an errata sheet will be prepared at a later date when
these numbers become available.
Only the 66 nonconventional pesticide pollutants previously mentioned
are included in the methods titles; however, both these pollutants and others
which can be analyzed by each method are listed under the "scope and
application" of all methods presented in this document. The methods in
Part A were developed/used by various members of the Pesticide Industry to
generate data which were supplied to the Agency in response to Information
requests made under the authority of Section 308 of the Act. The methods in
Part B were developed/used by the Agency contractors during the Pesticides
Industry Verification Program. The methods in Part C were developed/approved
by the Agency's Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio. The EMSL methods were not used for data generation for the proposed
pesticide effluent limitations and standards since data were collected prior
to their development.
In some cases methods from three sources (industry, contractor, and EMSL)
are available for one nonconventional pesticide pollutant (see Table 1A). The
Agency is presenting all available methods for each nonconventional pesticide.
The Agency then intends to seleict the most appropriate methods for
promulgation.
References
1. National Resources Defense Council, Inc., et al. , v. Train, 8 ERC 2120
(D.D.C. 1976), modified 12 ERC (D.D.C. 1979).
2. Guidelines Establishing Test Procedures for the Analysis of Pollutants, 40
Code of Federal Regulations! (CFR), Part 136, Published in Federal
Register, 44, 69464.
3. "Methods for Organic Chemical Analysis of Municipal and Industrial
Wastewater," July 1982, U.Ei. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268
(EPA-600/4-82-057).
4. Pesticide Chemicals Category Effluent Limitations Guidelines, Pretreatment
Standards, and New Source Performance Standards, 40 CFR, Part 455,
Published in Federal Register, 47, 53994.
5. "Development Document for Effluent Limitations Guidelines and Standards
for the Pesticides Point Source Category," November 1982, U.S.
Environmental Protection Agency Effluent Guidelines Division, Washington,
D.C. 20460 (EPA-440/l-82/079-b).
Vlll
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Proposed Test Procedures for Nonconventional Pesticides
Table 1A provides a list of proposed test procedures for 66 nonconventional
pesticide pollutants proposed for regulation in 40 CFR Part 455 (47 FR
53994).
IX
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xiii
-------
PART A
INDUSTRY ANALYTICAL METHODS
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, O.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ALACHLOR, BUTACHLOR, AND PROPACHLOR
IN WASTEWATER
METHOD 101
1. Scope and Application
1.1 This method covers the determination of certain acetanilides.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Alachlor 77825 15972-60-8
Butachlor 77860 23184-66-9
Propachlor 77729 1918-16-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in wastewater
streams.
1.3 The method detection limit (MDL) for each parameter is 0.5 ug/g
(ppm).
2. Summary of Method
2.1 An aliquot of wastevater (100 g) is extracted with chloroform.
The extract is drained through a wad of cotton and collected in a
vial containing 2 ml of internal standard solution. The
chloroform extract is concentrated and analyzed by GC with flame
ionization detector.
3. Interferences
Not Available.
101-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5 . Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Glenco liquid syringe 5 or 10 mL or equivalent.
5.3 Hewlett Packard 5830 A Gas Chromatograph or Equivalent
5.4 Hewlett Packard 18850 A GC Terminal or Equivalent
6. Reagents
6.1 N,N-Dibutylaniline, Eastman Kodak Co. Reagent grade or
equivalent
6.2 Chloroform, certified ACS, Fisher Scientific company or
equivalent
6.3 Alachlor (Lasso), Monsanto Co., known % purity
6.4 Propachlor (Ramrod), Monsanto Co., known % purity
6.5 Butachlor (Machete), Monsanto Co., known % purity
7. Calibration
7.1 GC Conditions
GC Conditions are summarized in Table 1.
7.2 Calibration Procedure
7.2.1 Into a 100-mL volumetric flask weigh 0.1000 g of each
expected pollutant (Alachlor, Butachlor, and Propachlor).
7.2.2 Dilute to mark with chloroform.
7.2.3 Make a further dilution by diluting 10 mL of this solution
to 100 mL in a 100-mL volumetric flask. This solution will
be used to make up calibration standards.
7.2.4 Into a 100-mL volumetric flask place one small drop of N,N-
dibutylaniline and dilute to mark with chloroform. This
will be the internal standard solution for calibration
standards and samples.
7.2.5 Into a 4-dram vial pipet 2 mL of the internal standard
solution.
101-02 January 1983
-------
7.2.6 Into the same 4-dram vial pipet 10 mL of the calibration
standard.
7.2.7 On a hot plate evaporate this solution to 0.5 mL using
moderate heat and a jet of air.
7.2.8 Place concentrated standard in a 1-mL serum cap injection
vial (Teflon-lined cap).
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Weigh 100 g of waste sample into a 125-mL separatory funnel.
10.2 Using four 10-mL aliquots of chloroform extract the organics from
the water. Shake for one minute each time and allow complete
separation of the layers.
10.3 Filter the chloroform through a wad of cotton in a small funnel
into a 4-dram vial containing 2 mL of internal standard solution
prepared in Step 7.2.4 above.
10.4 Using a hot plate and air jet, start concentrating the first
chloroform extract in a hood.
10.5 Continue with the remaining three extracts in Step 10.2.
10.6 Finally, concentrate the chloroform to 0.5 mL and place in a
serum cap injector vial (Teflon-lined cap).
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Inject a 1-uL sample into the GC which is set up with the columns
and conditions given in Table 1.
13. Calculations
_ t (Wt. Component)(Area Internal STD)(1,000)
Factor = : '
Area Component
(Factor)(Area Component)
(Area Internal Standard)
101-03 January 1983
-------
14. Method Performance
14.1 Precision expected is +. 10% relative error.
14.2 The detection limit for each parameter is 0.5 ug/g (ppm).
15. References
15.1 "Alachlor, Propachlor, and Butachlor in Wastewater Streams by
GC," Standard Test Method, Monsanto Agricultural Products, St.
Louis, MO.
101-04 January 1983
-------
Table 1
Chromatographic Conditions
o
Injector Temperature 250 C
Detector Temperature 300 C
Column Temperature Program 60°C to 220°C at 10 C/min
Carrier Flow 80 mL/min
Run Time 20 min
Column: 1/4 in x 2 ft 316 Stainless Steel column packed with 10% DCSD 710
on 80/100 Gas Chrom Q.
Note: Columns must be carefully matched or bleed will cause drift
problems. Some compensation for bleed can be made by adjusting the
carrier flow of the reference columns.
101-05 January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ALACHLOR, BDTACHLOR, AND PROPACHLOR
IN WASTEWATER
METHOD 102
1. Scope and Application
1.1 This method covers the determination of certain acetanilides.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Alachlor 77825 15972-60-8
Butachlor 77860 23184-66-9
Propachlor 77729 1918-16-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in waters and treated
aqueous effluents at acetanilide herbicide manufacturing
facilities.
1.3 The method detection limit (MDL) for each parameter is 0.2 ug/L
when using a 1-liter sample.
1.4 The allowable quantities of acetanilides in aqueous waste from
herbicide manufacturing sites are limited by both State and
Federal environmental protection agencies. In order to monitor
acetanilides at anticipated levels of interest, a parts-per-
billion (ug/L) method for their quantitation is necessary.
2. Summary of Method
2.1 A volume of water sample (1000 mL) is spiked with an internal
standard and then extracted with methylene chloride. The
extracts are combined and concentrated to 5 mL. The methylene
chloride solvent is exchanged for a toluene/methanol mixture.
Analysis for the acetanilides is by packed column chromatography
using a nitrogen selective detector routinely and an electron
capture detector as necessary to deal with interferences.
Component identification is based upon elution times and has been
102-01 Jamiarv
-------
confirmed by gas chromatographic-infrared and mass spectroscopy
techniques.
2.2 If the sample NPD chromatogram contains peaks interfering with
alachlor, butachlor, propachlor, or the internal standard, the
sample should also be analyzed similarly using an electron
capture detector. This change in detector specificity may allow
better quantitation.
3. Interferences
3.1 Accurate analyses for parts-per-billion (ug/L) concentrations
require rigorous controls to prevent external contamination from
the laboratory environment, glassware, reagents, or cross-
contamination between samples.
3.2 The laboratory should be maintained at a positive pressure
relative to its surroundings, and a suitable air intake
filtration system should be used. In addition, to protect the
laboratory from contamination carried on the clothing and shoes
of manufacturing, formulation, and other laboratory personnel,
access to the laboratory where this and other trace analyses are
performed should be strictly limited.
3.3 Laboratory Glassware: To avoid contamination from greater than
parts-per-billion (ug/L) concentrations of acetanilides, the
glassware used for specific parts of the method should be
dedicated to just those parts. For example, the equipment for
concentrated stock solutions and those for more dilute
calibration solutions should not be interchanged.
3.3.1 Glassware cleaning: A rigorous procedure was followed
during the development of this method. That procedure was
successful since no cross contamination was detected.
Since copious volumes of acetone are involved, gloves
should be used.
3.3.1.1 Initially glassware is washed with acetone and
water until all visible contaminants are
removed. Soap is not employed since it too may
be a source of interference. The visibly
cleaned glassware then is rinsed 10 times with
acetone and 30 times by distilled water.
Following each 10 water rinses are 5 acetone
rinses and finally an extra 5 acetone rinses
before the glassware is drained to dryness.
Clean glassware is stored in a closed cabinet.
3.3.1.2 Hypodermic syringes are rinsed with acetone
immediately after being used. Then, under
vacuum, 40 "beads" of water are sucked through
the syringe; after each 10 water rinses, 5
acetone rinses are done. Clean syringes are
stored in glass test tubes.
102-02 January 1983
-------
3.4 Gas Chromatography: To avoid contamination, the gas
chromatograph and analytical columns are dedicated to this
analysis.
3.5 Reagents: The best available pesticide grade reagents are
employed to assure minimum interferences from this source. It is
recommended that solvents from each newly opened reagent
container be tested by the analytical procedure to assure that
interferences are absent.
3.6 Acekanilide adsorption on glassware: At parts-per-billion (ug/L)
levels, tests indicate that certain acetanilides are adsorbed on
the surfaces of glassware. A significantly improved recovery can
be attained when solutions contain about 20 percent methanol.
Similar results were reported for polycyclic aromatics by Ogan,
Katz, and Slavin (15.2).
4. Safety
4.1 The solvents needed in this procedure should be used with
appropriate precautions and adequate ventilation.
4.2 See the EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
5.1.1 Samples are taken in 1-gallon, amber, glass bottles having
a Teflon disk cap insert. Plastic containers cannot be
used since components from the container may be dissolved
into the sample or sample constituents may be absorbed into
the container walls.
5.2 Glassware and Other Equipment
5.2.1 Graduated cylinders, 500 mL and 25 mL.
5.2.2 Separatory funnel, 1000 mL, with Teflon stopcock and
stopper.
5.2.3 Evaporative concentrator (modified Kuderna-Danish — Figure
1).
5.2.4 Disposable glass pipets.
5.2.5 Hypodermic syringe, 10 microliter.
5.2.6 Centrifuge tubes, 5 mL, graduated to 0.1 mL (Fisher
Scientific Co., 5-538-35A).
5.2.7 Volumetric flasks, 500 and 1000 mL.
5.2.8 Pipets, 5 and 25 mL.
102-03 January 1983
-------
5.3 Steam bath
5.4 Boiling chips
5.5 Gas Chromatograph: A modern gas chromatograph should be used.
It should be equipped for glass columns and on-column injection.
In the development of this method, a Hewlett Packard model 5840A
gas chromatograph was used.
5.5.1 Analytical column: A glass, 6-ft, 1/4-in O.D., 2 mm-I.D.
column packed with 10% OV-11 on Chromosorb W-HP, 100-120
mesh was used.
5.5.2 Gas chromatographic detector: An alkali nitrogen sensitive
detector is employed to maximize the sensitivity and
selectivity for acetanilides. In the development of this
method the Hewlett Packard NPD detector with associated
electronics and pneumatics was used.
Reagents
6.1 High Grade Distilled in Glass Solvents — Burdick and Jackson,
distilled in glass, pesticide grade
6.1.1 Toluene
6.1.2 Methyler.e chloride
6.1.3 Methane!
6.2 Acetone — Fisiher Scientific Co., certified ACS grade, A-18
6.3 Analytical Standards
6.3.1 Alachlor
6.3.2 Butachlor
6.3.3 Propachlor
Calibration
7.1 GC conditions
The GC analysis is performed isothermally at 200 C for 2 min.,
followed by programming at 6 C/min to 250 C and holding for 10
min. The operating parameters shown in Table 1 were used on the
HP 5840A to provide satisfactory sensitivity, resolution, and
quantitation.
7.2 Calibration Procedure
7.2.1 Internal Standard
102-04 January 1983
-------
"Hexoxy Alachlor," analytical standard grade [2-chloro-
2'6'-diethyl-N-(hexoxymethyl)-acetanilide], was used.
7.2.2 Calibration and Standardization
The text below describes the preparation of two ranges of
standard solutions for use in the two general concentration
ranges (0.2-10 ug/L and 20-300 ug/L) for which the method
has been validated. The composition of standard solutions
used for calibration is as follows for analysis of water
and effluents at low levels (0.2 to 10.0 ug/L):
Solution A: Components: Accurately weigh into a 1000-mL
volumetric flask 0.02 g of each of the three
components to be analyzed for and add methanol
to volume. This solution contains 20 ug/mL
of each of the materials.
Solution B: Components: Pipet 5 mL of Solution A into a
1000-mL volumetric flask and add methanol to
volume. This solution contains 0.1 ug/mL of
each of the materials.
Solution C: Internal Standard: Accurately weigh into a
500-mL volumetric flask 0.2 g of "hexoxy
alachlor"; add methanol to volume. This
solution contains 0.4 mg/mL of the internal
standard.
Solution D: Internal Standard: Pipet 5 mL of Solution C
into a 1000-mL volumetric flask and add
methanol to volume. This solution contains 2
ug/mL of the internal standard.
For waters and effluents where higher levels (20-300 ug/L) of the
acetanilide herbicides may be detected and quantitated, standard
solutions are prepared such that levels in the calibration
samples approximate those occurring in the actual samples. The
composition of those standard solutions is as follows:
Solution E: Components: Accurately weigh into a 100-mL
volumetric flask 0.02 g of propachlor, 0.20 g
of alachlor, and 0.10 g of butachlor. Add
methanol to volume. This solution contains
200, 2000, and 1000 ug/mL, respectively, of the
three acetanilides.
Solution F: Components: Pipet 5-mL of Solution E into a
100 mL volumetric flask; add methanol to
volume. This solution contains 10, 100, and 50
ug/mL, respectively, of the three acetanilides.
Solution G: Internal Standard: Accurately weigh into a
100-mL volumetric flask 0.14 g of hexoxy
alachlor; add methanol to volume. This
102-05
January 1983
-------
solution contains 1400 ug/mL of the internal
standard.
Solution H: Internal Standard: Pipet 5 mL of Solution G
into a 100-mL volumetric flask; add methanol
to volume. This solution contains 70 ug/mL of
the internal standard.
7.2.3 Instrument: and Method Calibration Samples — To compensate
for possible interferences and variable extraction
efficiencies, an instrument calibration sample is carried
through the same extractive and concentration steps used
for a test: sample.
7.2.3.1 Measure 500 mL of deionized water into a 1000-
mL separatory funnel.
7.2.3.2 To the above, add by pipet 25 mL of Solution B
(For low level calibration). If calibration at
a higher level is necessary, add the
appropriate level of standard, solution. The
spiking level of the calibration sample should
approximate as closely as possible the levels
to be measured in the actual sample.
7.2.3.3 To the above, add by pipet 5 mL of Solution D.
(Note that for the higher level range, Solution
H would be used.)
7.2,3.4 Add to the above an additional 470 mL of
deionized water.
7.2.3.5 Stopper the separatory funnel and mix the
contents. This aqueous calibration sample
solution contains 2.5 ug/L of each of the
components and 10 ug/L of the internal
standard. This solution is recommended for use
in the analysis of samples of varying
composition. During continued analysis of
samples of fairly constant composition, a
calibration solution more closely reflecting
sample composition may produce the best
results.
7.2.3.6 Extract the solution prepared in Step 7.2.3.5
with 20 mL of methylene chloride by shaking
vigorously for one minute.
7.2.3.7 After the phases separate, dra,w off the lower
methylene chloride layer into the evaporative
concentrator.
7.2.3.8 Repeat Steps 7.2.3.6 and 7.2,3.7 three
additional times; however, use 7 mL of
methylene cloride for each of these
extractions.
102-06 January 1983
-------
7.2.3.9 Add a boiling chip (previously extracted in
methylene chloride) to the evaporative
concentrator; under a stream of nitrogen and
with heating by a steam bath, concentrate the
volume to approximately 3 mL. This solution
must not go dry if accuracy is to be
maintained.
7.2.3.10 When the volume is reduced to approximately 3
mL, rinse down the upper flask with
approximately 2 mL of toluene and continue
heating this solution. The majority of the
solvent exchange is indicated by a dramatic
decrease in the size of the bubbles. Continue
heating approximately 5 minutes to decrease the
methylene chloride concentration to a
negligible amount. (Solvent exchange is
necessary since halogenated solvents cause a
marked decrease in the sensitivity of an NPD
detector.)
7.2.3.11 Rinse down the walls of the upper flask with
approximately 1 mL of methanol.
7.2.3.12 Dry the outside of the ground glass joint, and
with a new disposable pipet transfer the
concentrate to a 5-mL centrifuge tube.
7.2.3.13 Using no more than 2 mL of toluene, rinse the
interior of the lower part of the evaporative
concentrator; add these rinses to the 5-mL
centrifuge tube.
7.2.3.14 Under a stream of nitrogen, evaporate the
solution in the 5-mL centrifuge tube to
approximately 0.2 mL.
7.2.3.15 Rinse down the walls of the 5-mL centrifuge
tube with 0.1 mL of methanol. Stopper the tube
and mix its contents.
7.2.3.16 Inject 3 uL of this solution into the gas
chromatograph. Calibrate the instrument;
triplicate
calibration runs are recommended.
7.2.3.17 The solution from Step 7.2.3.15 is stable for
about 5 days. New calibration samples should
be prepared weekly. Instrument calibration
should be done daily.
8. Quality Control
8.1 Duplicate analysis should be done.
8.2 A 1000-mL distilled water sample, or other suitable blank sample,
102-07 January 1983
-------
is analyzed by this procedure regularly and whenever new
containers of solvents are employed.
8.3 See the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Samples are taken in 1-gallon, amber, glass bottles having a
Teflon disk cap insert. Plastic containers cannot be used since
components from the container may be dissolved into the sample or
sample constituents may be absorbed into the container walls.
9.2 Samples should be worked up and analyzed as soon as possible.
9.3 At ug/L levels, rigorous controls are required to prevent
inaccurate analyses. Special sample handling is needed to
prevent alteration of the sample before analysis and assure that
contamination does not occur during the analysis. Protocols
should be provided for sample taking, control of the laboratory
environment, glassware cleaning, and dedication of instruments
and glassware.
9.4 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Measure 1000 ml of aqueous sample into a 1000-mL separatory
funne1.
10.2 To the above, s.dd by pipet 5 mL of Solution D. (Note that for the
higher level rs.nge, Solution H would be used.)
10.3 Stopper the separatory funnel and mix the contents. This aqueous
solution contains the sample acetanilides and 10 ug/L of internal
standard.
10.4 Extract the acetanilides from the solution prepared in Step 10.4
using methylene chloride. Add 20 mL of methylene chloride and
extract by shaking vigorously for one minute.
10.5 After the phases separate, draw off the lower layer into the
evaporative concentrator. Do not draw off any emulsion layer
that may be at the solvent interface.
10.6 Repeat Steps 10.5 and 10.6 three additional times; however, use 7
mL of methylenc chloride for each of these extractions.
10.7 Add a boiling chip (previously extracted in methylene chloride)
to the evaporative concentrator; under a stream of nitrogen and
with heating by a steam bath, concentrate the volume to
approximately 3 mL. This solution must not go dry if accuracy is
to be attained.
102-08 January 1983
-------
10.8 When the volume is reduced to approximately 3 mL, rinse down the
upper flask with approximately 2 mL of toluene and continue
heating this solution.' The majority of the solvent exchange is
indicated by a dramatic decrease in the size of the bubbles.
Continue heating approximately 5 minutes to decrease the
methylene chloride concentration to a negligible amount.
(Solvent exchange is necessary since halogenated solvents cause a
marked decrease in the sensitivity of an NPD detector.)
10.9 Rinse down the walls of the upper flask with approximately 1 mL
of methanol.
10.10 Dry the outside of the ground glass joint, and with a new
disposable pipet transfer the concentrate to a 5-mL centrifuge
tube.
10.11 Using no more than 2 mL of toluene, rinse the interior of the
lower part of the evaporator concentrator; add these rinses to
the 5-mL centrifuge tube.
10.12 Under a stream of nitrogen, evaporate the solution in the 5-mL
centrifuge tube to approximately 0.2 mL.
10.13 Rinse down the walls of the 5-mL centrifuge tube with 0.1 mL of
methanol. Stopper the tube and mix its contents. The sample is
now ready for gas chromatographic analysis.
11. Cleanup and Separation
Not Available.
12. Gas Chromatoeraphv
12.1 Inject 3 uL of the solution from Step 10.13 into the gas
chromatograph and determine the amount of each acetanilide
present. Table 1 summarizes the recommended operating conditions
for the GC.
12.2 If the sample NPD chromatogram contains peaks interfering with
propachlor, alachlor, butachlor, or the internal standard, the
sample also should be analyzed similarly using an electron
capture detector. This change in detector specificity may allow
better quantitation.
13. Calculations
13.1 With the Hewlett Packard model 5840A, if the amount of
acetanilide in the calibratrion sample is incorporated in units
of ug/L, sample results calculated by the microprocessor are
reported in ug/L. Other recording/integrating systems may be
similarly calibrated for data output.
14. Method Performance
14.1 Range
102-09 January 1983
-------
The methodology has been validated over a range of 0.2-300 ug/L.
14.2 Sensitivity
The minimum level of detection is 0.2 ug/L when using a 1-liter
sample.
14.3 Accuracy
In the 0.2-300 ug/L concentration range, recoveries (accuracies)
of about 90-110% may be expected. Recoveries are summarized in
Table 3.
14.4 Precision
In the concentration range of 0.2 to 10.0 ug/L, the methodology
generally may be expected to give precision of +. 20% or better
(95% confidence level). In the range of 20 to 300 ug/L, the
methodology gives a precision of +. 12% or better (95% confidence
level). Table 3 summarizes the pooled coefficients of variation.
15. References
15.1 J.W. Worley, M.L. Rueppel, and F.L. Rupel, "Determination of
alpha-Chloroacetanilides in Water by Gas Chromatography and
Infrared Spectrometry," Anal. Chem. 52. 1845 (1980).
15.2 K. Ogan, E. Katz, and W. Slavin, "Concentration and Determination
of Trace Amounts of Several Polycyclic Aromatic Hydrocarbons in
Aqueous Samples," JN. Chrom. Sci. . 16. 517 (1978).
15.3 F.L. Rupel, J.W. Worley, and R.K. Beasley," Analytical Method for
the Determination of Propachlor, Alachlor, and Butachlor in
Waters and Effluents at the Parts Per Billion Level" Standard
Test Method, Validation Date 3-16-79, Monsanto Agricultural
Products Co., St. Louis, Mo.
102-10 January 1983
-------
Table 1
Chromatographic Conditions
Injector Temperature
NPD Detector Temperature
Chart Speed
Recorder Zero
Attenuation
Slope Sensitivity
Area Reject
Helium Carrier Flow
Column Temperature
250°C
300°C
1.00 in/min
10.0
2(7)
0.03
1000
25.0 mL/min
200°C, hold 2 min., then 6 C/min to
250°C, hold 10 min.
Footnote: Column - Glass, 6-ft x 1/4-in O.D., 2-mm ID packed with 10% OV-11
on Chromosorb W-HP, 100-120 mesh.
102-11
January 1983
-------
Table 2
Retention Times
Compound
Propachlor
Alachlor
Butachlor
Hexoxy Alachlor
Retention Time (Minutes)
3.9
7.7
10.6
13.9
102-12
January 1983
-------
Table 3
Accuracy and Precision
Component
Propachlor
Alachlor
Butachlor
Range, ug/L
0.2-10
20-30
0.2-10
200-300
0.2-10
100-150
Pooled Coeff. of Var.
0.09
0.05
0.09
0.06
0.10
0.03
Recovery. %
93
96
94
100
112
100
Coefficient of Variation = (standard deviation)/(mean).
102-13 January 1983
-------
1
Attachment 1
Evaporative Concentrator
(Modified Kuderna-Danish)
K-26500
Stopper No. 6
Metal
Spring
K-S700SO-042S
102-14
January 1983
-------
5.EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF AOP, ZAC, ZINEB,
AND ZIRAM IN WASTEWATER
METHOD 103
1. Scope and Application
1.1 This method covers the determination of certain dithiocarbamates.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
AOP
Ferbam — 14484-64-1
Niacide — 8011-66-3
ZAC —
Zineb — 142-14-3
Ziram — 137-30-4
1.2 This method is non-specific for individual dithiocarbamates and
results are reported as mg/L of carbon disulfide (CS-).
1.3 This is a spectrophotometric method applicable to drinking,
surface, and saline waters, and domestic and industrial waste
effluents.
2. Summary of Method
2.1 A volume of water sample (1-20 mL) is reacted with acid to
produce the acid decomposition of the dithiocarbamate followed by
the subsequent reaction of the evolved 082 with a color reagent,
and spectrophotometric determination.
3. Interferences
Not Available.
103-01 January 1983
-------
4. Safety
4.1 Follow the EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Flasks, round-bottom 1000 mL, 3-neck, 28/15, and 35/25
outside balljoints (See Figure 1).
5.2.2 Condenser, water-cooled, 12 inch, with bottom inside 28/15
balljoint and top 5-mm glass tubing extension tipped with
an inside 28/15 balljoint (See Figure 1).
5.2.3 Funnel, separatory, graduated, cylindrical, 150 mL with an
inside 28/15 balljoint and No. 2 stopcock.
5.2.4 Gas washing bottle, demountable, 150 x 25 mm with 24/40 top
joint.
5.2.5 Gas dispersion tubes, with medium fritted cylinders that
extend to within 10 mm of bottom of washing bottles. Glass
cylinders have one inside and one outside 12/5 balljoint.
5.2.6 Tubing, g,lass, 5-mm I.D.
5.2.7 Flask, volumetric, with stopper, 25 mL.
5.3 Stirrer, magnetic, "Magnestir"
5.4 Heating mantles, Glas-Col flask heaters, Hemispherical, 1000 mL
5.5 Magnetized stirring bars, Teflon R sealed, 5/16" x 1-1/2"
5.6 Transformers, variable, powerstat
5.7 Spectrophotometer, Beckman Model DB or equivalent
5.8 Tubing, Tygon, 1/4"
6. Reagents
6.1 Hydrochloric acid, 1.25N
6.2 Zinc acetate, 20% aqueous solution
6.3 Nitrogen, compressed gas
6.4 Color Reagent — To 0.004 g of cupric acetate monohydrate in a
250-mL volumetric flask, add 25 grams of diethanolamine and
103-02 January 1983
-------
dilute to volume with ethanol. This reagent is used for
determination of less than 150 ug of carbon disulfide.
7. Calibration
7.1 The optical densities of the standard solutions and standards
should be measured using a spectrophotometer operating at a
435-nm wavelength
7.2 Calibration Procedure
7.2.1 Carbon disulfide standard solution. Accurately weigh 1.0 g
of carbon disulfide into a 100-mL volumetric flask
containing 75 mL of absolute methanol and dilute to volume.
Dilute a 1.0-mL aliquot of above solution to 100 mL with
absolute methanol. This final solution contains 100 ug/mL
of CS2.
7.2.2 Prepare a standard solution containing 1 ug/mL of CS2 in
methanol. Add, into 25-mL volumetric flasks using a
syringe, aliquots of the standard solution equivalent to 25
to 150 ug of carbon disulfide and dilute to volume with a
50/50 v/v mixture of ethanol/color reagent A. Allow the
solutions to stand for at least 15 minutes. Measure the
optical densities of the solutions against a 50/50 v/v
mixture of ethanol/color reagent A using a spectro-
photometer operating at a 435-mu wavelength.
8. Quality Control
8.1. Follow the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Digestion
10.1 Assemble the decomposition-absorption apparatus as illustrated in
Figure 1. Fill trap I with 15 mL of a 20% aqueous zinc acetate
solution. Fill trap II with 12.5 mL of appropriate color
reagent.
10.2 Introduce a sample of wastewater through the 35/25 balljoint into
the 3-neck round bottom reaction flask. Immediately add 150 mL
of boiling 1.25 N hydrochloric acid through the addition funnel.
Apply nitrogen pressure and sweep the system at about 200 mL/min.
Perform all fortifications just prior to the addition of the
acid. Heat the contents to boiling temperature and continue the
digestion for 45 minutes.
103-03 January 1983
-------
10.3 Sample size of wastewater depends upon CSo concentration. Use 2
mL for 1-2 mg/L; 10 mL for 2-5 mg/L; and 1 mL for 10-15 mg/L.
11. Cleanup and Separation
Not Available.
12. Sample Analysis
12.1 After the digestion period, transfer the contents of trap II to a
25-mL volumetric flask using ethanol as wash liquid. Dilute the
contents of the flask to exactly 25 mL with ethanol, stopper,
shake, and allow to stand for at least 15 minutes. Measure the
absorbance of the sample vs. a 50/50 v/v mixture of ethanol/color
reagent spectrophotometrically at 435 mu using l-cm cuvettes.
13. Calculations
13.1 Plot the optical density readings vs. the concentration of carbon
disulfide. Figure 2 represents the standard curve for carbon
disulfide concentrations ranging from 25 to 150 ug. The standard
curve was linear for solutions containing between 25 and 150 ug
of carbon disulfide. Determine the micrograms/25 mL of carbon
disulfide using the standard curve:
CS./25 mL (from graph)
CS2 (mg/L) mL wastewater
14. Method Performance
Not Available.
15. References
15.1 "Spectrophotometric Determination of Dithiocarbamate in Waste
Water," Standard Test Method, FMC Corporation, Middleport, NY.
103-04 January 1983
-------
5 mm I.D.
12/51
Demountable
gas washing
bottle
3 mm bore
stopcock
FIGURE I. DECOMPOSITION-ABSORPTION APPARATUS
103-05
January 1983
-------
-. ~. uJ 5
January 1983
-------
SEFft
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENFLURALIN, ETHALFLURALIN, AND
ISOPROPALIN IN WASTEWATER
METHOD 104
1. Scope and Application
1.1 This method covers the determination of certain dinitroanilines.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Benfluralin 39002 1862-40-1
Ethalfluralin — 55283-68-6
Isopropalin — 33820-53-0
Trifluralin 39030 1582-09-8
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in water at the part-
per-billion level (ug/L), and in water collected at industrial
effluent discharge sites in the Wabash River.
1.3 The detection limit is less than 1.0 ug/L for all compounds.
2. Summary of Method
2.1 A measured volume of sample (500 ml) is extracted with chloroform
using a separatory funnel. The extract is evaporated to near
dryness and the residue is dissolved in benzene. Chromatographic
(GC) conditions are established which permit the measurement of
the compounds in the benzene extract by electron capture/gas
chromatograpby (GC/EC).
2.2 The method includes a Florisil cleanup procedure to aid in the
elimination of interferences.
3. Interferences
3.1 Accurate measurement of these chemicals at the ug/L level
requires that laboratory glassware and equipment be kept free
104-01 January 1983
-------
from contamination. The laboratory itself must be located in an
area that is secluded from high concentrations of organic
compounds. Solvents used in the procedures must be redistilled
to remove trace amounts of impurities that can interfere with the
detection of the chemicals. The sodium sulfate must also be
washed with methanol to remove interfering substances.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not available.
5.2 Glassware and Other Equipment
5.2.1 Redistillation apparatus for redistilling solvents.
5.2.2 Laboratory glassware (pipettes, beakers, flasks, funnels,
separatory funnels, and graduated cylinders).
5.2.3 Air-cooled condensing tubes, 24/40 taper, 22-mm O.D.
5.3 Refrigerator or chill room for storage of samples and standard
solutions
5.4 Rinco rotary vacuum evaporator with 50 C water bath
5.5 Hot plate with temperature control
5.6 Chromatography columns — 250-mm x 14-mm I.D. or 300-mm x 11-mm
I.D., equipped with stopcocks
5.7 Gas-liquid chromatograph equipped with electron capture detector
5.8 Gas-liquid chromatograph equipped with flame photometric detector
(optional)
6. Reagents
6.1 Solvents — reagent grade
6.1.1 Chloroform (redistilled)
6.1.2 Benzene (redistilled and stored over 5A molecular sieves
— McFaul Co., Cincinnati, OH)
6.1.3 Hexane (redistilled)
6.1.4 Carbon tetrachloride (redistilled)
104-02 January 1983
-------
6.1.5 Methane1
6.1.6 Pyridine
6.2 1% Isopropanol in acetonitrile
6.3 Concentrated HC1: methanol (1:1)
6.4 5% Sodium chloride solution
6.5 Methyl iodide (Matheson, Coleman, & Bell, 98% Stabilized
Industrial)
6.6 Sodium carbonate, anhydrous
6.7 Sodium sulfate, anhydrous, methanol washed
6.7.1 Washing Procedure for Sodium Sulfate — Connect a
2-liter separatory funnel to a large vacuum filtering
flask fitted with a rubber stopper. Fill the flask
4/5 full with sodium sulfate, and wash the sodium
sulfate with a total of about 2 liters of methanol
while pulling a vacuum. The sodium sulfate can then
be removed from the funnel and air dried or dried by
pulling a vacuum on a large Buchner funnel.
6.8 Phosphorous tribromide (practical)
6.9 5% Sodium hydroxide solution
6.10 Alumina, Alcoa F-20, deactivated with 4% water and sieved to
120/170 mesh
6.11 Florisil, 100/120 mesh, deactivated with 8% water
6.12 Trifluoroacetic anhydride (redistilled)
6.13 1.25N NaOH
6.14 Carborundum boiling chips
6.15 1.25N HC1
Calibration
7.1 Analyze the compounds by gas chromatography using electron
capture detection under the conditions given in Table 1
7.2 Calibration Procedure
7.2.1 Direct standard concentration — 0.05 ug/mL in benzene
7.2.2 Note: Direct standards for trifluralin and benfluralin
should be injected prior to each set of samples to ensure
104-03 January 1983
-------
that these two compounds can be distinguished by their
retention times.
8. Quality Control
8.1 Control samples and standard recovery samples should be carried
through the assay procedures for each set of experimental samples
if results are desired with an accuracy of about ± 10%. If only
approximate values (+. 25-30%) are acceptable, the control and
recovery samples can be omitted.
8.2 Assay a control sample and a recovery sample in exactly the same
manner as experimental samples. Use tap water for the control
sample and prepare a spiked recovery sample at a concentration of
0.001 mg/L by fortifying 500 mL of control tap water with 0.50 ug
of each compound being assayed.
8.3 See the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Transfer 500 ml of water to a 1-liter separatory funnel; add 50
mL of chloroform, shake for 30 seconds, and allow the phases to
separate.
10.2 Eliminate persistent emulsions by draining the lower chloroform
layer through a funnel containing sodium sulfate into a 250-mL
flat-bottom flask.
10.3 Repeat the extraction with two additional 50-mL portions of
chloroform and wash the sodium sulfate with 10 mL of chloroform.
10.4 Evaporate the chloroform just to drvness with a Rinco evaporator
and immediately dissolve the residue in 50.0 mL of benzene.
10.5 Transfer a 10.0-mL aliquot (Solution A) of the benzene to a 125-
mL flat-bottom flask.
10.6 The evaporating flasks must be removed from the Rinco immediately
after the chloroform has evaporated to prevent the loss of
volatile compounds such as trifluralin.
11. Cleanup and Separation
11.1 Evaporate Solution A just to drvness and dissolve the residue in
5 mL of hexane.
11.2 Follow the Florisil column procedure in Steps B-8 through B-14 of
Procedure 5801616 (Eli Lilly reference method, not enclosed
herein) with the following modification: In Step 12, add 120 mL
104-04 January 1983
-------
of hexane in order to elute ethalfluralin, isopropalin, and
benfluralin in addition to trifluralin.
Note: The Florisil must be deactivated and standardized prior to
initial use as described in Section D of Procedure 5801616 (Eli
Lilly reference method, not enclosed herein). The Florisil must
be standardized to include isopropalin, as well as trifluralin.
11.3 Evaporate the sample just to dryness and dissolve the residue in
2.0.mL of benzene.
2. Gas Chromatoeraphv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 The electron capture detector has only a limited range in which
the peak response is linear with concentration. Therefore, the
experimental samples must be diluted to yield a response that is
similar in magnitude to that of the direct standard. Likewise,
samples analyzed by flame photometric detection may require
dilutions to yield a peak response that falls within the range of
the standard curve. The magnitude of the dilutions must then be
accounted for in the dilution factor (DF) of the calculations.
13. Calculations
13.1 Percent recovery in standard recovery sample:
v n PR (recovery sample) - PR (control) ,nn
% Recovery PR (direct standard) X 10°
where PR « peak response
13.2 Parts-per-million (mg/L) residue in water sample:
(ug/mL) x DF x AF x 100
ppm = SV x % Recovery
, , T PR (sample) n n- . .
where ug/mL = PR (8tandard)X 0'05 ug/mL
DF = dilution factor (2.0 mL, unless sample is further
diluted for analysis)
AF » aliquot factor (AF=5, since each aliquot was 1/5
of the extract)
SV » sample volume (500 mL)
14. Method Performance
Not Available.
15. References
104-05 January 1983
-------
15.1 "Determination of Trifluralin and Other Blanco Herbicides in
Water," Procedure 5801665, Eli Lilly and Company, Lilly Research
Laboratories, Greenfield, IN.
104-06 January 1983
-------
Table 1
Chromatograpbic Conditions
Temperature - Oven 120 C
- Flash heater 210°C
- Detector 290°C
Carrier gas Argon/methane (90:10), flow rate - 50 mL/min
Electrometer - Range 1
- Attenuation 32
Injection volume 3.0-5.0 microliters
Column 122-cm x 3-mm I.D. glass packed with "Aue" Carbowax 20M
Note: The "Aue" packing was specially prepared by Eli Lilly personnel.
104-07 January 1983
-------
v-xEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENOM^'L AND CARBENDAZIM
IN WASTEWATER
Method 105
1• Scope and Application
1.1 This method covers the determination of benomyl and carbendazim.
Parameter Storet No. CAS No.
Benomyl — 17804-35-2
Carbendazim — 10605-21-7
1.2 Benomyl cannot be determined directly by this method. Both
compounds are measured and reported as carbendazim.
1.3 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of the compounds listed above in
aqueous waste effluents.
1.4 The method detection limit (MDL) is 0.08 ug/g (ppm) carbendazim.
2. Summary of Method
2.1 A measured volume of sample (100 mL) is acidified to hydrolyze
benomyl to carbendazim. The total carbendazim is extracted with
ethyl acetate. Determination is by high-speed cation exchange
liquid chromatograpby.
3. Interferences
Not Available.
4. Safety
4.1 Methanol is a colorless liquid with a faint alcoholic odor. The
threshold limit value (TLV) is 200 ppm, according to the American
Conference of Governmental Industrial Hygienists (ACGIH). This
105-01 January 1983
-------
material is flammable and toxic, if ingested, causing blindness
or death. The odor threshold is 100 ppm.
4.2 Ethyl acetate is a flammable liquid with a TLV of 400 ppm
(ACGIH) .
4.3 Sodium hydroxide is a strongly alkaline,, corrosive solid with a
TLV of 2 mg/m (ACGIH). The minimum protection of Butal-sol
rubber gloves, rubber sleeves, a rubber apron, and a face shield
should be worn. In case of contact, flush with large quantities
of water.
4.4 Benomyl is a stable, light tan powder with moderate toxicity
having a TLV of 10 mg/m (ACGIH).
4.5 Acetone is a colorless, highly flammable liquid which has a sweet
pungent odor. Its vapors form explosive mixtures; its TLV is 750
ppm (ACGIH).
4.6 Hydrochloric acid is a colorless, fuming liquid with a pungent
odor. It is highly corrosive to body tissue. Its TLV is 5 ppm
(ACGIH). Vapors may cause severe burns to the upper respiratory
tract and eyes, Butal-sol rubber golves, rubber sleeves, a face
shield, and rubber apron should be worn.
4.7 Carbendazim is a fungicide with very low oral toxicity (LDcQ for
rats, oral, is >11,000 mg/kg).
4.8 Ammonium hydroxide is a colorless liquid with a TLV of 25 ppm
(ACGIH). It is strongly alkaline by nature. The minimum
protective equipment which should be worn is Listed above.
4.9 Phosphoric acid is a viscous, light-colored liquid with a TLV of
1 mg/m3 (ACGIH). It :
treated with caution.
3
1 mg/m (ACGIH). It is a corrosive material and should be
4.10 n-Hexane is a colorless, volatile liquid with a faint, peculiar
odor. Handle in a hood and avoid breathing the vapors. It is
flammable.
4.11 Consult safety sheets and handbooks for further information
regarding these materials.
4.12 See EPA safety procedure found in Part D of this document.
Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
105-02 January 1983
-------
5.2.1 Evaporative concentrators, Cat. No. K659300, Kontes,
Vine land, NJ.
5.2.2 Micropipets, disposable "9 in", Cat. No. P5200-2,
Scientific Products, Obetz, OH.
5.2.3 Syringe, 1-mL B-D Yale Luer-Lok[tm] tip tuberculin, Cat.
No. 14-820-16, Fisher Scientific, King of Prussia, PA
19406.
5.3 Fluoropore filters, 0.5 urn, Cat. No. FHLP01300, Millipore Corp.,
Bedford, MA 01730
5.4 Filter assembly, Swinney, SS, 13 mm, Cat. No. 3001200, Millipore
Corp., Bedford, MA 01730
5.5 Guard column kit, CXHC Pellionex, SCX Guard Column kit, Whatman,
Inc., Clifton, NJ 07014
5.6 Tube heater, standard model, Cat. No. K720000, Kontes, Vineland,
NJ
5.7 Hewlett-Packard Model 1084B, liquid chromatograph, equipped with
auto sampler and variable wavelength UV detector, Hewlett-Packard
Co., Avondale, PA 19311
5.7.1 Partisil SCX LC column, PXS-1025, Cat. No. 4227-104, Anspec
Company, Ann Arbor, MI 48107
6. Reagents
6.1 Ethyl acetate, distilled-in-glass, pesticide grade, Burdick and
Jackson Labs, Muskegan, MI
6.2 n-Hexane, distilled-in-glass (UV), Burdick and Jackson Labs,
Muskegan, MI
6.3 Methyl alcohol, distilled-in-glass (UV) , liquid chromatography
quality, Fisher Scientific, Parkersburg, WV
6.4 Ammonium hydroxide, concentrated
6.5 Phosphoric acid, 85%, MCB
6.6 Hydrochloric acid, concentrated
6.7 Sodium hydroxide, 50%, MCB
6.8 Benomyl standard, Experimental Station, Dupont, Wilmington, DE
6.9 Carbendazim standard, Experimental Station, Dupont, Wilmington,
DE
6.10 2-Aminobenzimidazole standard, Experimental Station, Dupont,
Wilmington, DE
105-03 January 1983
-------
7. Calibration
7.1 Establish EPLC operating parameters equivalent to those in Table
1
7.2 Calibration Procedure
7.2.1 Weigh approximately 0.5 g (± 0.0001) of methyl-2-benzimi-
dazole carbamate (MBC) into a clean, dry 1000-mL volumetric
flask. Add 100 mL of methanol to dissolve and dilute to
the mark with 0.1 N H PO, (Step 7.2.4). Mix well. This
prepares a 500-ug/mL MBC standard.
7.2.2 Pipet 10.00 ml of the standard stock solution prepared
above into a clean, dry, 100-mL volumetric flask and dilute
to the mark with 0.1 N. H PO, . This represents a 50-ug/mL
MBC standard (Step 7.2.3).
7.2.3 Depending on the sample concentration, further dilutions
may be necessary. The standard concentration should be
matched with the sample concentration.
7.2.4 Prepare 0.1 N H3P04 by diluting 5.00 mL of 85% H3PC>4 to
2000.0 ml with liquid chromatographic grade water.
8. Quality Control
8.1 Follow the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Tare weigh a 250-mL beaker on a top-loading pan balance.
10.2 Add approximately 100 mL of the well-mixed wastewater sample to
the beaker and weigh to the nearest 0.01 g. Record the weight.
The sample should be adjusted to pH 4-5 with sodium hydroxide or
acid prior to preparation for extraction.
10.3 Add 2 mL of concentrated hydrochloric acid to the sample
contained in the 250-mL beakers, verify the pH with pH test paper
(should be pH = 2, if not, add more acid), cover with a watch
glass and heat on a 90 C hot plate or steam bath for 30 minutes.
10.4 Using an ice bath, cool and then transfer the sample to a 500-mL
separatory funnel with the aid of several small deionized water
washings.
105-04 January 1983
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10.5 Employing the 250-mL beaker in which the sample was heated, add
100 mL of pesticide grade n-hexane to the separatory funnel.
Shake for 2 minutes, allow the phases to separate, collect the
aqueous phase (lower) in the 250-mL beaker and discard the hexane
layer (top).
10.6 Repeat step 10.5 above.
10.7 Add 20 mL of 6.5 N. NaOH to the aqueous layer, verify the pH
(should be approximately pH = 10, if not basic, add more
caustic), and extract immediately with four 100-mL portions of
pesticide quality ethyl acetate. Use 2-minute shaking periods
for each extraction. Use the 250-mL beaker as before (Step
10.5).
10.8 Allow the phases to separate and filter the 100-mL portions of
ethyl acetate (top layers) through about 50 g of anhydrous sodium
sulfate into a 500-mL round -bottomed flask.
10.9 For each sample, concentrate the combined ethyl acetate extracts
to about 10 mL using a rotary vacuum evaporator with a bath
temperature of 60 C.
10.10 Quantitatively transfer the concentrated extracts to a 12-mL
Kontes evaporator tube using a 9-inch pasteur pipette and several
small volumes of ethyl acetate as wash.
10.11 Using a Kontes tube heater with a nitrogen bath over the samples,
further concentrate the extract to about 2 mL. Add 1 mL of 1.0 N.
H.FO, and continue to evaporate the remaining ethyl acetate.
10.12 Quantitatively transfer the aqueous solution remaining to a 2-mL
volumetric flask using a pasteur pipette and washes of 0.1 .N
10.13 Dilute to volume with 0.1 N. HoPO, and mix thoroughly.
10.14 Filter each sample solution through a 0.5-um millipore filter,
using a syringe and Swinney adaptor, into the 2-mL glass vial.
Cap and label the vial.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Dilute 1.00 mL of concentrated ammonium hydroxide to 2000.0 mL in
a volumetric flask. Adjust the pH to 1.7 (+. 0.04) using a pH
meter and concentrated phosphoric acid. Filter through 0.5-um
millipore filter paper.
105-05 Jannarv 1981
-------
12.2 Equilibrate the liquid chromatograph under the conditions shown
in Table 1 .
12.3 Inject standard and samples and allow chromatograms to develop.
Under these conditions the uncorrected retention times of MBC and
2AB (Step 12.4) are approximately 8 and 12 minutes, respectively.
12.4 2-Aminobenzimidazole (2AB) may be employed in the standard as a
method for assessing the column performance. 2AB may be prepared
in the same manner as MBC (Step 7.2.1) and both diluted to 100 ml
in the same volumetric flask (Step 7.2.2).
13. Calculations
13.1 Measure and record the peak height (mm) of MBC for each sample
and standard.
13.2 Calculate and report the concentration of MBC found in ug/g as
follows :
ug/g MBC - (AF>
Where:
, .. [MBCj standard (ug/mL)
CF = calibration factor = - , , . , . .. . , — r
peak height Std. (mm)
This should be an average of all standards analyzed,
corrected to the same attenuation.
PH = peak height of MBC in sample (mm) corrected to attenuation
s
for which standard was analyzed.
AF = aliquot: factor = final volume to which sample was diluted
after extraction (mL)
SW = weight of sample (g)
14. Method Performance
14.1 Based on non-consecutive replicate analyses, the precision is
approximately 2% relative at the 50-ug/g MBC level. Average
recovery for MBC was 88% at the 0.4-ug/g level. Using a 100-g
sample the theoretical sensitivity is 0.08 ug/g MBC.
15. References
15.1 American Conference of Governmental Industrial Hygienists,
"Documentation of the Threshold Limit Values for Substances,"
1979.
105-06 January 1983
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15.2 "Determination of Benomyl/Carbendazim, Liquid Chromatographic
Technique," Method No. W28.047(R), 4/14/81, E.I. duPont de
Nemours and Co. (inc.) HQ, TX.
105-07 January 1983
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Table 1
Chromatographic Conditions
Solvent temperature B (Methanol)
Solvent temperature A (Buffer)
Flow
% B (Methanol)
% A (0.007 N NH4H2P04 buffer)
Maximum pressure
Minimum pressure
Oven temperature
Wavelength sample: Reference
Chart speed
Zero
Area Rej.
Slope sensitivity
Attenuation
Injection volume
35°C
60°C
3 mL/min.
60
40
400 bar
1 bar
40°C
274 nm:0 nm
0.5 cm/min.
5
0
0.3
2 8 (AU x 10~4)
50 uL
Column: Partisil SCX LC column, PXS-1025, Cat. No. 4227-104, Anspec
Company, Ann Arbor, MI 48107
105-08
January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENOMYL AND CARBENDAZIM
IN WASTEWATER
Method 106
1. Scope and Application
1.1 This method covers the determination of benomyl and carbendazim.
Parameter Storet No. CAS No.
Benomyl — 17804-35-2
Carbendazim — 10605-21-7
1.2 Benomyl cannot be determined directly by this method. Benomyl is
hydrolized to carbendazim, and both compounds are measured and
reported as carbendazim.
1.3 This is a high performance liquid chromatographic (HFLC) method
applicable to the determination of the compounds listed above in
aqueous waste effluent.
1.4 The method detection limit (MDL) is 0.01 ug/g (ppm).
2. Summary of Method
2.1 An aliquot of sample (100 g) is acid hydrolized, extracted with
ethyl acetate, and subsequently analyzed by high speed cation
exchange liquid chromatograpby.
3. Interferences
Not Available.
4. Safety
4.1 Follow the EPA safety procedure found in Part D of this document.
106-01 January 1983
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5. Apparatus and Materials
5.1 Sampling Equipment
Not available.
5.2 Glassware and Other Equipment
5.2.1 Beakers, 250 ml
5.2.2 Separatory Funnel, 500 mL
5.2.3 Fine tip dropper
5.3 Steam bath
5.4 Rotary Evaporator
5.5 Liquid Chromatograph
6. Reagents
6.1 Concentrated Hydrochloric Acid
6.2 n-Hexane
6.3 Ethyl Acetate
6.4 6.5 N NaOH
6.5 .IN Phosphoric Acid
6.6 Benomyl Standard
6.7 Carbendazim Standard
7. Calibration
7.1 Chromatographic Conditions are given in Reference 15.1.
7.2 Calibration Procedure — Measure the peak height for MBC and
determine the micrograms of this compound in the aliquot using a
previously prepared calibration curve (Reference 15.1).
8. Quality Control
8.1 Follow the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
106-02 January 1983
-------
10.1 Weigh a 100-g sample into a 250-mL beaker, add 2 mL of
concentrated HC1, cover, and heat on a steam bath for about 30
minutes.
10.2 Cool and transfer to a 500-mL separatory funnel using several
small water washes. Add 100 mL n-hexane, shake for 2 minutes,
and allow the phases to separate. Discard the hexane layer.
Repeat the hexane wash using a second 100-mL portion of n-hexane.
10.3 Make the aqueous phase strongly basic by adding 15 mL of 6.5 N
NaOH and extract immediately with four 100-mL portions of ethyl
acetate using 2-minute shaking periods for each extraction.
10.4 Allow the phases to separate and filter the ethyl acetate phase
through anhydrous sodium sulfate into a 500-mL round-bottomed
flask.
10.5 Concentrate the combined ethyl acetate extracts to about 10 mL
using a vacuum rotary evaporator at 60 C.
10.6 Quantitatively transfer the concentrated extract to a 30-mL
beaker using several small volumes of ethyl acetate as wash.
Continue to concentrate the solvent to about 3-5 mL. Add 1 mL of
0.1 N H,PO, and continue the evaporation until all of the ethyl
acetate is removed.
10.7 Quantitatively transfer the aqueous solution to a 2-mL volumetric
flask using a fine tip dropper and several small washes of 0.1 N
H PO,. Dilute to volume with 0.1 N H-PO, and mix thoroughly.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Equilibrate the liquid chromatograph and inject an aliquot of the
prepared extract as described in the reference method (Reference
15.1).
13. Calculations
13.1 Calculate the amount of carbendazim present in ug/g (ppm) by
dividing the micrograms of MBC found, corrected for aliquot
factor, by the sample weight in grams. To express the residue as
ppm benomyl, divide the micrograms of MBC found, corrected for
the molecular weight conversion (1.53) a-d aliquot factors, by
the sample weight in grams.
14. Method Performance
14.1 Using a 100-g sample, the method has a sensitivity of 0.01 ug/g
(ppm).
106-03 January 1983
-------
15. References
15.1 Kirkland, J.J., Holt, R.F., Pease, H.L. Determination of Benomyl
Residues in Soils and Plant Tissues by High-Speed Cation Exchange
Liquid Chromatography, J. Agr. Food Chem., 21, 368 (1973).
15.2 Pease, H.L., Gardiner, J.A. Fluorometric and Colorimetric
Procedures for Determining Residues of Benomyl, J. Agr. Food
Chem., 17, 267 (1969).
15.3 "Determination of Benomyl/Carbendazim in Plant Aqueous Waste
Effluent," Attachment 3, E.I. duPont de Nemours and Company,
Belle, WV Plant.
106-04 January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENTAZON (BAS 351-H)
IN WASTEWATER
METHOD 107
1. Scope and Application
1.1 This method covers the determination of bentazon (BAS-351-H).
Parameter Storet No. CAS No.
Bentazon (BAS 351-H) — 25057-89-0
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
1.3 The method detection limit (MDL) is 10 ug/L.
2. Summary of Method
2.1 An aliquot of sample (lOOg) is acidified and extracted with ethyl
acetate. The extract is dried, concentrated to 1-2 ml, and
methylated with diazomethane. The methylated extracts are
analyzed by gas chromatography with flame photometric detection.
3. Interferences
Not Available.
4. Safety
4.1 Follow the EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not available.
107-01 January 1983
-------
5.2 Glassware and Other Equipment
5.2.1 Separator;/ funnels, 500 mL, Kimax or equivalent.
5.2.2 Graduated concentrator tubes, 35 mL, Kimble, 24/40
stoppers.
5.2.3 Flash Evaporator, Buchi or equivalent.
5.3 Diazomethane Generator, available from Aldrich Chemical Company
5.4 Gas Chromatograph, Tracer-Microtek, Model MT 220 equipped with a
Flame Photometric detector
5.4.1 Gas Chromatographic Column:
5.4.1.1 10% SE-30 on Chromosorb G, HP or Gas Chrom Q,
80/100 mesh 4-ft x 1/4-in I.D.
5.4.1.2 3% Carbowax 20M on Gas Chrom Q, 60/80 mesh, 4-
ft x 1/4-in I.D.
6. Reagents
6.1 Ethyl Acetate, MCB, pesticide quality
6.2 Sodium Chloride, Baker reagent
6.3 Sodium Sulfate, Baker anhydrous reagent (prewash with chloroform
to remove extraneous sulfur peaks)
6.4 Ethyl Ether, absolute, pesticide quality
6.5 Diazomethane solution: Prepare from Diazald reagent as specified
by the manufacturer, Aldrich Chemical Co.
6.6 Hydrochloric Acid, 12 N, reagent
6.7 Acetone, pesticide quality, MCB
6.8 Bentazon (BAS 351-H), Analytical standard, BASF Wyandotte
Corporation
7. Calibration
7.1 Sample residues are dissolved in acetone or benzene and injected
into a gas chromatograph equipped with a Flame Photometric
detector operating in the sulfur-specific mode (394-nm filter),
operating at the conditions given in Table 1 for methylated
Bentazon (BAS 351-H). Gas chromatographie separations are made
using a 10% SE-30 or 3% Carbowax 20M column on Gas Chrom Q or
Chromosorb G packing.
107-02 January 1983
-------
7.2 Calibration Procedure
7.2.1 Prepare a stock standard solution by weighing 100.0 mg
of Bentazon (BAS 351-H) analytical standard, and
quantitatively transfer to a 100-mL volumetric flask.
Dilute the material to 100 mL with acetone. This
solution contains 1 ug/uL (stock solution A).
7.2.2 Transfer 10.0 ml of this stock solution A to a 35-mL
graduated concentrator tube and evaporate the acetone to
dryness.
7.2.3 Dissolve the Bentazon (BAS 351-H) residue in 10-mL ethyl
ether and add 20 mL diazomethane solution. Allow the
solutions to react for 10 minutes.
7.2.4 Concentrate the solvent to approximately 2 mL on an N-
EVAP.
7.2.5 Quantitatively transfer the methylated Bentazon (BAS
351-H) solution to a 10-mL volumetric flask. Wash the
35-mL concentrator tube with acetone and add this to the
10-mL volumetric flask. Adjust the volume to 10 mL with
acetone. This is stock solution B containing 1 ug/uL of
methylated Bentazon (BAS 351-H) standard.
7.2.6 Stock solution B may be used for preparing injection
standards by serial dilutions of microliter quantities.
7.2.7 A series of dilutions of methylated Bentazon (BAS 351-H)
(stock solution B) containing 0.5, 1, and 2 ng/uL are
made. Inject 2-10 uL of each diluted standard solution
to obtain a standard curve for 1.0 to 10.0 ng standards.
Higher ranges may be used if so desired.
7.2.8 Determine the peak height or area of each standard
injection.
7.2.9 Construct a standard curve by plotting detector response
(peak height or area) versus concentration (ng or ug) of
standard methylated Bentazon (BAS 351-H) injected. A
typical standard curve for the flame photometric
detector is shown in Figure 1. Note: Since the
response of the flame photometric detector is not
linear, standard curves may be plotted on log-log paper
to obtain a straight line curve.
7.2.10 Bracket every 3-5 samples analyzed with known amounts of
standard to maintain a continual check for any shifts in
sensitivity.
8. Quality Control
8.1 Follow the EPA Quality Control procedure found in Part D of this
document.
107-03
January 1983
-------
9. Sample Collection, Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 If samples contain no sediment, go to Step 10.2; however, if
sediment is present, shake the samples well to get a homogenous
sample aliquot.
10.2 Measure out 100 mL equivalent to 100 g of sattiule into a 500-mL
separatory funnel.
10.3 Dilute the sample to 250 mL by adding sodium chloride solution
(150 g in 2 L distilled water).
10.4 Make the resultant solution acidic (pH <1) by adding
concentrated hydrochloric acid dropwise.
10.5 Extract the acidified solution 3 times with 50 mL ethyl acetate.
10.6 Dry the extracts through a pad of sodium sulfate which has been
prewashed with chloroform to remove extraneous sulfur peaks, and
collect them in a 250-mL Erlenmeyer flask.
10.7 Concentrate the combined extracts to 1-2 mL.
10.8 Take the residue from 10.7 and add 10 mL of ethyl ether and 1 mL
of diazomethane solution.
10.9 Allow the mixture to react for 10 minutes at room temperature.
10.10 Quantitatively transfer the methylated residues to a 35-mL
graduated concentrator tube with repeated acetone washings.
10.11 Concentrate the solution to approximately 1 mL.
10.12 Adjust the volume to an appropriate level with acetone for final
determination by gas chromatography.
11. Cleanup and Separation
Not available.
12. Gas Chromatographv
12.1 Inject an appropriate aliquot into the gas chromatograph and
determine the peak height or area for methylated Bentazon (BAS
351-H).
12.2 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
13. Calculations
107-04 January 1983
-------
13.1 Directly compare the peak heights or areas of unknown samples
injected with the appropriate standard curve and extrapolate the
concentration of methylated Bentazon (BAS 351-H) in the injected
sample aliquot.
13.2 Determine the residue results in terms of ppm methylated BAS
351-H in the sample by the following:
a
ppm = ~; :—:—I~~_,b divided by (R), where
vv mg sample injected J
a. = concentration extrapolated from standard curve
mg aliquot ~ microliters injected
for analysis microliters final volume
R = Recovery factor based on fortified controls carried
through the procedure. R is expressed as a decimal (i.e.
100% = 1.00, 90% = 0.90, etc.)
14. Method Performance
14.1 Recoveries for water are tabulated in Table 2. They range from
84 to 116% in samples from Texas and Mississippi, involving four
field experiments.
15. References
15.1 LOTEL Report No. 133, October 2, 1973, "Determination of BAS
351-H Residues in Soil and Runoff Water," James Devine, Lake
Ontario Environmental Laboratory, Oswego, New York.
15.2 Lab Report dated April 1972, "Method of Analysis for the
Determination of Bentazon in Drinking and Leaching Water," BASF
Analytical Laboratories, Limburgerhof, Germany.
15.3 "Determination of BAS 351-H [3-isopropyl-lH-2.1.3-
benzothiadiazin-4(3H)-one-2,2-dioxide] Residues in Water by Gas
Chromatography," Analytical Method No. 8, January 23, 1974, BASF
Wyandotte Corporation, Geismar, LA.
107-05 January 1983
-------
Table 1
Gas Chromatographic Conditions for Flame Photometric Detection
of Methylated Bentazon (BAS 351-H) Residues in Water
Gas Chromatograph
Column
Temperatures:
Glass Inlet
Detector
Column
Gas Flows:
Carrier
Hydrogen
Oxygen
Air
Electrometer:
Sensitivity
Attenuation
Recorder:
Range
Chart Speed
Minimum Detection Limit
Retention Time
(Approx)
Tracer-Microtek MT 220 equipped with a
Flame Photometric detector operating in
the sulfur specific mode (394-nm filter)
a. 3% Carbowax 20M on Gas Chrom Q,
60/80 mesh, 6-ft x 1/4-in I.D.
b. 10% SE-30 on Chromosorb G, H.P. or Gas
Crom Q, 80/100 mesh, 4-ft x 1/4-in
I.D.
225°C
200°C
200°C
Nitrogen 80 mL/min
60 mL/min
20 mL/min
15 mL/min
-x2(io"8)
102 x 64
I mV
0.25 inches/minute
1 ng Methylated Bentazon (BAS 351-H)-
3.0 - 3.5 min
107-06
January 1983
-------
Table 2
Typical Recoveries of Methylated Bentazon (BAS 351-H) Residues in Water
Sample Fortification Level (ug/g)
Matrix Bentazon (BAS 351-H) Added % Recovery
Water 0.50 95
0.10 105
0.10 94
0.10 90
0.10 104
0.05 84
0.01 94
0.01 94
0.01 116
0.01 103
0.01 108
Average = 98.8%
107-07 January 1983
-------
FIGURE 1
TYPICAL STANDARD CURVE Of
METHYLATED DAS 351-11
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107-10
January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BOLSTAR IN WASTEWATER
METHOD 108
4.
Scope and Application
1.1 This method covers the determination of Bolstar.
Parameter Storet No. CAS No.
Bolstar — 35400-43-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastevater samples
containing 0.005 - 1 mg/L Bolstar.
Summary of Method
2.1 A measured aliquot (250 mL) of wastewater is extracted with
chloroform, oxidized to Bolstar sulfone using potassium
permanganate, and determined by GC using a phosphorus
(thermionic) detector.
Interferences
3.1 All glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENT.
4.1 Follow the EPA safety procedure found in Part D of this
document.
108-01 January 1983
-------
5. Apparatus and Materials
5.1 Sampling Equipment
Not available.
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 10 uL
5.2.2 Rotoevaporator, Buchi or equivalent
5.3 Water bath, 50-55°C
5.4 Gas chromatograph, Varian 1400 or equivalent, equipped with a
phosphorus detector (rubidium sulfate salt pellet), an 18-in x
1/8-in O.D. glass column packed with 10% DC-200 (12,500
centistokes) on Gas Chrom Q 80/100 mesh, and glass injector and
detector inserts. (If the column ends are long enough to be
used as inserts, the glass inserts are not necessary.)
NOTE: Use only Teflon-backed septa, Supelco No. 2-0459.
6. Reagents
6.1 Acetone, nanograde
6.2 Chloroform, nanograde
6.3 Hydrochloric acid, IN — Carefully add 21 mL of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.4 Magnesium sulfate, 20% — Weigh 41 g of magnesium sulfate
heptahydrate (MgSO,.7H20) into a 100-mL glass-stoppered
graduated cylinder. Add distilled water to the 100-mL mark,
stopper, and mix.
6.5 Mineral oil solution, 2.5% — Dilute 25 mL of mineral oil,
Fisher #0-120 or equivalent, to 1000 mL with nanograde
chloroform.
6.6 Bolstar sulfone (NTN 9306) standard solution, 0.10% — Weigh
0.095-0.115 g of Bolstar (NTN 9306) sulfone (95% purity) into a
100-mL glass-stoppered graduated cylinder. Dilute to volume
with acetone, stopper, and mix thoroughly. This solution may be
used for one week only.
6.7 Potassium permanganate, O.lM — Weigh 1.6 g of potassium
permanganate, ACS into a 100-mL glass-stoppered graduated
cylinder. Dilute to 100 mL with distilled water and mix
thoroughly.
6.8 Sodium hydroxide, IN
108-02 Januarv
-------
6.9 Sodium hydroxide, O.lN
6.10 Sodium sulfate, anhydrous, ACS
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Pipet a 1-mL aliquot of the 0.10% Bolstar (NTN 9306)
sulfone solution into a 100-mL volumetric flask, dilute to
volume with acetone, and mix thoroughly. Label this
solution "10 ng". Prepare fresh daily.
7.2.2 Pipet 5-, 10-, and 20-mL aliquots of the 10-ng standard
from Step 7.2.1 into separate 100-mL volumetric flasks.
Dilute to volume with acetone and mix. Label these
solutions 500 pg, 1000 pg, and 2000 pg, respectively.
Prepare fresh daily.
8. Quality Control
8.1 Follow the EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately
to Step 10.2.
10.2 Measure 250 mL of the well-mixed sample into a 500-mL separatory
funne1.
10.3 If the pH of the sample is below 6.5 or above 7.0, adjust it to
6.5-7.0 with IN sodium hydroxide or IN hydrochloric acid using
pH indicator paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel.
If an emulsion forms, centrifuge the emulsified layer and add
the clear chloroform layer, obtained by centrifuging, to the
second separatory funnel. Return the water layer to the first
separatory funnel.
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minute.
108-03 January 1983
-------
10.6 Filter the chloroform layer into a 300-mL 24/40 boiling flask
through a. funnel containing 4 to 5 g of anhydrous sodium
sulfate, retained by a small glass-wool plug (pre-rinsed with
10 ml of chloroform). Rinse the sodium sulfate three times
with 10 mL of chloroform, and add 10 mL of 2.5% mineral oil
solution into the flask.
10.7 Place the flask on a rotoevaporator and- strip off all of the
chloroform, using a water bath at 50 C. Remove the flask and
cool to room temperature.
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Add 5 mL of nanograde acetone into the sample flask. Swirl the
flask to wash the inside wall with the acetone but do not allow
the acetone to get on the joint or splash out.
10.10 Add 5 mL of distilled water and swirl to mix, then add 10 mL of
0.1N sodium hydroxide to the flask. Stopper, swirl to mix, and
allow the flask to sit at room temperature for 15 minutes.
10.11 Add 10 mL of the magnesium sulfate solution to the flask and
swirl to mix.
10.12 Add 25 mL of O.lM potassium permanganate solution and swirl.
Allow the sample to stand for 30 minutes, making sure there is
an excess of potassium permanganate (as indicated by the purple
color) the entire time. (Add the permanganate in 5-mL
increments if necessary to maintain an excess; increase the
standing time 5 minutes for each addition.)
10.13 Quantitatively transfer the sample solution from Step 10.12 into
a 500-mL separatory funnel, using five 40-mL portions of
distilled water.
10.14 Add 50 mL of chloroform into the separatory funnel, stopper and
shake vigorously for 1 minute.
10.15 Allow the layers to separate. Drain the chloroform layer into a
clean 250-mL separatory funnel.
10.16 Repeat Steps 10.14 and 10.15 twice for a total of 3 extractions.
10.17 Add 25 mL of distilled water to the chloroform extracts,
stopper, and shake for 30 seconds.
10.18 Filter the chloroform layer into a 300-mL boiling flask through
another funnel containing 4 to 5 g of anhydrous sodium sulfate
retained by a small glass-wool plug (pre-rinsed with 10 mL of
chloroform).
10.19 Rinse the sodium sulfate with three 10-mL portions of chloroform
and combine with the chloroform extract.
108-04 January 1983
-------
10.20 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at 50 C. Remove the flask and
cool to room temperature.
10.21 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.22 Pipet 5 mL of acetone into the flask from Step 10.21, stopper
immediately, and rotate the flask so that the acetone washes
down the inside of the flask. DO NOT allow the acetone to get
on the neck or stopper.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Continue the analysis according to TM A-54.12 (Mobay reference
procedure, not enclosed herein).
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
15.1 "Determination of Bolstar in Wastewater by GLC," Analytical
Method TM B-34.50, Chemagro Agricultural Division, Mobay
Chemical Corporation, Kansas City, MO.
108-05 January 1983
-------
Table 1
Chromatographic Conditions
Attenuation X32
Cell voltage Flame
Detector temperature 230-240 C
-12
Electrometer range 10 A
Gas flows:
Carrier gas, Helium 30 mL/min
Air 230 mL/min
Hydrogen 40 mL/min
Injector temperature 230-240 C
Oven temperature 230 C
Column: 10% DC-200 (12,500 Centistokes) on Gas Chrom Q 80/100 mesh
Note: Conditions above are for Varian 1400 GC.
108-06 January 1983
-------
xvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BROMACIL, HEXAZINONE, OXAMYL,
TERBACIL AND METHOMYL IN WASTEWATER
METHOD 109
Scope and Application
1.1 This method covers the determination of bromacil, hexazinone,
oxamyl, terbacil, and methorny1. The following parameters can be
determined by this method:
Parameter Storet No. CAS No.
Bromacil 82198 314-40-9
Hexazinone — 51235-04-2
Oxamyl — 23135-22-0
Terbacil — 5902-51-2
Methomyl 39051 16752-77-5
Diuron 39650 330-54-1
Linuron — 330-55-2
1.2 This is a liquid chromatographic (HPLC) method applicable to the
determination of the compounds listed above in wastevater.
1.3 The method detection limit (MDL) for each of the pesticides is in
the ug/L (ppb) range.
Summary of Method
2.1 Samples containing solids or a large number of interferences in
the chromatogram are extracted with methylene chloride.
Acetonitrile (1 mL) is added to the methylene chloride extract,
which is then evaporated down to 1 mL in a 40 C water bath using
a flash evaporator. The residual solution is transferred and
diluted in a volumetric flask with distilled water. This
solution is either injected on HPLC with UV detector or is taken
through a "Sep-Pak" cleanup technique prior to HPLC analysis.
109-01 January 1983
-------
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 All glassware must be cleaned thoroughly with a solvent or
chromic acid to remove any trace of contamination of pesticides
from the glass.
3.2 At the beginning of each day, wash the HPLC column system for ten
minutes with 100% acetonitrile.
4. Safety
4.1 Methorny1 is highly toxic when taken orally or inhaled as dust or
mist. Wash hands before smoking or eating.
4.2 Oxamyl is highly toxic. Avoid personal contact in any manner.
All pesticides should be handled with care and avoid skin
contact.
4.3 Acetonitrile has a TLV of 40 ppm.
4.4 Waste streams stiould be handled with care, avoiding any skin
contact.
4.5 See the EPA Safety procedure as found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Flash-Evaporator, Buckler Instruments, Fort Lee, NJ, which
includes the condenser glassware, flasks, roto-motor, and
thermostated water bath.
5.2.2 Disposable Transfer Pipets (Pasteur type), Curtin Matheson
Scientific, Inc., (5 3/4" long).
5.3 Sep-Pak C-18 Cartridges, Waters Associates, Inc., Milford,
MA
5.4 Sample injection valve equipped with a 3.0-mL sample loop,
Rheodyne, Berkeley, CA
5.5 Du Pont Model 850 Liquid Chromatograph unit with linear gradient
elution system, heated column compartment, and 254-nm UV
detector, Du Pont Instrument Products Division, Wilmington, DE
19898
5.5.1 Column — 4.6-mm ID x 25-cm prepacked "Zorbax" ODS reverse
109-02 January 1983
-------
phase column (no substitute), part number 850952702, Du
Pont Instrument Products Division, Wilmington, DE 19898.
5.5.2 Printer/Plotter — Integrator 3380A, Hewlett Packard,
Avondale, PA.
6. Reagents
6.1 Acetonitrile, Burdick & Jackson, chromatographic grade (distilled
in glass)
6.2 Methylene chloride, Burdick & Jackson, chromatographic grade
(distilled in glass)
6.3 Bromacil, Hexazinone, Oxamyl, Terbacil, Methomyl (Dupont
Biochemicals Dept.)
Calibration
7.1 Establish HPLC operation parameters similar to those indicated in
Table 1.
7.2 Calibration Procedures
7.2.1 Analytical Stock Standard Solution
7.2.1.1 Weigh 0.0100 g (±0.001 g) of the analytical
standards oxamyl, methomyl, bromacil,
hexazinone, terbacil, diuron, linuron into
separate 100-mL volumetric flasks (See Step
3.1). Note the exact weight of the standard.
7.2.1.2 Dilute to volume with distilled water. (For
diuron, linuron, bromacil, and terbacil, add 5
mL acetonitrile and dilute with water after
dissolution.)
7.2.1.3 Place each volumetric in an ultrasonic bath for
five minutes to complete dissolution.
7.2.1.4 Store at 4 C. These solutions contain
approximately 100 ug/mL of standard (solutions
are stable for 4 weeks).
7.2.2 Analytical Working Standard Solutions:
7.2,2.1 Prepare 100-mL volumes of working standard
solutions at 50, 250, 500, 1000, 5000, and
10,000 ug/L by diluting aliquots of the stock
standard solution.
7.2.2.2 Additional working standards may be required to
bracket the concentration of a pesticide in a
given sample.
109-03 January 1983
-------
7.2.2.3 Store at 4°C (stable 2 weeks).
7.2.3 At 0.01 AUFS detector sensitivity, run the 50-, 250-, and
500-ug/L standards in duplicate.
7.2.4 At 0.05 AITS recorder sensitivity run the 1000-, 5000-, and
10,000-ug/L standards in duplicate.
7.2.5 Draw calibration curves showing peak area versus
concentration of standard (ug/L).
7.2.6 Additional standards may need to be run at a given detector
sensitivity in order to bracket the concentration of
pesticide seen in a sample.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Clean a pH probe with acetone and with water.
9.2 Using concentrated sulfuric acid or 50% sodium hydroxide, adjust
the pH of the contents of each bottle to pH 6-8 within four hours
after sampling. Record the volume of acid or caustic used to
neutralize the sample. Final calculations should be corrected
for any significant dilution of sample during neutralization.
9.3 Refrigerate the neutralized samples. Oxamyl, methomyl, and
hexazinone will hydrolyze at high pH. Samples which are not
neutralized will show a gradual reduction in the concentration of
these pesticides. The stability of various pesticides in neutral
aqueous standards at room temperature exposed to light (see
Reference 1) was as follows:
ug/L ug/L Exposure Z
Added Measured Time. Days Recovery
Bromacil 1.0 1.00 7 100
Hexazinone 1.0 0.83 7 83
Oxamyl 10 8.2 14 82
Methomyl 100 -95 7 95
Terbacil 1.0 0.99 7 99
Diuron 10 9.3 7 93
Linuron 10 9.6 7 96
9.4 See the EPA sample collection procedure as found in Part D of this
document.
10. Sample Extraction
10.1 Mark the level of the water sample on the side of the glass
bottle (Step 10.14).
109-04 Januarv 1983
-------
10.2 Pour the contents of the bottle into a separatory funnel.
10.3 Wash the bottle with 60 mL of methylene chloride and transfer it
to the separatory funnel.
10.4 Shake the separatory funnel for two minutes. Avoid forming an
emulsion by shaking gently.
10.5 Transfer the methylene chloride layer into a clean 250-mL flash-
evaporator flask, leaving a small portion (approximately 1 mL) of
solvent in the funnel.
10.6 Repeat Steps 10.3, 10.4 and 10.5 two more times.
10.7 Discard the water layer.
10.8 Add 1 mL of acetonitrile to the flask containing the methylene
chloride extract.
10.9 Place the flask into the 40 C water ba
flash-evaporator. Evaporate until onl;
remains.
10.10 If the sample requires no Sep-Pak cleai
solution into a volumetric flask (Step
funnel and three 5-mL washes of the evs
distilled water.
10.11 Dilute to volume with distilled water.
for HPLC analysis. Record the volume
concentration factor.
10.12 If the sample requires Sep-Pak cleanup
water to the evaporator flask and proc
procedure.
10.13 Using a graduated cylinder, measure th
to refill the original sample bottle t
volume.
10.14 Sample Information
h and connect it onto the
1 mL of acetonitrile
up, transfer the residual
10.14) using a clean glass
porator flask with
This solution is ready
o calculate the
add 200 mL of distilled
ed with the Sep-Pak
volume of water required
the mark and record the
109-05
January 1983
-------
Sample
Identification
Outfall 101
Technique
Used
500 ml extracted with
methylene chloride,
final volume of 25
mL of water for injection
OQ HPLC
Detector
Sensitivity
(AUFS)
0.01 for
15 min.
0.05 to end
Attenuation
8
Lift Station
Effluent
(LSE)
Central Scrubbed
Incinerator Feed
(CSIF)
Central Scrubbed
Incinerator Purge
(CSIP)
500 mL extracted with
methylene chloride,
final volume of 25 mL
of water for injection
of HPLC
100 mL extracted with
methylene chloride,
Sep-Pak cleaned, final
volume of 100 mL of water
for injection on HPLC
Direct injection of
this sample on HPLC
0.05
0.05
0.01
0.05
0.01
Chemical Oxidation Direct injection of
Feed this sample on HPLC
Chemical Oxidation Direct injection of
Discharge this sample on HPLC
11. Cleanup and Separation
11.1 Prepare the Sep-Pak cartridge train as follows:
11.1.1 Connect two Sep-Pak cartridges using a short piece of
polyethylene tubing. (Do not use Tygon tubing.) The
short end of one Sep-Pak should be connected to the
long end of the other Sep-Pak.
11.1.2 Connect the long end of the Sep-Pak train to a
disposable pipet. The Sep-Pak tubing will fit inside
the inner diameter of the disposable pipet.
11.1.3 Connect the short end of the Sep-Pak train to a
vacuum flask using flexible tubing.
11.1.4 To wash the Sep-Pak train, place the disposable pipet
into 10 mL of acetonitrile allowing the vacuum to
pull the solvent through the Sep-Pak.
11.1.5 Repeat Step 11.1.4 with 10-15 mL of distilled water.
The Sep-Pak train is now ready for use on a sample
cleanup.
109-06
January 1983
-------
11.2 Place the Sep-Pak train into the evaporator flask (See Step
10.12), allowing the vacuum to pull 200 mL of aqueous solution
through the Sep-Paks.
11.3 Turn the Sep-Pak train upside down to allow the vacuum to remove
as much water as possible from the train.
11.4 Disconnect the Sep-Pak train from the vacuum flask tubing.
11.5 Put the disposable pipet end of the train into a volumetric
flask.
11.6 Connect a 10-mL syringe with a Luer end fitting to the Sep-Pak
train.
11.7 Remove the plunger from the syringe. Pour 10 mL of acetonitrile
into the syringe barrel and replace the plunger. Push the
acetonitrile through the Sep-Pak train into the volumetric flask.
11.8 Repeat step 11.7 with air in the syringe to displace any residual
acetonitrile into the flask.
11.9 Place the volumetric flask in a water bath at 65 C-70 C. Use a
nitrogen purge to aid in evaporation of the acetonitrile down to
1 mL. Dilute to volume with distilled water. The sample is now
ready for HPLC. Record the volume to calculate the concentration
factor.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph
12.2 Run a gradient baseline at the beginning of each day
12.3 HPLC Injection Technique
12.3.1 With the sample valve in the LOAD position, inject 3-
4 mL of sample through the sample loop with a 5-mL
syringe.
12.3.2 Inject the sample by turning the sample valve to the
INJECT position. Depress the RUN button on the LC.
12.3.3 Depress the START/STOP button on the printer/plotter.
12.3.4 Settings on printer/plotter
a. Report - Mefhod
b. Start Delay - OFF
c. Stop Timer - 60 min
d. Area Reject - 100
e. Chart Speed - 0.5 cm/min
f. Chart - AUTO
g. Slope Sensitivity - 0.3 mV/min
109-07 January 1983
-------
h. Attenuation - See Step 10.14
12.3.5 Each sample or standard should be run in duplicate.
13. Calculations
13.1 From the calibration curves, determine the concentration (ug/L)
of pesticide in. the injected sample.
13.2 If the sample was concentrated by extraction or Sep-Pak cleanup,
determine the concentration (ug/L) of pesticide in the original
sample as follows:
„ ^,_. e i-,\ • • • i i ug/L in injected sample
Concentration (ug/L) in original sample = concentration factor
„ . .. mL of original sample
Concentration factor ~
mL of final volume after extraction
and/or cleanup
13.3 The concentrated sample may have to be diluted to bring the peak
area within the linear range of the calibration curve for some
pesticides. In this case, calculations are as follows:
Concentration (ug/L) in original sample =
ug/L in injected sample ,., . . ,.
—*• —Lr: K— x dilution factor
concentration factor
_., . , mL of final diluted volume
Dilution factor = ;—^ ~—~—7~:
mL of concentrate taken
14. Method Performance
14.1 A detector sensitivity of 0.01 to 0.05 AUFS was used depending on
which sample was being analyzed. The sensitivity for all of the
subject pesticides is in the ug/L range.
14.2 Precision and accuracy data have not been determined.
14.3 Methylene chloride extraction is used only if the sample
contained solids or if it requires "Sep-Pak" cleanup. The
extraction efficiencies for various pesticides are as follows
(see Reference 1):
Bromacil
Hexazinone
Oxamyl
Me thorny 1
Terbacil
Diuron
Linuron
ug/L
Added
1
1
10
100
1
10
10
ug/L
Measured
0.91
1.02
9.8
78
0.97
9.0
9.1
%
Recovery
91
102
98
78
97
90
91
109-08 January 1983
-------
14.4 The % recoveries of the subject pesticides using the "Sep-Pak"
cleanup technique are as follows:
% Recovery for a Given Amount of Pesticide
1.25 ug 25 ug 500 ug 2500 ug 5000 ug
Bromacil 103 91
Hexazinone 98 102 -
Oxamyl 101 98 - 87
Methomyl 81 76 -
Terbacil 126 101 - 106 92
Diuron 97 98 101
Linuron 92 98 98 -
15. References
15.1 Midwest Research Institute, Pesticide Analysis Methods. A
contract study conducted by MRI for the U.S. Environmental
Protection Agency.
15.2 "Determination of Houston Plant Pesticides, High Pressure Liquid
Chromatography (HPLC) Method," Method No. W28.303(R), April 29,
1980, E.I. duPont de Nemours & Co., Inc. Houston, TX.
ina-na
-------
Table 1
Chromatographic Conditions
Column temperature
Detector
Detector sensitivity
Flow rate
Maximum Pressure
Sample Size
Gradient
Water
Acetonitrile
a. Step 1
0% B to 40% B
b. Step 2
40% B to 100% B
c. Step 3
100% B to 100% B
d. Step 4
100% B to 0% B
e. Step 5
0% B to 0% B
35°C
254-nm fixed wavelength
0.01 to 0.05 Absorbance
Units Full Scale (AUFS)
2.0 mL/min
100 Bar
2.0 mL, nominally
Program
Solvent A
Solvent B
40 min
8 min
3 min
5 min
Column:
5 min
4.6-mm ID x 25-cm prepacked "Zorbax" ODS reverse phase column.
.Tanuarv 1983
-------
Compound
bromacil
hexazinone
oxamyl
methomy1
terbacil
diuron
Linuron
Table 2
Retention Times
Retention Time (Minutes)
31.6
33.2
15.5
16.6
32.7
41.8
46.7
109-11
January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BUSAN 4C, BUSAN 85, AND KN-METHYL
IN WASTEWATER
METHOD 110
1. Scope and Application
1.1 This method covers the determination of certain dithiocarbamates.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Buaan 40 — 51026-28-9
Busan 85 — 128-03-0
KN-Methyl
1.2 This method determines the amount of total dithiocarbamates
present. It cannot distinguish one compound from another.
1.3 This is a spectrophotometrie method applicable to the
determination of the compounds listed above in water.
2• Summary of Method
2.1 The absorbance of the sample is measured at 284 nm. The
absorbance value indicates the maximum level of dithiocarbamates
present, and a sufficiently low value shows that little or no
dithiocarbamates are present.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
110-01 January 1983
-------
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
Not Available.
Reagents
Not Available.
Calibration
7.1 Measure the absorbance of the sample solution at 284 nm in a 1-cm
cell.
7.2 For Busan 40, Busan 85, and KN-methyl, the following absorbance
values will indicate 1 ppm of analyte present in the solution:
A •
1 ppm. 284 nm. 1 cm
Busan 40 0.062
Busan 85 0.066
KN-methyl 0.071
7.3 Using these values, an absorbance value of 0.010 under the
indicated conditions will represent the following levels of
analyte:
ppm
Busan 40 0.16
Busan 85 0.15
KN-methyl 0.14
7.4 As previously indicated, these are maximum values and the method
of Lowen and Pease (1964) must be used if verification is
desired.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
110-02 January 1983
-------
10. Sample Extraction
Not Available.
11. Cleanup and Separation
Not Available.
12. Sample Analysis
12.1 Dithiocarbamates may be measured in water by measuring the
absorbance at 284 nm. the absorbance value will indicate the
maximum level of dithiocarbamate present, and a sufficiently low
value will show that little or no dithiocarbamate is present. If
significant levels are indicated, the sample must be analyzed by
the method described by Lowen and Pease in "Analytical Methods
for Pesticides, Plant Growth Regulators, and Food Additives,"
Volume III, Fungicides. Nematoe ides and Soil Fumigants.
Rodenticides. and Food and Feed Additives. Chapter 7, pages 69-
77, edited by G. Sweig, Academic Press, New York, 1964.
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
15.1 "Analytical Methods for Pesticides, Plant Growth Regulators, and
Food Additives," Vol III, Fungicides. Nematocides and Soil
Fumigants, Rodenticides. and Food and Feed Additives. Chapter 7,
pages 69-77, 1964.
15.2 "Analysis of Effluent for Dithiocarbamates," Standard Test
Method, Buckman Laboratories, Inc., Memphis, IN.
110-03 January 1983
-------
svEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CARBOFURAN
IN WASTEWATER
METHOD 111
1. Scope and Application
1.1 This method covers the determination of carbofuran.
Parameter Storet No. CAS No.
Carbofuran 81405 1563-66-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
2. Summary of Method
2.1 A measured aliquot of sample (100 ml) is extracted with methylene
chloride. The extract is concentrated to 10 mL on a hot water
bath with dry air or nitrogen. Analysis is by gas chromatography
with flame ionization detector (FID).
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
111-01 January 1983
-------
5.2.1 Separatory funnels, 250 mL, 60 funnels, 70-mm narrow stem
5.2.2 Erlenmeyer flasks, 250 mL, 500 mL
5.2.3 Volumetric flasks, 100 mL
5.2.4 Graduated Centrifuge tubes, 13 mL
5.3 Whatman Filter Paper, No. 1
5.4 Nuchar - Attaclay - Kensington Scientific Corp.
5.5 Hot Water Bath
5.6 Gas Chromatograph with FID detector
5.6.1 Column: 3-ft x 1/8-in O.D. SS packed with 5% OV-17 on
Chromes orb W-HP
5.6.2 HP 3380 A Integrator or similar
6. Reagents
6.1 Na2SO,, reagent grade anhydrous
6.2 Distilled in glass solvents from Burdick & Jackson or similar
6.2.1 Methylene Chloride
6.2.2 Chloroform
6.2.3 Ethyl Acetate
6.2.4 Hexane
6.3 Filtered dry air supply
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1 .
7.2 Calibration Procedure
7.2.1 Weigh 0.1000 g carbofuran standard into a 100-mL volumetric
flask. Dilute to volume with chloroform (1.0 ug/uL).
7.2.2 Pipet 10 mL of the 1-ug/uL standard into a 100-mL
volumetric flask and dilute to volume with chloroform (0.1
ug/uL) .
7.2.3 Make several 1-uL injections of the 0.1-ug/uL standard into
the gas chromatograph with FID detection system. Determine
the area of the carbofuran peak. This area represents 0.1
111-02 January 1983
-------
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Prewash Na.SO, with methylene chloride to remove contaminants.
10.2 Place No. 1 Whatman paper in 60° funnel and fill 3/4 full with
Na-SO,. Wash Na2SO, with three 25-mL portions of methylene
chloride. Discard the washings collected in the flask.
10.3 Extract 100 mL of water sample with three 25-mL portions of
methylene chloride, passing each portion through the prewashed
Na.SO, into the Erlenmeyer flask (500 ml).
10.4 Wash down the Na.SO, with another three 25-mL portions of
methylene chloride for a total of 125 mL in flask.
10.5 Concentrate the extract to 10 mL on a hot water bath with a
gentle stream of dry air or nitrogen.
10.6 Transfer the concentrate from the flask to a 13-mL graduated
centrifuge tube. Rinse the flask with 3 mL chloroform and
transfer to the centrifuge tube. Concentrate to less than 1.0 mL
using a gentle stream of dry air. Dilute the sample to a final
volume of exactly 1.0 mL with chloroform.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Inject a suitable aliquot (about 1-2 uL) of extracted water
sample.
