United States
Environmental Protection
Agency
EPA-821-R-93-01G-A
August 1993
Revision 1
SEPA Methods for the Determination
of Nonconventional Pesticides in
Municipal and Industrial Wastewater
Volume I
, - Printed on Hecyc'cd Pa.oe
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Acknowledgments
This methods compendium was prepared under the direction of Thomas E. Fielding, Ph.D., and
William A. Telliard of the Engineering and Analysis Division within EPA's Office of Water. This
document was prepared under EPA Contract No. 68-C9-0019 by the Environmental Services Division
of DynCorp Viar, Inc. The methods contained in this compendium were developed by the U.S. EPA
Environmental Monitoring Systems Laboratory in Cincinnati, Ohio.
Disclaimer
This methods compendium has been reviewed by the Engineering and Analysis 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.
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Introduction
The Environmental Protection Agency (EPA) is promulgating effluent limitations guidelines and stan-
dards for the Pesticide Chemicals Industry at 40 CFR Part 455 to control the discharge of pollutants,
including certain pesticide active ingredients, into the waters of the United States. This compendium of test
procedures (methods) supports this rulemaking.1 The purpose of publishing these methods in a compen-
dium is to create a single reference for analysts seeking to measure infrequently determined active
ingredients.
This volume contains most of the methods referenced in the proposed rule for the Pesticide Chemicals
Manufacturing Subcategory (57 FR 12560). Volume n (EPA-821-R-93-010-B) supplements this volume
and contains the remaining methods referenced in the proposal, except for those already promulgated at
40 CFR Part 136.
Many of the methods in this two-volume compendium were listed in Appendix E of EPA's original
promulgation of rules for the Pesticides Category (50 FR 40708). These methods were withdrawn as a part
of the remand of the pesticides rules in 1986 (51 FR 44911). Some of the methods that appeared in the
original promulgation have been updated to include more analytes and/or include additional performance
data.
Many of the methods in the original promulgation were also published by EPA's Effluent Guidelines
Division in 1983 as publication EPA 440/1-83/079-C. This publication is now out of print. The publica-
tion included industry methods and EPA-developed methods.
The test procedures in this two-volume compendium are methods developed by EPA's Environmental
Monitoring Systems Laboratory in Cincinnati, Ohio (EMSL-Ci), methods developed by EPA's Engi-
neering and Analysis Division (BAD; formerly the Industrial Technology Division and the Effluent
Guidelines Division) within EPA's Office of Science and Technology (formerly the Office of Water
Regulations and Standards), and an industry method for organotin compounds.
Volume I contains the 600-series and 1600-series methods and the one industry method for organotin.
The 600-series methods, written by EMSL-Ci, were developed in the late 1970's and early 1980's. Some
have been updated in the interim. The 1600-series methods, written by BAD, were developed to measure
active ingredients in support of the pesticides rulemaking, and have therefore been applied to the specific
wastewater for which they were intended. The industry method for total tin/triorganotin is Method EV-
024/EV-025. Volume n contains 13 of the 500-series methods and one 200-series method developed by
EMSL-Ci since the early 1980's for the determination of pesticide active ingredients. A summary of the
analytes that may be detected using the methods in this compendium is shown in the cross-reference list.
Some analytes can be detected by more than one method.
Questions about the content of this document or Volume n should be directed to:
W. A. Telliard
U.S. EPA (WH-552)
Office of Science and Technology
Engineering and Analysis Division
401 M Street, SW
Washington, DC 20460
(202) 260-5131
1 The methods in this volume were previously published in EPA 821 RR-92-002. This volume is a revision
of that publication and supersedes it.
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Questions, as well as requests for Volume II, may also be directed to the following address:
Sample Control Center
Operated by DynCorp Viar, Inc.
P.O. Box 1407
Alexandria, VA 22313
(703) 557-5040
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Contents
Method
No. Parameters Page
604.1 Hexachlorophene and Dichlorophen 1
608.1 Organochlorine Pesticides 19
608.2 Certain Organochlorine Pesticides 39
614 Organophosphorus Pesticides 57
614.1 Organophosphorus Pesticides 77
615 Chlorinated Herbicides 95
616 Certain Carbon-, Hydrogen-, and Oxygen-Containing Pesticides 115
617 Organohalide Pesticides and PCBs 135
618 Volatile Pesticides 167
619 Triazine Pesticides 183
620 Diphenylamine 203
622 Organophosphorus Pesticides 221
622.1 Thiophosphate Pesticides 243
627 Dinitroaniline Pesticides 263
629 Cyanazine 281
630 Dithiocarbamate Pesticides 297
630.1 Dithiocarbamate Pesticides 311
631 Benomyl and Carbendazim 325
632 Carbamate and Urea Pesticides 341
632.1 Carbamate and Amide Pesticides 361
633 Organonitrogen Pesticides 375
633.1 Neutral Nitrogen-Containing Pesticides 393
634 Thiocarbate Pesticides 413
635 Rotenone 433
636 Bensulide 451
637 MBTS and TCMTB 467
638 Oryzalin 485
639 Bendiocarb _ 503
640 