13. Calculations
,, . Area (unk) ... , , .
IJ.A Area (std) x US = amount °f Carbofuran in aliquot (ug;
111-03 January 1983
-------
ug's of Carbofuran x Total extract vol. (uL)
Vol. injected (uL) = ug/g (ppm)
Sample wt. (100 g if 100 mL water extracted) Carbofuran
14. Method Performance
Not Available.
15. References
15.1 "Gas Chromatographic Analysis of Carbofuran in Water at PPM
Level," Standard Test Method, Middleport Control Laboratory, FMC
Agricultural Chemical Division, Middleport, NY.
111-04 January 1983
-------
Table 1
Chromatographic Conditions
175°C
200°C
250°C
Column Temperature
Injection Temperature
Detector Temperature
Flow Rate: Carrier
Detector
Air
Detector: Range
Attenuation
H.P. 3380 A Integrator or similar
Start Delay - 1 min.
Stop Delay - 10 min.
Area Reject - 100
Chart Speed - 0.5 cm/min.
Chart - Auto
Slope Sensitivity - .03
Attn - 1
Column: 3-ft x 1/8-in O.D. S.S. packed with 5% OV-17 on Chromosorb W-HP
He, 25-30 mL/min
H2, 25-30 mL/min
350-400 mL/min
10
4
111-05
January 1983
-------
SERA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CHLOROBENZILATE
IN WASTEWATER
METHOD 112
1. Scope and Application
1.1 This method covers the determination of chlorobenzilate.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Chloropropylate — 5836-10-2
Chlorobenzilate 39460 510-15-6
1.2 This is a thin-layer chromatographic (TLC) method applicable to
the determination of the compounds listed above in wastewater.
1.3 The method detection limit (MDL) is 0.5 mg/L.
2. Su™"arv of Method
2.1 A measured volume of water sample (50 ml) is extracted with
methylene chloride. The extract is analyzed by silica gel TLC.
Spot development is with AgNO, and visualization is by UV light.
3. Interferences
Not Available.
4. Safety
4.1 Plate spraying and exposure to UV radiation should be carried out
in a hood. Special glasses must be used when working with UV
light. Eyes and skin must be protected from UV radiation.
4.2 See EPA safety procedure found in Part D of this document.
112-01 January 1983
-------
5. Apparatus and Materials
5.1 Sampling equipmemt
Not Available.
5.2 Plate: Silica Gel G, neutral (50% Merck Silica Gel G, 50% Bio
Rad Bio Sil-A) on 200- x 200-mm glass plates coated at a
thickness of 200 u.
5.3 Chamber: Saturated
Chamber size - 25 cm x 29 cm x 10 cm
6. Reagents
6.1 Silica Gel G
6.2 Chloroform
6.3 Chloropropylate
6.4 Chlorobenzilate
6.5 Eluent n-hexane/3-A alcohol (anhydrous) 95/5 (by volume)
7. Calibration
7.1 Normal principles of thin-layer chromatography apply for this
analysis. Table 1 gives TLC conditions.
7.2 Calibration Procedure
7.2.1 Weigh O.O.LOO g each of chloropropylate and chlorobenzilate
of 99+% purity into a 100-mL volumetric flask. Dilute this
stock standard to the mark with CHC1- and carry out a
serial dilution to the third dilution. These solutions are
equivalent to 4, 2, 1, and 0.5 mg/L when compared to
samples extracted 5:1 with samples applied 5 x and
standards applied 1 x.
7.2.2 Five applications of samples (approximately 35 uL total)
drying between increments. One application of standards
using open-end capillary tubes (7-8 uL). Capillary tubes
from Kimax size 1.6-1.8 x 100 mm.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
112-02 January 1983
-------
10. Sample Extraction
10.1 Extract a 50-mL sample with 10 ml of chloroform in a 125-mL
separatory funnel. Isolate the chloroform layer for spotting.
11. Cleanup and Separation
Not Available.
12. Thin-Layer Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for TLC.
12.2 Calibrate the system as described in Section 7.
12.3 After elution, air dry plate to remove solvents. Then spray with
0.02N AgNCL and expose to 1400-watt, 366-nm maximum wavelength
ultraviolet light until maximum contrast is achieved. (DANGER -
PROTECT EYES AND SKIN FROM UV RADIATION.) Estimations are made
by visual comparisons between samples and appropriate standards.
12.4 A fresh eluent should be prepared each day and allowed to
equilibrate 1 hour before use.
12.5 Fresh standard solutions should be prepared once per month.
13. Calculations
Not Available.
14. Method Performance
14.1 A statement of "not detected" (ND) for a component indicates only
that the component is less than 0.5 ug/L.
14.2 The standard deviation of an analysis is approximately 30%
relative to actual concentration.
15. References
15.1 "TLC Determination of Chloropropylate and Chlorobenzilate in
Wastewater," ETM-36-1, June 6, 1977, Ciba-Geigy, AL.
112-03 January 1983
-------
Table 1
Chromatographic Conditions
Eluent ri-hexane/3-A alcohol (anhydrous) 95/5 (by volume)
Chamber:
Saturated Chamber size 25 cm x 29 cm x 10 cm
Distance 15 cm
Time 40 minutes
112-04 January 1983
-------
Table 2
Typical Separation Rf
Rf Compound
0.6 Chloropropylate
0.5 Chlorobenzilate
0.0 Point of origin
112-05 January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CHLORPYRIFOS AND CHLORPYRIFOS METHYL
IN WASTEWATER
METHOD 113
1. Scope and Application
1.1 This method covers the determination of chlorpyrifos [0,0-diethyl
0-(3,5,6-trichloro-2-pyridyl) phosphorothioate] and chlorpyrifos
methyl.
Parameter Storet No. CAS No.
Chlorpyrifos 81403 2921-88-2
Chlorpyrifos methyl — 5598-13-0
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in wastewater.
1.3 The method detection limit (MDL) for chlorpyrifos is 0.001 ug/g
(ppm).
2. Summary of Method
2.1 A measured water sample (50 mL) is extracted with methylene
chloride. The methylene chloride extract is evaporated to
dryness with a jet of dry air. The residue is redissolved in a
known volume of acetone, and an aliquot is taken to analyze for
the presence of chlorpyrifos by gas chromatography, with flame
photometric detection.
3. Interferences
Not Available.
4. Safety
4.1 Acetone is flammable and should be used in well ventilated areas
away from an ignition source.
113-01 January 1983
-------
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Syringe, 10-uL, Hamilton brand No. 701N or equivalent.
5.2.2 Separatory funnels, 125-mL, with stopcock made of Teflon
resin and glass stoppers, Corning No. 6402.
5.2.3 Round bottles, 2-oz, with Polyseal caps.
5.3 Evaporation manifolds (see Figure 1)
5.4 Strip chart recorder, Hewlett-Packard Model 7137 or equivalent
5.5 Gas chromatograph, Micro Tek brand, Model MT-160 or equivalent,
equipped with a flame photometric detector, Tracor, Inc., Austin,
TX 78221
5.5.1 Flame photometric detector, Melpar brand, Model FPD100AT
with a 526-mu interference filter (selective, sensitive
response to phosphorus). Tracor, Inc., Austin, TX.
5.5.2 Gas chroma.tographic column: loop-shaped borosilicate glass,
6-ft x 5/32-in I.D. x 1/4-in O.D.
•5.5.3 The glass heat shields (windows) in the flame photometric
detector should be checked at least once a month for
clearness and changed as needed
6. Reagents
6.1 Methylene chloride, distilled in glass, Burdick & Jackson
Laboratories, Inc., Muskegon, MI
6.2 Acetone, nanograde, Mallinckrodt Chemical Works, St. Louis, MO
6.3 OV-17 and QF-1 (mixed phase), 11% on 80/100 mesh Gas-Chrom Q;
Cat. No. 12970, Applied Science Laboratories, Inc., P.O. Box 440,
State College, PA 16801
6.4 Chlorpyrifos, 0,0-diethyl 0-(3,5,6-trichloro-2-pyridyl)
phosphorothioate, analytical standard, Dow Chemical U.S.A.,
Midland, MI
6.5 Prepurified nitrogen (carrier gas), Air Reduction Company.
6.6 Hydrogen (burner gas), Air Reduction Company
113-02 January 1983
-------
6.7 Oxygen (burner gas), Air Reduction Company
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Calibration and spiking standards — Transfer 0.1000 g of
chlorpyrifos analytical standard to a 100-mL volumetric
flask and dissolve in acetone. Dilute to the 100-mL mark
to obtain a 1000-ug/mL stock solution. Prepare working
standards as shown in Table 2.
7.2.2 Injection Technique
7.2.2.1 Fill the 10-uL syringe with the solution to be
injected, eliminating all air bubbles with
rapid up-down strokes.
7.2.2.2 Inject the sample into the chromatographic
column with rapid injection rate and rapid
withdrawal of the needle from the column.
7.2.2.3 Turn the 4-port valve to the vent position for
approximately 25 seconds immediately after
sample injection in order to vent solvent and
prevent extinguishing flame, then close.
7.2.2.4 Change septum in the injection part of the
chromatograph daily when in use.
7.2.2.5 The glass insert in the injection part should
be replaced with a clean, dry insert at least
once a week when in daily use.
7.2.3 Inject a 10-uL aliquot of the chlorpyrifos standard
solutions, covering the concentration range from 0.05 to
0.5 ug/mL, into the chromatograph and record the resulting
peak heights. Plot peak heights on the ordinate as percent
full-scale deflection vs nanograms of chlorpyrifos on the
abscissa. A typical standard curve is presented in Figure
2.
8. Quality Control
8.1 Mix a freshly-thawed control sample of water.
8.2 Pipet 50-mL aliquots of the water sample into a series of 125-mL
separatory funnels.
8.3 Use a portion of the weighed samples as controls, and fortify the
remaining samples by adding 0.5-mL aliquots of 0.1-, 1-, 10-, and
100-ug/mL standard solutions of chlorpyrifos to obtain
113-03 January 1983
-------
concentrations ranging from 0.001 to 1.0 ppm. Prepare each
sample in duplicate.
8.4 Add 25 mL of me:thylene chloride to each sample contained in a
125-mL separatory funnel.
8.5 Shake each sepa.ratory funnel vigorously .by hand for 1 minute,
allow the separatory funnel to stand for 30 seconds with venting,
and again shake vigorously by hand for 1 minute.
8.6 Allow the phases to separate and drain the lower methylene
chloride layer into a 2-oz bottle.
8.7 Repeat steps 8.4, 8.5, and 8.6 three more times using 20, 15, and
10 mL of methylene chloride, and collect each extraction in the
same 2-oz bottle.
8.8 Evaporate the combined extractions to dryness with a gentle jet
of dry air and a water bath at about 50 C.
8.9 Dissolve the residue in 1 mL of acetone and cap the 2-oz bottle
with a Polyseal cap. Shake the bottle to dissolve the residue.
8.10 Chromatograph s. 10-uL aliquot of the acetone solution as
described in Step 7.2.2. (Dilute and re-inject if necessary.)
Measure the height of the peak obtained for chlorpyrifos in terms
of percent full-scale deflection and determine the weight of
chlorpyrifos injected (in nanograms) by reference to a standard
curve derived on the same day (see Step 8.13).
8.11 Correct for blctnk, if any, by subtracting the concentration of
apparent chlorpyrifos in the control sample from that of the
treated sample.
8.12 Calculate perceint recovery of chlorpyrifos from each of the
fortified samples and average the results.
8.13 Chlorpyrifos elutes from the gas chromatography column with a
retention time of about 1.4 minutes. The lower detection limit
of chlorpyrifos is about 0.25 ng. The most accurate calibration
technique is to bracket each sample injection with an injection
of a standard that closely approximates its concentration and
then average the responses of the standard injections to
calculate the concentration of the sample solution.
8.14 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
113-04 January 1983
-------
10.1 Analyze 50-g samples of water from the control and treated
samples as described in Section 8, Steps 8.1 through 8.9.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for GC.
12.2 Fill the GC column with packing with the aid of a slight vacuum.
Place a piece of cheesecloth between the effluent end of the
column and the vacuum hose to hold the packing in the column.
Tap the sides of the column gently until no further settling
occurs. Remove the packing from the first 1/2 inch of each end
of the column with a wire and insert a small glass-wool plug in,
close each end. Condition the column at 230 C overnight with a
nitrogen flow of above 200 mL/min and the 4-port valve in the
vent position.
12.3 Calibrate the system as described in Section 7.
12.4 Inject the sample extract into the GC as described in Step 7.2.2
12.5 Record the peak heights for each sample.
12.6 Typical chromatograms of chlorpyrifos standards and extracts of
unfortified and fortified water are shown in Figures 3 through 5
13. Calculations
13.1 Determine the ppm chlorpyrifos in each treated sample as follows:
i \ i 1 n~3 / \ xFinal Dilution. m!N
(ng)(10 ug/ng)
Gross ppm =
g
Net ppm = gross ppm in sample - gross ppm in control
Apparent ppm -% 0y * 100
14. Method Performance
14.1 Recovery of chlorpyrifos from deionized water fortified over the
range of 0.001 to 1.0 mg/L averaged 92 ±_ 5% at 95% confidence
limits for the mean. See Table 3.
15. References
15.1 Wetters, J.H. and Dishburger, H.J., "Determination of Residue of
0,0-diethyl 0-(3,5,6-trichloro-2-pyridyl) phosphorothioate in
Water by Gas Chromatography with Flame Photometric Detection,"
ACR 71.21, Dow Chemical U.S.A., Midland, MI.
113-05 January 1983
-------
Table 1
Chromatographic Conditions
Column temperature
Injection block temperature
Detector temperature
Carrier gas and make-up gas
Burner gas
Burner gas
Recorder
Electrometer sensitivity
Chart speed
205°C
220°C
215°C
prepurified N2 at 200 mL/min
H at 200 mL/min
0. at 30 mL/min
0-1 mv.
3
input attenuator 10 ,
output attenuator 64.
0.25 in/min.
113-06
January 1983
-------
Table 2
Preparation of Working Standards
Cone, of Standard,
ug/mL
Alq.» ml
Diluted to
Volume, mlt
1000
100
10
10
10
10
10
1
1
10
10
10
5
4
3
2
10
5
100
100
100
100
100
100
100
100
100
Cone, of Solution,
ug/mL
100
10
1
0.5
0.4
0.3
0.2
0.1
0.05
113-07
January 1983
-------
Table 3
Recovery of Chlorpyrifos from Water SampJ.es
Fortified with Known Amounts of the Compound
mg/L (ppm)
Added
mg/L (ppm)
Found
Recovery
0.001
0.001
0.001
0.001
0.01
0.01
0.01
0.01
1.0
1.0
0.00089
0.00093
0.00097
0.00110
0.0085
0.0087
0.0069
0.0089
0.097
0.097
0.092
0.089
1.01
0.94
89
93
97
110
85
87
69
89
97
97
92
89
101
94
92 + 5*
* 95% confidence limits for the mean.
113-08
January 1983
-------
FIGURE 1. EVAPORATION MANIFOLDS
113-09
January 1583
-------
Weight Injected
FIGURE 2. TYPICAL STANDARD CURVE FOR CHLORPYRIFOS.
113-10
January 1983
-------
RECORDER RESPONSE, % F.S.
I I
+44
rr
_LL
I I
*' * j
O» C Ou.
C HI
•H GO
^ 10 O
> o
•H • CO
so z
cr o
u M
8
113-11
January 1983
-------
60r-qF
50
UJ
00
o
Q_
-------
60
50
C/3
LlJ
00
oo
LU
UJ
CJ
£
10
'0
Vi
i±r
TPr
ffif
. i
J.T.
•»-*•
its
r
ruBt
1
.ti
•rri
S
-<
m
^1
01234 5
Deionized Water
0.01 ppm Added
100 mg of Sample
87* Recovery
01
TIME, MINUTES
Deionized Water
0 . 1 ppm Added
33.3 mg of Sample
97% Recovery
01 2345
Deionized Water
1.0 ppm Added
5 mg of Sample
101% Recovery
FIGURE 5. TYPICAL CJIROMATQGRAMS OF WATER FORTIFIED WITH
CHLORPYRIFOS.
113-13
January 1983
-------
oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF COOMAPHOS
IN WASTEWATER
METHOD 114
1. Scope and Application
1.1 This method covers the determination of coumaphos.
Parameter Storet No. CAS No.
Coumaphos 81293 56-72-4
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
2. Summary of Method
2.1 A measured volume of water sample (250 mL) is extracted with
chloroform. The extract is concentrated by rotary evaporation
and analyzed by gas chromatography with alkali flame detector.
3. Interferences
3.1 All glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENTS.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5 . Apparatus and Materials
5.1 Sampling Equipment
Not Available.
114-01 January 1983
-------
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 10-uL
5.2.2 Rotoevaporator, Buchi or equivalent
5.3 Water Bath, 50-55°C
5.4 Gas chromatograph, Varian Model 1400 or equivalent, equipped with
a phosphorus detector, an 18-in x 1/8-in O.D. glass column packed
with 10% DC-200 (12,500 centistokes) on Gas Chrom Q 80/100 mesh,
and glass injector and detector inserts. (If the ends of the
column are long enough to be used as inserts, the glass inserts
are not necessary.) Note: Use only Teflon-backed septums,
Supelco No. 2-0459.
6. Reagents
6.1 Acetone, nanograde
6.2 Chloroform, nanograde
6.3 Hydrochloric acid, IN — Carefully add 21 mL of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.4 Mineral oil solution, 2.5% — Dilute 25 mL of mineral oil, Fisher
#0-120 or equivalent, to 1000 mL with nanograde chloroform, and
mix thoroughly.
6.5 Sodium hydroxide, IN — Carefully dissolve 10 g of sodium
hydroxide pellets, ACS, in 250 mL of distilled water and mix
thoroughly.
6.6 Sodium sulfate, anhydrous, ACS
7. Calibration
7.1 Establish the GC operating parameters equivalent: to those
indicated in Table 1.
7.2 Calibration Procedures
7.2.1 Coumaphos, standard solution, 0.10% — Weigh 0.098-0.105 g
of coumaphos (98% minimum purity) into a 100-mL volumetric
flask. Dilute to volume with acetone, stopper, and mix
thoroughly. This solution may be used for one week only.
7.2.2 Pipet a 1-mL aliquot of the 0.10% coumaphos solution into a
100-mL volumetric flask, dilute to volume with acetone, and
mix thoroughly. Label this solution "10 ng". Prepare fresh
daily.
114-02 January 1983
-------
7.2.3 Pipet 5-, 10- and 20-mL aliquots from the 10-ng standard of
Step 7.2.2 into separate 100-mL volumetric flasks. Dilute
to volume with acetone and mix. Label these solutions 500,
1000, and 2000 pg, respectively. Prepare fresh daily.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately to
Step 10.2.
10.2 Measure 250 mL of the well-mixed sample into a 500-mL separatory
funnel.
10.3 If the pH of the sample is above 7.0 or below 6.5, adjust it to
6.5-7.0 with IN hydrochloric acid or IN sodium hydroxide using pH
indicator paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel.
If an emulsion forms, centrifuge the emulsified layer and add the
clear chloroform layer, obtained by centrifuging, to the second
separatory funnel. Return the water layer to the first
separatory funnel.
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minute.
10.6 Drain the chloroform layer through a funnel containing 4 to 5
g of anhydrous sodium sulfate retained on a small glass-wool plug
(pre-rinsed with 10 mL of chloroform), into a 300-mL 24/40
boiling flask. Rinse the sodium sulfate three times with 10--mL
portions of chloroform and add 10 mL of 2.5% mineral oil solution
into the flask.
10.7 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at 50 C. Remove the flask and
cool to room temperature.
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Pipet 5 mL of acetone into the flask from Step 10.8, stopper
immediately, and rotate the flask so that the acetone washes down
114-03 January 1983
-------
the inside of the flask. DO NOT allow the acetone to get on the
neck or stopper.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Continue the analysis according to TM A-54.12 (Mobay reference
procedure).
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
15.1 "Determination of Coumaphos in Wastewater by GLC," Analytical
Method TM B-34.51, Chemagro Agricultural Division, Mobay Chemical
Corporation, Kansas City, MO.
114-04 January 1983
-------
Table 1
Chromatographic Conditions
Varian 1400
Attenuation X32
Cell voltage Flame
Detector temperature, °C 210-230
-12
Electrometer range 10
Gas flows:
Carrier gas He, 30 mL/min
Air, 230 mL/min
H2, 40 mL/min
Injector temperature 230-250 C
Oven temperature 230 C
Column: 18-in x 1/8-in O.D., glass, packed with 10% DC-200 (12,500
centistokes) on Gas Chrom Q 80/100 mesh.
114-05 January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C, 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CYANAZINE
IN WASTEWATER
METHOD 115
Scope
1.1
and Application
This method covers the determination of
following parameters can be determined
Parameter Storet No.
Cyanazine
Simazine
Fropazine
77780
39055
39024
certain triazines. The
by this method:
CAS No.
21725-46-2
122-34-9
139-40-2
1.2 This is a thin-layer liquid chromatographic (TLC) method
applicable to the determination of the compounds listed above in
wastewater.
1.3 The method detection limit (MDL) is 1 mg/L for each parameter.
2. S'""""TV of Method
2.1 A measured aliquot of water sample (50 mL) is extracted with
chloroform. An aliquot of extract is used for spotting a TLC
plate which is eluted with tetrahydrofuran/ethyl acetate/n-
hexane. The plates are sprayed with AgNO-j solution and developed
by exposure to UV light.
3. Interferences
Not Available.
4. Safety
4.1 Plate spraying and exposure to UV radiation should be carried out
115-01 January 1983
-------
in a hood. Special glasses must be used when working with UV
light. Eyes and skin must be protected from UV radiation.
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Plate: Silica Gel G, neutral (50% Merck Silica Gel G 50%
Bio Rad Bio Sil-A) on 200- x 200-mm glass plates coated at
a thickness of 200 u.
5.2.2 Chamber: Supersaturated (filter paper lining)
Chamber size — 25 cm x 29 cm x 10 cm.
5.2.3 Capillary tube from Kimax, size 1.6-1.8 x 100 mm.
6. Reagents
6.1 Chloroform
6.2 Cyanazine Standard
6.3 Simazine Standard
6.4 Propazine Standard
6.5 Eluent: Tetrahydrofuran/ethylacetate/n-hexane 4%/16%/80% (by
volume)
7. Calibration
7.1 Normal principles of thin layer chromatography apply to this
analysis. TLC conditions are summarized in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 0.0160 g each of propazine, simazine, and cyanazine
of 99+% purity into a 100-mL volumetric flask. Dilute this
stock standard to the mark with chloroform and carry out a
serial dilution to the fifth dilution. These solutions are
equivalent to 32, 16, 8, 4, 2, and 1 mg/L when compared to
samples extracted 5:1.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
115-02 January 1983
-------
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Extract 50 mL of a water sample well with 10 ml of chloroform.
Isolate the chloroform layer for spotting.
11. Cleanup and Separation
Not Available.
12. Thin Layer Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for TLC.
12.2 Calibrate the system as described in Section 7.
12.3 , Perform one application of (7-8 uL) samples and standards using
open-tip capillary tubes. Capillary tubes from Kimax size 1.6-
1.8 x 100 mm.
12.4 After elution, air dry plate to remove solvents. Then spray with
0.02N AgNO, and expose to 1400-watt 366-mn wavelength ultraviolet
light until maximum contrast is achieved. (DANGER — PROTECT
EYES AND SKIN FROM UV RADIATION.) Estimations are made by visual
comparisons between samples and appropriate standards.
12.5 A fresh eluent should be prepared each day and should be allowed
to equilibrate 1 hour before use.
12.6 Fresh standard solutions should be prepared once per month.
13. Calculation
Not Available.
14. Method Performance
14.1 A statement of "not detected" (ND) for a component indicates that
the component is less than 1 mg/L (ppm).
14.2 The standard deviation of an analysis is i 30% relative to actual
concentration.
15. References
15.1 "TLC Determination of Propazine, Simazine, and Cyanazine in
Wastewater," ETM-54-1, June 6, 1977, Ciba-Geigy, AL.
115-03 January 1983
-------
Table 1
Chromatographic Conditions
Temperature Ambient
Distance 15 cm
Time 45 minutes
Eluent Tetrahydrofuran/ethylacetate/n-hexane 4%/16%/80% (by volume)
Plate Silica Gel G, neutral (50% Merck Silica Gel G 50% Bio Rad Bio Sil-A)
on 200- x 200-tnm glass plates coated at a thickness of 200 u.
Chamber Supersaturated (filter paper lining)
Chamber size — 25 cm x 29 cm x 10 cm
115-04 January 1983
-------
Table 2
Typical Separation, Rf
Rf Compound
0.3 Propazine
0.15 Simazine
0.1 Cyanazine
0.0 Point of origin
115-05 January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CYANAZINE [BLADEX(TM)]
AND STIROFOS [TETRACHLORVINPHOS, RABON(TM)]
IN WASTEWATER
METHOD 116
1. Scope and Application
1.1 This method covers the determination of cyanazine and stirofos.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Cyanazine (BLADEX) 77780 21725-46-2
Stirofos
(TetrachlorvinphosXRABON) — 961-11-5
Aldicarb 39053 16-06-3
Atrazine 39153 1912-24-9
1.2 This is a liquid chromatographic (HPLC) method applicable to the
determination of the compounds listed above in aqeuous effluent
samples.
1.3 The method detection limits (MDL) are 10 ug/L Aldicarb, 10 ug/L
tetrachlorvinphos (RABON), 50 ug/L cyanazine (BLADEX), and 50
ug/L Atrazine.
2. SU"""^TY of Method
2.1 A measured volume of water sample (500 mL) is extracted with
methylene chloride. The extract is concentrated to 0.5 mL and
analyzed by liquid chromatography with UV detection.
3. Interferences
Not Available.
116-01 January 1983
-------
4. Safety
4.1 Aldicarb Acetone Solution is highly toxic in contact with the
skin or eyes or if swallowed. Careful handling and special
precautions are essential. Wear face shield, rubber gloves,
rubber apron, and rubber footwear. If spillage occurs, flush
promptly with copious amount of water. Destroy residual amounts
of Aldicarb with 20-percent alcoholic sodium hydroxide. Do not
eat during or after handling the material until after a thorough
cleanup. Destroy all samples promptly after analysis by pouring
them into a container of 20-percent alcoholic sodium hydroxide,
which should be replenished daily.
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.3 Liquid Chromatograph, Varian Aerograph Model 4010-01, equipped
with ultraviolet photometric detector
5.3.1 Column, 25-cm x 1/8-in Micropak CN
5.3.2 Recorder, 1MV
6. Reagents
6.1 Chloroform, reagent grade
6.2 Aldicarb of known purity
6.3 Methylene chloride, pesticide grade
6.4 Heptane, reagent grade
6.5 Cyanazine (BLAD'EX) herbicide of known purity
6.6 Tetrachlorvinphos (BLADEX) insecticide of known purity
6.7 Atrazine of known purity
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 0.05 g Aldicarb, 0.02 g RABON, 0.10 g Cyanazine
(BLADEX) and 0.1 g Atrazine into a 100 mL volumetric flask.
Dilute to mark with methylene chloride. Dilute this
116-02 January 1983
-------
solution 1/10 in 10-mL volumetric flask. This solution
contains 50 ug/mL Aldicarb, 20 ug/mL RABON, 100 ug/mL
Cyanazine (BLADEX), and 100 ug/uL Atrazine.
7.2.2 The standards should be prepared fresh weekly. Sensitivity
could easily be lowered tenfold by adjusting parameters of
the method.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Extract 500 mL of sample according to procedure outlined in MLM
37 (Shell reference method).
10.2 Dilute the extract with 5 mL of methylene chloride and further
concentrate by evaporating to 0.5 mL in a graduated centrifuge
tube with a slow stream of air.
10.3 The chloroform/heptane should be vacuum stripped to remove the
entrained gas before use.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
12.3 Measure the peak height of the RABON, Aldicarb, BLADEX, and
Atrazine peaks and compare to the standard to determine the
amount present.
13. Calculation
13.1 Calculate the amount of each component present as follows:
/r r ^\ (P.H.a) x (ug/mL in std)
ug/L (ppb) - (p^
Where: P.H.a = peak height of sample
P.H.b = peak height of standard
116-03 January 1983
-------
14. Method Performance
14.1 Method detection limits by this method are: Aldicarb - 10 ug/L,
RABON - 10 ug/L, Cyanazine (BLADEX) - 50 ug/L, and Atrazine - 50
ug/L.
15. References
15.1 "Determination of Aldicarb Pesticide, RABON Insecticide, BLADEX
Herbicide and ATRAZINE Herbicide in Aqueous Effluent, LC Method,"
Standard Test Method, Shell, Axis, AL.
116-04 January 1983
-------
Table 1
Chromatographic Conditions
Mobile Phase
Pressure of Helium on mobile phase
Flow of mobile phase
Sample size
Range
Chart Speed
Column
65% Chloroform/35% Heptane
750 psi
1 mL/min.
10 uL
0.04
0.5 inches/min.
25-cm x 1/8-in Micropak CN
116-05
January 1983
-------
>-/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF 2,4-DB
IN WASTEWATER
METHOD 117
1. Scope and Application
1.1 This method covers the determination of certain chlorophenoxy
acids. The following parameters can be determined by this method:
Parameter Storet No. CAS No.
2,4-DB — 94-82-6
2,4-D 39730 94-75-7
MCPB — 94-81-5
MCPA — 94-74-6
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in treated process
and area drainage water.
2. Summary of Method
2.1 A measured volume of water sample (100 mL) is extracted with
ethyl ether and evaporated to near dryness. The extract residue
is esterified and injected into a gas chromatograph with flame
ionization detection.
3. Interferences
3.1 Chlorophenols, solvents, and alcohols are the impurities that
would be extracted if present. Water-soluble organics such as
glycolic acid do not extract and, in addition, also have short
retention times. Isomers of phenoxyalkanoic acid will interfere
with acids of other phenoxyacetic acids in some cases. If this
occurs, different GLC columns will be needed to separate the
isomers.
117-01 January 1983
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4. Safety
4.1 The use of ethyl ether is hazardous, as the material is highly
flammable and the vapors are explosive. Evaporation is performed
in a hood. Acetone is handled with care to prevent getting on
hands (rubber gloves available when using) and inhaling vapors.
Methanol esterification is performed in the hood. Steam baths
are used instead of electrical heating to avoid sparking.
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnels, 250 mL
5.2.2 Erlenmeyer flasks, 250 ml
5.2.3 Glass-stoppered graduates, 10 mL
5.2.4 Syringes with 4-inch needles, 1 mL
5.2.5 Centrifuge tubes, 15 mL
5.3 Hewlett Packard 5710A Gas Chromatograph with Flame lonization
Detectors
5.3.1 Autolab peak area Integrator
5.3.2 0V 101 3% on HMDS Chromosorb S, 1/8-in x 10-ft
6. Reagents
6.1 Standards - Recrystallized acid (3x) from benzene and methylene
chloride
6.2 Sulfuric Acid (ACS) - 10% solution
6.3 Ethyl Ether (ACS)
6.4 0.1 N NaOH
6.5 Distilled water
6.6 Methanol - HC1 esterification reagent. Prepared by adding Acetyl
Chloride slowly to pesticide grade methanol.
6.7 Acetone (ACS)
6.8 Sodium Chloride (table salt grade)
117-02 January 1983
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7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Make up standards by weighing 40 mg of the chlorophenoxy
alkanoic acid in a glass weighing boat and transfer into a
1-liter volumetric flask. Use 5 - 10 mL icetone to rinse
the glass boat into the flask. Add 200 mL of 0.1 N NaOH
and warm on steam bath to dissolve acid(s). Cool and
dilute to volume with 0.1 N NaOH. When 5 mL of this
solution is diluted to 100 mL, the concentration is 2
ug/mL.
7.2.2 Add 95 mL of distilled water to a separatory funnel and 5
mL of the standard solution listed in Step 7.2.1. If area
drainage water is to be analyzed, proceed to Step 7.2.3.
If process water is to be analyzed, add approximately 5
g sodium chloride,shake until in solution, then continue
with Step 7.2.3.
7.2.3 Add 5 mL of 10% H-SO, solution and swirl to mix.
7.2.4 Extract with 35-, 20-, and 20-mL portions of ether. Any
interface is left with the ether layer. Combine ether
layers and interface wash twice with 10 mL of distilled
water, leaving interface with ether, and save drainings.
Pour ether extract into a 250-mL erlenmeyer flask from the
top; interface should adhere to flask. Rinse top of
separatory funnel with 2 - 3 mL of ether back into flask or
into erlenmeyer. Add water wash drainings into separatory
funnel and 10 mL of ether. Shake and drain water. Again
pour ether from the top of separatory funnel into the
erlenmeyer flask.
7.2.5 Put a boiling chip into the erlenmeyer and evaporate ether
on a steam bath slowly. Use bath in a hood. Evaporate to
3 to 4 mL. Add enough salt to form a water-salt mass that
will not flow. With a 1-mL syringe, transfer the ether
layer to 15-mL centrifuge in a warm water bath. Add 2-3
mL of ether via the syringe-to the flask rinsing down the
sides. Swirl to break up salt mass and tip flask so that
salt agglomerates in the one side; tip slowly in the
opposite direction to drain ether. Transfer ether to 15-mL
centrifuge tube. Repeat with another 1 mL of ether.
7.2.6 Evaporate contents of centrifuge tube to near dryness and
add 5 mL of methanol-HCl reagent. Place on steam bath
until HC1 fumes cease to evolve (approximately 10 minutes).
7.2.7 After esterification is complete, transfer to 10-mL
graduate with the 1-mL syringe using acetone to wash down
117-03 January 1983
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sides and rinsing tube. Use enough acetone to bring total
to 10 mL in the graduate. Stopper and, shake.
7.2.8 Inject 4 uL in the GLC at the conditions indicated.
Integrate the peak area. Retention times of the acids are
shown below:
Parameter Seconds
MCPA 290
2,4-D 370
MCPB 655
2,4-DB 825
7.2.9 The uL injection size is equivalent to 0.08 ug of acid.
Record data for calculations with sample.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Measure out 100 mL of well-stirred sample into separatory funnel
(do not filter if there is any suspended material).
10.2 Add 5 mL of 10% H-SO, solution, stopper, and shake. If CO-
evolves when adding I^SO,, swirl without stopper until evolution
ceases. Test solution with pH paper to make sure pH is 1 or
less. Add more 10% H-SO,, if necessary.
10.3 Proceed as in Steps 7.2.4 through 7.2.8.
11. Cleanup and Separation
11.1 Cleanup methods (column type) are not needed as the effluents are
fairly consistent and do not contain much organic matter that
would interfere.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
13. Calculations
117-04 January 1983
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13.1 o p x 2.0 = mg/L phenoxyalkanoic acid in sample.
Standard Peak Area -a r j r
13.2 Calculation is based on the above 2.0 ug/mL (ppm) standards,
solution volumes, and injection sizes. If different, make
appropriate corrections.
14. Method Performance
14.1 The procedure is quantitative to 1 mg/L (ppm). Detectability is
less than 0.5 mg/L (ppm).
15. References
15.1 "Determination of Chlorophenoxy Alkanoic Acids in Plant
Effluent," Standard Test Method, Rhodia, Inc., Portland, OR.
117-05 January 1983
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Table 1
Chromatographic Conditions
Temperature 190°C
Flowrate (Nitrogen) 60 mL/min
(Hydrogen) 15 psig
Electrometer Flange 1, Attenuation 4
Column: 0V 101 3% on HMDS Chromosorb W, 1/8-in x 10-ft
Single Channel Mode
117-06 January 1983
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Table 2
Retention Times
Parameter Retention Time (seconds)
MCPA 290
2,4-D 370
MCPB 655
2,4-DB 825
117-07 January 1983
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SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF DEBT IN WASTEWATER
METHOD 118
1• Scope and Application
1.1 This method covers the determination of Deet.
Parameter Storet No. CAS No.
DEET ~ 134-62-3
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
2. Summary of Method
2.1 The sample is extracted with 15% methylene chloride in hexane.
The extract is concentrated under a slow nitrogen stream to a
final volume of 5 mL. Analysis is by GC-FID.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Wide-mouth glass jars such as the Mason type are recommended as
suitable sample containers when the sample is to be 2 L or less.
If the sample is of greater volume than 2 L, the 1-gallon glass
bottles in which acetone, hexane, or petroleum ether are normally
sold provide excellent sample containers. Furthermore, the
latter require no special precleaning before use. The glass
containers must be scrupulously cleaned and rinsed with some of
118-01 January 1983
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the same solvent used for subsequent pesticide extraction. All
bottle or jar caps should be Teflon- or foil-lined to prevent
contamination of the sample with trace quantities of impurities
which may be present in laminated paper liners or in the
composition of the material used for the seal in Mason jar lids.
5.2 Glassware and Other Equipment
5.2.1 Modified micro-Snyder Columns, 19/22, Kontes stock #K-
569251.
5.2.2 Glass bead, 3-mm plain, Fisher #11-312 or equivalent.
5.2.3 Modified micro-Snyder column, 19/22 I joint, Kontes #K-
569251.
5.2.4 Pipet, 4 mL.
5.2.5 Separatory funnels, 2 L with Teflon stopcocks.
5.2.6 Filter tubes, 150 x 24 mm, Corning 9480 or equivalent.
5.2.7 Kuderna-Danish concentrator fitted with graduated
evaporative concentrator tube. These are available from
the Kontes Glass Company, each component bearing the
following stock numbers.
5.2.7.1 Flask, 500 mL, stock #K-570001
5.2.7.2 Snyder column 3-ball, stock #K503000
5.2.7.3 Steel springs, 1/2-in stock #K-662750
5.2.7.4 Concentrator tubes, 10 mL, Size 1025, stock
#K580050
5.3 Water bath capable of maintaining 95° to 100°C.
5.4 Gas Chromatograph fitted with electron capture, flame
photometric, and electrolytic conductivity detectors. GLC
columns to be two of the three specified in Step 12.6, with all
operating parameters as specified in Step 12.6.
5.4.1 Chromatographic columns, 22-mm I.D. x 300-mm length, with
Teflon stopcock, without frits.
6. Reagents
6.1 Hexane, pesticide quality, distilled in glass.
6.2 Isooctane, pesticide quality.
6.3 Diethyl ether, AR grade, peroxide free. The ether must contain
2% (v/v) absolute ethanol. Most of the AR grade ethyl ether
contains 2% ethanol, added as a stabilizer, and it is therefore
118-02 January 1983
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unnecessary to add ethanol unless it is found necessary to remove
peroxides. The ether should be tested for the absence of
peroxides by a suitable method.
6.4 Petroleum ether, pesticide quality, redistributed in glass, b.p.
30-60°C.
6.5 Methylene chloride, pesticide quality.
6.6 Methylene chloride/hexane, 15% v/v.
6.7 Eluting mixture (6%), 60 mL of diethyl ether is diluted to 1000
mL with petroleum ether, and approximately 15 g of anhydrous
Na-SO, is added to insure freedom from moisture.
6.8 Eluting mixture (15%), 150 mL of diethyl ether is diluted to 1000
mL with petroleum ether, and approximately 15 g anhydrous Na2SO,
is added.
6.9 Eluting mixture (50%), 500 mL of diethyl ether is diluted to 1000
mL with petroleum ether, and approximately 15 grams anhydrous
Na-SO, is added.
Note: None of the eluting mixtures should be held longer than 24
hours after mixing.
6.10 Anhydrous sodium sulfate, reagent grade, granular, Mallinkrodt
stock #8024, or equivalent. Test for purity.
6.11 Florisil, 60/100 mesh, PR grade.
Calibration
Not Available.
Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
Sample Collection. Preservation, and Handling
9.1 The sampling location and the method of drawing the sample will,
to a great extent, be dictated by the objectives of the sample
data. If the objective is to determine the highest pesticide
pollution present in a stream or lake, a grab sample might be
drawn at the point of highest pollution introduction. If, on the
other hand, the objective is an average residue profile of the
entire body of water, the final sample would preferably be a
composite of a number of subsamples taken at various locations
and water depths.
9.2 As implied by the name, a grab sample would be a surface water
sample generally taken by simply filling the sample container by
118-03 January 1983
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immersing and allowing the bottle or jar to fill up. For
sampling at selected depths, devices such as a Precision sewage
water sampler or an Esmarch sampler may be utilized. Both
devices consist of a metal outer container with a glass bottle
inside as the sample collection vessel.
9.3 The Precision sampler, in which the interior of the collection
bottle has free access to the exterior by means of an open tube,
can be used to draw a composite depth sample. As soon as the
device if immersed, collection of the sample is started. By
premeasuring the rate of lowering the device to collect a given
amount of water, an approximately uniform amount of water can be
collected throughout the entire depth sampled.
9.4 The Esmarch sampler may be manually opened and closed by means of
a chain attached to the bottle stopper. This permits a sample or
subsample to be drawn from any given depth simply by lowering the
device with the stopper closed, opening it at the proper sampling
depth to permit filling of the collection bottle, then closing
the stopper and raising the device to the surface.
9.5 The size of sample is dictated primarily by the expected residue
levels. For example, if the sample is collected from a waterway
where pesticide levels are expectedly high (such as agricultural
run-off), a sample size of 500 to 1,000 mL may be sufficient. If
the sample is drawn in connection with a monitoring program where
no especially high residues would be expected, a sample size of 2
L or more may be indicated.
9.6 Ideally, analysis of the sample should be conducted within a
matter of hours from the time of sampling. However, this is
frequently impractical in terms of the distance from sampling
site to laboratory, and/or the laboratory workload. Samples
being examined solely for organochlorine residues may be held up
to a week under refrigeration at 2 to 4 C. Those intended for
organophosphorous or carbamate analysis should be frozen
immediately after drawing sample and should be extracted no more
than 4 days after sampling. These classes of pesticides undergo
degradation rapidly in the aqueous medium.
9.7 Every effort should be made to perform the solvent extraction
step at the earliest possible time after sampling, irrespective
of the class of pesticides suspected as being present. The
resulting extracts may then be held for periods of up to three or
four weeks at -15 to -20 C before conducting the adsorbent
partitioning and determinative portions of the analysis (see Table
1 of reference 15.5, not enclosed herein). These data show the
degradation rate of 29 pesticides in water at ambient temperature
in sealed containers.
9.8 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
118-04 January 1983
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10.1 Transfer 2 L of sample (or a lesser volume, if indicated) to a 4-
L separatory funnel and add 120 mL of 15% methylene chloride/
hexane.
Notes: 1. If, on the basis of prior analysis of a given
waterway, the residue levels may be expected to run
high, a sample of 500 mL or 1 L may be indicated. In
this event, the size of the separatory funnel should
be 2 L and the extraction solvent volumes given as
120 mL should be redrced to 100 mL.
2. A 500-mL graduated cylinder is a suitable measuring
device for the initial sample. Any measuring
discrepancy up to 5.0 mL would result in an error no
greater than 1.0%.
10.2 Stopper funnel and shake vigorously 2 minutes. Allow layers to
separate and draw off aqueous layer into a second 2 L separatory
funne1.
10.3 Add another 120 mL of 15% methylene chloride/hexane to the
aqueous phase in separatory funnel #2, stopper, and shake
vigorously another 2 minutes.
10.4 Prepare a 2-inch column of anhydrous, granular Na^SO, in a 150-x
24-mm filter tube with a small wad of pre-extracted glass-wool at
the bottom. Position this over a 500-mL K-D flask to which is
attached a 10-mL concentrator tube with one 3-mm glass bead in
the bottom.
10.5 Filter the methylene chloride/hexane extract in separatory funnel
#1 through the Na2SO, column into the flask.
10.6 Draw off the aqueous layer in separatory funnel #2 into empty
separatory funnel #1.
10.7 Add 120 mL of straight hexane to the aqueous solution in
separatory funnel #1, stopper, and shake again for 2 minutes.
Draw off and discard the aqueous layer.
10.8 Filter the solvent extracts in both separatory funnels through
the Na.SO, into the flask, rinsing down the filter tube with
three 10-mL portions of hexane.
10.9 Attach a 3-ball Snyder column to the K-D flask, place assembly in
a boiling water bath, and concentrate extract to approximately 5
mL.
10.10 Remove K-D assembly from bath, cool, and rinse I joint between
tube and flask with a small volume of hexane; also rinse down
walls of tube. Rinse should be delivered with 2-mL Mohr pipet
and should not exceed 3 mL.
118-05 January 1983
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10.11 Place tube under a slow nitrogen stream at ambient temperature
and reduce extract volume to approximately 0.5 ml. Using a
disposable pipet, carefuly add hexane to adjust volume to exactly
1.0 mL in the tube tip. Then, with a 4-mL pipet, add 4 mL of
hexane. DO NOT rely on the accuracy of the tube graduation at
the 5-mL mark.
10.12 Stopper concentrator tube and mix vigorously on Vortex mixer for
1 minute.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 The chromatographic approach should be made in accordance with
the guidelines presented in Steps 12.6.1 through 12.6.6, and by
applying the instrumental operating parameters specified for the
respective GLC columns listed. By following the given protocol,
it should be possible to make some tentative compound
identification upon computation of relative retention times
(RRT.) of peaks in the preliminary chromatograms via electron
A
capture.
12.2 Full reliance should not be placed on the chromatographic data
obtained from one column. An alternate column of completely
different compound elution characteristics should be used to (1)
confirm a number of compounds tentatively identified on the first
column, and (2) isolate and tentatively identify any compound
pairs which may have eluted as single peaks on the first column.
Subsequent quantitations are conducted as outlined in Section
12.6.
Note: If the initial chromatogram indicates the presence of a
sufficient amount of interfering materials, it may prove
necessary to conduct a Florisil cleanup on the extract.
Based on the general experience of water chemists, this is
rarely necessary on most surface water samples. If it
should prove necessary, the cleanup should be carried out
as prescribed in Section 5, A (1), pages 6-9 of Reference
15.5, not enclosed herein. After the cleanup, another
exploratory injection is made followed by peak
identifications and quantitation.
12.3 If the electron capture data indicate the probable presence of
one or more chlorinated pesticide compounds, the chromatographer
would be well advised to conduct confirmation via electrolytic
conductivity detection in the reductive mode, even though
positive identifications were made on two columns via electron
capture. This extra step provides needed validation,
particularly when compounds are tentatively identified which
appear to be out of place in light of known supplemental data
concerning the: waterway sampled.
118-06 January 1983
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12.4 It is improbable that parent compounds in the organophosphorous
class will be detected in an average water sample. Compound
degradation is rather rapid in the aqueous medium. However, if
the waterway received heavy run-off from nearby agricultural land
undergoing current spray programs, the presence of these
residuals is possible.
12.5 In general, many of the organophosphorous compounds are f-ar less
responsive to electron capture detection than compounds in the
organochlorine glass. This factor, combined with their highly
diluted concentration in a waterway, make electron capture
detection a dubious matter. Therefore, specific detection is
preferable for identification and quantitation of these compounds
or their metabolites. Flame photometric detection provides a
suitable mode for this mission. Guidelines for the use of this
detector are provided in Step 12.6. It will undoubtedly be
necessary to use the original 5-mL extract (undiluted) because of
the expectedly low concentrations. In fact, it may prove
necessary to concentrate the extract even further if a
preliminary chromatogram via FPD gives any indication of peaks
that may be of insufficient size to meet the response criteria
given in Step 12.6.
12.6 Gas Chromatography-Electron Capture Columns
12.6.1 Column material shall be of borosilicate glass, 6-
ft long, 1/4-in O.D., 5/32-in I.D. As off-column
injection will be used, one side of the column shall
be 1 inch longer than the other. The Swagelok nut,
ferrule, and silicone "0" ring are assembled as in
Section 4, A, (6) of Ref. 15.5, not enclosed herein.
Complete column specifications for the Tracer MT-220
gas chromatograph are given in Section 4, A, (6) of
Ref. 15.5, not enclosed herein.
12.6.2 Column Selection — There is a wide variety of column
packing materials in the marketplace, some of which
are entirely suitable for use in pesticide analysis,
and others which are of limited value. In general,
the columns selected as a "working pair" should be
significantly different in polarity and in their
compound elution characteristics. One pair that has
proved very useful is given as A and C of Section 4,
A, (6) of Ref. 15.5, not enclosed herein. B provides
another alternate. The peak elution patterns for 13
chlorinated pesticidal compounds on each of these
columns are shown in Figures 1 through 3 of Section
4, A, (6) of Ref. 15.5, not enclosed herein.
12.6.2.1 1.5% OV-17/1.95% 0V -210 — Liquid phases
premixed and coated on silanized support,
80/100 mesh.
12.6.2.2 4% SE-30/6% OV-210 — Liquid phases premixed
and coated on silanized support, 80/100 mesh.
118-07 January 1983
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12.6.2.3 5% OV-210 — coated on silanized support,
100/120 mesh.
12.6.3 Packing the Column:
12.6.3.1 Make certain the column is actually 6 ft long.
A paper template tacked to the wall is a
convenient and quick means of checking. For
off-column injection in the Micro-Tek Model
220, one column leg should be 1 inch shorter
than the other.
12.6.3.2 With a china marking pencil, place a mark on
the long column leg 2 in from the end. Place a
similar mark 1-1/8 inch from the end of the
short leg.
12.6.3.3 Add the packing to the column through a small
funnel, approximately 6 in at a time, and
bounce the column repeatedly on a semihard
surface. Rapid tapping up and down the column
with a wooden pencil will promote settling of
the packing. The packing is added until it
reaches the mark on each leg and it is found
that additional tapping will not produce any
further settling.
Note: This operation should be done with great
care, tapping the column a sufficient
length of time to be certain that no
further settling is possible by manual
vibration. The use of mechanical
vibrators is not advised as the packing
can be packed too densely, thus
introducing the possibility of an
excessive pressure drop when carrier gas
is applied.
12.6.3.4 Pack silanized glass wool into both ends of the
column just tightly enough to prevent
dislodging the carrier flow. The glass wool
should completely fill the space from the top
of the packing to the end of the column.
Note: If the glass wool is manipulated by
hand, the hands should be carefully
prewashed with soap or detergent, rinsed
and dried. This minimizes the
possibility of skin oil contamination of
the glass wool.
12.6.4 Column Conditioning — The column is conditioned, or
made ready for use, in two operations: (1) by heat
curing, and (2) by silylation treatment.
118-08 January 1983
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12.6.4.1 Heat Curing
12.6.4.1.1 A Swagelok fitting is attached to the
inlet port at the top of the oven.
This is comprised of a 1/4-inch
Swagelok to AN adapter, part number
400-A-4ANF, connected to a 1/4-inch
male union, part number 400-6.
12.6.4.1.2 Before assembling, the bore of the
union must be drilled out with a 1/4-
in drill and burnished with a rat-
tailed file so that it will accept the
1/4-in O.D. column glass.
12.6.4.1.3 The short column leg is attached to the
fitting, with the end of the long leg
venting inside the oven. The nut,
ferrule, and "0" ring are assembled as
shown in Fig. 4 of Section 4, A, (6) of
Ref. 15.5, not enclosed herein. Make
sure the nut is tight, as the "0" ring
will shrink during the curing period,
thus allowing carrier gas to escape.
Note: The outset ports leading to the
transfer line should be sealed
off during the conditioning
period to prevent traces of
column effluent from seeping
through to the detector. This
is easily done by assembling a
1/4-inch Swagelok nut on a short
piece of 6-mm glass rod with
ferrule and "0" ring.
12.6.4.2 Silylating Treatment
12.6.4.2.1 Treatment with a silylating compound
such as Silyl 8 serves to block active
absorption sites, particularly
prevalent in a new column, thereby
somewhat improving efficiency and
resolution characteristics. The most
drastic effect is in the improvement of
endrin response and the near
elimination of on-column breakdown c f
endrin. Silyl 8 is available in 1- and
25-mL septum capped bottles from the
Pierce Chemical Company, P.O. Box 117,
Rockford, IL 61105.
12.6.4.2.2 At the end of the prescribed heat
curing period, adjust the oven tempset
and carrier gas flow controllers to the
appropriate settings to give the
118-09 January 1983
-------
approximate recommended operating
parameters for the given column. While
the temperature is dropping, open the
oven door and, wearing heavy gloves,
retighten the Swagelok nut which will
invariably loosen during heat curing.
Close the door and allow oven
temperature to equilibrate. Make four
consecutive injections of 25 uL each of
Silyl 8, spacing the injections
approximately 1/2 hour apart. Allow at
least three hours for the final
injection to elute off the column
before proceeding.
Notes: Syringe used for Silyl 8
injection should be used for no
other purpose and should be
flushed with benzene
immediately after use to avoid
plugging of the needle.
It is strongly advised that
Silyl 8 be discarded after one
year and that fresh material be
ordered; some troublesome side
effects have been noted in
electon capture GLC arising
from the use of old Silyl 8.
13. Calculation
Not Available.
14. Confirmation
14.1 It is assumed that final TLC and electrolytic conductivity
confirmation may be applied to supplement the information
obtained by electron capture detection. For this reason a larger
sample is used than would be necessary for electron capture
alone. Dilution of an aliquot of the final extract for electron
capture — GLC requires far less time than the extraction of
another sample for confirmatory purposes.
15. References
15.1 FEDERAL REGISTER, Vol 38, No. 125, 29 June 1973, Part II.
15.2 Manual of "Method for Organic Pesticides in Water and
Wastewater," 1971, EPA, NERC, Cincinnati, OH.
15.3 Eichelberger, J.W. and Lichtenberg, J.J., Persistence of
Pesticides in River Water, Envir. Sci. & Technol., 5_, No. 6, June
1971 (Table 1),,
118-10 January 1983
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15.4 "The Sampling and Analysis of. Water for Pesticides," Standard
Test Method, Hardwicke Chemical Company, Elgin, SC.
15.5 Manual of "Analytical Methods for the Analysis of Pesticide
Residues in Human and Environmental Samples," Dec. 1974, U.S.
Environmental Protection Agency, Environmental Toxicology
Division, Research Triangle Park, NC 27711.
118-11 January 1983
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oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MEVINPHOS [PHOSDRIN(TM)], DICHLORVOS [VAPONA(TM)],
NALED [DIBROM(TM)], AND STIROFOS [RABON(TM)] IN WASTEWATER
METHOD 119
1. Scope and Application
1.1 This method covers the determination of certain organophosphate
pesticides. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Stirofos [RABON(TM)] — 961-11-5
Naled [DIBROM(TM)] — 300-76-5
Mevinphos [PHOSDRIN(TM)] 39610 7786-34-7
Dichlorvos [VAPONA(TM)] — 62-73-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in wastewater.
1.3 The method detection limits (MDL) are listed in Table 1.
2. Summary of Method
2.1 A measured volume of water sample (100 mL) is passed through a
C.g-Sep Pak. The Sep Pak is subsequently eluted with ether and
analyzed after concentration to 1 mL. An optional cleanup is
effected by diluting the ether eluent with hexane and passing it
through a Silica Sep Pak, followed by ether elution and
concentration. Analysis is by gas chromatography with flame
ionization detector.
2.2 This method provides an optional cleanup procedure to aid in the
elimination of interferences which may be encountered.
119-01 January 1983
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3. Interferences
3.1 All glassware must be scrupulously clean to avoid contamination.
4. Safety
4.1 Ether, acetone, and hexane are extremely flammable. Use in well
ventilated area. Keep away from all flames and sources of
ignition. Use methylene chloride in a well ventilated area.
4.2 VAPONA, PHOSDRIN, and DIBROM are extremely toxic insecticides.
Extreme care must be used when handling these insecticides. See
Material Safety Data Sheet for handling instructions, not
enclosed herein. Impervious gloves must be worn.
4.3 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Class A pipets, 1 ml and 10 mL
5.2.2 Volumetric flasks, 100 mL and 1 L
5.2.3 Centrifuge tubes (10 mL) graduated in 0.1-mL increments
5.2.4 Glass syringes, 5, 10 and 100 mL
5.2.5 Hamilton syringe, 10 uL
5.3 C10 and Silica Sep-Pak cartridges
lo
5.4 Gas Chromatograph, Varian 3700 or equivalent, equipped with a
flame photometric detector with phosphorous filter
5.4.1 Chromatographic column, 1/4-in x 6-ft glass tubing packed
with 1:1 mixture of 3% OV-225 on 100/120 Gas Chrom Q and 3%
QF-1 on 80/100 Gas Chrom Q, or equivalent.
5.4.2 Data System, Sigma 10, or suitable equivalent or strip
chart recorder.
6. Reagents
6.1 Ether, reagent grade, ACS
6.2 Hexane, distilled in glass, suitable for gas chromatography and
residue analysis
119-02 January 1983
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6.3 Sodium sulfate, anhydrous, reagent ACS
6.4 Dichlorvos (VAPONA Insecticide), analytical standard of known
purity
6.5 Naled (DIBROM Insecticide), analytical standard of known purity
6.6 Stirofos (RABON Insecticide), analytical standard of known
purity
6.7 Mevinphos (PHOSDRIN Insecticide), analytical standard of known
purity
6.8 Acetone, distilled in glass, suitable for gas chromatography and
residue analysis
6.9 Methylene chloride, distilled in glass, suitable for gas
chromatography and residue analysis
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 2.
7.2 Calibration Procedure
7.2.1 Standard Stock Solution: Weigh to the nearest 0.1 mg,
approximately 0.5 g each of VAPONA, PHOSDRIN, DIBROM, AND
RABON into a 100-mL volumetric flask. Dilute to the mark
with methylene chloride and mix well. Note: If other
pesticides are not to be analyzed by liquid chromatography,
acetone may be used as the solvent in this step. Correct
for the purity and calculate the concentration of each
component in mg/mL. (This solution will contain
approximately 5 mg/mL of each insecticide.)
7.2.2 Intermediate Standard Solution: Pipet 1.0 ml of the
Standard Stock Solution into a 100-mL volumetric flask.
Dilute to the mark with hexane and mix well. Calculate the
concentration of each component in mg/mL. (This solution
will contain approximately 0.05 mg/mL of each insecticide.)
7.2.3 Working Standard Solution: Pipet 10.0 mL of the
Intermediate Standard Solution into a 100-mL volumetric
flask. Dilute to the mark with hexane and mix well.
Calculate the concentation of each component in ug/mL to
the nearest 0.2 ug/mL. (This solution will contain
approximately 5 ug/mL of each insecticide.)
7.2.4 Inject 5> uL of the VAPONA, PHOSDRIN, DIBROM, and RABON
Working Standard Solution onto the gas chromatograph under
the chromatographic conditions shown in Table 2.
Note: All standard solutions should be prepared fresh
monthly.
119-03 January 1983
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7.2.5 Allow the chromatogram to develop. Figure 1 is a typical
chromatogram.
7.2.6 If a reporting integrator is used, calibrate the integrator
for each component by entering 10 times its actual ug/mL
value determined in Step 7.2.3. Example: 5.3 ug/mL x 10 =
53. This corrects for concentrating the sample 100 times,
and the reporting integrator will now report values in
actual ug/L (ppb) concentration.
7.2.7 Inject 5 uL of the 1.0-mL ether concentrate and allow
chromatogram to develop.
8. Quality Control
8.1 Spike Stock Solution: Weigh, to the nearest 0.1 mg,
approximately 0.5 g each of VAPONA, PHOSDRIN, DIBROM, and RABON
into a 100-mL volumetric flask. Dilute to the mark with acetone.
Correct the concentration for purity.
8.2 Spike Working Solution: Pipet 1.0 mL of the Spike Stock Solution
into a 100-mL volumetric flask and dilute to the mark with
acetone.
8.3 Spiked Sample: Pipet 1.0 mL of the Spike Working Solution into a
1-liter volumetric flask and dilute to the mark wtih the sample.
This spiked sample contains approximately 50 mg/mL of each spike
and should be handled exactly as the unspiked sample.
8.4 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Clean each C.R Sep Pak by eluting 10 mL of reagent grade acetone
through the Sep Pak and then follow with 20 mL of water to
activate the Sep Pak.
10.2 Attach the C Sep Pak to a 100-mL graduated glass syringe. Pour
J.O
100 mL of sample into the syringe. Attach to a vacuum and slowly
pull the sample; through the C „ Sep Pak. Discard the eluate.
Place the C Sep Pak on vacuum for 5 to 10 minutes to remove as
lo
much water as possible.
Note: If the sample is extremely "dirty," use the Silica Sep Pak
for further clean-up using the procedure in Section 11.
(This procedure is usually not necessary.) Otherwise
continue with Section 10.3.
119-04 January 1983
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10.3 Elute off the pesticides with 5 mL of ether into a 10 mL-
graduated centrifuge tube.
10.4 Using a heating block, concentrate the 5 mL of ether eluate to
1.0 mL with low heat and a gentle stream of air. The original
sample has now been concentrated 100 times and is ready for
analysis by GC.
11. Cleanup and Separation
11.1 Clean each Silica Sep Pak by eluting 10 mL of reagent grade ether
through the Sep Pak and then follow with 20 mL of reagent grade
dry hexane to activate the Sep Pak.
11.2 Using the C^_ Sep Pak prepared in Step 10.2 which contains the
adsorbed sample, elute off the pesticides with 5 mL of ether into
a 100-mL stoppered graduated cylinder.
11.3 Add approximately 95 mL of dry hexane and 2 to 3 g of Na2SO,.
Shake well.
11.4 Attach the Silica Sep Pak to a 100-mL graduated glass syringe and
pour the 100-mL hexane/ether solution into the syringe. Attach to
vacuum and slowly pull the sample through. Discard the eluate.
11.5 Continue as written in Step 10.3.
12. Gas Chromatographv
12.1 Table 2 summarizes the recommended operating conditions for the
liquid chromatograph. Included in Table 1 are the estimated
retention times and method detection limits.
12.2 Calibrate the system as described in Section 7.
13. Calculations
13.1 If a reporting integrator is used, ug/L (ppb) concentration may
be read directly from the report.
If only a strip recorder is available and peaks are well
chromatographed, measure peak heights (PH) to the nearest
millimeter and calculate as below:
pesticide, ng/mL =
PH of component in sample „ ug of component in std ., 1.000 ng „ 1 mL
PH of component in std mL 1 ug 100 mL
or
pesticide, ug/L (ppb) =
119-05 January 1983
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PH of component in sample v 1n _ • T , . .
^r— c —: —,— X 10 X ug/mL of component in std
PH of component in std e r
14. Method Performance
14.1 Detection limits by this method are:
PHOSDRIN - 2 ug/L, VAPONA - 2 ug/L, DIBROM - 10 ug/L, and
RABON - 2 ug/L.
14.2 Other ranges of sensitivity may be determined by this method by
varying concentrations of standards and instrument parameters.
14.3 Limited accuracy and precision data are available for this
method.
Plant effluent was spiked and analyzed on ten different days to
indicate method accuracy. Results are presented in Table 3.
14.4 Low recovery of VAPONA is expected because of hydrolysis. Low
recovery of DIBROM can be attributed to both hydrolysis and
thermal degradation in the injection port.
Plant effluent was spiked and analyzed on three different days in
duplicate by two technicians to indicate method precision.
Results are presented in Table 3.
14.5 Generally, duplicate analyses should agree within 15% of the mean
value.
15. References
(n\ (n) (o) (D)
15.1 "Determination of Phosdrin , Vapona , Dibrom , and Rabon *"'
Insecticides in Aqueous Effluent, Gas Chromatograph (GC) - Flame
Photometric Detector (FPD) Method," Standard Test Method, Shell
Chemical Company, Axis, AL.
119-06 January 1983
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Table 1
Retention Times and Detection Lmits
Parameter
stirof os
naled
mevinphos
dichlorvos
Retention Time (min)
12.5
7.65
6.02
1.84
Detection Limit
2
10
2
2
(ue/L)
119-07 January 1983
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Table 2
Chromatographic Conditions
Injection Port Temperature
Initial Oven Temperature, Time
Final Temperature, Rate, Time
Detector Temperature
Hydrogen Flow Rate
Air #1 Flow Rate
Air #2 Flow Rate
Helium Flow Rate
Range
Attenuation
Recorder Speed
Recorder Attenuation
150U
120°C, 3 min.
230°C, 10°C/min, 10 min.
250°C
140 mL/min
80 mL/min
170 mL/min
70 mL/min
io-9
8
0.5 cm/min
as required for optimum signal
Column: 1/4-in x 6-ft glass packed with 1:1 mixture of 3% OV-225 on 100/120
Gas Chrom Q and 3% QF-1 on 80/100 Gas Chrom Q.
119-08
January 1983
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Table 3
Method Accuracy
Pesticide
dichlorvos
mevinphos
naled
stirophos
Spike
ug/L
54
50
49
50
Mean Recovery
%
79.0
93.7
40.5
92.4
Standard
Deviation
7.3
4.9
15.0
8.5
Method Precision
ug/L
Pesticide
dichlorvos
mevinphos
naled
stirophos
Sample #1 Sample #2 Sample #3
Determination Determination Determination
#1 #2 #1 #2 #1 #2
51
36
11
15
49 47
41 53
11 <10
15 42
51 48
53 49
<10 12
48 39
54
50
14
43
119-09
January 1983
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FIGURE 1
PESTICIDE STANDARD
PEPIKIN EI. Mi.:n
I-..
§
VI
O
a
•H
M
T)
V)
O
£.
Du
O
3
TIME
119-10
January 1983
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&EPA
United States . Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MEVINPHOS [PHOSDRIN(TM)], DICHLORVOS [VAPONA(TM)],
NALED [DIBROM(TM)], AND STIROFOS [GARDONA(TM)]
IN WASTEWATER
METHOD 120
1. Scope and Application
1.1 This method covers the determination of certain organophosphorus
pesticides. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Dichlorvos [VAPONA(TM)] — 62-73-7
Mevinphos [PHOSDRIN(TM)] 39610 7786-34-7
Naled [DIBROM(TM)] — 300-76-5
stirofos [GARDONA(TM)] — 961-11-5
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in aqueous effluent.
1.3 The method detection limits (MDL) for many of the parameters are
listed in Table 1.
2. Summary of Method
2.1 The organic extractables are dissolved in 5 ml of methylene
chloride. The sample is concentrated by reducing the volume to
0.5 mL by use of a slow stream of air.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
120-01 January 1983
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5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Hamilton 10-uL syringe.
5.3 Gas chromatographic, Tracor Model 550 or equivalent, equipped
with a flame photometric detector with phosphorous filter.
5.3.1 Chromatographic column, 1/4-in x 4-ft glass tubing packed
with 3% QF-1 on Chromosorb W, AW-DMCS treated.
6. Reagents
6.1 Methylene Chloride, Reagent grade
6.2 Dichlorvos (VAPONA) Insecticide, known quality
6.3 Naled (DIBROM) Insecticide, known quality
6.4 Stirophos (GARDONA) Insecticide, known quality
6.5 Mevinphos (PHOSDRIN) Insecticide, known quality
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 2.
7.2 Calibration Procedure
7.2.1 Weigh 0.025 g Dichlorvos (VAPONA), 0.020 g Stirofos
(GARDONA), 0.50 g Naled (DIBROM), and 0.20 g Mevinphos
(PHOSDRIN) into a 10-mL volumetric flask and dilute to the
mark with methylene chloride.
7.2.2 Pipet 0.1 mL of this stock solution into another 10-mL
volumetric flask and dilute to the mark with methylene
chloride. This solution contains 25 ug/mL Dichlorvos, 20
ug/mL Stirofos, 500 ug/mL Naled, and 200 ug/mL Mevinphos.
7.2.3 Inject 2 uL of the standard solution onto the gas
chromatograph column.
7.2.4 Determine peak height in centimeters of the standards.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
120-02 January 1983
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9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 After determining the organic extractables as outlined in MLM 37
(Shell reference method), dissolve the extractables in 5 mL of
methylene chloride. Further concentrate the sample by reducing
the volume to 0.5 mL in a graduated centrifuge tube by use of a
slow stream of air.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 2 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Inject 2 uL of the concentrate onto the gas chromatograph column.
12.4 Measure peak height in centimeters of components in sample.
13. Calculations
13.1 Calculate the amount of each insecticide present as follows:
Insecticide, ug/L =
(Peak height of component in sample)(ug/mL component in standard)
(Peak height of component in Standard)
14. Method Performance
14.1 Other ranges of sensitivity may be determined by this method by
varying concentrations of standards and instrument parameters.
14.2 Detection Limits by this method are : PHOSDRIN-100 ug/L, VAPONA-
10 ug/L, DIBROM-100 ug/L, and GARDONA-10 ug/L.
15. References
(R) (R) (R)
15.1 "Determination of Phosdrin , Vapona , Dibrom , and
(R)
Gardona Insecticides in Aqueous Effluents, GC-FPD Method,"
Standard Test Method, Shell Co., Axis, AL.
120-03 January 1983
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Table 1
Detection Limits
Method Detection
Parameter Limit (ue/L)
Dichlorvos (VAPONA) 10
Mevinphos (PHOSDRIN) 100
Naled (DIBROM) 100
Stirophos (Tetrachlorvinphos)(GARDONA) 10
120-04 January 1983
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Table 2
Chromatographic Conditions
Injection Port Temperature 250
Oven Temperature 140°C - 200°C (after elution of DIBROM)
Detector Temperature 250 C
Hydrogen Flow Rate 28 mL/min
Nitrogen Flow Rate 70 mL/min
Air Flow Rate 180 mL/min
Attenuation 32 x 10
Recorder Speed 0.5 in/min
Column: 1/4-in x 4-ft glass packed with 3% QB-1 on Chromosorb W, AW-DMCS
treated.
120-05 January 1983
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FIGURE 1
PESTICIDES STANDARDS
Q
Z
120-06
January 1983
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v>EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF DINOSEB (2-SEC-BUTYL-4,6-DINITROPHENOL)
IN WASTEWATER
METHOD 121
1. Scope and Application
1.1 This method covers the determination of dinoseb (DNBP).
Parameter Storet No. CAS No.
Dinoseb — 88-85-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in water.
2. Summary of Method
2.1 Acidified wastewater samples are extracted with diethyl ether,
and the extract is cleaned up on alumina. The aqueous eluate
from the alumina column is re-acidified and re-extracted with
diethyl ether before derivatization with diazomethane. The
derivatized extract is evaporated to dryness, dissolved in 1 ml
of hexane, and cleaned up on an acidic alumina column. The
column is eluted with diethyl ether, and the eluate is analyzed
by GC with electron capture detector.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3. Interferences
Not Available.
4. Safety
4.1 Diazomethane is toxic, can cause specific sensitivity, and is
potentially explosive. Do not breathe vapor or expose to skin
or eyes. Preparation of diazomethane reagent should be
121-01 January 1983
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performed in a hood. Ground glass joints and sharp edges should
be avoided. Thus, all connections should be made with rubber
stoppers, glass tubing should be fire polished, and etched or
scratched glassware should not be used. Diazomethane solutions
should not be exposed to direct sunlight or strong artificial
light. (Ref. 15.1)
4.2 Benzene is flammable. LDcn orally in rats is 5.7 g/kg.
Methanol is flammable. Poisoning in humans may occur from
ingestion, inhalation, or percutaneous absorption. Diethyl
ether is flammable. Chloroform is toxic. These reagents should
be used in well ventilated areas away from an ignition source.
4.3 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Syringe - 10 ml (Hamilton No. 701N)
5.2.2 Capsule vials, screw cap, No. 4 - 4-1/4 in, Kimble No.
60957, 40-mL capacity
5.2.3 Graduated cylinder, glass-stoppered, 100 mL
5.2.4 Borosilicate glass column; about 1 x 14 cm, fitted with
coarse fritted disc
5.2.5 Volumetric flask, 10 mL
5.2.6 4 oz., square, amber bottles
5.3 Filter-eel filter-aid, Johns-Manville Corporation
5.4 Mechanical shaker
5.5 Clinical centrifuge
5.6 Lourdes Model MM-1 Multi-Mix Homogenizer.
5.7 Rotary evaporator
5.8 Gas chromatograph system equipped with an electron capture
detector. A Barber-CoIman Model 5000 gas chromatograph.
5.8.1 Gas chromatograph column; U-shaped borosilicate glass, 74-
in x 3-tom I.D.
121-02 January 1983
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90
5.8.2 Barber-Colman Sr ionization detector, Model A-4150.
Operating voltage for the detector was derived from a 90-
volt dry-cell battery connected in parallel with a 10-turn
300-K potentiometer. A 2000-uF capacitor was connected
between the anode of the detector cell and chassis ground
to reduce baseline noise.
Reagents
6.1 Benzene, glass distilled
6.2 Methanol, glass distilled
6.3 Chloroform, ACS
6.4 Diethyl ether, Analytical Reagent
6.5 2-sec-butyl-4,6-dinitrophenol (DNBP), analytical grade;
obtainable from Agricultural Department, The Dow Chemical
Company, Midland, MI
6.6 2-sec-butyl-4,6-dinitrophenol, methyl ether (DNME); obtainable
from Sampling Coordinator, Agricultural Department, The Dow
Chemical Company, Midland, MI
6.7 Dow Corning 200 fluid 12,000 cps. Dow Corning Corporation,
Midland, MI
6.8 Gas-Chrom Z, solid support, 80-100 mesh, Applied Science
Laboratories, Inc., 140 North Barnard Street, State College,
PA
6.9 Alumina; Woelm basic and acidic alumina, (kept at 130 C);
Alupharm Chemicals, P.O. Box 30628, New Orleans, LA 70130
6.10 Phosphoric acid, 85%
6.11 Sodium chloride, ACS
6.12 Sulfuric acid, 6 N
6.13 Prepurified nitrogen (carrier gas), Air Reduction Co.
6.14 2-(2-Ethoxyethoxy)ethanol, J.T. Baker Chemical Co.,
Phillipsburg, NJ
6.15 Diazald N-methl-N-nitroso-p-toluenesulfonamide, Aldrich Chemical
Company, Inc., 2369 North 29th Street, Milwaukee, WI
6.16 Pipets, disposable, capillary, 5-3/4 inch long. Scientific
Products #P5205-2.
6.17 Diazomethane solution (2): Approximately 18 mg per mL in ether.
Add 35 mL of 2-(ethoxy-ethoxy)ethanol and 10 mL of ether to a
solution of 6 g of KOH in 10 mL of distilled water contained in
121-03 January 1983
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a 100-mL long-necked distilling flask. Place a magnetic
stirring bar in the flask and mount in a water bath on top of a
hot plate magnetic stirrer. Attach a dropping funnel and an
efficient condenser connected to two receiving flasks in series.
A 350-mL Erlenmeyer serves as the first flask, followed by a 50-
mL Erlenmeyer flask containing 25 mL of. ether. The inlet tube
of the second receiver should dip below the surface of the
ether. Cool both receivers to 0°C. Place a solution of 21.5
g of Diazald reagent in 140 mL of ether in the dropping funnel.
While stirring the contents of the flask, heat the water to
65°C. As the distillation of ether begins, add the Diazald over
a 20-minute period. Follow with 20 mL of ether and interrupt the
distillation when the distillate appearing in the condenser is
nearly colorless. Combine the contents of the two receivers and
store in culture tubes with foil-lined screw caps. The reagent
is stable for several weeks if kept below 0 C, in full, closed
tubes.
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Dissolve 0.053 g 2-sec-butyl-4,6-dinitrophenol, methyl
ether (DNME) in 500 mL of benzene to give a solution
equivalent to 100 ug/mL DNBP. Make appropriate volumetric
dilutions of this solution with glass-distilled benzene to
obtain standard solutions covering the concentration range
from 0.01 to 0.05 ug/mL.
7.2.2 Inject 4 uL aliquots of DNME standard solutions, covering
the concentration range from 0.01 to 0.05 ug/mL into the
chromatograph and record the resulting peak heights. Plot
peak heights on the ordinate as percent full-scale
deflection vs. corresponding concentrations of DNME on the
abscissa.
8. Quality Control
8.1 Prepare 2-sec-butyl-4,6-dinitrophenol (DNBP) methanolic spiking
solutions (spiking standard). Dissolve 0.050 g of DNBP in 500
mL of benzene to obtain a 100-ug/mL solution. Dilute 1, 3, 5,
and 10 mL of benzene standard to 100 mL with methanol to equal
1, 3, 5, and 10 ug/mL methanolic spiking solutions.
8.2 Measure out 25-mL portions of water samples into a series of 4-
oz bottles.
8.3 Use one of the samples as a control, and fortify the remaining
samples by adding appropriate aliquots of methanolic standard
solutions of DNBP. Prepare each sample in duplicate.
121-04 January 1983
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8.4 Treat all the samples as follows:
8.4.1 Add 0.5 ml concentrated phosphoric acid and 8 g of sodium
chloride.
8.4.2 Partition the sample two times for 3 minutes with 20 mL
diethyl ether each time. Centrifuge and combine the ether
phase to 40 mL total volume.
8.4.3 Prepare a 1- x 3-cm basic al'imina column by placing 3 cm
in a 1-cm-wide glass column and place a small plug of
glass wool on top.
8.4.4 Pour 20 mL of the ether solution from Step 8.4.3 on the
alumina column and allow it to run completely through.
Discard the ether eluate.
8.4.5 Place a capsule vial under the column and elute with 5 mL
then 10 mL of pH-8 water. A slight air pressure may be
applied.
8.4.6 Acidify the eluate with 1 mL of 5% phosphoric acid
solution.
8.4.7 Add 5 mL of diethyl ether. Cap the tube and shake for 3
minutes, then centrifuge.
8.4.8 Pipet the ether phase into a 50-mL beaker.
8.4.9 Add 1 mL of diazomethane.
8.4.10 After 30 minutes, evaporate the ether to near dryness
with a gentle stream of air.
8.4.11 Take up the residue in 1 mL of hexane.
8.4.12 Prepare an acidic alumina column by placing a small plug
of glass wool in a disposable capillary pipet and adding
1 inch of acidic alumina.
8.4.13 Add the hexane solution from Step 8.4.12 to the prepared
column.
8.4.14 Discard the hexane eluate and elute the column with 5 mL
of ethyl ether into a 5-mL volumetric flask.
8.4.15 Adjust the volume to 5 mL.
8.4.16 Chromatograph 4 uL of the ether solution as described in
Section 12. Determine the apparent ppm DNBP in the
fortified samples by means of the standard curve.
Correct for blank, if any, by subtracting ppm of apparent
DNBP of the control sample from that of the treated
sample.
121-05 January 1983
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8.4.17 Calculate the percent recovery of DNBP from each of the
fortified samples and average the results.
8.5 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Analyze 25-mL samples of water as described in Section 8 (Step
8.4), and record peak heights for each sample.
10.2 Determine the ppm DNBP in each sample by referring to the
standard curve described in Section 7. Correct for blank, if
any, by subtracting ppm of apparent DNBP of the control sample
from that of the treated sample.
11. Cleanup and Separation
Not Available.
12. Gas Chromatoeraphv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
12.3 Gas Chromatograph Column
12.3.1 Dissolve 0.75 g DC 200 in 80 mL of chloroform in a round-
bottom flask. Add 15 grams 80-100 mesh Gas-Chrom Z, and
boil off most of the chloroform on a steam bath. Dry the
column packing on the rotary evaporator over a 50 C water
bath. The liquid phase loading on the support is 5% DC
200 by weight.
12.3.2 Fill the column with packing and tamp on a solid surface
such as the floor until no further settling occurs. When
finished, the packing in the inlet arm should be 3 in
below the injection septum. Insert a small glass-wool
plug over the packing at the effluent end of the column,
but not in the inlet side. Condition the column at 200 C
overnight with an N2 flow of about 100 mL/minute.
12.3.3 Injection Technique — Fill the needle of the 10-uL
syringe with benzene, eliminating all air bubbles with a
rapid up-down stroke. Draw 4 uL of solution into the
syringe, and inject onto the chromatographic column.
121-06 January 1983
-------
13. Calculations
13.1 Calculate the corrected apparent residue in the sample as
follows:
_.__ Net ppm DNBP .nn
ppm DNBP = ~ ~~T x 100
vv Percent Recovery
The final 5 mL of ether solution represents 2.5 g of original
sample. Therefore, 0.4 times the concentration of that
solution, expressed as ug/mL, is equivalent to parts-per-million
residue in the water sample.
mg/L (ppm) in original sample * ug/mL found x 0.4 x any
additional dilution
14. Method Performance
Not Available.
15. References
15.1 Organic Synthesis, Col. Vol. IV, p. 250, John Wiley and Sons,
Inc. (1963).
15.2 These directions are adapted from the procedure published by the
Aldrich Chemical Company, Inc., 2369 North 29th Street,
Milwaukee 10, WI.
15.3 R.L. McKellar, "Determination of Residues of DNBP (2-sec-butyl-
4,6-dinitrophenol) in Alfalfa by Gas Chromatography: Supplement
for Water," ACR 70.5.99, February 13, 1973, Dow Chemical
Company, MI.
121-07 January 1983
-------
Table 1
Chromatographic Conditions
Column temperature
Injector block temperature
Detector batb temperature
Detector operating voltage
Carrier gas
Recorder
Electrometer sensitivity
Chart speed
188°C
211°C
218°C
13 volts
prepurified N2 at 85 mL/min
0 - 5 mv
—10
3.3 x 10 amp, full scale
20 in/hr
Column - U-shaped glass, 74-in x 3-mm, packed with 80/100 mesh Gas Chrom Z,
5% DC 200
121-08
January 1983
-------
xvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF DINOSEB
IN WASTEWATER
METHOD 122
1. Scope and Application
1.1 This method covers the determination of dinoseb. The following
parameters can be determined by this method:
Parameter Storet No. CAS No.
Atrazine 39033 1912-24-9
Dinoseb — 88-85-7
Methyl parathion 39600 298-00-0
Toxaphene 39400 8001-35-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in total plant
effluent waters.
1.3 The method detection limit (MDL) for atrazine, dinoseb, and
methyl parathion is 0.1 mg/L. The MDL for a specific wastewater
may differ from those listed, depending upon the nature of
interferences in the sample matrix.
2. Summary of Method
2.1 A measured volume of water sample (1000 mL) is adjusted to pH 2
and extracted several times with chloroform. The combined
extracts are evaporated to dryness on a rotary evaporator using a
55 -50 C water bath. The residue is dissolved in exactly 5 mL of
acetone. Analysis is by gas chromatography using a flame
ionization detector.
2.2 A rotary evaporator.is used for sample concentration rather than
the K-D apparatus specified in Ref. 15.2. The reason for the
122-01 January 1983
-------
switch is that Atrazine precipitates in the K-D receiver which
prevents quantitative transfer of the sample.
3. Interferences
3.1 Glassware used in this and all other trace pesticides analyses is
dedicated and undergoes rigorous cleanup and meticulous handling.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5 . Apparatus and Materials
5.1 S amp1ing Equ ipmen t
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Precision liquid syringe, 10 uL
5.2.2 Standard laboratory glassware (chromic acid washed)
5.3 Rotary Evaporator, Rotavapor R or equivalent
5.4 Gas Chromatograph with FID-Tracor 550 or equivalent
5.4.1 Column: 6-ft x 1/4-in glass column packed with 1.95% OV-17
+ 1.5% QF-1 on 80/100 mesh Gas Chrom Q
5.4.2 Integrator, Varian CDS-111 or equivalent
6. Reagents
6.1 Acetone, pesticide quality
6.2 Chloroform, pesticide quality
6.3 H SO,, reagent grade
6.4 Atrazine, Dinoseb, Methyl Parathion standards
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh exactly 0.1 g Dinoseb, 0.1 g Methyl Parathion, and
0.3 g Atrazine into a 250-mL volumetric flask. Dilute to
volume with acetone.
122-02 January 1983
-------
7.2.2 Using a precision syringe, inject exactly 1, 2, 4, and 6 uL
standard solution on the GC. Record the integration counts
and the volume of each injection for each compound. Graph
the integration counts versus ug for each pesticide.
8. Quality Control
8.1 Quality control checks are performed by sample spiking. Results
show 98% recovery. Also, each analysis is performed with a
standard curve.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Adjust pH of exactly 1 liter effluent to pH = 2 using
concentrated sulfuric acid, and place in 2-L separatory funnel.
10.2 Extract 3 times using 100-mL aliquots of chloroform. Combine
chloroform layers in a 500-mL round-bottom flask and evaporate to
o o
dryness on a Rotavapor using a 55 -60 C water bath.
10.3 Dissolve the residue in exactly 5 mL Acetone.
11. Cleanup and Separation
Not Available.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
12.3 Inject 2-6 uL sample solution. Adjust sample size so that each
pesticide value will fall on its standard curve. Note ug of each
pesticide.
13. Calculations
,, , ,, (ug pesticide from standard curve)(5)
13.1 mg/L = —B—c :————
mL injected
14. Method Performance
14.1 The detection limit is approximately 0.1 mg/L for all three
pesticides.
122-03 January 1983
-------
15. References
15.1 "Manual of Analytical Quality Control for Pesticides in Human and
Environmental Media," by the U.S. Environmental Protection Agency
(Publication Number EPA-600/1-76-017).
15.2 "Federal Register," Volume 38, Number 75, Part II, as the
"National Pollutant Discharge Elimination System, Appendix A."
15.3 "Determination of Atrazine, Dinoseb, and Methyl Parathion in
Plant Effluent," Standard Test Method, Vicksburg Chemical
Company, Vicksburg, MS. Includes an addendum for Toxaphene.
122-04 January 1983
-------
Table 1
Chromatographic Conditions
Column Temperature
Injection Temperature
Detector Temperature
Carrier Gas
Flow Rate
190°C
250°C
220°C
Nitrogen
90 mL/min
Column - 6-ft x 1/4-in glass packed with 1.95% OV-17 +1.5% QF-1 on 80/100
mesh Gas Chrom Q.
122-05
January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ETHION[TM]
IN WASTEWATER
METHOD 123
1. Scope and Application
1.1 This method covers the determination of ethion.
Parameter Storet No. CAS No.
Ethion 39398 563-12-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in vastewater.
2. Summary of Method
2.1 A measured volume of water sample (50 mL) is extracted with
hexane. The combined extract is dried over sodium sulfate, and
filtered into an Erlenmeyer flask. The extract is concentrated
to less than 1 mL and analyzed by gas chromatography with flame
photometric detector in the phosphorus mode.
3. Interferences
3.1 The use*of the detector specific for phosphorus greatly reduces
the problems of interference. The detector does not respond to
general organic background.
3.2 The flame photometric detector can be adjusted to reponc to
sulfur by a simple change of optical filters. Since Ethion[TM]
contains sulfur, the question arises of how sulfur detection
would work for traces of EthionfTM]. It has been found that
small amounts of sulfur compounds are present to a much greater
extent than volatile phosphorus compounds in the general
environment. Interference problems are therefore much greater
with sulfur detection.
123-01 January 1983
-------
4. Safety
4.1 Ethion[TM] is a toxic substance that should be handled with care.
It is a cholinesterase inhibitor that can be absorbed through the
skin. The main hazard is contact. The vapor pressure is
negligible at room temperature. The odor associated with
technical EthionfTM] is not the compound- itself, but traces of
organic sulfur compounds.
4.2 Protective gloves should be worn when handling Ethion[TM] or
EthiontTM] solutions. Do not allow spills to remain on bench
tops; clean them up immediately. Rinse out glassware that has
contained EthionlTM] with organic solvents.
4.3 Exposure to ethion[TM] can be detected by regular measurement of
blood cholinesterase levels.
4.4 All persons who become contaminated by organo-phosphates are
instructed to wash thoroughly, or take a shower for fifteen
minutes (if more than hands have come in contact with the
chemicals), and change clothing. If there are no symptoms
following the "spill," the test for cholinesterase is performed
approximately 24 hours later.
Apparatus and Materials
5.1 Sampling Equipment
Not Available,,
5.2 Glassware and Other Equipment
5.2.1 Usual laboratory glassware
5.2.2 Small sample vials
5.2.3 Tuberculin syringe, 1 mL, graduated in 1/100 mL, BD No.
2004
5.2.4 Cannulas, 23 gauge, 2-in long, BD No. 1788
5.2.5 Microsyringe, Hamilton 701 N
5.2.6 Separatory funnels, 125 mL
5.2.7 Filter funnel
5.2.8 Centrifuge tubes
5.3 Filter paper
5.4 Reducing valve and needle valve controls for nitrogen system
5.5 Fume hood
123-02 January 1983
-------
5.6 Steam bath
5.7 Gas chromatograph, equipped with flame photometric detector in
the phosphorus mode
5.7.1 A Hewlett-Packard Model 5830A with reporting integrator was
used for this work so that operating instructions are given
for that type of equipment. Any equivalent GC can be used.
5.7.2 GC column. A Teflon column, 1/4-in O.D. by 4-ft long
packed with 10Z SE30 on Chromasorb W, 80/100 mesh.
Reagents
6.1 Ethion[TM] standard of known purity
6.2 Hexane, distilled in glass
6.3 Sodium sulfate, ACS, anhydrous. Destroy traces of adsorbed
organic contamination by heating in a furnace at about 500 -550 C
overnight. A platinum or porcelain dish should be used to hold
the salt while in the oven. Store treated reagent in air tight
containers. Several small containers are preferable to a single
large one.
6.4 Methanol, distilled in glass
6.5 Distilled water. Pass through activated carbon bed to remove
traces of organic contamination.
Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh approximately 0.1 g of Ethion[TM] into a 100-mL
volumetric flask. Record the exact weight to 0.1 mg. Add
hexane to the mark, and mix flask contents well.
7.2.2 Pipet 1 mL of the concentrate, prepared in Step 7.2.1 into
a 100-mL volumetric flask. Dilute to the mark with hexane,
and mix well.
7.2.3 Pipet 10 mL of the solution, prepared in Step 7.2.2, into a
100-mL volumetric flask. Dilute to the mark with hexane,
and mix well. This will be a concentration of about 1000
ug/L. The exact concentration in micrograms per liter will
be numerically equal to the exact weight (in grams) from
Step 7.2.1, multiplied by 10,000.
7.2.4 Calibrate the GC by injecting 10 uL of the hexane standard
from Step 7.2.3. Run according to the chromatographic
conditions given in Table 1.
123-03 January 1983
-------
For Model 5830A, run the sample by "Area %," enter the
external standard (ESTD) mode, 5% retention time window,
the retention time and the amount (AMI). The amount will
be the concentration of the standard expressed in ug/L.
Rerun the standard as a sample to check the calibration.
For other instruments, determine the concentration/peak
area ratio. If the same size injection (in this case, 10
uL) is used, consistently the peak area for EthiontTM] will
be proportional to the concentration.
8. Quality Control
8.1 All of the usual precautions and standard practices required for
trace analyses must be followed.
8.2 Blank analyses, which include all reagents and operations except
an actual sample, must be run at frequent intervals. It is
difficult to control trace contamination in a laboratory where
Ethion [TM] is handled. It is possible, however, to obtain a
flat GC baseline.
8.3 Known water samples must be run at frequent intervals. The
following procedure is provided for this purpose.
8.3.1 Weigh approximately 0.1 g of Ethion[TMJ into a 100-mL
volumetric flask. Record the exact weight to 0.1 mg. Add
methanol to the mark, and mix the flask contents well.
This will be a concentration of about 1000 ug/mL . The
exact concentration in ug/mL will be equal to the exact
weight (in grams) multipliud by 10,000.
8.3.2 Pipet 1 mL of the 1000-ug/mL concentrate into a 1-liter
volumetric flask. Dilute to the mark with water. This will
be a concentration of 1/1000 of the exact concentration
from Step 8.3.1 or approximately 1 ug/raL.
8.3.3 Pipet 10 mL of the 1-ug/mL solution into a 1-liter
volumetric flask. Dilute to the mark with water. This
will be a concentration of 1/100 of the exact concentration
from Step 8.3.2 or approximately 10 ug/L.
8.4 A fixed routine must be followed for the GC procedure to assure
dependable results.
8.4.1 Inject two 10-uL samples of hexane. A flat baseline must
be obtained to be certain that the syringe is clean.
8.4.2 Inject 10 uL of the standard in hexane. If the
chromatogram appears normal, proceed with the calibration.
8.4.3 Rinse the syringe well with hexane.
8.4.4 Inject 10 uL of hexane.
123-04 January 1983
-------
8.4.5 Inject 10 uL of sample.
8.4.6 Rinse the syringe well with hexane.
8.4.7 Inject 10 uL of hexane.
8.4.8 Inject 10 uL of the next sample.
8.4.9 Continue with this sequence until all samples have been
run.
8.4.10 After all samples have been run, inject 10 uL of the
standard in hexane.
8.5 Glassware used for this operation must be kept segregated. It
should be cleaned by soaking in alcoholic caustic, rinsing with
water, and oven drying.
Caution: Alcoholic caustic is irritating to skin and eyes. Wear
goggles when using the bath. Use tongs to place
glassware in the bath, or to remove it.
8.6 Keep the bench top and general area well scrubbed. Do not use
any phosphorus-containing detergents.
8.7 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow the EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Extract 50 mL of wastewater sample with 50 mL of hexane. Drain
the aqueous layer into a second separator funnel. Drain the
extract into a 250-mL Erlenmeyer flask.
10.2 Extract the aqueous layer with a second 50-mL portion of hexane.
Drain the aqueous layer into the first funnel. Combine the
extract with the previous extract in the Erlenmeyer flask.
10.3 Extract the aqueous layer with a third 50-mL portion of hexane.
Discard the aqueous layer. Combine the extract with the previous
extracts in the Erlenmeyer flask.
10.4 Add 5 g to 10 g of sodium sulfate to the combined extracts.
Swirl the flask gently. Filter through paper into a clean dry
250-mL Erlenmeyer flask. Rinse the solid and filter with 10 mL
of hexane. Add the rinsings to the combined extracts.
10.5 Place the flask with the dried extract on a steam bath in the
hood. Direct a stream of nitrogen into the flask to help carry
away the hexane vapors. Concentrate to a volume of about 5 mL.
123-05 January 1983
-------
10.6 Transfer the concentrate to a centrifuge tube. Use hexane to
rinse the flask, adding the rinsings to the tube.
10.7 Heat the tube on the steam bath, and use nitrogen to speed up the
evaporation process. Concentrate to a little less than 1 mL.
10.8 Measure the volume of concentrate. Fit -a cannula on a 1-mL
syringe. Draw the concentrate into the syringe. Draw a small
amount of air into the syringe behind the liquid. Read the
volume of the column of liquid from the graduations on the barrel
of the syringe.
10.9 Transfer the concentrate to a small vial. Cap tightly, label
well, and hold for GC analysis.
11 . Cleanup and Separation
Not Available.
12. Gas Chroma tog rap by
12.1 Table 1 summarizes the recommended operating conditions for the
GC.
12.2 Calibrate the system as described in Section 7.
12.3 For Model 5803A, enter the "XF" factor for the individual sample.
See Step 13.1, "Calculations."
12.4 After the instrument has been calibrated for ESTD, or the
concentration/ peak area ratio has been obtained, inject 10 uL of
concentrate as prepared in Step 10.9.
13. Calculations
13.1 For Model 5830A
13.1.1 Calculate the "XF" factor. When the integrator is
supplied with this factor, it will automatically correct
the concentration results for the concentration change
effected by the hexane extraction and evaporation. Enter
the factor in the integrator individually for each sample.
XF _ Cone
XF 50
Where: Vol. Cone = Volume in mL, of concentrate from
Step 10.8.
50 = Volume, in mL, of original water sample.
13.1.2 The integrator will report the concentration of Ethion[TM] in
the wastewater in ug/L. Although it is not precisely correct
to do so, these results can be referred to as "PPB" for most
practical purposes .
123-06 January 1983
-------
13.2 For Other Instruments
13.2.1 Calculate the Ethion[TMj concentration in the injected
concentrate.
_ (Constd) x (Samparea)
Concon = ~—i c
Stdarea
Where:
Concon = Concentration of the concentrate, in ug/L
Constd = Concentration of the standard, from Step 7.2.3
Samparea - Area of sample peak
Stdarea = Area of standard peak
13.2.2 Correct results for the concentration change during
extraction and evaporation.
nun (Concon) x (Vol Concj
PPB = 5£)
Where:
PPB » Concentration, in ug/L, of Ethion in wastewater
sample
Concon = from Step 13.2.1
Vol Cone «• Volume, in mL, of concentrate from Step 10.8
50 = Volume, in mL, of original water sample
14. Method Performance
14.1 The detection limit for Ethion[TM] in water is approximately 0.5
ug/L with the use of this method of analysis.
15. References
(R)
15.1 "Ethion in Wastewater," Test Method 30.3, FMC Corporation,
Agricultural Chemical Division, Baltimore, MD.
123-07 January 1983
-------
Table 1
Chromatographic Conditions
Column Temperature 235 C
Injector Temperature 225 C
Auxiliary Temperature 225 C
o
Oven Max. Temperature 250 C
Chart Speed 1.00 cm/min
Attn 2 8
Aux sgnl B
Sip sens 1.00
Area rej. 100
Flow A 20 (not in use)
Flow B 67
Optn. 0
Column: Teflon, 1/4-in x 4-ft, packed with 10% SE30 on Chromasorb W, 80/100
mesh.
123-08 January 1983
-------
00-
FIGURE 1
Hexane Blank
s
i-<
•
t
w
Retention Time (min)
n
t
ca
FIGURE 2
Ethion Standard in Hexane
1082 Micrograms/liter
123-09
RETENTION TIME (min)
January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ETRIDIAZOLE [TERRAZOLE(TM)]
IN WASTEWATER
METHOD 124
1. Scope and Application
1.1 This method covers the determination of certain organochlorine
pesticides. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Etridiazole [Terrazole(TM)] — 2593-15-9
PCNB [Terrachlor(TM)] 39029 82-68-8
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above, manufacturing
impurities, and related metabolites in water.
2. Sunmary of Method
2.1 A measured volume of water sample (100 ml) is extracted with
hexane. The extract is dried over sodium sulfate and analyzed by
gas chromatography (GC) using an electron capture detector.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
124-01 January 1983
-------
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnel with Teflon stopcocks, 250 mL.
5.3 Perkin Elmer 3920 B gas chromatograph equipped with Ni-63
electron capture detector or equivalent.
5.3.1 Column: 6 ft of glass tubing (1/4-in O.D., 2-mm I.D.)
packed with 15% SP2100 on Supelcoport 80-100 mesh. Column
conditioned at 275°C with N2 flow of 25 mL/min for about 2
hours in column conditioner.
6. Reagents
6.1 n-Hexane: Distill reagent-grade hexane (high purity) over sodium
dispersed in mineral oil. Use approximately L mL of 50%
dispersed sodium in mineral oil per liter of hexane.
6.2 Anhydrous Na_SO, (granular).
6.3 Etridiazole [TERRAZOLE(TM)], Technical Grade, 01in Corporation,
minimum 95% assay.
6.4 Pentachlorobenzene (PCB): Olin Corporation, obtained from PCNB
process and recrystallized. Structure confirmed by infrared
spectroscopy.
6.5 Hexachlorobenzene (HCB) obtained in similar manner as
pentachlorobenze.
6.6 PCNB: Olin Technical Grade, 98% PCNB.
6.7 Pentachloroaniline (PCA): prepared from PCNB by reduction, with
zinc in ethanol - HC1, and recrystallized from ethanol.
6.8 Methyl pentachlorophenyl sulfide: prepared from PCNB by reacting
with Na-S followed by CH.,1 with subsequent recrystallization from
ethanol; MP 95-96°C. Structure confirmed by mass spectroscopy.
6.9 3-Dichloromethyl-5-Ethoxy-l,2,4-Thiadiazole; prepared from
TERRAZOLE[TM] with Na2S20^. Distilled under reduced pressure,
47°C, and 0.2 mm Hg. Structure confirmed by mass spectroscopy.
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
124-02 January 1983
-------
7.2.1 Suitable concentrations are PCS at 0.0001 ug/mL; HCB and
PCNB at 0.0002 ug/mL; PGA, methyl pentachlorophenyl
sulfide, TERRAZOLE[TM] and 3-Dichloromethyl-5-Ethoxyl-
1,2,4-Thiadiazole at 0.0004 ug/mL. These concentrations
are in the linear range of peak heights versus uL injected
when 3- to 8-uL injection volumes are employed. Solutions
of methyl pentachlorophenyl sulfide decompose with time;
therefore, when the peak height begins to drop 15 percent,
then a new diluted standard should be prepared.
8. Quality Control
8.1 Follow Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 The water sample is shaken well and a 100-mL aliquot is added to
a 250-mL separatory funnel.
10.2 Extract the water with 25 mL of hexane.
10.3 The hexane layer is dried over sodium sulfate.
10.4 Analyze the hexane extract by gas chromatography.
11'. Cleanup and Separation
Not Available.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
12.3 Inject 3-8 uL of the sample hexane extract and compare versus
standards run before each sample injection.
13. Calculations
13.1 Calculate the concentration of the unknown per the following
equation.
r VS x CS Ru
Cu is" x vTT x vt + w
where Cu = Apparent ppm component in sample
VS = Microliters of standard injected
124-03 January 1983
-------
CS = Micrograms of component per mL in standard
RS - Peak height in millimeters for VS
Ru = Peak height in millimeters for unknown
Vu = Microliters of unknown injected
Vt = Volume of extracted solution
W = Volume of sample extracted
Corrected ppm component in treated sample:
100
CR " Cu x £s.
where CR = corrected ppm component in sample
Cu = ppm component in sample
R.S. • % recovery of component added to blank sample.
14. Method Performance
Not Available.
15. References
(R) (R)
15.1 "Determination of Terrachlor and Terrazole in Water
Samples," Standard Analytical Method, Olin Corporation.
124-04 January 1983
-------
Table 1
Chromatographic Conditions
Interface 200°C
Injection Port 200 C
Detector 225°C
Column 180 C
Carrier Gas/Flow 95% Argon/5% Methane, 45 mL/min
Attenuator x 4
Standing Current 2.0
Chart Speed 1 cm/min
Column - 1/4-in x 6-ft glass, packed with 15% SP2100 on Supelcoport 80-100
mesh.
124-05 January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF FENSULFOTHION [DASAHIT(TM)]
IN WASTEWATER
METHOD 125
1. Scope and Application
1.1 This method covers the determination of fensulfothion.
Parameter Storet No. CAS No..
Fensulfothion — 115-90-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in vastevater.
2. Summary of Method
2.1 A measured volume of water sample (250 mL) is adjusted to pH 6.5-
7.0 and extracted with chloroform. The extract is partitioned
with distilled water, dried, and evaporated to dryness. The
residue is brought up in 5 mL acetone and analyzed by gas
chromatography with a phosphorus-specific alkali flame detector.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 Fenthion sulfoxide interferes and, if present, will appear as
fensulfothion.
3.2 All glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENTS.
125-01 January 1983
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4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 10 uL
5.3 Rotoevaporator, Buchi or equivalent
5.4 Water bath, 50°-55°C
5.5 Gas chromatograph, Varian Model 1400 or equivalent, equipped with
a phosphorus detector, a 3-ft x 1/8-in O.D. glass column packed
with 3% OV-225 on Gas Chrom Q 80/100 mesh, and glass injector and
detector inserts. (If the ends of the column are long enough to
be used as inserts, the glass inserts are not necessary.) Note:
Use only Teflon-backed septa, Supelco No. 2-0459.
6. Reagents
6.1 Acetone, nanograde
6.2 Chloroform, nanograde
6.3 Hydrochloric acid, IN — Carefully add 21 mL of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.4 Mineral oil solution, 2.5% — Dilute 25 mL of mineral oil, Fisher
#0-120 or equivalent, to 1000 mL with nanograde chloroform and
mix thoroughly.
6.5 Sodium hydroxide, IN
6.6 Sodium sulfate, anhydrous, ACS
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Fensulfothion (DASANIT), 0.10% — Weigh 0,110 - 0.115 g of
Fensulfothion (DASANIT) (88% minimum purity) into a 100-mL
volumetric flask. Dilute to volume with acetone, stopper,
125-02 January 1983
-------
and mix thoroughly. This solution may be used for one week
only.
7.2.2 Pipet a 1-mL aliquot of the 0.10% Fensulfothion
(DASANIT)solution into a 100-mL volumetric flask, dilute to
volume with acetone, and mix thoroughly. Label this
solution "10 ng." Prepare fresh daily.
7.2.3 Pipet 5-, 10- and 20-mL aliquots of 10-ng standard of Step
7.2.2 into separate 100-mL volumetric flasks. Dilute to
volume with acetone and mix. Label these solutions 500,
1000, and 2000 pg, respectively. Prepare fresh daily.
7.2.4 Continue analysis according to TM A-54.12 (Mobay reference
method, not enclosed herein).
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately to
Step 10.2.
10.2 Measure 250 mL of the well-mixed sample into a 500-mL separatory
funnel.
10.3 If the pH is above 7.0 or below 6.0, adjust it to pH 6.5-7.0 with
IN hydrochloric acid or IN sodium hydroxide using pH indicator
paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel.
If an emulsion forms, centrifuge the emulsified layer and add the
clear chloroform layer, obtained by centrifuging, to the second
separatory funnel. Return the water layer to the first
separatory funnel.
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minute.
10.6 Filter the chloroform layer through a funnel containing a glass-
wool plug and 4 to 5 g of anhydrous sodium sulfate (pre-rinsed
with 10-mL of chloroform), into a 300-mL 24/40 boiling flask.
Rinse the sodium sulfate three times with 10 mL of chloroform.
Add 10 mL of the mineral oil solution to the boiling flask.
125-03 January 1983
-------
10.7 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at 50°C. Remove the flask and
cool to room temperature.
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Pipet 5 mL of acetone into the flask from Step 10.8, stopper
immediately, and rotate the flask so that the acetone washes down
the inside of the flask. DO NOT allow the acetone to get on the
neck or stopper.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Continue the analysis according to TM A-54.12 (Mobay reference
method, not enclosed herein).
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
(R)
15.1 "Determination of Dasanit in Waste Water by GLC," Analytical
Method TM 13-34.38, Chemagro Agricultural Division, Mobay
Chemical Corporation, Kansas City, MO.
125-04 January 1983
-------
Table 1
Chromatographic Conditions
Attenuation X32
Cell voltage Flame
Detector temperature 210-230°C (do not exceed 230 C)
-12
Electrometer range 10
Gas flows:
Carrier gas Helium, 20 mL/min
Air 230 mL/min
Hydrogen 40 mL/min
Injector temperature 200°-215°C (do not exceed 215°C)
o
Oven temperature 230 C
Column - 3-ft x 1/8-in O.D. glass, packed with 3% OV-225 on Gas Chrom Q
80/100 mesh.
125-05 January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF FENTHION
IN WASTEWATER
METHOD 126
1• Scope and Application
1.1 This method covers the determination of fenthion.
Parameter Storet No. CAS No.
Fenthion — 55-38-9
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
2. Summary of Method
2.1 A measured volume of water sample (250 mL) is adjusted to pH 6.5-
7.0 and extracted with chloroform. The extract is partitioned
with distilled water, dried, and evaporated to dryness. The
residue is brought up in 5 mL acetone and analyzed by gas
chromatography with a phosphorus-specific alkali flame detector.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 BAY 25064 interferes and, if present, will appear as fenthion.
3.2 AH, glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENTS.
126-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 10-uL
5.2.2 Rotoevaporator, Buchi or equivalent
5.3 Water bath, 50°-55°C
5.4 Gas chromatograph, Varian Model 1400 or equivalent, equipped with
a phosphorus detector, a 3-ft x 1/8-in O.D. glass column packed
with 15% OV-17 on Chromosorb W, DMCS 60/80 mesh, and glass
injector and detector inserts. (If the ends of the column are
long enough to be used as inserts, the glass inserts are not
necessary.) Note: Use only Teflon-backed septa, Supelco No. 2-
045.
6. Reagents
6.1 Acetone, nanograde
6.2 Chloroform, nanograde
6.3 Hydrochloric acid, IN — Carefully add 21 mL of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.4 Mineral oil solution, 2.5% — Dilute 25 mL of mineral oil, Fisher
#0-120 or equivalent, to 1000 mL with nanograde chloroform and
mix thoroughly.
6.5 Sodium hydroxide, IN — Carefully dissolve 10 g of sodium
hydroxide pellets,"ACS, in 250 mL of distilled water and mix
thoroughly.
6.6 Sodium sulfate, anhydrous, ACS
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
126-02 January 1983
-------
7.2.1 Fenthion, 0.10% — Weigh 0.100 - 0.110 g of fenthion (95%
minimum purity) into a 100-mL glass-stoppered graduated
cylinder. Dilute to 100 mL with acetone, stopper, and mix
thoroughly. This solution may be used for one week only.
7.2.2 Pipet a 1-mL aliquot of the 0.10% fenthion solution into a
100-mL volumetric flask, dilute to'volume with acetone, and
mix thoroughly. Label this solution "10 ng." Prepare fresh
daily.
7.2.3 Pipet 5-, 10- and 20-mL aliquots from the 10-ng standard of
Step 7.2.2 into separate 100-mL volumetric flasks. Dilute
to volume with acetone and mix. Label these solutions 500,
1000, and 2000 pg, respectively. Prepare fresh daily.
7.2.4 Continue the analysis according to TM A-54.12 (Mobay
reference method, not enclosed herein).
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately to
Step 10.2.
10.2 Measure 250 mL of well-mixed sample into a 500-mL separatory
funne1.
10.3 If the pH is above 7.0 or below 6.0 adjust it to 6.5 - 7.0 with
IN hydrochloric acid or IN sodium hydroxide using pH indicator
paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel.
If an emulsion forms, centrifuge the emulsified layer and add the
clear chloroform layer, obtained by centrifuging, to the second
separatory funnel. Return the water layer to the first
separatory funnel.
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minutte.
10.6 Drain the chloroform layer through a funnel containing a glass-
wool plug and 4 to 5 g of anhydrous sodium sulfate (pre-rinsed
with 10 mL of chloroform), into a 300-mL 24/40 boiling flask.
Rinse the sodium sulfate three times with a 10-mL portion of
126~03 January 1983
-------
chloroform, and add 10 mL of 2.5% mineral oil solution into the
flask.
10.7 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at 50°C. Remove the flask and
cool to room temperature.
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Pipet 5 mL of acetone into the flask from Step 10.8, stopper
immediately, and rotate the flask so that the acetone washes down
the inside of the flask. DO NOT allow the acetone to get on the
neck or stopper.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Continue the analysis according to TM A-54.12 (Mobay reference
method, not enclosed herein).
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
15.1 "Determination of Fenthion in Waste Water by GI.C," Analytical
Method TM B-34.37, Chemagro Agricultural Division, Mobay
Chemical Corporation, Kansas City, MO.
126-04 January 1983
-------
Table 1
Chromatographic Conditions
Attenuation X32
Cell voltage Flame
Detector temperature 230°-240°C (do not exceed 250°C)
Electrometer range 10*^
Gas flows:
Carrier gas Ee> 20 mL/Min
Alr 230 mL/min
Hydrogen 40 mL/min
Injector temperature 230°-240°C (do not exceed 250°C)
Oven temperature 235°C
Column: 3-ft x 1/8-in glass, packed with 15% OV-17 on Chromosorb W, DMCS
60/80 mesh.
126-05 T ,001
January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF GLYPHOSATE
IN WASTEWATER
METHOD 127
1. Scope and Application
1.1 This method covers the determination of glyphosate.
Parameter Storet No. CAS No.
Glyphosate 39941 1071-83-6
1.2 This is a high performance liquid chroma tographic (HPLC) method
applicable to the determination of glyphosate in industrial
effluent.
1.3 The method detection limit (MDL) is 50 ug/L (ppb) of glyphosate
standard with 52 of full-scale deflection. The MDL for a specific
vastevater may be different depending upon the nature of
interferences in the sample matrix.
2. SiMnmft yy Q f Method
2.1 A known volume of glyphosate industrial effluent, including
noncontact cooling waters, is applied to AG50V-X8 (Bio-Rad)
cation exchange column and eluted with two bed volumes of water.
The collected effluent is preconcentrated and/or injected into a
HPLC using a Technicon-based ninhydrin/ post-column detector. The
peak height of the resulting glyphosate is determined and
compared with peak heights obtained from standards.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3 . Interferences
3.1 Ammonia, as well as primary and secondary amines, will have a
significant response with ninhydrin reagent, and, therefore,
127-01 January 1983
-------
prevent detection of the presence of the glyphosate peak in the
chromatogram.
3.2 A sample clean-up using packed AG50W-X8 strong cation exchange
column (Bio-Rad Inc.) will eliminate the interferences of amines,
organic amines, and amino acid derivatives. A preconcentration
factor of 20 (10 mL of waste effluent concentrated to 0.5 mL) for
dilute industrial effluents associated with glyphosate production
is unequivocally free of interferences.
3.3 Equipment used in this analysis (columns, filters, glassware,
etc.) should be limited to trace analysis only. This will
eliminate any possibility of residual contamination at these low
levels.
3.4 The employment of 0.04-0.06% NaOCl for washing the glassware is
to remove any remaining trace levels of glyphosate. The
glassware should be rinsed with copious amounts of distilled
water before application. Routine change of vacuum pump oil is
necessary if contamination has been observed.
Safety
4.1 Follow EPA Safety procedure found in Part D of this document.
Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Pipets, 10-100 uL, 100-1,000 uL, and 1-5 mL delivery pipets
with disposable tips.
5.2.2 Volumetric flasks, 100 mL or convenient sizes for making
standards or dilution.
5.3 Buffer pump, Waters 6000A pump, flow rate 0.6 mL/min.
5.4 Injector, Waters Intelligent Sample Processor (WISP).
5.5 Ninhydrin Reagent Technicon proportionation pump.
5.6 Reactor, Technicon research cartridge or oil bath controlled at
95°C.
5.7 Detector, Technicon Single Channel Colorimeter with 570-nm
optical interference filter and 1.5-mm x 55-mm flow cell.
5.8 Electronic Filter — Spectrum 1021 filter and amplifier.
5.9 Recorder, Fisher, lOOmv-range.
127-02 January 1983
-------
5.10 HPLC Column, DuPont Zorbax SAX, 15-cm x 4.6-mm (853952703).
5.11 Vacuum Evaporation Setup, including pump, condenser, rotator,
heating plant, and receiving flask.
5.12 CDC 1700 Computer System.
5.13 AG50W-X8, 100-200 mesh, Econo Column (Bio-Rad, 731-6213).
6. Reagents
6.1 Dimethysulfoxide (Pierce, 20687)
6.2 Ninhydrin (Pierce, 21001)
6.3 Lithium Acetate pHix[TM] Buffer, 4M Li ; pH 5.20 (Pierce, 27203)
6.4 Hydrindantin Dihydrate (Pierce, 24000)
6.5 Methanol (Burdick and Jackson)
6.6 H3P04 (85%, Fisher A-242)
6.7 KH2P04 (Mallinckrodt, 7100)
6.8 Brij[TM] 35, 30% aqueous solution (Fisher CS-285-2)
6.9 Argon
6.10 NaOCl (4-6%, Fisher SOS-290)
6.11 H2S04 (Fisher, A-300)
6.12 Preparation of Reagent
6.12.1 HPLC Buffer Solution -- Dissolve 10 g KH2P04 in 3.8 L
of 160 mL methanol/deionized water; adjust the
solution to pH 2.3 with 85% H3P04. Normal HPLC
degassing/filtration procedures are followed.
6.12.2 Ninhydrin Solution — Combine 1,100 mL of
dimethylsulfoxide (DMSO), 800 mL of deionized water,
400 mL of 4.0M (pH 5.2) lithium acetate, and bubble
argon through the solution for approximately 15
minutes. Add 32 g of ninhydrin to the solution with
stirring. In a separate beaker dissolve 1.6 g of
hydrindantin solution in two portions of 50 mL DMSO
with a f'.w mL of previously prepared ninhydrin
solution, and add together with argon bubbling. An
additional 15 minutes for argon deoxygenation is
required before application.
127-03 January 1983
-------
6.12.3 Wetting Reagent for Autoanalyzer — Dissolve 10 mL
Brij in 800 mL of deionized water; a few drops of
concentration EJ50, are added to make acidic
solution.
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Tab le 1.
7.2 Calibration Procedure
7.2.1 A stock solution of 1,000 ug/mL (ppm) is prepared by
dissolving 100 mg of 3X recrystallized analytical grade of
glyphosate in 100 mL deionized water and stored in the
refrigerator.
7.2.2 A series of working standards (0.2, 0.4, 0.6, 0.8, and 1.0
ug/mL) is prepared weekly by appropriate dilutions.
7.2.3 A series of external standards (0.2-1.0 ug/mL) is prepared
and analyzed under the same HPLC conditions and on the same
day as unknown samples. A blank industrial effluent sample
and water also have to be included in the sample
pretreatment and HPLC analysis to ensure the absence of
glyphosate contamination via sample manipulation. It is
recommended that only the lowest (0.2 ug/mL) concentration
of glyphosate standard be employed for the determination of
glyphosate less than 30 ug/L in the waste sample. This
effectively eliminates any contamination due to syringe and
needle carryover of standards.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 The AG50W-X8 resin column is washed with 15 mL of deionized water
before application.
10.2 For glyphosate concentrations greater than 300 ug/L, 1.0 mL of
sample is applied to the column and then eluted with 5 mL of
deionized water. The collected eluent (6 mL) is ready for HPLC
injection after complete mixing.
10.3 For glyphosate concentrations less than 300 ug/L, 10.0 mL of
sample is applied to the column and then eluted with 5 mL of
deionized water. The collected eluent is evaporated to dryness
127-04 January 1983
-------
under vacuum at 50 -60 C. Deionized water (0.5 mL) is added to
the residual, which is then ready for HPLC injection.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Construction of Ninhydrin Reactor — Refer to Figure 1.
12.4 HPLC Injection — An alternating injection between sample and
standard is carried out throughout the analysis.
12.5 A typical chromatogram of glyphosate standard, blank, and waste
effluent sample is included in Figure 2. It has been observed
that the retention time of glyphosate in waste effluent samples
is approximately 60 seconds less than the immediately preceding
glyphosate standard.
13. Calculations
13.1 For glyphosate concentrations greater than 0.4 mg/L (ppm), the
actual concentration of glyphosate in waste sample mg/L (ppm) =
(ppm glyphosate from calibration curve) x 6 (dilution factor)
13.2 For glyphosate concentrations less than 300 ug/L (ppb), the
actual concentration of glyphosate in waste sample ug/L (ppb) =
(peak height of sample) 2QQ /,
(average peak height of 200-ug/L standard)
14. Method Performance
14.1 The method was validated over the range of 5-30 ug/L with a.
preconcentration factor of 20, and 0.3-10 mg/L with a dilution
factor of 6. The probable linear range is 0.05 to 10 mg/L with
glyphosate standards.
14.2 The detection limit is 50 ug/L of glyphosate standard with 5% of
full scale (S/N=2).
14.3 The laboratory validation of this method was carried out using
synthetic glyphosate industrial waste sample. The components of
the mixture are listed in Table 2. This was necessary due to
currently large (ppm) quantities of glyphosate being discharged
and so that future effects of pretreatment technology on
glyphosate industrial effluent could be taken into account.
127-05 January 1983
-------
14.4 The pooled coefficient of variation (CV) for the analytical
method in the range of 5-30 ug/L and 0.3-10 mg/L range of
glyphosate in 002 sump is 0.0788 and 0.0366, respectively.
14.5 The average values obtained in the 5-30 ug/L and 0.3-10 mg/L
range of glyphosate in the appropriate industrial effluent were
20 and 5 percent less than the "true" values, respectively. The
difference between the "found" and "true" concentrations is not
due to a bias in the sampling and analytical method but rather
due to a random variation of the experimentally determined "true"
concentration. Therefore, no recovery should be applied to the
final results in Section 13.
14.6 The HPLC/ninhydrin reaction post-column detector provides the
required selectivity and sensitivity for trace-level glyphosate
determination.
14.7 The disadvantage of the method is the manual sample cleanup and
evaporation/preconcentration step. Therefore, the precision of
the method is limited by the reproducibility of each operational
step.
15. References
15.1 "Method for the Determination of Glyphosate in Industrial
Effluents," Monsanto Agricultural Products, Co., St. Louis, MO.
127-06 January 1983
-------
Table 1
Chromatographic Conditions
Buffer Flow Rate
WISP Operation Parameters
Injection Volume
Run Time
Number of Injections
Detector - DAMP 2, Std. Cal. 1.00
Electronic Filter, Cutoff Frequency
Attenuation
Recorder - Chart Speed
0.6 mL/min
200 uL
24 minutes
1
0.01.
2.0.
0.25 cm/znin
127-07
January 1983
-------
1.
2.
3.
Table 2
Composition of the Synthetic Glyphosate
Industrial Waste Sample
Component
Glyphosate Intermediate
Raw Material
PO
3-
NH.,
Composition (ppm)
30
30
100
4. NO,
5. SO
2-
6. Cl
7. Na+
8. Ca
9. Mg2+
10. Monoethanolamine
100
200
200
200
50
10
10
127-08
January 1983
-------
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January 1983
-------
f/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MANCOZEB [DITHANE M-45(TM)]
IN WASTEWATER
METHOD 128
1. Scope and Application
1.1 This method covers the determination of mancozeb (Dithane M-
45[TM]) and other carbamates.
Parameter Storet No^ CAS No.
Mancozeb [Dithane M-45(TM)] ~ 8018-01-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above and other
dithiocarbamates in various samples, including wastewater streams
from manufacturing operations.
1.3 The method detection limit (MDL) for mancozeb is 10 ug/L. The
MDL for a specific wastewater may differ from this value
depending upon the nature of interferences in the sample matrix.
2. Summary of Method
2,1 A known weight of sample is quantitatively decomposed to carbon
disulfide (CS2) by refluxing the sample in dilute HC1 in the
presence of stannous chloride (SnCl-). The liberated CS- is
swept by an air stream through a series of purification traps and
absorbed in ethanol at dry-ice temperature. The CSj is measured
by gas chromatcgraphy using a flame-photometric detector in the
sulfur mode. For confirmatory CS2 analysis an aliquot of the
ethanol solution is injected into a GC-MS system. The CS»
digestion apparatus of the standard Keppel colorimetric method is
used with a series of trapping tubes containing MallcosorbfTM],
lead acetate, sulfuric acid, and ethanol.
128-01 January 1983
-------
2.2 This method provides selected cleanup procedures to aid in the
elimination of interferences which may be encountered.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Decomposition - distillation - absorption train (See Figure
1).
5.2.2 Gas chromatograph - equipped with a Tracor flame
photometric detector with sulfur filter (394 nM) and 6-ft
x 4-mm I.D. coiled glass column packed with Tenax GC 60-80
mesh.
6. Reagents
6.1 Mancozeb (Dithane M-45) Standard — Commercial Dithane M-45 (use
assay value from official formulation analysis to calculate
active ingredient weight to take for preparation of spiking
standard).
6.2 Na, EDTA Solution — Dissolve 100 g of ethylenedinitrilo
tetraacetic acid, tetrasodium salt (MCB #E x 550) in 800 mL
distilled water and dilute to 1L with distilled water.
6.3 Water, distilled and deaerated — Deaerate approximately 4 liters
of distilled water by bubbling nitrogen gas through it at 50 to
100 mL per minute for 10 to 15 minutes.
6.4 95% Ethanol — Denatured, commercial formula 3-A (Publicker
Industries, Inc.).
6.5 First Absorber - Mallcosorb absorbent, 30/50 mesh, (Mallinckrodt
#6077) for removal of acidic gases (C02> H2S» etc')-
6.6 Second Absorber - 30% lead acetate solution for removal of
remaining H2S. Add 30 g lead acetate trihydrate (Fisher
Scientific #L-33) to distilled H20 in a 100-mL volumetric flask
and dilute to volume.
128-02 January 1983
-------
6.7 Third Absorber — Concentrated sulfuric acid (ACS reagent grade)
for removal of alkaline gases and water vapor (water appearing in
cryogenic capillary tubing of Absorber #4 will block air flow).
6.8 Fourth Absorber — 95% ethanol held in bath of dry-ice/3-A
ethanol.
6.9 Stannous chloride reagent — Weigh 40 g of stannous chloride
dehydrate (Fisher Scientific #T-142) and transfer it to a 100-mL
volumetric flask. Dissolve and dilute to the mark with distilled
water. Note: Prepare this solution within two aours of use, and
discard remainder after use.
7. Calibration
7.1 Establish GC operating conditions equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Carbon disulfide standard solution for preparing GLC
standard curve. Make up 95% ethanol solutions of 0.4, 0.3,
0.2, 0.15, and 0.0 ug/mL of carbon disulfide for standard
curve developed using 10-uL injections.
7.2.2 Inject 10 uL of standard CS, solutions.
7.2.3 Read the amount of CS- from a calibration curve prepared by
plotting peak height (chart units) versus varying nanogram
(or alternatively ug/mL) levels of CS2 standards injected.
(Establish a standard curve before each series of sample
injections.)
8. Quality Control
8.1 It is advisable to run a reagent blank (standard run with all
reagents but no sample) before starting a series of analyses, to
demonstrate clean apparatus and guard against false positive
results. A reagent blank should also be run following an
abnormally high sample. From time to time the apparatus should
be disassembled, cleaned, adsorbents replaced, etc., especially
if reagent blanks give positive results.
8.2 Freshly prepare a stock standard of 500 ug/mL mancozeb (Dithane
M-45) in 10% w/v aqueous tetrasodium ethylenediaminetetraacetate
(Na.EDTA). Make appropriate dilutions with distilled deaerated
water to obtain the desired level of the spiking standard.
Example: To prepare a 5-ug/mL standard, dissolve 0.05 g active
ingredient (the acutal weight will be slightly more than 0.05 g
depending on the percent active ingredient in 100 mL of 10% w/v
aqueous Na^ EDTA, and dilute 1 mL of this solution to 100 mL with
distilled deaerated H20. Add the appropriate spiking aliquot to
sample just prior to beginning decomposition step. [Note: EBDC
128-03 January 1983
-------
in Na EDTA solutions should not be stored due to decomposition
(Ref. 16.4).]
8.3 Determine recovery by adding an aliquot of mancozeb in Na, EDTA
spiking standard to the weighed sample in the digestion flask.
Correct recovery for control when the corresponding control or
unfortified sample produces a significant value.
8.4 See EPA Quality Control procedure found in Part D of this
document .
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document .
10. Sample Extraction
10.1 Sample Preparation
10.1.1 No sample preparation is necessary for wastewater
except to stir or agitate so that a representative
sub sample is taken for analysis. The standard
subsample is 50 g.
10.2 Decomposition-Distillation-Absorption
10.2.1 Transfer the sample to the 1,000-mL digestion flask
using distilled and deaerated water. Add sufficient
water as needed to give a volume of about 400 mL.
Add boiling chips, 40 mL of concentrated HC1, and 10
mL of 40% aqueous SnC solution.
10.2.2 In the first absorber tube place 30 cc of Mallcosorb
powder. To the second absorption tube add 10 mL of
30% aqueous lead acetate reagent, and to the third,
add 5 mL of concentrated H2SO^. Add 5 mL of 95%
ethanol to the fourth adsorber (CS2 trap). Cool the
CS» trapping tube by immersing in a bath of dry-
ice/3-A ethanol.
10.2.3 Connect the equipment, start water flow through
reflux condenser, and apply gentle vacuum to outlet
of CS- trap to draw air through the system at a rate
of about 60 mL/min. Turn on heating mantles and
bring flask contents to boiling. Continue refluxing
for about 45 minutes.
10.2.4 Turn off heating mantle. Disconnect the CS2 trap
from the vacuum and the dry-ice bath and let stand at
room temperature for about 1 hour before diluting
sample to 5 mL using 95% ethanol.
128-04 January 1983
-------
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
12.3 By syringe remove a 10-uL aliquot of final ethanol sample and
inject into the gas chromatograph set at the described
conditions. Measure the height of the peak of CSj in the
chromatogram and calculate the concentration from the standard
curve.
13. Calculations
13.1 The amount of mancozeb (Dithane M-45) in the sample is calculated
from the following equations:
i\ oo ic \ 5 ml (EtOH trap) . ,0 MW mancozeb
1) ng CS2 (from curve) x 10 uL (Inj. VolJ X 1J* 2 MW CS2
™ Total ug mancozeb (Dithane M-45) found in sample taken for
digestion
2) ug/g (ppm) mancozeb (Dithane M-45)=
ug mancozeb (Dithane M45) found
g of sample taken for digestion x recovery factor
14. GC7MS Confirmation
14.1 For CS_ confirmation, inject an aliquot of the final ethanol
sample into a GC/MS system having similar GC column operating
parameters as those described for the flame photometric GC
system. (Semi-quantitative MS estimates are made based on single
ion monitoring of sample vs. standard ions at mass 75.9).
15. Method Performance
15.1 Recoveries — Table 2 presents recovery data by the described
method. Example chromatograms (Figures 2 and 3) and the
corresponding calibration curve (Figure 4) are also presented.
15.2 Limit of Detection — The minimum detectable amount is about 0.5
ng of CS2 in 10 uL of injected solution. Based on a 5-mL final
volume of ethanol sample, the detection limit is about 0.5 ug of
total Mancozeb (Dithane M-45) in the sample taken for digestion.
Based on these parameters the limit of detection is 0.010 ug/g
for a 50-g sample. The limit of detection can be lowered, if
necessary, by increasing the size of the sample. Conversely, if
128-05 January 1983
-------
high concentrations are expected it may be desirable to use a
smaller sample, or to dilute the ethanol solution of trapped CS_
before injection into the gas chromatograph.
16. References
16.1 G.E. Keppel, J.A.O.A.C. .54, 528-532 (1974).
16.2 Tokyo Organic Chemical Industries, Ltd., TOCIL Memo TR-78-9-11,
T.ISO to H. Kuyama, 9/11/78, Anal. Method for Dithane M-45
Residues.
16.3 "A Gas Chromatographic Method for Measurement of Dithane M-45 or
other Dithiocarbamate Fungicides in Wastewater or Natural
Waters," TR36F-82-20, Rohm and Haas Comapny, Spring House, PA.
16.4 B.D. Ripley, Bull Envir. Cont. Tox. 22_, 182-189 (1979).
128-06 January 1983
-------
Table 1
Chromatographic Conditions
Column temperature 140 C
Injection temperature 140 C
Detector temperature 190 C
Carrier Gas/Flow nitrogen, 30 mL/min
Air 80 mL/min
Oxygen 28 mL/min
Hydrogen 110 mL/min
Recorder Speed 5 cm/min
Range 100 mv f.s.
Column: 6-ft x 4-mm I.D. coiled glass, packed with Tenax BC 60-80 mesh
128-07 January 1983
-------
Table 2
Recovery of Fortifications of Mancozeb (Dithane M-45)
Recovery
Concentration Added ug/g (ppm) 2
1.0 85.4
0.5 87.2
5.0 71.6
0.1 71.2
Average 79 i 8
128-08 January 1983
-------
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January 1983
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128-11
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''S3 -'WJ&O'J-Vty January 1983
-------
oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MANEB
IN WASTEWATER
METHOD 129
1. Scope and Application
1.1 This method covers the determination of maneb.
Parameter Storet No. CAS No.
Maneb — 12427-38-2
1.2 This is a spectrophotometrie method applicable to the
determination of maneb in aqueous waste effluents.
2. Suminarv of Method
2.1 Maneb is determined in wastewater using a carbon disulfide
evolution procedure. The carbon disulfide formed by the
hydrolysis of maneb is measured colorimetrically after it is
reacted with an alcoholic solution containing amines and cupric
acetate to form cupric diethyldithiocarbamate.
2.2 This method provides selected cleanup procedures to aid in the
elimination of interferences which may be encountered.
2.3 The method detection limit (MDL) for maneb is 0.02 ug/g (ppm)
based on a 1000-g sample.
3. Interferences
3.1 This method is not compound-specific since CS2 or any compounds
that are capable of hydrolyzing to CS2 will analyze as maneb. If
unusually or surprisingly high results are obtained, additional
analytical studies will be necessary to identify the source
and/or to determine maneb specifically.
129-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Distilling flask, 500 mL
5.2.2 Decomposition Absorption Apparatus (See Figure 1)
5.3 Spectrophotometer, Beckman Model B or equivalent:
6. Reagents
6.1 Color reagent — To 0.012 g cupric acetate monohydrate in 250-mL
volumetric flask, add 25 g diethanolamine. Dilute to volume with
EtOH and mix.
6.2 NaOH solution — 10%, w/v.
6.3 Carbon disulfide standard solution — To accurately tared 25-mL
volumetric flask containing 5 mL ethanol, pipet approximately 0.1
mL CS^. Close flask at once and reweigh to obtain weight of CS^
by difference. Dilute to mark with ethanol and mix well. Dilute
2 mL of this solution to 100 mL with ethanol and mix (standard
solution). Calculate ug CS2/mL.
6.4 Stannous chloride — 40%, w/v.
7. Calibration
7.1 Determine the absorbance at 435 nm
7.2 Calibration Procedure
7.2.1 To series of 25-mL volumetric flasks, add amounts of
standard CS2 solution varying from 0.1 to 3 mL, using 10-mL
buret. To each flask, add 15 mL color reagent. Dilute to
mark with ethanol and mix. Let stand 15 minutes and read
absorbances at 435 nm against mixture of 15 mL color
reagent and 10 mL ethanol as reference. Plot absorbances
against ug CS^.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
129-02 January 1983
-------
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Fart D of this document.
10. Sample Extraction
10.1 Transfer a 250-g aliquot of the sample into the 500-mL distilling
flask of the Decomposition-Absorption apparatus described in
Reference 15.1. Assemble the-equipment, and using the reagents
and procedure outlined in Reference 15.2, continue the analysis.
At the end of the boiling period, cool the 500-mL distillation
flask containing the 250-mL sample and replace it with a second
500-mL flask containing a second 250-mL aliquot of the sample.
Do not change the absorption towers. Collect the evolved €82
from the second run in the same color reagent trap. Continue to
analyze the sample using a third and a fourth 250-mL aliquot and
collecting the evolved CS2 from all 4 runs in the sample trap.
The total sample weight is 1000 g. It is not possible to analyze
a 1000-g single sample with the existing equipment because of
handling difficulties, i.e. pressure build-up. Scaled-up
equipment can be used, however, if desired.
10.2 At the conclusion of the fourth run, drain the CS2 trap contents
into a 250-mL volumetric flask, rinse with a small volume of
ethyl alcohol, dilute to bring to volume with ethyl alcohol, and
mix thoroughly.
11. Cleanup and Separation
Not Available.
12. Sample Analysis
12.1 Determine the absorbance at 435 nm of the solution in Step 10.2
and calculate the amount of maneb in ppm using a calibration
curve previously prepared using analytical standard maneb.
13. Calculations
13.1 Calculate ug dithiocarbamate from equivalent CS2 found. If the
dithiocarbamate(s) present is unknown, calculate as maneb by
following formula:
,„ „„ K _ „ ro Y maneb (g molecular weight)
ug Maneb - ug CS2 X 2 x cs(g molecular
= ug CS2 X 1.81
14. Method Performance
14.1 The method has a sensitivity of 0.02 ppm using a 1000-g sample.
129-03 January 1983
-------
15. References
15.1 Determination of Dithiocarbamate Fungicide Residues, by H.L.
Pease, JAOAC, 40, 1113 (1957).
15.2 Collaborative Study of the Determination of Dithiocarbamate
Residues by a Modified Carbon Disulfide Evolution Method by
George E. Keppel, JAOAC, 54, 528 (1971).
15.3 "Determination of Maneb in Plant Aqueous Waste Effluent,"
Standard Test Method, DuPont Company, Laporte, IX.
129-04 January 1983
-------
PEASE: DITHIOCARBAMATE FUNGICIDE RESIDUES
FIGURE 1.—Decomposition-absorption apparatus for dithiocarbamate determinations.
129-05
January 1983
-------
v>EPA
United States Effluent Guidelines Division (WH 554
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MEPHOSFOLAN [CYTROLANE(TM)], PHORATE (THIMET(TM)],
AND TERBUFOS [CODNTER(TM)]
IN WASTEWATEH.
METHOD 130
1. Scope and Application
1.1 This method covers the determination of certain organophosphorus
compounds. The following parameters can be determined by this
method.
Parameter Storet No. CAS No.
Mephosfolan (CYTROLANE) — 950-10-7
Phorate (THIMET) — 298-02-2
Terbufos (COUNTER) 82088 13071-79-9
Malathion 39530 121-75-5
Dimethoate (CYGON) — 60-51-5
Famphur — —
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in process wastewater
streams.
1.3 The method detection limit (MDL) for the pesticides covered by
this method is in the range of 5 to 25 mg/L (based on the
original waste sample) when a flame ionization detector is
employed, and 5-25 ug/L when an alkali flame ionization detector
is employed.
2. S|tiiminrv. of Method
2.1 A measured volume of sample (1-liter)is extracted with several
portions of chloroform. The combined chloroform extracts are
concentrated and analyzed by gas chromatography with flame
ionization and/or alkali flame ionization detector.
130-01 January 1983
-------
3. Interferences
3.1 All solvents, reagents, glassware, sample handling equipment, and
instrumentation must be demonstrated to be free from pesticides
and/or artifacts which could cause interference or
misinterpretation of gas chromatograms.
3.2 Some organic compounds present in mixed plant waste streams cause
interferences in the analysis of organophosphorus pesticides by
gas chromatography, using a flame ionization detector. These
interferences can often be overcome by the use of an alkali flame
(phosphorus specific) ionization detector.
4.
4.1 All normal laboratory safety equipment and procedures should be
used.
4.2 Personal protective clothing should be worn, including safety
glasses, laboratory coat, and rubber gloves.
4.3 All solvent handling should be performed in a well ventilated
laboratory hood.
4.4 All gas chromatographic work should be performed by an
experienced analyst.
4.5 Some organophosphate pesticides are very toxic and must be
handled carefully, e.g., phorate exhibits an L/ftcn of
approximately 1 mg/kg (rat oral).
4.6 If solution containing organophosphate pesticide is spilled onto
the skin or splashed into the eyes, wash the affected area with
tap water for a minimum of 5 minutes. In severe cases of
exposure, seek medical attention. All contaminated clothing must
be removed immediately.
4.7 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Microliter syringes (Series No. 700, Hamilton Company), 10
uL
5.2.2 Separatory funnels, 100 mL
5.2.3 Beakers, 150 mL
130-02 January 1983
-------
5.2.4 Watch glasses, Speedyvap Ace Scientific Cat. No. 14-9420
5.2.5 Volumetric flasks, (10 mL, 25 mL, 50 mL, 100 mL and 200 mL)
5.2.6 Graduated cylinders (25 mL, 100 mL, 500 ml)
5.2.7 Glass wool, silane-treated (No. 14502, Applied Science
Labs)
5.3 Gas Chromatograph — An instrument designed for use with dual
glass columns and equipped with an on-column injection system,
and high-sensitivity flame ionization detectors or alkali flame
ionization detectors and an electrometer having a sensitivity of
-12
at least 10 amperes driving a 1-mv strip chart recorder, and
having a drift of less than 1% per hour (a totally solid state
amplifier with an FET input) is recommended. Electronic digital
integration or computer-based area measurements may also be
employed. The integrator should have independent controls for the
selection of up and down slope sensitivities so that start and
stop integration points can be selected. An automated sample
injection system also contributes significantly, particularly to
analytical precision. The Hewlett-Packard Model 7600 and the
Varian Model 2800 are suitable when equipped as described. Other
equivalent instrumentation may be used but may require
modification of operating conditions in order to obtain good peak
shape, adequate resolution, and appropriate retention times.
5.3.1 Gas Chromatographic Column — 122-cm borosilicate glass
tube (4-mm I.D., 6-mm O.D.) bent to fit the chromatograph
and packed with 3% OV-25 on Gas Chrom Q (100/120 mesh). A
prepared packing can be purchased from Supelco, Inc.,
Supelco Park, Bellefonte, PA 16823, or Alltech Associates,
202 Campus Drive, Arlington Heights, IL 60004.
6. Reagents
6.1 Chloroform, reagent grade
6.2 Pesticide standards — High purity pesticides: Malathion,
THIMET, COUNTER, CYGON, CYTROLANE, and Famphur, may be obtained
from American Cyanamid Company, Agricultural Division, Princeton,
NJ 08540.
6.3 Di(2-ethyIhexyl) sebacate: Applied Science Laboratories, Inc.,
State College, PA 16801.
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Gas chromatographic operating conditions are considered
acceptable if the response to malathion is at least 50% of
130-03 January 1983
-------
full scale when £0.6 ug is injected for flame icmization
detection and £0.6 ng is injected for alkali flame
ionization detection. For all quantitative measurements,
the detector must be operated within its linear response
range.
7.2.2 Standards should be injected frequently to check the
stability of the operating conditions.
7.2.3 Either external standardization or internal standardization
methods may be used. When the internal standardization
method is employed with flame ionization, di-(2-ethylhexyl)
sebacate is used as the internal standard.
8. Quality Control
•
8.1 Duplicate sample injections should be used as quality control
checks. In addition, known "standard" samples should be injected
during each run as an additional check.
8.2 A method blank of distilled water should be extracted and carried
through all analytical steps for each set of samples.
8.3 See EPA Quality Control procedure found in Part: D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Non-persistent pesticide compounds hvdrolvze in water solutions;
therefore, wastewater should be extracted immediately.
9.2 If it is necessary to store wastewater samples for even a short
period of time, the samples should be refrigerated.
9.3 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Depending on the level of detection required in the waste sample,
200 to 600 mL of sample is required for analysis.
10.2 Mix the sample thoroughly, and quantitatively transfer the
required amount to a 1000-mL separatory funnel.
10.3 Add 15 mL of chloroform to the sample in the separatory funnel
and shake vigorously for two minutes.
10.4 Allow the solvent to separate from the sample, then drain the
solvent phase into a 50-mL volumetric flask (if concentration is
not required) or into a 150-mL beaker (if concentration is
required).
10.5 Perform second and third extractions in the same manner.
130-04 January 1983
-------
10.6 Add a specific amount of di-(2-ethylhexyl) sebacate to the 50-mL
volumetric flask if the internal standardization method is to be
used, and fill the flask to the mark with chloroform.
10.7 Analyze the extract by gas chromatography.
10.8 The external standardization method is employed if a low
detection limit is required. The chloroform extract is
concentrated by covering the 1 50-mL beaker of extract with a
watch glass and allowing the extract to evaporate to dryness in a
well-ventilated hood overnight. Do not use heat to evaporate the
extract because some pesticide compounds exhibit a high vapor
pressure. In addition, uncontrolled heat may cause degradation
of the non-persistent pesticides.
10.9 Carefully rinse the extract residue from the 150-mL beaker into a
lOmL volumetric flask with small portions of chloroform and fill
the flask to the mark.
10.10 Analyze the concentrated extract by gas chromatography.
11 . Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chroma tograph.
12.2 Calibrate the system as described in Section 7.
13. Calculations
13.1 Determine the pesticides concentration by using either external
standardization or internal standardization.
13.2 In the external standardization method, the gas chromatographic
response of the sample extract is compared to the response of a
pesticide standard.
13.2.1 The pesticides concentration of the sample is
calculated as follows:
_ _
micrograms/liter -- (KF) (Vs)
where Ai = Area of the pesticide component in the
extract injected.
Vs = Volume (liters) of wastewater sample
extracted.
KF = Calibration factor for the pesticide
component.
130-05 January 1983
-------
717 _ (Ac)
KF " (Cc)
where Ac = Area of pesticide standard in the
calibration standard injected.
Cc = Amount of the pes-ticide in the calibration
standard (micrograms) .
13 .3 In the internal standardization method the gas chromatographic
response of the sample extract is quantified by the addition of a
known internal standard; this response is compared to response of
a pesticide standard containing the same internal standard.
13.3.1 The pesticide concentration of the sample is
calculated as follows:
•11- / , • (IS) (KF) (Ai)
milligram/liter - (Vg)
where IS = Internal standard amount, (milligrams)
Ai = Area of the pesticide component in extract
injected
Vs = Volume of wastewater sample extracted
(liters)
Ais = Area of the internal standard in the
extract injected
KF = Calibration factor of the pesticide
component
_ (Cc) (Ais)
KF ~ (A) (Cis)
where Cc = Amount of pesticide component in the
calibration sample (milligrams)
Cis = Amount of internal standard in the
calibration sample (milligrams)
Ais = Area of the internal standard in the
calibration sample
A = Area of the pesticide component in the
calibration sample
13.4 Report results in milligrams per liter (mg/L) or micrograms per
liter (ug/L) without correction for recovery data.
14. Method Performance
14.1 The detection limit for the pesticides covered by the method is
130-06 January 1983
-------
14.1 The detection limit for the pesticides covered by the method is
in the range of 5 to 25 mg/L (based on the original waste sample)
when a flame ionization detector is employed.
14.2 The detection limit for the pesticides covered by the method is
in the range of 5 to 25 ug/L (based on the original waste sample)
when an alkali flame ionization detector-is employed.
15. References
15.1 Methods for Analysis of American Cyanamid Company.
15.2 Zweig, G & Sherma, J, Analytical Methods for Pesticides and Plant
Growth Regulators. Vol. VI, Gas Chromatoeraphic Analysis,
Academic Press, NY, 1972.
15.3 "Developmental Laboratory Procedure for the Detection of American
Cyanamid Company Non-Persistent Organophosphorus Pesticides in
Wastewater," Standard Test Method, American Cyanamid Company,
Linden, NJ.
130-07 January 1983
-------
Table 1
Chromatographic Conditions
Instrumentation Operation Parameters
I. The following operating parameters are used with the Hewlett-Packard
Model 7600:
Operation Mode Dual Column
Detector Flame lonization
Column-oven, programmed
temperature 130°C - 250°at 4°C/min
Injection port temperature 230 C
Detector temperature 270 C
Helium flowrate 75 mL/min
Hydrogen flowrate 50 mL/min
Air flowrate 500 mL/min
Range 10
Attenuation 1
Chart Speed 1/4 inch/min
II. The following operating parameters are used with the Varian Model
2800:
Operating Mode Single Column
Detector Alkali Flame lonization
Column-oven, programmed
temperature 170°C - 250°C at 8°C/min
Injection port temperature 230 C
Detector temperature 270 C
Helium flowrate 65 mL/min
Hydrogen flowrate 24 mL/min
Air flowrate 250 mL/min
Range 10~12
Attenuation Variable
Injection Volume 2 uL
Chart Speed 40 cm/hr
130-08 January 1983
-------
Table 2
Retention Times
Condition I
Parameter Retention Time (min)
THIMET (phorate) 8.7
COUNTER (terbufos) 9.5
CYGON 13.3
MALATHION 16.7
CYTROLANE (mephosfolan) 22.5
Famphur 25.8
Internal Standard 28.3
Condition II
Parameter Retention Time (min)
TIMET (phorate) 3.3
COUNTER (terbufos) 4.3
CYGON 6.7
Malathion 7.7
CYTROLANE(mephosfolan) 12.3
Famphur 13.3
13°-°9 January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF METHAM [VAPAM(TM)]
IN WASTEWATER
METHOD 131
1. Scope and Application
1.1 This method covers the determination of metham as its degradation
product, methyl isotbiocyanate.
Parameter Storet No. CAS No.
Metham — 137-42-8
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater. This
method assumes that complete degradation of metham to methyl
isothiocyanate has occurred prior to analysis.
1.3 The method detection limit (MDL) for the degradation product,
methyl isothiocyanate, is 2 ug/L, which corresponds to
approximately 4 ug/L as metham. The MDL for a specific wastewater
nay differ from those listed, depending upon the nature of
interferences in the sample matrix.
2. S'Tv of Method
2.1 A measured volume of water sample is extracted with chloroform or
ethyl acetate. Sample volume is dependent on the extraction
solvent used. Methyl isothiocyanate in the extract is determined
by gas chromatography using a flame photometric detector in the
sulfur mode.
3. Interferences
Not Available.
131-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnels, 250-mL and 1-liter with Teflon
stopcocks.
5.2.2 Syringe, 10-uL Hamilton No. 701 or equivalent
5.3 Gas Chromatograph — Hewlett Packard Model 5711A or equivalent,
equipped with a flame photometric detector in the sulfur mode.
5.3.1 Gas Chromatographic Column — 1.8-m x 2-mm I.D. Pyrex,
packed with 100/120 mesh Supelcoport coated with 10% SP-
2100, conditioned overnight at 235 C using a low nitrogen
flow.
6. Reagents
6.1 Chloroform, nanograde or equivalent
6.2 Ethyl Acetate, nanograde or equivalent
6.3 Sodium Sulfate, anhydrous, reagent grade
6.4 Sodium Chloride, reagent grade
6.5 Methyl Isothiocyanate, available from Aldrich Chemical Co.,
Milwaukee, WI
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Prepare a stock solution of 1000 ug/mL methyl
isothiocyanate in chloroform or ethyl acetate (see Step
10.2). From this solution, prepare a calibration solution
to be used in the analysis.
7.2.2 Using the analytical conditions stated above, chromatograph
5 uL of a calibration solution of methyl isothiocyanate
with a concentration approximately the same as that of the
sample extract. Measure the peak height of the methyl
isothiocyanate peak. Since the response of the flame
131-02 January 1983
-------
photometric detector in the sulfur mode is not linear,
reference standard concentrations must be adjusted so that
approximately equal peak heights are obtained when equal
volumes of sample and reference solutions are analyzed.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Use either of the following procedures
10.1.1 Measure 50 mL of water sample (Vj) into a 4-oz bottle
that contains 20 g dry NaCl. Pipet into the bottle
2.5 mL ethyl acetate. Cap the bottle with a foil-
lined lid and shake it vigorously for 20 min. Remove
the upper (ethyl acetate) phase via a disposable
pipet and measure its volume (72). Dry the extract
over anhydrous sodium sulfate.
10.1.2 Chloroform Solvent — Measure 400 mL of water sample
(V,) into a 1-liter separatory funnel and extract
twice with 10-mL portions of chloroform. Measure the
volume of the combined extracts ^2). Dry the
combined extracts over anhydrous sodium sulfate.
10.2 Choice of solvent — Either chloroform or ethyl acetate can be
used. Use of ethyl acetate is preferable because it burns
cleaner in the flame photometric detector. Use of chloroform can
cause rapid fogging of the windows in this detector.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system daily as described in Section 7.
13. Calculations
13.1 Calculate the concentration of methyl isothiocyanate in water
using the following equation:
131-03 January 1983
-------
H x C x V-
ppm = __s 2
HrXVl
H = Peak height from sample, cm
s
H = Peak height from reference standard, cm
C = Concentration of methyl isothiocyanate in reference
standard, ug/mL
V. = Volume of water sample, mL
V- - Total volume of chloroform or ethyl acetate extract, mL
14. Method Performance
14.1 Recoveries of methyl isothiocyanate from water are shown in Table
2. They are 2. 80% at all fortification levels tested.
14.2 Using this method, the detection limit is 2 ug/L of methyl
isothiocyanate. This level corresponds to approximately 4 ug/L
of metham (Vapam).
15. References
15.1 "Determination of Methyl Isothiocyanate in Water by Gas
Chromatography," Provisional Method, Stauffer Chemical Company,
Westport, CT.
131-04 January 1983
-------
Table 1
Chromatographic Conditions
The instrument parameters listed below are for a Hewlett Packard Model
5711A gas chromatograph.
Injector temperature 200 C
Detector temperature 250 C
o
Column temperature 60 C
Carrier N-j 30 mL/min
Hydrogen 50 mL/min
Air 50 mL/min
Oxygen 0 mL/min
Volume injected 5 uL
Column: 1.8-m x 2-mm Pyrex packed with 100/120 mesh Supelcoport coated with
10% SP-2100.
Under these conditions, methyl isothiocyanate has a retention time of 3.5
min.
131-05 January 1983
-------
Table 2
Methyl Isothiocyanate Recoveries from Water
Solvent
Chloroform
Ethyl acetate
Ethyl acetate
Ethyl acetate
Fortification
Level. ug/L
50
10
5
2
Recovery. %
88
83
81
85
Average 84.3 ± 3.0
131-06
January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF METHOMYL [NUDRIN(TM)]
IN WASTEWATER
METHOD 132
1. Scope and Application
1.1 This method covers the determination of certain insecticides.
The following parameters can be determined by this method:
Parameter Storet No. CAS No.
Methomyl [NUDRIN(TM)] 39051 16752-77-5
Monocrotophos [AZODRIN(TM)] 81890 6923-22-4
Dicrotophos [BIDRIN(TM)] — 141-66-2
1.2 This is a high-performance chromatographic (HPLC) method
applicable to the determination of the compounds listed above in
aqueous plant waste streams.
1.3 The method detection limit (MDL) for each of the parameters above
is 0.1 mg/L (ppm). The MDL for a specific wastewater may differ
from that listed, depending upon the nature of interferences in
the sample matrix.
2. Summary of Method
2.1 A measured volume of wastewater sample (250 mL) is adjusted to a
pH of 6.5-8.0 and extracted with ethylene chloride. The extract
is analyzed by two separate normal phase HFLC methods. The first
method is used to ultimately determine the quantity of methorny 1
(NUDRIN) from the total of monocrotophos (AZODRIN) and methorny 1
(NDDRIN) insecticides and to indicate the presence of dicrotophos
(BIDRIN)insecticide. If any of these insecticides are found to
be present, the second method is employed to determine the
monocrotophos (AZODRIN) and dicrotophos (BIDRIN) contents. The
methomy1 (NDDRIN) content is calculated as a difference between
the total monocrotophos (AZODRIN) and methomy1 (NUDRIN) found by
the first method and the monocrotophos (AZODRIN) determined by
132-01 January 1983
-------
the second method. In all cases, the determinations are made by
comparison of chromatographic peak heights with those obtained
from standards of known concentration.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Volumetric glassware, including 50-mL, 25-mL, 10-mL, and 5-
mL volumetric flasks; 15-mL pipet; and 250-mL and 1,000-mL
graduated cylinders.
5.2.2 Separatory funnel, 500 ml.
5.2.3 Vacuum filtration apparatus, equipped with an 0.8-micron
millipore membrane filter and a 500-mL vacuum flask.
5.2.4 Microsyringe, 50 uL.
5.2.5 Hydrometer, 1.000 to 1.200 specific gravity.
5.2.6 Round-bottom flask, 500-mL equipped with a reflux
condenser.
5.3 Hot water bath, controlled at 90 C.
5.4 pH Meter
5.5 Liquid Chromatograph (1) capable of operating at 1,500 psi; (2)
equipped with a variable wavelength UV detector capable of
operating at 215 and 230 nanometers; (3) a loop injector valve
with a 500-microliter loop; and (4) a 250-mm x 3.2-mm Partisil
(Whatman), 10-micron PAC column.
6. Reagents
6.1 n-Heptane, distilled in glass, Burdick-Jackson or equivalent
6.2 Isopropanol, distilled in glass, Burdick-Jackson or equivalent
6.3 Tetrahydrofuran (THF), UV grade, distilled in glass, Burdick-
Jackson or equivalent
132-02 January 1983
-------
6.4 Ethylene chloride (1,2-dichloroethane), distilled in glass,
Burdick-Jackson or equivalent
6.5 Methanol, distilled in glass, Burdick-Jackson or equivalent
6.6 Sulfuric acid, 15%w
6 .7 Glass wool
6.8 Sodium sulfate, anhydrous
6.9 (Monocrotophos) AZODRIN Insecticide (SD 9129) of known purity*
6.10 (Dicrotophos) BIDRIN Insecticide (SD 3562) of known purity*
6.11 (Methomyl) NUDRIN Insecticide (SD 14999-W) of known purity*
6.12 Sodium Hydroxide Solution, 50%w
* Available upon request from Shell Chemical Company, One Shell Plaza,
HS&E Product Safety and Compliance, P.O. Box 4320, Houston, TX 77210
7. Calibration
7.1 Calibration Procedure
7.1.1 Weigh 25 mg (+.2 mg) to 0.1 mg of each of the insecticides
into separate 5-mL volumetric flasks. Dilute to the mark
with methanol. These are stock solutions of the standards.
7.1.2 Prepare a working solution containing monocrotophos
(AZODRIN) and dicrotophos (BIDRIN) by carefully measuring
50-uL of these stock solutions into a 50-mL volumetric
flask using a 50-uL microsyringe. Dilute to the mark with
ethylene chloride. This solution contains approximately
0.25 mg each of monocrotophos (AZODRIN) and dicrotophos
(BIDRIN).
7.1.3 Prepare a working solution of methomyl (NUDRIN) in the same
manner as in Step 7.1.2.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Measure 250 mL of sample into a 500-mL separatory funnel.
132-03 January 1983
-------
10.2 Measure the specific gravity and check its pH. Adjust if
necessary with 15%w sulfuric acid to a pH of 6.3 to 8.0.
10.3 Pipet 15 mL of ethylene chloride into the separatory funnel and
shake vigorously for one minute. Allow phases to separate. If
good separation occurs, skip to step 10.6. If poor separation
occurs, empty and clean the separatory funnel and proceed with
step 10.4, etc.
10.4 Filter greater than 250 mL of sample through an 0.8-micron
membrane filter by vacuum filtration. Repeat step 10.3 with the
filtered sample.
10.5 Withdraw the ethylene chloride phase into a 50-mL volumetric
flask. If phase separation is still poor, the ethylene chloride
phase should be passed through a small wad of glass wool in a
funnel before entering the volumetric flask.
10.6 Repeat the extraction with two more 15-mL portions of ethylene
chloride. (If glass wool was used, return it to the separatory
funnel before the next extraction.)
10.7 Dilute the combined ethylene chloride extracts to 50 mL with
ethylene chloride. If a large quantity of water is floating in
the flask, remove it with a dropper and add sodium sulfate to dry
the solution.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Table 1 summarizes the recommended HFLC operating conditions.
12.2 Instrument performance should be checked by injecting on the HPLC
column 500 uL of the standard AZODRIN-BIDRIN mixture prepared in
Step 7.1.2 under the chromatographic conditions (I) described in
Table 1. Peaks should be present at 8.7 and 11.7 minutes.
12.3 For analysis, inject 500 uL of the sample extract prepared in
Step 10.7 into the LC using chromatographic conditions (I)
described in Table 1.
12.4 If no peaks appear at either 8.7 or 11.7 minutes, it may be
assumed that neither AZODRIN, BIDRIN, nor NUDRIN is present at
•greater than 0.1 mg/L (ppm) in the sample (see Figures 1 and 2).
12.5 If a peak appears at 8.7 minutes in the sample, inject 500 uL of
the NUDRIN standard solution prepared in Step 7.1.3 and record
the NUDRIN peak.
12.6 Place a fresh 250-mL portion of the sample in a 500-mL round-
bottom flask. Using the pH meter, add 50%w sodium hydroxide
solution dropwise until a pH of >12 is reached.
132-04 January 1983
-------
12.7 Connect a reflux condenser to the flask and place it in a 90°C
hot water bath for one hour. This will destroy AZODRIN and
NUDRIN.
12.8 Cool and add 15%w sulfuric acid dropwise until a pH of 6-8 is
reached.
12.9 Extract contents of flask with ethylene chloride as in Section
10.
12.10 Repeat step 12.3 with this extract. Mark chromatogram as "LAB
HYDROLYZED." If the peak at 8.7 minutes remains the same as in
the original extract, the peak was not due to NUDRIN or AZODRIN.
If the peak at 8.7 minutes disappeared or was significantly
reduced, determine the AZODRIN as follows.
12.11 Change the LC conditions to chromatographic conditions (II) as
described in Table 1.
12.12 Rerun the original sample extract from Step 10.7 and the AZODRIN-
BIDRIN mixture prepared in Step 7.1.2 (See Figures 3 and 4). If
the peak appeared at 11.7 minutes in Step 12.4, BIDRIN is present
and should be quantitated by using the conditions in step 12.12.
13. Calculation
13.1 To determine BIDRIN Insecticide, draw base lines for the 18.6-
minute peak as shown in Figure 3 and 4. Measure the peak height
of the sample and standard peaks. Calculate as follows:
BIDRIN, ppm = S oc5^ „ y
HSTD X 25 X ° X E
where H and H = peak heights of the sample and standard,
& b LU
respectively
C0__ = concentration of insecticide in standard
o 11)
stock solution in mg/5 mL (Step 7.1.1)
D = sample density, g/mL
E = extraction coefficient =0.5
13.2 To determine methomyl (AZODRIN) Insecticide, draw base lines for
the 16.7-minute peak as shown in Figure 3 and 4. Measure the
peak heights for the sample and standard peaks. Calculate as
follows:
H x C
S STD
(methomyl) AZODRIN, ppm = ~
x 25 x D x E
132-05 January 1983
-------
where E, the extraction coefficient = 0.5
13.3 To determine NUDRIN Insecticide, draw a base line for the 8.7-
minute peak of the chromatogram run at 230 nm as in Figure 1.
Measure the peak height; this is due to AZODRIN and NUDRIN. The
portion of this peak due to AZODRIN is obtained by multiplying
the AZODRIN peak height for this sample,.found in Step 13.2, by
1.08. This response factor is the ratio of peak heights for a
given AZODRIN sample determined by the two methods. Subtract
this value from the total peak height measured in this step to
get the peak height due to NUDRIN.
13.4 Calculate the NUDRIN Insecticide as follows:
HSC X CSTD
NUDRIN, ppm ~
HST]) x 25 x 4 x 4
where Hgc is the corrected sample peak height from step 13.3.
E is the extraction coefficient "0.7
14. Method Performance
Not Available.
15. References
15.1 "Determination of NUDRIN, AZODRIN, and BUDRIN Insecticides in
Aqueous Waste Streams by Liquid Chromatography," JH-LC-14A/80,
Shell Chemical Company, Denver, CO.
132-06 January 1983
-------
Table 1
Chromatographic Conditions
I
Column
Mobile Phase
Flow Rate
Detection
10-micron Partisil-PAC 250- x 3.2-mm
15% v/v isopropanol/heptane
2 mL/min
UV at 230 nm
Retention Times: methorny1 (NUDRIN)
monocrotophos (AZODRIN)
dicrotophos (BIDRIN)
8.7 min
8.7 min
11.7 min
Column
Mobile Phase
Flow Rate
Detection
II
10-micron Partisil-PAC 250- x 3.2-mm
5%/15%/80% volume ratio
isopropanol/tetrahydrofuran/heptane
2 mL/min
UV at 215 nm
Retention Times: monocrotophos (AZODRIN)
dicrotophos (BIDRIN)
16.7 min
18.6 min
132-07
January 1983
-------
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January 1983
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January 1983
-------
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132-10
January 1983
-------
301 , : I I I • 0 « i I : • : Q
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132-11
Januarv 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF METHOMYL
IN WASTEWATER
METHOD 133
1. Scope and Application
1.1 This method covers the determination of methomy1.
Parameter Storet No. CAS No.
Methomyl 39051 16752-77-5
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater.
1.3 The method detection limit (MDL) for methorny1 is 0.01 ug/g (ppm)
using a 100-g sample. The MDL for a specific wastewater may
differ from that listed, depending upon the nature of
interference in the sample matrix.
2. S""""TY of Method
2.1 A 100-g aliquot of wastewater (100 mL) is extracted with hexane.
The hexane layer is discarded. The aqueous phase is hydrolized
with NaOH, acidified, and extracted with ethyl acetate. The
extract is dried and concentrated to a final volume of 1 mL by
rotary evaporation. Analysis is by gas chromatography with flame
photometric detector in the sulfur mode.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences.
3. Interferences
3.1 This method is not compound specific since an oximino compound,
methyl N-hydroxythioacetimidate, or any compounds capable of
hydrolyzing to methyl N-hydroxythioacetimidate will analyze as
methomyl. If unusually or surprisingly high results are
133-01 January 1983
-------
obtained, additional analytical studies will be necessary to
identify the source and/or to determine methomy1 specifically.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnel, 250 mL
5.2.2 Beaker, 30 mL and 400 mL
5.2.3 Vacuum rotary evaporator
5.2.4 Volumetric flask, 1 mL
5.3 Steam bath
5.4 Gas Chromatograph with flame photometric detector
6. Reagents
6.1 Hexane
6.2 IN NaOH
6.3 IN H2S04
6.4 Ethyl acetate
6.5 Triethylamine
7. Calibration
7.1 GC conditions
7.1.1 Establish chromatographic conditions as presented in
references 15.1 and 15.2.
7.2 Calibration Procedure
7.2.1 Calibration procedures are given in references 15.1 and
15.2.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
133-02 January 1983
-------
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Transfer a 100-g sample into a 250-mL separatory funnel, add 50
mL of n-hexane, shake for 2 minutes, and allow the phases to
separate. Discard the hexane layer. Repeat the hexane wash using
a second 50-mL portion of n-hexane. Transfer the aqueous phase
to a 400-mL beaker. Add 10 mL of IN NaOH, cover, and heat on a
steam bath for 20 minutes. Cool, acidify with 15 mL of IN t^SO,,
and quantitatively transfer the solution to a 250-mL separatory
funnel using several small volumes of water as wash.
10.2 Extract the aqueous hydrolyzate with three 100-mL portions of
ethyl acetate using 2-minute shaking periods for each extraction.
Allow the phases to separate and filter the ethyl acetate phase
through anhydrous sodium sulfate into a 500-mL round-bottomed
flask.
10.3 Add 0.3 mL of triethylamine to the combined extracts and
concentrate the solvent to about 10 mL using a vacuum rotary
evaporator at 16 C. Quantitatively transfer the concentrated
extract to a 30-mL beaker using several small volumes of ethyl
acetate as wash. Add 0.1 mL more of triethylamine and carefully
concentrate the solution to about 0.5 mL by evaporation at room
temperature in a well-ventilated hood. [Do not allow the solvent
to go to dryness.3 Transfer the concentrated extract to a 1-mL
volumetric flask using a fine-tip dropper and several small
washes of ethyl acetate. Dilute to volume with ethyl acetate and
mix thoroughly.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Equilibrate the gas chromatograph (the flame photometric detector
is preferred) and inject an aliquot of the prepared extract as
described in the reference methods (15.1 and 15.2). Measure the
peak height of the oximino fragment and determine the micrograms
of this material in the aliquot using a previously prepared
calibration curve.
13. Calculations
13.1 Calculate the amount of methomyl present in ug/g (ppm) by
dividing the micrograms found, corrected for the molecular weight
conversion (1.54), and aliquot factors by the sample weight in
grams.
133-03 January 1983
-------
14. Method Performance
14.1 The method has a sensitivity of 0.01 ug/g (ppm) using a 100-gram
sample.
15. References
15.1 Determination of Methorny1 Residue Using Microcoulometric Gas
Chromatography by H.L. Pease and J.J. Kirkland, J. Agr. Food
Chem., 16, 554 (1968).
15.2 Modification of the Published Gas Chromatographic Method for
Determination of Methorny1 Residues: Use of a Flame Photometric
Detector by H.L. Pease, Unpublished, 1969.
15.3 "Determination of Methomyl in Plant Aqueous Waste Effluent,"
Standard Test Method, Vertac Chemical Corporation, Memphis, TN.
133-04 January 1983
-------
f/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF MEVINPHOS
IN WASTEWATER
METHOD 134
1. Scope and Application
1.1 This method covers the determination of mevinphos.
Parameter Storet No. CAS No.
Mevinphos 39610 298-01-1
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater and
plant effluent.
1.3 The method detection limit for mevinphos is 100 ug/L (ppb). The
MDL for a specific wastewater may differ from that listed,
depending upon the nature of interferences in the sample matrix.
2. s»tfiiniarv of Method
2.1 A measured volume of water sample (100 mL) is diluted with
saturated NaCl solution and extracted with isopropyl acetate.
The extract is brought to 100 mL, dried, and analyzed by gas
chromatography with electron capture detection.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
134-01 January 1983
-------
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Graduated cylinder, 500 mL
5.2.2 Assorted volumetric glassware and pipets as needed for
samples and standards.
5.3 Gas Chromatograph
5.3.1 Hewlett Packard 5710A with electron capture detector, 2-ft
x 2-mm glass column containing 10% EGSS-X coated on 100/120
mesh Chromosorb W-HP.
6. Reagents
6.1 Isopropyl acetate, nanograde
6.2 Sodium sulfate
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1
7.2 Calibration Procedure
Not Available.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 The amount of solution was measured and recorded. A 100-mL
aliquot was placed in a 250-raL separatory funnel. Ten mL
saturated NaCl solution was added, and 50 mL isopropyl acetate
was added for extraction. The water layer was drained, and the
isopropyl acetate layer was placed in a 100-mL glass-stoppered
graduated cylinder. The water layer was then reextracted twice
with 20 mL isopropyl acetate. The extracts were combined in the
graduate. The extracts were brought to volume, and sufficient
sodium sulfate was added to dry the solvent completely.
11. Cleanup and Separation
134-02 January 1983
-------
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommmended operating conditions for the
gas chromatograph.
13. Calculations
Not Available.
14. Method Performance
14.1 Recoveries were in excess of 90 percent.
14.2 Limits of detection 100 ug/L (ppb).
15. References
15.1 "Mevinphos in Waste Water," Standard Test Method, Amvac Chemical
Corporation, Los Angeles, CA.
134-03 January 1983
-------
table 1
Chromatographic Conditions
Column Temperature 150 C
Detector Temperature 300 C
o
Injector Temperature 200 C
Carrier/Flow Rate N2, 50 mL/min
Column: 2-ft x 2-mm glass, 10% EGSS-X coated on 100/120 mesh Chromosorb W-
HP
Retention Time (Mevinphos) 51 sec
134-04 January 1983
-------
V-/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF PROFLURALIN
IN WASTEWATER
METHOD 135
Scope and Application
1.1 This method covers the determination of profluralin.
Parameter Storet No. CAS No,
Profluralin — 26399-36-0
1.2 This is a thin-layer chromatographic (TLC) method applicable to
the determination of the compound listed above in wastewater.
1.3 The method detection limit (MDL) for profluralin is 6 mg/L
(ppm). The MDL for a specific wastewater may differ from that
listed, depending upon the nature of interferences in the sample
matrix.
Summary of Method
2.1 An aliquot of sample (50 mL) is extracted with chloroform. The
chloroform layer is isolated and used for spotting pre-coated TLC
plates. The spots are eluted with benzene/chloroform/ethyl
acetate (21 ill). Detection is by air drying, exposure to
chlorine gas, and spraying with a starch solution.
4.
4.1 Special precautions must be taken to avoid exposure to Cl» gas.
Spraying must be done in a hood. The TWA for benzene is 1 ppm in
135~01 January 1983
-------
air and is a suspected human carcinogen. Precautions must be
taken to preclude exposure to benzene vapors.
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Capillary tubes — Kimax 1.6, 1.8- x 100-mm
5.2.2 Chamber (25 cm x 29 cm x 10 cm)
5.2.3 Plates 200- x 200-mm glass
5.3 Plate: Silica Gel G, neutral (50Z Merck Silica Gel G, 50Z Bio
Rad Bio Sil-A) on 200-x 200-mm glass plates coated at a thickness
of 20 u.
6. Reagents
6.1 Silica Gel G, neutral (50Z Merck Silica Gel G, 50Z Bio Rad Bio
Sil-A)
6.2 Chloroform
6.3 Profluralin Standard
6.4 Benzene
6.5 Ethyl alcohol
6.6 Starch
6.7 Potassium iodide
7. Calibration
7.1 Establish TLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 0.1000 g of profluralin of 99+2 purity into a 100-mL
volumetric flask. Dilute this stock standard to the mark
with CHCL, and carry out a serial dilution to the fifth
dilution. These solutions are equivalent to 200, 100, 50,
25, 12.5, and 6 mg/L when compared to samples extracted
5:1.
135-02 January 1983
-------
7.2.2 Fresh standard solutions should be prepared once per month.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Extract 50-mL samples with 10 mL CHC13 in a 125-mL separatory
funnel. Isolate the CHC1, layer for spotting.
11. Cleanup and Separation
Not Available.
12. Thin Layer Chromatographv
12.1 Normal principles of thin-layer chromatography apply for this
analysis. Table 1 summarizes the recommended operating
conditions for TLC.
12.2 Make one application using sample and standards using open-tip
capillary tubes (7-8 uL), capillary tubes from Kimax (size 1.6,
1.8- x 100-mm).
12.3 After elution, air dry plate and expose to chlorine gas in a
saturated chamber for 30 seconds. Remove plate and after 2
minutes spray with a starch solution prepared as follows:
12.3.1 Weigh 1.5 g starch into a beaker and add 50 mL water.
12.3.2 Weigh 0.5 g potassium iodide into another beaker and
add 50 mL water.
12.3.3 Measure 20 mL 3A alcohol.
12.3.4 Heat solution A until starch goes into solution, then
add KI solution and alcohol.
13. Calculations
Not Available.
14. Method Performance
14.1 The standard deviation of an analysis is approximately 30%
relative to actual concentration.
135-03 January 1983
-------
14.2 A statement of "not detected" (ND) for profluralin indicates only
that the profluralin content is less than 6 ppm.
15. References
15.1 "TLC Determination of Profluralin in Wastewater," Method No. ETM-
154-1, June 6, 1977, Ciba-Geigy, AL.
135-04 January 1983
-------
Table 1
Chromatographic Conditions
Eluent Benzene/chloroform/ethylacetate 2/2/1 (by volume)
Chamber: Supersaturated (filter paper lining)
Chamber size - 25 cm x 29 cm x 10 cm
Plate: Silica Gel G, neutral (50% Merck Silica Gel G, 50% Bio Rad Bio-Sil-
A), 200-mm x 200-mm, 20-micron
Temperature Ambient
Distance 15 cm
Time 35 minutes
Rf (profluralin) 0.7
Point of Origin (Rf) 0.0
Note: A fresh eluent should be prepared each day and should be allowed to
equilibrate 1 hour before use.
135-05 January 1983
-------
SEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF SIMETRYN
IN WASTEWATER
METHOD 136
1. Scope and Application
1.1 This method covers the determination of triazine pesticides. The
following parameters can be determined by this method.
Parameter Storet No. CAS No.
Simetryn 39054 1014-70-6
Prometryn 39057 7287-19-6
Ametryn — 834-12-8
1.2 This is a thin-layer chromatographic (TLC) method applicable to
the determination of the compounds listed above in wastewater.
1.3 The method detection limit (MDL) for each of the parameters is 1
mg/L (ppm). The MDL for a specific wastewater may differ from
that listed, depending upon the nature of interferences in the
sample matrix.
2. Summary of Method
2.1 An aliquot of sample (50 mL) is extracted with chloroform.- The
chloroform layer is isolated and used for spotting on a pre-
coated silica gel plate. Elution is with benzene/chloroform/
ethyl acetate (2/2/1). Detection is by air drying, exposure to
chlorine gas, and spraying with a starch solution.
3. Interferences
Not Available.
136-01 January 1983
-------
4. Safety
4.1 Special precautions must be taken to avoid exposure to C12 gas.
Plate spraying must be done in a hood. The time weighted average
for benzene is 1 ppm in air and benzene is a suspected human
carcinogen. Precautions must be taken to preclude exposure to
benzene vapors.
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnel, 125 mL
5.2.2 Chamber (25 cm x 29 cm x 10 cm)
5.2.3 Capillary tubes (Kimax 1.6 - 1.8 x 100 mm)
5.2.4 Glass plates (200 x 200 mm)
5.3 Plate: Silica Gel G, neutral (50% Merck Silica Gel G, 50% Bio
Rad Bio Sil-A) on 200- x 200-mm glass plates coated at a
thickness of 200 u.
6. Reagents
6.1 Chloroform
6.2 Silica Gel G, neutral (50% Merck Silica Gel G, 50% Bio Rad Bio
Sil-A)
6.3 Prometryn, Ametryn, and Simetryn Standards
6.4 Benzene
6.5 Ethyl Acetate
7. Calibration
7.1 Establish TLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 0.0160 g each of prometryn, ametryn, and simetryn of
99+% purity into a 100-mL volumetric flask. Dilute this
stock standard to the mark with CHCl. and carry out a
serial dilution to the fifth dilution. These solutions are
136-02 January 1983
-------
7.2.2 Fresh standard solutions should be prepared once per month.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly extract 50 mL water sample with 10 mL CHCl^ in a 125-
mL separatory funnel. Isolate the CHC1, layer for spotting.
11. Cleanup and Separation
Not Available.
12. Thin Layer Chromatographv
12.1 Table 1 summarizes the recommended operating conditions of the
TLC.
12.2 Prepare calibration standards as discussed in Section 7.
12.3 Make one application of samples and standards using open-tip
capillary tubes (7-8 u), using capillary tubes from Kimax (size
1.6-1.8 x 100 mm).
12.4 After elution, air dry plate and expose to chlorine gas in a
saturated chamber for 30 seconds. Remove plate and after 2
minutes spray with a starch solution prepared as follows:
12.4.1 Weigh 1.5 g starch into a beaker and add 50 mL water.
12.4.2 Weigh 0.5 g potassium iodide into another beaker and
add 50 mL water.
12.4.3 Measure 20 mL 3A alcohol.
12.4.4 Heat solution A until starch goes into solution; then
add KI solution and alcohol.
13. Calculations
Not Available.
14. Method Performance
14.1 A statement of "not detected" (ND) for a component indicates only
that the component is less than 1 ppm.
136-03 January 1983
-------
14.2 The standard deviation of an anlaysis is 30% relative to actual
concentration.
15. References
15.1 "TLC determination of Prometryn, Ametryn, and Simetryn in
Wastewater," Method No. ETM-51-1, June 6, 1977, Ciba-Geigy, AL.
136-04 January 1983
-------
Eluent
Chamber
Temperature
Distance
Time
Rf (simetryn)
Rf (ametryn)
Rf (prometryn)
Rf (point of origin)
Table 1
Chromatographic Conditions
Benzene/chloroform/ethylacetate 2/2/1 (by volume)
Supersaturated (filter paper lining) Chamber size
25 cm x 29 cm x 10 cm
Ambient
15 cm
35 min
0.2
0.3
0.4
0.0
Note: A fresh eluent should be prepared each day and should be allowed to
equilibrate 1 hour before use.
136-05
January 1983
-------
AEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF TRIADIMEFON [BAYLETON(TM)]
IN WASTEWATER
METHOD 137
1. Scope and Application
1.1 This method covers the determination of triadimefon.
Parameter Storet No. CAS No.
Triadimefon (BAYLETONtTMj) — 43121-43-3
1.2 This is a thin-layer chromatographic (TLC) method applicable to
the determination of the compound listed above in wastewater.
2. Su™narv of Method
2.1 A measured volume of water sample (25 mL) is extracted with
chloroform. The chloroform layer is spotted on pre-coated silica
gel plates. The chromatogram is developed with toluene/ethyl
acetate (9/1). The plate is sprayed with IN methanolic sodium
hydroxide and later with p-nitrobenzene diazonium fluoborate
(NBDF), 0.2Z metbanol-water 1:1. The plate is then immersed in
brown bromine fumes. Triadimefon is estimated by visual
comparison and known standards spots.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
137-01 January 1983
-------
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Syringes, 10 uL and 50 uL
5.2.2 Plates, silica gel, E. Merck F0254
5.2.3 Funnel, separatory, 60-mL
5.3 Heat gun
5.4 Oven, 100°C
5.5 Tank, bromine
5.6 Tank, chromatographic
5.7 Spray bottle
6. Reagents
6.1 Triadimefon (BAYLETON) standard, 0.1% in acetone
6.2 Bromine, elemental
6.3 Chloroform, ACS
6.4 Methanelie sodium hydroxide, IN — dissolve 4 g of ACS sodium
hydroxide pellets in 100 mL of methanol
6.5 Mobile solvent, toluene-ethyl acetate 9:1
6.6 p-Nitrobenzenediazonium fluoborate (NBDF), 0.2% in methanol-water
1:1 — prepare fresh before use
7. Calibration
7.1 Establish TLC operating conditions equivalent to those indicated
in Tab le 1.
7.2 Calibration Procedure
7.2.1 Apply 10-, 25-, 50-, and 100-uL portions of the chloroform
extract and the following amounts of standard to an E.
Merck F-254 silica gel plate. Use a heat gun on the cool
setting to keep the spots small.
137-02 January 1983
-------
0.1% BAYLETON Corresponding BAYLETON. pom, in Sample
uL
1.0
2.5
5.0
7.5
10.0
10 uL
20
50
100
150
200
25 uL
8
20
40
60
80
50 uL
4
10
20
30
40
100 uL
2
5
10
15
20
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Pipet 25 mL of the aqueous sample and 5 ml of chloroform into a
60-mL separatory funnel.
10.2 Stopper the funnel and shake vigorously for 3 minutes.
10.3 Allow the layers to separate; then drain the chloroform layer
into a 5-mL glass-stoppered graduated cylinder.
11. Separation and Cleanup
Not Available.
12. Thin Layer Chromatography
12.1 Develop the chromatogram with toluene-ethyl acetate 9:1 mobile
solvent until the front is near the top.
12.2 Remove the plate and allow it to dry in a hood.
12.3 Spray the plate with IN methanolic sodium hydroxide and allow the
plate to air dry for 15 minutes.
12.4 Spray the plate heavily with freshly prepared NBDF and allow the
plate to sit for 5 minutes.
12.5 Immerse the plate in brown bromine fumes.
12.6 Respray the plate with IN methanolic sodium hydroxide.
12.7 Respray the plate with NBDF, then heat it at 110 C for 10
minutes. Triadimefon (BAYLETON) appears as reddish spots at
approximately Rf 0.1.
137-03 January 1983
-------
13. Calculations
13.1 Estimate the BAYLETON in the sample by visual comparison with the
known standard spots.
13.2 If the BAYLETON content in the sample is higher than the highest
standard, dilute a portion of the chloroform extract from Step
10.3 at a 1:10 ratio with chloroform and repeat the test. In
this case, multiply the apparent content by 10.
14. Method Performance
Not Available.
15. References
(R)
15.1 "Determination of Bayleton in Waste Water," Analytical Method
TM B-34,45, Chemagro Agricultural Division, Mobay Chemical
Corporation, Kansas City, MO.
137-04 January 1983
-------
Table 1
Chromatographic Conditions
Eluent Toluene/ethyl acetate 9/1 (by volume)
Plate Silica-gel, E.- Merck F-254
Rf (triadimefon)• 0.1
137~05 January 1983
-------
xvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF TRICHLORONATE [AGRITOX(TM)]
IN WASTEWATER
METHOD 138
1. Application and Scope
1.1 This method covers the determination of trichloronate.
Parameter Storet No. CAS No.
Trichloronate [AGRITOX(TM)] — 327-98-0
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater samples
containing 0.005 - 1 ppm trichloronate.
2. Summary of Method
2.1 A measured volume of water sample (250 mL) is adjusted to pH 6.5
- 7.0 and extracted with chloroform. The extract is partitioned
with distilled water, dried, and rotary evaporated to dryness.
The residue is dissolved in acetone and reacted with potassium
permanganate. The mixture is diluted with water and extracted
with chloroform. The extract is then partitioned with distilled
water, dried, and rotary evaporated to dryness. The residue is
dissolved in acetone and analyzed by gas chromatography using a
phosphorus (thermionic) detector.
3. Interference
3.1 All glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENT.
138-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 10-uL
5.2.2 Rotoevaporator, Buchi or equivalent
5.3 Water bath, 50-55°C
5.4 Gas chromatograph, Varian 1400 or equivalent, equipped with a
phosphorus detector (rubidium sulfate salt pellet), a 3-ft x 1/8-
inch O.D. glass column packed with 3% OV-225 on Gas Chrom Q
80/100 mesh, and glass injector and detector inserts. (If the
column ends are long enough to be used as inserts, the
glass inserts are not necessary.) Note: Use only Teflon-backed
septa, Supelco No. 2-0459.
6. Reagents
6.1 Acetone, nanograde
6.2 Trichloronate (AGRITOX) standard, technical purity (82-85%)
6.3 Chloroform, nanograde
6.4 Hydrochloric acid, IN — Carefully add 21 ml of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.5 Magnesium sulfate, 20% — Weigh 41 g of magnesium sulfate
heptahydrate (MgSO,.7H20) into a 100-mL glass-stoppered graduated
cylinder. Add distilled water to the 100-mL mark, stopper, and
mix.
6.6 Mineral oil solution, 2.5% — Dilute 25 mL of mineral oil, Fisher
#0-120 or equivalent, to 1000 mL with nanograde chloroform.
6.7 Potassium permanganate, 0.1M — Weigh 1.6 g of potassium
permanganate, ACS into a 100-mL glass-stoppered graduated
cylinder. Dilute to 100 mL with distilled water and mix
thoroughly.
6.8 Sodium hydroxide, IN - TM A-28.3
138-02 January 1983
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6.9 Sodium sulfate, anhydrous, ACS
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 AGRITOX standard solution, 0.10Z — Weigh 0.120 - 0.130 g
of technical AGRITOX (82 - 85% purity) into a 100-mL glass-
stoppered graduated cylinder. Dilute to volume with
acetone, stopper, and mix thoroughly. This solution may be
used for one week only.
7.2.2 Pipet a 1-mL aliquot of the 0.10% AGRITOX solution into a
100-mL volumetric flask, dilute to volume with acetone, and
mix thoroughly. Label this solution "10 ng." Prepare
fresh daily.
7.2.3 Pipet 5-, 10- and 20-mL aliquots of the 10-ng standard from
Step 7.2.2 into separate 100-mL volumetric flasks. Dilute
to volume with acetone and mix. Label these solutions
500, 1000, and 2000 pg, respectively. Prepare fresh daily.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately to
Step 10.2.
10.2 Measure 250 mL of the well-mixed sample into a 500-mL separatory
funnel.
10.3 If the pH is below 6.5 or above 7.0, adjust it to 6.5 - 7.0 with
IN sodium hydroxide or IN hydrochloric acid using pH indicator
paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel.
If an emulsion forms, centrifuge the emulsified layer and add the
clear chloroform layer obtained by centrifuging to the second
separatory funnel. Return the water layer to the first
separatory funnel.
138-03 January 1983
-------
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minute.
10.6 Filter the chloroform layer through a funnel containing 4 to 5
g of anhydrous sodium sulfate retained by a small glass-wool plug
(pre-rinsed with 10 mL of chloroform), into a 300-mL 24/40
boiling flask. Rinse the sodium sulfate three times with 10 mL
of chloroform, and add 10 mL of 2.5% mineral oil solution into
the flask.
10.7 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water
cool to room temperature.
chloroform, using a water bath at 50 C. Remove the flask and
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Add 5 mL of nanograde acetone into the sample flask. Swirl the
flask to wash the inside wall with the acetone but do not allow
the acetone to get on the joint or splash out.
10.10 Add 10 mL of the magnesium sulfate solution to the flask and
swirl to mix.
10.11 Add 25 mL of 0.1M potassium permanganate solution and swirl.
Allow the sample to stand for 30 minutes, making sure that there
is an excess of potassium permanganate (as indicated by the
purple color) the entire time. (Add the permanganate in 5-mL
increments, if necessary, to maintain an excess; increase the
standing time 5 minutes for each addition).
10.12 Quantitatively transfer the sample solution from Step 10.11 into
a 500-mL separatory funnel, using five 40-mL portions of
distilled water.
10.13 Add 50 mL of chloroform into the separatory funnel, stopper and
shake vigorously for 1 minute.
10.14 Allow the layers to separate. Drain the chloroform layer into a
clean 250-mL separatory funnel.
10.15 Repeat Steps 10.13 and 10.14 twice for total of 3 extractions.
10.16 Add 25 mL of distilled water to the chloroform extracts, stopper,
and shake for 30 seconds.
10.17 Filter the chloroform layer through another funnel containing 4
to 5 g of anhydrous sodium sulfate and retained by a small glass-
wool plug (pre-rinsed with 10 mL of chloroform) into a 300-mL
boiling flask.
10.18 Rinse the sodium sulfate with three 10-mL portions of chloroform
and combine with the chloroform extract.
138-04 January 1983
-------
10.19 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at SO C. Remove the flask and
cool to room temperature.
10.20 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.21 Pipet 5 mL of acetone into the flask from Step 10.20, stopper
immediately, and rotate the flask so that the acetone washes down
the inside of the flask. DO NOT allow the acetone to get on the
neck or stopper.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
12.3 Continue the analysis according to TM A-54.12 (Mobay reference
method), programming immediately after each injection.
13. Calculations
Not Available.
14. Method Performance
Not Available.
15. References
(R)
15.1 "Determination of AGRITOX in Waste Water by GLC," Analytical
Method TM B-34, 48, Chemagro Agricultural Division, Mobay
Chemical Corporation, Kansas City, MO.
138-05 January 1983
-------
Table 1
Chromatographic Conditions
Attenuation
Cell voltage
Detector temperature
Electrometer range
Gas Flows:
Carrier gas
Air
Hydrogen
Injector temperature
Oven temperature
Initial
Final
Mode
Rate
Upper limit hold
Varian 1400
X32
Flame
230-240
io-12
He, 20 mL/min
230 mL/min
40 mL/min
230-240°C
130°C
240°C
Hold
15 C/min
15
Column: 3-ft x 1/8-in glass packed with 3% OV-225 on Gas Chrom Q
80/100 mesh.
138-06
January 1983
-------
x°/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management1
TEST METHOD
DETEBMINATION OF TRICYCLAZOLE
IN WASTEWATER
METHOD 139
1. Scope and Application
1.1 This method covers the determination of tricyclazole.
Parameter Storet No. CAS No.
Tricyclazole — 41814-78-2
1.2 This is a high-pressure liquid chroma tog rap hie (HPLC) method
applicable to the determination of the compound above in water.
1.3 The method detection limit (MDL) for tricyclazole is 20 ug/L
using direct aqueous injection and 1 ug/L using solvent
extraction. The MDL for a specific wastewater may differ from
those listed, depending upon the nature of interferences in the
sample matrix.
2 . Summary of Method
2.1 Water samples are filtered and injected directly into a high-
pressure liquid chromatograph (HPLC). Alternatively, the water is
acidified and extracted with dichloromethane. The extract is
evaporated and the residue is dissolved in an appropriate volume
of mobile phase (methanol/water , 50/50, v/v) for injection into
the HPLC. Detection is accomplished with a UV absorbance
detector at a wavelength of 254 nm.
3. Interferences
Not Available.
139-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.3 Analytical filter papers, folded, Schleicher and Schuell, 12.5 cm
(No. 588) or equivalent
5.4 Magnetic stirrer and stirring bar
5.5 A high-pressure liquid chromatograph consisting of the following
components (or equivalent models)
5.5.1 Waters Model 6000A Solvent Delivery System
5.5.2 Waters Model 440 Absorbance Detector (fixed wavelength, 254
run)
5.5.3 Water Intelligent Sample Processor (WISP), Model 710A
5.5.4 Houston Instruments Omni Scribe strip chart recorder, 1-10
mv
6. Reagents
6.1 Solvents
6.1.1 Methanol, HPLC grade, distilled in glass
6.1.2 Water, HPLC grade, free of organic impurities
6.1.3 Dichloromethane, reagent grade, redistilled
6.2 Solutions
6.2.1 HPLC mobile phase — methanol:water (50:50, v/v), filtered
and degassed
6.2.2 Hydrochloric acid, concentrated
6.3 pH paper, range 1-14
6.4 Sodium sulfate, anhydrous, methanol washed
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
139-02 January 1983
-------
7.2 Calibration Procedure
7.2.1 Preparation of Standard Solutions
7.2.1.1 Standard Solution A (tricyclazole 1.0 mg/mL).
Dissolve 100 mg of tricyclazole analytical
standard in methanol in a 100-mL volumetric
flask and dilute to volume.
7.2.1.2 Standard Solution B (tricyclazole 10.0 ug/mL).
Transfer a. 1.0-mL aliquot of Standard Solution
A to a 100-mL volumetric flask and dilute to
volume with HPLC quality water.
7.2.1.3 Standard Solution C (tricyclazole 1.0 ug/mL).
Transfer a 10.0-mL aliquot of Standard Solution
B to a 100-mL volumetric flask and dilute to
volume with HPLC-grade water.
7.2.1.4 Standard Solution D (tricyclazole 0.10 ug/mL).
Transfer a 10.0-mL aliquot of Standard Solution
C to a 100-mL volumetric flask and dilute to
volume with HPLC-grade water.
7.2.1.5 Standard Solution E (tricyclazole 1.0 ug/mL).
Transfer a 10.0-mL aliquot of Standard Solution
B to a 100-mL volumetric flask and dilute to
volume with methanol:water (50:50).
8. Quality Control
8.1 Prepare duplicate recovery samples by fortifying two 200-mL
aliquots of untreated water with 1.0 mL of Standard Solution C.
Prepare these samples using the solvent extraction technique
(Step 10.2) and analyze.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
Note: Samples may be prepared for analysis by one of the following
techniques, depending upon the detection limit required for the
assay.
10.1 Direct Injection Technique
10.1.1 Remove particulate matter from the sample by
filtering an aliquot of water through folded filter
paper into a 20-mL screw cap vial, and transfer
approximately 3 mL of filtered water into a WISP
139-03 January 1983
-------
sample vial. (Alternatively, filter directly into the
WISP sample vial.)
10.1.2 Cap the vial with a self-sealing septum cap and
analyze the samples using the HPLC conditions listed
in Table 1.
10.2 Solvent Extraction Technique
10.2.1 Transfer an aliquot of water (normally 200 mL) to a
250-mL separatory funnel. Acidify the water by
adding 0.5 mL of concentrated hydrochloric acid, and
check with pH paper to ensure that the pH of the
water is 3 or less.
10.2.2 Extract the water by partitioning with three 20-mL
aliquots of dichloromethane for at least 20 seconds
each.
10.2.3 Combine the dichloromethane (lower phase) extracts by
passing through a funnel containing sodium sulfate
into a 250-mL evaporating flask.
10.2.4 Evaporate the dichloromethane just to dryness on a
rotary vacuum evaporator with a 35 -45 C water bath.
10.2.5 Dissolve the residue in 2.0 mL of HPLC mobile phase
(normally methanol:water, 50:50) and analyze the
solutions using the HPLC conditions listed in Table
1.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph for both the direct injection and solvent
extraction techniques.
12.2 Calibrate the system as discussed in Section 7.
12.3 Measure the HPLC peak height (or area) for tricyclazole in water
samples using the instrumentation listed in Section 5 and the
parameters on Table 1. (Note: The parameters listed below may
be modified as needed to compensate for daily variations in
instrument performance. Also, the injection volume and/or the
absorbance sensitivity setting may be adjusted to keep peak
responses "on scale" for a given set of samples. The parameters
used for each set of samples should be recorded.)
13. Calculations
139-04 January 1983
-------
13.1 Calculations for Direct Injection Samples From Step 10.1
PR,
Pr
PRsa x C
mg/L (ppm) tricyclazole = ~ std
'std
2
where PR = peak response (cm height or cm area) for sample
Sfl. '
PR . " peak response for direct standard
C . - concentration (ug/mL) of direct standard
13.2 Calculations for Extracted Samples from Step 10.2
PR
11 t i T> .. n rec x C x V_ x 100 percent
13.2.1 Percent Recovery « rr std F *
IT 1C .
std __^
ug fortified
where PR = peak response for recovery sample
16C
VF = final volume (mL), including dilutions
PR
,, 9 0 ,. ( *. _ . , . §a_ x C x V_ x 100 percent
13.2.2 mg/L (ppm) tricyclazole * ^ std F
std
V, x Percent Recovery
where Vj - initial volume (mL) of water extracted
14. Method Performance
14.1 The method for the direct injection of water samples into the
HPLC has a detection limit of approximately 0.020 mg/L of
tricyclazole in water. If a lower detection limit (0.001 mg/L)
is required for the assay, the dichloromethane extraction
procedure should be employed. A summary of the recoveries
obtained during validation of the two methods is presented on
Table 2.
15. References
15.1 "Determination of Residue Levels of Tricyclazole in Water by High-
Pressure Liquid Chromatography," Method No. AM-AA-CA-J037-AB-755,
Eli Lilly and Company, Greenfield, IN.
139-05 January 1983
-------
Table 1
Cbromatographic Conditions
Parameters for Direct Injection (From Step 10.1)
Precolumn
Column
Mobile Phase
Flow rate
Injection volume
Absorbance sensitivity
Chart speed
Direct standard
CO-PELL ODS or equivalent
uBond pak C.g (10-uM particle size,
3.9-mm I.D. x 30 cm) or Lichrosorb RP-
18 (10-uM particle size, 4.6-mm ID x 25
cm)
methanol:water (50:50, v/v)
0.8-1.5 mL/min (adjust to achieve the
desired retention time)
1000 uL
0.01-0.02 AUFS
0.25 cm/min
Standard Solution D
Parameters for Concentrated Extracts (From Step 10.2)
Precolumn
Column
Mobile phase
Flow rate
Injection volume
Absorbance sensitivity
Chart Speed
Direct Standard
same as above
same as above
same as above
same as above
200 uL
0.02 AUFS
same as above
Standard Solution E
139-06
January 1983
-------
Table 2
Summary of Fortified Sample Recoveries
(mg/L) ppm Percent Recovery
Method Fortified N (mean + s.d)
direct injection 0.020 6 101.6 +.3.8
0.200 6 91.5 i 4.0
2.000 6 94.3 + 2.5
extraction 0.001 6 122.7 ±31.4
0.010 6 109.8 i 2.8
0.100 6 94.2 + 3.3
!39-07 January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF GLYPHOSATE
IN WASTEWATER
METHOD 140
1. Scope and Application
1.1 This method covers the determination of glyphosate.
Parameter Storet No. CAS No.
Glyphosate 39941 1071-83-6
1.2 This is a high-performance liquid chromatographic (HPLC) method
applicable to the determination of the compound listed above in
industrial effluent.
1.3 The method detection limit (MDL) is 10 ug/L with 5% of full-scale
deflection. The MDL for a specific wastewater may differ from
that given, depending upon the nature of interferences in the
sample matrix.
2. SiiirnnflTy of Method
2.1 A known volume of glyphosate industrial effluent is applied to a
packed AG50W-X8 (400 mesh, H form, Bio-Rad) cation exchange
primary column via an auto injector. The selected fraction of
column eluent is switched onto the analytical column. The
Technicon Auto Analyzer-based ninhydrin post column reactor is
employed as a detector.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 Ammonia, as well as primary and secondary amines, will have a
significant response with ninhydrin reagent, and, therefore,
140-01 January 1983
-------
prevent detection of the presence of the glyphosate peak in the
chromatogram.
3.2 A sample clean-up using packed AG50W-X8 strong cation exchange
primary column will eliminate the interferences of the above
mentioned species. The regeneration of the primary column is
feasible within the analysis time domain (40 min).
3.3 Equipment used in this analysis (columns, filters, glassware,
etc.) should be limited to trace analysis only to eliminate any
possibility of residual contamination at these low levels.
4. Safety
4.1 Follow EPA safety procedure found in Fart D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Pipets, 10-100 uL, 100-1,000 uL, and 1-5 raL delivery pipets
with disposable tips.
5.2.2 Volumetric flasks, 100 ml or convenient sizes for making
standards or dilution.
5.3 Two buffer pumps, Waters 6000A pump, flow rate 0.6 mL/min.
5.4 Injector, Waters Intelligent Sample Processor (WISP).
5.5 Technicon Proportionation Pump, Ninhydrin reagent reactor.
5.6 Post Column Reactor, Technicon research cartridge or oil bath
controlled at 95°C +. 0.1°C.
5.7 Detector, Technicon Single Channel Colorimeter with 570-nm
optical interference filter and 2.0-mm x 50-mm flow cell.
5.8 Electronic Filter, Spectrum 1021 filter and amplifier.
5.9 Recorder, Fisher, 100 mv, full scale.
5.10 HPLC Analytical Column, DuPont Zorbax SAX, 15 cm x 4.6 mm.
5.11 HPLC Primary Column, stainless steel tubing for LC, 15-cm x 4.0-
mm I.D. (Handy & Barman Tube Co., Whitehall Road, Morristown, PA
19404).
5.12 Bed support, 1/4-in diameter, 15-u pore size (Whatman 4334-2351).
140-02 January 1983
-------
5.13 Bottom Drilled Swagelok fitting, P.J. Coert, 40061-BD.
5.14 Six-Part valve, air activated Valco valve (Model CF-6-UHPa-N60).
5.15 Digital Valve Sequence Programmer, DVSP, switching sequence
controller.
5.16 Cation Exchange Resin for Primary Column, AG50W-X8, 400 mesh, H
form (Bio-Rad 142-1461).
5.17 CDC 1700 Computer System, Sample Injection, DVSP starter and data
collection.
6. Reagents
6.1 Dimethylsulfoxide (Pierce, 20687).
6.2 Ninhydrin (Pierce, 21001).
a
6.3 Lithium Acetate pHix Buffer, 4M Li ; pH 5.20 (Pierce, 27203).
6.4 Hydrindantin Dihydrate (Pierce, 24000).
6.5 Methanol (MCB reagent, MX 0488).
6.6 H3p04 (85%, Fisher Z-242).
6.7 KH2P04 (Mallinckrodt, 7100).
6.8 Brij 35, 30% aqueous solution (Fisher, CS-285-2).
6.9 Argon
6.10 H2S04 (Fisher, A-300).
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 A stock solution of 1,000 ug/mL (ppm) is prepared by
dissolving 100 mg of 3X recrystallized analytical grade of
glyphosate in 100 mL deionized water and stored in the
refrigerator.
7.2.2 A series of working standards of 20, 40, 60, 80, and 100
ug/L (ppb) is prepared weekly by appropriate dilutions of
10 ug/mL (ppm) glyphosate standard solution, which is
prepared from the dilution of 1,000 ug/mL (ppm) stock
solution.
140-03 January 1983
-------
7.2.3 A series of external standards (20-100 ug/L) is prepared
and analyzed under the same HPLC conditions and on the same
day as unknown samples. A blank industrial effluent sample
and water also have to be included to ensure the absence of
glyphosate contamination due to incomplete regeneration of
primary column during analysis.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Samples are filtered via syringe-type filter and ready for
injection.
11. Cleanup and Separation
11.1 A sample clean-up using packed AG50W-X8 strong cation exchange
primary column will eliminate the interferences of the above-
mentioned species. The regeneration of the primary column is
feasible within the analysis time domain (40 min).
11.2 The automated in situ sample clean-up via primary column
essentially eliminates the possible contamination introduced due
to sample manipulation (e.g., evaporation, etc.). The automatic
method provides the minimum sample handling time so a larger
number of samples can be analyzed.
12. Liquid Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the system as des'cribed in Section 7.
12.3 Primary Column Packing
12.3.1 Dimension—15-cm x 6.35-mm O.D. x 4.0-mm I.D.
12.3.2 Packing Material AG50W-X8, 400 mesh, H+ form (Bio-
Rad, 142-1461)
12.3.3 Packing Procedures
1) Prepare a slurry of AG50W-X8 in HPLC buffer solution
(Step 8.2). Decant the free suspension and let stand
overnight before application.
140-04 January 1983
-------
2) Cut 15 cm of stainless steel tubing. File both ends
of tubing and sand to remove burrs.
3) Place bed support in end fitting; insert one end of
column. Tighten nut and ferrule onto end fitting.
4) Fill column from open end with vacuum aspirator to
settle packing material.
5) Attach end fitting with bed support, nut, and ferrule
on open end.
6) Connect to LC pump and run buffer to settle packing.
7) Open column on inlet and check to see if there is a
void. If there is, repeat steps 4-6 until packing
material is packed tight and no voids remain in
column.
12.4 Preparation of Reagent
12.4.1 HPLC Buffer Solution. Dissolve 10 g KH2PO, in 3.8 L
of 160 mL methanol/deionized water; adjust the
solution to pH 2.3 with 85% H PO,. Follow normal
HPLC degassing/filtration procedures.
12.4.2 Ninhydrin Solution. Combine 1,100 mL of
dimethylsulfoxide (DMSO), 800 mL of deionized water,
400 mL of 4.0M (pH 5.2) lithium acetate, and bubble
argon through the solution for approximately 15
minutes. Add 32 g of ninhydrin to the solution with
stirring. In a separate beaker dissolve 1.6 g of
hydrindantin solution in two portions of 50 mL DMSO
with a few mL of previously prepared ninhydrin
solution; add together with argon bubbling. An
additional 15 minutes for argon deoxygenation is
required before the application.
12.4.3 Wetting Reagent for Autoanalyzer. Dissolve 10 mL
Brij in 800 mL of deionized water; add a few drops of
concentrated H^SO, to make the solution acidic.
12.5 Construction of Ninhydrin Reactor
Refer to Figure 1.
12.6 Interfacing Between WISP and DVSP — Refer to Figure 2.
12.7 Interfacing Between DVSP and Six Part Valve — Refer to Figure 3.
DVSP sequence timing is set as follows:
140-05 January 1983
-------
Sequence Time (Minutes)
1 2.0
2 3.0
12.8 Interfacing Between Colorimeter and Spectrum — Refer to Figure
4.
12.9 Configuration of Column Switching Instrumentation — Refer to
Figure 5 .
12.10 HPLC Injection
12.10.1 An alternating injection between sample and standard
is carried out through the analysis to ensure the
complete regeneration of the primary column.
12.11 A typical chromatogram of 100 ug/L glyphosate in 002 sump
synthetic waste is shown in Figure 6. The peak height of
glyphosate standard can be optimized by adjusting switching time
on DVSP as illustrated in Figure 7.
13. Calculations
13.1 The actual concentration of glyphosate in waste sample is
/-,/ v \ (ug/L glyphosate from calibration curve)
ug/L (ppb) - recovery)
13.2 The spiking recovery should be redetermined when a plant
industrial effluent sample is available.
14. Method Performance
14.1 The method was validated over the range of 20-100 ug/L. The
probable linear range is up to 10 mg/L with glyphosate standards.
14.2 The detection limit is 10 ug/L of glyphosate standard with 5% of
full-scale deflection (S/N = 2).
14.3 The laboratory validation of this method was carried out using a
synthetic glyphosate industrial waste sample. The components of
the mixture are listed in Table 2.
14.4 The pooled coefficient of variation (CV) for the analytical
method in the range between 20 and 100 ug/L glyphosate in 002
sump synthetic waste is 0.0775.
14.5 The average values obtained for glyphosate in 002 sump synthetic
waste are invariably 30% less than the "true value." The
difference between the "found" and "true" concentrations is not
due to a random variation of the experimentally determined "true"
concentration but rather due to the actual constant sampling
variation of glyphosate in the primary column. Therefore, the
recovery should be applied to the final results in step 10.
140-06 January 1983
-------
15.1 "Automated Method for the Determination of Glyphosate in
Industrial Effluents via Column Switching Technique," Standard
Test Method, Monsanto Agricultural Products Co., St. Louis, MO.
140-07 January 1983
-------
Table 1
Chromatographic Conditions
HPLC Operation Conditions for Both Columns
Buffer Flow Rate
WISP Operation Parameters
Injection Volume
Run Time
Number of Injections
Detector
Electronic Filter - Cutoff Frequency
Attenuation
Recorder
Primary Column
Analytical Column
Mobile Phase
0.6 mL/min
1,000 uL
25 minutes
1
DAMP 2, Std. Cal. 1.00
0.01
2.0
Chart Speed 0.25 cm/min
15-cm x 4.0-mm AG 50W-X8, 200-400 mesh,
H form
15-cm x 4.6-mm DuPont SAX
lOg KH2P04, 4% Methanol, pH = 2.3 with
85% HPO dissolved in 4L of water
140-08
January 1983
-------
Table 2
The Composition of the Synthetic Glyphosate
Industrial Waste Sample
Component
Glyphosate Intermediate Raw Material
NO,
SO
2-
Composition (ppm)
30
30
100
100
200
Cl
Na
Ca
2+
Mg
Monoethanolamine
200
200
50
10
10
140-09
January 1983
-------
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140-10
January .1983
-------
FIGURE 2.
Electrical Connections for Digital Valve Sequence
Programmer and WISP
DVSP
PIN 18 9
o PIN 34 (Ground)
PIN 35
o 6
INTEG
START
(Back)
WISP
140-11
January 1983
-------
FIGURE 3.
Electrical Connections for Six Port Valve and .Digital
Valve Sequence Programmer
Six Port Valve (White)
o
o
o
o
o
o
o
o
o
o
o
o
o
o
Six Port (Black)
o
o
o
o
o
o
o
o-
o
o
o
o
o
o
110 volts AC
140-12
January 1983
-------
FIGURE 4.
The Electrical Connections between Spectrum and
Colorimeter Recorder
4 Port Plug
Technicon
Colorimeter
Input
OiatAut
Spectrum
e e
Recorder
140-13
January 1983
-------
Figure 5
"The Configuration of Column Switching Instrumentation
HPLC
BUFFER
PUMP A
POSITION 1
WASTE
WISP
PRIMARY
COLUMN
HLPC
BUFFER
PUMP B
PCR
DETECTOR
ANALYTICAL
COLUMN
Q 6
DVSP
WASTE
HPLC
BUFFER
PUMP A
CDC 1700
POSITION 2
WISP
PRIMARY
COLUMN
HLPC
BUFFER
PUMP B
PCR
DETECTOR
ANALYTICAL
COLUMN
140-14
DVSP
WASTE
January 1983
-------
>e cc-ioo
HOUSTON iN
FIGURE 6.
Chromatogram of 100 ppb Glyphosate via
Column Switching Technique
140-15
January 1983
-------
J.... I .._•— J
: o , o
!. J ._.
cu
CO
o
c
o
rH
(0
0)
c
M
s
•H
EI
•H
4J
D)
3
Cu
0)
(0
c
o
a
CO
0)
10
0)
c
o
•H
Jj
N
•H
£
•H
CU
o
140-16
January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D,C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF HEXAZINONE, TERBACIL, AND BROMACIL
IN WASTEWATER
METHOD 141
1• Scope and Application
1.1 This method covers the determination of certain organonitrogen
pesticides. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Bromacil 82198 314-40-9
Hexazinone — 51235-04-2
Terbacil — 5902-51-2
1.2 This is a gas chromatographic-mass spectrometric (GC/MS) method
applicable to the determination of the compounds listed above in
effluent and waste streams.
1.3 The method detection limits (HDL) are 10 ug/L (bromacil) and 1
ug/L (hexazinone and terbacil). The MDL for a specific
wastewater may differ from those listed, depending upon the
nature of interferences in the sample matrix.
2. Summary of Method
2.1 A measured volume of water sample (1000 mL) is extracted with
methylene chloride. The extract is evaporated to 5 mL using a
stream of nitrogen and 45 -50 C heat from a hot plate. Analysis
is by GC/MS.
3. Interferences
Not Available.
141-01 January 1983
-------
4.
4.1 All pesticides should be handled with care, avoiding any skin
contact. Waste streams should be handled with care, avoiding any
skin contact.
4.2 Although these pesticides are not considered to be highly toxic
samples may irritate the eye, nose, throat, and skin:
. methoxychlor; LD in mice, 6000 mg/kg
. chloroneb; LD in rats, 11,000 mg/kg
. terbacil; LD in rats, 5000-7000 mg/kg
. hexazinone; LD in rats, 1690 mg/kg
. bromacil; LD,_ in rats, 5200 mg/kg
4.3 Therefore, avoid personal contact and breathing of dust or
contact with organic solutions. Perform all operations in a well
ventilated area or hood, and clean up all spills promptly.
4.4 See EPA safety procedure found in Part D of this document.
Apparatus and Materials
5.1 S amp1ing Equ ipmen t
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Graduated cylinder, 100 mL
5.2.2 Separatory funnel, 2000-mL capacity, pear-shaped with
Teflon stockcock.
5.2.3 Syringe, 10-uL capacity; Hamilton No. 801N., Supelco Inc.,
Bellafonte, PA 16823.
5.3 Hewlett-Packard Model 5992B — gas chromatograph/mass
spectrometer with flexible disc software. Hewlett-Packard,
Avondale, PA 19311 (No substitute).
5.3.1 Column: 6-ft, 1/4-in O.D. x 2-mm I.D., glass-packed with
3% OV-101 on 80/100 mesh high performance Chromosorb W.
Reagents
6.1 Methylene Chloride — Burdick & Jackson (distil le>d in glass).
141-02 January 1983
-------
Calibration
7.1 Establish GC/MS operating parameters equivalent to those
indicated in Table 1.
7.2 Calibration Procedure
7.2.1 Analytical Stock Standard Solution (150 ug/mL)
7.2.1.1 Weigh 0.015 g (+.0.001 g) of the analytical
standard methoxychlor, chloroneb, terbacil,
hexazinone, and bromacil (Du Pont Bio-chemical
Department) into separate 100-mL volumetric
flask. Note the exact weight of each standard.
7.2.1.2 Dilute to volume with spectral quality
methylene chloride.
7.2.1.3 Place the volumetric flasks in an ultrasonic
bath for 5 minutes to complete dissolution.
7.2.1.4 Store at 4°C.
7.2.2 Analytical Working Standard Solutions — Note: All working
standard solutions must be prepared fresh daily.
7.2.2.1 Solution "A" (1.5 ug/mL):
Add the following volumes of each analytical
stock solutions into a 100-mL volumetric flask:
methoxychlor, 1000 uL; chloroneb, 1000 uL;
terbacil, 1000 uL; hexazinone, 1000 uL;
bromacil, 1000 uL. Dilute to volume with
methylene chloride. Mix well.
7.2.2.2 Solution "B" (0.75 ug/mL):
Add the following volumes of each analytical
stock solutions into a 100-mL volumetric flask:
methoxychlor, 500 uL; chloroneb, 500 uL;
hexazinone, 500 uL; bromacil, 500 uL. Dilute
to volume with methylene chloride. Mix well.
7.2.2.3 Solution "C" (0.375 ug/mL):
Add the following volumes of each analytical
stock solutions into a 100-mL volumetric flask:
methoxychlor, 250 uL; chloroneb, 250 uL;
terbacil, 250 uL; hexazinone, 250 uL; bromacil,
250 uL. Dilute to volume with methylene
chloride. Mix well.
7.2.3 Inject 1.0 uL of standard solution "A" (run in duplicate).
141-03 January 1983
-------
7.2.4 Average the integrated area from the two runs and record.
7.2.5 Repeat steps 7.2.3 and 7.2.4 with standard solutions "8"
and "C".
7.2.6 Prepare a standard calibration curve of area response vs.
concentration.
7.2.7 Repeat steps 7.2.3 through 7.2.6 with each compound.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Transfer 1000 ml of wastewater sample to a 2000-mL separatory
funnel via a 1000-mL graduate cylinder.
10.2 Extract with 75 mL of methylene chloride (carefully vent the
separatory funnel).
10.3 Drain the methylene chloride layer (bottom) to a 250-mL beaker.
10.4 Repeat step 10.2 (twice) and combine the methylene chloride
layer, for a total of three portions of methylene chloride.
o
10.5 Evaporate the methylene chloride using a stream of N« and 45 -
50°C heat from a hot plate.
10.6 Concentrate to 5 mL and transfer to a 10-mL Kuderna-Danish (K-D)
graduated receiver flask. Rinse with 5 mL methylene chloride and
add to the K-D flask.
10.7 Evaporate to 1 mL using a stream of N..
11. Cleanup and Separation
Not Available.
12. GC/MS
12.1 Analyze the extract according to the conditions given in Table 1
for the desired compound. Run in duplicate.
12.2 Calibrate the system as described in Section 7.
141-04 January 1983
-------
12.3 Compare the SIM integrated area to the calibration curve to get
the apparent compound concentration in mg/L (ppm).
13. Calibration
13.1 Calculate the concentration (mg/L) of each component using the
following equation:
methoxychlor apparent concentration
mg/L (ppm) chloroneb » 1000
terbacil
hexazinone
bromacil
13.2 Report the concentrations of methoxychlor, chloroneb, terbacil,
hexazinone, and bromacil to two significant figures.
14. GC/MS Confirmation
14.1 GC/MS confirmation is included in the analysis method.
15. Method Performance
15.1 The 2s relative standard deviation of 50 ug/L standards from 10
analyses are shown on Table 2.
15.2 Typical detection limits are: terbacil, 1 ug/L; hexazinon, 1
ug/L; and bromacil, 10 ug/L.
16. References
16.1 "Determination of Methoxychlor, Chloroneb, Hexazinone, Terbacil,
and Bromacil, Gas Chromatography/Mass Spectrometry (GC/MS)
Method," Method No. W28.3032(R), September 25, 1980, E.I. duPont
de Nemours Co., Inc., Wilmington, DE.
141-05 January 1983
-------
Table 1
Gas Chromatography of Organonitrogen Pesticides
Conditions for Terbacil
Flow (helium): 25 mL/min
275°C
Injection port temp
Column temp.: 220°C
Solvent time out: 1.4 min
Run time: 3.0 min
Electron Multiplier volts: 2800 volts
Integration sensitivity: 0.01
Area threshold: 10
Dwell time: 150 milliseconds/mass
SIM window: 0.1 (AMU)
Ion Masses
1 - 161.0
2 - 160.0
2 - 117.0
Conditions for Hexazinone
Flow (helium): 25 mL/min
Injection port temp: 275 C
Column temp.: 275 C isothermal
Solvent time out: 0.8 min
Run time: 5.0 min
Electron Multiplier volts: 2800 volts
Integration sensitivity: 0.005
Area threshold: 1
Dwell time: 150 milliseconds/mass
SIM Window: 0.1 (AMU)
Ion Masses
1 - 171.0
2 - 83.0
3 - 128.0
Conditions for Bromacil
Flow (helium): 25 mL/min
Injection port temp.: 275 C
C
0.8 min
230°C
Column temp.:
Solvent time out:
Run time: 5.0 min
Electron Multiplier volts: 2800 volts
Integration sensitivity: 0.005
Area threshold: 1
Dwell time: 150 milliseconds/mass
SIM window: 0.1 (AMU)
Ion Masses
1 - 205.0
2 - 207.0
3 - 162.0
141-06
January 1983
-------
Table 2
Relative Standard Deviation and Recovery
Pesticide Relative Std. Dev..% Recovery. %
terbacil 4.8 91
hexazinone 6.2 82
bromacil 3.1 87
141-07 January 1983
-------
>>EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ZIRAM
IN WASTEWATER
METHOD 142
1• Scope and Application
1.1 This method covers the determination of ziram. The following
parameter can be determined by this method:
Parameter Storet No. CAS No.
Ziram — 137-30-4
1.2 This is a titration method applicable to the determination of the
compound listed above in water.
2. Suimnarv of Method
2.1 The sample is decomposed in an acid medium with €82 evolution.
The evolved gas is passed through a lead acetate trap, removing
H«S and SO.. The scrubber gas is collected in methanelie KOH,
forming potassium Xanthate. The solution is neutralized to a.
phenolphthalein end point and then titrated with iodine solution.
3. Interferences
3.1 Large amounts of copper compounds
3.2 Calcium and sodium arsenates
3.3 Ethylene thiourea
3.4 H2S, S02, and COS
142-01 January 1983
-------
4. Safety
4.1 Acetic acid vapors are irritating to the eyes and respiratory
system. The liquid readily burns the skin. It is flammable and
emits toxic fumes when heated to decomposition temperatures.
Safety glasses and rubber gloves are mandatory when handling this
chemical.
4.2 Dithiocarbamate samples may be hazardous.
4.3 Sulfuric Acid—when making dilute solutions, never add water to
the acid, but always add (slowly) sulfuric acid to the water.
This is a highly exothermic addition and care must be taken to
avoid splattering due to heat. Sulfuric acid is very corrosive,
causing severe burns to the skin. Contact with the eyes may
result in total blindness. In case of eye contact, wash with
copious quantities of water—SECONDS COUNT. After thoroughly
washing the eyes (hold the lids apart), get immediate
transportation to a hospital. Safety goggles and rubber gloves
are mandatory when handling acid.
4.4 If heated too rapidly the sample may react forcibly with the
sulfuric acid in the reaction flask.
4.5 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 See Figure 1
6. Reagents
6.1 30% acetic acid, aqueous
6.2 O.lN Iodine. For preparation and standardization, see Method No.
601.0 (reference procedure, not enclosd herein).
6.3 10% Lead acetate, aqueous
6.4 1% Phenolphthalein indicator, alcoholic, Reagent No. 606.0
6.5 2 N Potassium hydroxide, methanolic
6.6 Starch indicator, stabilized or laboratory-prepared, Reagent No.
607.0.
6.7 25% Sulfuric acid, Reagent No. 608.0
7. Calibration
142-02 January 1983
-------
Not Available.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Fart D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 Follow EFA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Refer to Figure 1. Fill the first two scrubbers about 2/3 full
with lead acetate solution and insert glass-wool plugs in the
towers to break up the bubble streams. Add (graduate) 100 ml of
menthanolic potassium hydroxide to the last two scrubbing towers.
Make sure that the towers are dry before adding the KOH. Attach
a vacuum to the fourth tower.
10.2 Weigh (to the nearest mg) an approximately 0.1-g sample and
transfer to a 250-mL round-bottomed reaction flask. The sample
size must be increased for lower concentrations. Add several
glass beads to prevent bumping and attach the flask to the
reaction train.
10.3 Turn on the water condensor, and slowly add the sulfuric acid
solution into the reaction flask. Adjust the vacuum to give slow
bubbling in the towers.
10.4 Heat the reaction flask with a gas burner as rapidly as possible.
Watch the reaction closely for a few minutes, for when the acid
is boiling vigorously problems with foaming usually occur.
Control the reaction by removing the flame as necessary and
adjusting the vacuum to prevent solution from backing up in the
tower.
10.5 After about 10 minutes or so, when the solvent is boiling
steadily in the reaction flask, adjust the vacuum to give the
least bubbling to prevent solution back-up. Occasional checking
only is required from this point.
10.6 Continue boiling for 1/2 to 1-1/2 hours or until the acid
solution looks clear. Remove the heat, disconnect the reaction
flask and the towers.
10.7 Rinse contents of the third and fourth scrubbing towers into
separate 500-mL Erlenmeyer flasks using about a total of 250-mL
demineralized water on each. Do not combine the two flasks.
11. Cleanup and Separation
Not Available.
142-03 January 1983
-------
12. Titration
12.1 Put a Teflon-coated stirring bar in the flasks, and place them on
magnetic stirrers. Start the solutions swirling, and add 3 drops
phenolphthalein indicator to each.
12.2 To each flask add acetic acid just until' the red color
disappears. Immediately titrate with standardized iodine
solution; when near the end point, add 5 cc of starch indicator
and titrate to a faint, but definite, color change.
12.3 Prepare a blank by pipetting a 100-mL KOH solution into a 500-mL
Erlenmeyer flask, adding 250 mL water and neutralizing with
acetic acid solution as in step 12.2. Add the starch indicator
at this point and continue with step 12.2.
13. Calculations
13.1 Factors
Compound Factor
Amobam 42 12.300
Amobam 48
Amobam M 19.330
Amobam T 25.537
Butyl Ziram 23.705
DBTD 22.315
Diram 14.325
DMTD 13.800
Ethyl Ziram 18.005
Methyl Ziram 15.285
PTD 15.019
Rocure-10 33.260
RZ-100 33.260
13.2 = percent Dithiocarbamate
where: A * sample titrant
B = blank titrant
C a normality of iodine
D » factor from step 13.1
E = weight of sample taken in step 10.2
14. Method Performance
Not Available.
142-04 January 1983
-------
15. References
15.1 FTM 36.2/11/80
15.2 USDA 765.1, 4/55
15.3 "Dithiocarbamate Assay by CS2 Evolution/1 Analytical Method No.
105.0-R1, Revised 7/28/82, Fike Chemicals, Inc., Nitro, WV.
142-05 January 1983
-------
ANALYTICAL METHOD NO. 105.0-R1
LEAD ACETATE
SCRUBBERS
KOH
RECEIVER
TO VACUUM SOURCE
FIGURE NO. 1
DITHIOCARBAMATE REACTION TRAIN
142-06
January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF PROPACHLOR
IN WASTEWATER
METHOD 143
1. Scope and Application
1.1 This method covers the determination of prqpachlor (2-chlor-N-
isopropylacetanilide).
Parameter Storet No. CAS No.
Propachlor 77729 1918-16-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in industrial streams
at the mg/L (ppm) level and higher.
1.3 The sensitivity of this procedure is usually dependent upon the
level of interferences rather than instrumental limitations.
2• Summar? of Method
2.1 A measured volume of water sample (10 mL) is extracted with 10 ml
of methylene chloride. ' The extract is analyzed without
concentration using gas chromatography with a flame ionization
detector.
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 by running blanks.
3.2 Interferences coextracted from the sample will vary considerably
from source to source, depending upon the diversity of the stream
being sampled.
143-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
5.1.1 Grab sample bottle—Amber glass washed and solvent-rinsed
before use to minimize interferences.
5.1.2 Threaded screw-on bottle caps. Caps must be lined with
Teflon.
5.1.3 Compositing equipment—Automatic or manual compositing
system, incorporating glass sample containers for the
collection of a minimum of 250 mL. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing or fittings may be used in the system.
5.1.4 Clean 5-dram vial with Teflon-lined screw caps.
5.2 Glassware and Other Equipment
5.2.1 Gas Chromatograph—Analytical system complete with gas
chromatograph with flame ionization detector, suitable for
on-column injection and all column supplies, recorder,
gases, and syringes. A data system for measuring peak
areas is recommended.
6. Reagents
6.1 Methylene chloride, pesticide quality or equivalent
6.2 Propachlor standards
7. Calibration
7.1 Assemble the necessary gas chromatographic apparatus and
establish operating parameters equivalent to those indicated in
Table 1. By injecting calibration standards, establish the
sensitivity limit of the detector and the linear range of the
analytical system.
7.2 Calibration Procedure
7.2.1 Stock standards—Prepare stock standard solutions at a
concentration of 1.00 ug/uL by dissolving 0.100 g of
assayed reference material in pesticide-quality acetone or
other appropriate solvent and diluting to volume in a 100-
mL glass-stoppered volumetric flask. The stock solution is
transferred to the glass-stoppered reagent bottles, stored
in a refrigerator, and checked frequently for signs of
degradation or evaporation, especially just prior to
preparing working standards.
143-02 January 1983
-------
7.2.2 Prepare calibration standards that contain the compound of
interest. The standards should be prepared at
concentrations covering two or more orders of magnitude
that will completely bracket the working range of the
chromatographic system.
7.2.3 Calibrate the system daily with a minimum of three
injections of calibration standards.
8. Quality Control
8.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water blank that all
glassware and reagents are interference free.
8.2 Standard quality assurance practices should be used with this
procedure. Where doubt exists over the identification of a peak
on the chromatograph, confirmatory techniques such as mass
spectroscopy should be used.
8.3 Recoveries of spiked samples should be greater than 70% (actual
recoveries averaged 90% for two samples).
8.4 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Grab samplers must be collected in glass containers.
Conventional sampling practices should be followed, except that
the bottle must not be prewashed with sample before collection.
Composite samples should be collected in refrigerated glass
containers in accordance with the requirements of the program.
Automatic sampling equipment must be free of tygon and other
potential sources of contamination.
9.2 The samples must be iced or refrigerated from the time of
collection until extraction.
9.3 See EPA sample, collection, preservation, and handling procedure
found in Part D of this document.
10. Sample jSxtraction
10.1 Using a 10-mL graduate, add 10 mL of sample to a 5-dram vial.
Using the same graduate, add 10 mL of methylene chloride to the
vial. Cap and shake either manually or mechanically for two
minutes. After the phases separate, analyze aliquots from the
lower (methylene chloride) layer.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
143-03 January 1983
-------
12.1 Table 1 summarizes the recommended gas chromatographic column
materials and operating conditions for the instrument.
12.2 Inject 2 to 5 uL of the sample extract. Smaller volumes can be
injected if automatic devices are employed. Record the volume
injected to the nearest 0.05 uL and the resulting peak size.
12.3 If the peak size exceeds the linear range of the system, dilute
the extract and reanalyze.
12.4 If the peak measurement is prevented by the presence of
interferences, other techniques such as gas chromatography/mass
spectrometry are required.
13. Calculations
13.1 Determine the concentrations of Fropachlor according to the
formula:
Concentration (mg/L) = (A) (B) (C)
(D) (E)
where A - concentration of standard in ug/mL
B » sample peak response
C » total mL of extract
D = standard peak response
E » total mL of sample extracted
13.2 Report results in milligrams per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
all data obtained should be reported.
14. Method Performance
Not Available.
15. References
15.1 "Propachlor (2-chloro-N-isopropylacetanilide)."Standard Method,
Dow Chemical Co., Midland, MI.
143-04 January 1983
-------
Table 1
Chromatographic Conditions
Column Temperature
Carrier Gas
Detector
Injection Port Temperature
Sample Size
Column:
150 C isothermal
N» at 40 mL/min
Flame ionization at 220°C
210°C
2 Ul
1.8-m x 2-mm I.D. glass-packed with
Permabond DECS on Chromosorb WAW
143-05
January 1983
-------
v>EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF FLUOMETDRON
IN WASTEWATERS
METHOD 144
1• Scope and Application
1.1 This method covers the determination of fluometuron.
Parameter Storet No.
Fluometuron —
1.2 This is a thin-layer chromatographic (TLC) method applicable to
the determination of the compound listed above in effluent
streams.
1.3 The method detection limit (MDL) is 1 mg/L (ppm). A statement of
"not detected" (ND) indicates only that the fluometuron is less
than 1 mg/L (ppm).
2. S"Tnn'n.ry of Method
2.1 A measure volume of effluent sample (50 mL) is extracted with 10
mL of chloroform in a separatory funnel. The chloroform extract
is isolated for spotting on pre-coated silica gel plates, which
are then eluted with toluene/acetone (85/15, v,v). The plates
are air dried to remove solvents, then exposed to chlorine gas in
a saturated chamber for 45 seconds. The plates are removed, air
dried, and sprayed with starch/KI solution.
3. Interferences
Not Available.
4. Safety
4.1 Plate exposure to chlorine and spraying with starch/KI are
performed in a hood.
144-01 January 1983
-------
4.2 See EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Chambers: 23 cm x 28 cm x 7.5 cm from Analtech or
equivalent.
5.2.2 Plate: Silica Gel G from Analtech 20 x 20 cm, 250-micron
layer.
5.2.3 Capillary tubes, Kimax size 1.6-1.8 x 100 mm.
6. Reagents
6.1 Chloroform, reagent grade
6.2 Toluene
6.3 Acetone
6.4 Starch
6.5 Potassium Iodide (Kl)
6.6 Distilled Water
6.7 Ethanol
6.8 Chlorine Gas
7. Calibration
7.1 Establish TLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 0.016 g +. 0.0005 g (to the nearest tenth milligram)
of fluometuron standard into a 100-mL volumetric flask.
Dilute this stock standard to the mark with CHClj. This
stock solution is equivalent to 32 mg/L when compared to
samples extracted 5:1. Dilutions of the stock solution are
made such that additional standards are equivalent to 16,
8, 4, 2, and 1 mg/L when compared to samples extracted 5:1.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
144-02 January 1983
-------
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Extract 50 mL of the effluent sample with 10 mL CHClj in a 125-mL
separatory funnel. Isolate the CHC1- layer for spotting.
11. Cleanup and Separation
Not Available.
12. Thin Layer Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for
TLC.
12.2 Calibrate the system as described in Section 7.
12.3 Eluent—One hundred milliter (100 mL) of eluent is prepared by
mixing 85 mL of toluene and 15 mL of acetone by volume. The
eluent should be prepared each day and allowed to equilibrate 1
hour before use. The eluent chamber is supersaturated and is
equipped with a U-shaped filter paper lining.
12.4 Starch/KI Solution—To 1.5 g starch add 50 mL water. Heat and
stir to the boiling point. To a second flask add 0.5 g potassium
iodide plus 50 mL ethanol. Combine the starch, KI solutions, and
ethanol into a TLC spray bottle.
12.5 One application of standards and samples is made with open-end
capillary tubes (7 to 8 uL) onto a silica gel plate. The plate
is placed in the chamber and the eluent allowed to migrate for 40
minutes to a distance of about 18 cm.
12.6 After elution, air dry plate to remove solvents. Then expose the
plate to chlorine gas in a saturated chamber for 45 seconds.
Remove plate and air dry 3 minutes or more. Spray the plate to
produce a good contrast with starch/KI solution.
13. Calculations
13.1 Visual estimates of impurity concentrations in the sample are
made based on spot size and intensity compared to the standards.
14. Method Performance
14.1 Detection Limit—A statement of "not detected" (ND) indicates
only that the fluometuron is less than 1.0 ppm.
14.2 Relative Standard Deviation—The relative standard deviation of
an analysis is approximately 30% relative to the actual
concentration.
144-03 January 1983
-------
15. References
15.1 "TLC Determination of Fluometuron in Effluent Streams," ETM-95-1,
June, 1982, Ciba-Geigy Corp., Mclntosh, AL.
144-04 January 1983
-------
Table 1
Chromatographic Conditions
Eluent Toluene/acetone, 85/15 by volume
Chamber 23 cm x 28 cm x 8.5 cm, supersaturated
(filter paper lining)
Plate Silica Gel G, Analtech 20 x 20 cm,
250-micron layer
Temperature Ambient
Distance 18 cm
Time 40 min
Rf (fluometuron) 0.22
Rf (point of origin) 0.0
144-05 January 1983
-------
x-/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF METRIBUZIN [SENCOR(TM)]
IN WASTEWATER
METHOD 145
I. Scope and Application
1.1 This method covers the determination of metribuzin.
Parameter Storet No. CAS No,
Metribuzin [SENCOR(TM)] 39051 21087-64-9
1.2 This is a high performance liquid chromatographic (HFLC) method
applicable to the determination of the compound listed above in
wastevater samples at concentrations of 1 mg/L or less.
2. Summary of Method
2.1 A measured volume of water sample (250 ml) is extracted with
chloroform. The extract is partitioned with water, then
evaporated to dryness. The residue is dissolved in methanol,
filtered, and, analyzed by HFLC with UV detector.
3. Interferences
3.1 All glassware used for analyzing wastewater samples must be pre-
rinsed with chloroform and not previously used for any analysis
other than wastewater. DO NOT USE PLASTIC WASH BOTTLES FOR ANY
SOLVENTS.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
145-01 January 1983
-------
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Microsyringe, 50-uL with blunt-end needle (Adapted for use
with a Valco valve)
5.2.2 Syringe, 1-mL
5.3 Water bath, 50° - 55°C
5.4 Filters, 13-mm diameter, Millipore Fluoropore #FHLP01300 or
equivalent
5.5 Filter holder, Swinney, 13-mm stainless steel
5.6 Liquid chromatograph, Waters ALC 200 or equivalent, equipped with
a uBondapak C.g column and a UV detector capable of measuring
absorbances at 313 nm
6. Reagents
6.1 Acetonitrile, Burdick and Jackson "Distilled in Glass" or
equivalent, degassed for 3 minutes under a vacuum in an
ultrasonic bath
6.2 Chloroform, nanograde
6.3 Hydrochloric acid, IN—Carefully add 21 mL of concentrated
hydrochloric acid into a 250-mL glass-stoppered graduated
cylinder containing approximately 200 mL of distilled water.
Dilute to 250 mL with distilled water and mix thoroughly.
6.4 Methanol, ACS
6.5 Metribuzin (SENCOR) standard, technical grade (90-93% purity)
6.6 Sodium hydroxide, IN—Carefully dissolve 10 g of sodium hydroxide
pellets, ACS, in 250 mL of distilled water. Mix thoroughly.
6.7 Sodium sulfate, anhydrous, ACS
6.8 Water, deionized, filtered through activated carbon and a final
0.2-um filter and degassed for 3 minutes under vacuum in an
ultrasonic bath
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 SENCOR standard solution, 0.10%—Weigh 0.11 - 0.12 + 0.0001
g of technical SENCOR (90 - 93% purity) into a 100-mL
145-02 January 1983
-------
volumetric flask. Dilute to volume with methanol, stopper,
and mix thoroughly. This solution may be kept for one week
only.
7.2.2 Pipet a 1-mL aliquot of the 0.10% SENCOR solution into a
10-mL volumetric flask, dilute to volume with methanol, and
mix thoroughly. Label this solution 0.4 ppm SENCOR. (This
is not the actual concentration, but is an equivalent
value.) Prepare fresh daily.
7.2.3 Inject 10 uL of the 0.4-ppm SENCOR standard solution and
continue to inject the standard until the peak heights are
reproducible to within _+ 5% of the previous injections.
SENCOR has an approximate retention time of 35-42 minutes.
8. Quality Control
8.1 Follow EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample by shaking, then proceed immediately to
step 10.2.
10.2 Measure 250 mL of the well-mixed sample into a 500-mL separatory
funnel.
10.3 If the pH is above 7.0 or below 6.5, adjust it to 6.5 - 7.0 with
IN hydrochloric acid or IN sodium hydroxide using pH indicator
paper or a pH meter.
10.4 Extract three times by vigorously shaking for 1 minute each time
with fresh 50-mL portions of nanograde chloroform. Collect the
three chloroform extracts in another 250-mL separatory funnel. If
an emulsion forms, centrifuge the emulsified layer and add the
clear chloroform layer, obtained by centrifuging, to the second
separatory funnel. Return the water layer to the first separatory
funnel.
10.5 Add 50 mL of distilled water to the combined chloroform extracts
in the second separatory funnel and shake for one minute.
10.6 Drain the chloroform layer into a 300-mL 24/40 boiling flask
through a funnel containing 4 to 5 grams of anhydrous sodium
sulfate retained on a small glass-wool plug (pre-rinsed with 10
mL of chloroform). Rinse the sodium sulfate three times with 10-
mL portions of chloroform.
145-03 January 1983
-------
10.7 Place the flask on a rotoevaporator and strip off all of the
chloroform, using a water bath at 50 C. Remove the flask and cool
it to room temperature.
10.8 Remove any last traces of chloroform with a stream of dry air at
room temperature.
10.9 Pipet 1 ml of methanol into the flask from step 10.8, stopper
immediately, and rotate the flask so that the methanol washes the
inside of the flask. DO NOT allow the methanol to get onto the
neck or stopper.
10.10 Put a Fluoropore filter into a Swinney filter holder, then attach
the filter holder to a 1-mL syringe. Remove the syringe plunger
and hold the tip of the filter holder in the mouth of a 1-mL
volumetric flask.
10.11 Using a disposable pipet, transfer the sample solution into the
syringe barrel, replace the syringe plunger, and force the sample
solution through the filter and into a 1-mL flask. Stopper the
flask.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the instrument as described in Section 7.
12.3 Inject 10 uL of the sample solution from step 10.11 and
immediately push the "Run" button on the programmer. Allow the
sample to complete the entire program cycle to elute all of the
nonpolar compounds.
12.4 If there is any doubt as to which peak in the sample scan is
SENCOR, inject 7.5 uL of sample solution spiked with 2.5 uL of
0.4 ppm SENCOR standard solution. Compare the spiked scan with
the scan from step 12.3 to identify the SENCOR peak in the sample
scan.
12.5 If the SENCOR peak in the sample scan is greater than the
standard scan (off scale), repeat steps 12.2 through 12.4 with a
range setting of 0.1 on the Waters ALC 202 or at attenuation 4 on
the P-E Series 3. NOTE: The standard should be about full scale.
12.6 Measure the peak height of the standard and sample to the nearest
0.1 mm; record as S and A, respectively.
13. Calculations
145-04 January 1983
-------
13.1 SENCOR, mg/L (ppm) «
14. Method Performance
Not Available.
15. References
15.1 "Determination of SENCORtTM] in Waste Water by HPLC," Analytical
Method TM B-34.62, Mobay Chemical Corp, Agricultural Chemical
Div., Kansas City, MO.
145-05 January 1983
-------
Table 1
Chromatographic Conditions
Waters ALC 200
Range, AUFS
Injector loop, uL
Solvent A
Solvent B
Flow rate, mL/min
Initial conditions (Solvent B, %)
Final conditions (Solvent B, Z)
Time, minutes
Wavelength, nm
Curve
0.05
10
Water, deionized (see Reagents)
Acetonitrile (see Reagents)
2.0
13
100
50
313
11
P-E Series 3
Attenuation
Flow, mL/min
ZA
T. , min
Tl, ZA
Tl, curve
T2, min
Tpurge, min
Tpurge, flow, ml/Min
Tpurge, ZA
Tequil, min
Tend
Time constant
Lamp
Filter
Injector loop, uL
Solvent A
Solvent B
1
2.0
13.0
50.0
13.0
0
0
10.0
2
99.9
5
999
1.0
DV
0
10
Acetonitrile (see Reagents)
Water, deionized (see Reagents)
145-06
January 1983
-------
PART B
CONTRACTOR ANALYTICAL METHODS
-------
x-xEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF AOP, FERBAM, NIACIDE, ZAC,
ZINEB, AND ZIRAM IN WASTEWATER
METHOD 401
1. Scope and Application
1.1 This method covers the determination of certain dithiocarbamate
herbicides. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
AOP — —
Ferbam — 14484-64-1
Niacide — 15339-36-3
ZAC —
Zineb — 12122-67-7
Ziram — 137-30-4
1.2 This is a class determination. The total dithiocarbamates are
expressed as ug/L as Ziram. Dithiocarbamates and thiuram
disulfides have been shown to react along the pathway chosen for
this determination.
1.3 This is a spectrophotometrie method applicable to the
determination of the compounds listed above in water and
industrial waste.
1.4 The method detection limit (MDL) for these parameters is
approximately 200 ug/L. The MDL for a specific wastewater may
differ from this value, depending upon the nature of
interferences in the sample matrix.
2. Summary of Method
2.1 The sample is decomposed in a strong acid resulting in the
evolution of CS_. CS- is reacted with alcoholic potassium
401-01 January 1983
-------
hydroxide to form xanthates, which are measured
spectrophotometrically at 380 nm.
3. Interferences
3.1 Only dithiocarbamates and thiuram disulfides have been shown to
undergo the decomposition reaction to produce 0829 but the
analyst should be aware that any compound containing the S=C-S-
moiety will undergo the decomposition reaction and thus produce
interference.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Volumetric flasks, 10.0 mL, Class A, serialized
5.2.2 Capillary micro-pipets; 10-, 50-, and 100-uL sizes
5.2.3 Miscellaneous laboratory glassware
5.3 CS. generator (Figure 1 and 2), consisting of (in each set):
5.3.1 One 125-mL long-neck flask with X 19/22 female neck joint
5.3.2 One 250-mL Erlenmeyer flask with S 19/22 female neck joint
5.3.3 One 500-mL Erlenmeyer flask with * 19/22 female neck joint
5.3.4 One j-stem addition funnel with I 19/22 male joint
5.3.5 One condenser/funnel adapter with S 15 side joint
5.3.6 Three M-receivers (micro) (Figure 2)
5.3.7 One hose adapter
5.4 Analytical balance — Mettler H78AR or equivalent
5.5 HV-Vis spectrophotometer — Beckman DU-2 is suitable, operated at
380 nm, using W source
5.5.1 Spectrophotometer cells, Pyrex with path length adjustment
inserts to allow path lengths of 10 mm, 3 mm, and 1 mm
6. Reagents
401-02 January 1983
-------
6.1 Ziram Analytical Standard, U.S. EPA, Research Triangle Park, NC
6.2 Potassium hydroxide, analytical reagent grade
6.3 Methanol, 99.9%, spec trophotome trie grade, Aldrich Chemical
Company, Milwaukee, WI
6.4 Sulfur ic Acid, analytical reagent grade
6.5 Water, distilled in glass, boiled prior to use
6 .6 Solutions
6.6.1 0.5 KOH in methanol — to 500 mL methanol, add 28 g KOH.
Stir to effect solution. Make the solution up to 1 liter
with more methanol. Mix thoroughly and store in a plastic
vessel.
6.6.2 30% V/V H2S04 — to 600 mL water, slowly add 300 mL
concentrated H-SO,. Mix well, and store, when cool, in a
reagent bottle. Caution; Pour the acid into the water!
Dilution of HjSO, produces much heat. Use thick-walled
glassware, and take suitable safety precautions.
7 . Calibration
7.1 The sample is analyzed spectrophotometrically at 380 nm
7.2 Calibration Procedure
7.2.1 Make a solution of CS_ in methanol to contain 0.1000 g
This is the master stock solution. Due to the
fugitive nature of carbon disulfide, this solution must be
kept frozen when it is not in actual use, and it should be
remade no less often than weekly. All dilutions must be
remade daily.
7.2.2 Dilute 0.1 mL of the master stock solution with methanol in
a 10-mL volumetric flask. Withdraw 0.1 mL for further
dilution and freeze this solution immediately. Label the
flask "C-dilution."
7.2.3 Dilute 0.1 mL of the first dilution to the mark in another
10-mL volumetric flask with methanol. Freeze this solution
immediately. Label the flask "E-dilution."
7.2.4 Thaw the C-dilution. Pipet 1 .0 mL of the solution into a
culture tube containing 4.0 mL of 0.5N KOH in methanol.
7.2.4.1 Refreeze the dilution. Swirl the culture tube
to mix the solutions.
401-03 January 1983
-------
7.2.5 After 5 minutes, determine the absorbance. of the mixture at
380 nm in the spectrophotometer.
7.2.6 Using 4-cycle semi-logarithmic graph paper, plot weight of
CS« (ordinate) versus absorbance (abscissa).
7.2.7 Repeat Steps 7.2.4 through 7.2.6 using the E-dilution.
7.2.8 Prepare dilutions using 0.2, 0.4, 0.6, and 0.8 ml of the
master stock solution, as described in Step 7.2.1. Carry
them through Steps 7.2.3 through 7.2.7.
8. Quality Control
8.1 The analyst should verify method recovery by fortifying samples
with a dithiocarbamate standard in appropriate amounts. No less
than 10% of the samples should be duplicate, /in additional 5% of
the samples should be rerun as recovery spikes. Each lot of
reagents should be screened for interferences in the blank.
8.2 Standardization of the decomposition and verification of recovery
process are accomplished by the following:
8.2.1 Accurately weigh 0.01000 g of technical Ziram into a
polypropylene weighing boat.
8.2.2 Transfer the material quantitatively to the
decomposition flask, using approximately 50 mL of
zinc-free water.
8.2.3 Arm the apparatus with its dropping, funnel and
absorber bulb. Add 30 mL of 30% Aqueous H^O^ to the
dropping funnel before attaching the absorber bulb.
8.2.4 Start the magnetic stirrer and heat, the flask.
8.2.5 Start the aspirator and open the needle valve
slightly.
8.2.6 Add the acid to the flask.
8.2.7 Adjust the stopcock of the addition funnel and the
aspirator needle valve until the bubbling in the
receivers is gentle and steady.
8.2.8 Bring the liquid in the flask to a gentle boil.
8.2.9 Continue the boiling and aspiration for
approximately 15 minutes. Use cooling water in the
condenser to avoid distilling any of the acidified
water.
8.2.10 When 15 minutes have elapsed, cool the flask,
continuing aspiration until boiling ceases.
401-04 January 1983
-------
8.3 Remove the dropping funnel assembly from the apparatus, then stop
the aspirator.
8.4 Dismount the receiver tube and combine its contents in a 10-mL
volumetric flask.
8.4.1 Rinse the receiver tubes with methanol and transfer
the rinsings to the volumetric flask.
8.4.2 Make the contents of the volumetric flask to the mark
with methanol.
8.5 From the plot prepared in Step 7.2.6, determine the weight of CSj
in the solution obtained in Step 8.4.2.
8.6 Calculate the efficiency of the decomposition from the standard
as follows:
64 x (Weight of Ziram)(76/306) „. . , c P0 / T A
s ° r ' *- = theoretical g of CS2/mL = A
(weight of CS2 from step 8.2.1) * 5 * actual g of CS2/mL = B
Percent efficiency = 100 B/A
8.7 See reference listed in Step 15.1.
8.8 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 See reference listed in Step 15.2.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Preparation
10.1 Assemble the CS_ generation apparatus (Figure 1).
10.2 Attach the two micro-receivers in series, clamping the ball
joints with the appropriate clamps.
10.3 Attach the aspiration apparatus to the last receiver.
10.4 Add approximately 2 mL of 0.5N ROH in methanol to each receiver.
10.5 Warm the stored sample to room temperature.
10.6 Agitate the sample thoroughly to effect resuspension of the heavy
metal salts of dithiocarbamoic acids.
10.7 Using a 100-mL graduate cylinder, transfer 100 mL of the sample
to a 250-mL decomposition flask.
401-05 January 1983
-------
10.8 Carry out the decomposition as described in Steps 8.2.3 through
8.4.2.
11. Cleanup and Separation
Not Available.
12. Analysis
12.1 Determine the absorbance of the sample at 380-nm wavelength,
using 0.5N KCH in methanol as a reference.
12.2 Determine the weight of CS_ in the sample from the standard curve
prepared in Section 7.
13. Calculations
13.1 Calculate the weight of Ziram equivalent to the weight of CS2
detected as follows:
[(weight of CS, evolved in ug)/72] x 306
: : :—~. = dithiocarbamates ug/L
sample volume in liters
as Ziram
14. Method Performance
14.1 The method detection limit is 200 ug/L.
14.2 Table 1 presents precision and accuracy data for this method.
15. References
15.1 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewater prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
15.2 Environmental Science and Engineering, Inc. (ESE). 1979
Pesticide BAT Review Verification Sampling Protocol. Prepared
for the U.S. EPA, Research Triangle Park, North Carolina.
15.3 "Procedure for the Determination of Dithiocarbamates and Related
Compounds," ESE Method 1200, February 16, 1979. Environmental
Science and Engineering, Inc. Gainesville, FL.
401-06 January 1983
-------
Table 1
Precision/Accuracy Data
Parameter
Ziram
Ziram
Ziram
Ziram
Sample
Type
DW
DW
WW
WW
mg/L
Theoretical
11.33
28.67
18.33
22.33
mg/L
Recovered
7.43
20.88
14.79
17.67
% Recovery
65.7
72.9
80.7
79.0
DW = Reagent Water
WW - Wastewater
401-07 January 1983
-------
FINAL ASSEMBLY OF ESE MODEL NO. 1 CS2 EVOLUTION APPARATUS
Figure 1.
w^^Sic'''*"'"-** ''.T'^1- ."^ »-" • - -
S & M CS2 RECEIVER SHOWING FILLING LEVEL
Figure 2.
401-08
January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENOMYL AND GARBENDAZIM
IN WASTEWATER
METHOD 402
1. Scope and Application
1.1 This method covers the determination of benomyl and carbendazim.
The following parameters can be determined by this method:
Parameter Storet No. CAS NO.
Benomyl — 17804-35-2
Carbendazim — 10605-21-7
1.2 This method will not separate carbendazim and benomyl from each
other and, if both of these compounds are suspected to be
present, the carbendazim results should be considered to be due
to the sum of the contributions of each of these compounds.
1.3 The method detection limit (MDL) for carbendazim under the
conditions used is 100 ug/L based upon a 5% response at 0.005
AUFS. The MDL for a specific wastewater may differ from those
listed, depending upon the nature of the interferences in the
sample matrix.
2. Summary of Method
2.1 Carbendazim is determined in wastewater by direct aqueous
injection HFLC using a reverse-phase column. No cleanup or
pretreatment was used.
3. Interferences
Not Available.
402-01 January 1983
-------
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Sample Filter: 5-mL Hamilton gas-tight syringe with
Swinney Filter Apparatus and 5-u Nucleopore polyester
filter.
5.2.2 High Pressure Liquid Chromatograph: Altex Model 322 or
equivalent.
5.2.3 Injection valve: Rheodyne 7120 or equivalent
6. Reagents
6.1 HPLC Mobile Phase — Dissolve 4.1 g of sodium acetate in 450 mL
of HPLC-grade water. Add 50 mL glacial acetic acid. Mix
thoroughly. Add 500 mL of HPLC grade methanol and mix
thoroughly.
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
Not Available.
8. Quality Control
8.1 See reference listed in Step 15.1.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 See reference listed in Step 15.2.
9.2 Follow EPA sample collection, preservation, and handling
procedure found in Part D of this document.
10. Sample Extraction
10.1 Bring the sample to room temperature.
402-02 January 1983
-------
10.2. Thoroughly mix the sample to ensure homogeneity.
10.3 Prefilter the sample thorugh a 0.5-um polyester filter using a.
suitable syringe and Svinney Adapter.
10.4 High-level samples (greater that 500 ppm) should be diluted
appropriately to the analytical working -range (0.1 - 500 ppm)
with the HPLC mobile phase.
10.5 Sample is now ready for injection.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
13. Calculations
Not Available.
14. Method Performance
14.1 The limit of detection for carbendazim under the conditions used
is 100 ug/L based upon a 5% response at 0.0005 AUFS.
14.2 Accuracy/Precision data are presented in Table 2.
15. References
15.1 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters Prepared for the
U.S. EPA, Process Measurements Branch, Industrial
Environmental Research Laboratory, Research Triangle Park, North
Carolina. Research Triangle Institute, Research Triangle Park,
North Carolina.
15.2 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide Review Verification Sampling Protocol. Prepared for
the U.S. EPA, Research Triangle Park, North Carolina.
15.3 "Determination of Carbendazim in Industrial Wastewater," ESE
.Method 1700, April 19, 1979, Environmental Science and
Engineering, Inc., Gainesville, FL.
402-03 January 1983
-------
Table 1
Chromatographic Conditions
HPLC Conditions
Column 25-cm x 4.6-nan I.D. packed with Zorbax ODS, 6-8 u
Pre-Column 5-cm x 2.1-mm I.D. packed with Whatman Co! Pell ODS
Flow Rate 1.0 mL/min
Injection Volume 50 uL
Temperature Ambient
Detector 254-nm fixed wavelength U.V.
Mobile Phase 50-percent methanoI/50-percent - 10-percent v/v
glacial acetic acid, 0.10 M sodium acetate
402-04 January 1983
-------
Table 2
Precis ion/Accuracy Data
Parameter
Carbendazim +
Carbendazim +
Carbendazim +
Carbendazim +
Benomyl
Benomy 1
Benomyl
Benomyl
Sample
Type
WW
WW
WW
WW
ug/L
Theoretical
50
2.0
4.0
2.0
ug/L
Recovered
47.8
2.1
3.5
1.86
% Recovery
95.6
105
87.5
93
Parameter
Carbendazim +
Carbendazim +
Carbendazim +
Carbendazim +
Benomyl
Benomyl
Benomyl
Benomy 1
Sample
Type
WW
WW
WW
WW
ug/L
Replicate 1
2410
8.16
2.66
0.89
ug/L
Replicate 2
2430
8.24
2.66
0.91
WW - wastewater
402-05
January 1983
-------
oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CARBOFURAN
IN WASTEWATER
METHOD 403
Scope and Application
1.1 This method covers the determination of carbofuran. The
following parameter can be determined by this method:
Storet No. CAS NO.
— 1563-66-2
1.2 This is a high-performance liquid chromatograpbic (HPLC) method
applicable to the determination of the compound listed above in
water and industrial waste.
2 . Su1™"*!^ of Method
2.1 Carbofuran and its metabolites (hydroxycarbofuran, keto-7-
carbofuran, and carbofuran phenol) are determined by direct
aqueous injection. Some samples require pre-concentration and
all samples are filtered before determination by HPLC with DV
detection.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
403-01 January 1983
-------
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Syringe, 100 mL gas tight, with Luer-Lok tip
5.2.2 Syringe, 5 mL gas tight, with Luer-Lok tip
5.2.3 Syringe, 100 uL, for filling the sampling loop.
5.3 Sample filtration kit, syringe adapter with 0.5-um polyester
membrane filters, available from Altex, Inc., Berkeley, CA
5.4 Sep-Pak C._ Cartridges, Waters Associates, Milford, MA
5.5 High-Pressure Liquid Chromatograph — Altex Model 322 or
equivalent (Gradient Elution system is not necessary)
5.5.1 254-nm Ultraviolet Absorbance Detector with 8-uL flow cell,
Altex Model 153 or equivalent
5.5.2 High pressure injection valve, capable of withstanding
5,000 psi, with 50 uL loop, Rheodyne 7 or equivalent
5.5.3 Zorbax ODS column, 4.6-mm I.D. x 25 cm
5.5.4 Guard column, 2.1-mm I.D. x 5 cm, packed with Whatman
Co:pell-ODS. Obtained as Whatman "Column Survival Kit."
5.5.5 Potentiometric strip chart recorder, 1 mV and, 1- mV full
ranges
6. Reagents
6.1 Water, Fisher HPLC Grade or equivalent
6.2 Methanol, Fisher HPLC Grade or equivalent
6.3 Glacial Acetic Acid, Analytical Reagent
6.4 Solutions
6.4.1 Acidic Water — To 500 mL of HPLC-grade water in a 1-L
volumetric flask, add 2.0 mL of glacial acetic acid.
Dilute to the mark with HPLC-grade water and mix
thoroughly.
6.4.2 HPLC Eluant — (55% Methanol/44.8% Water/0.2% Acetic Acid)-
To 300 mL of HPLC-grade methanol in a 1-L graduated
cylinder, add 270 mL of acidified water and mix well. This
403-02 January 1983
-------
mixture may be degassed if bubbles occur in the HFLC
detector.
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Prepare stock solutions of the various analyte standards in
HPLC-grade methanol.
7.2.2 Prepare the appropriate dilute standards to bracket the
concentration range of the samples to be analyzed by
dilution of the stock solutions with the HPLC eluant.
7.2.3 Determine the average response factor for each analyte by
measuring the peak height or peak area of the standard
peaks in two standard solutions which bracket the sample
concentration.
-i o / n TJ peak area or height
7.2.4 Response Factor = : . _ T^ ,
r concentration of standard
8. Quality Control
8.1 See reference listed in Step 15.1.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation., and Handling
9.1 See reference listed in Step 15.2.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Concentrations greater than 1 ug/mL
10.1.1 Bring the sample to room temperature.
10.1.2 Place a filter holder containing a 0.5-um membrane
filter on the Luer-Lok of a 5-mL gas-tight syringe.
10.1.3 Remove the plunger and add 5 mL of the sample.
10.1.4 Replace the plunger, and force the sample through the
filter.
10.1.5 Collect the filtrate in a small beaker. The sample
is now ready for chromatography.
403-03 January 1983
-------
10.1.6 Note: Samples with concentrations of carbofuran and
related compounds greater than 1000 ug/mL should be
diluted (1:10) with the HPLC eluant before filtration
in order to ensure the solubilities of the analytes.
10.2 Concentrations less than 1 ug/mL
10.2.1 Attach a Sep-Pak cartridge to a 5-mL Luer-Lok
syringe.
10.2.2 Prepare the Sep-Pak cartridge for the sample as
follows.
10.2.2.1 Force about 2-.0 mL of HPLC-grade methanol
through the cartridge.
10.2.2.2 Force about 10 mL of HPLC-grade water through
the cartridge.
10.2.2.3 The cartridge is now prepared for the sample.
10.2.3 Measure 100 mL of sample into a 250 mL graduated
cylinder.
10.2.4 Add 0.1 mL glacial acetic acid and mix well.
10.2.5 Place a prepared Sep-Pak cartridge on the Luer-Lok of
a 100-mL gas-tight syringe.
10.2.6 Remove the plunger from the syringe and pour 100 mL
of sample into the barrel.
10.2.7 Rinse the wall of the graduated cylinder with a few
mL of HPLC-grade water and add the rinsing to the
syringe barrel.
10.2.8 Replace the syringe plunger.
10.2.9 Force the liquid through the Sep-Pak cartridge,
discarding the effluent.
10.2.10 Repeat steps 10.2.2 through 10.2.9 twice, for a total
of 300 mL of sample.
10.2.11 Samples with high solids content may clog the
cartridge after only 200 mL of sample has been
processed. Such samples may either be pre-filtered
or a concentration factor of 200:1 may be used.
10.2.12 Remove the cartridge from the 100-mL syringe and
place it on the Luer-Lok of a 5-mL gas-tight syringe
containing 2.0 mL of methanol.
10.2.13 Force the methanol through the cartridge.
403-04 January 1983
-------
10.2.14 Collect 1.0 mL of the eluate in a calibrated receiver
(a Kuderna-Danish receiver or volumetric flask is
adequate).
10.2.15 The sample is now ready for chromatography.
10.2.16 Determine carbofuran and its. related compounds on
high-pressure liquid chromatography using the
conditions listed in Table 1.
11. Cleanup and Separation
11.1 Some degree of clean-up is provided by the Sep-Pak concentration
procedure.
12. Liquid Chromatoeraphv
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the system as described in Section 7.
13. Calculations
13.1 Concentration of analyte =
peak area or height of analvte peak
Average Response Factor
14. Method Performance
14.1 Organic-free water was spiked with several different
concentrations of carbofuran and its metabolites and passed
through the Sep-Pak Cartridges. Results are given in Table 2.
The concentration factors were 300:1.
14.2 Precision and accuracy data for this method are given in Table 3.
15. References
15.1 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of
Quality Control/Quality Assurance for the Determination of
Priority Pollutants in Pesticide Industry Wastewaters. Prepared
for U.S. EPA, Process Measurements Branch, Industrial
Environmental Research Laboratory, Research Triangle Park, North
Carolina. Research Triangle Institute, Research Triangle Park,
North Carolina.
15.2 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide BAT Review Verification Sampling Protocol. Prepared
for U.S. EPA, Research Triangle Park, North Carolina.
15.3 "Procedure for the Determination of Carbofuran and Metabolites in
Water and Industrial Wastes," ESE Method 1800, April 10, 1979,
Environmental Science and Engineering, Inc., Gainesville, FL.
403-05 January 1983
-------
Table 1
Chromatographic Conditions
Pre-column 2.1-mm I.D. x 5-cm Whatman Co:Pell ODS
Column 4.6-mm I.D. x 25-cm Zorbax ODS
Mobile Phase 55% Methanol/44.8% Water/0.2% Acetic Acid, isocratic
Flow Rate 1.5 mL
Pressure 4300 psi
Injection Volume 50 uL
Detector 254 nm, 0.01 AUFS
403-06 January 1983
-------
Table 2
Recovery Study In Laboratory Water
Retention
Time (min) Compound
Cone. (ug/L) % Recovery (Ave of 3 exp.)
3.1
4.3
6.9
8.3
Hydroxy-Carbofuran
Keto-7-Carbofuran
Carbofuran
Carbofuran Phenol
3.9
38.6
580
8.1
81.2
1220
8.9
89
1330
69%
59%
83%
99%
99%
83%
99%
95%
93%
85%
78%
403-07
January 1983
-------
Table 3
Freeision/Accuracy Data
Parameter
Carbofuran
Carbofuran
Carbofuran
Sample
Type
WW
WW
DW
Theoretical
(ue/L)
11,400
114,000
417.6
Acutal
(ua/L)
11,400
111,000
429.2
%
Recovery
100
97.3
102.7
Parameter
Carbofuran
Carbofuran
Carbofuran
Carbofuran
Sample
Type
WW
WW
WW
WW
Replicate 1
(ua/L)
89
<25
<25
124,000
Replicate 2
(ue/L)
85
<25
<254
141,000
WW • wastewater
DW = reagent water
403-08
January 1983
-------
xvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CHLOROBENZILATE, TERBUTRYN, AND
PROFLURALIN IN WASTEVATER
METHOD 404
1. Scope and Application
1.1 This method covers the determination of certain triazines and
other pesticides. The following parameter's can be determined by
this method:
Parameter Storet No. CAS No.
Ametryn — 834-12-8
Chlorobenzilate 39460 510-15-6
Profluralin — 26399-36-0
Terbutryn — 886-50-0
Diazinon 39570 333-41-5
Atrazine 39033 1912-24-9
1.2 This is a gas chromatographic method (GC) applicable to the
determination of the compounds listed above in wastewater.
2. Summary of Method
2.1 A 100-mL aliquot of sample is pH adjusted to pH 6.5 to 7.5 and
extracted with hexane. The combined extract is dried and
analyzed by gas chromatography with electron capture or
thermionic detection.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
404-01 January 1983
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5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnel, 2-liter
5.2.2 Kuderna-Danish apparatus
5.2.3 Micro-Snyder Column
5.3 Gas Chromatograph
5.3.1 Electron Capture Detector
5.3.2 Thermionic Detector
5.3.3 Column — 1.8-m x 2-mm glass-packed with 3% STAP on 80/100
mesh Chromosorb WHF
6. Reagents
6.1 50% sodium hydroxide
6.2 50% sulfuric acid
6.3 Hexane, distilled-in-glass, Burdick and Jackson or equivalent
6.4 Anhydrous sodium sulfate
6.5 Dry nitrogen
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 The gas chromatograph is calibrated by the analysis of
standard solutions in hexane of the compounds listed above.
The range of standard concentrations should span the range
of linear instrument response for each of the compounds.
8. Quality Control
8.1 The establishment and maintenance of quality control in the
analyses of the environmental samples should include the
determination of samples spiked with the compounds listed above,
the determination of distilled water samples spiked with the
compounds, and the determination of duplicate wastewater samples.
404-02 January 1983
-------
8.2 See reference listed in Step 15.2.
8.3 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 See reference listed in Step 15.3.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Thoroughly mix the sample and transfer 100 mL of the wastewater
sample to a 2-liter separatory funnel. Add 1400 mL of distilled,
deionized water to the funnel and mix well. (The dilution of the
sample prevents the formation of an emulsion during the
extraction step.)
10.2 Adjust the pH of the diluted sample aliquot to 6.5 to 7.5 with
50% sodium hydroxide or 50% sulfuric acid.
10.3 In the separatory funnel extract the sample three times with 50
mL of ultrapure hexane. High pollutant concentrations may
require use of a methylene chloride/hexane extracting solvent
with a subsequent solvent transfer step to hexane prior to GC
analysis.
10.4 Combine the three hexane extracts with hexane washings of the
glassware. Remove traces of water from the mixture by passing it
through a 75- to 100-mm by 12-mm O.D. column of anhydrous sodium
sulfate. Dilute the dry hexane extract to 200 mL. Reserve 1-5
mL for analysis by gas chromatography.
10.5 Some samples may require additional concentration. Evaporate the
remainder of the combined extracts to 5 to 10 mL in a 500-mL
Kuderna-Danish (K-D) apparatus fitted with a 3-ball macro-Snyder
column and a 10-mL calibrated receiver tube. Allow the K-D to
cool to room temperature. Remove the receiver, add fresh boiling
chips, attach a two-chamber macro-Snyder column and carefully
evaporate to 1.0 mL (or when active distillation ceases). If the
volume is greater than 1 mL, remove the micro-Snyder column and
carefully evaporate to 1.0 mL with dry nitrogen.
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
404-03 January 1983
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13. Calculations
Not. Available.
14. Method Performance
14.1 Precision and recovery data are given in Table 3.
15. References
15.1 This procedure was adapted from the "Method for Triazine
Pesticides in Industrial Effluents," in Federal Register. 38.
No. 75, Part II.
15.2 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters. Prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
15.3 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide BAT Review Verification Sampling Protocol. Prepared
for U.S. EPA, Research Triangle Park, North Carolina.
15.4 "Analytical Procedures, Manufactured Pesticides and Related
Compounds," Southern Research Institute Method., Southern Research
Institute, Birmingham, AL.
404-04 January 1983
-------
Table 1
Chromatographic Conditions
Detectors:
(1) Electron capture detector (BCD), Ni
(2) Thermionic detector (TD)
Operating conditions: ECD and TD
(1) Column temperature:
. Isothermal — 190°C
o o
. Program — maintain at 90 C for 5 min, program from 90 C to
225°C at 10°/min, maintain at 225°C for 14 min
(2) Injection port temperature: 170 C
(3) Detector temperature: ECD-275C; TD-225°C
(4) Carrier gas flow rate: 45 mL/min ultra high purity nitrogen
(5) Auxiliary gas flow rates (or pressure settings): ECD — 100
mL/min ultra high purity nitrogen purge gas
(6) Injection volume, 1 uL
Column: 1.8-m x 2-mm I.D. glass column packed with 3% STAP on 80/100 mesh
Chromosorb WHP.
404-05 January 1983
-------
Program
Table 2
Retention Times and Detection Limits
Propazine 18.2
Profluralin 15.1
Diazinon 15.8
Atrazine 19.3
Terbutryn 20.1
Ametryn 20.6
Chlorobenzilate 27.6
Estimated
Instrumental Detection
*
Co Xumu Xsiflp
Isothermal
Compound
Prof luralin
Diazinon
Atrazine
Terbutryn
Ametryn
Chlorobenzilate
Retention
Time, Min
1.8
2.5
8.5
10.8
12.2
38.8
Limit .
BCD
2
40
-
300
pe/uL
TD
20
1
30
50
90
10
100
200
40
100
2
40
40
40
The concentration in hexane that is estimated to yield a peak height
that is twice the background noise level in the absence, of
interferences.
404-06
January 1983
-------
Table 3
Precision/Recovery Data
Compound
Profluralin
Terbutryn
Chlorobenzilate
Profluralin
Terbutryn
Chlorobenzilate
Compounds
Profluralin
Terbutryn
Chlorobenzilate
Profluralin
Terbutryn
Chlorobenzilate
Sample
Type
WW
WW
WW
WW
WW
WW
Sample
Type
WW
WW
WW
WW
WW
WW
Spike Level
(ue/L)
428
848
83
10,000
2,120
2,070
Level
(ue/L)
12,100
144
488
92.4
716
60.4
Recovery
58.6
126
104
200
102
149
RSD
12.4%
5%
8%
13.5%
21%
3%
WW - vastewater
404-07
January 1983
-------
xvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF 2,4-DB, 2,4-DB ISOBDTYL ESTER (2,4-DB IBE), AND
2,4-DB ISOOCTYL ESTER (2,4-DB IOE) IN WASTEWATER
METHOD 405
1. Scope and Application
1.1 This method covers the determination of certain phenoxy alkanoic
acids and their esters. The following parameters can be
determined by this method:
Parameter Storet No. CAS No.
2,4-DB — 94-82-6
2,4-DB IBE — 51550-64-2
2,4-DB IOE — 1320-15-6
1.2 This is a gas chromatographic (GC) method applicable to the
compounds listed above in water and industrial effluents.
2. Summary of Method
2.1 An aliquot of sample is made basic with sodium carbonate and
extracted with methylene chloride to isolate the phenoxy alkanoic
esters. The sample is'then acidified and extracted with
methylene chloride to isolate the phenoxy alkanoic acids. The
ester fraction is concentrated to a suitable small volume and
analyzed by gas chromatography using electron capture or flame
ionization detection. The acid fraction is evaporated to
dryness, and the residue is methylated with diazomethane in
ether. The excess diazomethane is evaporated. The methylated
compound is analyzed by gas chromatography using electron capture
or flame ionization detection.
3. Interferences
Not Available.
405-01 January 1983
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4. Safety
4.1 Diazomethane (CE^2^ is an extremely powerful me thy la ting agent.
It is toxic and highly explosive. It should be used only by
experienced chemists in a well-equipped laboratory. It must not
be generated or used in glassware with ground joints, or
glassware which is etched or scratched, or has unpolished edges.
Diazomethane should be generated and used only in a high-draft
hood. Its solutions are moderately stable if no active protons
are present and if they are kept in the dark at -20 C.
4.2 Excess diazomethane and its solutions can be destroyed by mixing
with aqueous copper (II) sulfate and discarding the resulting
solutions in a flammable waste can. Failure to exercise good
laboratory techniques and observe proper precautions can lead to
intoxication by exposure to the gas or its solutions, or to
explosion of the generator or other glassware. (See
Manufacturing Chemists' Association Handbook of Laboratory
Safety.)
4.3 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnels, 1-liter, all Teflon
5.2.2 Filtering funnels, 60 C with long stems
5.2.3 Kuderna-Danish Concentrators, with 500-mL flasks, 10-mL
receivers, and 3-ball Snyder columns
5.2.4 Pasteur pipets
5.2.5 2-mL septum-sealing vials, with Teflon-lined septum seals
5.2.6 Diazomethane generator, 0.1-mole size, Aldrich Chemical Co.
210,025-0
5.2.7 Culture tubes with Teflon-lined caps, 15-mL size
5.2.8 Miscellaneous laboratory glassware
5.3 Glass wool, Pyrex
5.4 Funnel Racks
5.5 Analytical balance, Mettler H-78AR
405-02 January 1983
-------
5.6 500-mL heating mantle with variable transformer
5.7 Magnetic stirrer
5.8 High-draft hood, face velocity in excess of 50 cfm. This hood
should be explosive-proof and provided with water and a drain, as
well as 110-VAC electrical service.
5.9 Gas Chromatograph
5.9.1 Column — 1.8-m x 2.0-mm glass packed with 15% OV-17 +
1.95% OV-210 on 100/120 mesh Chromosorb W-HP, or 1.8-m x
2.0-mm glass packed with 3% OV-101 on 100/120 mesh
Chromosorb W-HP, or 1.8-m x 2.0-mm glass packed with a 3%
OV-7 on 100/120 mesh Chromosorb W-HP. At least two of
these columns should be used to allow confirmation of
identification.
5.9.2 Electron Capture Detector
5.9.3 Flame lonization Detector
6. Reagents
6.1 Methylene Chloride — distilled in glass (Fisher "Pesticide
Residue Grade" is acceptable)
6.2 Sulfuric Acid — Analytical Reagent Grade
6.3 Sodium Carbonate — Analytical Reagent Grade
6.4 Sodium Sulfate — technical
6.4.1 Sodium sulfate, as received, is ignited at 600 C in a
muffle furnace for 8 hours. It is cooled in a desiccator
and stored in a glass reagent bottle.
6.5 Diazald (N-methyl, N-nitroso, p-toluenesulfonamide) Aldrich
Chemical Co., D-2800-0
6.6 2- (2-ethoxy) ethanol, Aldrich Chemical Co., E-455-0
6.7 Diethyl Ether, absolute, Aldrich Chemical Co., 17296-4
6.8 Potassium Hydroxide, Analytical Reagent Grade
6.9 Standards of phenoxyalkanoic acids and esters
6.10 30% Sulfuric acid, aqueous — 300 mL of concentrated sulfuric
acid is added to 500 mL of demineralized water in a 2,000-mL
heavy Pyrex beaker. When the solution has cooled somewhat, 200 mL
of water is added. The mixture is mixed well and stored in a
glass reagent jar when cool.
405-03 January 1983
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6.11 Ethereal Diazomethane. In a high-draft hood, assemble the
diazomethane generator. Attach the condensor lines and start the
flows of cooling water. If the temperature of the cooling water
is judged to be too high, a cooling coil may be needed to provide
the necessary efficiency. A temporary cooling coil can be made
by coiling the I/4-inch copper tubing around a small fire
extinguisher, and connecting this coil upstream of the condensor.
The coil should be immersed in an ice bath to provide cooling.
Provide the reaction pot with magnetic stirring. Add 30 mL of 2
(2-ethoxyethoxy)-ethanol to the pot through the addition funnel.
Begin heating the reaction pot. Start the magnetic stirrer. Add
10 mL of 50% aqueous KOH through the addition funnel. Add 30 mL
of ether through the addition funnel. Weigh out 21.5 g (0.1 mol)
Diazald. In a 250-mL beaker, add the Diazald to 150 mL of ether.
Stir the solution well and place the majority of the solution in
the addition funnel. When distillation of the ether begins,
start addition of the ether solution of Diazald at a rate
approximating the rate of distillation. Cool the receivers in an
ice bath. A greenish-yellow hue in the distillate indicates
evolution of diazomethane. Continue addition of Diazald until
2.5 g has been added. Rinse the beaker and addition funnel into
the pot with small amounts of ether. Continue distillation until
the distillate is water-white. When this occurs, cool the pot
and rinse the contents into a waste down the drain. Combine the
contents of the receivers and store, in amber glass bottles with
Teflon-lined caps, in a freezer at -20 C until used or destroyed.
Diazomethane solutions such as those generated by this method are
stable under the storage conditions indicated for at least four
weeks.
6.12 50% Potassium Hydroxide, aqueous — add 10 g KOH to 10 mL of
demineralized water. Stir the solution well and use immediately.
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 The identity of each compound should be determined by
comparison of the adjusted retention times of the unknown
and an authentic standard.
7.2.2 Quantitation should be accomplished from a regression line
of at least five points.
7.2.3 Each compound should be reported without correction for
recovery but with recovery stated. Acids should be
reported as the free acid.
8. Quality Control
8.1 It is well known that reagents and glassware can contribute
discrete artifacts which can interfere with determination.
405-04 January 1983
-------
Glassware and reagents must be shown to be free of interfering
artifacts by the determination of a blank with each preparative
"run."
8.2 Recovery of the analytes must be demonstrated in each sample
matrix by fortifying the sample with appropriate concentrations
of authentic standards. Not less than 5% of all samples should
be fortified in this matter.
8.3 No less than 10% of all samples should be run in duplicate.
8.4 The esterification of the phenoxy alkanoic acids by diazomethane
is quantitative. Low recoveries may result from evaporation of
esters if the solvent is removed too rapidly or to dryness.
8.5 See reference listed in Step 15.1.
8.6 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 See reference listed in Step 15.2.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Bring the samples to room temperature and agitate thoroughly.
10.2 Measure 500 mL of the sample into a 1-liter Teflon separatory
funnel, using a graduated cylinder. Rinse the walls of the
cylinder with methylene chloride, holding the rinsings
temporarily in the cylinder.
10.3 Adjust the pH of the sample, in the funnel, to pH 10 or 11 using
sodium carbonate.
10.4 When effervescence has ceased, add 100 mL of methylene chloride.
10.5 Cap the funnel and invert it. Vent the pressure to the hood.
10.6 Shake the funnel vigorously, taking care to vent the pressure to
the hood periodically.
10.7 Allow the layers to separate. Drain the organic layer (lower)
through sodium sulfate into a Kuderna-Danish concentrator.
10.8 Add another 100-mL portion of methylene chloride to the sample.
10.9 Repeat Steps 10.5 through 10.8 twice, so that the sample will
have been extracted three times.
10.10 Combined extracts will contain phenoxyalkanoic esters. The
405-05 January 1983
-------
extracts are concentrated to 1.0 mL, transferred to septum capped
glass vials, and stored at 4°C for later.
10.10.1 If electron-capture detection is to be used during
chromatographic analysis, replace the solvent with
iso-octane before storing the extract, using the
following method.
10.10.2 Add 5 mL of ether to the sample in its Kuderna-Danish
receiver.
10.10.3 Attach a micro-Snyder column and heat the apparatus
in a hot water bath until the volume is reduced to
about 0.5 mL.
10.10.4 Add another 5 mL of ether to the micro-concentrator
and allow it to reflux until the volume is reduced to
about 0.5 mL.
10.10.5 Add several drops of iso-octane to the concentrator
and remove the last of the ether by heating the
concentrator gently.
10.10.6 Make the sample to 1.0 mL and store it in a septum-
sealing vial to await analysis. Label this extract
"Ester Fraction."
10.10.7 If the ester fraction requires further cleanup, use
modified Florisil Chromatography.
10.11 To the aqueous phase from Step 10.9, add 30% sulfuric acid
(Step 6.11) until the pH of the sample is about 2.
10.12 Swirl the funnel until effervescence ceases.
10.13 Add 100 mL of methylene chloride to the sample. Cap the funnel
and shake gently. Vent the pressure to the hood.
10.14 Agitate the funnel vigorously, venting the pressure to the hood.
10.15 Allow the layers to separate. Drain the organic layer (lower)
directly into a Kuderna-Danish apparatus. Do not pass this
extract over a drying agent, as low recoveries of the acids may
result (through formation of the metal salts of the acids).
10.16 Repeat steps 10.13 through 10.15 twice.
10.17 Concentrate the combined acid extracts to about 2 mL in the
Kuderna-Danish concentrator.
10.18 Transfer the concentrate quantitatively to a 15~mL culture tube.
Transfer any crystals that have formed in the concentrate.
Remove the last of the solvent by heating the sample in a water
bath.
405-06 January 1983
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10.19 Add 5 to 8 ml of ethereal diazomethane. Cap the tube with a
Teflon-lined cap and agitate it.
10.19.1 If the yellow color disappears, add more
diazomethane.
10.19.2 Loosen the cap of the tube and gently heat it in a
water bath to reduce the volume.
10.19.3 If the yellow color disappears, add more
diazomethane.
10.19.4 When the color of the diazomethane remains for about
3 minutes, remove the residual diazomethane by
heating the tube in a water bath. Add more ether if
the volume is reduced too far (i.e., less than 0.5
mL). DO NOT allow the sample to go to dryness, as
the analytes will be almost entirely lost.
10.20 Transfer the methylated sample to a 1-mL volumetric flask and
dilute it to the mark with either methylene chloride or iso-
octane. The choice of solvent will depend on the detector in use
in the determinative step. Label this fraction "Acid Fraction."
11. Cleanup and Separation
Not Available.
12. Gas Chromatographv
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
12.2 Calibrate the system as described in Section 7.
13. Calculations
Not Available.
14. Method Performance
14.1 Precision and recovery data for this method are summarized in
Table 2.
15. References
15.1 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters. Prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
405-07 January 1983
-------
15.2 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide BAT Review Verification Sampling Protocol. Prepared
for U.S. EPA, Research Triangle Park, North Carolina.
15.3 "Determination of Phenoxy Alkanoic Acids in Industrial
Effluents," ESE Method 200, Environmental Science and
Engineering, Inc., Gainesville, FL.
405-08 January 1983
-------
Table 1
Chromatographic Conditions
Electron-Capture detector ( Ni) temperature
Flame lonization detector temperature
Column Oven temperature
Injector temperature
Carrier gas (F.I.D.)
Carrier gas (E.C.D.)
Hydrogen (F.I.D.)
Air (F.I.D.)
300°C
300°C
180 C isothermal
200°C
N-, 30 mL/min
Ar/5% CH4, 30 mL/min
30 mL/min
240 to 300 mL/min,
optimized for the detector
405-09
January 1983
-------
Table 2
Freeision/Recovery Data
Compound
2,4-DB
2,4-DB
Sample
Type
WW
WW
Spike Level
(ue/L)
72,400
14,480
I Recovery
54
89
Compound
Sample
Type
Replicate 1
(ug/L)
Replicate 2
(ue/L)
2,4-DB
2,4-DB
WW
WW
506,000
304,414
540,000
319,800
WW = wastewater
405-10
January 1983
-------
xvEPA
United States " Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF DINOSEB
IN WASTEWATER
METHOD 406
1. Scope and Application
1.1 This method covers the determination of dinoseb.
Parameter Storet No. CAS No.
Dinoseb — 88-85-7
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above in wastewater and
effluents.
2. Summary of Method
2.1 A measured volume of water sample is adjusted to pH 6 and
extracted with methylene chloride. The combined extracts are
dried and concentrated to 10 mL. Analysis of the extracts is
performed by gas chromatography using a flame ionization
detector.
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
406-01 January 1983
-------
5.2 Glassware and Other Equipment
5.2.1 Separatory funnel, 2 liter
5.2.2 K-D flask
5.3 Water Bath
5.4 Gas Chromatograph
5.4.1 6-ft x 2-mm glass column packed with 1.5% OV-17 + 1.95% QF-
1 on Chromosorb W
5.4.2 FID Detector
6. Reagents
6.1 ION Sodium hydroxide
6.2 50% Sulfuric Acid
6.3 Methylene Chloride
6.4 Anhydrous Sodium Sulfate
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
Not Available.
8. Quality Control
8.1 See reference listed in Step 15.2.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 See reference listed in Step 15.3.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Quantitatively transfer a 1000-mL aliquot into a 2-liter
separatory funnel and adjust pH to 6 with ION sodium hydroxide or
50% sulfuric acid.
10.2 Add 60-mL methylene chloride to the sample in the separatory
funnel and shake vigorously for two minutes. Allow the solvent to
406-02 January 1983
-------
separate from the sample, then draw the organic layer into a 500-
mL flask.
10.3 Add a second 60-mL volume of solvent to the funnel and repeat the
extraction procedure. Perform a third extraction in the same
manner combining all the extracts.
10.4 Pre-dry with 1 to 2 g of sodium sulfate, then pass the organic
extract through a chromatographic column containing 3 inches
anhydrous sodium sulfate, and collect it in a 500-mL K-D flask
equipped with a 10-mL ampul.
10.5 Concentrate the extract to approximately 5 mL in a K-D evaporator
on a hot water bath.
10.6 Transfer the sample to a 10-mL volumetric flask and dilute to
volume. Analyze the concentrate by gas chromatography.
11. Cleanup and Separation
Not Available.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph.
13. Calculations
Not Available.
14. Method Performance
14.1 The detection limit of this procedure is approximately 200 ug/L.
14.2 Table 2 gives recoveries of fortified samples.
15. References
15.1 "Methods for Benzidine, Chlorinated Organic Compounds,
Pentachlorophenol and Pesticides in Water and Wastewater,"
EPA/EMSL, September 1978.
15.2 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters. Prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
15.3 Environmental Science and Engineering (ESE). 1979. Pesticide BAT
Review Verification Sampling Protocol. Prepared for U.S. EPA,
Research Triangle Park, North Carolina.
406-03 January 1983
-------
15.4. "Procedure for Extraction and Analysis of Atrazine and Dinoseb at
High Concentrations," TRW Method, TRW Systems Group.
406-04 January 1983
-------
Table 1
Chromatographic Conditions
Detector Temperature 190 C
o
Injector Temperature 190 C
Column Temperature 190 C
Carrier Gas Helium
Flow Rate 30 mL/min
Column: 1Z SP-1240DA on 100/120 Supelcoport (6-ft X 2-mm I.D. glass),
although a 1.95Z OV-17 + 1.5Z QF-1 column packing is acceptable.
406-05 January 1983
-------
Table 2
Spike Recoveries
Compound
Dinoseb
Dinoseb
Sample
Type
WW
WW
Spike Level
(ue/L)
215
320
%
Recovery
150
147
WW = wastewater
406-06 January 1983
-------
v=/EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF DINOSEB
IN WASTEWATER
METHOD 407
1. Scope and Application
1.1 This method covers the determination of dinoseb. The following
parameter can be determined by this method:'
Parameter Storet No. CAS Ho .
Dinoseb — 88-85-7
1.2 This is a gas chroma tographic-mass spectrometric (GC-MS) method
applicable to the determination of the compound above in
wastewater.
1.3 The method detection limit (MDL) for dinoseb is approximately 200
ug/L.
2 . Svi'"""'rv of Method
2.1 An aliquot of sample (pH adjusted to 2 or less) is extracted with
methylene chloride. The combined extracts are dried and
concentrated to 1 mL. An internal standard is added to the
extract which is then analyzed by GC-MS.
Interferences
3.1 Emulsions — The recovery of 852 of the added solvent will
constitute a working definition of a broken emulsion. (You may
correct the recovery of the first portion for water solubility of
methylene chloride.)
3.2 Any of the following techniques that meet these criteria are
acceptable.
407-01 January 1983
-------
3.2.1 Centrifugation of the emulsion layer after removal of any
separated solvent.
3.2.2 Passage of the emulsion through a column plugged with a
ball of methylene chloride-wet glass wool. The solvent
used to wet the wool and to wash it after emulsion must be
measured and subtracted from the total volume to determine
85% recovery.
3.2.3 Addition of excess extracting solvent sometimes breaks weak
emulsions. Use excess solvent in the blank.
3.2.4 Let the emulsion stand for up to 24 hours.
3.2.5 Draw off the small amount of free solvent that separates
and slowly drip it back in the top of the separatory funnel
and through the sample and emulsion.
3.2.6 Stir with a glass rod, heating on a steam bath, add
concentrated sodium sulfate solution, and sonicate.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Separatory funnels — 2- and 4-L with Teflon stopcock
5.2.2 Continuous liquid-liquid extractors — any such apparatus
designed for use with solvents heavier than water and
having a capacity of 2- to 5-L. Connecting joints and
stopcocks must be of Teflon or glass with no lubrication.
5.3 6C/MS with conditions as described in Table 1
6. Reagents
6.1 6N Hydrochloric acid
6.2 Methylene chloride
7. Calibration
7.1 Establish GC/MS parameters equivalent to those indicated in Table
1.
7.2 External standardization is used to determine instrument
response.
407-02 January 1983
-------
7.3 Inject varying dilutions of the standard mixure of acid/neutral
compounds repeatedly into the GC/MS. Great care should be
exercised to reproduce the injection volume. Prepare calibration
curves of selected mass chromatogram peak areas as a function of
the amount injected for each compound of interest. Use these
calibration curves to determine the amount of each compound in
the unknown wastewater sample extracts.
8. Quality Control
8.1 See reference listed in Step 15.2.
8,2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 See reference listed in Step 15.3.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Sample Preparation
10.1.1 Blend the composite sample to provide a homogeneous
mixture including a representative portion of the
suspended solids that are present. Stirring with
metal devices is acceptable for organic sampling.
10.1.2 Transfer the sample from the composite container
through a glass funnel into a 2-L graduated cylinder
and measure the volume. Then transfer to a 4-L
separatory funnel or a continuous extractor. Rinse
the cylinder with several portions of the first
volume of extracting solvent. (Note: Either
separatory funnel or continuous extraction is
acceptable for isolation of the organics.)
10.2 Acid-Neutral Extraction
10.2.1 Separatory funnel extraction — Adjust the pH of the
sample with 6 N HC1 to 2 or less. Use multirange pH
paper for the measurement. Serially extract with 250
x 100- x 100-mL portions of distilled-in-glass
methylene chloride. (About 40 mL of the first 250-mL
portion will dissolve in the sample and not be
recovered.) Shake each extract for at least 2
minutes.
10.2.2 Dry and filter the solvent extract by passing it
through a short column of sodium sulfate.
Concentrate the solvent by Kuderna-Danish (K-D)
evaporation (distillation). The sodium sulfate
should be prewashed in the column with methylene
407-03 January 1983
-------
chloride. (Note: Check sodium sulfate blank and, if
necessary, heat in 500°C oven for 2 hours to remove
interfering organics.) After drying the extract,
rinse the sodium sulate with solvent and add to the
extract.
10.2.3 Evaporate the extract to 5-lt) mL in a 500-mL K-D
apparatus fitted with a 3-ball macro-Snyder column
and a 10-mL calibrated receiver tube. Allow the K-D
to cool to room temperature. Remove the receiver,
add fresh boiling chips, attach a two-chamber micro-
Snyder column and carefully evaporate to 1.0 mL or
when active distillation ceases. If required, remove
the micro-Snyder column and carefully evaporate to
1.0 mL using an inert gas stream. Add the internal
standard: 10 uL of 2 ug/uL d.Q-anthracene (per each
mL of extract). Mix thoroughly.
10.2.4 If it is to be overnight or longer before the extract
is run by GC/MS, transfer it from the R-D ampul with
a disposable pipet to a solvent tight container. The
recommended container is a standard 2-mL serum vial
with a Teflon Mininert valve. Store the extracts in
the dark and at refrigerator or freezer temperatures.
10.3 Continuous Extractions — If 85% solvent recovery cannot be
achieved, start with a fresh aliquot of sample and extract by
continuous extraction.
10.3.1 Adjust the pH of the sample as appropriate, pour into
the extractor, and extract for 24 hours. When
extracting a 2-L sample, using the suggested
equipment, 2-L of blank water must be added to
provide proper solvent recycle.
10.3.2 For operation, place 200-300 mL of solvent in the
extractor before the sample is added and charge the
distilling flask with 500 mL of solvent. At the end
of the extraction remove the solvent from the
distilling flask only, evaporate, and treat as
described in the base-neutral extract section.
10.3.3 An analysis of a blank sample is required to evaluate
the effect of the reagents and solvents used in the
extraction and sample work up. All of the operations
performed on the samples must be duplicated with the
blank. Organic-free, distilled water is suitable for
this blank sample.
11. Cleanup and Separation
Not Available.
12. GC/MS Analysis
407-04 January 1983
-------
12.1 Table 1 summarizes the recommended operating conditions for the
GC/MS.
12.2 A computer system should be interfaced to the mass spectrometer
to allow acquisition of continuous mass spectral scans for the
duration of the chromatographic program. The computer system
should also be equipped with mass storage devices for saving all
data from GC-MS runs. There should be computer software available
to allow searching any GC-MS run for specific ions and plotting
the intensity of the ions with respect to time or scan number.
The ability to integrate the area under any specific ion plot
peak is essential for quantification.
12.3 Three conditions must be met to show the presence of a compound
by GC/MS. First, the characteristic ions for the compound must be
found to maximize in the same spectrum. Second, the time at
which the chromatographic peak elutes must be within a specified
window (usually +. 1 minute) for the retention time of the
suspected component. Finally, the ratios of the three mass
spectral peak heights must agree with the relative intensities of
the standard spectrum of the suspected component with +. 20%.
12.4 For example, hexachlorobenzene elutes at 19.4 minutes. The
characteristic ions are 284 (100%), 142 (30%), and 249 (24%).
A plot of the intensities of these ions versus time or scan
number in the 18.4 to 20.4-minute window is examined. If all
three ions are present, the ratio of the peak areas is checked to
verify that it is 100:30:24 ± 20 percent. If these three
criteria are met, hexachlorobenzene has been identified in the
sample.
13. Calculations
13.1 When a compound has been identified, the quantification of that
compound will be based on the integrated area from the specific
ion plot. Quantification will be performed by the internal
standard method using deuterated anthracene. Response factors,
therefore, must be calculated to compare the MS response for
known quantities of each compound of interest with the internal
standard. The Response Ratio (R) may be calculated as follows:
A C
R - -£• X -*-
R A X C
a c
where AC is the integrated area of the characteristic ion from
the specific ion plot for a known concentration, C . A and C
C 3 a
are the corresponding values for the deuterated anthracene,
internal standard. The relative Response Ratio should be known
for at least two amounts that range over a factor of 10 (40 mg
and 400 mg). Those compounds that do not respond at either of
these levels may be run at concentrations appropriate to their
response.
407-05 January 1983
-------
13.2 The concentration of the compound may now be calculated using:
A C
C _ _C. jr _J1
C A X R
a
where C is the concentration of a component; A is the integrated
area of the characteristic ion from the specific ion plot; R is
the response ratio for this component; A is the integrated area
of the characteristic ion in the specific ion plot, for
deuterated anthracene; and C is the concentration of deuterated
antracene in the injected contract.
14. Method Performance
14.1 The analytical recovery data presented in Table 2 were determined
by spiking actual wastewater samples at levels approximately
equal to those found in the neat samples or expected based on
prior analysis. Percent recovery is calculated according to the
formula
Recovery, % = A ~Q B X 100%
where:
A = Observed concentration (ug/L) in the spiked sample
B = Observed concentration (ug/L) in the neat sample
C = Concentration (ug/L) resulting only from the spike
14.2 Recovery for Dinoseb at 1060 ug/L was adequate and presented no
problem. However, the GC/FID procedure has a detection limit at
such a level that when sample dilution factors are included, no
level below 200 ug/L can be accurately quantified. The spike
level on the effluent sample was only 106 ug/L. These values
obtained for the neat and spiked samples are not accurately known
and subtracting these values from one another is meaningless.
15. References
15.1 "Sampling and Analysis Procedures for Screening of Industrial
Effluents and Priority Pollutants," EPA-EMSL, April 1977.
15.2 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters. Prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
407-06 January 1983
-------
15.3 Environmental Science and Engineering, Inc. (ESE). Pesticide BAT
Review Verification Sampling Protocol. Prepared for U.S. EPA,
Research Triangle Park, North Carolina.
15.4 "Method for GC/MS Analysis of Acid/Neutral Priority Pollutants,"
TRW Method, TRW Systems Group.
January 1983
-------
Table 1
Chromatographic Conditions
Packing 1% SP-1240DA on 100/120 mesh Supelcoport
Program 80°C -185°C at 8°C/min hold at 185°C for 15 min
Injector 190°C
Jet Separator 300°C
Carrier Gas He at 30 mL/minute
Injection Size 1 uL
Column — 6-ft x 2.0-mm I.D. glass
407-08 January 1983
-------
Table 2
Recovery Data
Compound
Dinoseb
Dinoseb
Sample Type
WW
WW
Spike Level (ue/L)
1060
106
% Recovery
91
Not Calcula
See Step 14.2
WW = wastewater
407-09 January 1983
-------
>-,EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF METHOMYL
IN WASTEWATER
METHOD 408
1. Scope and Application
1.1 This method covers the determination of methorny1.
Parameter Storet No. CAS No.
Methomyl 39051 16752-77-5
1.2 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of the compound listed above in
water.
1.3 The method detection limit (MDL) for methomy1 in wastewater is
approximately 1 ug/g; for "clean" water samples the MDL is
approximately 0.01 ug/g. The MDL for a specific wastewater may
differ from those given, depending upon the nature of
interferences in the sample matrix.
2. Su™narv of Method
2.1 A measured volume of water sample (100 ml) is passed through two
C.g Sep-Pak cartridges in series. The Sep-Paks are then eluted
with acetonitrile, which is concentrated to 5 mL by a nitrogen
stream. The extract is then analyzed by high performance liquid
chromatography using an ultraviolet detector.
3. Interferences
Not Available.
4. Safety
408-01 January 1983
-------
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Syringe (10 mL)
5.2.2 Separatory funnel
5.3 Water Bath
5.4 Waters Associates Liquid Chromatograph
5.4.1 Model 6000-A pump
5.4.2 Model 440 DV detector
5.5 C-18 Sep-Pak Cartridge
6. Reagents
6.1 Acetonitrile, HPLC grade
6.2 Dry nitrogen
7. Calibration
7.1 Establish the HPLC conditions as described in Section 12.
7.2 Calibration Procedure
Not Available.
8. Quality Control
8.1 Two control samples (a blank" and a fortified spike at 10 ppm)
are run with the samples.
8.2 See reference listed in Step 16.3.
8.3 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 See reference listed in Step 16.4.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
408-02 January 1983
-------
10. Sample Extraction
10.1 Clean the Sep-Pak cartridge by pumping 20 mL HPLC-grade
acetonitrile through the cartridge using a 10-mL syringe.
10.2 Pump 10 mL distilled water through the cartridge using a 10-mL
syringe.
«
10.3 Using apparatus set up as described in Figure 1, pour 100 g of
the sample (10 g of the influent sample) into the separatory
funnel. Open the stopcock and allow the water to drop into the
long-stem funnel slowly enough that the funnel does not overflow.
Use just enough vacuum to keep the water moving through the
apparatus.
10.4 When all of the water has passed through the cartridges,
disconnect the cartridges from the system.
10.5 Pump 10 mL HPLC-grade acetonitrile through the first cartridge
into a 15 mL-centrifuge tube. Concentrate to <5 mL in a water
bath using a stream of dry nitrogen. Repeat procedure with the
second cartridge, eluting into the same centrifuge tube.
Concentrate to 5 mL. Concentration is 100 g (10 g)/5 mL or 20 mg
(2 mg)/uL.
10.6 Sample is ready for injection on LC.
11. Cleanup and Separation
Not Available.
12. Liquid Chromatograohv
12.1 Primary analysis was made using a Waters Associates Liquid
Chromatograph equipped with a Model 6000-A pump and a Model 440
UV detector operated at a wavelength of 254 nanometers. A
BONDAPAK Clg was used. The mobile phase was 40% CH2CN/60% H20 at
1.0 mL/min.
12.2 Residues were detected and measured as the "parent" methorny1
compound; methorny1 oxime (methyl-N-hydroxythioacetimidate)
was also detected.
13. Calculations
Not Available.
14. GC/MS Confirmation
14.1 The residues were confirmed by GC-MS after conversion of the
methomyl to methomyl oxime according to the procedure by Pease
and Kirkland, (Journal of Agricultural and Food Chemistry, 16.,
554-557, (1968) as modified in 1969.
408-03 January 1983
-------
14.2 Qualitative confirmation was also obtained by flame photometric
detector.
15. Method Performance
15.1 The minimum detectable level for wastewater samples was
approximately 1 ug/g (ppm); for "clean" water samples the MDL
is approximately 0.01 ug/g .
15.2 Prior to running the samples, three controls were run to
determine an approximate recovery for methomyl. Recoveries for
the spiked controls were 41% for 1 ug/g and 66% for 10 ug/g. The
"blank" control was equivalent to 0.02 ug/g (ppm).
15.3 Recovery of methomyl from a fortified sample spike at 10 ug/g was
70%.
15.4 Precision of this method is 41%.
16. References
16.1 "Journal of Agricultural and Food Chemistry", 16_, 554-557 (1968),
Pease and Kirkland.
16.2 The extraction procedure is based on a technique developed by
Waters and Associates.
16.3 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of
Quality Control/Quality Assurance for the Determination of
Priority Pollutants in Pesticide Industry Wastewaters. Prepared
for U.S. EPA, Process Measurements Branch, Industrial
Environmental Research Laboratory, Research Triangle Park, North
Carolina. Research Triangle Institute, Research Triangle Park,
North Carolina.
16.4 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide BAT Review Verification Sampling Protocol. Prepared
for U.S. EPA, Research Triangle Park, North Carolina.
16.5 "Methomyl in Water," Pesticides Monitoring Laboratory Method,
NASA/NSTL, Bldg. 1105, Bay St. Louis, MS 39529..
408-04 January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CYANAZINE
IN WASTEWATER
METHOD 409
1. Scope and Application
1.1 This method covers the determination of certain triazine
compounds. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Atrazine 39033 1912-24-9
Cyanazine 77780 21725-46-2
Simazine 39055 122-34-9
Propazine 39024 139-40-2
1.2 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of the compounds listed above in
industrial wastewater.
1.3 The method detection limit (MDL) for the above listed parameters
is 0.14 mg/L. The MDL for a specific wastewater may differ from
that listed, depending upon the nature of interferences in the
sample matirx.
2. Summary pf Method
2.1 An aliquot of sample is pH adjusted (to 7) and passed through a
Sep-Pak C,g cartridge rinsed with methanol. The triazine
compounds are trapped on the cartridge. The triazine compounds
are eluted from the cartridge using an eluant of methanol/water
(702/302). Analysis is by high performance liquid chromatography
with an ultraviolet detector at 254 nm.
409-01 January 1983
-------
3. Interferences
Not Available.
4. Safety
4.1 Follow EPA safety procedure found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
5.1.1 Syringes, 5-mL glass, gas-tight with Leur-Lock tip
5.1.2 Syringe, 100-uL or greater for filling sample loop
5.2 Sep-Pak C1Q cartridges, available from Waters Associates,
J.O
Milford, MA
5.3 Altex Model 322 MP high-pressure liquid chromatograph with
gradient capability or equivalent
5.3.1 Ultraviolet absorption detector — 254-nm Eixed waavelength
with 8-uL flow cell, Altex model 153 or equivalent.
5.3.2 High pressure injection valve, capable of 5,000 psi, with
50-uL sample loop — Rheodyne 7120 or equivalent.
5.3.3 Column: Zorbax ODS, C10, 4.6-mm I.D. x 25-cm, available
18
from DuPont, Wilmington, DE.
5.3.4 Guard column: 2.1-mm I.D. x 5-cm stainless steel column
packed with Whatman Co:Pell ODS, available from Whatman,
Clifton, NJ
6. Reagents
6.1 Methanol, distilled-in-glass, Burdick and Jackson or equivalent
6.2 Acetonitrile, UV-grade, distilled-in-glass, Burdick and Jackson
or equivalent
6.3 Water, purified, or HPLC grade
6.4 10% v/v methanol/water solution. Prepare by adding 6 mL of
methanol to 54 mL of water. Mix thoroughly.
6.5 70% v/v methanol/water solution. Prepare by adding 42 mL of
methanol to 18 mL of water. Mix thoroughly.
7. Calibration
7.1 Establish HPLC parameters equivalent to those indicated in Table
1.
409-02 January 1983
-------
7.2 Calibration Procedure
7.2.1 Prepare stock solutions of the various analytes in
methanol.
7.2.2 Prepare appropriate dilute standards to bracket the
concentration range of the samples- to be analyzed by
dilution of the stock solutions with methanol.
7.2.3 Determine the linear range of the detector.
8. Quality Control
8.1 See reference listed in Step 15.1.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection, Preservation, and Handling
9.1 See reference listed in Step 15.2.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 The sample is brought to room temperature.
10.2 The sample is thoroughly, mixed to ensure uniformity.
10.3 Adjust pH of sample with 6N NaOH solution or 6N HC1 solution to
neutral pH-7. Check pH with pH paper.
10.4 Highly concentrated samples should be diluted to ensure complete
sample dissolution and good recoveries through clean-up step.
10.5 Rinse the Sep-Pak C,g cartridge with 3 mL of methanol by
attaching the long end of the cartridge to the Leur-lock end of a
5-mL gas-tight glass syringe and adding methanol to the barrel.
Replace the plunger and pass the methanol through cartridge.
10.6 Rinse the cartridge with two 3-mL portions of organic-free water.
10.7 Remove the plunger from the barrel of a clean 5-mL gas-tight
glass syringe and attach the long end of the cartridge to the
Leur-lock tip.
10.8 Pipet 3.0 mL of sample into the syringe barrel.
10.9 Replace the syringe plunger carefully and invert syringe. Force
trapped air out of syringe.
10.10 Slowly pass sample through cartridge with manual pressure at a
409-03 January 1983
-------
rate of approximately 1 mL/minute or alternatively attach syringe
to syringe pump and pump at rate of 1 mL/minute.
10.11 Discard eluate. Carefully remove cartridge from syringe, so as
not to lose the residual sample trapped on the cartridge, and
place on clean surface.
10.12 Remove plunger from the syringe and replace the cartridge on the
syringe tip.
10.13 Rinse the syringe barrel with approximately 1 mL of organic-free
water and force water through cartridge by replacing plunger and
employing manual pressure.
11. Cleanup and Separation
11.1 Pipet 1.0 mL of 10% v/v methanoI/water into the syringe barrel
of the syringe attached to the sample cartridge.
11.2 Replace the plunger and force the 10% v/v methanol/water solution
through the cartridge.
11.3 Carefully remove cartridge from syringe and remove the plunger.
11.4 Replace the cartridge and pipet 3.0 mL of the 70% v/v
methanol/water solution into the syringe barrel.
11.5 Replace syringe plunger, slowly pass the solution through the
cartridge, and collect the eluate in a 10- or 25-mL Kuderna-
Danish receiver.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph.
12.2 Calibrate the system as described in Section 7.
13. Calculations
13.1 Calculate the average response factor for each analyte by
measuring the peak heights or peak areas of the standard peaks in
two standard solutions which approximate and bracket the sample
concentration.
noo c peak area (or height)
13.2 Response factor = concentration of standard
, _ _ . ,. , peak area (or heighO of analvte
13.3 Concentration of analyte = average response factor
14. Method Performance
409-04 January 1983
-------
14.1 Organic-free water was spiked with various concentrations of
cyanazine, simazine, atrazine, and propazine. Three ml of this
solution was passed through the clean-up procedure and analyzed
by HPLC. Results are summarized in Table 2.
15. References
15.1 Jayanty, R.K. and Gutknecht, W.F. 1979. A Program of Quality
Control/Quality Assurance for the Determination of Priority
Pollutants in Pesticide Industry Wastewaters. Prepared for U.S.
EPA, Process Measurements Branch, Industrial Environmental
Research Laboratory, Research Triangle Park, North Carolina.
Research Triangle Institute, Research Triangle Park, North
Carolina.
15.2 Environmental Science and Engineering, Inc. (ESE). 1979.
Pesticide BAT Review Verification Sampling Protocol. Prepared
for U.S. EPA, Research Triangle Park, North Carolina.
15.3 "Determination of Cyanazine, Simazine, Atrazine, and Propazine in
Industrial Wastewater (Non-concentrative approach using Sep-Pak
Cleanup)," ESE Method 710, Environmental Science and Engineering,
Inc., Gainesville, FL.
409-05 January 1983
-------
Table 1
Chromatographic Conditions
Injection volume
Column
Flow Rate
Temperature
Mobile phase:
A.
B.
C.
D.
50 uL
Zorbax ODS, C,g, 4.6-mm I.D. x 25 cm
1.0 ml/minute
Amb ient
To determine cyanazine and simazine only, isocratic, 40%
acetonitrile/60% water.
To determine cyanazine, simazine, atrazine:, and propazine
. Isocratic, 40% acetonitrile/60% water, for 19 minutes.
. To 70% acetonitrile/30% water in 5 minutes.
. Hold for 6 minutes.
The column should be flushed of extraneous peaks by
changing mobile phase to 70% acetonitrile/30% water or
100% acetonitrile for a short period of time.
Sufficient and reproducible equilibration time should be
employed before the next injection.
409-06
January 1983
-------
Table 2
Retention Times*
Retention
Parameter Time (min)
Cyanazine 9.6
Simazine 10.1
Atrazine 17
Propazine 27.8
* Under Condition B above
409-07 January 1983
-------
Compound
Table 3
Recovery Study in Laboratory Water
Spiked
Concentration
(mg/L)
Percent Recovery
(Avg. of 2 exp)
Cyanazine
Simazine
Atrazine
Propazine
3.24
3.26
3.04
3.65
96.4
97.1
97.0
94.0
409-08
January 1983
-------
PART C
EMSL ANALYTICAL METHODS
-------
vvEPA
United States
Environmental Protection
Agency
Effluent Guidelines Division (WH 552)
Washington, O.C. 20460
Water and Waste Management
TEST METHOD
DETERMINATION OF PENTACHLOROPHENOL SALT
IN WASTEWATER
METHOD 604
1 . Scope and Application
1.1 This method covers the determi-
nation of phenol and certain substituted
phenols. The following parameters may
be determined by this method:
Parameter
4-Chloro-3-methylphenol
2-Chlorophenol
2.4-Oichlorophenol
2,4-Oimethylphenol
2,4-Dinitrophenol
2-Methyl-4,6-dimtrophenol
2-Nitropheno!
4-Nitrophenol
Pentachlorophenol
Phenol
2.4,6-Tnchlorophenol
Pentachlorophenol Salt
STORE! No.
34452
34586
34601
34606
34616
34657
34591
34646
39032
3.4694
34621
—
CAS No.
59-50-7
95-57-8
1 20-83-2
105-67-9
51-28-5
534-52-1
88-75-5
100-02-7
87-86-5
108-95-2
88-06-2
131-52-2
1.2 This is a gas chromatographic
(GC) method applicable to the
determination of the compounds listed
above m municipal and industrial
discharges as provided under 40 CFR
136.1. When this method is used to
analyze unfamiliar samples for any or
all of the compounds above,
compound identifications should be
supported by at least one additional
qualitative technique. This method
describes analytical conditions for
derivatization, cleanup and electron
capture gas chromatography that can
be used to confirm measurements
made by flame lonization. Method 625
provides gas chromatograph/mass
spectrometer (GC/MS) conditions ap-
propriate for the qualitative and
quantitative confirmation of results for
all of the parameters listed above, us-
ing the extract produced by this
method.
1.3 The method detection limit
(MDL. defined in Section 14.1) "' for
each parameter is listed in Table 1.
The MDL for a specific wastewater
may differ from those listed,
depending upon the nature of
interferences in the sample matrix.
The MDL listed in Table 1 for each
parameter was achieved with a flame
lonization detector. Comparable
results were achieved when the
derivatization cleanup and the
electron capture detector were
employed (See Table 2).
1.4 Any modification of this method,
beyond that expressly permitted, shall
be considered a major modification
subject to application and approval of
alternate test procedures under 40
CRF 136.4 and 136.5.
1.5 This method is restricted to use
by or under the supervision of
analysts experienced in the use of gas
chromatography and in the
interpretation of gas chromatograms.
Each analyst must demonstrate the
604-1
January 1983
-------
ability to generate acceptable results
with this method using the procedure
described in Section 8 2.
2. Summary of Method
2.1 A 1 -liter sample of wastewater
is acidified and extracted with
methylene chloride using separatory
funnel techniques. The extract is dried
and concentrated to a volume of 10
mL or less During the concentration
step, the solvent is exchanged to
2-propanol. Flame ionization gas
chromatographic conditions are
described which allow for the
measurement of the compounds in
the extract '*'.
2.2 A preliminary sample wash
under basic conditions can be
employed for samples having high
general organic and organic base
interferences
2.3 The method also provides for the
preparation of pentafluorobenzyl
bromide (PFBB) derivatives for
electron capture gas chromatography
as an additional cleanup procedure to
aid in the elimination of inter-
ferences l2-3'
3. Interferences
3.1 Method interferences may be
caused by contaminants in solvents.
reagents, glassware, and other
sample processing hardware that lead
to discrete artifacts and/or elevated
baselines in gas chromatograms. All of
these materials must be routinely
demonstrated to be free from
interferences under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5.
3.7.7 Glassware must be
scrupulously cleaned141. Clean all
glassware as soon as possible after
use by rinsing with the last solvent
used in it. This should be followed by
detergent washing with hot water,
and rinses with tap water and distilled
water It should then be drained dry,
and heated in a muffle furnace at
400°C for 15 to 30 minutes. Some
thermally stable materials, such as
PCBs, may not be eliminated by this
treatment. Solvent rinses with
acetone and pesticide quality hexane
may be substituted for the muffle
furnace heating. Volumetric ware
should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and
stored in a clean environment to
prevent any accumulation of dust or
other contaminants Store inverted or
capped with aluminum foil.
3.1.2 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.
3.2 Matrix interferences may be
caused by contaminants that are
co-extracted from the sample. The
extent of matrix interferences will
vary considerably from source to
source, depending upon the nature
and diversity of the industrial complex
or municipality being sampled. The
cleanup procedure in Section 12 can
be used to overcome many of these
interferences, but unique samples
may require additional cleanup
approaches to achieve the method
detection limits listed in Tables 1 and
2.
3.3 The basic sample wash (Section
10.2) may cause significantly
reduced recovery of phenol and
2,4-dimethylphenol. The analyst must
recognize that results obtained under
these conditions are minimum
concentrations
4. Safety
4.1 The toxicity or carcinogenicity of
each reagent used in this method has
not been precisely defined; however,
each chemical compound should be
treated as a potential health hazard.
From this viewpoint, exposure to
these chemicals must be reduced to
the lowest possible level by whatever
means available. The laboratory is
responsible for maintaining a current
awareness file of OSHA regulations
regarding the safe handling of the
chemicals specified in this method. A
reference file of material data
handling sheets should also be made
available to all personnel involved in
the chemical analysis Additional
references to laboratory safety are
available and have been identified I5~71
for the information of the analyst.
4.2 Special care should be taken in
handling pentafluorobenzyl bromide,
which is a lachrymator, and 18 crown
6 ether, which is highly toxic.
5. Apparatus and Materials
5.1 Sampling equipment, for
discrete or composite sampling
5.1.1 Grab sample bottle - Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with Tef-
lon Foil may be substituted for Teflon
if the sample is not corrosive If amber
bottles are not available, protect
samples from light The container and
capliner must be washed, rinsed with
acetone or methylene chloride, and
dried before use to minimize con-
tamination.
5.7.2 Automatic sampler (optional)
- The sampler must incorporate glass
containers for the collection of a
minimum of 250 mL of sample
Sample containers must be kept
refrigerated at 4°C and protected from
light during compositing. If the
sampler uses a peristaltic pump, a
minimum length of compressible
silicone rubber tubing may be used.
Before use, however, the
compressible tubing must be
thoroughly rinsed with methanol,
f' Mowed by repeated rinsings with
distilled water to minimize the
potential for contamination of the
sample. An integrating flow meter is
required to collect flow proportional
composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included tor illustration only).
5.2.7 Separatory funnel - 2000-mL.
with Teflon stopcock.
5.2.2. Drying column -
Chromatographic column 400-mm
long x 19-mm ID with coarse frit.
5.2.3. Chromatographic column -
100-mm long x 10-mm ID, with Teflon
stopcock.
5.2.4. Concentrator tube,
Kuderna-Danish - 10-mL, graduated
(Kontes K-570050-1025 or
equivalent). Calibration must be
checked at the volumes employed in
the test. Ground glass stopper is used
to prevent evaporation of extracts.
5.2.5 Evaporative flask,
Kuderna-Danish - 500-mL (Kontes
K-570001 -0500 or equivalent). Attach
to concentrator tube with springs.
5.2.6 Snyder column,
Kuderna-Danish - three-ball macro
(Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column,
Kuderna-Danish - two-ball micro
(KontesK-569001-0219or
equivalent).
5.3 Vials - Amber glass, 10- to 15-
ml_ capacity, with Teflon-lined
screwcap.
5.4 Reaction flask - Pyrex glass, 15-
to 25-mL round bottom flask with
standard tapered joint, fitted with a
water cooled condenser and U-shaped
drying tube containing granular
calcium chloride
5.5 Boiling chips - Approximately
10/40 mesh. Heat to 400°C for 30
minutes or Soxhlet extract with
methylene chloride.
5.6 Water bath - Heated, with
concentric ring cover, capable of
604-2
January 1983
-------
temperature control (± 2°C) The bath
should be used in a hood
5.7 Balance - Analytical, capable of
accurately weighing 00001 g
5.8 Gas chromatograph - An
analytical system complete with a
temperature programmable gas
chromatograph suitable for on-column
injection and all required accessories
including syringes, analytical columns,
gases, detector, and strip-chart
recorder A data system is
recommended for measuring peak
areas
5.8.1 Column for undenvatized
phenols -18m long x 2 mm ID
glass, packed with 1% SP-1240 DA
on Supelcoport (80/100 mesh) or
equivalent This column was used to
develop the method performance
statements in Section 14 Guidelines
for the use of alternate column
packings are provided in Section
11 1
5 8.2 Column for denvatized
phenols - 1.8 m long x 2 mm ID glass
column packed with 5% OV-17 on
Chromosorb W-AW-DMCS (80/100
mesh). The carrier gas is 5%
methane/95% Argon at a flow rate of
30 mL/mm. The column temperature
is 200°C This column has proven
effective in the analysis of
wastewaters for denvatization
products of the parameters listed in
the scope (Section 1 1), and was used
to develop the method performance
statements m Section 14. Guidelines
for the use of alternate columns are
provided in Section 11 1
5.8.3 Detectors - flame lonization
and electron capture. The flame
lonization is used when determining
the parent phenols The electron cap-
ture is used when determining the
denvatized phenols Guidelines for
use of alternate detectors are provided
in Section 11 1
6. Reagents
6.1 Reagent water - Reagent water
is defined as a water in which an
interferent is not observed at the
MDL of each parameter of interest.
6.2 Sodium hydroxide solution (10
N) - (ACS) Dissolve 40g NaOH m
reagent water and dilute to 100 ml
6.3 Sodium hydroxide solution (1 N)
- (ACS) Dissolve 4g NaOH in reagent
water and dilute to 100 ml
6.4 Sodium sulfate - (ACS)
Granular, anhydrous Purify by
heating at 400°C for four hours in a
shallow tray
6.5 Sodium thiosulfate - (ACS)
Granular
6.6 Sulfuric acid solution (1 + 1) -
(ACS) Slowly, add 50 mL H2SO< (sp.
gr. 1 84) to 50 mL of reagent water.
6.7 Sulfunc acid(1 N) -(ACS)
Slowly, add 29 mL H2SO< (ACS, sp.
gr 1.84) to reagent water and dilute
to one liter.
6.8 Potassium carbonate - (ACS)
powdered.
6.9 Pentafluorobenzyl bromide (a-
Bromopentafluorotoluene) - 97%
minimum purity. NOTE: This chemical
is a lachrymator (See Section 4.2.)
6.10 18-crown-6 ether
(1,4,7,10,13,16 -
Hexaoxacyclooctadecane) - 98%
minimum purity. NOTE. This chemical
is highly toxic.
6.11 Denvatization reagent -Add
one mL pentafluorobenzyl bromide
and one gram 18 crown 6 ether to a
50-mL volumetric flask and dilute to
volume with 2-propanol. Prepare fresh
weekly. This operation should be car-
ried out in a hood. Store 4°C and pro-
tect from light.
6.12 Acetone, hexane, methanol,
methylene chloride, 2-propanol,
hexane, toluene - Pesticide quality or
equivalent
6.13 Silica gel - Davison chemical,
grade 923 (100/200 mesh) or
equivalent Activate at 130°C
overnight and store in a desiccator.
6.14 Stock standard solutions (1.00
(jg/(jL) - Stock standard solutions may
be prepared from pure standard
materials or purchased as certified
solutions.
6.14.1 Prepare stock standard
solutions by accurately weighing
about 0.0100 grams of pure material.
Dissolve the material m pesticide
quality 2-propanol and dilute to
volume in a 10-mL volumetric flask.
Larger volumes may be prepared at
the convenience of the analyst. If
compound purity is certified at 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
6.14.2 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. Quality control
check standards that can be used to
determine the accuracy of calibration
standards, will be available from the
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio. 45268
6.14.3 Stock standard solutions
must be replaced after six months, or
sooner if comparison with check
standards indicates a problem
7. Calibration
7.1 To calibrate the FIDGC for the
analysis of underivatized phenols.
establish gas chromatographic
operating parameters equivalent to
those indicated m Table 1 The gas
chromatographic system can be
calibrated using the external standard
technique (Section 7 2) or the internal
standard technique (Section 7.3).
7.2 External standard calibration
procedure for FIDGC.
7.2.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with 2-
propanol. One of the external
standards should be at a
concentration near, but above, the
MDL and the other concentrations
should correspond to the expected
range of concentrations found in real
samples or should define the working
range of the detector.
7.2.2 Using injections of 2 to 5 fjL
of each calibration standard, tabulate
peak height or area responses against
the mass injected. The results can be
used to prepare a calibration curve for
each compound. Alternatively, if the
ratio of response to amount injected
(calibration factor) is a constant over
the working range (< 10% relative
standard deviation, RSD), linearity
through the origin can be assumed
and the average ratio or calibration
factor can be used in place of a
calibration curve
7.2.3 The working calibration curve
or calibration factor must be verified
on each working day by the
measurement of one or more
calibration standards. If the response
for any parameter varies from the
predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound
604-3
January 1983
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7.3 Internal standard calibration
procedure for FIDGC To use this
approach, the analyst must select one
or more internal standards that are
similar in analytical behavior to the
compounds of interest The analyst
must further demonstrate that the
measurement of the internal
standard is not affected by method or
matrix interferences Because of these
limitations, no internal standard can
be suggested that is applicable to all
samples
7.3.7 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
To each calibration standard, add a
known constant amount of one or
more internal standards, and dilute to
volume with 2-propanol One of the
standards should be at a
concentration near, but above, the
MDL and the other concentrations
should correspond to the expected
range of concentrations found in real
samples or should define the working
range of the detector
7.3.2 Using injections of 2 to 5 fjL
of each calibration standard, tabulate
peak height or area responses against
concentration for each compound and
internal standard, and calculate
response factors (RF) for each
compound using equation 1.
Eq. 1 RF = (A,C,»)/(A,, C,)
where'
A, = Response for the parameter to be
measured.
A,,= Response for the internal
standard.
C,j = Concentration of the internal
standard, (/jg/L)
C, = Concentration of the parameter
to be measured, (fjg/L).
If the RF value over the working range
is a constant « 10% RSD). the RF can
be assumed to be nonvanant and the
averge RF can be used for
calculations Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/As, vs .RF
7 3.3 The working calibration curve
or RF must be verified on each
working day by the measurement of
one or more calibration standards If
the response for any parameter varies
from the predicted response by more
than ±10%, the test must be repeated
using a fresh calibration standard
Alternatively, a new calibration curve
must be prepared for that compound
7.4 To calibrate the ECGC for the
analysis of phenol derivatives,
establish gas chromatographic
operating parameters equivalent to
those indicated in Table 2.
7.4.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest
by adding volumes of one or more
stock standards to a volumetric flask
and diluting to volume with 2-
propanol One of the external
standards should represent a
concentration near but above the MDL
and the other concentrations should
correspond to the expected range of
concentrations found in real samples
or should define the working range of
the detector
7.4.2 Each time samples are to be
denvatived, simultaneously treat a
one-mL aliquot of each calibration
standard as described in Section 12.
7.4.3 After denvatization, inject 2 to
5 fjL of each column eluate collected
and tabulate peak height or area
responses against the calculated
equivalent mass of undenvatized
phenol injected The results can be
used to prepare a calibration curve for
each compound
7.5 Before using any cleanup
procedure, the analyst must process a
series of calibration standards through
the procedure to validate elution
patterns and the absence of
interferences from the reagents.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program The minimum
requirements of this program consist
of an initial demonstration of
laboratory capability and the analysis
of spiked samples as a continuing
check on performance The laboratory
is required to maintain performance
records to define the quality of data
that is generated Ongoing
performance checks must be
compared with established
performance criteria to determine if
the results of analyses are within
accuracy and precision limits expected
of the method
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision
with this method This ability is
established as described in Section
82
8.12 In recognition of the rapid
advances that are occurring in
chromatography, the analyst is
permitted certain options to improve
the separations or lower the cost of
measurements Each time such
modifications are made to the method.
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike and
analyse a minimum of 10% of all
samples to monitor continuing
laboratory performance. This
procedure is described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform
the following operations.
8.2.1' Select a representative spike
concentration for each compound to
be measured. Using stock standards,
prepare a quality control check sample
concentrate m 2-propanol 1000 times
more concentrated that the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate to each
of a minimum of four 1000-mL
aliquots of reagent water. A
representative wastewater may be
used in place of the reagent water,
but one or more additional aliquots
must be analyzed to determine
background levels, and the spike level
must exceed twice the background
level for the test to be valid. Analyze
the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard
deviation of the percent recovery (s),
for the results. Wastewater
background corrections must be made
before R and s calculations are
performed.
8.2.4 Using Table 3, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s >
2p or |X-R| > 2p, review potential
problem areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish per-
formance criteria for R and s based
upon the results of mterlaboratory
testing When they become available,
these criteria must be met before any
samples may be analyzed
8.3 The analyst must calculate
method performance criteria and
define the performance of the
laboratory for each spike
concentration and parameter being
measured.
604-4
January 1983
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8.3.1 Calculate upper and lower
control limits for method performance
Upper Control Limit (UCL) R + 3 s
Lower Control Limit (LCL) - R - 3 s
where R and s are calculated as in
Section 8 2.3 The UCL and LCL can
be used to construct control charts'8'
that are useful in observing trends in
performance. The control limits above
must be replaced by method
performance criteria as they become
available from the U.S Environmental
Protection Agency
8.3.2 The laboratory must develop
and maintain separate accuracy
statements of laboratory performance
for wastewater samples. An accuracy
statement for the method is defined
as R ± S. The accuracy statement
should be developed by the analysis of
four aliquots of wastewater as
described in Section 8 2.2, followed
by the calculation of R and s.
Alternately, the analyst may use four
wastewater data points gathered
through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly181
8.4 The laboratory is required to
collect a portion of their samples in
duplicate to monitor spike recoveries.
The frequency of spiked sample
analysis must be at least 10% of all
samples or one sample per month,
whichever is greater. One aliquot of
the sample must be spiked and
analyzed as described in Section 8.2.
If the recovery for a particular
parameter does not fall within the
control limits for method performance,
the results reported for that parameter
in all samples processed as part of the
same set must be qualified as
described in Section 13.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagents interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as
a safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance practices for use with this
method. The specific practices that
are most productive depend upon the
needs of the laboratory and the nature
of the samples Field duplicates may
be analyzed to monitor the precision
of the sampling technique When
doubt exists over the identification of
a peak on the chromatogram,
confirmatory techniques such as gas
chromatography with a dissimilar
column, specific element detector, or
mass spectrometer must be used
Whenever possible, the laboratory
should perform analysis of standard
reference materials and participate in
relevant performance evaluation
studies
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices'91 should be
followed, except that the bottle must
not be prewashed with sample before
collection Composite samples should
be collected in refrigerated glass
containers m accordance with the
requirements of the program
Automatic sampling equipment must
be as free as possible of Tygon tubing
and other potential sources of
contamination
9.2 The samples must be iced or
refrigerated at 4°C from the time of
collection until extraction Fill the
sample bottle and at time of collection
if residual chlorine is present, add 80
mg of sodium thiosulfate and mix
well U.S Environmental methods
330 4 and 330 5 may be used for
measurement of residual chlorine110'
Field test kits are available for this
purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction121
10. Sample Extraction
10.1 Mark the water meniscus on
the side of the sample bottle for later
determination of sample volume Pour
the entire sample into a two-liter
separatory funnel
10.2 For samples high inorganic
content, the analyst may solvent wash
the sample at a basic pH as
prescribed in Section 1021 and
1022 to remove potential method
interferences Prolonged or exhaustive
contact with solvent during the wash
may result in low recovery of some of
the phenols, notably phenol and 2,4-
dimethyl phenol For relatively clean
samples, the wash should be omitted
and the extraction, beginning with
Section 10 3, should be followed
10.2.1 Adjust the pH of the sample
to 12 0 or greater with 10 N sodium
hydroxide
10.2.2 Add 60 mL of methylene
chloride to the sample by shaking the
funnel for one minute with periodic
venting to release vapor pressure
Discard to solvent layer The wash
can be repeated up to two additional
times if significant color is being
removed
10.3 Adjust the sample to a pH of 1
to 2 with sulfunc acid (1 + 1)
10.4 Add 60 mL of methylene
chloride to the sample bottle, seal,
and shake 30 seconds to rinse the
inner walls Transfer the solvent to
the separatory funnel and shake for
two minutes Allow the solvent to
separate from the sample and collect
the methylene chloride in a 250-mL
Erlenmeyer flask If the emulsion
interface between layers is more than
one-third the size of the solvent layer,
the analyst must employ mechanical
techniques to complete the phase
separation The optimum technique
depends upon the sample, but may
include stirring, filtration of the
emulsion through glass wool, or
centrifugation
10.5 Add a second 60-mL volume of
methylene chloride to the sample
bottle and complete the extraction
procedure a second time, combining
the extracts in the Erlenmeyer flask.
Perform a third extraction in the same
manner
10.6 Assemble a Kuderna-Danish
(K-D) concentrator by attaching a 10-
mL concentrator tube to a 500-mL
evaporative flask Other concentration
devices or techniques may be used in
place of the KD if the requirements of
Section 8 2 are met
10.7 Pour the combined extract
through a drying column containing
three to four inches of anhydrous
sodium sulfate, and collect m the K-D
concentrator Rinse the Erlenmeyer
flask and column with 20 to 30 mL
methylene chloride to complete the
quantitative transfer
10.8 Add one to two clean boiling
chips to the flask and attach a three-
ball Snyder column Prewet the
Snyder column by adding about 1 mL
methylene chloride to the top Place
the K-D apparatus on a hot water
bath (60 to 65°C) so that the con-
centrator tube is partially immersed in
the hot water, and the entire lower
rounded surface of the flask is bathed
in vapor Adjust the vertical position
of the apparatus and the water
temperature as required to complete
the concentration in 1 5 to 20
minutes. At the proper rate of distilla-
tion the balls of the column will
actively chatter but the chambers will
604-5
January 1983
-------
not flood When the apparent volume
of liquid reaches 1 mL, remove the K-
D apparatus and allow it to dram for
at least 10 minutes while cooling
10.9 Increase the temperature of
the hot water bath to 95 to 100°C
Remove the Snyder column and rinse
the flask and its lower joint into the
concentrator tube with 1 to 2 mL of 2-
propanol A 5-mL syringe is
recommended for this operation
Attach a micro-Snyder column to the
concentrator tube and prewet the
column by adding about 0 5 mL of 2-
propanol to the top Place the micro-
K-D apparatus on the water bath so
that the concentrator tube is partially
immersed in the hot water Adjust the
vertical position of the apparatus and
the water temperature as required to
complete concentration in 5 to 10
minutes At the proper rate of
distillation, the balls of the column
will actively chatter but the chambers
will not flood. When the apparent
volume of the liquid reached 2.5 mL,
remove the K-D apparatus and allow
it to dram for at least 10 minutes
while cooling Add an additional 2 mL
of 2-propanol through the top of the
micro-Snyder column and resume
concentrating as before When the
apparent volume of liquid reaches 0 5
mL, remove the K-D apparatus and
allow it to dram for at least 10
minutes while cooling. Remove the
micro-Snyder column and rinse its
lower joint into the concentrator tube
with a minimum amount of 2-
propanol Adjust the extract volume to
1 0 mL. Stopper the concentrator tube
and store in refrigerator at 4°C, if
further processing will not be per-
formed immediately. If the sample ex-
tract requires no further cleanup, pro-
ceed with flame lonization gas
chromatographic analysis (Section
11) If the sample requires further
cleanup, proceed to Section 12. If the
extracts will be stored longer than two
days, they should be transferred to
Teflon-sealed screw-cap vials
10.10 Determine the original
sample volume by refilling the sample
bottle to the mark and transferring the
liquid to a 1000-mL graduated
cylinder. Record the sample volume to
the nearest 5 mL
11. Gas Chromatography -
Flame lonization Detector
11.1 Table 1 summarizes the
recommended gas chromatographic
column and operating conditions This
Table includes retention times and
MDL obtained under these conditions.
An example of the parameter separa-
tion achieved by this column is shown
in Figure 1 Other packed columns,
chromatographic conditions, or de-
tectors may be used if the require-
ments of Section 8 2 are met
Capillary (open-tubular) columns may
also be used if the relative standard
deviations of responses for replicate
injections are demonstrated to be less
than 6% and the requirements of Sec-
tion 8.2 are met
11.2 Calibrate the system daily as
described in Section 7 1
11.3 If the internal standard
approach is used, the standard must
be added to the sample extract and
mixed thoroughly immediately before
injection into the instrument
11.4 Inject 2 to 5 /^L of the sample
extract using the solvent-flush
technique111 Smaller (1 O/^L) volumes
may be injected if automatic injectors
are employed. Record the volume
injected to the nearest 0 05 fjL and
the resulting responses in peak area
or peak height units. If the response
for the peak exceeds the working
range of the system, dilute the extract
and reanalyze.
11.5 The width of the retention time
window used to make identifications
should be based upon measurements
of actual retention time variations of
standards over the course of a day.
Three times the standard deviation of
a retention time for a compound may
be used to calculate a suggested
window size, however, the experience
of the analyst should weigh heavily in
the interpretation of chromatograms.
11.6 If the measurement of the peak
response is prevented by the presence
of interferences, an alternate gas
chromatographic procedure is
required. Section 1 2 describes a
denvatization and column
chromatographic procedure which has
been tested and found to be a
practical means of analyzing phenols
in complex extracts
12. Derivatization and
Electron Capture Gas
Chromatography
12.1 Pipet a 1.0-mL aliquot of the 2-
propanol solution of standard or
sample extract into a glass reaction
vial. Add 1.0-mL of denvatizmg
reagent (Section 611) This is a
sufficient amount of reagent to
denvatize a solution whose total
phenolic content does not exceed 0.3
mg/mL
12.2 Add about 3 mg of potassium
carbonate to the solution and shake
gently.
12.3 Cap the mixture and heat it for
four hours at 80°C in a hot water
bath
12.4 Remove the solution from the
hot water bath and allow it to cool
12.5 Add 10 mL of hexane to the
reaction flask and shake vigorously for
one minute Add 3 0 mL of distilled,
deionized water to the reaction flask
and shake for two minutes Decant a
portion of the organic layer into a
concentrator tube and cap with a
glass stopper
12.6 Pack a 10-mm ID
chromatographic column with 4 0
grams of activated silica gel After
settling the silica gel by tapping the
column, add about two grams of
anhydrous sodium sulfate to the top.
12.7 Pre-elute the column with 6
mL of hexane. Discard the eluate and
just prior to exposure of the sulfate
layer to air, pipet onto the column 2.0
mL of the hexane solution (Section
12.5) that contains the denvatized
sample or standard. Elute the column
with 10.0 mL of hexane (Fraction 1)
and discard this fraction. Elute the
column, in order, with 10 0 mL of
15% toluene in hexane (Fraction 2);
10 0 rnl. of 40% toluene in hexane
(Fraction 3), 10.0 mL 75% toluene in
hexane (Fraction 4); and 10.0 mL 15%
2-propanol in toluene (Fraction 5). All
elution mixtures are prepared on a
volume:volume basis. Elution patterns
for the phenolic derivatives are shown
in Table 2. Fractions may be combined
as desired, depending upon the
specific phenols of interest or level of
interferences.
12.8 Analyze the fractions by
electron capture gas Chromatography.
Table 2 summarizes the
recommended gas chromatographic
column and operating conditions This
Table includes retention times and
MDL obtained under these conditions.
An example of the parameter separa-
tion achieved by this column is shown
in Figure 2
12.9 Calibrate the system daily with
a minimum of three aliquots of
calibration standards, containing each
of the phenols of interest that are
denvatized according to the procedure
(See Section 7 4)
12.10 Inject 2 to 5 fjL of the column
fractions using the solvent-flush tech-
nique. Smaller (1 0^/L) volumes can
be injected if automatic devices are
employed Record the volume injected
to the nearest 0 05 /jL, and the result-
ing peak size, in area units or height.
If the peak response exceeds the
604-6
January 1983
-------
linear range of the system, dilute the
extract and reanalyze
13. Calculations
13.1 Calculate ihe concentration of
individual compounds in the sample
determined by the flame lonization
procedure (without denvatization) as
indicated below
13.1.1 If the external standard
calibration procedure is used.
calculate the amount of material
injected from the peak response using
the calibration curve or calibration
factor in Section 7 2.2 The
concentration m the sample can be
calculated from equation 2:
Eq. 2. Concentration.
(A) (V.)
IV,) (V.)
where
A - Amount of material injected, in
nanograms
V, = Volume of extract injected (j/L).
Vi - Volume of total extract (jjL).
V, = Volume of water extracted (ml).
13.1.2 If the internal standard
calibration procedure was used,
calculate the concentration in the
sample using the response factor (RF)
determined in Section 7.3.2 and
equation 3.
Eq.3.Concentration./yg/L-
(A,,)(RF)(V0)
where:
A, = Response for the parameterto be
measured
A,, - Response for the internal
standard.
I, = Amount of internal standard
added to each extract (/ug).
V0 = Volume of water extracted, in
liters.
13.2 Calculate the concentration of
individual compounds in the sample
when determined by the derivatization
and electron capture procedure
according to Equation 4.
Eq. 4 Concentration, /jg/L =
(V,) (V,) (C) (D)
where:
A = Mass of undenvatized phenol
represented by area of peak in
sample chromatogram,
determined from calibration
curve m Section 7 4.3, in
nanograms
B Total volume of 2-propanol
extract after concentration
in mL
V, - Volume of eluate injected (//g/L).
Vi - Total volume of column eluate
U/g/L).
V, - Volume of water extracted (mL)
C Volume of hexane sample
solution added to cleanup
column, in mL.
D Volume of 2-propanol extract
used for derivatization in mL
13.3 Report results in micrograms
per liter without correction for
recovery data When duplicate and
spiked samples are analyzed, report-
all data obtained with the sample
results.
13.4 For samples processed as part
of a set where the laboratory spiked
sample recovery falls outside of the
control limits in 8.4, data for the
affected parameters must be labeled
as suspect.
14. Method Performance
14.1 The method detection limit
(MDU is defined as the minimum
concentration of a substance that can
be measured and reported with 99%
confidence that the value is above
zero'" The MDL concentrations listed
m Tables 1 and 2 were obtained using
reagent water"2'. Similar results were
achieved using representative
wastewaters.
14.2 In a single laboratory (IT.
Enviroscience, Inc.). using spiked
reagent water and wastewater
samples, the average recoveries
presented m Table 3 were obtained
using a flame lonization detector1121
The standard deviation of the percent
recovery is also included in Table 3.
14.3 The U.S. Environmental Protec-
tion Agency is in the process of con-
ducting an mterlaboratory method
study to fully define the performance
of this method.
References
1. See Appendix A.
2. "Determination of Phenols in
Industrial and Municipal
Wastewaters," Report for EPA
Contract 68-03-2625. In
preparation
3. Kawahara, F.K., Analytical
Chemistry. 40. 1009 (1968).
4. ASTM Annual Book of Standard,
Part 31, D3694, "Standards
Practice for Preparation of Sample
Containers and for Preservation,"
American Society for Testing and
Materials, Philadelphia, PA, p.
679, 1980
5. "Carcinogens - Working With
Carcinogens," Department of
Health, Education, and Welfare,
Public Health Service, Center for
Disease Control, National Institute
for Occupational Safety and
Health, Publication No 77-206,
Aug 1977
6 "OSHA Safety and Health
Standards, General Industry," (29
CFR 1910), Occupational Safety
and Health Administration, OSHA
2206, (Revised. January 1976).
7. "Safety in Academic Chemistry
Laboratories," American Chemical
Society Publication, Committee on
Chemical Safety, 3rd Edition,
,979
8. "Handbook for Analytical Quality
Control m Wastewater and
Wastewater Laboratories," EPA-
600/4-79-019, U.S
Environmental Protection Agency,
Environmental Monitoring and
Support Laboratory - Cincinnati,
Ohio 45268, March 1979.
9. ASTM Annual Book of Standards,
Part 31, D3370, "Standard
Practice for Sampling Water,"
American Society for Testing and
Materials, Philadelphia, PA, p. 76,
1980.
10. "Methods 330 4 (Titrimetric, DPD-
FAS) and 330.5
(Spectrophotometric, DPD) for
Chlorine, Total Residual."
Methods for Chemical Analysis of
Water and Wastes, EPA 600/4-
79-020, U.S. Environmental
Protection Agency. Environmental
Monitoring and Support
Laboratory - Cincinnati, Ohio
45268. March 1979.
11. Burke, J.A.. "Gas
Chromatography for Pesticide
Residue Analysis; Some Practical
Aspects," Journal of the
Association of Official Analytical
Chemists. 48. 1037 (1965).
12. "Development of Detection Limits,
EPA Method 604, Phenols."
Special letter report for EPA
contract 68-03-2625.
Environmental Monitoring and
Support Laboratory - Cincinnati,
Ohio 45268.
13. "Methods for Organic Chemicals
Analyses in Municipal and
Industrial Wastewater," July 1982,
U.S. Environmental Protection
Agency, Environmental Monitoring
and Support Laboratory,
Cincinnati, Ohio 45668.
604-7
January 1983
-------
Table 1. Chromatographic Conditions and
Method Detection Limit
Parameter
Retention Method
Time Detection Limit
(mm.) ffjg/U
2-Chlorophenol
2-Nitrophenol
Phenol
2.4-Dimethylphenol
2. 4-Dichlorophenol
2.4.6- Trichlorophenol
4-Ch/oro-3-methylphenol
2. 4-Dimtrophenol
2-Methyl-4. 6-dinitrophenol
Pentachlorophenol
4-Nitrophenol
1.70
2.00
301
403
430
6.05
750
10.OO
10.24
12.42
2425
0.31
0.45
0.14
0.32
0.39
0.64
0.36
13.0
16.0
7.4
2.8
Column conditions: Supelcoport (80/100 mesh) coated
with 1% SP-1240 DA in 1 8 m long x 2 mm ID glass
column with nitrogen carrier gas at a flow rate of 30
mL/min flow rate. Column temperature was 80° C at
injection, programmed immediately at 8°C/mm to
150°C final temperature. Method detection limits were
determined with a flame ionization detector
Table 2. Silica Gel Fractionation and Electron Capture
Gas Chromatography of PFBB Derivatives
Election Capture
Recovery /%/ by Fraction'
Parent Compound 1
2-Chlorophenol
2-Nttrophenol
Phenol
2.4-Dimethylphenol
2.4-Dichlorophenol
2.4.6-Tnchlorophenol
4 Chloro-2-methyphenol
Pentachlorophenot
4-Nitrophenol
2 3
90
90
95
95
50 50
84
75 20
4 5
1
9 90
10
7
t
14
t 90
Retention
Time
(mm I
33
9 1
t a
29
58
70
48
288
140
Method
Detection
Limit /ug/L>
058
077
22
063
O68
058
18
059
070
"fluting solvent compositions as given in Section 12 7
Column conditions Chromosorb W-AW-DMCS 180/100 mesh) coated with 5% OV-17 packed in a I 8
m long x 20 mm ID glass column with 5% methane/95% argon carrier gas at a flow rate of 30
mL/min Column temperature isothermal at 20O°C
Table 3. Single Operator Accuracy and Precision
Parameter
4-Chloro-3-methylphenol
2-Chlorophenol
2.4-Otchlorophenol
2.4-Dimethylphenot
2.4-Dinilropheno/
2 Methyl-4,6-dinitrophenol
2-Nttrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2,4,6-Tnchlorophenol
Average
Percent
Recovery
82
67
74
51
74
86
67
45
79
41
71
Standard
Deviation
%
1SO
148
11 4
140
165
124
129
79
88
84
145
Spike
Range
lng/L!
070
0 74
t 03
082
287
346
080
159
21 0
076
1 20
35
3 7
52
- 4 1
40
38
• 60
Number
of
A na/yses
21
21
21
21
14
21
21
21
21
21
2!
Matrix
Types
3
3
3
3
2
3
3
3
3
3
3
604-8
January 1983
-------
o
-------
-------
&EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CHLOROBENZILATE, ETRIDIAZOLE, PROPACHLOR,
DIBROMOCHLOROPROPANE (DBCP) IN WASTEWATER
METHOD 608.1
1. Scope and Application
1.1 This method covers the determination of certain organochlorlne
pesticides. The following parameters can be determined by this
method:
Parameter STORET No. CAS No.
Chlorobenzilate 39460 510-15-6
Chloroneb — 2675-77-6
Chloropropylate — 5836-10-2
Dibromochloropropane ~ 96-12-8
Etridiazole — 2593-15-9
PCNB — 82-68-8
Propachlor — 1918-16-7
1.2 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in industrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15)
for each parameter is listed in Table 1. The MOL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 This method presents an extension in scope of Method 608. Further,
the sample extraction and concentration steps in this method are
essentially the same as several others in the 600-method series.
Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is
required, the concentration levels must be high enough to permit
selecting aliquots, as necessary, in order to apply appropriate
cleanup procedures. Under Gas Chromatography, the analyst is
allowed the latitude to select chromatographic conditions
appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
608.1-01 January 1983
-------
1.5 This method is restricted to use by or under the supervision of
analysts experienced 1n the use of gas chromatography and 1n the
Interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described 1n Section 8.2.
1.6 When this method 1s used to analyze unfamiliar samples for any or
all of the compounds above, compound Identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas
chrocnatographlc column for chlorobenzllate and chloropropylate that
can be used to confirm measurements made with the primary column.
Section 14 provides gas chromatograph/mass spectrometer (GC/MS)
criteria appropriate for the qualitative confirmation of compound
Identifications.
2. Summary of Method
271A measured volume of sample, approximately 1 liter, is solvent
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and exchanged to hexane during
concentration to a volume of 10 mL or less. Gas chromatographic
conditions are described which permit the separation and measure-
ment of the compounds in the extract by electron capture (EC) gas
chromatography,'
2.2 This method provides an optional Florisil column cleanup procedure
to aid in the elimination or reduction of interferences which may
be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon 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 15 to-30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
608.1-02 January 1983
-------
3.1.2 The use of high purity reagents and solvents helps to
minimize Interference problems. Purification of solvents by
distillation 1n all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem 1n
pesticide analysis when the EC detector 1s used. These compounds
generally appear 1n the chromatogram as large late eluting peaks,
especially 1n the 15 and 50% fractions frtim the Flor1s1l column
cleanup. Common flexible plastics contain varying amounts of
phthalates. These phthalates are easily extracted or leached from
such materials during laboratory operations. Cross contamination
of clean glassware occurs when plastics are handled during
extraction steps, especially when solvent wetted surfaces are
handled. Interferences from phthalates can be minimized by
avoiding the use of plastics in the laboratory. Exhaustive cleanup
of reagents and glassware may be required to eliminate background
phthalate contamination.3.4 The interferences from phthalate
esters can be avoided by using a mlcrocoulometric or electrolytic
conductivity detector.
3.3 Matrix interferences may be caused by contaminants that are co-.
extracted from the sample. The extent of matrix Interferences will
vary considerably from source to source, depending upon the nature
and diversity of the industrial complex or municipality sampled.
The cleanup procedure in Section 11 can be used to overcome many of
these interferences, but unique samples may require additional
cleanup approaches to achieve the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
.has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest" possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 5-7 for the information of the analyst.
4.2 The following parameters covered by this method have been
tentatively classified as known or suspected, human or mamalian
carcinogens: chlorobenzilate; dibromochloropropane; and PCNB.
Primary standards of these toxic compounds should be prepared in a
hood.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
608-1-03 January 1983
-------
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-1 Her or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
1f the sample 1s not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
Uner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must Incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
sillcone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Vials - Amber glass, 10 to 15 mL capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
608.1-04 January 1983
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5.4 Water bath - Heated, with concentric ring cover, capable of temper-
ature control (± 2°C). The bath should be used 1n a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column Injection and' all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system 1s recommended for measuring peak
areas.
5.6.1 Column 1 - 180 cm long x 2 mm ID glass, packed with 1.5%
SP-2250/1.95X SP-2401 on Supelcoport (100/120 mesh) or
equivalent. This column was used to develop the method
performance statements in Section 15. Alternative columns
may be used in accordance with the provisions described in
Section 12.1.
5.6.2 Column 2 - 180 cm long x 2 mm ID glass, packed with
Ultrabond 20M (100/120 mesh) or equivalent.
5.6.3 Detector - Electron capture. This detector has proven
effective in the analysis of wastewaters for the parameters
listed in the scope and was used to develop the method
performance statements in Section 15. Alternative
detectors, including a mass spectrometer, may be used in
accordance with the provisions described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride, methanol -
Pesticide quality or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. PI 126-8, and
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 mL ethyl
alcohol preservative must be added to each liter of ether.
6.4 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
608.1-05 January 1983
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6.5 Flor1s1l - PR grade (60/100 mesh). Purchase activated at 1250°F
and store In dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.6 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.6.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality isooctane and dilute to volume
in a 10-mL volumetric flask. Larger volumes may be used at
the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards may be used
at any concentration if they are certified by the
manufacturer or by an independent source.
6.6.2 Transfer the stock standard solutions into
TFE-fluorocarbon-sealed screw cap vials. Store at 4°C and
protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation,
especially just prior to preparing calibration standards
from them.
6.6.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
isooctane. One of the external standards should be
representative of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates or should define the working range of
the detector.
608.1-06 January 1983
-------
7.2.2 Using Injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
Injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass Injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less-than 10X over the working
range, the average calibration factor can be used In place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration stan-
dard. Alternatively, a new calibration curve or calibration
factor must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst'must
further demonstrate that the measurement of the Internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three concen-
tration levels for each parameter of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with isooctane. Cne of the standards should be
representative of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF = (AsCis)/(Ais Cs)
where:
As = Response for the parameter to be measured.
Ais * Response for the internal standard.
C-js = Concentration of the Internal standard in ug/L.
Cs = Concentration of the parameter to be measured in
ug/L.
608'1-07 January 1983
-------
If the RF value over the working range is constant, less
than 10X relative standard deviation, the RF can be assumed
to be Invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, AS/AIS against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10X, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 The cleanup procedure 1n Section 11 utilizes Florisil chromato-
graphy. Flor1s1l from different batches or sources may vary 1n
adsorptlve capacity. To standardize the amount of Florisil which
is used, the use of laurlc add value is suggested. This
procedure** determines the adsorption from hexane solution of
lauric add, 1n mg, per g of Florisll. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absense of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an Initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The.laboratory is required to maintain performance records
to define the quality of data that 1s generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
608.1-08
January 1983
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8.2 To establish the ability to generate acceptable acr«r*cy and
precision, the analyst must perform t*« f-rJ«*1ng operations.
8.2.1 Select a representative SP^C concentration for each com-
pound to be measured, using stock standards, prepare a
quality control check sample concentrate 1n acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL allquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method perfor-
mance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) « R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts^
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.'
608.1-09
January 1983
-------
8.4 The laboratory 1s required to collect 1n duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10X of all samples or one spiked
sample per month, whichever 1s greater. One aliquot of the sample
must be spiked and analyzed as described 1n Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter 1n all samples processed as part of the same set
must be qualified as described 1n Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that 1t
remains at or below 5X.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-Hter aliquot of reagent water that all
glassware and reagents Interferences are under control. Each time
a set of samples 1s extracted or there 1s a change In reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dis-
similar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant
performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices'^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
608'1-10 January 1983
-------
10.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 s to rinse the Inner walls. Transfer the solvent to the separa-
tory funnel and extract the sample by shaking the funnel for 2 m1n
with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
m1n. If the emulsion Interface between layers ts more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may Include stir-
ring, filtration of the emulsion through glass wool, centrifuga-
tlon, or other physical methods. Collect the methylene chloride
extract 1n a 250-mL Erlenmeyer flask.
10.3 Add a second 60-ml volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts 1n the Erlenmeyer flask. Perform a third extraction
1n the same manner.
10.4 Assemble a Kuderna-Danlsh (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-D 1f the
requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 ml methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
10.7 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 mi of hexane and a new
boiling chip and reattach the Snyder column. Pour about 1 ml of
hexane into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be
5 to 10 m1n. When the apparent volume of liquid reaches 1 ml,
remove the K-D apparatus and allow it to drain and cool for at
least 10 min.
608.1-11
January 1983
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10.8 Remove the Snyder column and rinse the flask and Its lower joint
Into the concentrator tube with 1 to 2 mL of hexane and adjust the
volume to 10 mL. A 5-mL syringe 1s recommended for this operation.
Stopper the concentrator tube and store refrigerated 1f further
processing will not be performed Immediately. If the extracts will
be stored longer than two days, they should be transferred to
TFE-fluorocarbon-sealed screw-cap vials. If the sample extract
requires no further cleanup, proceed with.gas chromatographlc
analysis. If the sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-rnL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 85%.
11.2 The following Florisil column cleanup procedure has been
demonstrated to be applicable to the four organochlorine pesticides
listed in Table 3. It should also be applicable to the cleanup of
extracts for PCNB.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a chromatographic
column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 mL of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 mL with hexane and
transfer it from the K-D concentrator tube to the Florisil
column. Rinse the tube twice with 1 to 2 mL hexane, adding
each rinse to the column.
11.2.3 Place a 500-mL K-D flask and clean concentrator tube under
the chromatography column. Drain the column into the flask
until the sodium sulfate layer is nearly exposed. Elute the
column with 200 mL of 6% ethyl ether in hexane (V/V)
(Fraction 1) using a drip rate of about 5 mL/min. Remove
the K-D flask and set aside for later concentration. Elute
the column again, using 200 mL of 15* ethyl ether in hexane
608.1-12 January 1983
-------
(Y/V) (Fraction.2), Into a second K-D flask. Perform a
third elutlon using 200 ml of 50% ethyl ether 1n hexane
(V/V) (Fraction 3), Into a separate K-D flask. The elutlon
patterns for four of the pesticides are shown 1n Table 3.
11.2.4 Concentrate the eluates by standard K-D techniques (Section
10.6), substituting hexane for the glassware rinses and
using the water bath at about 85°C. Adjust final volume
to 10 ml with hexane. Analyze by gas chromatography.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included 1n this table are estimated retention
times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met.
Capillary (open-tubular) columns may also be used if the relative
standard deviations of responses for replicate injections are
demonstrated to be less than 6% and the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 \ii of the sample extract using the solvent-flush
technique." Record the volume injected to the nearest 0.05 yL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
,•608.1-13
January 1983
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13. Calculations
13.1 Determine the concentration of Individual compounds In the sample.
13.1.1 If the external standard calibration procedure 1s used,
calculate the amount of material Injected from the peak
response using the calibration curve or calibration factor
1n Section 7.2.2. The concentration 1n the sample can be
calculated as follows:
(A)(Vt)
Concentration, yg/L *
where:
A » Amount of material injected, in nanograms.
Vj » Volume of extract injected in y!_.
Vt * Volume of total extract in yl.
Vs « Volume of water extracted in ml.
13.1.2 If the Internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
Concentration, yg/L s JT-
where:
AS - Response for the parameter to be measured.
ATS ~ Response for the internal standard.
Is = Amount of internal standard added to each extract
in yg.
V0 = Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
irv the electron impact ionization mode. A GC to MS interface
constructed of all-glass or glass-lined materials is recommended.
608.1-14 T lno,
January 1983
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A computer system should be Interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chroma-
tographic tailing factors of less than 5.0 must be achievedJ?
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotrlphenyl phosphlne (DFTPP) performance criteria are
achieved.'^
14.4 To confirm an Identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section >11).
15. Method Performance
15,1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero.14 The MDL
608.1-15 January 1983
-------
concentrations listed 1n Table 1 were estimated from the response
of an electron capture detector to each compound. The estimate 1s
based upon the amount of material required to yield a signal five
time the GC background noise, assuming a 5-M«- injection from a
10-mL final extract of a 1-liter sample.
15.2 In a single laboratory (west cost Technical Services, Inc.), using
effluents from pesticide manufacturers and publicly owned treatment
works (POTW), the average recoveries presented in Table 2 were
obtained after Florisil cleanup.1 The standard deviations of the
percent recoveries of these measurements are also included in Table
2.
References
1. "Pesticide Methods Evaluation," Letter Report #17 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. Giam, D.S., Chan, H.S. and Nef, G.S., "Sensitive method for
Determination of Phthalate Ester Plasticizers in Open-Ocean Biota
Samples," Analytical Chemistry. 47, 2225, (1975).
4. Giam, C.S., Chan, H.S., "Control of Blanks in the Analysis of Phthalates
in Air and Ocean Biota Samples," National Bureau of Standards (U.S.),
Special Publication 442, pp. 701-708, 1976.
5. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, 03086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
9. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
608'1-16 January 1983,
-------
10. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
11. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
12. McNalr, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
• Printing, Berkeley, California, p. 52, 1969.
13. Eichelberger, J.W., Harris, I.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry. 47, 995 (1975).
14. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, J5f 1426 (1981).
15. "Determination of Organochlorine Pesticides in Industrial and Municipal
Wastewater," Method 608-1, EPA No. 600/4-82-003, NTIS No. PB82-155979,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
608'1-17 January 1983
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TABLE 1
CHROMATOGRAPHIC CONDITIONS AND ESTIMATED METHOD DETECTION LIMITS
Parameter
Dlbromochloropropane
EtrldUzole
Chloroneb
Propachlor
PCNB
Chloropropylate
Chi orobenz Hate
Column 1
Temperature Retention
°C Time (Min)
100
140
150
150
160
215
215
3.1
1.3
2.0
3.8
2.4
3.6
3.8
Column 2
Retention
Time (Min)
—
—
—
—
8.4
10.7
Estimated
MDL
(ug/L)
0.04
0.04
0.04
1.0
0.06
0.2
0.2
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5%
SP-2250/1.95* SP-2401 packed in a 1.8 m long x 2 mm ID glass column with
nitrogen carrier gas at a flow rate of 30 mL/min. Column temperatures are
listed above. An electron capture detector was used with this column to
estimate the MDL.
Column 2 conditions: Ultrabond 20M (100/120 mesh) packed in a 1.8 m long x
2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/min.
Column temperature is 200°C.
608.1-18
January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Chlorobenzilate
Chloroneb
Chloropropylate
Dibromochloro-
propane
Etridiazole
PCNB
Propachlor
Sample
Type
MW
MW
MW
MW
IW
IW
MW
MW
MW
MW
IW
IW
MW
MW
MW
MW
IW
MW
Mean
Background Spike Recovery
ug/L ug/L (X)
NO
NO
NO
NO
0.84
110
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
21.3
NO
10.5
52.5
18.1
181
6.1
484
10.0
50.0
1.9
24
1.9
24
0.50
9.9
1.0
20.0
179
895
74
97
92
93
53
97
78
96
83
70
61
55
144
91
100
91
87
83
Standard
Deviation
(X)
7.2
3.2
2.9
7.7
38.*
18.*
8.6
3.3
12.4
6.5
._
1.2*
9.9
1.7
11.0
3.1
3.8
3.8
Number of
Replicates
6
7
7
7
2
2
6
7
7
7
1
2
7
7
7
7
7
7
NO = Not detected
MW = Municipal wastewater
IW = Industrial wastewater, pesticide manufacturing
*For duplicate analyses range is listed.
608.1-19
January 1983
-------
TABLE 3
DISTRIBUTION OF CHLORINATED PESTICIDES
INTO FLORISIL COLUMN FRACTIONS
Percent Recovery by Fraction
Parameter Fraction 1 Fraction 2 Fraction 3
Chlorobenzilate 0 15 70
Chloroneb 93
Chloropropylate 0 32 61
Etr1d1azole 100
Eluant composition by fraction:
Fraction 1 - 200 mL of 6% ethyl ether in hexane
Fraction 2 - 200 mL of 15% ethyl ether in hexane
Fraction 3 - 200 mL of 50% ethyl ether in hexane
608.1-20 January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D C 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF ETHION
IN WASTEWATER
METHOD 614
1. Scope and Application
1.1 This method covers the determination of certain organophosphorus
pesticides. The following parameters can be determined by this
method:
Parameter STORET No. CAS No.
Azinphos methyl 39580 86-50-0
Oemeton 39560 8065-48-3
Diazinon 39570 333-41-5
Disulfoton 39010 298-04-4
Ethion ~ 563-12-2
Malathion 39530 121-75-5
Parathion ethyl 39540 56-38-2
Parathion methyl 39600 298-00-0
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compounds listed above in industrial and
municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted, sh~a?1
be considered a major modification subject to application and
approval of alternate test procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for several
parameters are listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are
essentially the same as in Method 617. Thus, a single sample may
be extracted to measure the parameters included in the scope of
both of these methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as
necessary, in order to apply appropriate cleanup procedures. Under
Gas Chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous
measurement of combinations of these parameters (see Section 12).
614-01 Janyary 1983
-------
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography 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 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas
chromatographic column that can be used to confirm measurements
made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for
the qualitative confirmation of compound identifications.
2. Summary of Method
2.1- A measured volume of sample, approximately 1-liter, is solvent
extracted with 15% methylene chloride in hexane using a separatory
funnel. The extract is dried and concentrated to a volume of 10 ml
or less. Gas chromatographic conditions are described which permit
the separation and measurement of the compounds in the extract by
flame photometric or thermionic bead gas chromatography.
2.2 Method 614 represents an editorial revision of a previously
promulgated U.S. EPA method for organophosphorus pesticides.^
While complete method validation data is not presented herein, the
method has been in widespread use since its promulgation, and
represents the state of the art for the analysis of such materials.
2.3 This method provides selected cleanup procedures to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane may be substituted for the
614-02
January 1983
-------
heating. After drying and cooling, seal and store glassware
1n a clean environment to prevent any accumulation of dust
or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to
minimize Interference problems. Purification of solvents by
distillation 1n all-glass systems may be required.
3.2 Matrix Interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix Interferences
will vary considerably from source to source, depending upon the
nature and diversity of the Industrial complex or municipality
sampled: The cleanup procedure in Section 11 can be u~*ed to
overcome many of these Interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in
Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 3-5 for tne information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
614-03
January 1983
-------
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An Integrating flow meter 1s required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 125-mL, 1000-mL and 2000-mL, with
TFE-fluorocarbon stopcock, ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Snyder column, Kuderna-Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.8 Pipet, disposable - 140 mm long x 5 mm ID.
5.2.9 Vials - Amber glass, 10 to 15 mL capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balanc§ - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all required
614-04
January 1983
-------
accessories Including syringes, analytical columns, gases, detector
and stripchart recorder. A data system is recommended for
measuring peak areas.
5.6.1 Column 1 - 180 cm long x 4 mm ID glass, packed with 3% OV-1
on Gas Chrom Q (100/120 mesh) or equivalent. This column
was used to develop the method performance statements in
Section 15. Alternative columns may be used in accordance
with the provisions described in Section 12.1.
5.6.2 Column 2 - 180 cm long x 4 mm ID glass, packed with 1.5%
OV-17/1.95X QF-1 on Gas Chrom Q (100/120 mesh) or equivalent.
5.6.3 Detector - Phosphorus specific: Flame photometric (FPD)
(526 nm filter) or thermionic bead detector in the nitrogen
mode. These detectors have proven effective in the analysis
of wastewaters for the parameters listed in the scope. The
FPD was used to develop the method performance statements in
Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions
described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride - Pesticide quality
or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. P1126-8, and
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips, After cleanup, 20 ml ethyl
alcohol preservative must be added to each liter of ether.
6.4 Acetom'trile, hexane-saturated - Mix pesticide quality acetonitrile
with an excess of hexane until equilibrium is established.
6.5 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.6 Sodium chloride solution, saturated - Prepare saturated solution of
NaCl in reagent water and extract with hexane to remove impurities.
614-05
January 1983
-------
6.7 Alumina - Woelm, neutral; deactivate by pipeting 1 ml of distilled
water into a 125-mL ground glass-stoppered Erlenmeyer flask.
Rotate flask to distribute water over surface of glass.
Immediately add 19.0 g fresh alumina through small powder funnel.
Shake flask containing mixture for 2 h on a mechanical shaker.
6.8 Florlsil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.9 Stock standaru solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality isooctane or acetone and
dilute to volume in a 10-mL volumetric flask. Larger
volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight
may be used without correction to calculate the
concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.9.2 Transfer the stock standard solutions into
TFE-fluorocarbon-sealed screw cap vials. Store at 4°C and
protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation,
especially just prior to preparing calibration standards
from them.
6.9.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
614-06 January 1983
-------
Isooctane or other suitable solvent. One of the external
standards should be representative of a concentration near,
but above, the method detection limit. The other
concentrations should correspond to the range of
concentrations expected 1n the sample concentrates or should
define the working range of the detector.
7.2.2 Using Injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
Injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any .
parameter varies from the predicted response by more than
±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels for each parameter of interest by
adding volumes of one or more stock standards to a
volumetric flask. To each calibration standard, add a known
constant amount of one or more internal standards, and
dilute to volume with isooctane or other suitable solvent.
One of the standards should be representative of a
concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of
concentrations expected in the sample concentrates, or
should define the working range of the detector.
7.3.2 Using injections of 1 to 5 yL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
614-07
January 1983
-------
Calculate response factors (RF) for each compound as follows:
RF - (AsC1s)/(A1s Cs)
where:
As * Response for the parameter to be measured.
A-jS « Response for the Internal standard.
C-js « Concentration of the internal standard 1n ug/L.
Cs * Concentration of the parameter to be measured in
ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for
calculations. Alternatively, the results may be used to
plot a calibration curve of response ratios, As/A-js
against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil
chromatography. Florisil from different batches or sources may
vary in adsorptive capacity. To standardize the amount of Florisil
which is used, the use of lauric acid value is suggested. This
procedure^ determines the adsorption from hexane solution of
lauric acid, in mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absense of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on
performance. The laboratory is required to maintain performance
records to define the quality of data that is generated.
<
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
614-°8 January 1983
-------
8.1.2 In recognition of the rapid advances occurring In
chromatography, the analyst 1s permitted certain options to
Improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the
analyst 1s required to repeat the procedure 1n Section 8.2.
8.1.3. The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing- laboratory performance.
This procedure Is described 1n Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Table 2 provides single operator recovery and precision for
diazinon, parathion methyl and parathion ethyl. Similar
results should be expected from reagent water for all
organophosphorus compounds listed in this method. Compare
these results to the values calculated in Section 8.2.3. If
the data are not comparable, review potential problem areas
and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts7
that are useful in observing trends in performance.
614-°9 January 1983
-------
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method 1s defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.'
8.4 The laboratory 1s required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a
dissimilar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant
performance evaluation studies.
q. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
614_10 January 1983
-------
9.2 The samples must be Iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 ml 15% methylene chloride In hexane (V:V) to the sample
bottle, seal, and shake 30 s to rinse the inner walls. Transfer
the solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 min with periodic venting to release
excess pressure. Allow the organic layer to separate from the
water phase for a minimum of 10 min. If the emulsion Interface
between layers is more than one third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete
the phase separation. The optimum technique depends upon the'
sample, but may include stirring, filtration of the emulsion
through glass wool, centrifugation, or other physical methods.
Drain the aqueous phase into a 1000-mL Erlenmeyer flask and collect
the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase
to the separatory funnel.
10.3 Add a second 60-mL volume of 15% methylene chloride in hexane to
the sample bottle and repeat the extraction procedure a second
time, combining the extracts in the 250-mL Erlenmeyer flask.
Perform a third extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other
concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 mL of hexane to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 ml methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 80 to 85°C, so that the concentrator tube is
partially immersed, in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
614-11
January 1983
-------
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
1t to drain and-cool for at least 10 m1n.
10.7 Remove the Snyder column and rinse the flask and Its lower joint
Into the concentrator tube with 1 to 2 ml of hexane and adjust the
volume to 10 ml. A 5-mL syringe 1s recommended for this operation.
Stopper the concentrator tube and store refrigerated 1f further
processing will not be performed Immediately. If the extracts will
be stored longer than two days, they should be transferred to
Teflon-sealed screw-cap bottles. If the sample extract requires no
further cleanup, proceed with gas chromatographic analysis. If the
sample requires cleanup, proceed to Section 11.
10.8 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-rnL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 85%.
11.2 Acetonltrile partition - The following acetonitrile partitioning .
procedure may be used to isolate fats and oils from the sample
extracts. The applicability of this procedure to organophosphorus
pesticides 1s indicated in Table 3.
11.2.1 Quantitatively transfer the previously concentrated extract
to a 125-mL separatory funnel with enough hexane to bring
the final volume to 15 ml. Extract the sample four times by
shaking vigorously for 1 min with 30-mL portions of hexane-
saturated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-liter
separatory funnel and add 650 ml of reagent water and 40 mL
of saturated sodium chloride solution. Mix thoroughly for
30 to 45 s. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 s.
11.2.3 Combine the hexane extracts in a 1-liter separatory funnel
and wash with two 100-mL portions of reagent water. Discard
the water layer and pour the hexane layer through a drying
column containing 7 to 10 cm of anhydrous sodium sulfate
614-12
January 1983
-------
Into a 500-mL K-D flask equipped with a 10-mL concentrator
tube. Rinse the separatory funnel and column with three
10-mL portions of hexane.
11.2.4 Concentrate the extracts to 6 to 10 ml 1n the K-D as
directed 1n Section 10.6. Adjust the extract volume to 10
mL with hexane.
11.2.5 Analyze by gas chromatography unless a need for further
cleanup 1s Indicated.
11.3 Flor1s1l column cleanup - The following Flor1s1l column cleanup
procedure has been demonstrated to be applicable to the seven
organophosphorus pesticides listed 1n Table 3. It should also be
applicable to the cleanup of extracts for ethlon.
11.3.1 Add a weight of Florlsll (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a chromatographic
column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 ml of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.3.2 Adjust the sample extract volume to 10 mL with hexane and
transfer it from the K-D concentrator tube to the Florisil
column. Rinse the tube twice with 1 to 2 mL hexane, adoing
each rinse to the column.
11.3.3 Place a 500-mL K-D flask and clean concentrator tube under
the chromatography column. Drain the column into the flask
until the sodium-sulfate-Jayeiv is nearly exposed. Elute the
column with 200 mL of 6% ethyl ether in hexane (V/V)
(Fraction 1) using a drip rate of about 5 mL/min. Remove
the K-D flask and set aside for later concentration. Elute
the column again, using 200 mL of 15% ethyl ether in hexane
(V/V) (Fraction 2), into a second K-D flask. Perform a
third elution using 200 mL of 50% ethyl ether in hexane
(V/V) (Fraction 3) and a final elution with 200 mL of 100%
ethyl ether (Fraction 4), into separate K-D flasks. The
elution patterns for seven of the pesticides are shown in
Table 3.
11.3.4 Concentrate the eluates by standard K-D techniques (Section
10.6), using the water bath at about 85°C (75°C for.
Fraction 4). Adjust final volume to 10 mL with hexane.
Analyze by gas chromatography.
614-13
January 1983
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11.4 Removal of sulfur (9) - Elemental sulfur will elute 1n Fraction 1
of the Flor1s11 cleanup procedure. If a large amount of sulfur is
present 1n the extract, 1t may elute 1n all fractions. If so each
fraction must be further treated to remove the sulfur.
11.4.1 Add 1 or 2 boiling chips to the 10-mL hexane solution
contained 1n a concentrator tube. Attach a mlcro-Snyder
column and concentrate the extract to about 0.2 ml 1n a hot
water bath at 85°C. Remove the micro K-0 from the bath,
cool, and adjust the volume to 0.5 ml with hexane.
11.4.2 Plug a disposable plpet with a small quantity of glass
wool. Add enough alumina to produce a 3-crn column after
settling. Top the alumina with a 0.5-cm layer of anhydrous
sodium sulfate.
11.4.3 Quantitatively transfer the concentrated extract to the
alumina mlcrocolumn using a 100-yL syringe, Rinse the ampul
with 200-uL of hexane and add to the microcolumn.
11.4.4 Elute the microcolumn with 3 ml of hexane and discard the
eluate.
11.4.5 Elute-the column with 5 ml of 10% hexane in methylene
chloride, and collect the eluate in a 10-mL concentrator
tube. Adjust final volume to 10 ml with hexane. Analyze by
gas chromatography.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met.
Capillary (open-tubular) columns may also be used if the relative
standard deviations of responses for replicate injections are
demonstrated to be less than 6% and the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as described ir. Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 yL of the sample extract using the solvent-flush
technique.^0 Record the volume injected to the nearest 0.05 uL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
614~14 January 1983
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12.5 The width of the retention time window used to make Identifications
should be based upon measurements of actual retention time
variations of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily 1n the interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup 1s required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(V )
Concentration, ug/l = —rn
where:
A = Amount of material injected, in nanograms.
V-j = .Volume of extract injected in \ii.
Vt = Volume of total extract in uL.
Vs = Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
'(A MI )
Concentration, ug/L = (A. )(RF)(V )
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract
in ug.
vo - Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
614-15 T lno,
January 1983
-------
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be-capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS Interface
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices.
Chromatographic tailing factors of less than 5.0 must be
achieved Jl
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.'2
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25-nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
614-16
January 1983
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14.4.3 Compounds that have very similar mass spectra can be
explicitly Identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionlzation mass spectra may be employed
to aid 1n the qualitative Identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reahalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99< confidence that the value is above zero.'3 The MDL concen-
trations listed in Table 1 were obtained using reagent water.'4
15.2 In a single laboratory, Susquehanna University, using spiked tap
water samples, the average recoveries presented in Table 3 were
obtained. The standard deviation of the percent recovery is.also
included in Table 3.14
References
1. "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol
and Pesticides in Water and Wastewater," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory - Cincinnati,
Ohio 45268, September 1978.
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
614-17
January 1983
-------
7. "Handbook for Analytical Quality Control 1n Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. Law, L. M. and D. F. Goerlitz, "Microcolumn Chromatographic Cleanup for
the Analysis of Pesticides 1n Water," Journal of the Association of
Official Analytical Chemists, 53_, 1276, (1970).
10. Burke," J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists. 48, 1037 (1965).
11. McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry, 47_, 995 (1975).
13. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology. J_5_, 1426 (1981).
14. McGrath, T. F., "Recovery Studies of Pesticides From Surface .and
Drinking Waters," Final Report for U.S. EPA Grant R804294, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
15. "Determination of Organophosphorus Pesticides in Industrial and Municipal
Wastewater," Method 614, EPA No. 600/4-82-004, NTIS No. PB82-155987,
January 1982, National Technical Information Center, 5285 Port Royal Road,
Springfield, VA 22165.
January 1983
-------
TABLE T
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Retention Time Method
Parameter (min.) Detection Limit
Column 1 Column 2 (ytg/L)
Diazinon
Disulfoton
Demeton
Parathion methyl
Ma lath ion
Parathion ethyl
Ethion
Azinphos methyl
1.8
1.9
2.3
2.5
2.9
3.1
5.8
14.5
1.8
2.1
2.1
3.7
3.9
4.5
9.1
29.9
0.012
NO
ND
0.012
ND
0.015
ND
ND
ND = Not determined
Column 1 conditions: Gas-Chrom Q (100/120 mesh) coated with 3% OV-1 packed
in a 1.8 m long x 4 mm ID glass column with nitrogen carrier gas at a flow
rate of 60 mL/min. Column temperature, isothermal at 200°C. A flame
photometric detector was used with this column to determine the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.5%
OV-17+1.95£ QF-1 packed in a 1.8 m long x 4 mm ID glass column with nitrogen
carrier gas at 70 mL/min flow rate. Column temperature, isothermal at
215°C.
614~19 January 1983
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TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Average
Percent
Recovery
Standard
Deviation
(*)
Spike
Range
(ug/D
Number
of
Analyses
Matrix
Types
Diazinon
Parathion methyl
Parathion ethyl
94
95
102
5.2
3.2
4.1
0.04-40
0.06-60
0.07-70
27
27
27
4
4
4
614-20 January 1983
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TABLE 3
FLORISIL FRACTIONATION PATTERNS
AND ACETONITRILE PARTITION APPLICABILITY
Flor1s1l Fractlenaction Pattern Acetonitrile
Parameter Percent Recovery by Fraction Partition
No. 1No. 2 No. 3No7~4 Applicability
Demeton
Disulfoton
Diazinon
Malathion
Parathion ethyl
Parathion methyl
Azinphos methyl
Ethion
100
100
ND
100
5
100
100
ND
95
20
ND
ND
ND
Yes
Yes
Yes
Yes
80 ND
ND Yes
ND = Not determined
Florisil eluate composition by fraction
Fraction 1 - 200 mL of 6% ethyl ether in hexane
Fraction 2 - 200 mL of 15% ethyl ether in hexane
Fraction 3 - 200 mL of 50% ethyl ether in hexane
Fraction 4 - 200 mL of ethyl ether
614-21 January 1983
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United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF 2,4-DB, 2,4-DB ISOBUTYL ESTER (2,4-DB IBE), AND
2,4-DB ISOOCTYL ESTER (2,4-DB IOE), AND DINOSEB
IN WASTEWATER
METHOD 615
1. Scope and Application
1.1 This method covers the determination of certain chlorinated herbi
cides and their esters. The following parent acids can be
determined by this method:
Parameter STORE! No. CAS No.
2 4-0 - 397361 94-75-7
oilaSon - g-M-0
2 4-08 — 94-82-6
Dicamba - 1918-00-9
Oichlorprop - *7
Oinoseb - °8 -35-7
— 94-74-6
MPPP — — jj— O3— c
2 4 5-T 39740 93-76-5
zXs-TP (Silvex) 39760 93-72-1
1 2 This method is also applicable to the determination of salts and
esters of these compounds. This includes, but is not limited to:
the isobutyl and isooctyl esters of 2,4-0; the isobutyl and
isooctyl esters of 2,4-08; the isooctyl ester of MCPA; and the
isooctyl ester of 2,4,5-TP. The actual form of each acid is not
distinguished by- this method. Results are calculated and reported
for each listed parameter as total free acid.
13 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in industrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.4 The method detection limit (MOL, defined in Section 15} for each
parameter 1s listed in Table 1. The MDL for a specific wastewater
may differ from those listed, depending upon the nature of inter-
ferences in the sample matrix.
1 5 This method 1s restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography and in the
615-01 January 1983
-------
Interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described 1n Section 8.2.
1.6 When this method Is used to analyze unfamiliar samples for any or
all of the compounds above, compound Identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for alternative gas chroma-
tographlc columns that can be used to confirm measurements made
with the primary column. Section 15 provides gas chromatograph/
mass spectrometer (GC/MS) criteria appropriate for the qualitative
confirmation of compound Identifications.
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, 1s acidified.
The add herbicides and their esters and salts are extracted with
ethyl ether using a separatory funnel. The derivatives are hydro-
lyzed with potassium hydroxide and extraneous organic material is
removed by a solvent wash. After acidification, the acids are ex-
racted and converted to their methyl esters using diazomethane as
the derivatizing agent. Excess reagent is removed, and the esters
are determined by electron capture (EC) gas chromatography.'
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all glass-
ware as soon 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 dilute
acid, tap and reagent water. Drain dry, and heat 1n an oven
or muffle furnace at 400°C for 15 to 30 min. Do not heat
volumetric ware. Thermally stable materials such as PCBs,
might not be eliminated by this treatment. Thorough rinsing
with acetone and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation 1n all-glass systems may be required.
3.2 The acid forms of the herbicides are strong organic: acids, which
615-02 January 1983
-------
react readily with alkaline substances and can be lost during
analysis. Glassware and glass wool must be acid-rinsed with (1+9)
hydrochloric add and the sodium sulfate must be acidified with
sulfurlc add prior to use to avoid this possibility.
3.3 Organic adds and phenols, especially chlorinated compounds, cause
the most direct Interference with the determination. Alkaline
hydrolysis and subsequent extraction of the basic solution remove
many chlorinated hydrocarbons and phthalate esters that might
otherwise Interfere with the electron capture analysis.
3.4 Matrix Interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix Interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to
overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in
Table 1.
4.1 The toxicity or carcinogenidty of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 3-5 for the Information of the analyst.
4.2 Dlazomethane is a toxic carcinogen and can explode under certain
conditions. The following precautions must be followed:
4.2.1 Use only a well ventilated hood - do not breath vapors.
4.2.2 Use a safety screen.
4.2.3 Use mechanical pipetting aides.
4.2.4 Do not heat above 90°C - EXPLOSION may result.
4.2.5 Avoid grinding surfaces, ground glass joints, sleeve
bearings, glass stirrers - EXPLOSION may result.
4.2.6 Store away from alkali metals - EXPLOSION may result.
4.2.7 Solutions of diazomethane decompose rapidly in the presence
of solid materials such as copper powder, calcium chloride,
615-03 January 1983
-------
and boiling chips.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosllicate or flint glass,
1-11ter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
If the sample 1s not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
Uner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must Incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnels - 60-mL and 2000-ml, with TFE-fluoro-
carbon stopcocks, ground glass or TFE stoppers.
5.2.2 Concentrator tube, Kuderna-Oanish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed 1n the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.3 Evaporative flask, Kuderna-Dam'sh - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.4 Snyder column, Kuderna-Dam'sh - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.5 Snyder column, Kuderna-Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.6 Erlenmeyer flask - Pyrex, 250 ml with 24/40 ground glass
joint.
5.2.7 Vials - Amber glass, 10 to 15 ml capacity with TFE-fluoro-
carbon lined screw cap.
615-04
January 1983
-------
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
m1n or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of temper-
ature control (± 2°C). The bath should be used 1n a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Dlazomethane generator - assemble from two 20 x 150 mm test tubes,
two Neoprene rubber stoppers and a source of nitrogen. The gener-
ator assembly 1s shown 1n Figure 1.
5.7 Glass wool - Add washed (Supelco 2-0383 or equivalent).
5.8 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column Injection and all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system is recommended for measuring peak
areas.
5.8.1 Column 1 - 180 cm long x 4 mm ID glass, packed with 1.5X
SP-2250/1.95X SP-2401 on Supelcoport (100/120 mesh) or
equivalent. This column was used to develop the method
performance statements in Section 16. Alternative columns
may be used in accordance with the provisions described in
Section 13.1.
5.8.2 Column 2 - 180 cm long x 4 mm ID glass, packed with 5%
OV-210 on Gas Chrom Q (100/120 mesh) or equivalent.
5.8.3 Column 3 - 180 cm long x 2 mm ID g7ass, packed with 0.1X
SP-1000 on Carbopak C (80/100 mesh) or equivalent.
5.8.3 Detector - Electron capture. This detector has proven
effective in the analysis of wastewaters for the parameters
listed in the scope and was used to develop the method
performance statements in Section 15. Alternative
detectors, including a mass spectrometer, may be used in
accordance with the provisions described in Section 13.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, methanol - Pesticide quality or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. P1126-8, and
January 1983
-------
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 ml ethyl
alcohol preservative must be added to each liter of ether.
6.4 Sodium sulfate - (ACS) Granular, acidified, anhydrous. Heat treat
1n a shallow tray at 400°C for a minimum of 4 h to remove
phthalates and other Interfering organic substances. Alterna-
tively, heat 16 h at 450-500°C 1n a shallow tray or Soxhlet
extract with methylene chloride for 48 h. Acidify by slurrylng 100
g sodium sulfate with enough ethyl ether to just cover the solid.
Add 0.1 ml concentrated sulfurlc add and mix thoroughly. Remove
the ether under vacuum. Mix 1 g of the resulting solid with 5 ml
of reagent water and measure the pH of the mixture. It must be
below pH 4. Store at 130°C.
6.5 Hydrochloric add (1+9) - (ACS) Add one volume of concentrated add
to 9 volumes reagent water.
6.6 Potassium hydroxide solution - 372 aqueous solution (W:V).
Dissolve 37 g ACS grade potassium hydroxide pellets In reagent
water and dilute to 100 ml.
6.7 Sulfuric acid solution (1+1) - Slowly add 50 ml ^$04 (sp. gr.
1.84) to 50 ml of reagent water.
6.8 Sulfurlc acid solution (1+3) - Slowly add 25 mL H2$04 (sp. gr.
1.84) to 75 ml of reagent water. Maintain at 4°C.
6.9 Carbitol - (Diethylene glycol monoethyl ether), ACS. Available
from Aldrich Chemical Co.
6.10 Diazald - (N-methyl-N-nitroso-p-toluenesulfonamide), ACS.
Available from Aldrich Chemical Co.
6.11 Silicic acid - Chromatographic grade, nominal 100 mesh. Store at
130°C.
6.12 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
can be prepared from pure standard materials or purchased as
certified solutions.
6.12.1 Prepare stock standard solutions by accurately weighing
about 0.0100 grams of pure acids. Dissolve the material in
pesticide quality ethyl ether and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the
convenience of the analyst. If compound purity is certified
at 96X 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.
615~°6 January 1983
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6.12.2 Transfer the stock standard solutions Into Teflon-sealed
screw-cap vials. 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.
6.12.3 Stock standard solutions must be replaced after one week or
sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system must be
calibrated using the external standard technique.
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare working standards of
the free acids at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a 10-mL volumetric flask containing 1.0 ml
methanol and diluting to volume with ethyl ether. One of
the external standards should be representative of a concen-
tration near, but above, the method detection limit. The
other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should
define the working range of the detector.
7.2.2 Prepare calibration standards by esterification of 1.00 mL
volumes of the working standards as described in Section
11. Using injections of 2 to 5 uL of each calibration
standard, tabulate peak height or area responses against the
mass of free acid represented by the injection. The results
can be used to prepare a calibration curve for each para-
meter. Alternatively, the ratio of the response to the mass
injected, defined as the calibration factor (CF), can be
calculated for each parameter at each standard concentra-
tion. If the relative standard deviation of the calibration
factor is less than 10% over the working range, the average
calibration factor can be used in place of a calibration
curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the preparation of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration stan-
dard. Alternatively, a new calibration curve or calibration
factor must be prepared for that parameter.
7.3 Before using any cleanup procedure, the analyst must process a
615-07
January 1983
-------
series of calibration standards through the procedure to validate
elutlon patterns and the absence of Interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an Initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that 1s generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability 1s established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to Improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure 1n Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10* of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound (acid or ester) to be measured. Using stock
standards, prepare a quality control check sample
concentrate in acetone 1000 times more concentrated than the
selected concentrations.
8.2.2 Using a pipet, add .1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water, A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
615-08 January 1983
-------
method, and compare these results to the values calculated
In Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) « R * 3 s
Lower Control Limit (LCL) « R - 3 s
where R and s are calculated as In Section 8.2.3.
The UCL and LCL can be used to construct control charts5
that are useful 1n observing trends 1n performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method 1s defined as R ± s.
The accuracy statement should be developed by the analysis
of four allquots of wastewater as described 1n Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control 1n Section 8.4. The accuracy statements should be
updated regularly."
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10X of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 14.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5X.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
615-09 January 1983
-------
confirmatory techniques such as gas chromatography with a dis-
similar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate 1n relevant
performance evaluation studies.
9. Sample Collection. Preservation, and Handling
9.1 Grab samples must be collected 1n glass containers. Conventional
sampling practices7 should be followed; however, the bottle must
not be prerlnsed with sample before collection. Composite samples
should be collected 1n refrigerated glass containers 1n accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be Iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel. Check the pH with wide-range pH paper
and adjust to pH less than 2 with sulfuric acid (1+1).
10.2 Add 150 ml ethyl ether to the sample bottle, cap the bottle and
shake 30 s to rinse the walls. Transfer the solvent to the
separatory funnel and extract the sample by shaking the funnel for
2 min with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layers is more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, centrifu-
gation or other physical means. Drain the aqueous phase into a
1000-mL Erlenmeyer flask and collect the extract in a 250-mL
ground-glass Erlenmeyer flask containing 2 mL of 37% potassium
hydroxide solution. Approximately 80 ml of the ethyl ether will
remain dissolved in the aqueous phase.
10.3 Add a 50-mL volume of ethyl ether to the sample bottle and repeat
the extraction a second time, combining the extracts in the
Erlenmeyer flask. Perform a third extraction in the same manner.
10.4 Add 15 ml reagent water and 1 or 2 clean boiling chips to the
250-mL flask and attach a three-ball Snyder column. Prewet the
Snyder column by adding 1 ml ethyl ether to the top. Place the
615-10 January 1983
-------
apparatus on a hot water bath (60 to 65°C), such that the bottom
of the flask 1s bathed 1n the water vapor. Although the ethyl
ether will evaporate 1n about 15 min, continue heating for a total
of 60 m1n, beginning from the time the flask 1s placed on the
water bath. Remove the apparatus and let stand at room tempera-
ture for at least 10 m1n.
10.5 Transfer the solution to a 60-mL separatory funnel using 5 to 10
ml of reagent water. Wash the basic solution twice by shaking for
one m1n with 20-mL portions of ethyl ether. Discard the organic
phase. The free adds remain 1n the aqueous phase.
10.6 Acidify the contents of the separatory funnel to pH 2 by adding 2
ml of cold (4°C) sulfurlc add (1+3). Test with pH Indicator
paper. Add 20 mL ethyl ether and shake vigorously for 2 m1n.
Drain the aqueous layer Into the 250-mL Erlenmeyer, then pour the
organic layer into a 125-mL Erlenmeyer flask containing about 0.5
g of acidified anhydrous sodium sulfate. Repeat the extraction
twice more with 10-mL aliquots of ethyl ether, combining all
solvent 1n the 125-mL flask. Allow the extract to remain in
contact with the sodium sulfate for approximately 2 h.
10.7 Assemble a Kuderna-Danlsh (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-0 if the
requirements of Section 8.2 are met.
10.8 Pour the combined extract through a funnel plugged with acid
washed glass wool, and collect the extract 1n the K-D in concen-
trator. Use a-glass rod to crush any caked sodium sulfate during
the transfer. Rinse the Erlenmeyer flask and column with 20 to 30
mL of ethyl ether to complete the quantitative transfer.
10.9 Add 1 to 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL ethyl ether to the top. Place the K-D apparatus on a
hot water bath, 60 to 65°C, so that the concentrator tube 1s
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the verti-
cal position of the apparatus and the water temperature as
required to complete the concentration in 15 to 20 min. At the
proper rate of distillation the balls of the column will actively
chatter but the chambers will not flood. When the apparent volume
of liquid reaches 1 mL, remove the K-D apparatus and allow it to
drain and cool for at least 10 min.
10.10 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 mL of ethyl ether. A 5-mL
syringe is recommended for this operation. Add a fresh boiling
chip. Attach a micro-Snyder column to the concentrator tube and
prewet the column by adding about 0.5 mL of ethyl ether to the
top. Place the micro K-D apparatus on the water bath so that the
615-11 January 1983
-------
concentrator tube 1s partially Immersed 1n the hot water. Adjust
the vertical position of the apparatus and the water temperature
as required to complete concentration 1n 5 to 10 mln. When the
apparent volume of liquid reaches 0.5 ml, remove the micro K-D
from the bath and allow 1t to drain and cool. Remove the micro
Snyder column and add 0.1 ml of methanol. Rinse the walls of the
concentrator tube while adjusting the voTume to 1.0 ml with ethyl
ether.
It. Ester if 1 cation of Adds
11.1 Assemble the dlazomethane generator (See Figure 1) 1n a hood using
two 20 x 150 mm test tubes. Use neoprene rubber stoppers with
holes drilled 1n them to accommodate glass delivery tubes. The
exit tube must be drawn to a point to bubble diazornethane through
the sample extract.
11.2 Add 5 ml of ethyl ether to the first test tube. Add 1 ml of ethyl
ether, 1 ml of carbltol, 1.5 ml of 37% aqueous KOH,, and 0.1 to 0.2
g Dlazald to the second test tube. Immediately place the exit
tube Into the concentrator tube containing the sample extract.
Apply nitrogen flow (10 mL/m1n) to bubble diazomethane through the
extract for 10 m1n or until the yellow color of dlazomethane
persists.
11.3 Remove the concentrator tube and seal 1t with a Neoprene or Teflon
stopper. Store at room temperature in a hood for 20 min.
11.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 g silicic
acid to the concentrator tube. Allow to stand until the evolution
of nitrogen gas has stopped. Adjust the sample volume to 10.0 ml
with hexane. Stopper the concentrator tube and store refrigerated
1f further processing will not be performed immediately. It is
recommended that the methylated extracts be analyzed immediately
to minimize any transesterification and other potential reactions
that may occur. Analyze by gas chromatography.
11.5 Determine the original sample volume by refilling the sample
bottle to the marie and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest 5 mL.
12. Cleanup and Separation
12.1 No cleanup procedures were required to analyze the wastewaters
described 1n Section 16. If particular circumstances demand the
use of a cleanup procedure, the analyst must determine the elution
profile and demonstrate that the recovery of each compound of
interest for the cleanup procedure is no less than 85%.
13. Gas Chromatography
13.1 Table 1 summarizes the recommended operating conditions for the
615-12
January 1983
-------
gas chromatograph. Included 1n this table are estimated retention
times and method detection limits that can be achieved by this
method. Examples of the separations achieved for the methyl
esters are shown in Figures 2 to 4. Other packed columns,
chromatographlc conditions, or detectors may be used if the
requirements of Section 8.2 are met. Capillary (open-tubular)
columns may also be used if the relative standard deviations of
responses for replicate Injections are demonstrated to be less
than 6X and the requirements of Section 8.2 are met.
13.2 Calibrate the system dally as described 1n Section 7.
13.3 Inject 1 to 5 uL of the sample extract using the solvent-flush
technique.8 Record the volume injected to the nearest 0.05 yL,
and the resulting peak size 1n area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
13.4 The width of the retention time window used to make identifica-
tions should be based upon measurements of actual retention time
variations of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chroma-
tograms.
13.5 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
13.6 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
14. Calculations
14.1 Determine the concentration of individual compounds in the sample.
Calculate the amount of free acid injected from the peak response
using the calibration curve or calibration factor in Section
7.2.2. The concentration in the sample can be calculated as
follows:
(A)(Vt)
Concentration, yg/L = —jr.
where:
A = Amount of material injected, in nanograms.
V-j = Volume of extract injected in yL.
V^ = Volume of total extract in yL.
Vs - Volume of water extracted in ml_.
14.2 Report results in micrograms per liter as acid equivalent without
correction for recovery data. When duplicate and spiked samples
615-13
January 1983
-------
are analyzed, report all data obtained with the sample results.
14.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits 1n Section 8.3,
data for the affected parameters must be labeled as suspect.
15. GC/MS Confirmation
15.1 It 1s recommended that GC/MS techniques be judiciously employed to
support qualitative compound Identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the methyl ester of the add herbicide. The Instrument must be
capable of scanning the mass range at a rate to produce at least 5
scans per peak but not to exceed 7 s per scan utilizing a 70 V
(nominal) electron energy 1n the electron Impact 1on1zat1on mode.
A GC to MS Interface constructed of all-glass or glass-lined
materials 1s recommended. A computer system should be Interfaced
to the mass spectrometer that allows the continuous acquisition and
storage on machine readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
15.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chromato-
graphic tailing factors of less than 5.0 must be achieved.9
15.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved. "0
15.4 To confirm an identification of a compound, the background
corrected mass spectrum of the methyl ester must be obtained from
the sample extract and compared with a mass spectrum from a stock
or calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
15.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10X. For example, if the relative abundance of an ion is
30X in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
15.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
615-14
January 1983
-------
15.4.3 Compounds that have very similar mass spectra can be
explicitly Identified by GC/MS only on the basis of
retention time data.
15.5 Where available, chemical 1on1zat1on mass spectra may be employed
to aid 1n the qualitative Identification process.
15.6 Should these MS procedures fall to provide satisfactory results,
additional steps may be taken before reanalysls. These may Include
the use of alternate packed or capillary GC columns or additional
cleanup.
16. Method Performance
16.1 The method detection limit (MOL) 1s defined as the minimum concen-
tration of a substance that can be measured and reported with 99*
confidence that the value 1s above zero.1' The MDL concentra-
tions listed 1n Table 1 were obtained from reagent water with an
electron capture detector.'
16.2 In a single laboratory (West Cost Technical Services, Inc.), using
reagent water and effluents from publicly owned treatment works
(POTW), the average recoveries presented in Table 2 were
obtained.1 The standard deviations of the percent recoveries of
these measurements are also included in Table 2.
References
1. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
2. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for
Preparation of Sample Containers and for Preservation, M American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
615-15 January 1983
-------
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
8. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists. 48, 1037 (1965).
9. McNalr, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
10. Elchelberger, J.W., Harris, I.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement 1n Gas Chromatography-Mass
Spectrometry," Analytical Chemistry. 4£, 995 (1975).
11. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, ]S_, 1426 (1981).
12. "Determination of Chlorinated Herbicides in Industrial and Municipal
Wastewater," Method 615, EPA No. 600/4-82-005, NTIS No. PB82-155995,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
615-16 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Parameter
(as methyl ester)
Dicamba
2,4-D
2,4,5-TP
2,4,5-T
2,4-DB
Dalapon
MCPP
MCPA
Dichlorprop
Dinoseb
Column
1.2
2.0
2.7
3.4
4.1
—
3.4
4.1
4.8
11.2
Retention Time
1 Column 2 Column 3
1.0
1.6
2.0
2.4
—
5.0
._
_.
—
— — •••
Method
Detection
Limit yg/L
0.27
1.2
0.17
0.20
0.91
5.8
192
249
0.65
0.07
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5* SP-2250/
1.95* SP-2401 packed in a 1.8 m long x 4 mm ID glass column with 95%
argon/5% methane carrier gas at a flow rate of 70 mL/min. Column
temperature: isothermal at 185°C, except for MCPP, MCPA, dichlorprop and
dinoseb, where the column temperature was held at 140°C for 6 min and then
programmed to 200°C at 10°/min. An electron capture detector was used
to measure MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 5% OV-210
packed in a 1.8 m long x 4 mm ID glass column with 95% argon/5% methane
carrier gas at a flow rate of 70 mL/min. Column temperature: isothermal at
185°C.
Column 3 conditions: Carbopak C (80/100 mesh) coated with 0.1% SP-1000
packed in a 1.8 m long x 2 mm ID glass column with nitrogen carrier gas at a
flow rate of 25 mL/min. Column temperature: programmed at injection from
100°C to 150°C at 10°/min.
615-17
January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION*
Parameter
2,4-0
Dalapon
2,4-DB
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
2.4,5-T
2,4,5-TP
Sample
Type
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
Spike
(ug/L)
10.9
10.1
200
23.4
23.4
468
10.3
10.4
208
1.2
1.1
22.2
10.7
10.7
213
0.5
102
2020
2020
21400
2080
2100
20440
1.1
1.3
25.5
1.0
1.3
25.0
Mean
Recovery
(X)
75
77
65
66
96
81
93
93
77
79
86
82
97
72
100
86
81
98
73
97
94
97
95
85
83
78
88
88
72
Standard
Deviation
(<)
4
4
5
8
13
9
3
3
6
7
9
6
2
3
2
4
3
4
3
2
4
3
2
6
4
5
5
4
5
*A11 results based upon seven replicate analyses.
DW = Reagent water
MW = Municipal water
615-18
January 1983
-------
o»
^c
2
3
5 (7-
*\
0
=>
f"« « -
V * \
CS
J_
O
r-;° ?«oA ^ s
' T> ) -O a,
r-— ^ 3 c
o>
V
c
_Q
3
HJ
•I—
O
I
en
615-19
January 1983
-------
Figure 2.
01 2345
Retention Time (Min)
Gas chromatogram of methyl esters of chlorinated
herbicides on Column 1. For conditions, see Table 1
615-20
January 1983
-------
4 6.8
Retention Time (Hin.)
10
12
Figure 3.
Gas chromatogram of methyl esters of chlorinated
herbicides on Column 1. For conditions, see
Table 1.
615-21
January 1983
-------
o
Q.
(O
Figure 4.
0246
Retention Time (Min.)
Gas chromatogram of methyl ester of dalapon on
Column 3. For conditions, see Table 1.
615-22
January 1983
-------
SEPA
United States
Environmental Protection
Agency
Effluent Guidelines Division (WH 552)
Washington, D.C. 20460
Water and Waste Management
TEST METHOD
DETERMINATION OF CARBOPHENOTHION
IN WASTEWATE5
METHOD 617
1. Scope and Application
1.1 This method covers the determination of certain organohalide
pesticides and PCBs. The following parameters can be determined by
this method:
Parameter
Aldrin
cx-BHC
8-8HC
S-BHC
Y-8HC (Lindane)
Captan
Carbophenothion
Chlordane
4,4'-ODD
4,4'-OOE
4,4'-DOT
Dichloran
Oicofol
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Isodrin
Methoxychlor
Mi rex
PCN8
Perthane
Strobane
Toxaphene
Trifluralin
PCB-1016
PCB-1221
PCB-1232
PCB-1242
STORE! No.
39330
39337
39338
39259
39340
39640
39350
39310
39320
39300
39780
39380
34356
34361
34351
39390
34366
39410
39420
39430
39480
39755
39029
39034
39400
39030
34671
39488
39492
39496
CAS No.
309-00-2
319-84-6
319-85-7
319-86-8
58-89-9
133-06-2
786-19-6
5103-74-2
72-54-8
72-55-9
50-29-3
99-30-9
115-32-2
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
76-44-8
1024-57-3
465-73-6
72-43-5
2385-85-5
82-68-8
72-56-0
8001-50-1
8001-35-2
1582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
617-01
January 1983
-------
Parameter STORE! No. CAS No,.
PCB-1248 39500 12672-29-6
PCB-1254 39504 11097-69-1
PCB-1260 39508 11096-82-5
1.2 This 1s a gas chromatographic (GC) method applicable to the
determination of the compounds listed above 1n Industrial and
municipal discharges as provided under 40 CFR 136.1. Any
modification of this method beyond those expressly permitted, shall
be considered a major modification subject to application and
approval of alternate test procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for many of
the parameters are listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are
essentially the same as in Method 614. Thus, a single sample may
be extracted to measure the parameters included in the scope of
both of these methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as
necessary, in order to apply appropriate cleanup procedures. Under
Gas Chromatography, the analyst is allowed the latitude to select
chromatographic conditions appropriate for the simultaneous
measurement of combinations of these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of
analysts experienced 1n the use of gas Chromatography 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 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas
chromatographic column that can be used to confirm measurements
made with the primary column. Section 14 provides gas
chromatograph/mass spectrometer (GC/MS) criteria appropriate for
the qualitative confirmation of compound identifications.
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with 15% methylene chloride in hexane using a separatory
funnel. The extract is dried and concentrated to a volume of 10 mL
or less. Gas chromatographic conditions are described which permit
the separation and measurement of the compounds in the extract by
electron capture gas Chromatography.
6l7-°2 January 1983
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2.2 Method 617 represents an editorial revision of two previously
promulgated U.S. EPA methods for pesticides and for PCBs.1 While
complete method validation data is not presented herein, the method
has been 1n widespread use since its promulgation, and represents
the state of the art for the analysis of such materials.
2.3 This method provides selected cleanup procedures to aid in the
elimination of interferences which may be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.' Clean all
glassware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem in
pesticide analysis when the EC detector is used. These compounds
generally appear in the chromatogram as Targe late eluting peaks,
especially in the 15 and 50% fractions from the Florisil column
cleanup. Common flexible plastics contain varying amounts of
phthalates. These phthalates are easily extracted or leached from
such materials during laboratory operations. Cross contamination
of clean glassware occurs when plastics are handled during
extraction steps, especially when solvent wetted surfaces are
handled. Interferences from phthalates can be minimized by
avoiding the use of plastics in the laboratory. Exhaustive cleanup
of reagents and glassware may be required to eliminate background
phthalate contamination.3.4 The interferences from phthalate
esters can be avoided by using a microcoulometric or electrolytic
conductivity detector.
617-03 January 1983
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3.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix Interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to
overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in
Table 1.
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 5-7 for the information of the analyst.
4.2 The following parameters covered by this method have been
tentatively classified as known or suspected, human or mammalian
carcinogens: aldrin, benzene hexachlorides, chlordane, heptachlor,
PCNB, PCBs and toxaphene. Primary standards of these toxic
materials should be prepared in a hood.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
617-04 January 1983
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5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 125-mL, 1000-mL and 2000-mL, with
TFE-fluorocarbon stopcock, ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 500-ml (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Shaker - Laboratory, reciprocal action.
5.7 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all required
accessories including syringes, analytical columns, gases, detector
and stripchart recorder. A data system is recommended for
measuring peak areas.
5.6.1 Column 1 - 180 cm long x 4 mm ID glass, packed with 1.5%
SP-2250/1.95X SP-2401 on Supelcoport (100/120 mesh) or
equivalent. This column was used to develop the method
performance statements in Section 15. Alternative columns
may be used 1n accordance with the provisions described in
Section 12.1.
617-°5 January 1983
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5.6.2 Column 2 - 180 cm long x 4 mm ID glass, packed with 3% OV-1
on Supelcoport (100/120 mesh) or equivalent.
5.6.3 Detector - Electron capture. This detector has proven
effective in the analysis of wastewaters for the parameters
listed in the scope and was used to develop the method
performance statements in Section 15. Alternative
detectors, including a mass spectrometer, may be used in
accordance with the provisions described in Section 12.1.
6; Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride - Pesticide quality
or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. P1126-8, and
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 ml ethyl
alcohol preservative must be added to each liter of ether.
6.4 Acetonitrile, hexane-saturated - Mix pesticide quality acetonitrile
with an excess of hexane until equilibrium is established.
6.5 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.6 Sodium chloride solution, saturated - Prepare saturated solution of
NaCl in reagent water and extract with hexane to remove impurities.
6.7 Sodium hydroxide solution (ION) - Dissolve 40 g ACS grade NaOH in
reagent water and dilute to 100 ml.
6.8 Sulfuric acid solution (1+1) - Slowly add 50 mL H2S04 (sp. gr.
1.84) to 50 ml of reagent water.
6.9 Mercury - Triple distill.
6.10 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
617~06 January 1983
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6.11 Stock standard solutions (KOO ug/yL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.11.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material 1n pesticide quality isooctane and dilute to volume
1n a 10-mL volumetric flask. Larger volumes may be used at
the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight may be used withc-'Jt
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards may be used
at any concentration if they are certified by the
manufacturer or by an independent source.
6.11.2 Transfer the stock standard solutions into
TFE-fluorocarbon-sealed screw cap vials. Store at 4°C and
protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation,
especially just prior to preparing calibration standards
from them.
6.11.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
isooctane. One of the external standards should be
representative of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates or should define the working range of
the detector.
7.2.2 Using injections of 1 to 5 yL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
617-07 January 1983
-------
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any
parameter varies from the predicted response by more than
±10X, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7,3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels for each parameter of interest by
adding volumes of one or more stock standards to a
volumetric flask. To each calibration standard, add a known
constant amount of one or more internal standards, and
dilute to volume with isooctane. One of the standards
should be representative of a concentration near, but above,
the method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF - (AsC1s)/(A1s Cs)
where:
As * Response for the parameter to be measured.
Ais « Response for the internal standard.
C-js * Concentration of the internal standard in yg/L.
Cs a Concentration of the parameter to be measured in
yg/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for
calculations. Alternatively, the rasults may be used to
plot a calibration curve of response ratios, As/A-jS
against RF.
617-08 January 1983
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7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10<, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 The cleanup procedure 1n Section 11 utilizes Flor1s1l
chromatography. Florisil from different batches or sources may
vary 1n adsorptive capacity. To standardize the amount of Florisil
which is used, the use of lauric acid value is suggested. This
procedure** determines the adsorption from hexane solution of
lauric acid, 1n mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
7.6 The multipeak materials included in this method present a special
calibration problem. Recommended procedures for calibration,
separation and measurement of PCBs is discussed in detail in the
previous edition of this method.' Illustrated methods for the
calibration and measurement of chlordane and strobane/toxaphene are
available elsewhere.9
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on
performance. The laboratory is required to maintain performance
records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in
chromatography, the analyst is permitted certain options to
improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the
analyst is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
617-09 January 1983
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8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 mL of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Table 2 provides single operator recovery and precision for
many of the organohalide pesticides. Similar results should
be expected from reagent water for all parameters listed in
this method. Compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) =• R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts'0
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.10
617~10 January 1983
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8.4 The laboratory 1s required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10* of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It 1s recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a
dissimilar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant
performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices1' should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction. Chemical preservatives should not be
used in the field unless more than 24 hours will elapse before
delivery to the laboratory. If the samples will not be extracted
with,in 48 hours of collection, the sample should be adjusted to a
pH range of 6.0 to 8.0 with sodium hydroxide or sulfuric acid.
Record the volume of acid or base used.
617-11 January 1983
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9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 ml 15X methylene chloride in hexane (V:V) to the sample
bottle, seal, and shake 30 s to rinse the inner walls. Transfer
the solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 min with periodic venting to release
excess pressure. Allow the organic layer to separate from the
water phase for a minimum of 10 min. If the emulsion interface
between layers is more than one third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete
the phase separation. The optimum technique depends upon the
sample, but may include stirring, filtration of the emulsion
through glass wool, centrifugation, or other physical methods.
Drain the aqueous phase into a 1000-mL Erlenmeyer flask and collect
the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase
to the separatory funnel.
10.3 Add a second 60-mL volume of 15% methylene chloride in hexane to
the sample bottle and repeat the extraction procedure a second
time, combining the extracts in the 250-mL Erlenmeyer flask.
Perform a third extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other
concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of hexane to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 80 to 85°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
617-12 January 1983
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10.7 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexane and adjust the
volume to 10 ml. A 5-mL syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will
be stored longer than two days, they should be transferred to
Teflon-sealed screw-cap bottles. If the sample extract requires no
further cleanup, proceed with gas chromato'graphic analysis. If the
sample requires cleanup, proceed to Section 11.
10.8 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 85%.
11.2 Acetonitrile partition - The following acetonitrile partitioning
procedure may be used to isolate fats and oils from the sample
extracts. This procedure is applicable to all of the parameters in
this method except mirex.
11.2.1 Quantitatively transfer the previously concentrated extract
to a 125-mL separatory funnel with enough hexane to bring
the final volume to 15 ml. Extract the sample four times by
shaking vigorously for 1 min with 30-mL portions of hexane-
saturated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-liter
separatory funnel and add 650 ml of reagent water and 40 ml
of saturated sodium chloride solution. Mix thoroughly for
30 to 45 s. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 s.
11.2.3 Combine the hexane extracts in a 1-liter separatory funnel
and wash with two 100-mL portions of reagent water. Discard
the water layer and pour the hexane layer through a drying
column containing 7 to 10 cm of anhydrous sodium sulfate
into a 500-mL K-D flask equipped with a 10-mL concentrator
tube. Rinse the separatory funnel and column with three
10-mL portions of hexane.
11.2.4 Concentrate the extracts to 6 to 10 mL in the K-D as
directed in Section 10.6. Adjust the extract volume to 10
mL with hexane.
617~13 January 1983
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11.2.5 Analyze by gas chromatography unless a need for further
cleanup 1s indicated.
11.3 Florisil column cleanup - The following Florisil column cleanup
procedure has been demonstrated to be applicable to most of the
organochlorlne pesticides and PCBs listed in Table 3. It should
also be applicable to the cleanup of extracts for PCNB, strobane
and trifluralln.
11.3.1 Add a weight of Florisil (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a chromatographic
column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 ml of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.3.2 Adjust the sample extract volume to 10 ml with hexane and
transfer it from the K-D concentrator tube to the Florisil
column. Rinse the tube twice with 1 to 2 ml hexane, adding
each rinse to the column.
11.3.3 Place a 500-mL K-D flask and clean concentrator tube under
the chromatography column. Drain the column into the flask
until the sodium sulfate layer is nearly exposed. Elute the
column with 200 mL of 6% ethyl ether in hexane (V/V)
(Fraction 1) using a drip rate of about 5 mL/min. Remove
the K-D flask and set aside for later concentration. Elute
the column again, using 200 ml of 15% ethyl ether in hexane
(V/V) (Fraction 2), into a second K-D flask. Perform a
third elution using 200 ml of 50% ethyl ether in hexane
(V/V) (Fraction 3) into a separate K-D flask. The elution
patterns for the pesticides and PCBs are shown in Table 3.
11.3.4 Concentrate the eluates by standard K-D techniques (Section
10.6), using the water bath at about 85°C. Adjust final
volume to 10 mL with hexane. Analyze by gas chromatography.
11.4 Removal of sulfur - Elemental sulfur will elute in Fraction 1 of
the Florisil cleanup procedure. If a large amount of sulfur is
present in the extract, it may elute in all fractions. If so, each
fraction must be further treated to remove the sulfur. This
procedure cannot be used with heptachlor, endosulfans, or endrin
aldehyde.
11.4.1 Pipet 1.00 ml of the concentrated extract into a clean
concentrator tube or a vial with a TFE-fluorocarbon seal.
Add 1 to 3 drops of mercury and seal.
617"14 January 1983
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11.4.2 Agitate the contents of the vial for 15 to 30 s.
11.4.3 Place the vial 1n an upright position on a reciprocal
laboratory shaker and shake for up to 2 h.
11.4.4 If the mercury appears shiny after-this treatment, analyze
the extract by gas chromatography. If the mercury is black,
decant the extract into a clean vial and repeat the cleanup
with fresh mercury.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met.
Capillary (open-tubular) columns may also be used if the relative
standard deviations of responses for replicate injections are
demonstrated to be less than 6% and the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 uL of the sample extract using the solvent-flush
technique.'2 Record the volume injected to the nearest 0.05 yL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used. Multipeak materials present a special
analytical problem beyond the scope of this discussion.
Illustrated procedures for calibration and measurement are
available for PCBs' and pesticides.9
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time
variations of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
617 15 January 1983
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13. Calculations
13.1 Determine the concentration of Individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(V )
Concentration, yg/L * —rr
where:
A » Amount of material injected, in nanograms.
Vj = Volume of extract injected in uL.
V-t = Volume of total extract in yl_.
Vs * Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(A,)(I.)
Concentration, yg/L = (A1s)(ftF)(V0)'
where:
As = Response for the parameter to be measured.
A-JS ~ Response for the internal standard.
Is " Amount of internal standard added to each extract
in yg.
V0 = Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. 6C/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface
617-16 January 1983
-------
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices.
Chromatographic tailing factors of less than 5.0 must be
achieved.T3
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.14
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
617~17 January 1983
-------
15. Method Performance
15.1 The method detection limit (MDL) 1s defined as the minimum
concentration of a substance that can be measured and reported with
99* confidence that the value is above zero. '5 yne MDL concen-
trations listed in Table 1 were obtained using reagent water.16
15.2 In a single laboratory, Susquehanna University, using spiked tap
water samples, the average recoveries presented in Table 2 were
obtained. The standard deviation of the percent recovery is also
included in Table 2.16
References
1. "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol
and Pesticides in Water and Wastewater," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory •• Cincinnati,
Ohio 45268, September 1978.
2. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. Giam, D.S., Chan, H.S. and Nef, G.S., "Sensitive method for
Determination of Phthalate Ester Plasticizers 1n Open-Ocean Biota
Samples," Analytical Chemistry, 47, 2225, (1975).
4. Giam, C.S., Chan, H.S., "Control of Blanks in the Analysis of Phthalates
in Air and Ocean Biota Samples," National Bureau of Standards (U.S.),
Special Publication 442, pp. 701-708, 1976.
5. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
6. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
617-18 January 1983
-------
9. "Pesticide Analytical Manual Volume 1," U.S. Department of Health and
Human Services, Food and Drug Administration.
10. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
11. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
12. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
13. McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
14. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry. 47, 995 (1975).
15. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology. J5_, 1426 (1981).
16. McGrath, T. F., "Recovery Studies of Pesticides From Surface and
Drinking Waters," Final Report for U.S. EPA Grant R804294, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
17. "Determination of Organohalide Pesticides & PCB's in Industrial and
Municipal Wastewater," Method 617, EPA No. 600/4-82-006, NTIS No.
PB82-156001, January 1982, National Technical Information Center,
5285 Port Royal Road, Springfield, VA 22165.
617-19 January 1983
-------
Parameter
TABLE 1
GAS CHROMAT06RAPHY OF PESTICIDES AND PCBs*
Retention Time (min)
Column 1 Column 2
Method
Detection Limit
(ug/L)
Aldrin
a-BHC
B-BHC
6-BHC
Y-BHC
Captan
Carbophenothion
4, 4 '-ODD
4,4'-DDE
4, 4' -DDT
Dichloran
Dicofol
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Isodrin
Methoxychlor
Mi rex
PCNB
Trifluralin
2.40
1.35
1.90
2.15
1.70
6.22
10.90
7.83
5.13
9.40
1.85
2.86
5.45
4.50
8.00
14.22
6.55
11.82
2.00
3.50
3.00
18.20
14.60
1.63
0.94
4.10
1.82
1.97
2.20
2.13
5.00
10.90
9.08
7.15
11.75
2.01
4.59
7.23
6.20
8.28
10.70
8.10
9.30
3.35
5.00
4.83
26.60
15.50
2.01
1.35
0.009
0.004
ND
ND
0.002
ND
ND
0.012
0.004
0.032
ND
ND
0.011
0.011
0.017
ND
ND
ND
0.004
0.003
ND
0.176
0.015
0.002
0.013
*For multipeak materials, see Figures 2 to 10 for chromatographic conditions
and retention patterns.
ND = Not Determined.
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5%
SP-2250/1.95% SP-2401 1n a 1.8 m long x 4 mm ID glass column with 95%
argon/5% methane carrier gas at a flow rate of 60 mL/min. Column
temperature: isothermal at 200°C. An electron capture detector was used
with this column to determine the MDL.
Column'2 conditions: Supelcoport (100/120 mesh) coated with 3% OV-1 packed
in a 1.8 m long x 4 mm ID glass column with 95% argon/5% methane carrier gas
at a flow rate of 60 mL/min. Column temperature: isothermal at 200°C.
617-20
January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
FOR TAP WATER
Parameter
Aldrln
6-BHC
Y-BHC
4,4'-DDD
4, 4 '-ODE
4, 4 '-DDT
Dieldrin
Endosulfan I
Endosulfan II
Heptachlor
Heptachlor epoxide
Methoxychlor
Mirex
PCNB
Trifluralin
Average
Percent
Recovery
78.1
95.3
95.1
94.4
89.8
91.0
98.2
101.0
92.9
84.4
93.7
96.6
89.1
82.6
94.3
Standard
Deviation
(«)
5.4
8.9
7.2
5.0
3.7
4.5
4.9
7.6
4.8
6.4
3.9
6.7
4.8
6.2
10.5
Spike
Range
(yg/L)
0.03-3.0
0.01-1.0
0.01-1.0
0.08-8.0
0.05-5.0
0.2-20
0.06-6.0
0.05-5.0
0.09-9.0
0.02-2.0
0.03-3.0
0.6-60
0.2-20
0.01-1.0
0.03-3.0
Number
of
Analyses
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
617-21
January 1983
-------
Parameter
TABLE 3
DISTRIBUTION AND RECOVERY OF CHLORINATED PESTICIDES
AND PCBs USING FLORISIL COLUMN CHROMATOGRAPHY
Percent Recovery by Fraction
No. 1 No. 2 No. 3
Aldrin
a-BHC
e-BHC
6-BHC
Y-BHC
Captan
Carbofenthion
Chlordane
4,4'-ODD
4,4'-DDE
4,4'-DDT
Dicofol
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
Isodrin
Methoxychlor
Mi rex
Perthane
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
100
100
97
98
100
+
100
100
99
98
100
+
0
37
0
0
4
0
100
100
100
100
100
100
96
97
97
95
97
103
90
95
1100
64
7
0
96
68
91
106
26
"""Compound occurs in both 6% and 15% fractions.
Florisil eluate composition by fraction.
Fraction 1 - 200 mL of 65 ethyl ether in hexane.
Fraction 2 - 200 mL of 15% ethyl ether in hexane.
Fraction 3 - 200 mL of 50% ethyl ether in hexane.
617-22
January 1983
-------
COLUMN: 1.5% SP-2250*
1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200*C.
DETECTOR: ELECTRON CAPTURE
LU
a
04 8 12 16
RETENTION TIME-MINUTES
Figure 1. Gas chromatogram of pesticides
617-23
January 1983
-------
COLUMN: 1.5% SP-2250*
1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200*C.
DETECTOR: ELECTRON CAPTURE
4 8 12
RETENTION TIME-A/IINUTES
16
Figure 2. Gas chromatogram of chlordane
617-24
January 1983
-------
COLUMN: 1.5% SP-2250*
1.95K SP-2401 ON SUPELCOPORT
TEMPERATURE: 200'C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18 22
RETENTION TIME-MINUTES
Figure 3. Gas chromatogram of toxaphene
26
617-25
January 1983
-------
COLUMN: 1.5% SP-2250+ 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160*C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 4. Gas chromatogram of PCS-1016
22
617-26
January 1983
-------
COLUMN: 1.5% SP-2250* 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160*C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 5. Gas chromatogram of PCB-1221
22
617-27
January 1983
-------
COLUMN: 1.5% SP-2250* 1.95''.
TEMPERATURE: 160'C.
DETECTOR: ELECTRON CAPTURE
SP-2401 ON SUPELCOPORT
10 14 18
RETENTION TIME-MINUTES
22
24
Figure 6. Gas chromatogram of PCB-1232
617-28
January 1983
-------
COLUMN: 1.5% SP.2250+1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160*C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 7. Gas chromatogram of PCB-1242
22
617-29
January 1983
-------
COLUMN: 1.5% SP-2250 + 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 160'C.
DETECTOR: ELECTRON "CAPTURE
10 14 18
RETENTION TIME-MINUTES
22
26
Figure 8. Gas chromatogram of PCB-1248
617-30
January 1983
-------
COLUMN: 1.5% SP-2250* 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200*C.
DETECTOR: ELECTRON CAPTURE
2 6 10 14 18
RETENTION TIME-MINUTES
Figure 9. Gas chromatogram of PCB-1254
22
617-31
January 1983
-------
COLUMN: 1.5% SP-2250+ 1.95% SP-2401 ON SUPELCOPORT
TEMPERATURE: 200*C.
DETECTOR: ELECTRON CAPTURE
I I I
10 14 18 22
RETENTION TIME-MINUTES
26
Figure 10. Gas chromatogram of PC8-1260
617-32
January 1983
-------
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, O.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF SIMETRYN AND TERBDTRYN
IN WASTEWATER
METHOD 619
1. Scope and Application
1.1 This method covers the determination of certain triazine pesti-
cides. The following parameters can be determined by this method:
Parameter STORE! No. CAS Mo.
Ametryn — 834-12-3
Atraton — 1610-17-9
Atrazine 39033 1912-24-9
Prometon 39056 1610-18-0
Prometryn 39057 7287-19-6
Propazine 39024 139-40-2
Secbumeton — 26259-45-0
Simetryn 39054 1014-70-6
Simazine 39055 122-34-9
Terbuthylazine — 5915-41-3
Terbutryn — 886-50-0
1.2 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in inaustrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15)
for each parameter is listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are
essentially the same as several others in the 600-method series.
Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is
required, the concentration levels must be high enough to permit
selecting aliquots, as necessary, 1n order to apply appropriate
cleanup procedures. Under Gas Chromatography, the analyst is
allowed the latitude to select chromatographic conditions
619-01 January 1983
-------
appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography 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 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas chromato-
graphic column that can be used to confirm measurements made with
the primary column. Section 14 provides gas chromatograph/mass
spectrometer (GC/MS) criteria appropriate for the qualitative
confirmation of compound identifications.
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with 15% methylene chloride using a separatory funnel.
The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 ml or less. Gas chromato-
graphic conditions are described which permit the separation and
measurement of the compounds in the extract by gas chromatography
with a thermionic bead detector in the nitrogen modeJ»2
2.2 Method 619 represents an editorial revision of a previously promul-
gated U.S. EPA method for organophosphorus pesticides.3 While
complete method validation data is not presented herein, the method
has been in widespread use since its promulgation, and represents
the state of the art for the analysis of such materials.
2.3 This method provides an optional Florisil column cleanup procedure
to aid in the elimination or reduction of interferences which may
be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.** Clean all
glassware as soon 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
619-02 January 1983
-------
furnace at 400°C for 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane may be substituted for the
heating. After drying and cooling, seal and store glassware
1n a clean environment to prevent any accumulation of dust
or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation 1n all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are co-
extracted from the sample. The extent of matrix interferences will
vary considerably from source to source, depending upon the nature
and diversity of the industrial complex or municipality sampled.
The cleanup procedure in Section 11 can be used to overcome many of
these interferences, but unique samples may require additional
cleanup approaches to achieve the MDL listed in Table 1.
4.1 The toxlcity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 5-7 for the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-Hter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
619-03 January 1983
-------
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory fun.iel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Oanish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of temper-
ature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system is recommended for measuring peak
areas.
5.6.1 Column 1 - 180 cm long x 2 mm ID glass, packed with 5%
Carbowax 20M-TPA on Supelcoport (80/100 mesh) or
619-04 January 1983
-------
equivalent. This column was used to develop the method
performance statements 1n Section 15. Alternative columns
may be used in accordance with the provisions described in
Section 12.1.
%
5.6.2 Column 2 - 180 cm long x 4 mm ID glass, packed with 1.0%
Carbowax 20M on Gas Chrom Q (100/120" mesh) or equivalent.
5.6.3 Detector - Thermionic bead in the nitrogen mode. This
detector has proven effective in the analysis of wastewaters
for the parameters listed in the scope and was used to
develop the method performance statements in Section 15.
Alternative detectors, including a mass spectrometer, may be
used in accordance with the provisions described in Section
12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, methylene chloride, methanol - Pesticide quality
or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips. (Avail-
able from Scientific Products Co., Cat. No. P1126-8, and other
suppliers.) Procedures recommended for removal of peroxides are
provided with the test strips. After cleanup, 20 ml ethyl alcohol
preservative must be added to each liter of ether.
6.4 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.5 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.6 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.6.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality hexane or other suitable
solvent and dilute to volume in a 10-mL volumetric flask.
Larger volumes may be used at the convenience of the
619-05 January 1983
-------
analyst. If compound purity is certified at 96% or greater,
the weight may be used without correction to calculate the
concentration of the stock standard. Commercially prepared
stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source,,
6.6.2 Transfer the stock standard solutions into TIFE-fluorocarbon
sealed screw cap vials. Store at 4°C and protect from
light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.6.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
hexane or other suitable solvent. One of the external
standards should be representative of a concentration near,
but above, the method detection limit. The other concen-
trations should correspond to the range of concentrations
expected in the sample concentrates or should define the
working range of the detector.
7.2.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
619-06 January 1983
-------
the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three concen-
tration levels for each parameter of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with hexane or other suitable solvent. One of the
standards should be representative of a concentration near,
but above, the method detection limit. The other concen-
trations should correspond to the range of concentrations
expected in the sample concentrates, or should define the
working range of the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF » (AsCis)/(A1s Cs)
where:
As » Response for the parameter to be measured.
» Response for the internal standard.
- Concentration of the internal standard in ug/L.
Cs = Concentration of the parameter to be measured in
ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/A-js against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromato-
graphy. Florisil from different batches or sources may vary in
adsorptive capacity. To standardize the amount of Florisil which
6l9-°7 January 1983
-------
is used, the use of laurlc acid value is suggested. This
procedure8 determines the adsorption from hexane solution of
1 auric acid, in mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
619-08 January 1983
-------
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the data from Table 2, estimate the recovery and
single operator precision expected for the method, and
compare these results to the values calculated 1n Section
8.2.3. If the data are not comparable, review potential
problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) » R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts9
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.'
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 102 of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all glass-
ware and reagents interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
619-09 January 1983
-------
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dis-
similar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of quality control materials and participate in relevant
performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices'" should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the separ-
atory funnel and extract the sample by shaking the funnel for 2 min
with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layars is more than one
third the volume of the solvent layer, the analyst: must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stir-
ring, filtration of the emulsion through glass wool, centrifuga-
tion, or other physical methods. Collect the methylene chloride
extract in a 250-ml Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
619-10
-------
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-D if the
requirements of Section 8.2 are met.
10,5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed With hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
10.7 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 ml of hexane and a new
boiling chip and reattach the Snyder column. Pour about 1 ml of
hexane into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be
5 to 10 min. When the apparent volume of liquid reaches 1 mL,
remove the K-0 apparatus and allow it to drain and cool for at
least 10 min.
10.8 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexane and adjust the
volume to 10 mL. A 5-mL syringe is recommended for this operation.
(Note: Precipitation of triazines in the hexane may occur if the
concentration in the original sample exceeded 500 ug/L. If this
occurs, redissolve the triazines in methylene chloride and analyze
the extract using flame ionization gas chromatography.) Stopper
the concentrator tube and store refrigerated if further processing
will not be performed immediately. If the extracts will be stored
longer than two days, they should be transferred to TFE-fluoro-
carbon sealed screw-cap vials. If the sample extract requires no
further cleanup, proceed with gas chromatographic analysis. If the
sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 mL.
619-11 January 1983
-------
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 85%.
11.2 The following Florisil column cleanup procedure has been
demonstrated to be applicable to the nine triazine pesticides
listed in Table 3.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a chromatographic
column. Settle the Florisil by tapping the.column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 ml of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 ml with hexane and
transfer it from the K-D concentrator tube to the Florisil
column. Rinse the tube twice with 1 to 2 mL hexane, adding
each rinse to the column.
11.2.3 Drain the column until the sodium sulfate layer is nearly
exposed. Elute the column with 200 ml of 6% ethyl ether in
hexane (V/V) (Fraction 1) using a drip rate of about
5 mL/min. This fraction may be discarded. Place a 500-mL
K-D flask and clean concentrator tube under the chromato-
graphy column. Elute the column into the flask, using 200
ml of 15% ethyl ether in hexane (V/V) (Fraction 2). Perform
a third elution using 200 ml of 50% ethyl ether in hexane
(V/V) (Fraction 3), and a final elution with 200 ml of 100%
ethyl ether (Fraction 4), into separate K-D flasks. The
elution patterns for nine of the pesticides are shown in
Table 3.
11.2.4 Concentrate the eluates by standard K-D techniques (Section
10.6), substituting hexane for the glassware rinses and
using the water bath at about 85°C. Adjust final volume
to 10 ml with hexane. Analyze by gas chromatography.
12. Gas Chromatoqraphy
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
619-12 January 1983
-------
method. An example of the separation achieved by Column 1 is shown
in Figure 1. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met.
Capillary (open-tubular) columns may also be used if the relative
standard deviations of responses for replicate injections are
demonstrated to be less than 6% and the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 uL of the sample extract using the solvent-flush
technique.'1 Record the volume injected to the nearest 0.05 uL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(Vt)
Concentration, ug/L = —r^
where:
A = Amount of material injected, in nanograms.
Vj = Volume of extract injected in uL.
Vt = Volume of total extract in uL.
Vs = Volume of water extracted in mL.
619-13 January 1983
-------
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(A.KIJ
Concentration, ug/L * -rr-.-
where:
As » Response for the parameter to be measured.
A-js * Response for the internal standard.
Is * Amount of internal standard added to each extract
in ug.
V0 = Volume of water extracted, in"liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chromato-
graphic tailing factors of less than 5.0 must be achieved.^
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.'3
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
619-14 January 1983
-------
conditions. It 1s recommended that at least 25 nanograms of
material be Injected Into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All Ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, 1f the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that 1on in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within € seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero.'4 The MDL concen-
trations listed in Table 1 were estimated from the response of the
thermionic bead nitrogen detector to each compound. The estimate
1s based upon the amount of material required to yield a signal
five times the GC background noise, assuming a 5-uL injection from
a 10-mL final extract of a 1-liter sample.
15.2 In a single laboratory (either West Cost Technical Services, Inc.
or Midwest Research Institute), using effluents from pesticide
manufacturers and publicly owned treatment works (POTW), the
average recoveries presented in Table 2 were obtained after
Florisil cleanup.''2 The standard deviations of the percent
recoveries of these measurements are also included in Table 2.
References
1. "Pesticide Methods Evaluation," Letter Report #11 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
619-15 January 1983
-------
2. "Development of Analytical Test Procedures for Organic Pollutants in
Wastewater-Application to Pesticides," EPA Report 600/4-31-017, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268. PB#82 132507,
National Technical Information Service, Springfield, Va.
3. "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol
and Pesticides in Water and Wastewater," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory - Cincinnati,
Ohio 45268, September 1978.
4. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
5. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
6. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
8. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
9. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
10. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, ?A, p. 76, 1980.
11. Burke, 0. A., "Gas Chromatograpny for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
12. McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
13. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry, 47_, 995 (1975).
14. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, 15, 1426 (1981).
15. "Determination of Triazine in Industrial and Municipal Wastewater,"
Method 619, EPA No. 600/4-82-007, NTIS No. PB82-156019, January 1982,
National Technical Information Center, 5285 Port Royal Road, Springfield,
VA, 22165.
619-16 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Parameter
Retention Time
(min.)
Column 1 Column 2
Method
Detection Limit
(yg/L)
Prometon
Atraton
Propazine
Terbuthylazine
Secbumeton
Atrazi ne
Prometryn
Terbutryn
Simazine
Ametryn
Simetryn
6.9
—
9.2
10.2
—
12.4
13.8
15.4
16.3
17.7
23.0
4.9
6.3
6.7
7.3
8.3
9.4
10.3
—
12.7
14.0
~
0.03
ND
0.03
0.03
ND
0.05
0.06
0.05
0.06
0.06
0.07
ND * Not determined
Column 1 conditions: Supelcoport (80/100 mesh) coated with 5% Carbowax
20M-TPA packed in a 1.8 m long x 2 mm ID glass column with helium carrier
gas at a flow rate of 30 mL/min. Column temperature, isothermal at 200°C.
A thermionic bead detector was used with this column to determine the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.05J Carbowax
20 M packed in a 1.8 m long x 4 mm ID glass column with helium carrier gas
at 80 mL/min flow rate. Column temperature, isothermal at 155°C.
619-17
January 1983
-------
TABLE 2
SINGLE LABORATORY ACCURACY AND PRECISION
Sample
Parameter Type*
Ametryn
Atrazlne
Prometon
Prometryn
Propazine
Simatryn
Simazine
Terbuthylazlne
Terbutryn
3
3
3
1
1
2
3
3
1
3
3
3
1
3
3
3
1
2
Spike
(wg/L)
4,000
2,000
300
1,000
130
260
2,000
50
516
15
30
15
115
10
100
15
968
169
Number of
Replicates
2
2
2
7
7
7
2
2
7
2
2
2
7
2
2
2
7
7
Mean
Recovery
(<)
104
118
108
177
67
51
76
110
54
116
183
182
152
99
114
100
83
89
Standard
Deviation
(«)
--
__
15.2
3.9
3.0
—
-_
6.5
__
-.
._
24.3
..
—
.-
10.0
24.0
*Sample Type
1 - Industrial process water
2 - Industrial effluent
3 - 80% Industrial process water/20% industrial effluent
619-18
January 1983
-------
TABLE 3
FLORISIL FRACTIONATION PATTERNS
Parameter
NoTl
Percent Recovery by Fraction
RoTl NoTl
No74
Propazi ne
Terbuthylazine
Atrazlne
Ametryn
Prometryn
Simazine
Atraton
Secbumeton
Prometon
0
0
0
90
30
20
10
70
80
100
TOO
100
100
100
100
Florist! eluate composition by fraction
Fraction 1 - 200 ml of 6% ethyl ether 1n hexane
Fraction 2 - 200 ml of 15% ethyl ether in hexane
Fraction 3 - 200 ml of SOU ethyl ether in hexane
Fraction 4 - 200 ml of ethyl ether
619-19
January 1983
-------
o
20
25
05 10 15
Minutes
Figure 1. Gas chromatogram of triazine pesticides on Column 1
For conditions, see Table 1.
619-20
January 1983
-------
oEPA
United States
Environmental Protection
Agency
Effluent Guidelines Division (WH 552)
Washington, O.C. 20460
Water and Waste Management
TEST METHOD
DETERMINATION OF BOLSTAR, CHLORPYRIFOS, CHLORPYRIFOS METHYL,
COUMAPHOS, DICHLORVOS, FENSDLFOTHION, FENTHION, MEVINPHOS,
NALED, PHORATE, RONNEL, STIROFOS, AND TRICHLORONATE
IN WASTEWATER
METHOD 622
1. Scope and Application
1.1 This method covers the determination of certain organophosphorus
pesticides. The following parameters can be determined by this
method:
Parameter
Azinphos methyl
Bo1 star
Chlorpyrifos
Chlorpyrifos methyl
Coumaphos
Demeton
Oiazinon
Dichlorvos
Oisulfoton
Ethoprop
Fensulfothion
Fenthion
Merphos
Mevinphos
Naled
Parathion methyl
Phorate
Ronnel
Stirofos
Tokuthion
Trichloronate
STORET No.
39580
81293
39560
39570
39010
39016
39019
39600
39023
39357
CAS No.
86-50-0
35400-43-2
2921-38-2
5598-13-0
56-72-4
8065-48-3
333-41-5
62-73-7
298-04-4
13194-48-4
115-90-2
55-38-9
150-50-5
7786-34-7
300-76-5
298-00-0
298-02-2
299-84-3
961-11-5
34643-46-4
327-98-0
1.2 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in industrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15)
for each parameter is listed in Table 1. The MOL for a specific
622-01 January 1983
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wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are
essentially the same as several others in the 600-method series.
Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is
required, the concentration levels must be high enough to permit
selecting aliquots, as necessary, in order to apply appropriate
cleanup procedures. Under Gas Chromatography, the analyst is
allowed the latitude to select chromatographic conditions appro-
priate for the simultaneous measurement of combinations of these
parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas Chromatography 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 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique.
Section 14 provides gas chromatograph/mass spectrometer (GC/MS)
criteria appropriate for the qualitative confirmation of compound
identifications.
Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with 15% methylene chloride using a separatory funnel.
The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 ml or less. Gas chromato-
graphic conditions are described which permit the separation and
measurement of the compounds in the extract by gas Chromatography
with a thermionic bead or flame photometric detector in the
phosphorus mode.'
Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon 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
622-02 January 1983
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furnace at 400°C for 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to
minimize ir.terference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. Unique samples may require special clean-up approaches or
selective GC detectors to achieve the MDL listed in Table 1. Use
of a flame photometric detector in the phosphorus mode will mini-
mize interferences from materials that do not contain phosphorus.
Elemental sulfur, however, may interfere with the determination of
certain organophosphorus pesticides by flame photometric gas
chromatography. A halogen specific detector (electrolytic conduc-
tivity or microcoulometric) is very selective for the halogen
containing pesticides and has been shown to be effective in the
analysis of wastewater for dichlorvos, naled and stirofos.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 3-5 for the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
622-03 January 1983
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chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
sillcone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contemination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 nro long x 19 mm
ID with coarse fritted disc.
5.2.3 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
1s used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.5 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system is recommended for measuring peak
areas.
622-04 January 1983
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5.6.1 Columns - These columns were used to develop the method
performance statements 1n Section 15. Alternate columns may
be used 1n accordance with the provisions described in
Section 12.1.
5.6.1.1 Column 1 - 180 cm long x 2 mm ID glass, packed with
5% SP-2401 on Supelcoport (100/120 mesh) or equiva-
lent.
5.6.1.2 Column 2 - 180 cm long x 2 mm ID glass, packed with
3% SP-2401 on Supelcoport (100/120 mesh) or equiva-
lent.
5.6.1.3 Column 3 - 50 cm long x 1/8 inch OD Teflon, packed
with 15* SE-54 on Gas Chrom Q (80/100 mesh) or
equivalent.
5.6.2 Detector - Thermionic bead or flame photometric in the
phosphorus mode. These detectors have proven effective in
the analysis of wastewaters for the parameters listed in the
scope and were used to develop the method performance state-
ments in Section 15. Alternative detectors, including a
mass spectrometer, may be used in accordance with the
provisions described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent Is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, methylene chloride - Pesticide quality or
equivalent.
6.3 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.5 Stock standard solutions (1.00 ug/yL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.5.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality hexane or other suitable
solvent and dilute to volume in a 10-mL volumetric flask.
Larger volumes may be used at the convenience of the
analyst. If compound purity is certified at 96% or greater,
the weight may be used without correction to calculate the
concentration of the stock standard. Commercially prepared
622-05 January 1983
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stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.5.2 Transfer the stock standard solutions into TFE-fluoro-
carbon sealed screw cap vials. Store at 4°C and protect
from light. Frequently check stock standard solutions for
signs of degradation or evaporation., especially just prior
to preparing calibration standards'from them.
6.6.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
hexane or other suitable solvent. One of the external
standards should be representative of a concentration near,
but above, the method detection limit. The other concentra-
tions should correspond to the range of concentrations
expected in the sample concentrates or should define the
working range of the detector.
7.2.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration stan-
dard. Alternatively, a new calibration curve or calibration
factor must be prepared for that parameter.
622-06 January 1983
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7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of Interest. The analyst must
further demonstrate that the measurement of the Internal standard
1s not affected by method or matrix interferences. Due to these
limitations, nq Internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three concen-
tration levels for each parameter of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with hexane or other suitable solvent. One of the
standards should be representative of a concentration near,
but above, the method detection limit. The other concentra-
tions should correspond to the range of concentrations
expected in the sample concentrates, or should define the
working range of the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF - (AsC1s)/(A1s Cs)
where:
As * Response for the parameter to be measured.
Response for the internal standard.
Concentration of the internal standard in ug/L.
Concentration of the parameter to be measured in
ug/l.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/A-js against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
622-07 January 1983
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8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To.establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
622-08 January 1983
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8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) * R + 3" s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts6
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.°
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dis-
similar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
622-09 January 1983
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analysis of quality control materials and participate 1n relevant
performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected 1n glass containers. Conventional
sampling practices7 should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected 1n refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample Into a
2-Hter separatory funnel.
10.2 Add 60 ml methylene chloride to the sample bottle,, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the separa-
tory funnel and extract the sample by shaking the funnel for 2 min
with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layers is more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include stir-
ring, filtration of the emulsion through glass wool, centrifuga-
tion, or other physical methods. Collect the methylene chloride
extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 Assemble a Kuderna-Oanish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-Q if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
622-10 January 1983
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10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mi methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
10.7 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 ml of hexane and a new
boiling chip and reattach the Snyder column. Pour about 1 ml of
hexane into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be
5 to 10 min. When the apparent volume of liquid reaches 1 ml,
remove the K-D apparatus and allow it to drain and cool for at
least 10 min.
10.8 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexane and adjust the
volume to 10 ml. A 5-mL syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will
be stored longer than two days, they should be transferred to
TFE-fluorocarbon-sealed screw-cap vials. If the sample extract
requires no further cleanup,- proceed with gas chromatographic
analysis. If the sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix and were not required for the analysis of the
wastewaters reported in Section 15. If particular circumstances
demand the use of a cleanup procedure, the analyst must determine
the elution profile and demonstrate that the recovery of each
compound of interest for the cleanup procedure is no less than 85%.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. Naled is partially converted to dichlorvos on GC Columns 1
and 2 but not on Column 3. Therefore, if naled is to be measured
622-11 January 1983
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1n the sample, GC analysis for dlchlorvos and naled must be
performed using Column 3. Examples of the separations achieved are
shown 1n Figures 1 to 4. Other packed columns, chromatographlc
conditions, or detectors may be used 1f the requirements of Section
8.2 are met. Capillary (open- tubular) columns may also be used 1f
the relative standard deviations of responses for replicate injec-
tions are demonstrated to be less than 6% .and the requirements of
Section 8.2 are met.
12.2 Calibrate the system daily as described 1n Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
Instrument. Mix thoroughly.
12.4 Inject 1 to 5 uL of the sample extract using the solvent-flush
technique. ° Record the volume injected to the nearest 0.05 uL,
and the resulting peak size 1n area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(Vt)
Concentration, ug/L s — /..
where:
A = Amount of material injected, in nanograms.
V-j a Volume of extract injected in uL.
Vt = Volume of total extract in uL.
V = Volume of water extracted in ml.
622-12 January 1983
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13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(AS)(IS)
Concentration, ug/L * -rj-
where:
As » Response for the parameter to be measured.
A^s * Response for the internal standard.
Is » Amount of Internal standard added to each extract
in ug.
V0 » Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this method.
The mass spectrometer should be capable of scanning the mass range
from 35 amu to a mass 50 amu above the molecular weight of the
compound. The instrument must be capable of scanning the mass
range at a rate to produce at least 5 scans per peak but not to
exceed 7 s per scan utilizing a 70 V (nominal) electron energy in
the electron impact ionization mode. A GC to MS interface con-
structed of all-glass or glass-lined materials is recommended. A
computer system should be interfaced to the mass spectrometer that
allows the continuous acquisition and storage on machine readable
media of all mass spectra obtained throughout the duration of the
chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chromato-
graphic tailing factors of less than 5.0 must be achieved.9
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorgtriphenyl phosphine (DFTPP) performance criteria are
achieved.10
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
622-13 January 1983
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conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MDL) is defined as the minimum concen-
tration of a substance that can be measured and reported with 99%
confidence that the value is above zero." The MDL concentra-
tions listed in Table 1 were estimated from the response of the
detector to each compound. The estimate is based upon the amount
of material required to yield a signal five times the GC background
noise, assuming a 5-uL injection from a 10-mL final extract of a
1-liter sample.
15.2 In a single laboratory (West Cost Technical Services, Inc.), using
effluents from pesticide manufacturers and publicly owned treatment
works (POTW), the average recoveries presented in Table 2 were
obtainedJ The standard deviations of the percent recoveries of
these measurements are also included in Table 2.
622-14 January 1983
-------
References
1. "Pesticide Methods Evaluation," Letter Reports #6, 12A, and 14 for EPA
Contract No. 68-03-2697. Available from U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio 45268.
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testinc and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Anal)tical Quality Control in Water and Wastewater
Laboratories," EPA-500/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
8. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
9. McNair, H.M. and Bcnelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abuncance Measurement in Gas Chromatography-Mass
Spectrometry," Anal/tical Chemistry, 47, 995 (1975).
11. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technolocy, J_5, 1426 (1981).
12. "Determination of Dinitroaniline in Industrial and Municipal Wastewater,"
Method 622, EPA No. 600/4-82-008, NTIS No. PB82-156027, January 1982,
National Technical Information Center, 5285 Port Royal Road, Springfield,
VA 22165.
622-15 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND ESTIMATED METHOD DETECTION LIMITS
Parameter
Demeton
Phorate
Dlsulfoton
Trichloronate
Pent hi on
Tokuthlon
Bo 1 star
Fensulfothion
Azinphos methyl
Coumaphos
Dichlorvos
Mevlnphos
Stirofos
Ethoprop
Parathion methyl
Ronnel
Chlorpyrlfos methyl
Chlorpyrifos
Merphos
Dlazinon
Dichlorvos
Naled
Stirofos
GC
Column
la
la
la
la
la
la
la
la
la
la
Ib
Ib
Ib
2
2
2
2
2
2
2
3
3
3
Retention
Time
(Min)
1.16, 2.53
1.43
2.10
2.94
3.12
3.40
4.23
6.41
6.30
11.6
0.8
2.41
8.52
3.02
3.37
5.57
5.72
6.16
7.45
7.73
1.50
3.28
5.51
Estimated
MDL
(uq/L)
0.25
0.15
0.20
0.15
0.10
0.5
0.15
1.5
1.5
1.5
0.1
0.3
5.0
0.25
0.3
0.3
0.3
0.3
0.25
0.6
0.1
0.1
5.0
Column la Conditions: Supelcoport (100/120 mesh) coated with 5* SP-2401
packed in a 180 cm long x 2 mm ID glass column with helium carrier gas at a
flow rate of 30 mL/min. Column temperature, programmed: initial 150°C,
hold for 1 min, then program at 25°C/min to 220°C and hold. A flame
photometric detector was used with this column to estimate the MDL.
Column Ib Conditions: Same as Column la, except nitrogen carrier gas at a
flow rate of 30 ml/min. Temperature, programmed: initial 170°C, hold 2
min, then program at 20°C/min to 220°C and hold.
Column 2 Conditions: Supelcoport (100/120 mesh) coated with 3% SP-2401
packed in a 180 cm long x 2 mm ID glass column with helium carrier gas at a
flow rate of 25 mL/min. Column temperature, programmed, initial 170°C,
hold for 7 min, then program at 10°C/min to 250°C and hold.. A
thermionic bead detector was used with this column to estimate the MDL.
622-16
January 1983
-------
TABLE 1. (Continued)
Column 3 Conditions: Gas Chrom Q (100/120 mesh) coated with 15% SE-54
packed in a 50 cm long x 1/8 in. 00 Teflon column with nitrogen carrier gas
at a flow rate of 30 mL/min. Temperature, programmed: initial 100°C,
then program immediately at 25°C/min to 200°C and hold. An electrolytic
detector in the halogen mode was used with this column to estimate the MOL.
622-17 January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Azinphos methyl
Bo 1 star
Chlorpyrifos
Coumaphos
Oemeton
Oiazinon
Dichlorvos
Dlsulfoton
Ethoprop
Fensulfothion
Fenthion
Merphos
Mevinphos
Naled
Parathion methyl
Phorate
Ronnel
Stlrofos
Tokuthion
Trichloronate
Average
Percent
Recovery
72.7
64.6
98.3
109.0
67.4
67.0
72.1
81.9
100.5
94.1
68.7
120:7
56.5
78.0
96.0
62.7
99.2
66.1
64.6
105.0
Standard
Deviation
rt)
18.8
6.3
5.5
12.7
10.5
6.0
7.7
9.0
4.1
17.1
19.9
7.9
7.8
8.1
5.3
8.9
5.6
5.9
6.8
18.6
Spike Number
Range of
(yg/L) Analyses
21-250
4.9-46
1.0-50.3
25-225
11. 9-314
5.6
15.6-517
5.2-92
1.0-51.5
23.9-110
5.3-64
1.0-50
15.5-520
25.8-294
0.5-500
4.9-47
1.0-50 •
30.3-505
5.3-64
20
17
17
18
17
17
7
16
17
18
17
17
18
16
16
21
17
18
16
17
3
Matrix
Types
3
3
3
3
3
1
3
3
3
3
3
3
3
3
3
3
3
3
3
1
622-18
January 1983
-------
soqdeumoo
622-19
January 1983
-------
soj.cu.i4s
CO
I/I
o
o
u
s.
O
o
o
o
in
-------
vt
O
o
2
to
a.
vt
O
•s I
C O
I
1
8
Figure 3.
234567
Minutes
Gas chromatogram of organophosphorus pesticides on
Column 2. For conditions, see Table 1.
622-21
January 1983
-------
4>EPA
United States
Environmental Protection
Agency
Effluent Guidelines Division (WH 552)
Washington, O.C. 20460
Water and Waste Management
TEST METHOD
DETERMINATION OF PENTACHLOROPHENOL SALT
IN
METHOD 625
1. Scope and Application
1.1 This method covers the determi-
nation of a number of organic
compounds that are partitioned into an
organic solvent and are amenable to
gas chromatography. The parameters
listed in Tables 1 and 2 may be
qualitatively and quantitatively
determined using this method.
1,2 The method may be extended to
include the parameters listed in Table 3.
Benzidine can be subject to oxidative
losses during solvent concentration.
cr-SHC. r-BHC, endosulfan I and II. and
endrin are subject to decomposition
under the alkaline conditions of the
extraction step. Hexachlorocyclopenta-
diene is subject to thermal decomposi-
tion in the inlet of the gas chromato-
graph, chemical reaction in acetone
solution and photochemical decompo-
sition. N-nitrosodimethylamine is
difficult to separate from the solvent
under the chromatographic conditions
described. N-nitrosodiphenylamine
decomposes in the gas chromato-
graphic inlet and cannot be separated
from diphenylamine. The preferred
method for each of these parameters is
listed in Table 3.
1.3 This is a gas chromatography/
mass spectrometry (GC/MS) method
applicable to the determination of the
compounds listed in Tables 1, 2, and 3
in municipal and industrial discharges
as provided under 40 CFR 136.1. Until
the U.S. Environmental Protection
Agency establishes performance cri-
teria based upon the results of inter-
laboratory testing, any alternative
GC/MS method which meets the per-
formance criteria described in Section
8.2 will be permitted. Performance
must be verified for such modification
by analyzing wastewater as described
in Section 8.2.2. In addition, the
laboratory must successfully partici-
pate in the applicable performance
evaluation studies.
1.4 The method detection limit (MOL,
defined in Section 16)'1' for each
parameter is listed in Tables 4 and 5.
The MOL for a specific wastewater
differ from those listed, depending
upon the nature of interferences in the
sample matrix.
1.5 This method is restricted to use
by or under the supervision of analysts
experienced in the operation of gas
chromatograph/mass spectrometers
and skilled in the interpretation of mass
spectra. Each analyst must demon-
strate the ability to generate accept-
able results with this method using the
procedure described in Section 8.2.
2. Summary of Method
2.1 A measured volume of sample,
approximately one-liter, is serially
extracted with methylene chloride at a
pH greater than 11 and again at pH
less than 2 using a separatory funnel or
a continuous extractor. The methylene
chloride extract is dried and
concentrated to a volume of 1 mL.
Chromatographic conditions are
described which permit the separation
and measurement of the parameters in
the extract. Qualitative identification is
performed using the retention time and
the relative abundance of three
characteristic ions. Quantitative
analysis is performed using either
625-1
January 1983
-------
external or internal standard techniques
with a single characteristic ion.
3. Interferences
3.1 Method interferences may be
caused by contaminants in solvents,
reagents, glassware, and other sample
processing hardware that lead to
discrete artifacts and/or elevated
baselines in the total ion current
profiles. All of these materials must be
routinely demonstrated to be free from
interferences under the conditions of
the analysis by running laboratory
reagent blanks as described in Section
8.5.
3.1.1 Glassware must be scrupulously
cleaned'3'. Clean all glassware as soon
as possible after use by rinsing with the
last solvent used in it. This should be
followed by detergent washing with
hot water, and rinses with tap water
and reagent water. It should then be
drained dry, and heated in a muffle
furnace at 400 °C for 1 5 to 30
minutes. Some thermally stable
materials, such as PCBs, may not be
eliminated by this treatment. Solvent
rinses with acetone and pesticide
quality hexane may be substituted for
the muffle furnace heating. Volumetric
ware should not be heated in a muffle
furnace. After drying and cooling,
glassware should be sealed and stored
in a clean environment to prevent any
accumulation of dust or other
contaminants. Store it inverted or
capped with aluminum foil.
3.1.2 The use of high purity reagents
and solvents helps to minimize inter-
ference problems. Purification of
solvents by distillation in all-glass
systems may be required.
3.2 Matrix interferences may be
caused by contaminants that are
coextracted from the sample. The
extent of matrix interferences will vary
considerably from source to source,
depending upon the nature and diver-
sity of the industrial complex or munici-
pality being sampled.
3.3 The base-neutral extraction may
cause significantly reduced recovery of
phenol, 2-methylphenol, and
2,4-dimethylphenol. The analyst must
recognize that results obtained under
these conditions are minimum
concentrations.
3.4 The packed gas chromatographic
columns recommended for the basic
fraction may not exhibit sufficient
resolution for certain isomeric pairs.
These include anthracene and phenan-
threne; chrysene and benzo(a)anthra-
cene; and benzo(b)fluoranthene and
benzo(k)fluoranthene. The gas
chromatograph retention time and
mass spectra are not sufficiently
different to make an unambiguous
distinction between these compounds.
Alternative techniques should be used
to identify and quantify these specific
compounds. See method 610.
3.5 In samples that contain an
inordinate number of interferences, the
use of chemical ionization (CD mass
spectrometry may make identification
easier. Tables 6 and 7 give
characteristic Cl ions for most of the
compounds covered by this method.
The use of Cl mass spectrometry to
support electron ionization (El) mass
spectrometry is encouraged but not
required.
4. Safety
4.1 The toxicity or carcinogenicity of
each reagent used in this method has
not been precisely defined; however,
each chemical compound should be
treated as a potential health hazard.
From this viewpoint, exposure to these
chemicals must be reduced to the
lowest possible level by whatever
means available. The laboratory is
responsible for maintaining a current
awareness file of OSHA regulations
regarding the safe handling of the
chemicals specified in this method. A
reference file of material data handling
sheets should also be made available to
all personnel involved in the chemical
analysis. Additional references to
laboratory safety are available and
have been identified for the information
of the analyst<4-6).
4.2 The following parameters
covered by this method have been
tentatively classified as known or
suspected, human or mammalian
carcinogens; benzo(a)anthracene,
benzidine, 3,3 '-dichlorobenzidine,
benzo(a)pyrene, o-BHC, /J-BHC, <5-BHC,
y-BHC, dibenzo(a,h) anthracene, Ni-
nitrosodimethylamine, 4,4'-DDT and
polychlorinated biphenyls.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete
or composite sampling.
5.1.1 Grab sample bottle —Amber
glass, one-liter or one-quart volume,
fitted with screw caps lined with
Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If
amber bottles are not available, protect
samples from light The container must
be washed, rinsed with acetone or
methylene chloride, and dried before
use to minimize contamination.
5.1.2 Automatic sampler (optional) —
Must incorporate glass sample
containers for the collection of a
minimum of 250 ml_. Sample
containers must be kept refrigerated at
4 °C and protected from light during
compositing. If the sampler uses a
peristaltic pump, a minimum length of
compressible silicone rubber tubing
may be used. Before use, however, the
compressible tubing should be
thoroughly rinsed with methanol,
followed by repeated rinsings with
distilled water to minimize the potential
for contamination of the sample. An
integrating flow meter is required to
collect flow proportional composites.
5.2 Glassware (All specifications are
suggested. Catalog numbers are
included for illustration only).
5.2.1 Separatory funnel —2000-mL,
with Teflon stopcock.
5.2.2 Drying column—1 9 mm ID
chromatographic column with coarse
frit.
5.2.3 Concentrator tube, Kuderna-
Danish— 10-mL, graduated (Kontes
K-570050-1025 or equivalent).
Calibration must be checked at the
volumes employed in the test. Ground
glass stopper is used to prevent
evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-
Danish —500-mL (Kontes
K-570001-0500 or equivalent).
Attach to concentrator tube with
springs.
5.2.5 Snyder column, Kuderna-
Danish — Three-ball macro (Kontes
K-503000-01 21 or equivalent).
5.2.6 Snyder column, Kuderna-
Danish —Two-ball micro (Kontes
K-569001-021 9 or equivalent).
5.2.7 Vials—Amber glass, 10-to
1 5-mL. capacity, with Teflon-lined
screw cap.
5.2.8 Continuous liquid-liquid
extractors —Equipped with Teflon or
glass connecting joints and stopcocks
requiring no lubrication. (Hershberg-
Wolf Ex'tractor-Ace Glass Company,
Vineland, N.J. P/N 6841-10 or
equivalent.)
5.3 Boiling chips —approximately
1 0/40 mesh. Heat to 400 °C for 30
minutes or Soxhlet extract with
methylene chloride.
5.4 Water bath—Heated, with
concentric ring cover, capable of
temperature control ( ± 2 °C). The bath
should be used in a hood.
625-2
January 1983
-------
5.5 Balance—Analytical, capable of
accurately weighing 0.0001 g.
5.6 GC/MS system.
5.6.1 Gas chromatograph —An
analytical system complete with a
temperature programmable gas
chromatograph and all required
accessories including syringes,
analytical columns, and gases. The
injection port must be designed for on-
column injection when using packed
columns and for splitless injection
when using capillary columns.
5.6.2 Column for Base Neutrals —1.8
m long x 2 mm ID glass, packed with
Supelcoport (100/120 mesh) coated
with 3% SP-2250 or equivalent. This
column was used to develop the
accuracy and precision statements in
Table 6 and the MDL data in Table 4.
Guidelines for the use of alternate
column packings are provided in
Section 13.1.
5.6.3 Column for Acids—1.8 m long
x 2 mm ID glass, packed with
Supelcoport (100/120 mesh) coated
with 1 % SP-1 240 DA or equivalent.
This column was used to develop the
accuracy and precision statements in
Table 7, and the MDL data in Table 5.
Guidelines for the use of alternate
column packings are given in Section
13.1.
5.6.4 Mass Spectrometer—Capable
of scanning from 35 to 450 amu every
seven seconds or less utilizing a 70
volt (nominal) electron energy in the
electron impact ionization mode and
producing a mass spectrum which
meets all the criteria in Table 9 when
50 ng of decafluorotriphenyl phosphine
(DFTPP; bis(perfluorophenyl) phenyl
phosphine) is injected through the gas
chromatographic inlet. Any gas
chromatograph to mass spectrometer
interface that gives acceptable
calibration points at 50 ng per injection
for each compound of interest in
Tables 1 through 3 and achieves all
acceptable performance criteria
(Section 1 2) may be used. Gas
chromatograph to mass spectrometer
interfaces constructed of all glass or
glass lined materials are recommended.
Glass can be deactivated by silanizing
with dichlorodimethylsilane.
5.6.5 A computer system must be
interfaced to the mass spectrometer
that allows the continuous acquisition
and storage on machine readable media
of all mass spectra obtained
throughout the duration of the
chromatographic program. The
computer must have software that
allows searching any GC/MS data file
for ions of a specific mass and plotting
such ion abundances versus time or
scan number. This type of plot is
defined as an Extracted Ion Current
Profile (EICP). Software must also be
available that allows integrating the
abundance in any EICP between
specified time or scan number limits.
6. Reagents
6.1 Reagent water—Reagent water is
defined as a water in which an inter-
f erent is not observed at the method
detection limit of each parameter of
interest.
6.2 Sodium hydroxide solution (10
N) —Dissolve 40g NaOH in reagent
water and dilute to 100 ml.
6.3 Sodium thiosulfate-(ACS)
Granular.
6.4 Sulfuric acid solution
(1 + 1 (-Slowly add 50 ml of H2S04
(sp. gr. 1.84) to 50 ml of reagent
water.
6.5 Acetone, methanol, methylene
chloride —Pesticide quality or
equivalent.
6.6 Sodium sulfate—(ACS) Granular,
anhydrous. Purify by heating at 400 °C
for four hours in a shallow tray.
6.7 Stock standard solutions (1.00
^g/^D — Stock standard solutions can
be prepared from pure standard
materials or purchased as certified
solutions.
6.7.1 Prepare stock standard
solutions by accurately weighing about
0.0100 g of pure material. Dissolve
the material in pesticide quality
acetone or other suitable solvent and
dilute to volume in a 10-mL volumetric
flask. Larger volumes may be used at
the convenience of the analyst. If
compound purity is assayed at 96% or
greater, the weight may be used
without correction to calculate the
concentration of the stock standard.
Commercially prepared stock standards
may be used at any concentration if
they are certified by the manufacturer
or by an independent source.
6.7.2 Transfer the stock standard
solutions into Teflon-sealed screw-cap
bottles. Store in 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 cali-
bration standards from them. Quality
control check samples, that can be
used to determine the accuracy of
calibration standards will be available
from the U.S. Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
6.7.3 Stock standard solutions must
be replaced after six months or sooner
if comparison with quality control
check samples indicate a problem.
6.8 Surrogate standard spiking
solutions —select a minimum of three
surrogate compounds from Table 8.
Prepare a surrogate standard spiking
solution at a concentration of 100
^ig/1.00 ML in acetone. Addition of
1.00 mL of this solution to 1000-mL
of sample is equivalent to a concentra-
tion of 100 ng/L of each surrogate
standard. Store the spiking solutions at
4 °C in Teflon-sealed containers. The
solutions should be checked frequently
for stability. These solutions must be
replaced after six months, or sooner if
comparison with quality control check
samples indicate a problem. Surrogate
standard spiking solutions, appropriate
for use with this method will be
available from the U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
6.9 DFTPP standard-Prepare a 25
ng/^L solution of DFTPP in acetone.
7. Calibration
7.1 Establish gas chromatographic
operating parameters equivalent to
those indicated in Tables 4 or 5, The
GC/MS system can be calibrated using
the external standard technique
(Section 7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration
procedure:
7.2.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding volumes of one or more stock
standards to a volumetric flask and
diluting to volume with acetone. One
of the external standards should be at a
concentration near, but above, the
MDL and the other concentrations
should correspond to the expected
range of concentrations found in real
samples or should define the working
range of the GC/MS system.
7.2.2 Analyze 2 to 5 jjL of each
calibration standard and tabulate the
area responses of the primary
characteristic ion of each standard
(Tables 4 and 5) against the mass
injected. The results may be used to
prepare a calibration curve for each
compound. Alternatively, if the ratio of
response to amount injected
(calibration factor) is a constant over
625-3
January 1983
-------
the working range «10% relative
standard deviation, RSD), linearity
through the origin may be assumed and
the average ratio or calibration factor
may be used in place of a calibration
curve.
7.2.3 The working calibration curve
or calibration factor must be verified on
each working day by the measurement
of one or more calibration standards. If
the response for any parameter varies
from the predicted response by more
than ± 10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
or calibration factor must be prepared
for that compound.
7.3 Internal standard calibration
procedure. To use this approach, the
analyst must select one or more
internal standards that are similar in
analytical behavior to the compounds
of interest. The analyst must further
demonstrate that the measurement of
the internal standard is not affected by
method or matrix interferences. Table
8 lists some recommended internal
standards. Phenanthrene-d-io has been
used for this purpose. Use the base
peak ion as the primary ion for
quantification of the standards. If
interferences are noted, use the next
two most intense ions as the
secondary ions.
7.3.1 Prepare calibration standards
at a minimum of three concentration
levels for each parameter of interest by
adding appropriate volumes of one or
more stock standards to a volumetric
flask. To each calibration standard or
standard mixture, add a known
constant amount of one or more
internal standards, and dilute to volume
with acetone. One of the calibration
standards should be at a concentration
near, but above, the MDL and the other
concentrations should correspond to
the expected range of concentrations
found in real samples or should define
the working range of the GC/MS
system.
7.3.2 Analyze 2 to 5 fiL of each
calibration standard and tabulate the
area of the primary characteristic ion
(Tables 4 and 5) against concentration
for each compound and internal
standard, and calculate response
factors (RF) for each compound using
equation 1.
Eq. 1 RF = (ASC1S)/(A,SCS)
where:
As = Area of the characteristic ion
for the parameter to be
measured.
A1S = Area of the characteristic ion
for the internal standard.
CIS = Concentration of the internal
standard, (\tgl\-).
Cs = Concentration of the
parameter to be measured,
(M9/D.
If the RF value over the working range
is a constant «1 0% RSD), the RF can
be assumed to be invariant and the
average RF can be used for
calculations. Alternatively, the results
can be used to plot a calibration curve
of response ratios, As/Ais, vs. RF.
7.3.3 The working calibration curve
or RF must be verified on each working
day by the measurement of one or
more calibration standards. If the
response for any parameter varies from
the predicted response by more than
±10%, the test must be repeated
using a fresh calibration standard.
Alternatively, a new calibration curve
must be prepared.
8. Quality Control
8.1 Each laboratory that uses this
method is required to operate a formal
quality control program. The minimum
requirements of this program consist of
an initial demonstration of laboratory
capability and the analysis of spiked
samples as a continuing check on
performance. The laboratory is required
to maintain performance records to
define the quality of data that is
generated. Ongoing performance
checks must be compared with estab-
lished performance criteria to
determine if the results of analyses are
within accuracy and precision limits
expected of the method.
8.1.1 Before performing any
analyses, the analyst must
demonstrate the ability to generate
acceptable accuracy and precision with
this method. This ability is established
as described in Section 8.2.
8.1.2 In recognition of the rapid
advances that are occurring in chroma-
tography, the analyst is permitted
certain options to improve the
separations or lower the cost of
measurements. Each time such
modifications are made to the method,
the analyst is required to repeat the
procedure in Section 8.2.
8.1.3 The laboratory must spike all
samples with surrogate standards to
monitor continuing laboratory
performance. This procedure is
described in Section 8.4.
8.2 To establish the ability to
generate acceptable accuracy and
precision, the analyst must perform the
following operations.
8.2.7 Select a representative spike
concentration for each parameter to be
measured. Using stock standards,
prepare a quality control check sample
concentrate in acetone 1000 times
more concentrated than the selected
concentrations. Quality control check
sample concentrates, appropriate for
use with this method, will be available
from the U.S Environmental Protection
Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio
45268.
8.2.2 Using a pipet, add 1.00 mL of
the check sample concentrate and 1.0
ml of the surrogate standard dosing
solution (Section 6.8) to each of a
minimum of four 1 000-mL aliquots of
reagent water. A representative
wastewater may be used in place of
the reagent water, but one or more
additional aliquots must be analyzed to
determine background levels, and the
spike level must exceed twice the
background level for the test to be
valid. Analyze the aliquots according to
the method beginning in Section 10.
8.2.3 Calculate the average percent
recovery, (R), and the standard devia-
tion of the percent recovery (s), for all
parameters and surrogate standards.
Wastewater background corrections
must be made before R and s
calculations are performed.
5.2.4 Using Table 6 or 7, note the
average recovery (X) and standard
deviation (p) expected for each method
parameter. Compare these to the
calculated values for R and s. If s > p or
(X-R| > p, review potential problem
areas and repeat the test.
8.2.5 The U.S. Environmental Pro-
tection Agency plans to establish
performance criteria for R and s based
upon the result of interlaboratory
testing. When they become available,
these criteria must be met before any
samples may be analyzed.
8.3 The analyst must calculate
method performance criteria for each
of the surrogate standards.
8.3.1 Calculate upper and lower
control limits for method performance
for each surrogate standard, using the
values for R and s calculated in Section
8.2.3:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCD = R - 3s
The UCL and LCL can be used to
construct control charts171 that are
useful in observing trends in
performance. The control limits above
must be replaced by method perfor-
mance criteria as they become avail-
625-4
January 1983
-------
able from the U.S. Environmental
Protection Agency.
8.3.2 For each surrogate standard,
the laboratory must develop and
maintain separate accuracy statements
of laboratory performance for
wastewater samples. An accuracy
statement for the method is defined as
R ± s. The accuracy statement should
be developed by the analysis of four
aliquots of wastewater as described in
Section 8.2.2, followed by the calcula-
tion of R and s. Alternately, the analyst
may use four wastewater data points
gathered through the requirement for
continuing quality control in Section
8.4. The accuracy statements should
be updated regularly.I7'
8.4 The laboratory is required to
spike all samples with the surrogate
standard spiking solution to monitor
spike recoveries. If the recovery for any
surrogate standard does not fall within
the control limits for method
performance, the results reported for
that sample must be qualified as
described in Section 1 5.3. The
laboratory should monitor the
frequency of data so qualified to
ensure that it remains at or below 5%.
8.5 Before processing any samples,
the analyst should demonstrate
through the analysis of a one-liter
aliquot of reagent water, that all
glassware and reagent interferences
are under control. Each time a set of
samples is extracted or there is a
change in reagents, a laboratory
reagent blank should be processed as a
safeguard against laboratory
contamination.
8.6 It is recommended that the
laboratory adopt additional quality
assurance practices for use with this
method. The specific practices that are
most productive depend upon the
needs of the laboratory and the nature
of the samples. Field duplicates may be
analyzed to monitor the precision of
the sampling technique. Whenever
possible, the laboratory should perform
analysis of standard reference
materials and participate in relevant
performance evaluation studies.
9. Sample Collection,
Preservation, and Handling
9.1 Grab samples must be collected
in glass containers. Conventional
sampling practices'8' should be
followed, except that the bottle must
not be prewashed with sample before
collection. Composite samples should
be collected in refrigerated glass
containers in accordance with the
requirements of the program.
Automatic sampling equipment must
be as free as possible of Tygon and
other potential sources of
contamination.
9.2 The samples must be iced or
refrigerated at 4 °C from the time of
collection until extraction. Fill the
sample bottles and, if residual chlorine
is present, add 80 mg of sodium
thiosuifate per each liter of water. U.S.
Environmental Protection Agency
methods 330.4 and 330.5 may be
used to measure the residual
chlorine<9l. Field test kits are available
for this purpose.
9.3 All samples must be extracted
within 7 days and completely analyzed
within 40 days of extraction.
10. Separatory Funnel
Extraction
10.1 Samples are usually extracted
using separatory funnel techniques. If
emulsions will prevent achieving
acceptable solvent recovery with
separatory funnel extractions,
continuous extraction (Section 11)
may be used. The separatory funnel
extraction scheme described below
assumes a sample volume of one-liter.
When sample volumes of two liters are
to be extracted, use 2 50-, 1 00-, and
100-ml volumes of methylene chloride
for the serial extraction of the base/
neutrals and 200-, 100-, and 100-mL
volumes of methylene chloride for the
acids.
10.2 Mark the water meniscus on the
side of the sample bottle for later
determination of sample volume. Pour
the entire sample into a two-liter
separatory funnel. Pipet 1.00 mL
surrogate standard spiking solution into
the separatory funnel and mix well.
Check the pH of the sample with wide-
range pH paper and adjust to pH > 1 1
with 10 N sodium hydroxide.
10.3 Add 60 ml methylene chloride
to the sample bottle, seal, and shake
for 30 seconds to rinse the inner
surface. Transfer the solvent to the
separatory funnel and extract the
sample by shaking the funnel for two
minutes with periodic venting to
release excess pressure. Allow the
organic layer to separate from the
water phase for a minimum of 10
minutes. If the emulsion interface
between layers is more than one-third
the volume of the solvent layer, the
analyst must employ mechanical
techniques to complete the phase
separation. The optimum technique
depends upon the sample, but may
include stirring, filtration of the
emulsion through glass wool, centrifu-
gation, or other physical methods.
Collect the methylene chloride extract
in a 250-ml Erlenmeyer flask. If the
emulsion cannot be broken (recovery of
less than 80% of the methylene
chloride, corrected for the water
solubility of methylene chloride),
transfer the sample, solvent, and
emulsion into the extraction chamber
of a continuous extractor and proceed
as described in Section 11.3.
10.4 Add a second 60-mL volume of
methylene chloride to the sample bottle
and repeat the extraction procedure a
second time, combining the extracts in
the Erlenmeyer flask. Perform a third
extraction in the same manner. Label
the combined extract as base/neutral
fraction.
10.5 Adjust the pH of the aqueous
phase to less than 2 using sulfuric acid
(1 +1). Serially extract three times
with 60-mL aliquots of methylene
chloride. Collect and combine the
•extracts in a 250-mL Erlenmeyer flask
and label the combined extract as the
acid fraction.
10.6 For each fraction, assemble a
Kuderna-Danish (K-D) concentrator by
attaching a 10-mL concentrator tube
to a 500-mL evaporative flask. Other
concentration devices or techniques
may be used in place of the K-D if the
requirements of Section 8.2 are met.
10.7 For each fraction, pour the
combined extract through a drying
column containing about 10 cm of
anhydrous sodium sulfate, and collect
the extract in the K-D concentrator.
Rinse the Erlenmeyer flask and column
with 20 to 30 mL of methylene
chloride to complete the quantitative
transfer.
10.8 To the evaporative flask for
each fraction, add one or two clean
boiling chips and attach a three-ball
Snyder column. Prewet the Snyder
column by adding about 1 mL
methylene chloride to the top of the
column. Place the K-D apparatus on a
hot water bath (60 ° to 65 °C) so that
the concentrator tube is partially
immersed in the hot water, and the
entire lower rounded surface of the
flask is bathed with hot vapor. Adjust
the vertical position of the apparatus
and the water temperature as required
to complete the concentration in 1 5 to
20 minutes. At the proper rate of
distillation the balls of the column will
actively chatter but the chambers will
not flood with condensed solvent.
When.the apparent volume of liquid
reaches 1 mL, remove the K-D
apparatus from the water bath and
625-5
January 1983
-------
allow it to drain and cool for at least 1 0
minutes. Remove the Snyder column
and rinse the flask and its lower joint
into the concentrator tube with 1 to 2
ml of methylene chloride. A 5-mL
syringe is recommended for this
operation.
10.9 Add another one or two clean
boiling chips to the concentrator tube
and attach a two-ball micro Snyder
column. Prewet the Snyder column by
adding about 0.5 mL of methylene
chloride to the top of the column. Place
the K-D apparatus on a hot water bath
(60° to 65 °C) so that the
concentrator tube is partially immersed
in the hot water. Adjust the vertical
position of the apparatus and the water
temperature as required to complete
the concentration in 5 to 10 minutes.
At the proper rate of distillation the
balls of the column will actively chatter
but the chambers will not flood with
condensed solvent. When the apparent
volume of liquid reaches about 0.5 mL,
remove the K-D apparatus from the
water bath and allow it to drain for at
least 10 minutes while cooling.
Remove the Snyder column and rinse
its the flask and its lower joint into the
concentrator tube with 0.2 mL of
acetone or methylene chloride. Adjust
the final volume to 1.0 mL with the
solvent. Stopper the concentrator tube
and store refrigerated if GC/MS
analysis will not be performed
immediately. If the extracts will be
stored longer than two days, they
should be transferred to Teflon-sealed
screw-cap bottles and labeled
base/neutral or acid fraction as
appropriate.
10.10 Determine the original sample
volume by refilling the sample bottle to
the mark and transferring the water to
a 1000-mL graduated cylinder. Record
the sample volume to. the nearest 5
mL.
11. Continuous Extraction
11.1 When experience with a sample
from a given source indicates that a
serious emulsion problem will result or
an emulsion is encountered in Section
10.3, using a separatory funnel, a
continuous extractor should be used.
11.2 Mark the water meniscus on the
side of the sample bottle for later
measurement of the sample volume.
Check the pH of the sample with wide-
range pH paper and adjust to pH 11
with 10 N sodium hydroxide. Transfer,
the sample to the continuous extractor
and using a pipet, add 1.00 mL of
surrogate standard spiking solution and
mix well. Add 60 mL of methylene
chloride to the sample bottle, seal and
shake for 30 seconds to rinse the inner
surface. Transfer the solvent to the
extractor.
11.3 Repeat the sample bottle rinse
with an additional 50- to 100-mL
portion of methylene chloride and add
the rinse to the extractor.
11.4 Add 200 to 500 mL of
methylene chloride to the distilling
flask, add sufficient reagent water to
ensure proper operation, and extract
for 24 hours. Allow to cool, then
detach the boiling flask, and dry,
concentrate and seal the extract as in
Section 10.6 through 10.9. Hold the
aqueous phase for acid extraction (See
Section 1 1.5).
11.5 Charge a clean distilling flask
with 500 mL of methylene chloride and
attach it to the continuous extractor.
Carefully, adjust the pH of the aqueous
phase to less than 2 using sulfuric acid
(1 +1). Extract for 24 hours. Dry,
concentrate and label and seal the
extract as described in Sections 10.6
through 10.9.
12. Daily GC/MS Performance
Tests
12.1 At the beginning of each day
that analyses are to be performed, the
GC/MS system must be checked to see
that acceptable performance criteria
are achieved for DFTPP. Each day that
benzidine is to be determined, the
tailing factor criterion described in
Section 1 2.4 must be achieved. Each
day the acids are to be determined, the
tailing factor criterion in Section 12.5
must be achieved.
12.2 These DFTPP performance test
require the following instrumental
parameters.
Electron Energy 70 volts (nominal)
Mass Range 35 to 450 amu
Scan Time to give at least 5
scans per peak but
not to exceed 7
seconds per scan.
12.3 DFTPP performance testdO)-
At the beginning of each day, inject
2^L (50 ng) of DFTPP standard
solution. Obtain a background cor-
rected mass spectra of DFTPP and
check that all the key ion criteria in
Table 9 are achieved. If all the criteria
are not achieved, the analyst must
retune the mass spectrometer and
repeat the test until all criteria are
achieved. The performance criteria
must be achieved before any samples,
blanks, or standards are analyzed. The
tailing factor tests in Section 1 2.4 and
12.5 may be performed simultaneously
with the test.
12.4 Column performance test for
base/neutrals —At the beginning of
each day that the base-neutral fraction
is to be analyzed for benzidine, the
benzidine tailing factor must be
calculated. Inject 100 ng of benzidine
either separately or as a part of a stan-
dard mixture that may contain DFTPP
and calculate the tailing factor. The
benzidine tailing factor must be less
than 3.0. Calculation of the tailing
factor is illustrated in Figure 13.HD
Replace the column packing if the
tailing factor criterion cannot be
achieved.
12.5 Column performance for
acids—At the beginning of each day
that the acids are to be determined,
inject 50 ng of pentachlorophenol
either separately or as a part of a
standard mix that may contain DFTPP.
The tailing factor for pentachlorophenol
must be less than five. Calculation of
the tailing factor is illustrated in Figure
1 3'111, Replace the column packing if
the tailing factor criterion cannot be
achieved.
13. Gas Chromatography/
Mass Spectrometry
13.1 Table 4 summarizes the
recommended gas chromatographic
operating conditions for the
base/neutral fraction. Table 5
summarizes the recommended gas
chromatographic operating conditions
for determination of the acid fraction.
These tables include retention times
and MDL. that were achieved under
these conditions. Examples of the
parameter separations achieved by
these columns are shown in Figures 1
through 1 2. Other packed columns or
chromatographic conditions may be
used if the requirements of Section 8.2
and Section 1 2 are met. Capillary
(open-tubular) columns may also be
used if the relative standard deviations
of responses for replicate injections are
demonstrated to be less than 6% and
the requirements of Section 8.2 and
Section 12 are met.
13.2 After the GC/MS performance
requirements of Section 12, calibrate
the system daily as described in
Section 7.
13.3 If the internal standard
approach is being used, the internal
standard must be added to sample
extract and mixed thoroughly, imme-
diately, before injection into the
instrument. This minimizes losses due
to adsorption, chemical reaction or
evaporation.
13.4 Inject 2 to 5 jiL of the sample
extract using the solvent-flush
625-6
January 1983
-------
technique'12). Smaller (1.0 ^U volumes
may be injected if automatic devices
are employed. Record the volume
injected to the nearest 0.05 ^L.
13.5 If the response for any ion
exceeds the working range of the
GC/MS system, dilute the extract and
reanalyze.
13.6 Perform all qualitative and
quantitative measurements as
described in Sections 14 and 1 5.
When the extracts are not being used
for analyses, store them at 4 °C pro-
tected from light in screw-cap vials
equipped with unpierced Teflon-lined
septa.
14. Qualitative Identification
14.1 Obtain an EICP for the primary
ion and the two other ions listed in
Tables 4 and 5. See Section 7.3 for
ions to be used with internal and
surrogate standards. The following
criteria must be met to make a
qualitative identification.
14.1.1 The characteristic ions for
each compound of interest must
maximize in the same or within one
scan of each other.
14.1.2 The retention time must fall
within ± 30 seconds of the retention
time of the authentic compound.
14.1.3 The relative peak heights of
the three characteristic ions in the
EICP's must fall within ± 20% of the
relative intensities of these ions in a
reference mass spectrum. The refer-
ence mass spectrum can be obtained
by a standard analyzed in the GC/MS
system or from a reference library.
14.2 Structural isomers that have
very similar mass spectra and less than
30 seconds difference in retention
time, can be explicitly identified only if
the resolution between authentic
isomers in a standard mix is acceptable.
Acceptable resolution is achieved if the
baseline to valley height between the
isomers is less than 25% of the sum of
the two peak heights. Otherwise,
structural isomers are identified as
isomeric pairs.
15. Calculations
15.1 When a compound has been
identified, the quantitation of that
compound will be based on the inte-
grated abundance from the EICP of the
primary characteristic ion in Tables 4
and 5. Use the base peak ion for
internal and surrogate standards. If the
sample produces an interference for
the first listed ion, use a secondary ion
to quantitate. Quantitation will be per-
(As)(ls)
formed using external or internal
standard techniques.
15.1.1 If the external standard
calibration procedure is used, calculate
the amount of material injected from
the area of the characteristic ion using
the calibration curve or calibration
factor in Section 7.2.2. The concentra-
tion in the sample can be calculated1
from equation 2:
Eq. 2. Concentration, yg/L =
\VjJ\Vgi
where:
A = Amount of material injected,
in nanograms.
V; = Volume of extract injected
V, = Volume of total extract (^L).
Vs = Volume of water extracted
(mL).
15.1.2 If the internal standard cali-
bration procedure was used, calculate
the concentration in the sample using
the response factor (RF) determined in
Section 7.3.2 and equation 3.
Eq. 3
Concentration, ug/L =
w (Ais)(RF)(V0)
where:
As = Area of the characteristic ion
for the parameter to be
measured.
Ais = Area of the characteristic ion
for the internal standard.
ls = Amount of internal standard
added to each extract (yg).
V0 = Volume of water extracted
(liters).
15.2 Report results in micrograms
per liter without correction for recovery
data. When duplicate and spiked
samples are analyzed, report all data
obtained with the sample results.
15.3 If the surrogate standard
recovery falls outside the control limits
in Section 8.3, data for all parameters
in that fraction of the sample must be
labeled as suspect.
16. Method Performance
16.1 The method detection limit
(MDL) is defined as the minimum
concentration of a substance that can
be measured and reported with a 99%
confidence that the value is above
zero'11. The MDL concentrations listed
in Tables 4 and 5 were obtained using
reagent water" 3).
16.2 The average recoveries and the
average standard deviations of the
percent recoveries, presented in Table
5, were the result of a study of the
accuracy and precision of this method
by several laboratories. The values
listed represent the results from two to
four laboratories'14'.
16.3 The U.S. Environmental
Protection Agency is in the process of
conducting an interlaboratory method
study to fully define the performance
of this method.
17. Screening Procedure for
2,3.7.8-TCDD
17.1 If the sample must be screened
for the presence of 2,3,7,8-TCDD, it is
recommended that the reference mate-
rial not be handled in the laboratory
unless extensive safety precautions are
employed. It is sufficient to analyze the
base/neutral extract by selected ion
monitoring (SIM) GC/MS techniques,
as follows:
17.1.1 Concentrate the base/neutral
extract to a final volume of 0.2 ml.
17.1.2 Adjust the temperature of the
base/neutral column (Section 5.6.2) to
220°C.
17.1.3 Operate the mass spec-
trometer to acquire data in the SIM
mode using the ions at m/e 257, 320
and 322 and a dwell time no greater
than 333 milliseconds per ion.
17.1.4 Inject 5 to 7 pL of the base/
neutral extract. Collect SIM data for a
total of 10 minutes.
17.1.5 The possible presence of
2,3,7,8-TCDD is indicated if all three
ions exhibit simultaneous peaks at any
point in the selected ion current
profiles.
17.1.6 For each occurrence where
the possible presence of 2,3,7,8-
TCDD is indicated, calculate and retain
the relative abundances of each of the
three ions.
17.2 False positives to this test may
be caused by the presence of single or
coeluting combinations of compounds
whose mass spectra contain all of
these ions.
17.3 Conclusive results of the
presence and concentration level of
2,3,7,8-TCDD can be obtained only
from a properly equipped laboratory
through the use of method 613 of
other approved alternate test
procedures.
625-7
January 1983
-------
References
1. See Appendix A.
2. "Sampling and Analysis Procedures
for Screening of Industrial Effluents for
Priority Pollutants." U.S. Environmental
Protection Agency, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1977,
Revised April 1977. Available from
Effluent Guidelines Division,
Washington, DC 20460.
3. ASTM Annual Book of Standards,
Part 31, D 3694. "Standard Practice
for Preparation of Sample Containert
and for Preservation," American
Society for Testing and Materials,
Philadelphia, PA, p. 679, (1980).
4. "Carcinogens —Working with
Carcinogens," Department of Health,
Education, and Welfare, Public Health
Service, Center for Disease Control,
National Institute for Occupational
Safety and Health, Publication No.
77-206, Aug. 1977.
5. "OSHA Safety and Health
Standards, General Industry,"
(29CFR1 910), Occupational Safety
and Health Administration, OSHA
2206, (Revised, January 1976).
6. "Safety in Academic Chemistry
Laboraties," American Chemical
Society Publication, Committee on
Chemical Safety, 3rd Edition, 1979.
7. "Handbook of Analytical Quality
Control in Water and Wastewater
Laboratories," EPA-600/4-79-019,
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268,
March 1979.
8. ASTM Annual Book of Standards,
Part 31, D 3370, "Standard Practice
for Sampling Water," American
Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. "Methods 330.4 (Titrimetric, DPD-
FAS) and 330.5 (Spectrophotometric,
DPD) for Chlorine, Total Residual/'
Methods for Chemical Analysis of
Water and Wastes, EPA 600-4/79-020,
U.S. Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268,
March 1979.
1 0. Eichelberger, J.W., Harris, L.E.,
and Budde, W.L., "Reference Com-
pound to Calibrate Ion Abundance
Measurement in Gas Chromatography-
Mass Spectrometry," Analytical
Chemistry, 47, 995 (1975).
1 1. McNair, H.M. and Bonelli, E.J.,
"Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52,
1969.
1 2. Burke, J.A., "Gas Chromatography
for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the
Association of Official Analytical
Chemists, 48, 1037 11965).
1 3. "Method Detection Limit for
Methods 624 and 625," Olynyk, P.,
Budde, W.L, Eichelberger, J.W.,
unpublished report October, 1 980.
14. Kloepfer, R.D., "POTW Toxic
Study, Analytical Quality Assurance
Final Report," U.S. Environmental
Protection Agency, Region VII, Kansas
City, Kansas 66115, 1981.
15. "Methods for Organic Chemicals
Analyses in Municipal, and Industrial
Wastewater," July 1982, U.S.
Environmental Protection Agency,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45668.
625-8
January 1983
-------
Table 1, Base/Neutral Extractables
Parameter
STORETNo.
CAS No.
Acenaphthene
Acenaphthylene
Anthracene
Aldrin
Benzo (a tan thracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Benzolghi)perylene
Benzyl butyl phthalate
P-BHC
6-BHC
Bis(2-chloroethyl)ether
Bis(2-chloroethoxylmethane
Bis(2-ethylhexyl)phthalate
Bis(2-chloroisopropyl)ether
4-Bromophenyl phenyl ether
Chlordane
2-Chloronaphthalene
4-Chlorophenyl phenyl ether
Chrysene
4,4' -ODD
4, 4 '-DDE
4, 4 '-DDT
Dibenzo(a,hlanthracene
Di-n-butylphthalate
1 , 3-Dichlorobenzene
1 , 2 -Dichlorob enzene
1 , 4-Dichlorobenzene
3, 3 '-Dichlorobenzidine
Dieldrin
Diethyl phthalate
Dimethyl phthalate
2, 4-Dinitrotoluene
2, 6-Dinitrotoluene
Di-n-octylphthalate
Endosulfan sulfate
Endrin aldehyde
Fluoranthene
Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachloroethane
Indeno (1,2, 3-cdlp yrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propylamine
PCB-1016
PCB-1221
PCB- 1232
PCB-1242
PCB- 1248
PCB- 12 54
PCB- 1260
Phenanthrene
Pyrene
Toxaphene
1 ,2,4-Trichlorobenzene
34205
34200
34220
39330
34526
34230
34242
34247
34521
34292
39338
34259
34273
34278
39100
34283
34636
39350
34581
34641
34320
39310
39320
39300
34556
39110
34566
34536
34571
34631
39380
34336
34341
34611
34626
34596
34351
34366
34376
34381
39410
39420
39700
34391
34396
34403
344O8
34696
34447
34428
34671
39488
39492
39496
39500
39504
39508
34461
34469
394OO
34551
83-32-9
208-96-8
120-12-7
309-00-2
56-55-3
205-99-2
207-08-9
50-32-8
191-24-2
85-68-7
319-85-7
319-86-8
1 1 1-44-4
111-91-1
117-81-7
108-60-1
101-55-3
57-74-9
91-58-7
7005-72-3
218-01-9
72-54-8
72-55-9
50-29-3
53-70-3
84-74-2
541-73-1
95-50-1
106-46-7
91-94-1
60-57-1
84-66-2
131-11-3
121-14-2
606-20-2
1 1 7-84-O
1031-07-8
7421-93-4
206-44-O
86-73-7
76-44-8
1024-57-3
118-74-1
87-68-3
67-72-1
193-39-5
78-59-1
91-2O-3
98-95-3
621-64-7
12674-11-2
1 1 104-28-2
11141-16-5
53469-2 1-9
12672-29-6
11097-69-1
1 1096-82-5
85-01-8
129-OO-0
8001-35-2
120-82-1
625-9
January 1983
-------
Table 2. Acid Extractables
Parameter
STORETNo.
CAS No.
4-Chloro-3-methylphenol
2-Chlorophenol
2. 4-Dichlorophenol
2, 4-Dimethylphenol
2. 4-Oinitrophenol
2-Methyl-4, 6-dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2. 4, 6- Trichlorophenol
Pentachlorophano! Salt
34452
34586
34601
34606
34616
34657
34591
34646
39032
34694
34621
—
59-5O-7
55-57-5
/ 20-83-2
105-67-9
51-28-5
534-52-1
88-75-5
100-02-7
87-86-5
108-95-2
88-06-2
131-52-2
Table 3. Additional Ex tractable Parameters'
Parameter STORETNo.
Benzidine
a-BHC
r-BHC
Endosulfan 1
Endosulfan II
Endrin
Hexachloncyclopentadiene
N-Nitrosodimethylamine
N-Nitrosodiphenylamine
39120
39337
39340
34361
34356
39390
34386
34438
34433
CAS No.
92-87-5
319-84-6
58-89-8
959-98-3
33213-65-9
72-20-8
77-47-4
62-75-9
86-30-6
Method
6O5
608
608
608
608
608
612
605
605
•See Section 1.2 of method
Table 4. Chromatognphic Conditions, Method Detection Limits and Characteristic Ions for Base/Neutral Extractables
Parameter
1, 3-Dichlorobenzene
1 ,4-Dichlorobenzene
Hexachloroethane
Bis(2-chloroethyl)ether
1,2-Oichlorobenzene
Bis(2-chloroisopropyl)ether
N-Nitrosodi-n-propyl amine
Nitrobenzene
Hexachlorobutadiene
1,2,4- Trichlorobenzene
Isophorone
Naphthalene
Bis(2-chloroethoxy)njethane
Hexachlorocydopentadiene *
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2, 6-Dinitrotoluene
Fluorene
4-Chlonphenyl phenyl etner
2,4-Oinitmtoluene
Diethylphthalate
N-Nitrosodiphenylamine *
Hexachlorobenzene
a-BHC'
4-Bromophenyl phenyl ether
y-BHC'
Phenenthrene
Anthracene
13-BHC
Heptachlor
6-BHC
Aldrin
naieniion
Time
(min.J
7.4
7.8
8.4
8.4
8.4
9.3
11. 1
11.4
11.6
11.9
12.1
12.2
13.9
15.9
17.4
17.8
18.3
18.7
19.5
19.5
19.8
20.1
20. 5
21.0
21.1
21.2
22.4
22.8
22.8
23.4
23.4
23.7
24.0
ivieinua
Detection
Limit (ng/U
1.9
4.4
1.6
5.7
1.9
5.7
1.9
0.9
1.9
2.2
1.6
5.3
1.9
3.5
1.9
1.6
1.9
1.9
4.2
5.7
22
1.9
1.9
1.9
5.4
1.9
4.2
1.9
3.1
1.9
Electron Impact
Primary
146
146
117
93
146
45
130
77
225
18O
82
128
93
237
162
152
154
163
165
166
204
165
149
169
284
183
248
183
178
178
181
100
183
66
Secondary
148
148
201
63
148
77
42
123
223
182
95
129
95
235
164
151
153
194
89
165
206
63
177
168
142
181
25O
181
179
179
183
272
109
263
113
113
199
95
113
79
101
65
227
145
138
127
123
272
127
153
152
164
121
167
141
182
150
167
249
109
141
109
176
176
109
274
181
220
Chemical lonization
(Methane)
146
146
199
63
146
77
124
223
181
139
129
65
235
163
152
154
151
183
166
183
177
169
284
249
178
178
148
148
201
107
148
135
152
225
183
167
157
107
237
191
153
155
163
211
167
211
223
170
286
251
179
179
150
150
203
109
ISC
137
164
227
209
178
169
137
239
203
181
183
164
223
195
223
251
198
288
277
207
207
625-10
January 1983
-------
Table 4. (Continued)
Parameter
Dibutyl phthalate
Heptachlor epoxkle
Endosulfan 1*
Fluoranthene
Dieldrin
4.4' -DDE
Pyrene
Endrin *
Endosulfan II*
4,4' -ODD
Benzidine *
4, 4' -DDT
Endosulfan sulfate
Endrin aldehyde
Butyl benzyl phthalate
Bis(2-ethylhexyl) phthalate
Chrysene
Benzo(a)anthracene
3, 3 '-Dichlorobenzidine
Di-n-octylphthalate
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Indenol 1, 2, 3-c,d)pyrene
Dibenzo(a,h)anthracene
Benzo(ghi)perylene
N-Nitrosodimethyl amine *
Chlordane * *
Toxaphene * *
PCB 1016"
PCB 1221'
PCB 1232*
PCB 1242*
PCB 1248*
PCB 1254*
PCB 1260*
neittrmuri
Time
(min.)
24.7
25.6
26.4
26.5
27.2
27.2
27.3
27.9
28.6
28.6
28.8
29.3
29.8
—
29.9
30.6
31.5
31.5
32.2
32.5
34.9
34.9
36.4
42.7
43.2
45.1
_
19 to 30
25 to 34
18 to 30
15 to 30
15 to 32
15 to 32
12 to 34
22 to 34
23 to 32
ivieuiuu
Detection
Limit i\ng/U
2.5
2.2
—
2.2
2.5
5.6
1.9
—
_
2.8
44
4.7
5.6
—
2.5
2.5
2.5
7.8
16.5
2.5
4.8
2.5
2.5
3.7
2.5
4.1
—
—
—
—
30
—
—
—
36
—
Electron Impact
Primary
149
353
237
202
79
246
202
81
237
235
184
235
272
67
149
149
228
228
252
149
252
252
252
276
278
276
42
373
159
224
190
190
224
294
294
330
Secondary
150
355
339
101
263
248
101
263
339
237
92
237
387
345
91
167
226
229
254
253
253
253
138
139
138
74
375
231
260
224
224
260
330
330
362
104
351
341
100
279
176
100
82
341
165
185
165
422
250
206
279
229
226
126
125
125
125
277
279
277
44
377
233
294
260
260
294
362
362
394
Chemical lonization
(Methane)
149
203
203
185
149
149
228
228
252
252
252
276
278
276
205
231
231
213
299
229
229
253
253
253
277
279
277
279
243
243
225
327
257
257
281
281
281
305
307
305
*See Section 1.2.
* * These compounds are mixtures of various isomers. (See Figures 2 to 12)
Gas Chromatographic conditions: Glass column 1.8m long x 2 mm ID packed with Supelcoport (100/120 mesh) coated with
3% SP-2250. Carrier gas: helium at a flow rate of 30 mL per min.
Temperature: Isothermal at 50 °C for 4 min., then 8 ° per min to 270°C. Hold at 2 70 °C for 30 min.
Table 5. Chromatographic Conditions, Method Detection Limits and Characteristic Ions for Acid Extractables
Parameter
2-Chlorophenol
2-Nitrophenol
Phenol
2, 4-Dimethylphenol
2, 4-Dichlorophenol
2, 4, 6- Trichlorophenol
4-Chloro-3-methylphenol
2, 4-Dinitrophenol
2-Methyl-4, 6-dinitrophenol
Pentachlorophenol
4-Nitrophenol
nctv/itiun
Time
(min.)
5.9
6.5
8.0
9.4
9.8
11.8
13.2
15.9
16.2
17.5
20.3
IVItSillUU
Detection
Limit (pg/L)
3.3
3.6
1.5
2.7
2.7
2.7
3.0
42
24
3.6
2.4
Electron Impact
Primary
128
139
94
122
162
196
142
184
198
266
65
Chemical lonization
Secondary
64
65
65
107
164
198
107
63
182
264
139
130
109
66
121
98
200
144
154
77
268
109
129
140
95
123
163
197
143
185
199
267
140
(Methane)
131
168
123
151
165
199
171
213
227
265
168
157
122
135
163
167
201
183
225
239
269
122
Chromatographic conditions: 1.8m long x 2 mm ID glass column packed with Supelcoport (100/120 mesh) coated with 1%
SP-1240. Carrier gas: helium at a flow rate of 30 mL per min. Column temperature, isothermal at 70°Cfor2min, then 8° per
min, to 200°.
625-11
January 1983
-------
Table 6. Accuracy and Precision for Base/Neutral Extractables
Reagent Water
Parameter
Acenaphthene
A cenaph th ylene
Aldrin
Anthracene
Benzo (a)anthracene
Benzo (b) fluoranthene
Benzo(kl fluoranthene
Benzo tghilperylene
Benzofajpyrene
Benzidine
Butyl benzyl phthalate
p-BHC
6-BHC
Bis{2-chloroethoxy)methane
Bisl2-chloroethyl)ether
Bis<2- chloroisoprop yl)e ther
Bis 12-ethylhexyl) phthalate
4-Bromophenyl phenyl ether
2-Chloronaph thalene
4-Chlorophenyl phenyl ether
Chrysene
4, 4 '-ODD
4, 4 '-DDE
4, 4 '-DDT
Dibenzota, hlanthracene
Di-n-butyl phthalate
1 ,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
3, 3 '-Dichlorobenzidine
Diethylphthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2, 6-Dinitrotoluene
Di-n-octylphthalate
Endosulfan sulfate
Fluoranthene
Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachlorocyclopentadiene
Hexachloroethane
Indeno (1 ,2,3-cd) pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propylamine
N-Nitrosodiphenylamine
PCB-1221
PC B- 12 54
Phenanthrene
Pyrene
1 ,2,4- Tnchlorobenzene
Average
Percent
Recovery
77
78
72
84
83
96
96
80
90
87
47
69
56
84
56
71
129
80
73
45
83
80
69
63
82
70
59
55
61
184
42
25
83
79
97
79
89
77
69
82
79
46
27
46
65
75
6
72
68
84
77
80
84
86
64
Standard
Deviation
(%)
23
22
6
14
19
68
68
45
22
61
32
25
18
33
36
33
50
17
24
11
19
9
20
15
39
25
27
28
31
174
28
33
32
18
37
29
19
16
6
7
20
25
25
21
37
33
32
31
39
24
11
13
14
15
16
Wastewater
Average
Percent
Recovery
83
82
—
76
75
41
47
68
43
63
74
—
—
82
72
71
82
75
79
—
75
—
—
—
70
93
62
54
63
143
48
35
79
79
89
—
80
80
-
—
71
48
12
52
81
77
75
82
76
86
—
—
76
80
69
Standard
Deviation
(%>
29
23
—
22
28
21
27
40
21
55
43
—
—
74
37
39
63
20
27
—
28
—
—
—
40
51
28
24
35
145
28
36
34
25
62
—
26
20
-
—
22
28
12
26
43
42
35
54
45
31
—
—
22
23
26
Spiked between 5 to 2400 \tg/L.
625-12
January 1983
-------
Table 7. Accuracy and Precision for Acid Extractables
Reagent Water
Parameter
4- Chloro -3-methylphenol
2-Chlorophenol
2,4-Dichlorophenol
2,4-Dimethytphenot
2, 4-Dinitrophenol
2-Methyl-4, 6-dinitrophenol
4-Nitrophenol
2-Nitrophenol
Pen tachlorophenol
Phenol
2, 4, 6- Trichlorophenol
Average
Percent
Recovery
79
70
74
64
78
83
41
75
86
36
77
Standard
Deviation
{%)
18
23
24
25
21
18
20
25
20
14
20
Was re wafer
Average
Percent
Recovery
75
71
80
58
108
90
43
75
66
36
81
Standard
Deviation
<%)
21
25
21
26
56
35
16
27
36
21
20
Spiked from 10 to 1500
Table 8. Suggested Internal and Surrogate Standards
Base/Neutral Fraction
Acid Fraction
Aniline-d5 2-Fluorophenol
Anthracene-djo Pentafluorophenol
Benzo(a)anthracene-d,2 Phenol-ds
4,4 '-Dibromobiphen yl 2 -Perfluorome th yf phenol
4,4 '-Dibromooctafluorobiphenyl
Decafluorobiphenyl
2.2 '-Difluorobiphen yl
4-Fluoroaniline
1 -Fluoronaphthylene
2 -Fluoronaph th ylene
Naphthalene-d s
Nitrobenzene-d 5
2,3,4,5,6-Pentafluorobiphenyl
Phenanthrene-d, 0
Pyridine-d5
Table 9. DFTPP Key Ions and Ion Abundance Criteria
Mass
51
68
70
127
197
198
199
275
365
441
442
443
Ion Abundance Criteria
30-60% of mass 198
less than 2% of mass 69
less than 2% of mass 69
4O-60%ofmass 198
less than 1% of mass 198
base peak, 100% relative abundance
5-9% of mass 198
10-30% of mass 198
greater than 1% of mass 198
present but less than mass 443
greater than 40% of mass 198
1 7-23% of mass 442
625-13
January 1983
-------
Column: 3% SP-2250 on Supelcoport
« Program: 50°C. 4 min. 8° per min to 270°C
-2 Detector: Mass spectrometer
2.4-Dinitrotoluene J N-Nitroso Diphenylamine
10
15
20 25 30
Retention time, minutes
35
40
45
Figure 1. Gas chroamatogram of base/neutral fraction.
Column: 1% SP-1240DA on Supelcoport
Program: 70°C-2 min. 8%/min to 20O°C
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C-4 min. 8°/minute
to 270°C
Detector: Mass spectrometer
u
Q
V )
0246 8 10 12 14 16 18 20 22
Retention time, minutes
Figure 2. Gas chromatogram of acid fraction.
625-J 4
10 15 20
Retention time, minutes
25
30
Figure 3. Gas chromatogram of pesticide fraction.
January 1983
-------
Column: 3% SP-2250 on Supelcoport
Program: 5O°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.. 8° per min. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32 34 36
Retention time, minutes
Figura 4. Gas chromatogram of chlordane.
22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 5. Gas chromatogram of toxaphene.
625-15
January 1983
-------
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.. 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min., 8° per min. to 270°C.
Detector: Mass spectrometer
VJ VJ \J
m/i-35 to 450
m/z=260
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 6. Gas chromatogram of PCB-1016.
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 7. Gas chromatogram of PCB-1221.
625-16
January 1983
-------
Column: 3% SP-22SO on Supelcoport
Program: SO°C. 4 min.. 8° per min. to 270°C.
Detector: Mass spectrometer
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.. 8° per min. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 8. Gas chromatogram of PCB-1232.
18 20 22 24 26 28 30
Retention time, minutes
Figure 9. Gas chromatogram of PCB-1242.
32
625-17
January 1983
-------
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.. 8° per im. to 270°C.
Detector: Mass spectrometer
18 20 22 24 26 28 30 32
Retention time, minutes
Figure 10. Gas chromatogram of PCB-1248.
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min..8° per min to 270°C.
Detector: Mass spectrometer
m/z=35 to 450 A/\J «
m/z=362
m/z=330
m/z=294
\M
18 20 22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 11. Gas chromatogram of PCB-1254.
Column: 3% SP-2250 on Supelcoport
Program: 50°C. 4 min.. 8° per min. to 270°C.
Detector: Mass spectrometer
m/z=394
m/z=362
m/z=330
625-18
18 20 22 24 26 28 30 32 34 36 38
Retention time, minutes
Figure 12. Cs chromatogram of PCB-1260.
January 1983
-------
Tailing Factor =
BC
AB
Example calculation:
Peak Height = Of = 100mm
10% Peak Height = BD=10 mm
Peak Width at 10% Peak Height = AC = 23 mm
AB =11 mm
BC = 12 mm
Therefore: Tailing Factor = —— =/. /
Figure 13. Tailing factor calculation.
625-19
January 1983
-------
United States
Environmental Protection
Agency
Effluent Guidelines Division (WH 552)
Washington, D.C. 20460
Water and Waste Management
TEST METHOD
DETERMINATION OF BENFLUBALIN, ETHALFLURALIN, ISOPROPALIN
AND PROFLURALIN IN WASTEWATER
METHOD 627
1. Scope and Application
1.1 This method covers the determination of certain dinitroaniline
pesticides. The following parameters can be determined by this
method:
1.2
1.3
1.4
1.4
Parameter
Benfluralin
Etna! flora 11 n
Isopropalin
Profluralin
Trifluralin
STORET No.
39002
39030
CAS No.
1861-40-1
55283-68-6
33820-53-0
25399-36-0
1582-09-8
627-01
This method fails to distinguish between benfluralin, ethalfluralin
and trifluralin. When more than one of these materials may be
present 1n a sample, the results are reported as trifluralin.
This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in industrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
The method detection 1-imits (MDL, defined in Section 15) for four
of the parameters are listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
The sample extraction and concentration steps in this method are
essentially the same as several others in the 600-method series.
Thus, a single sample may be extracted to measure the parameters
included in the scope of each of these methods. When cleanup is
required, the concentration levels must be high enough to permit
selecting aliquots, as necessary, in order to apply appropriate
cleanup procedures. Under Gas Chromatography, the analyst is
allowed the latitude to select chromatographic conditions
appropriate for the simultaneous measurement of combinations of
these parameters (see Section 12).
January 1983
-------
1.6 This method is restricted to use by or under the supervision of
analysts experienced 1n the use of gas chromatography and 1n the
Interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described 1n Section 8.2.
1.7 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound Identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas chromato-
graphic column that can be used to confirm measurements made with
the primary column within the limitations described in Section
1.2. Section 14 provides gas chromatograph/mass spectrometer
(6C/MS) criteria appropriate for the qualitative confirmation of
compound Identifications.
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with 15% methylene chloride using a separatory funnel.
The methylene chloride extract is dried and exchanged to hexane
during concentration to a volume of 10 ml or less. Gas chromato-
graphic conditions are described which permit the separation and
measurement of the compounds in the extract by electron capture
(EC) gas chromatography.'
2.2 This method provides an optional Florisil column cleanup procedure
to aid in the elimination or reduction of interferences which may
be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all glass-
ware as soon as possible after use by thoroughly rinsing
with the last solvent used in 1t. 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
627-02 January 1983
-------
3.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Interferences by phthalate esters can pose a major problem in
pesticide analysis when the EC detector is used. These compounds
generally appear in the chromatogram as large late eluting peaks.
Common flexible plastics contain varying amounts of phthalates.
These phthalates are easily extracted or leached from such mater-
ials during laboratory operations. Cross contamination of clean
glassware occurs when plastics are handled during extraction steps,
especially when solvent wetted surfaces are handled. Interferences
from phthalates can be minimized by avoiding the use of plastics in
the laboratory. Exhaustive cleanup of reagents and glassware may
be required to eliminate background phthalate contamination.3>4
3.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. Unique samples may require special cleanup approaches to
achieve the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 5-7 for the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample 1s not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
627-03 January 1983
-------
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
sllicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.5 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.6 Vials - Amber glass, 10 to 15 mL capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system is recommended for measuring peak
areas.
5.6.1 Column 1 - 180 cm long x 2 mm ID glass, packed with 1.5%
OV-17/1.95* OV-210 on Gas Chrom Q (100/120 mesh) or
627-04 January 1983
-------
equivalent. This column was used to develop the method
performance statements in Section 15. Alternative columns
may be used in accordance with the provisions described in
Section 12.1.
5.6.2 Column 2 - 180 cm long x 2 mm ID glass, packed with
Ultrabond 20M (100/120 mesh) or equivalent.
5.6.3 Detector - Electron capture. This detector has proven
effective in the analysis of wastewaters for the parameters
listed in the scope and was used to develop the method
performance statements in Section 15. Alternative
detectors, including a mass spectrometer, may be used in
accordance with the provisions described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, methylene chloride - Pesticide quality or
equivalent.
6.3 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.4 Stock standard solutions (1.00 ug/yL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.4.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in pesticide quality hexane and dilute to volume in
a 10-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified
at 96% or greater, the weight may be used without correction
to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or
by an independent source.
6.4.2 Transfer the stock standard solutions into TFE-fluorocarbon-
sealed screw cap vials. Store at 4°C and protect from
light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to
preparing calibration standards from them.
627-05
January 1983
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6.4.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. These parameters do not adequately
resolve benfluralin, ethalfluralin and trifluralin. When more than
one of these compounds may be present in a sample, the instrument
must be calibrated with trifluralin. The gas chromatographic
system may be calibrated using either the external standard
technique (Section 7.2) or the internal standard technique (Section
7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration stan-
dards at a minimum of three concentration levels by adding
accurately measured volumes of one or more stock standards
to a volumetric flask and diluting to volume with hexane.
One of the external standards should be representative of a
concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should
define the working range of the detector.
7.2.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve or calibration factor
must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
. analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
627-06 January 1983
-------
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels for each parameter of interest by
adding volumes of one or more stock standards to a
volumetric flask. To each calibration standard, add a known
constant amount of one or more internal standards, and
dilute to volume with hexane. One of the standards should
be representative of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 1 to 5 yL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF ° (AsC1s)/(A1s Cs)
where:
As = Response for the parameter to be measured.
A-js * Response for the internal standard.
CTS = Concentration of the Internal standard in yg/L.
Cs a Concentration of the parameter to be measured in
ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/AiS against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10*, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
8. Quality Control
8.1. Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
January 1983
-------
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each com-
pound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 mL of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Table 2 provides single operator recovery and precision for
isopropalin, profluralin and trifluralin. Similar results
should be expected for benfluralin and ethalfluralin.
Compare these results to the values calculated in Section
8.2.3.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
627-08 January 1983
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8.3.1 Calculate upper and lower control limits for method perfor-
mance as follows:
Upper Control Limit (UCL) » R + 3 s
Lower Control Limit (LCL) - R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts^
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.8
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5S».
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all glass-
ware and reagents interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissimi-
lar Qolumn, specific element detector, or mass spectrometer must be
used. Whenever possible, the laboratory should perform analysis of
quality control materials and participate in relevant performance
evaluation studies.
627-09 January 1983
-------
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and' other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the
separatory funnel and extract the sample by shaking the funnel for
2 min with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layers is more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The opti-
mum technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool, centrifugation, or
other physical methods. Collect the methylene chloride extract in
a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-D if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
627~10 January 1983
-------
about 1 ml methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially Immersed In the hot water, and the entire lower rounded
surface of the flask 1s bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
10.7 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 ml of hexane and a new
boiling chip and reattach the Snyder column. Pour about 1 ml of
hexane into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be
5 to 10 min. When the apparent volume of liquid reaches 1 ml,
remove the K-D apparatus and allow it to drain and cool for at
least 10 min.
10.8 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexane and adjust the
volume to 10 ml. A 5-mL syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will
be stored longer than two days, they should be transferred to
TFE-fluorocarbon-sealed screw-cap vials. If the sample extract
requires no further cleanup, proceed with gas chromatographic
analysis. If the sample requires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. If particular circumstances demand the use of a
cleanup procedure, the analyst must determine the elution profile
and demonstrate that the recovery of each compound of interest for
the cleanup procedure is no less than 85%.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included 1n this table are estimated retention
, times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 is
shown in Figure 1. Other packed columns, chromatographic
conditions, or detectors may be used if the requirements of Section
8.2 are met. Capillary (open-tubular) columns may also be used if
627-n January 1983
-------
the relative standard deviations of responses for replicate
Injections are demonstrated to be less than 6% and the requirements
of Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7. Since the
gas chromatographic conditions provided do- not adequately separate
benfluralin, ethalfluralin and trifluralin, calibrate with
trifluralin if more than one of these materials may be present in a
sample.
12.3 If the Internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 pL of the sample extract using the solvent-flush
technique.10 Record the volume injected to the nearest 0.05 pL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(V )
Concentration, pg/L = r\j
where:
A * Amount of material injected, in nanograms.
V-j * Volume of extract injected in uL.
V^ * Volume of total extract in uL.
Vs * Volume of water extracted in ml.
627 12 January 1983
-------
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration 1n the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
,(
Concentration, ug/L * -r^—)(Rp
where:
As * Response for the parameter to be measured.
ATS * Response for the Internal standard.
Is s Amount of internal standard added to each
extract in ug.
V0 s Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results. Results for
benfluralin and ethalfluralin must be reported as trifluralin
unless the sample has been characterized beyond the capabilities
provided in this method.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chromato-
graphic tailing factors of less than 5.0 must be achievedJl
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.12
627-13 January 1983
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14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It 1s recommended that at least 25 nanograms of
material be Injected into the GC/MS. The .criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion 1n the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fall to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MDL) is defined as the minimum concen-
tration of a substance that can be measured and reported with 99%
confidence that the value is above zeroJ^ The MDL concentra-
tions listed in Table 1 were obtained using reagent water.1
15.2 In a single laboratory (West Cost Technical Services, Inc.), using
reagent water and effluents from pesticide manufacturers and the
average recoveries presented in Table 2 were obtained1. The
standard deviations of the percent recoveries of these measurements
are also included in Table 2.
References
1. "Pesticide Methods Evaluation," Letter Report #5 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
627~14 January 1983
-------
2. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. 61am, O.S., Chan, H.S. and Nef, 6.S., "Sensitive method for
Determination of Phthalate Ester Plasticizers 1n Open-Ocean Biota
Samples," Analytical Chemistry, 47, 2225, (1975).
4, 61am, C.S., Chan, H.S., "Control of Blanks in the Analysis of Phthalates
1n A1r and Ocean Biota Samples," National Bureau of Standards (U.S.),
Special Publication 442, pp. 701-708, 1976.
5. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
6. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
8. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
10. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists. 48, 1037 (1965).
11. McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry, 47, 995 (1975).
13. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology. J5_, 1426 (1981).
14. "Determination of Dinitroaniline in Industrial and Municipal Wastewater,"
Method 627, EPA No. 600/4-82-009, NTIS No. PB82-156035, January 1982,
National Technical Information Center, 5285 Port Royal Road, Springfield,
VA 22165.
627-15 January 1983
-------
Parameter
TABLE 1
GAS CHROMATOGRAPHY AND METHOD
DETECTION LIMITS OF DINITROANILINES
Retention Time
(min)
Column 1 Column 2
Method
Detection Limit
(yg/L)
Trifluralin
Benfluralin
Ethalfluralin
Profluralin
Isopropalin
1.6
1.6
1.6
2.3
6.4
2.2
2.3
2.3
3.4
6.3
0.03
ND
ND
0.14
0.02
ND = Not determined.
Column 1 conditions: Gas Chrom Q (100/200 mesh) coated with 1.5%
OV-17/1.95% OV-210 packed in a 1.8 m long x 2 mm ID glass column with 95%
argon/5% methane carrier gas at a flow rate of 30 mL/min. Column
temperature: isothermal at 190°C.
Column 2 conditions: Ultrabond 20M (100/120 mesh) packed in a 1.8 m long x
2 mm ID glass column with nitrogen carrier gas at a flow rate of 30 mL/min.
Column temperature: held at 160°C for 2 min, then programmed to 200°C at
10°C/min.
627-16
January 1983
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TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Benfluralin
Isopropalin
Profluralin
Trifluralin
Sample
Type
IW
DW
IW
DW
IW
DW
IW
Spike
Range
(pq/L)
2.0
0.5
2.2
0.5
2.04
0.5
2.08
Number
of
Replicates
2
7
7
7
7
7
7
Average
Percent
Recovery
93
93
88
99
73
97
77
Standard
Deviation
%
—
1.1
13.2
9.0
5.8
1.8
20.0
IW = Industrial wastewater, pesticide manufacturing
DW = Reagent water
627-17
January 1983
-------
Q.
O
Q.
O
v)
J I I I i_ I I I
0 123 45 678
Minutes
Figure 1. Gas chromatogram of dinitroaniline pesticides on Column 1
For conditions, see Table 1.
627-18
January 1983
-------
oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CYANAZINE
IN WASTEWATER
METHOD 629
1. Scope and Application
1.1 This method covers the determination of cyanazine. The following
parameter can be determined by this method:
Parameter STORET No. CAS No.
Cyanazine — 21725-46-2
1.2 This is a high performance liquid chromatographic (HLPC) method
applicable to the determination of the compound listed above in
industrial and municipal discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly
permitted, shall be considered a major modification subject to
application and approval of alternate test procedures under 40 CFR
136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15)
for cyanazine is 6 ug/L. The MOL for a specific wastewater may
differ from those listed, depending upon the nature of inter-
ferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in the
interpretation of liquid chromatograms. Each analyst must demon-
strate the ability to generate acceptable results with this method
using the procedure described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for
cyanazine, compound identifications should be supported by at least
one additional qualitative technique.
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and exchanged to methanol
during concentration to a volume of 10 ml or less. HPLC conditions
are described which permit the separation and measurement of
cyanazine in the extract by HPLC with a UV detector.1
629-01 January 1983
-------
2.2 This method provides an optional Florisil column cleanup procedure
to aid in the elimination or reduction of interferences which may
be encountered.
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all glass-
ware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to over-
come many of these interferences, but unique samples may require
additional cleanup approaches to achieve the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 3-5 for the information of the analyst.
629-02 January 1983
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5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
Uner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Oanish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Vials - Amber glass, 10 to 15 mL capacity with
TFE-fluorocarbon lined screw cap.
629~03 January 1983
-------
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover,, capable of temper-
ature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Filtration apparatus - As needed to filter chromatographic solvents
prior to HPLC.
5.7 Liquid chromatograph - High performance analytical system complete
with high pressure syringes or sample injection loop, analytical
columns, detector and strip chart recorder. A guard column is
recommended for all applications.
5.7.1 Gradient pumping system, constant flow.
5.7.2 Column - 25 cm long x 2.6 mm ID stainless steel packed with
Spherisorb ODS (10 urn) or equivalent. This column was used
to develop the method performance statements in Section 14.
Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.7.3 Detector - Ultraviolet, 254 nm. This detector has proven
effective in the analysis of wastewaters for cyanazine and
was used to develop the method performance statements in
Section 14. Alternative detectors may be used in accordance
with the provisions described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, hexane, methylene chloride - Pesticide quality or equiva-
lent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips.
(Available from Scientific Products Co., Cat. No. P1126-8, and
other suppliers.) Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 ml ethyl
alcohol preservative must be added to each liter of ether.
6.4 Methanol - HPLC/UV quality.
629-04 January 1983
-------
6.5 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.6 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.7 Stock standard solution (1.00 ug/uL) - A stock standard solution
may be prepared from pure standard material or purchased as a
certified solution.
6.7.1 Prepare a stock standard solution by accurately weighing
approximately 0.0100 g of cyanazine. Dissolve the material
in UV quality methanol and dilute to volume in a 10-mL
volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified
at 96% or greater, the weight may be used without correction
to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or
by an independent source.
6.7.2 Transfer the stock standard solution into a TFE-fluoro-
carbon-sealed screw cap vial. Store at 4°C and protect
from light. Frequently check the stock standard solution
for signs of degradation or evaporation, especially just
prior to preparing calibration standards from it.
6.7.3 The stock standard solution must be replaced after six
months or sooner if comparison with a check standard
indicates a problem.
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1. The HPLC system may be calibrated using either the
external standard technique (Section 7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 Prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes
of stock standard to volumetric flasks and diluting to
volume with methanol. One of the external standards should
be representative of a concentration near, but above, the
method detection limit. The other concentrations should
629-05 January 1983
-------
correspond to the range of concentrations expected in the
sample concentrates or should define the working range of
the detector.
7.2.2 Using injections of 10 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for cyanazine. Alternatively,' the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated at each standard concen-
tration. If the relative standard deviation of the
calibration factor is less than 105J over the working range,
the average calibration factor can be used in place of a
calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response varies from the
predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve or calibration factor must be
prepared.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select an internal standard similar in analytical
behavior to cyanazine. The analyst must further demonstrate that
the measurement of the internal standard is not affected by method
or matrix interferences. Due to these limitations, no internal
standard applicable to all samples can be suggested.
7.3.1 Prepare calibration standards at a minimum of three concen-
tration levels by adding volumes of stock standard to
volumetric flasks. To each calibration standard, add a
known constant amount of internal standard, and dilute to
volume with methanol. One of the standards should be
representa- tive of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 10 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for both cyanazine and internal standard.
Calculate response factors (RF) as follows:
RF = (AsCis)/(AiS Cs)
where:
As = Response for cyanazine.
~ Response for the internal standard.
= Concentration of the internal standard in ug/L.
Cs = Concentration of cyanazine in ug/L.
629-06 January 1983
-------
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcu-
lations. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/AiS against RF.
7.3.3 The working calibration curve or RF.must be verified on each
working shift by the measurement of one or more calibration
standards. If the response varies from the predicted
response by more than ±10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new cali-
bration curve must be prepared.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromato-
graphy. Florisil from different batches or sources may vary in
adsorptive capacity. To standardize the amount of Florisil which
is used, the use of lauric acid value is suggested. This
procedure^ determines the adsorption from hexane solution of
lauric acid, in mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
629-°7 January 1983
-------
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration. Using stock
standard, prepare a quality control check sample concentrate
in methanol 1000 times more concentrated than the selected
concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or mere additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration
being measured.
8.3.1 Calculate upper and lower control limits for method perfor-
mance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) * R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts7
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.7
629-08 January 1983
-------
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 102 of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery of cyanazine does not fall within the control limits for
method performance, the results reported for cyanazine in all
samples processed as part of the same set must be qualified as
described in Section 13.3. The laboratory should monitor the
frequency of data so qualified to ensure that 1t remains at or
below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all glass-
ware and reagents interferences are under control. Each time a set
of samples is extracted or there is a change in reagents, a labora-
tory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram
as cyanazine, confirmatory techniques such as chromatography with a
dissimilar column, or ratio of absorbance at two or more wave-
lengths must be used. Whenever possible, the laboratory should
perform analysis of quality control materials and participate in
relevant performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9,1 Grab samples must be collected in glass containers. Conventional
sampling practices^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
629~09 January 1983
-------
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the
separatory funnel and extract the sample by shaking the funnel for
2 min with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If the emulsion interface between layers is more than one
third the volume of the solvent layer, the-analyst must employ
mechanical techniques to complete the phase separation. The
optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, centrifu-
gation, or other physical methods. Collect the methylene chloride
extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 It is necessary to exchange the extract solvent to hexane if the
Florisil cleanup procedure is to be used. For direct HPLC
analysis, the extract solvent must be changed to methanol. The
analyst should only exchange a portion of the extract to methanol
if there is a possibility that cleanup may be necessary.
10.5 Assemble a Kuderna-Danish (K-0) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-0 if the
requirements of Section 8.2 are met.
10.6 Pour a measured fraction or all of the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate,
and collect the extract in the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20 to 30 ml of methylene chloride
to complete the quantitative transfer.
10.7 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 ml methylene chloride to the top. Place the K-0 apparatus
on a hot water bath, 60 to 65°C, so-that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 mL, remove the K-D apparatus and allow
it to drain and cool for at least 10 min.
10.8 Increase the temperature of the hot water bath to about 80°C.
Momentarily remove the Snyder column, add 50 ml of hexane or
methanol and a new boiling chip and reattach the Snyder column.
629-10 January 1983
-------
Pour about 1 ml of solvent into the top of the Snyder column and
concentrate the solvent extract as before. Elapsed time of
concentration should be 5 to 10 min. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to
drain and cool for at least 10 min.
10.9 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexane or methane1
and adjust the volume to 10 ml. A 5-mL syringe is recommended for
this operation. Stopper the concentrator tube and store
refrigerated if further processing will not be performed
immediately. If the extracts will be stored longer than two days,
they should be transferred to TFE-fluorocarbon-sealed screw-cap
vials. If the sample extract requires no further cleanup, proceed
with HPLC analysis. If the sample requires cleanup, proceed to
Section 11.
10.10 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of cyanazine for
the cleanup procedure is no less than 85%.
11.2 The following Florisil column cleanup procedure has been demon-
strated to be applicable to cyanazine.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined
by calibration (Section 7.4 and 7.5), to a chromato-
graphic column. Settle the Florisll by tapping the
column. Add anhydrous sodium sulfate to the top of the
Florisil to form a layer 1 to 2 cm deep. Add 60 ml of
hexane to wet and rinse the sodium sulfate and Florisil.
Just prior to exposure of the sodium sulfate to air, stop
the elution of the hexane by closing the stopcock on the
chromatography column. Discard the eluate.
11.2.£ Adjust the sample extract volume to 10 ml with hexane and
transfer it from the K-0 concentrator tube to the
Florisil column. Rinse the tube twice with 1 to 2 ml
hexane, adding each rinse to the column.
629-11 January 1983
-------
11.2.3 Drain the column until the sodium sulfate layer is nearly
exposed. Elute the column with 200 ml of 6% ethyl ether
in hexane (V/V) (Fraction 1) and with 200 ml of 15% ethyl
ether in hexane (V/V) (Fraction 2) using a drip rate of
about 5 mL/min. These fractions may be discarded. Place
a 500-mL K-0 flask and clean concentrator tube under the
chromatograpy column. Elute the column with 200 ml of
50* ethyl ether in hexane (V/V) (Fraction 3), into the
K-D flask. Cyanazine elutes quantitatively in Fraction 3.
11.2.4 Concentrate the eluate by standard K-D techniques
(Section 10.7), exchanging the solvent to methanol.
Adjust final volume to 10 ml with methanol. Analyze by
HPLC.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph. Included in this table are the estimated
retention time and method detection limit that can be achieved by
this method. An example of the separations achieved by this
column is shown in Figure 1. Other HPLC columns, chromatographic
conditions, or detectors may be used if the requirements of
Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 10 ul of the sample extract. Record the volume injected to
the nearest 0.05 uL, and the resulting peak size in area or peak
height units.
12.5 The width of the retention time window used to make identifi-
cations should be based upon measurements of actual retention time
variations of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
629-12 January 1983
-------
13. Calculations
13.1 Determine the concentration of cyanazine in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(VJ
Concentration, ug/L a —rr
where:
A » Amount of cyanazine injected, in nanograms.
V.j * Volume of extract injected in uL.
V^ * Volume of total extract in uL.
Vs * Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(AJUJ
Concentration, ug/L - (Al8)
-------
14.2 In a single laboratory (West Cost Technical Services, Inc.), using
effluents from pesticide manufacturers and publicly owned treatment
works (POTW), the average recoveries presented in Table 2 were
obtained.' The standard deviations of the percent recoveries of
these measurements are ail so included in Table 2.
References
1.. "Pesticide Methods Evaluation," Letter Report for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
7. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, ]£, 1426 (1981).
10. "Determination of Cyanazine in Industrial and Municipal Wastewater,"
Method 629, EPA No. 600/4-82-010, NTIS No. PB82-156043, January 1982,
National Technical Information Center, 5285 Port Royal Road, Springfield,
VA 22165.
629-14 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND ESTIMATED DETECTION LIMIT
Retention Time Estimated MDL
Parameter (min) (ug/L)
Cyanazine 10.0 6
Column conditions: Spherisorb ODS (10 urn) packed in a 25 cm long x 2.6 mm
ID stainless steel column with a mobile phase flow rate of 1.0 rnL/min.
Mobile phase: linear gradient from 50% solvent B to TOO* solvent B in 2
min, where solvent A is 25% methanol in water and solvent B is 50% methanol
in water.
629-15 January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Cyanazine
Sample
Type
DW
MW
PW
IW
Spike
ug/L
121
60.8
10100
10100
No. of
Replicates
7
7
3
2
Average
Percent
Recovery
100.0
85.5
94.3
78.0
Standard
Deviation
X
8.9
3.9
__
--
DW » Reagent water.
MW » Municipal waste'water.
PW » Process water, pesticide manufacturing.
IW » Industrial wastewater, pesticide manufacturing.
629-16
January 1983
-------
0)
c
•f~
IM
10
10
15
Minutes
Figure 1. Liquid chromatogram of cyanazine in process water extract on
Column 1. For conditions, see Table 1.
629-17
January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF AOP, BUSAN 40, BUSAN 85, CARBAM-S,
FERBAM, KN METHYL, MANCOZEB, MANEB, METHAM, NABAM
NIACIDE, ZAC, ZINEB, AND ZIRAM
IN WASTEWATER
METHOD 630
1. Scope and Application
1.1 This method covers the determination of dithiocarbamate pesticides.
The following parameters can be determined by this method:
Parameter STORE! No. CAS. No.
Amobam — 3566-10-7
AOP
Busan 40
Busan 85
Ferbam — 14484-64-1
KN Methyl
Mancozeb -- 8065-67-6
Manei> - 12327-38-2
Metham — 137-42-8
Nabam — 142-59-6
Niacide — 8011-66-3
Polyram — 9006-42-2
Sodium dimethyldithiocarbamate — 128-04-1
Thiram - 137-26-8
ZAC
Zineb - 12122-67-7
Ziram — 137-30-4
1.2 This method fails to distinguish between the individual dithiocar-
bamates. The compounds above are reduced to carbon disulfide and
the total dithiocarbamate concentration is measured. Unless the
sample can be otherwise characterized, all results are reported as
Ziram. Carbon disulfide is a known interferent.
1.3 This is a colorimetric method applicable to the determination of
the compounds listed above in industrial and municipal discharges
as provided under 40 CFR 136.1. Any modification of this method
beyond those expressly permitted, shall be considered a major
modification subject to application and approval of alternate test
procedures under 40 CFR 136.4 and 136.5.
630-01 January 1983
-------
1.4 The method detection limit (MDL, defined in Section 12) for maneb,
metham and ziram are listed in Table 1. The MDL for a specific
dithiocarbamate or wastewater may differ from those listed,
depending upon the nature of interferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in trace organic analyses. Each analyst must
demonstrate the ability to generate acceptable results with this
method using the procedure described in Section 8.2.
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, is digested
with acid to yield carbon disulfide by hydrolysis of the dithiocar-
bamate moiety. The evolved CS? is purged from the sample and
absorbed by a color reagent. The absorbance of the solution is
measured at 380 and 435 ran using a UV-visible spectrophotometer.'
3. Interferences
3.1 Method interferences may be caused by contaminants in reagents,
glassware and other sample processing hardware that lead to high
blank values and biased results. All of these materials must be
routinely demonstrated to be free from interferences under the
conditions of the analysis by running laboratory reagent blanks as
described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 After each use,
rinse the decomposition flask and condenser with 4N NaOH and
reagent water. Overnight soaking in 4N NaOH may be neces-
sary. Clean the HgS scrubber between each use with 0.1N
HC1 in methanol, rinse three times with methanol and bake at
200°C for 15 min. Rinse the C$2 trap with methanol
three times between each use and follow by heating for 15
min at 200°C. Should it become difficult to force the
color reagent through the glass frit of the CSg trap,
clean in the same manner as the HjS scrubber. After
cooling, store glassware sealed to prevent any accumulation
of dust or other contaminants.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems.
3.2 Carbon disulfide may be a significant direct interferent in
wastewaters. Its elimination or control i«; not addressed in this
method. If correction for background carbon disulfide is required,
the C$2 should be measured by an independent procedure, such as
direct aqueous injection gas chromatography.
3.3 Additional matrix interferences may be caused by contaminants that
are codistilled from the sample. The extent of matrix interfer-
630-02 January 1983
-------
ences will vary considerably from source to source, depending upon
the nature and diversity of the industrial complex or municipality
being sampled. The cleanup provided by the HgS trap will
eliminate or reduce some of these interferences, but unique samples
may require additional clean-up approaches to achieve the MDL
listed in Table 1.
4.1 The toxicity or carcinogeniclty of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
1n this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
available and have been identified^ for the information of the
analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect f .'ow
proportional composites.
5.2 Dithiocarbamate hydrolysis apparatus (Figure 1) - (Available from
Southern Scientific Inc., Box 83, Micanopy, Florida 32267).
Apparatus includes the following or equivalent components:
5.2.1 Hot plate with magnetic stirrer.
630-03 January 1983
-------
5.2.2 Hydrolysis flask - 2 L, flat bottom with ground glass
joints, 2 necks.
5.2.3 Condenser - low internal volume, ground glass joints,
Liebig (Kontes K-447000, 100 mm or equivalent).
5.2.4 Gas washing bottles - 125 ml, with extra-coarse porosity
(Kontes K-657750 or equivalent).
5.2.5 Addition funnel - 60 ml, ground glass joint to fit
hydrolysis flask, with long stem to reach at least 2 cm
below the liquid level in the hydrolysis flask.
5.2.6 Dust trap (Adapter) - to fit top of addition funnel (Kontes
K-174000 or equivalent).
5.2.7 Vacuum source - stable pressure with needle valve for
control.
5.3 UV-Visible spectrophotometer - Double beam with extended cell path
length capability of 1.0 and 4.0 cm cells.
5.4 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 gram. The preparation of calibration standards for some
dithiocarbamates (e.g. metham) requires the use of a balance
capable of weighing 10 ug.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest. Prepare by boiling distilled water 15 min
immediately before use.
6.2 Acetonitrile, diethanolamine, methanol - ACS grade.
6.3 Ethanol - 95%.
6.4 Cupric acetate - Monohydrate, ACS grade.
6.5 Hydrochloric acid - Concentrated.
6.6 Hydrochloric acid, 0.1 N in methanol - Slowly add 8.3 mL cone. HC1
to methanol and dilute to 100 ml.
6.7 Sodium hydroxide, 4 N - Dissolve 16 g ACS grade NaOH pellets in
reagent water and dilute to 100 ml.
6.8 Stannous chloride - SnCl2 • 2H20, ACS grade.
6.9 Zinc acetate solution, 20* - Dissolve 20 g ACS grade
• 2H20 in reagent water and dilute to 100 ml.
630-04 January 1983
-------
6.10 Color reagent - Add 0.012 g cupric acetate monohydrate to 25 g
diethanolamine. Mix thoroughly while diluting to 250 ml with
ethanol. Store in amber bottle with TFE-fluorocarbon-lined cap.
6.11 Decomposition reagent - Dissolve 9.5 g stannous chloride in 300 ml
cone, hydrochloric acid. Prepare fresh daily.
6.12 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.12.1 Prepare a stock standard solution for ziram by accurately
weighing approximately 0.0100 g of pure material. Dissolve
the material in acetonitrile and dilute to volume in a 10-mL
volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified
at 96% or greater, the weight may be used without correction
to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or
by an independent source.
6.12.2 Transfer the stock standard solution into a
TFE-fluorocarbon-sealed screw cap vial. Store at 4°C and
protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation,
especially just prior to preparing calibration standards
from them.
6.12.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
6.12.4 When using other dithiocarbamates for calibration, such as
maneb or metham, it may be necessary to weigh microgram
amounts of the pure material into small aluminum foil boats
and place them directly in the hydrolysis flask.
7. Calibration
7.1 Use ziram as the standard for total dithiocarbamates when a mixture
of dithiocarbamates 1s likely to be present. Use the specific
dithiocarbamate as a standard when only one pesticide is present
and its identity has been established.
7.2 With the apparatus assembled and reagents in place. Section 10,
pour 1500 ml of reagent water into each decomposition flask, add 30
ml of decomposition reagent and start aspiration.
7.3 Spike the water in each flask with an accurately known weight of
dithiocarbamate standard. Use a series of weights equivalent to
5-200 ug of CS2« Follow the procedure outlined Section 10.
630-05 January 1983
-------
7.4 Prepare calibration curves at a minimum of three concentrations by
plotting absorbance vs. weight of dithiocarbamate. A separate
curve is prepared from readings taken at 435 nm and at 380 nm for
each cell path length used. Normally the 435 nm curve is used for
calibration above 30 ug ziram (4 cm cell), and the 380 nm curve is
used for calibration below 30 ug ziram. The choice of which curve
to use is left to the discretion of the analyst. It is recommended
that the curves be transformed into mathematical equations using
linear least squares fit for the data from 435 nm and quadratic
least squares fit for data from the 380 nm.
7.5 The working calibration curve must be verified on each working
shift by the measurement of one or more calibration standards. If
the response varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve must be prepared.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 The laboratory must spike and analyze a minimum of 105» of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured.
8.2.2 Add the known amount of dithiocarbamate standard to each of
a minimum of four 1000-mL aliquots of reagent water. A
representative wastewater may be used in place of the
reagent water, but one or more additional aliquots must be
analyzed to determine background levels, and the spike level
must exceed twice the background level for the test to be
valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
630-06 January 1983
-------
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 1, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) » R + 3 s
Lower Control Limit (LCL) • R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts^
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alternatively,
the analyst may use four wastewater data points gathered
through the requirement for continuing quality control 1n
Section 8.4. The accuracy statements should be updated
regularly.*5
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 11.3. The laboratory
should monitor the frequency of data so qualified to ensure that 1t
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
630-07 January 1983
-------
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique.
Whenever possible, the laboratory should perform analysis of
quality control materials and participate in relevant performance
evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices7 should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be analyzed within seven days of collection.
10. Sample Analysis
10.1 Assemble the hydrolysis apparatus as follows (See Figure 1):
10.1.1 Place the hydrolysis flask on the hot plate.
10.1.2 Place the addition funnel in one of the necks of the
hydrolysis flask and the dust trap in the top of the funnel.
10.1.3 Place the condenser in the other neck and attach two gas
washing bottles in succession to the condenser outlet.
10.1.4 Attach a vacuum line with a flow valve to the second
scrubber .
10.2 Allow the sample to warm to room temperature. Mark the water
meniscus on the side of the sample bottle for later determination
of sample volume. Pour the entire sample into the 2-liter
hydrolysis flask. Rinse the bottle four times with 100-mL aliquots
of reagent water, addinn the washes to the hydrolysis flask. Bring
the volume in the hydrolysis flask to approximately 1,500 ml with
reagent water.
10.3 Place 15.0 ml of color reagent into the C$2 trap (second gas
washing bottle). Place 9 ml of zinc acetate solution into the
H2$ scrubber (first gas washing bottle). Add 2 mL of ethanol to
the H?S scrubber. Place a magnetic stirring bar in the
hydrolysis flask and place the flask on the hot plate/magnetic
630-08 January 1983
-------
stirrer (ambient at this time). Assemble the apparatus providing
adequate support for all glassware. The addition funnel stem
opening must be below the water level. Ground glass joints may be
slightly coated with silicone grease.
10.4 Start the stirrer, begin water flow through the condenser, and turn
on hot plate and begin heating the flask. Open the needle valve
slightly and start the aspirator. By closing the needle valve,
adjust the airflow through the absorption train until the proper
flow is attained. (The column of bubbles extends to the bottom of
the spherical expansion chamber at the top of the C$2 trap.) Add
30 ml of decomposition reagent to the flask. (Note: The analyst
must ensure that the sample pH is less than 2 during hydrolysis.)
10.5 Bring the liquid in the flask to a gentle boil. Continue the
boiling for 60 minutes, then remove the heat. Continue aspiration
until boiling ceases.
10.6 Transfer the contents of the C$2 trap into a 25.0 mL volumetric
flask by forcing the liquid through the glass frit and out of the
inlet arm with pressure from a large pipet bulb. Ensure quantita-
tive transfer by rinsing the trap three times with ethanol. Bring
the colored solution to volume with ethanol. Mix thoroughly and
allow the color to develop for at least 15 min but not more than
two h before determining the absorbance.
10.7 Determine the absorbance of the sample at 435 nm and 380 nm using a
1-cm cell or a 4-cm cell as necessary. Determine the weight of
dithiocarbamate from the appropriate calibration curve prepared in
7.4.
10.8 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1,000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml. If a
smaller measured aliquot of sample was used to remain within the
range of the color reagent, this step may be omitted.
11. Calculations
11.1 Determine the concentration of total dithiocarbamates in the sample
as ziram directly from the calibration curve. When a specific
dithiocarbamate is being measured, quantitate in terms of the
selected pesticide.
11.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
11.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in 8.3, data
for the affected parameters must be labeled as suspect.
630-09 January 1983
-------
12. Method Performance
12.1 The method detection limit (MDL) is defined as the minimum concen-
tration of a substance that can be measured and reported with 99%
confidence that the value is above zero.° the MDL concentrations
listed in Table 1 were determined using wastewater, and are
expressed in concentration units of the spiked materialsJ
12.2 In a single laboratory, Environmental Science and Engineering,
using spiked wastewater samples, the average recoveries presented
in Table 1 were obtained. The percent standard deviation of the
recovery is also included in Table 1.' All recoveries are based
on calibrations using the specific dithiocarbamate instead of zirara.
References
1. "Pesticides Methods Development," Report for EPA Contract 68-03-2897 (In
preparation).
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, 03370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
8. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, _15_, 1426 (1981).
9. "Determination of Dithiocarbamate in Industrial and Municipal Wastewater,"
Method 603, EPA No. 600/4-82-011, NTIS No. PB82-156050, January 1982,
National Technical Information Center, 5285 Port Royal Road, Springfield,
VA 22165.
630-10 January 1983
-------
TABLE 1
METHOD PERFORMANCE
Parameter
Maneb
Metham
Ziram
Method
Detection Limit Sample
(ug/L) Type*
15.3 1
3.7 2
3
1.9 4
5
Number
of Spike
Replicates (yg/L)
7
7
7
8
3
31.5
20.1
250
32.2
1050
Mean 5tandard
Recovery Deviation
(X) (%)
97.1
94.5
65.2
100
96.2
15.5
5.9
2.8
2.0
10.0
*Sample type:
1 = Municipal wastewater.
2 » Mixture of 13* Industrial (pesticide manufacturing) wastewater and 87%
municipal wastewater.
3 * Industrial wastewater, pesticide manufacturing.
4 = Mixture of 4056 industrial and 60% municipal wastewater.
5 a 7% industrial process water, 7% industrial wastewater, 86% municipal
wastewater.
630-11
January 1983
-------
Figure 1. Dithiocarbamate hydrolysis apparatus.
630-12
January 1983
-------
v>EPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BENOMYL AND CARBENDAZIM
IN WASTEWATER
METHOD 631
1. Scope and Application
1.1 This method covers the determination of benomyl and carbendazim.
The following parameters can be determined by this method:
Parameter STORET No. CAS No.
Bencmyl -- 17804-35-2
Carbendazim - 10605-21-7
1.2 Benomyl cannot be determined directly by this method. Benomyl is
hydrolyzed to carbendazim, and both compounds are measured and
reported as carbendazim.
1.3 This is a high performance liquid chromatographic (HPLC) method
applicable to the determination of the compounds listed above in
industrial and municipal discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly
permitted, shall be considered a major modification subject to
application and approval of alternate test procedures under 40 CFR
136.4 and 136.5.
1.4 The method detection limit (MDL, defined in Section 15) for each
parameter is 8.7 ug/L. The MDL for a specific wastewater may
differ from those listed, depending upon the nature of inter-
ferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in the
interpretation of liquid chromatograms. Each analyst must
demonstrate the ability to generate acceptable results with this
method u^ing the procedure described in Section 3.2.
1.6 When this method is used to analyze unfamiliar samples for either
of the compounds above, compound identifications should be
supported by at least one additional qualitative technique.
631-01 January 1983
-------
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, is acidified if
necessary to hydrolyze benomyl to carbendazim. The total carben-
dazim is extracted with methylene chloride using a separatory
funnel. The extract is dried and exchanged to methanol during
concentration to a volume of 10 ml or less. HPLC conditions are
described which permit the separation and.measurement of total
carbendazim in the extract by HPLC with a UV detector."»2
3. Interferences
3.1 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"liquid chromato-
grams. 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.3 Clean all
glassware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. Unique samples may require cleanup approaches to achieve
the MDL listed in Section 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. Prom this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
631-°2 January 1983
-------
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 4-6 f0r the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarfaon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination,
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 250-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.5 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
631-03 January 1983
-------
5.2.6 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat-at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Liquid chromatograph - High performance analytical system complete
with high pressure syringes or sample injection loop, analytical
columns, detector and strip chart recorder. A guard column is
recommended for all applications.
5.6.1 Column - 30 cm long x 4 mm ID stainless steel, packed with
u Bondapak C-\Q (10 jam) or equivalent. This column was
used to develop the method performance statements in Section
14. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Detector - Ultraviolet, 254 nm. This detector has proven
effective in the analysis of wastewaters and was used to
develop the method performance statements in Section 14.
Alternative detectors may be used in accordance with the
provisions described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Methylene chloride, methanol - Pesticide quality or equivalent.
6.3 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.4 Sodium hydroxide solution (ION) - Dissolve 40g NaOH in reagent
water and dilute to 100 mL.
6.5 Sulfuric acid solution (1+1) - Slowly add 50 ml H2S04 (sp. gr.
1.84) to 50 ml of reagent water.
6.6 Mobile phase - Methanol/water (1+1). Mix equal volumes of HPLC/UV
quality methanol and reagent water.
631-04 January 1983
-------
6.7 Stock standard solution (1.00 ug/uL) - The stock standard solution
may be prepared from a pure standard material or purchased as a
certified solution.
6.7.1 Prepare the stock standard solution by accurately weighing
approximately 0.0100 g of pure carbendazim. Dissolve the
material in HPLC/UV quality methanol and dilute to volume in
a 10-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified
at 96% or greater, the weight may be used without correction
to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or
by an independent source.
6.7.2 Transfer the stock standard solution into a TFE-fluorocarbon-
sealed screw cap vial. Store at 4°C and protect from
light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.6.3 The stock standard solution must be replaced after six
months or sooner if comparison with a check standard
indicates a problem.
7. Calibration
7,1 Establish HPLC operating parameters equivalent to those indicated
in Table 1. The HPLC system may be calibrated using either the
external standard technique (Section 7.2} or the internal standard
technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 Prepare calibration standards at a minimum of three
concentration levels by adding accurately measured volumes
of carbendazim stock standard to volumetric flasks and
diluting to volume with methanol. One of the external
standards should be representative of a concentration near,
but above, the method detection limit. The other concentra-
tions should correspond to the range of concentrations
expected in the sample concentrates or should define the
working range of the detector.
7.2.2 Using injections of 10 yL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for carbendazim. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for carbendazim at each
standard concentration. If the relative standard deviation
631-°5 January 1983
-------
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any
parameter varies from the predicted response by more than
±1055, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or
calibration factor must be prepared.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select an internal standard similar to carbendazim in
analytical behavior. The analyst must further demonstrate that the
measurement of the internal standard is not affected by method or
matrix interferences. Due to these limitations, no internal stan-
dard applicable to all samples can be suggested.
7.3.1 Prepare calibration standards at a minimum of three
concentration levels of carbendazim by adding volumes of
stock standard to volumetric flasks. To each calibration
standard, add a known constant amount of internal standard,
and dilute to volume with methanol. One of the standards
should be representative of a concentration near, but above,
the method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 10 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF * (AsCis)/(Ais Cs)
where:
As = Response for carbendazim.
Ais = Response for the internal standard.
C-Js = Concentration of the internal standard in ug/L.
Cs = Concentration of carbendazim in ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/A-js against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
631-06 January 1983
-------
standards. If the response for carbendazim varies from the
predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared.
7.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each com-
pound to be measured. Using stock standards, prepare a
quality control check sample concentrate of either benomyl
or carbendazim in methanol 1000 times more concentrated than
the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-ml aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
631-07 January 1983
-------
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts7
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively, the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.7
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10# of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for benomyl or carbendazim does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagent interferences are under control. Each time
631-08 January 1983
-------
a set of samples Is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram
as carbendazim, confirmatory techniques such as chromatography with
a dissimilar column, or ratio of absorbance at two or more wave-
lengths may be used. Whenever possible, the laboratory should
perform analysis of standard reference materials and participate in
relevant performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Using a 250-mL graduated cylinder, measure 150 ml of well-mixed
sample into a 250-mL Erlenmeyer flask. If benomyl is a
potentiality in the sample, continue with Section 10.2. If only
carbendazim is to be measured, proceed directly to Section 10.3.
10.2 Carefully add 2 mL of 1+1 sulfuric acid and a TFE-fluorocarbon
covered magnetic stirring bar to the sample. Check the sample with
wide-range pH paper to insure that the pH is less than 1.0. Stir
at room temperature for 16 to 24 h.
10.3 Adjust the sample pH to within the range of 6 to 8 with sodium
hydroxide. Pour the entire sample into a 250-mL separatory funnel.
10.4 Add 60 mL methylene chloride to the separatory funnel and extract
the sample by shaking the funnel for 2 min with periodic venting to
release excess pressure. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion interface
631-09
January 1983
-------
between layers is more than one third the volume of the solvent
layer, the analyst must employ mechanical techniques to complete
the phase separation. The optimum technique depends upon the
sample, but may include stirring, filtration of the emulsion
through glass wool, centrifugation, or other physical methods.
Collect the methylene chloride extract in a 250-mL Erlenmeyer
flask.
10.5 Add a second 60-mL volume of methylene chloride to the separatory
funnel and repeat the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
10.6 Assemble a Kuderna-Oanish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concen-
tration devices or techniques may be used in place of the K-D if
the requirements of Section 8.2 are met.
10.7 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20
to 30 ml of methylene chloride to complete the quantitative
transfer.
10.8 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL methylene chloride to the top. Place the K-D apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube
is partially immersed in the hot water, and the entire lower
rounded surface of the flask is bathed with hot vapor. Adjust the
vertical position of the apparatus and the water temperature as
required to complete the concentration in 15 to 20 min. At the
proper rate of distillation, the balls of the column will actively
chatter but the chambers will not flood with condensed solvent.
When the apparent volume of liquid reaches 1 ml, remove the K-D
apparatus and allow it to drain and cool for at least 10 min.
10.9 Increase the temperature of the hot water bath to 85 to 90°C.
Momentarily remove the Snyder column, add 50 ml of methanol and a
new boiling chip and reattach the Snyder column. Pour about 1 mL
of methanol into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should
be 5 to 10 min. When the apparent volume of liquid reaches 1 mL,
remove the K-0 apparatus and allow it to drain and cool for at
least 10 min.
10.10 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of methanol and adjust
the volume to 10 mL. A 5-mL syringe is recommended for this
operation. Stopper the concentrator tube and store refrigerated if
further processing will not be performed immediately. If the
631-10 January 1983
-------
extracts will be stored longer than two days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap vials. Proceed
with HPLC analysis.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. If particular circumstances demand the use of a
cleanup procedure, the analyst must determine the elution profile
and demonstrate that the recovery of each compound of interest for
the cleanup procedure is no less than 85%.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph. Included in this table are the estimated
retention time and method detection limit that can be achieved by
this method. An example of the separation achieved by this column
is shown in Figure 1. Other HPLC columns, chromatographic
conditions, or detectors may be used if the requirements of Section
8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 10 uL of the sample extract. Record the volume injected to
the nearest 0.05 uL, and the resulting peak size in area or peak
height units.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of carbendazim in the sample.
631-11 January 1983
-------
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
Concentration, ug/L
where:
A » Amount of carbendazim injected, in nanograms.
V-j * Volume of extract injected in uL.
V-t = Volume of total extract in uL.
Vs » Volume of water extracted in mL.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(AJdJ
Concentration, ug/L =
(A1s)(RF)(VQJ
where:
As = Response for carbendazim.
A-jS s Response for the internal standard.
Is = Amount of internal standard added to
each extract in ug.
V0 = Volume of water extracted, in liters.
13.2 If the sample was treated to hydrolyze benomyl, report the results
as benomyl (measured as carbendazim). If the hydrolysis step was
omitted, report results as carbendazim. Report results in
micrograms per liter without correction for recovery data. When
duplicate and spiked samples are analyzed, report all data obtained
with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. Method Performance
14.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero.9 The MDL
concentrations listed in Table 1 were determined by extracting
1000-mL aliquots of reagent water with three 350 mL volumes of
methylene chlorideJ
14.2 In a single laboratory (West Cost Technical Services, Inc.), using
reagent water and effluents from publicly owned treatment works
631-12 January 1983
-------
(POTW), the average recoveries presented in Table 2 were
obtainedJ The standard deviations of the percent recoveries of
these measurements are also included in Table 2. All results were
obtained using the same experimental scale described in Section
14.1.
References
1.- "Pesticide Methods Evaluation," Letter Report #17 for EPA Contract No.
68-03-2697. Available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
2. "Development of Analytical Test Procedures for Organic Pollutants in
Wastewater-Application to Pesticides," EPA Report 600/4-81-017, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268. PB #82 132507,
National Technical Information Service, Springfield, Va.
3. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
4. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
7. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268,
March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
9. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, J5_, 1426 (1981).
10. "Determination of Benomyl and Carbendazim in Industrial and Municipal
Wastewater," Method 631, EPA No. 600/4-82-012, NTIS No. PB82-156068,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
631-13 January 1983
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TABLE 1
CHROMATOGRAPHIC CONDITIONS AND
METHOD DETECTION LIMITS
Method
Retention Time Detection Limit
Parameter (min) (ug/L)
Benomyl (as carbendazim) — 25.0
Carbendazim 8.1 8.7
Column conditions: u Bondapak CIQ (10 ym) packed in a 30 cm long x 4 mm
ID stainless steel column with a mobile phase flow rate of 2.0 mL/min at
ambient temperature.
Mobile phase: methanol/water (1+1)
631-14 January 1983
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TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Sample
Type
Number
of
Replicates
Spike
(uq/L)
Average
Percent
Recovery
Standard
Deviation
(%)
Benomyl (as carbendazim)
Carbendazim
ow
MW
MW
DW
MW
MW
7
7
7
7
7
7
51,
51,
103
50
50
100
70
78
99
106
117
108
15.5
8.8
6.4
5.5
18.5
11.3
DW * Reagent water
MW - Municipal wastewater
631-15
January 1983
-------
carbendazim
10
Mi nutes
Figure 1. Liquid chromatogram of carbendazim on Column 1. For
conditions, see Table 1.
631-16
January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, O.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF CARBOFURAN, FLUOMETURON, METHOMYL, AND OXAMYL
IN WASTEWATER
METHOD 632
1. Scooe and Application
1.1 This method covers the determination of certain carbamate and urea
pesticides. The following parameters can be determined by this
method:
Parameter STORET No. CAS No.
Aminocarb — 2032-59-9
Barban — 101-27-9
Carbaryl 39750 63-25-2
Carbofuran 81405 1563-66-2
Chlorpropham — 101-21-3
Diuron 39650 330-54-1
Fenuron — 101-42-8
Fenuron-TCA - 4482-55-7
Fluometuron — 2164-17-2
Linuron — 330-55-2
Methiocarb -- 2032-65-7
Methomyl 39051 16752-77-5
Mexacarbate — 315-18-4
Monuron — 150-68-5
Monuron-TCA — 140-41-0
Nefauron — 555-37-3
Oxamyl- — 23135-22-0
Propham 39052 122-42-9
Propoxur — 114-26-1
Siduron — 1982-49-6
Swep ' - 1918-18-9
1.2 This method cannot distinguish monuron from monuron-TCA and fenurcn
from fenuron-TCA. Results for the paired parameters are reported
as monuron and fenuron respectively.
1.3. This is a high performance liquid chromatographic (HPLC) method
applicable tc the determination of the compounds listed above in
industrial and municipal discharges as provided under 40 CFR
136.1. Any modification of this method beyond those expressly
632-01 January 1983
-------
pennitted, shall be considered a major modification subject to
application and approval of alternate test procedures under 40 CFR
136.4 and 136.5.
1.4 The method detection limit (MDL, defined in Section 15) for many of
the parameters are listed in Table 1. The MDL for a specific
wastewater may differ from those listedt depending upon the nature
of interferences in the sample matrix.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of liquid chromatography and in the
interpretation of liquid chromatograms. Each analyst must demon-
strate the ability to generate acceptable results with this method
using the procedure described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique.
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and concentrated to a volume of
10 ml or less. HPLC chromatographic conditions are described which
permit the separation and measurement of the compounds in the
extract by HPLC with a UV detector.1'2
2.2 This method provides an optional Florisil column cleanup procedure
to aid in the elimination or reduction of interferences which may
be encountered.
3. Interferences
3.1 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 liquid chromato-
grams. 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.3 Clean all glass-
ware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane may be substituted for the
heating. After drying and cooling, seal and store glassware
632~02 January 1983
-------
in a clean environment to prevent any accumulation of dust
or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to
overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MOL listed in
Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 4-6 for the information of the analyst.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
632-03 January 1983
-------
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with coarse
fritted disc at bottom and TFE-fluorocarbon stopcock (Kontes
K-420540-0224 or equivalent).
5,2.4 Flask, round bottom - 500-mL, with standard taper to fit
rotary evaporator.
5.2.5 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Rotary evaporator.
5.4 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Filtration apparatus - As needed to filter Chromatographic solvents
prior to HPLC.
5.7 Liquid chromatograph - High performance analytical system complete
with high pressure syringes or sample injection loop, analytical
columns, detector and strip chart recorder. A guard column is
recommended for all applications.
5.7.1 Gradient pumping system, constant flow.
5.7.2 Column - 30 cm long x 4 mm ID stainless steel packed with
u Bondapak C]s (10 uro) or equivalent. This column was
used to develop the method performance statements in Section
14. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.7.3 Detector - Ultraviolet, capable of monitoring at 254 nm and
280 nm. This detector has proven effective in the analysis
of wastewaters and was used to develop the method
performance statements in Section 14. Alternative
632-04 January 1983
-------
detectors may be used in accordance with the provisions
described in Section 12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, acetonitrile, hexane, methylene chloride, methanol -
Pesticide quality or equivalent.
6.3 Ethyl ether - Nanograde, redistilled in glass if necessary. Must
be free of peroxides as indicated by EM Quant test strips. (Avail-
able from Scientific Products Co., Cat. No. P1126-8, and other
suppliers.) Procedures recommended for removal of peroxides are
provided with the test strips. After cleanup, 20 ml ethyl alcohol
preservative must be added to each liter of ether.
6.4 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
6.5 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in dark in glass container with ground glass stopper or
foil-lined screw cap. Before use activate each batch at least 16 h
at 130°C in a foil covered glass container.
6.6 Acetic acid - Glacial.
6.7 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure mate'rial. Dissolve the
material in pesticide quality acetonitrile or methanol and
dilute to volume in a 10-mL volumetric flask. Larger
volumes may be used at the convenience of the analyst. If
compound purity is certified at 96% or greater, the weight
may be used without correction to calculate the concentra-
tion of the stock standard. Commercially prepared stock
standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into TFE-fluorocarbon-
sealed screw cap vials. Store at 4°C and protect from
light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to
preparing calibration standards from them.
632-05 January 1983
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6.7.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish HPLC operating parameters equivalent to those indicated
in Table 1. The HPLC system may be calibrated using either the
external standard technique (Section 7.2) or the internal standard
technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration
standards at a minimum of three concentration levels by
adding accurately measured volumes of one or more stock
standards to a volumetric flask and diluting to volume with
acetonitrile or methanol. One of the external standards
should be representative of a concentration near, but above,
the method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates or should define the working range of
the detector.
7.2.2 Using injections of 10 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any
parameter varies from the predicted response by more than
±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or
calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
632-06 January 1983
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7.3.1 Prepare calibration standards at a minimum of three concen-
tration levels for each parameter of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with acetonitrile or methanol. One of the standards
should be representative of a concentration near, but above,
the method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 10 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF = (AsC1s)/(A1s Cs)
where:
As = Response for the parameter to be measured.
Ajs - Response for the internal standard.
C-Js * Concentration of the internal standard in ug/L.
Cs s Concentration of the parameter to be measured in
ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/A-jS against RF.
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromato-
grapny. Florisil from different batches or sources may vary in
adsorptive capacity. To standardize the amount of Florisil which
is used, the use of lauric acid valuo is suggested. This
procedure? determines the adsorption from hexane solution of
lauric acid, in mg, per g of Florisil. The amount of Florisil to
be used for each column is calculated by dividing this factor into
110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
632-07 January 1983
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8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to ma-intain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetonitrile or
methanol 1000 times more concentrated than the selected
concentrations.
8.2.2 Using a pipet, add 1.00 mL of the check sample concentrate
to each of a minimum of four 1000-ml aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
3.2.4 Table 2 provides single operator recovery and precision for
most of the carbamate and urea pesticides. Similar results
should be expected from reagent water for all compounds
listed in the method. Compare these results to the values
632-08 January 1983
-------
calculated in Section 8.2.3. If the data are not compar-
able, review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) « R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts^
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater
samples. An accuracy statement for the method is defined as
R ± s. The accuracy statement should be developed by the
analysis of four aliquots of wastewater as described in
Section 8.2.2, followed by the calculation of R and s.
Alternatively, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.°
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It is recommended that the laboratory adopt additional quality
assurance practices for use with this method. The-specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
632-°9 January 1983
-------
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as chromatography with a dissimilar
column, or ratio of absorbance at two or more wavelengths may be
used. Whenever possible, the laboratory should perform analysis of
quality control materials and participate in relevant performance
evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices^ should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the separ-
atory funnel and extract the sample by shaking the funnel for 2 min
with periodic venting to release excess pressure. Allow the organ-
ic layer to separate from the water phase for a minimum of 10 min.
If the emulsion interface between layers is more than one third the
volume of the solvent layer, the analyst must anploy mechanical
techniques to complete the phase separation. The optimum technique
depends upon the sample, but may include stirring, filtration of
the emulsion through glass wool, centrifugation, or other physical
methods. Collect the methylene chloride extract in a 250-mL
Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 It is necessary to exchange the extract solvent to hexane if the
Florisil clean up procedure is to be used. For direct HPLC
analysis the extract solvent must be exchanged to a solvent (either
632-10 January 1983
-------
methanol or acetonitrile) that is compatible with the mobile
phase. The analyst should only exchange a portion of the extract
to HPLC solvent if there is a possibility that cleanup may be
necessary.
10.5 Pass a measured fraction or all of the combined extract through a
drying column containing about 10 cm of .anhydrous sodium sulfate
and collect the extract in a 500-mL round bottom flask. Rinse the
Erlenmeyer flask and column with 20 to 30 ml of methylene chloride
to complete the quantitative transfer.
10.6 Attach the 500-mL round bottom flask containing the extract to the
rotary evaporator and partially immerse in the 50°C water bath.
10.7 Concentrate the extract to approximately 5 ml in the rotary
evaporator at a temperature of 50°C. Other concentration
techniques may be used if the requirements of Section 8.2 are met.
10.8 Add 50-mL of hexane, methanol, or acetonitrile to the round bottom
flask and concentrate the solvent extract as before. When the
apparent volume of liquid reaches approximately 5 ml remove the
500-mL round bottom flask from the rotary evaporator and transfer
the concentrated extract to a 10-mL volumetric flask, quantita-
tively washing with 2 mL of solvent. Adjust the volume to 10 mL.
10.9 Stopper the volumetric flask and store refrigerated at 4°C if
further processing will not be performed immediately. If the
extracts will be stored longer than two days, they should be
transferred to TFE-fluorocarbon-sealed screw-cap bottles.
10.10 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the water to a 1000-mL
graduated cylinder. Record the sample volume to the nearest 5 mL.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various industrial and municipal
effluents. If particular circumstances demand the use of an
alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest for the cleanup procedure is no less than 35%.
11.2 The following Florisil column cleanup procedure has been demon-
strated to be applicable to the five pesticides listed in Table
3. It should also be applicable to the cleanup of extracts for
the other carbamate and urea pesticides listed in the scope of
this method.
632-11 January 1983
-------
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by
calibration (Section 7.4 and 7.5), to a'chromatographic
column. Settle the Florisil by tapping the column. Add
anhydrous sodium sulfate to the top of the Florisil to form
a layer 1 to 2 cm deep. Add 60 ml of hexane to wet and
rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate to-air, stop the elution of
the hexane by closing the stopcock on the chromatography
column. Discard the eluate.
11.2.2 Adjust the sample extract volume to 10 ml with hexane and
transfer it from the volumetric flask to the Florisil
column. Rinse the flask twice with 1 to 2 ml hexane, adding
each rinse to the column.
11.2.3 Drain the column until the sodium sulfate layer is nearly
exposed. Elute the column with 200 ml of 20% ethyl ether in
hexane (V/V) (Fraction 1) using a drip rate of about 5
mL/min. Place a 500-mL round bottom flask under the chroma-
tography column. Elute the column again, using 200 ml of 6%
acetone in hexane (V/V) (Fraction 2), into a second flask.
Perform a third elution using 200 ml of 15% acetone in
hexane (V/V) (Fraction 3), and a final elution with 200 ml
of 50/5 acetone in hexane (V/V) (Fraction 4), into separate
flasks. The elution patterns for five of the pesticides are
shown in Table 3.
11.2.4 Concentrate the eluates to 10 ml with a rotary evaporator as
described in Section 10.7, exchanging the solvent to
acetonitrile or methanol as required.
12. Liquid Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the
liquid chromatograph. Included in this table are estimated
retention times and method detection limits that can be achieved by
this method. An example of the separations achieved by this column
is shown in Figure 1. Other HPLC columns, chromatographic condi-
tions, or detectors may be used if the requirements of Section 8.2
are met.
12.2 Calibrate the system daily as described in Section 7. The
standards and extracts must be in the solvent (acetonitrile or
methanol) compatible with the mobile phase.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 10 uL of the sample extract. Record the volume injected to
the nearest 0.05 uL, and the resulting peak size in area or peak
height units.
632-12 January 1983
-------
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response 1s prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(VJ
Concentration, ug/L »
(Vi)(Vs)
where:
A a Amount of material injected, in nanograms.
V-j = Volume of extract injected in uL.
Y£ a Volume of total extract in uL.
Vs a Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(AJ(IJ
Concentration, ug/L = (A.
.
I S
where:
As = Response for the parameter to be measured.
A-js * Response for the internal standard.
Is a Amount of internal standard added to each
extract in ug.
V0 a Volume of water extracted, in liters.
13.2 Calculate and report fenuron-TCA as fenuron and monuron-TCA as
monuron. Report results in micrograms per liter without correction
for recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
632-13 January 1983
-------
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 3.3,
data for the affected parameters must be labeled as suspect.
14. Method Performance
14.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero JO The MDL
concentrations lifted in Table 1 were obtained using reagent water
or river water.*»'I
14.2 In a single laboratory, the average recoveries presented in Table 2
were obtained using this method.2*11 The standard deviations of
the percent recoveries of these measurements are also included in
Table 2.
References
1. "Development of Analytical Test Procedures for Organic Pollutants in
Wastewater-Application to Pesticides," EPA Report 600/4-81-017, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268. PB#82 132507,
National Technical Information Service, Springfield, Va.
2. Farrington, D.S., Hopkins, R.G. and Ruzicka, J.H.A. "Determination of
Residues of Substituted Phenylurea Herbicides in Grain, Soil, and River
Water by Use of Liquid Chromatography," Analyst, 102, 377-381 (1977).
3. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation,, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
4. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
7. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3,
"Standardization of Florisil Column by Weight Adjustment Based on
Adsorption of Laurie Acid," American Society for Testing and Materials,
Philadelphia, PA, p 765, 1980.
632-14 January 1983
-------
8. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
9. ASTM Annual Book of Standards, Part 31, 03370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
10. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology. JJ3, 1426 (1981).
11. "Pesticide Methods Evaluation," Letter Reports #12B, 18, 19, 20, 22 and
23 for EPA Contract No. 68-03-2697. Available from U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
12. "Determination of Carbamate and Urea Pesticides in Industrial and
Municipal Wastewater," Method 632, EPA No. 600/4-82-014, NTIS No. PB82-
156084, January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
632-15 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Parameter
Mexacarbate
Propoxur
Monuron
Carbaryl
Propham
Dluron
Llnuron
Methiocarb
Chlorpropnam
Barb an
Neburon
Propoxur
Methomyl
Carbaryl
Diuron
Linuron
Propoxur
Carbofuran
Fluorometuron
Mobile Phase*
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
C
C
C
Retention
Time (Min)
8.7
14.3
14.4
17.0
17.2
19.5
21.0
21.4
21.8
22.3
24.3
2.0
6.5
14.1
15.5
17.9
1.7
3.5
3.6
UV
Wavelength
fnm)
254
280
254
280
254
254
254
254
254
254
254
280
254
280
254
254
280
280
254
Method
Detection
Limit
(ua/L)
0.52
o.n
0.003
0.02
0.07
0.009
O.OOS
0.02
0.03
0.05
0.012
o.n
8.9
0.02
0.009
0.009
o.n
3.2
11.1
Oxamyl
3.2
254
9.2
*Mobile Phase:
A Methanol/1% acetic acid, programmed linearly from 5 to 95% methanol at
2.0 mL/min flow rate and at ambient temperature.
B Acetonitrile/water, programmed linearly from 10% to 100% acetonitrile •
30 min at a flow rate of 2.0 mL/min.
C 50% acetonitrile in water at a flow rate of 2.0 mL/min.
D 35% methanol in water at a flow rate of 2.0 mL/min.
Column: 'u Bondapak C-\Q (10 urn) packed in a 30 cm long x 4 mm ID stainless
steel column, with a Whatmann Co. PELL ODS (30-38 urn) guard column, 7 cm
long x 4 mm ID.
632-16
January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Fluorometuron
Propoxur
Ox any 1
Methomyl
Diuron
Linuron
Carbofuran
Barban
Carbaryl
Chlorpropham
MethiocartD
Mexacarbate
Monurcn
Neburon
Prooham
* = Samnle Type
Sample
T^pe*
1
2
4
1
3
4
5
1
2
2
1
3
2
2
1
3
2
2
5
1
3
2
2
5
1
4
5
5
^
5
f
r-
5
•>
Spike
(yg/L)
50
50
1724
550
2200
550
0.5
TOO
53
1080
100
30660
100
1960
10
500
10
400
0.05
10
4000
10
210
0.05
37
143
0.3
0.1
0.2
0.2
4.0
0.05
0.05
0.3
No. of
Analyses
7
7
7
7
3
7
5
7
7
7
4
4
7
7
4
4
7
7
5
4
4
7
7
5
7
7
5
5
5
5
5
5
5
5
Average
Percent
Recovery
93.9
80.0
99
94.5
105
87.2
93
87
84.9
89.8
74.4
48.2
91.8
94.4
89.8
56.1
90.0
95.7
98
95.0
72.2
93.0
103
99
87.8
99.3
98
101
95
95
96
97
96
88
Standard
Deviation
%
7.0
7.2
11.6
1.7
3.0
7.3
6.0
8.4
5.5
2.7
2.4
2.8
2.8
1.9
1.0
5.0
2.5
3,2
4.7
3.4
5.1
1.5
4.6
4.7
2.7
1.4
4.1
4.1
3.9
2.6
3.5
1.7
6.6
5.9
1 = Reagent Water
2 = Municipal wastewater
3 = Industrial
A = Industrial
5 = River Water
process water, pesticide
wastewater,
manufactu -ing
pesticide manuf acturirr
632-17
January 1983
-------
TABLE 3
FLORISIL FRACTIONATION PATTERNS
Percent Recovery by Fraction
Parameter
Dluron
Linuron
Methomyl
Oxamyl
Propachlor
No. 1
0
0
0
0
0
No. 2
0
13
0
0
94
No. 3
24
82
0
92
0
No. 4
58
0
84
0
0
Florisil eluate composition by fraction
Fraction 1 - 200 ml of 20% ethyl ether in hexane
Fraction 2 - 200 ml of 6% acetone in hexane
Fraction 3 - 200 ml of 15% acetone in hexane
Fraction 4 - 200 ml of 50% acetone in hexane
632-18 January 1983
-------
1
3
|
10
Minutes
15
20
Figure 1. Liquid chromatogram of diuron, linuron and
methomyl on Column 1. For conditions, see Table 1
632-19
January 1983
-------
so^ou
00
c
o
o
u
o
u.
tr>
I/)
01
•o
632-20
January 1983
-------
I I 1 I
f I
7
8
Figure 3.
123456
Minutes
Gas chromatogram of organophosphorus pesticides on
Column 2. For conditions, see Table 1.
632-21
January 1983
-------
(/I
O
s.
O
•o
0)
1
8
Figure 4.
2 34 567
Mi nutes
Gas chromatogram of organophosphorus pesticides on
Column 3. For conditions, see Table 1.
632-22
January 1983
-------
oEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, O.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETERMINATION OF BROMACIL, DEET, HEXAZINONE, METRIBUZIN,
TERBACIL, TRIAD IMEFON, AND TRICYCLAZOLE
IN WASTEWATER
METHOD 633
1. Scope and Application
1.1 This method covers the determination of certain organonitrogen
pesticides. The following parameters can be determined by this
method:
Parameter STORET No. CAS No.
Bromacil — 314-40-9
Deet — 134-62-3
Hexazinone — 51235-04-2
Metribuzin 81408 21087-64-9
Terbacil — 5902-51-2
Triadimefon — 43121-43-3
Tricyclazole — 41814-78-2
1.2 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in industrial and municipal
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted, shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for five of
the parameters are listed in Table 1. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography 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 3.2.
1.5 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique.
Section 14 provides gas chromatograph/mass spectrometer (GC/MS)
criteria appropriate for the qualitative confinnation of compound
identifications.
633~01 January 1983
-------
2. Summary of Method
2.1 A measured volume of sample, approximately 1-liter, is solvent
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and exchanged to acetone during
concentration to a volume of 10 mi or less. Gas chromatographic
conditions are described which permit the separation and measure-
ment of the compounds in the extract by gas chromatography with a
thermionic bead detector.'
3. Interferences
3.1 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 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon 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 15 to 30 min. Do not heat volumetric
ware. Thermally stable materials such as PCBs, might not be
eliminated by this treatment. Thorough rinsing with acetone
and pesticide quality hexane 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.
3.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
3.2 Matrix interferences may be caused by contaminants that are coex-
tracted from the sample. The extent of matrix interferences will
vary considerably from source to source, depending upon the nature
and diversity of the industrial complex or municipality sampled.
Unique samples may require special cleanup approaches to achieve
the MDL listed in Table 1.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
must be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible
633-02 January 1983
-------
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified 3-5 for the information of the analyst.
5.' Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass,
1-liter or 1-quart volume, fitted with screw caps lined with
TFE-fluorocarbon. Aluminum foil may be substituted for TFE
if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap
liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass sample
containers for the collection of a minimum of 250 ml.
Sample containers must be kept refrigerated at 4°C and
protected from light during compositing. If the sampler
uses a peristaltic pump, a minimum length of compressible
sHlcone rubber tubing may be used. Before use, however,
the compressible tubing must be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water
to minimize the potential for contamination of the sample.
An integrating flow meter is required to collect flow
proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with TFE-fluorocarbon stopcock,
ground glass or TFE stopper.
5.2.2 Drying column - Chromatographic column 400 mm long x 19 mm
ID with coarse fritted disc.
5.2.3 Concentrator tube, Kuderna-Danish - 10-mL, graduated (Kontes
K-570050-1025 or equivalent). Calibration must be checked
at the volumes employed in the test. Ground glass stopper
is used to prevent evaporation of extracts.
5.2.4- Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2:5 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
633-03 January 1983
-------
5.2.6 Vials - Amber glass, 10 to 15 ml capacity with
TFE-fluorocarbon lined screw cap.
5.3 Boiling chips - approximately 10/40 mesh. Heat at 400°C for 30
min or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of temper-
ature control (± 2°C). The bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas chromato-
graph suitable for on-column injection and all required accessories
including syringes, analytical columns, gases, detector and strip-
chart recorder. A data system is recommended for measuring peak
areas.
5.6.1 Column 1 - 180 cm long x 2 mm ID glass, packed with 3%
SP-2250DB on Supelcoport (100/120 mesh) or equivalent.
Operation of this column at high temperatures will seriously
reduce its useful period of performance. This column was
used to develop the method performance statements in Section
15. Alternative columns may be used in accordance with the
provisions described in Section 12.1.
5.6.2 Column 2 - 180 cm long x 2 mm ID glass, packed with 2%
SP-240l'on Supelcoport (100/120 mesh) or equivalent.
5.6.3 Detector - Thermionic bead in the nitrogen mode. This
detector has proven effective in the analysis of wastewaters
for the parameters listed in the scope and was used to
develop the method performance statements in Section 15.
Alternative detectors, including a mass spectrometer, may be
used in accordance with the provisions described in Section
12.1.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest.
6.2 Acetone, methylene chloride - Pesticide quality or equivalent.
6.3 Sodium sulfate - (ACS) Granular, anhydrous. Heat treat in a
shallow tray at 400°C for a minimum of 4 h to remove phthalates
and other interfering organic substances. Alternatively, heat 16 h
at 450-500°C in a shallow tray or Soxhlet extract with methylene
chloride for 48 h.
633-04 January 1983
-------
6.4 Stock standard solutions (1.00 ug/uL) - Stock standard solutions
may be prepared from pure standard materials or purchased as
certified solutions.
6.4.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material In pesticide quality acetone and dilute to volume
in a 10-mL volumetric flask. Larger volumes may be used at
the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight may be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards may be used
at any concentration if they are certified by the manufac-
turer or by an independent source.
6.4.2 Transfer the stock standard solutions into TFE-fluorocarbon-
sealed screw cap vials. Store at 4°C and protect from
light. Frequently check stock standard solutions for signs
of degradation or evaporation, especially just prior to
preparing calibration standards from thenu
6.4.3 Stock standard solutions must be replaced after six months
or sooner if comparison with check standards indicates a
problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 1. The gas chromatographic system may be
calibrated using either the external standard technique (Section
7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each parameter of interest, prepare calibration stan-
dards at a minimum of three concentration levels by adding
accurately measured volumes of one or more stock standards
to a volumetric flask and diluting to volume with acetone.
One of the external standards should be representative of a
concentration near, but above, the mathod detection limit.
The other concentrations should correspond to the range of
concentrations expected in the sample concentrates or should
define the working range of the detector.
7.2.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), may be calculated for each parameter at each
standard concentration. If the relative standard deviation
633-05 January 1983
-------
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any para-
meter varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration stan-
dard. Alternatively, a new calibration curve or calibration
factor must be prepared for that parameter.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three con-
centration levels for each parameter of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards,, and dilute to
volume with acetone. One of the standards should be
representative of a concentration near, but above, the
method detection limit. The other concentrations should
correspond to the range of concentrations expected in the
sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard,
tabulate the peak height or area responses against the
concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as follows:
RF * (AsC1s)/(Ais Cs)
where:
As * Response for the parameter to be measured.
A-js = Response for the internal standard.
* Concentration of the internal standard in ug/L.
= Concentration of the parameter to be measured
in ug/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF may be used for calcula-
tions. Alternatively, the results may be used to plot a
calibration curve of response ratios, As/A-js against RF.
633-06 January 1983
-------
7.3.3 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. If the response for any parameter varies from
the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. Alternatively,
a new calibration curve must be prepared for that compound.
7.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interference from the reagents.
8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on perfor-
mance. The laboratory is required to maintain performance records
to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate
the ability to generate acceptable accuracy and precision
with this method. This ability is established as described
in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in chromato-
graphy, the analyst is permitted certain options to improve
the separations or lower the cost of measurements. Each
time such modifications to the method are made, the analyst
is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in acetone 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the background level for the test to
be valid. Analyze the aliquots according to the method
beginning in Section 10.
633-07 January 1983
-------
8.2.3 Calculate the average percent recovery (R), and the standard
deviation of the percent recovery (s), for the results.
Wastewater background corrections must be made before R and
s calculations are performed.
8.2.4 Using the appropriate data from Table 2, determine the
recovery and single operator precision expected for the
method, and compare these results to the values calculated
in Section 8.2.3. If the data are not comparable, review
potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method perfor-
mance as follows:
Upper Control Limit (UCL) * R + 3 s
Lower Control Limit (LCL) - R - 3 s
where R and s are calculated as in Section 8.2.3.
The UCL and LCL can be used to construct control charts^
that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater samples.
An accuracy statement for the method is defined as R ± s.
The accuracy statement should be developed by the analysis
of four aliquots of wastewater as described in Section
8.2.2, followed by the calculation of R and s. Alterna-
tively,the analyst may use four wastewater data points
gathered through the requirement for continuing quality
control in Section 8.4. The accuracy statements should be
updated regularly.6
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one spiked
sample per month, whichever is greater. One aliquot of the sample
must be spiked and analyzed as described in Section 8.2. If the
recovery for a particular parameter does not fall within the
control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set
must be qualified as described in Section 13.3. The laboratory
should monitor the frequency of data so qualified to ensure that it
remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through
the analysis of a 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
633-08 January 1983
-------
laboratory reagent blank must be processed as a safeguard against
laboratory contamination.
8.6 It 1s recommended that the laboratory adopt additional quality
assurance practices for use with this method. The specific
practices that are most productive depend upon the needs of the
laboratory and the nature of the samples. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a dissim-
ilar column, specific element detector, or mass spectrometer must
be used. Whenever possible, the laboratory should perform analysis
of quality control materials and participate in relevant perfor-
mance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected 1n glass containers. Conventional
sampling practices7 should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 All samples must be extracted within seven days and completely
analyzed within 40 days of extraction.
10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
10.2 Add 60 ml methylene chloride to the sample bottle, seal, and shake
30 s to rinse the inner walls. Transfer the solvent to the separa-
tory funnel and extract the sample by shaking the funnel for 2 min
with periodic venting to release excess pressure. Allow the
organic layer to separate from the water phase for a minimum of 10
min. If'the emulsion interface between layers is more than one
third the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation. The opti-
mum technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool, centrifugation, or
other physical methods. Collect the methylene chloride extract in
a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
633-09 January 1983
-------
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 Assemble a Kuderna-Oanish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other concentra-
tion devices or techniques may be used in place of the K-D if the
requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the Snyder column by adding
about 1 ml methylene chloride to the top. Place the K-0 apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 min. At the proper rate of
distillation, the balls of the column will actively chatter but the
chambers will not flood with condensed solvent. When the apparent
volume of liquid reaches 1 ml, remove the K-D apparatus and allow
it to drain and' cool for at least 10 min.
10.7 Increase the temperature of the hot water bath to about 70°C.
Momentarily remove the Snyder column, add 50 ml of acetone and a
new boiling chip and reattach the Snyder column. Pour about 1 mL
of acetone into the top of the Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be
5 to 10 min. When the apparent volume of liquid reaches 1 ml,
remove the K-D apparatus and allow it to drain and cool for at
least 10 min.
10.8 Remove the Snyder column and rinse the flask and its lower joint
into the concentrator tube with 1 to 2 ml of hexarie and adjust the
volume to 10 ml. A 5-mL syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extracts will
be stored longer than two days, they should be transferred to
TFE-fluorocarbon-sealed screw-cap vials. Analyze by gas chromato-
graphy.
10.9 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. If particular circumstances demand the use of a
633-10 January 1983
-------
cleanup procedure, the analyst must determine the elation profile
and demonstrate that the recovery of each compound of interest for
the cleanup procedure is no less than 85%.
12. Gas Chromatography
12.1 Table 1 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. An example of the separations achieved by Column 1 is
shown in Figure 1. Other packed columns, chromatographic condi-
tions, or detectors may be used if the requirements of Section 8.2
are met. Capillary (open-tubular) columns may also be used if the
relative standard deviations of responses for replicate injections
are demonstrated to be less than 63. and the requirements of Section
8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
12.4 Inject 1 to 5 uL of the sample extract using the solvent-flush
technique.8 Record the volume injected to the nearest 0.05 uL,
and the resulting peak size in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chromato-
grams.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak-
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
633-11 January 1983
-------
Concentration, yg/L = -
where:
A » Amount of material injected, in nanograms.
Vi » Volume of extract injected in uL.
Vt 3 Volume of total extract in uL.
Vs * Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 7.3.2 as follows:
(As
Concentration, yg/L * -TV-
where:
As a Response for the parameter to be measured.
A-jS a Response for the internal standard.
Is « Amount of internal standard added to each
extract in ug.
V0 » Volume of water extracted, in liters.
13.2 Report results in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected parameters must be labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to
support qualitative compound identifications made with this
method. The mass spectrometer should be capable of scanning the
mass range from 35 amu to a mass 50 amu above the molecular weight
of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not
to exceed 7 s per scan utilizing a 70 V (nominal) electron energy
in the electron impact ionization mode. A GC to MS interface
constructed of all-glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer
that allows the continuous acquisition and storage on machine
readable media of all mass spectra obtained throughout the duration
of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for
• optimum separation and performance. The conditions selected must
be compatible with standard GC/MS operating practices. Chromato-
graphic tailing factors of less than 5.0 must be achieved.9
633-12 January 1983
-------
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC/MS system must be checked to see that all
decafluorotriphenyl phosphine (DFTPP) performance criteria are
achieved.10
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatograph.ic
conditions. It is recommended that at least 25 nanograms of
material be injected into the GC/MS. The criteria below must be
met for qualitative confirmation.
14.4.1 All ions that are present above 10% relative abundance in
the mass spectrum of the standard must be present in the
mass spectrum of the sample with agreement to plus or minus
10%. For example, if the relative abundance of an ion is
30% in the mass spectrum of the standard, the allowable
limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20% to 40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC/MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MOL) is defined as the minimum concen-
tration of a substance that can be measured and reported with 99%
confidence that the value is above zero.1' The MDL
concentrations listed in Table 1 were obtained using reagent
water.1
15.2 In a single laboratory (West Cost Technical Services, Inc.), using
effluents from pesticide manufacturers and publicly owned treatment
works (POTW), the average recoveries presented in Table 2 were
obtained.1 The standard deviations of the percent recoveries of
these measurements are also included in Table 2.
633-13 January 1983
-------
References
>. "Pesticide Methods Evaluation," Letter Reports #6, 12A and 14 for EPA
Contract No. 68-03-2697. Available from U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio 45268.
2.- ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for
Preparation of Sample Containers and for Preservation, " American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 220(5, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
6. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories," EPA-600/4-79-019, U. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio
45268, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
8. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
9. McNair, H.M. and Bonelli, E. J., "Basic Chromatography," Consolidated
Printing, Berkeley, California, p. 52, 1969.
10. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to
Calibrate Ion Abundance Measurement in Gas Chromatography-Mass
Spectrometry," Analytical Chemistry, 47, 995 (1975).
11. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental
Science & Technology, J5_, 1426 (1981).
12. "Determination of Organonitrogen Pesticides in Industrial and Municipal
Wastewater," Method 633, EPA No. 600/4-82-013, NTIS No. PB82-156076,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
633-14 January 1983
-------
TABLE 1
CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
Parameter
Terbacil
Bromacil
Hexazinone
Tricyclazole
Metrlbuzin
Triadimefon
Deet
GC
Column
la
la
la
Ib
2a
2a
2b
Retention
Time
(Min)
2.1
3.7
7.6
3.5
2.4
4.1
4.6
Method
Detection Limit
(ug/L)
NO
2.38
0.72
NO
0.46
0.78
3.39
ND - Not determined
Column la conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250DB
packed in a 180 cm long x 2 mm ID glass column with nitrogen carrier gas at
a flow rate of 30 mL/min. Column temperature, programmed: initial 210°C,
hold for 1 min, then program at 10°C to 250°C and hold. A thermionic
bead detector in the nitrogen mode was used to calculate the MDL.
Column Ib conditions: Same as Column la, except column temperature
isothermal at 240°C.
Column 2a conditions: Supelcoport (100/120 mesh) coated with 3% SP-2401
packed in a 180 cm long x 2 mm ID glass column with nitrogen carrier gas at
a flow rate of 30 mL/min. Column temperature, programmed: initial 160°C,
programmed at injection at 10°C/min to 230°C.
Column 2b conditions: Same as Column 2a, except temperature programmed:
initial 130°C, hold for 1 min, then program at 12°C/min to 200°C.
633-15
January 1983
-------
TABLE 2
SINGLE OPERATOR ACCURACY AND PRECISION
Parameter
Bromac i 1
Deet
Hexazinon
Metribuzin
Terbacil
Tr 1 admef on
Tricyclazole
Sample
Type
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
PW
IW
MW
MW
Spike
(uq/L)
5
11.1
333
5.8
5.2
515
4.9
10.1
369
5.2
32.8
656
5.2
515
154.5
12.3
303
Number
of
Replicates
7
7
7
7
7
7
7
7
7
6
7
7
6
4
7
7
7
Mean
Recovery
(*)
92.2
89
95
99.1
92.6
94.2
86.6
92.2
94.0
98.2
106.7
101
126
71.8
70.4
69
98
Standard
Deviation
(X)
13.9
3.9
0.8
18.4
5.9
2.2
4.1
5.3
1.9
2.7
3.6
1.2
6.0
4.5
3.8
1.9
1.2
DW s Reagent water
MW = Municipal wastewater
PW =* Process water, pesticide manufacturing
IW = Industrial wastewater, pesticide manufacturing
633-16
January 1983
-------
Terbacil
Hexazinone
I I
I I I » I 1
4 5
Minutes
8
Figure 1. Gas chromatogram of organonitrogen pesticides on Column 1
For conditions, see Table 1.
633-17
January 1983
-------
vvEPA
United States Effluent Guidelines Division (WH 552)
Environmental Protection Washington, D.C. 20460
Agency
Water and Waste Management
TEST METHOD
DETEBMINATION OF DICHLOFENTHION, DIOXATHION,
AND CABBOPHENOTHION IN WASTEWATER
METHOD 701
1. Scope and Application
1.1 This method covers the determination of certain organophosphorus
compounds. The following parameters can be determined by this
method:
Parameter Storet No. CAS No.
Dichlofenthion — 97-17-6
Dioxathion — 78-34-2
Carbophenothion 39786 786-19-6
Diazinon 39570 333-41-5
Methyl Parathion 39600 298-00-0
Malathion 39530 121-75-5
Parathion 39540 56-38-2
Methyl Trithion —
Ethion 39398 563-12-2
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the above listed compounds in water.
2 . Su"p*"TY of Method
2.1 Water samples (800-900 mL) are extracted with n-hexane. The
combined extracts are dried over sodium sulfate and concentrated
using a Kuderna-Danish (K-D) apparatus to 0.4 - 0.5 mL. The
extract is then cleaned up on a deactivated alumina microcolumn.
The eluates are analyzed by gas chroma tog rap by with flame
photometric detector.
2.2 This method provides a selected cleanup procedure to aid in the
elimination of interferences which may be encountered.
701-01 January 1983
-------
3. Interferences
3.1 Any compound or compounds having chemical and physical properties
similar to the pesticide of interest may cause interference. The
procedure incorporates a column chromatographic technique which
eliminates most extraneous material. Special precautions are
necessary to avoid contamination during -sampling and analysis.
4. Safety
4.1 Follow EPA safety procedare found in Part D of this document.
5. Apparatus and Materials
5.1 Sampling Equipment
Not Available.
5.2 Glassware and Other Equipment
5.2.1 Concentrating apparatus: Kuderna-Danish concentrator,
250-mL capacity with 1-ball Snyder column. Final
concentration is performed in the receiver using a 1-ball
Snyder micro-column. A calibrated 4.00-mL receiver tube
is used with the concentration apparatus.
5.2.2 Cleanup microcolumns: Disposable Pasteur pipets, 15-cm
long and 5-mm I.D., are used for the chromatographic
cleanup columns. The pipets are washed in warm detergent
solution, thoroughly rinsed with dilute hydrochloric acid
and organic-free distilled water, then heated to 300 C
overnight to remove any traces of organic matter. A
column is prepared by plugging the pipet with a small
amount of specially cleaned glass wool, adding enough
deactivated alumina through a microfunnel to fill 3 cm of
the column, followed by another 0.5 cm of anhydrous sodium
sulfate.
5.2.3 Separatory funnels: Squibb form, 1- or 2-liter capacity.
No lubricant is used on the stopcocks.
5.2.4 Microliter capillary pipets: Volumetric micropipets in
1-, 5-, 10-, and 25-uL sizes; disposable types are
satisfactory.
5.2.5 Microliter syringes: Three microsyringes having
capacities of 10, 50, and 100 uL, respectively, are used.
The syringe needle should be about 2-in long and have a
point shaped to prevent punching out a core when
penetrating the injection septum.
5.2.6 Volumetric glassware: Class A volumetric flasks in 5-,
10-, and 25-ml sizes. The stoppers should fit well
because volatile organic solvents are used for dilutions.
701-02 January 1983
-------
Volumetric ware (supplied by Kontes Glass Co. or
equivalent) is acceptable.
5.2.7 Sandbath, fluidized, Tecam, or equivalent.
5.2.8 Microbalance: A Cahn Gram Electro-balance or equivalent.
5.2.9 Integrating equipment: A compensating polar planimeter
readable to the nearest 0.001 square inch is acceptable.
Other instruments or methods of integration demonstrating
greater accuracy may be used.
5.2.10 Recorder: A 1-mv (millivolt) full-scale response, 10-sec
(second) pen speed, strip-recorder having a fixed or
selectable chart speed of 0.5-in inch per minute is
acceptable.
5.3 Gas Chromatograph
5.3.1 Flame-photometric gas chromatograph: A gas chromatograph
equipped with a Melpar flame-photometric detector having
filters for the specific detection of phosphorus or
sulfur. Such an instrument is the Micro-Tec Model MT-220
flame-photometric gas chromatograph. A provision for
venting solvent effluent between the column and the
detector should be specified.
5.3.2 Gas chromatographic columns: The gas chromatographic
columns are fabricated from 1.5-m lengths of Pyrex glass
tubing; 1.8-mm I.D. or 4-mm I.D. glass tubing may be used.
The smaller bore columns accept injection volumes up to 10
uL and the larger bore columns will accept volumes up to
80 uL. Gas Chrom Q support, 60/80 mesh, is used for the
preparation of two different column packings as follows:
with 5% by weight DC-200 silicone oil (viscosity 12,500
centistokes), 0.5% by weight QF-1 fluorinated silicone oil
(also designed FS 1265), and 0.5% by weight Carbowax 20 M.
The packing materials are loaded in the glass columns
using vibration and a vacuum to settle. The packing is
held in place by small plugs of "silanized" glass wool.
6. Reagents
6.1 Alumina, neutral aluminum oxide, activity grade I, Woelm. Weigh
19 g activated alumina into a 50-mL glass-stoppered Erlenmeyer
flask and quickly add 1.0 mL distilled water. Stopper the flask
and mix the contents thoroughly by tumbling. Allow 2 hours
before use. The deactivated alumina may be used for 1 week.
6.2 Benzene, distilled in glass, pesticide-analysis quality.
6.3 n-Hexane, distilled in glass, pesticide-analysis quality.
6.4 Sodium sulfate, anhydrous granular. Prepare by heating at 300 C
overnight; store at 130°C.
701-03 January 1983
-------
6.5 Water, distilled, obtained from a high-purity tin-lined still.
Feed water is passed through an activated carbon filter.
Distillate is collected in a tin-silver-lined storage tank, and
the water is constantly irradiated with ultraviolet light during
storage. A gravity delivery system is used, and no plastic
material other than Teflon is allowed to contact the distilled
water.
7. Calibration
7.1 Establish GC operating parameters equivalent to those indicated
in Table 1.
7.2 Calibration Procedure
7.2.1 Weigh 5.00 mg of reference pesticide into a 5.00-mL
volumetric flask and dilute to volume with benzene. Make
a series of appropriate nanogram standards from this
solution. [Example: Take 10.0 uL and dilute to 10.00 mL
with the solvent to be used in the analysis. The
concentration of pesticide in the resulting solution is
-9
1.0 X 10 g/uL, or 1.0 ng/uL (nanogram per microliter)].
7.2.2 A 5.0-uL volume of each of the appropriate standard
solutions is injected into the gas chromatograph. The
concentration of pesticide in the series of standard
solutions should calibrate either the full range of linear
detector response or the range of anticipated pesticide
concentration in the sample, whichever is less. The
injection should be made so that the solution enters the
injection port in a single volume and in a reproducible
manner. The volume injected should be measured by reading
the syringe before and after injection.
7.2.3 All information pertinent to the standardization should be
written directly on the recorder chart. Calibration
should be performed on both the DC-200 and the QF-1
columns.
7.2.4 During analysis, the standard curve must be checked by
running at least two standards at different concentrations
so correction can be made for day-to-day fluctuations.
7.2.5 Measurement of gas chromatogram peak areas by use of a
planimeter or by any method of equal or greater accuracy
is acceptable. If a planimeter is used, the average of at
least two measurements is taken as the peak area.
7.2.6 Using log-log graph paper, plot area of response, in
2
square inches (in ), against nanograms of pesticide
injected. Determine the least-squares regression line.
The two or more daily response check standards are used to
correct the slope of the standard curve, as follows:
701-04 January 1983
-------
Ac
where C = correction factor,
Ac = area of check standard obtained from the
standard curve, and
As = area obtained from the chromatogram of the check
standard.
The slope of the standard curve is corrected by
multiplying it by the correction factor.
8. Quality Control
8.1 All glassware, except volumetric flasks, should be washed in the
usual manner, rinsed in dilute hydrochloric acid and distilled
water, and heat treated at 300 C overnight. Instead of heat
treating, the volumetric ware may be solvent rinsed to remove
organic matter. A reagent and glassware blank should accompany
each analysis.
8.2 See EPA Quality Control procedure found in Part D of this
document.
9. Sample Collection. Preservation, and Handling
9.1 Samples should be collected according to the recommended practice
for the collection of samples for organic analysis. A 1-liter
bottle of water should be collected for each sample. No
preservative is used. Samples should be shipped promptly.
Unless analyzed within a few days, the water should be protected
from light and refrigerated. If the sample contains sediment,
the sediment must be analyzed separately. Remove the sediment by
centrifugation or filtration through a metal membrane filter.
9.2 See EPA sample collection, preservation, and handling procedure
found in Part D of this document.
10. Sample Extraction
10.1 Water samples (800-900 mL) are extracted with n-hexane in such a
manner that the water and the container itself are exposed to the
solvent. Weigh the uncapped bottle of water on a triple-beam
balance and pour the sample into a 1-liter separatory funnel.
Allow the bottle to drain for a few minutes, weigh again, and
record the weight of water to three significant figures.
10.2 Add 25 mL n-hexane to the empty sample bottle and gently swirl to
wash the sides of the container with the solvent. Pour the
contents of the sample bottle into the separatory funnel
containing the water. Stopper and shake the separatory funnel
vigorously for 1 full min, venting the pressure often. Allow the
contents to separate for 10 min and draw off the aqueous layer
701-05 January 1983
-------
into the original sample bottle. If the hexane layer emulsifies,
separate as much water as possible, then shake the contents of
the funnel vigorously so that the liquids contact the entire
inside surface of the vessel. (CAUTION: Vent often!) Allow the
layers to separate and add approximately 5 ml distilled water to
aid the separation, if necessary. Remove the water and pour the
extract from the top of the separatory funnel into a 125-mL
Erlenmeyer flask containing about 0.5 g anhydrous sodium sulfate.
10.3 Repeat a second and third extraction of the water sample in the
same manner using 25 mL n-hexane each time, and collect the
extracts in the 125-mL Erlenmeyer flask containing; the drying
agent. Cover the flask containing the extract with foil and set
aside for 30 min.
10.4 Filter the dried extract through glass wool into the Kuderna-
Danish apparatus. Add a sand-sized boiling stone and remove most
of the hexane by heating on a fluidized sandbath at 100 C in a
hood. When the ball in the Snyder column stops bouncing, remove
the apparatus from the heat and allow to cool. Add another small
boiling stone, fit the receiver with a Snyder microcolumn, and
reduce the volume to between 0.4 and 0.5 mL on the sandbath. Set
aside to cool. When changing columns, sand must be cleared from
the glass joint before opening.
11. Cleanup and Separation
11.1 Quantitatively transfer the contents of the Kuderna-Danish
receiver (0.4-0.5 mL) to the top of a deactivated alumina cleanup
microcolumn. Use a disposable pipet to transfer. Not more than
0.1-0.2 mL hexane should be needed for washing. Using hexane,
elute the extract from the column to a volume of 8.5 mL in a
calibrated 10.00-mL receiver. Add only enough hexane so that the
solvent level enters the column packing just as the 8.5-mL
elution level is reached. Change receivers and continue the
elution using 1:1 benzene-hexane solvent. Collect 8.5 mL of
eluate in a second receiver. The first fraction of eluate should
contain any chlorinated hydrocarbon insecticides, and
carbophentnion, methyl trithion, and dichlofenthion. The
remaining phosphorus-containing pesticides are eluted in the
benzene-hexane fraction. Reduce the volume of each eluate to 1.00
mL using a Kuderna-Danish micro-apparatus on the sandbath. NOTE:
The insecticides are separated chromatographically in a
predictable order on the micro-column, and this may be used to
augment gas chromatographic analysis. Although alumina is the
absorbent of choice for the majority of water and sediment
samples, occasionally a second pass through a different column is
needed for more difficult samples. For a more comprehensive
treatment of the cleanup procedure refer to 15.3.
12. Gas Chromatography
12.1 Analyze the eluates by gas chromatography under conditions
optimized for the particular gas chromatographic system used.
701-06 January 1983
-------
Run the analysis on the flame-photometric gas chromatographic
system on both the DC-200 and the QF-1 columns.
13. Calculations
13.1 The concentration of pesticides in water samples may be
determined using the following equation:
Concentration of pesticide (ug/L) « A x T~ X ^7 . X —-
2
where A = area of component (in )
2
Cm • corrected slope (in /ng)
Vext = volume of extract (mL)
Vinj * volume injected (mL)
Vs * volume of water sample (liters)
This equation may be used to calculate the concentration of
pesticides in sediment or soil by substituting the weight of
sample in kilograms for the sample volume (Vs) with the resulting
concentration expressed as ug/kg.
14. Method Performance
14.1 The results may vary as much as +. 15% for compounds in the 0.01-
to 0.10-ug/L concentration range. Recovery and precision data
are given in Table 3.
15. References
15.1 Lamar, W.L., Goerlitz, D.F., and Law, L.M. 1965 Identification
and measurement of chlorinated organic pesticides in water by
electron-capture gas chromatography: U.S. Geol. Survey Water-
Supply Paper 1817-B.
15.2 Lamar, W.L., Goerlitz, D.F., and Law, L.M. 1966 Determination of
organic insecticides in water by electron-capture gas
chromatography, in organic pesticides in the Environment. Am.
Chem. Soc., Advances in Chemistry, ser. 60.
15.3 Law, L.M. and Goerlitz, D.F. 1970. Microcolumn chromatographic
cleanup for the analysis of pesticides in water. Assoc. Official
Anal. Chemists Jour.,v. 53, no. 6.
15.4 (U.S.) Federal Water Pollution Control Administration 1969 FWPCA
method for chlorinated hydrocarbon pesticides in water and
wastewater. Cincinnati, Federal Water Pollution Control Adm.
15.5 Zweig, G. and Devine, J.M. 1969 Determination of
organophosphorus pesticides in water. Residue Rev., v. 26.
15.6 Donald S. Goerlitz and Eugene Brown "Methods for Analysis of
Organic Substances in Water," Book 5, Chapter A3 in Techniques
of Water Resources Investigations of the United States Geological
Survey, 1972.
701-07 January 1983
-------
Table 1
Chromatographic Conditions
Carrier gas flow 75 mL/min nitrogen
Column temperature 185 C
Detector flame photometric detector — phosphorus mode
Column—1.8-m x 4-mm I.D. 5% DC-200 or 5% QF-1 on 60/80 mesh Gas Chrom Q
701-08 January 1983
-------
Table 2
Retention Times
Relative Retention Time
Parameter
Dioxathion
Diazinon
Dichlof enthion
Methyl Parathion
*
Parathion
Methyl Trithion
Ethion
Carbophenothion
5% DC-200
0.50
0.55
0.71
0.72
1.00
2.2
2.6
2.9
5% OF-1
0.40
0.22
0.34
1.04
1.00
2.7
1.84
2.48
Parathion retention time: DC-200, 3.82 min; QF-1, 4.55 min
701-09 January 1983
-------
Table 3.
-lns*cttcid«s in w
Aldnn
0.019
82.0
113
... 90.1
92.1
.. 97.0
80.5
... 91.2
... tt.l
... 0s. r
... M.O
89. 9
... M.S
91.9
... #4.3
as. 8
... 06.4
... 98.4
... 84.2
93.1
49.3
T.OJ
-7.8
Si
P.M'-
DDD
0.080
92 .S
8D 1
96.0
94 S
1(5.0
90.5
105
00.5
94.0
94.0
03. S
93.0
87.8
»e. a
M.8
85. «
84.1
98.5
92.8
27.0
5.28
— 7.2
18
J>.p'-
DDE
0.040
86. S
94 3
93.5
93 i
93 2
92.1
93. «
98. 8
W.O
98.3
95 A
88.2
87 4
90.7
92.8
92.1
8».S
107
93 4
24 9
4. 99
—4 8
13
P.P'-
DDT
0.081
as o
97 0
103
101
98 0
98 0
99.0
W.O
102
98.0
97 5
92.6
93 6
93.1
93.1
88.6
92.6
104
96 7
17 1
4 14
— 3 3
12
nieldrm
0.019
S»8 8
104
99 «
104
106
97 7
104
105
103
103
99 4
94.9
99 3
IOS
104
102
104
110
102
12 9
3. $9
+2 0
9.J
Endnn
0.040
95.1
98 0
M.O
81 9
81 1
83.4
86.6
85.0
83.3
83.3
(M.O
83.3
89 9
88.2
89.0
00.6
02.4
82.5
87.2
23.0
4.80
-13
22
Hrpia-
rhlor
0.018
86 8
98 *
95.2
96 3
99 6
94 S
95 7
103
1 00
93.7
93 3
90.0
99 2
07.8
88.1
98.1
90.8
88.4
04 5
21 5
4.64
-4 S
U
Hepla-
rfilor
fpoxidr
0.021
94 0
94 9
99.2
98 I
103
94.2
90.1
101
98.3
98.5
92 1
»1.7
94.4
100
93.4
97.6
100
90.6
96 7
12.5
3.54
— 3 3
10
f.indniM*
0 021
90 1
101
99 0
107
97 S
109
103
115
101
111
99 8
94.5
94.4
98.5
108
102
101
117
103
51 1
7. IS
+3 0
17
M.U-
t Fiion
0.181
92 9
IOIS
120
89 3
108
9)1 3
10V
10!)
US
103
97 8
101
IOC!
80.0
M>. 6
103'
100'
107
103
63.4
7.96
+3.0
10
Methyl
paru-
thion
0 082
75 1
94 6
89. S
81 0
87 8
86 3
81.5
97.1
S1.7
99.0
96 6
S3. 4
88.8
9&.1
86.8
93.7
92.1
121
91
96.5
U.82
-9.0
29
P«r«-
thion
0.076
'J9 0
96 0
110
86 0
107
84 1
103
US
97.9
101
84.7
87.8
124
8A.7
88.4
80.4
95.6
110
98.3
139.7
11.3
-1.7
25
1 MeFuTpn. E. F., U«kv R. J.. and Pirkrr. J. H . 1970. Criterion for judciac the arrrptability nl analytical method*
p. 354-358.
\ul. ChemiMry. v. 42,
701-10
January 1983
-------
PART D
QUALITY ASSURANCE REQUIREMENTS
-------
PART D
Quality Assurance Requirements
Part D contains quality assurance (QA) requirements for collection,
preservation and handling of samples, quality control (QC), and safety.
Scope and Application
These requirements are designed for applicability to a wide variety of
analytical method types. QA for an analytical procedure may vary as a
function of method type. However, these requirements are applicable to all
nonconventional pesticide methods enclosed herein. The Agency has also
presented quality assurance information available from the industry,
contractors, and EMSL as shown in methods found in Parts A, B, and C of this
document.
QA requirements are presented in three procedures. The collection, preser-
vation, and handling of samples including collecting grab and composite
samples are included below under Procedure A. QC is a program developed to
assure the quality of data. It involves a rigorous format that includes a
control over performance of the laboratory and method analysis. It also
includes calibration of instruments, duplication of sample analyses to
determine precision, and spiking with a known concentration of compounds to
determine percent recoveries and the suitability of the method for the matrix
of concern. Details of this program are found in Procedure B below.
Laboratory safety procedures involved when using the analytical methods are
presented in Procedure C.
Procedure A—Requirements for Sample Collection, Preservation, and Handling
1. Grab samples must be collected in glass containers. Conventional
sampling practices should be followed; however, the bottle must not
be prerinsed with the sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with program requirements. Automatic sampling equipment must be as
free as possible of plastic tubing and other potential sources of
contamination.
2. All samples must be adjusted to pH between 5 and 9. When analysis
of chlorinated pesticides is to be made it should be preserved with
0.008 percent ^28203.
3. The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
4. All samples must be extracted within 7 days and completely analyzed
within 40 days of extraction for GC, HPLC, and TLC procedures.
5. All samples must be analyzed within 7 days of collection for
colorimetric and titrametric procedures.
Procedure B—Quality Control Requirements
1. The Agency requires that each laboratory operate a formal quality
control program. The minimum requirements of this program consist
D-l January 1983
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of an initial demonstration of laboratory capability and the analysis of
spiked samples as a continuing check on performance. The laboratory is
required to maintain performance records to define the quality of data
generated.
(a) Before performing any analyses, the analyst must demonstrate the
ability to generate acceptable accuracy and precision. This ability
is established as described below.
(b) In recognition of the rapid advances occurring in chromatography, the
analyst is permitted certain options to improve the separations cr
lower the cost of measurements. Each time such modifications to the
method are made, the analyst is required to repeat the accuracy and
precision procedure.
(c) The laboratory must spike and analyze a minimum of 10 percent of all
samples to monitor continuing laboratory performance.
To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
(a) Select a representative spike concentration for each compound to be
measured.
(b) Add the known amount of standard to each of a minimum of four 1,000-ml
aliquots of reagent water. A representative wastewater may be used in
place of the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike level must
exceed twice the background level for the test to be valid.
(c) Calculate the average percent recovery (R) and the standard deviation
of the percent recovery (s) for the results. Wastewater background
corrections must be made before R and s calculations are performed.
(d) Using average recovery and standard deviation expected for each method
parameter, compare these values to R and s. If the data are not
comparable, review potential problem areas and repeat the test.
The analyst must calculate method performance criteria and define the
performance of the laboratory for each spike concentration and parameter
being measured.
(a) Calculate upper and lower control limits for method performance as
follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as noted above. The UCL and LCL can be
used to construct control charts that are useful in observing trends
in performance.
(b) The laboratory must develop and maintain separate accuracy statements
of laboratory performance for wastewater samples. An accuracy
statement for the method is defined as R+s. The accuracy statement
should be developed by the analysis of four aliquots of wastewater,
followed by the calculation of R and s. Alternatively, the analyst
may use four wastewater data points gathered through the requirement
for continuing quality control. The accuracy statements should be
updated regularly.
D_2 January 1983
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4. The laboratory is required to collect in duplicate a portion of its
samples to monitor spike recoveries. The frequency of spiked sample
analysis must be at least 10 percent of all samples or one spiked sample
per month, whichever is greater. One aliquot of the sample must be spiked
and analyzed. If the recovery for a particular parameter does not fall
within the control limits for method performance, the results reported for
that parameter in all samples processed as part of the same set must be
qualified. The laboratory should monitor the frequency of data qualified
to ensure that it remains at or below 5 percent.
5. Before processing any samples, the analyst must demonstrate through the
analysis of a 1-liter aliquot of reagent water that all glassware and
reagents interferences are under control. Each time a set of samples is
extracted or there is a change in reagents, a laboratory reagent blank
must be processed as a safeguard against laboratory contamination.
6. It is recommended that the laboratory adopt additional quality assurance
practices for use with these method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of the
samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. When doubt exists over the identification of a peak
on a chromatogram, confirmatory techniques such as gas chromatography with
a dissimilar column, specific element detector, or mass spectrometer must
be used. Whenever possible, the laboratory should perform analysis of
quality control materials and participate in relevant performance
evaluation studies.
Procedure C—Safety Requirements
1. The toxicity or carcinogenicity of each reagent used in a method has not
been precisely defined; however, each chemical compound must be treated as
a potential health hazard. From this viewpoint, exposure to these
chemicals must be reduced to the lowest possible level by whatever means
available. The laboratory is responsible for maintaining a current
awareness file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of material data
handling sheets should also be made available to all personnel involved in
the chemical analysis.
2. Primary standards of toxic compounds should be prepared in a hood,
especially when classified or tentatively classified as known or suspected
human or mammalian carcinogens.
3. For those compounds classified as potential explosives the following
precautions must be followed:
(a) Use only a well ventilated hood—do not breathe vapors.
(b) Use a safety screen.
(c) Use mechanical pipetting aides.
(d) Do not heat above 90°C—EXPLOSION may result.
(e) Avoid grinding surfaces, ground glass joints, sleeve bearings, glass
stirrers—EXPLOSION may result.
(f) Store away from alkali metals—EXPLOSION may result.
(g) Solutions may decompose rapidly in the presence of solid materials
such as copper powder, calcium chloride, and boiling chips.
D-3 January 1983
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References
1. "Methods for Organic Chemicals Analyses in Municipal and Industrial
Wastewater," July 1982, U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio
45668.
2. Guidelines Establishing Test Procedures for the Analysis of Pollutants;
Proposed Regulations, 40 Code of Federal Regulations (CFR)< Part 136,
Published in Federal Register, 44, 69464.
3. "Determination of Organochlorine Pesticides in Industrial and Municipal
Wastewater," Method 608.1, EPA No. 600/4-82-003, NTIS No. PB82-155979,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
4. "Determination of Organophosphorus Pesticides in Industrial and
Municipal Wastewater," Method 614, EPA No. 600/4-82-004, NTIS No. PB82-
155987, January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
5. "Determination of Chlorinated Herbicides in Industrial and Municipal
Wastewater," Method 615, EPA No. 600/4-82-005, NTIS Mo. PB82-155995,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
6. "Determination of Organohalide Pesticides & PCB's in Industrial and
Municipal Wastewater," Method 617, EPA No. 600/4-82-006, NTIS No. PB82-
156001, January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
7. "Determination of Triazine in Industrial and Municipal Wastewater,"
Method 619, EPA No. 600/4-82-007, NTIS No. PB82-156019, January 1982,
National Technical Information Center, 5285 Port Royal Road,
Springfield, VA 22165.
8. "Determination of Organophosphorus Pesticides in Industrial and
Municipal Wastewater," Method 622, EPA No. 600/4-82-008, NTIS No. PB82-
156027, January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
9. "Determination of Dinitroaniline in Industrial and Municipal
Wastewater," Method 627, EPA No. 600/4-82-009, NTIS No. PB82-156035,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
10. "Determination of Cyanazine in Industrial and Municipal Wastewater,"
Method 629, EPA No. 600/4-82-010, NTIS No. PB82-156043, January 1982,
National Technical Information Center, 5285 Port Royal Road,
Springfield, VA 22165.
D-4 January 1983
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\
11. "Determination of Dithiocarbamate in Industrial and Municipal
Wastewater," Method 630, EPA No. 600/4-82-011, NTIS No. PB82-
156050,January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
12. "Determination of Benomyl and Carbendazim in Industrial and Municipal
Wastewater," Method 631, EPA No. 600/4-82-012,' NTIS No. PB82-156068,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
13. "Determination of Carbamate and Urea Pesticides in Industrial and
Municipal Wastewater," Method 632, EPA No. 600/4-82-014, NTIS No. PB82-
156084, January 1982, National Technical Information Center, 5285 Port
Royal Road, Springfield, VA 22165.
14. "Determination of Organonitrogen Pesticides in Industrial and Municipal
Wastewater," Method 633, EPA No. 600/4-82-013, NTIS No. PB82-156076,
January 1982, National Technical Information Center, 5285 Port Royal
Road, Springfield, VA 22165.
D~5 January 1983
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APPENDICES
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Appendix A
Definition and Procedure for the Determination
of the Method Detection Limit
The method detection limit (MOD is defined as the minimum concentration of a
substance that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero and determined from analysis of a
sample in a given matrix containing analyte.
Scope and Application
This procedure is designed for applicability to a wide variety of sample types
ranging from reagent (blank) water containing analyte to wastewater containing
analyte. The MDL for an analytical procedure may vary as a function of sample
type. The procedure requires a complete, specific and well defined analytical
method. It is essential that all sample processing steps of the analytical method be
included in the determination of the method detection limit.
The MDL obtained by this procedure is used to judge the significance of a single
measurement of a future sample.
The MDL procedure was designed for applicability to a broad variety of physical
and chemical methods. To accomplish this, the procedure was made device- or
instrument-independent.
Procedure
1. Make an estimate of the detection limit using one of the following:
(a) The concentration value that corresponds to an instrument signal/noise
ratio in the range of 2.5 to 5. If the criteria for qualitative identification of
the analyte is based upon pattern recognition techniques, the least
abundant signal necessary to achieve identification must be considered in
making the estimate.
(b) The concentration value that corresponds to three times the standard
deviation of replicate instrumental measurements for the analyte in
reagent water.
(c) The concentration value that corresponds to the region of the standard
curve where there is a significant change in sensitivity at low analyte
concentrations, i.e., a break in the slope of the standard curve.
(d) The concentration value that corresponds to known instrumental
limitations
It is recognized that the experience of the analyst is importarw to this process.
However, the analyst mustinclude the above considerations in the estimate
of the detection limit.
2. Prepare reagent (blank) water that is as free of analyte as possible. Reagent or
interference free water is defined as a water sample in which analyte and
mterferent concentrations are not detected at the method detection limit of
each analyte of interest. Interferences are defined as systematic errors in the
measured analytical signal of an established procedure caused by the
presence of interfering species (interferent). The mterferent concentration is
presupposed to be normally distributed in representative samples of a given
matrix
3. (a) If the MDL is to be determined in reagent water (blank), prepare a
laboratory standard (analyte in reagent water) at a concentration which is
at least equal to or in the same concentration range as the estimated MDL.
(Recommend between 1 and 5 times the estimated MDL.) Proceed to Step
4.
A-1 January 1983
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(b) If the MDL is to be determined in another sample matrix, analyze the
sample. If the measured level of the analyte is in the recommended range
of one to five times the estimated MDL, proceed to Step 4.
If the measured concentration of analyte is less than the estimated MDL,
add a known amount of analyte to bring the concentration of analyte to
between one and five times the MDL. In the case where an interference is
coanalyzed with the analyte-
If the measured level of analyte is greater than five times the estimated
MDL, there are two options:
(1) Obtain another sample of lower level of analyte in same matrix if
possible.
(2) The sample may be used as is for determining the MDL if the analyte
level does not exceed 10 times the MDL of the analyte m reagent
water. The variance of the analytical method changes as the analyte
concentration increases from the MDL, hence the MDL determined
under these circumstances may not truly reflect method variance at
lower analyte concentrations.
4. (a) Take a minimum of seven aliquots of the sample to be used to calculate
the MDL and process each through the entire analytical method. Make all
computations according to the defined method with final results in the
method reporting units. If blank measurements are required to calculate
the measured level of analyte. obtain separate blank measurements for
each sample aliquot analyzed. The average blank measurement is
subtracted from the respective sample measurements.
(b) It may be economically and technically deirable to evaluate the estimated
MDL before proceeding with 4a. This will: (1) prevent repeating this entire
procedure when the costs of analyses are high and (2) insure that the
procedure is being conducted at the correct concentration. It is quite
possible that an incorrect MDL can be calculated from data obtained at
many times the real MDL even though the background concentration of
analyte is less than five times the calculated MDL To insure that the
estimate of the MDL is a good estimate, it is necessary to determine that a
lower concentration of analyte will not result in a significantly lower MDL.
Take two aliquots of the sample to be used to calculate the MDL and
process each through the entire method, including blank measurements
as described above in 4a Evaluate these data:
(1) If these measurements indicate the sample is in the desirable range for
determining the MDL, take five additional aliquots and proceed. Use
all seven measurements to calculate the MDL.
(2) If these measurements indicate the sample is not in the correct range,
reestimate the MDL, obtain new sample as in 3 and repeat either 4a or
4b.
5. Calculate the variance (S2) and standard deviation (S) of the replicate
measurements, as follows:
s-^
S = (S2)''2
where: the x,, i = 1 to n are the analytical results in the final method reporting
units obtained from the n sample aliquots and j- X,2 refers to the sum of
the X values from i = 1 to n. ' = '
6. (a) Compute the MDL as follows
MDL= tw-i. i-a. 991 (S)
A-2 January 1983
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where.
MDL = the method detection
tm-i. i-a = 991 = the students' t value appropriate for a 99% confidence
level and a standard deviation estimate with n-1 degrees
of freedom. See Table.
S = standard deviation of the replicate analyses.
(b) The 95% confidence limits for the MDL derived in 6a are computed
according to the following equations derived from percentiles of the chi
square over degrees of freedom distribution (XVdf) and calculated as
follows:
MOUct = 0.69 MDL
MDLucL = 1.92 MDL
where MDLLCL and MDLucL are the lower and upper 95% confidence limits
respectively based on seven aliquots.
7. Optional iterative procedure to verify the reasonableness of the estimated
MDL and calculated MDL of subsequent MDL determinations.
(a) If this is the initial attempt to compute MDL based on the estimated MDL
in Step 1, take the MDL as calculated in Step 6, spike in the matrix at the
calculated MDL and proceed through the procedure starting with Step 4.
(b) If the current MDL determination is an iteration of the MDL procedure for
which the spiking level does not permit qualitative identification, report the
MDL as that concentration between the current spike level and the
previous spike level which allows qualitative identification
(c) If the current MDL determination is an iteration of the MDL procedure and
the spiking level allows qualitative identification, use S2 from the current
MDL calculation and S2 from the previous MDL calculation to compute the
F ratio.
if |f < 3.05
SB
then compute the pooled standard deviation by the following equation:
12
S2
if -Tj > 3.05, respike at the last calculated MDL and process the samples
Sa
through the procedure starting with Step 4.
(c) Use the SPooi«
-------
determine the MDL must also be identified with the MDL value. Report the mean
analyte level with the MDL. If a laboratory standard or a sample that contained a
known amount analyte was used for this determination, report the mean recovery,
and indicate if the MDL determination was iterated.
If the level of the analyte in the sample matrix exceeds 10 times the MDL of the
analyte in reagent water, do not report a value for the MDL.
Reference
Glaser, J. A., Foerst, D. L, McKee, G. D., Quave, S. A., and Budde, W. L, "Trace
Analysis for Wastewaters," Environmental Science and Technology. 15. 1426
(1981).
Table of Students' t Values at the 99 Percent Confidence Level
Number of Degrees of Freedom
Replicates (n-1) fm-i, i-a . .MI
7 6 3.143
8 7 2.998
9 8 2.896
10 9 2.821
11 10 2.764
16 15 2.602
21 20 2.528
26 25 2.485
31 30 2.457
61 60 2.390
» « 2.326
A-4 January 1983
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APPENDIX B
ABBREVIATIONS, ACRONYMS, AND OTHER TERMS USED IN THIS DOCUMENT
Act - The Clean Water Act
Agency - The U.S. Environmental Protection Agency
AOP - Ambam oxidation product
BAT - The best available technology economically achievable under Section
301(b)(2)(E) of the Act
BBTAC - l,l'-(2-Butenylene)bis(3,5,7-triaza-l-azo niaadamantane chloride)
Cf - Confirmation
Clean Water Act - (CWA) The Federal Water Pollution Control Act Amendments
of 1972 (33 U.S.C. 1251 et seq.) as amended by the Clean
Water Act of 1977 (Public Law 95-217)
Conventional Pollutants - For the pesticide industry conventional pollutants
are defined as BOD, TSS, and pH.
BCD - Electron capture detector
Effluent Limitations - Any restrictions established by a state or the
Administrator on quantities, rates, and
concentrations of chemical, physical, biological, and
other constituents which are discharged from point
sources into navigable waters, the waters of the
contiguous zone, or the ocean, including schedules of
compliance
FID - Flame ionization detector
FPD - Flame photometric detector
GC - Gas chromatography
GC/MS - Gas chromatograpby/mass spectrometry
HPLC - High pressure liquid chromatography
Nonconventional Pollutants - For the pesticide industry nonconventional
pollutants are defined as nonpriority pollutant
pesticides, COD, ammonia, and manganese
B-l January 1983
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Nonconventional Pesticide Pollutants - All pesticides active ingredients
which are included under the scope of
effluent limitations and standards
for BAT, NSPS, and Pretreatment
Standards but were not defined as
"toxic" under the Clean Water Act.
Non-toxic Pollutants - All conventional or nonconventional pollutants which
are currently regulated or proposed to be regulated
NPD - Nitrogen phosphorus detector
NPDES permit - A National Pollutant Discharge Elimination system permit
issued under Section 402 of the Act
PCP - Pentachlorophenol
Pesticide - Any technical grade ingredient used for controlling, preventing,
destroying, repelling, or mitigation any pest
Pesticide Active Ingredient - The ingredient of a pesticide which is
intended to prevent, destroy, repel, or
mitigate any pest. The Active ingredients may
make up only a small percentage of the final
product which also consists of binders,
fillers, diluents, etc.
Pesticide Industry - The combined facilities which manufacture as well as
formulate and/or package pesticides.
Point Source - Any discernable, confirmed and discrete conveyance, including
but not limited to any pipe, ditch, channel, tunnel, conduit,
well, discrete fissure, container, rolling stock,
concentrated animal feeding operation, or vessel or other
floating craft, from which pollutants are or may be
discharged. This term does not include return flow from
irrigated agriculture.
Pretreatment Standards - Any restrictions established by the states or the
Administrator on quantities, rates and
concentrations of chemical, physical, biological
and other constituents which are discharged to
Publically Owned Treatment Works.
Priority Pollutants - See Toxic Pollutants
QA - Quality assurance. In this document quality assurance pertains to
requirements for sample collection, preservation, and handling; quality
control; and safety
QC - Quality control
SPM - Spectrometry
B-2 January 1983
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308 Survey - A questionnaire drafted by EPA, approved by the National
Agricultural Chemicals Association and the Office of Management
and Budget (OMB #158-R0160), and subsequently distributed to
pesticide manufacturers in July 1978. The primary purpose of
the survey was to obtain basic data concerning manufacturing,
disposal, and treatment, as well as potential sources of toxic,
conventional, and nonconventional pollutants.
TLC - Thin layer chromatography
TLV - Threshhold limit value
Toxic Pollutants - Those 65 pollutants and classes of pollutants specified
as an outgrowth of the 1976 Settlement Agreement and
declared by Congress to be "toxic" under Section 307(a)
of the Clean Water Act of 1977.
TTN - Titration
TWA - Time weighted average
UV - Ultraviolet absorbance
ZAC - Zinc ammonium carbamate
Verification Program - A sampling and analysis project conducted by private
contractors to the Agency at selected plants in the
pesticide industry. The purpose of the program was
to verify the presence of the toxic, conventional,
and nonconventional pollutants identified during the
screening program and to determine the levels of
these pollutants present in process wastewaters prior
to and after application of the various control and
treatment technologies employed in the industry.
** •* *U.S. GOVERNMENT PRINTING OFFICE : 1983 0-381-545/3801 JaUUary 1983
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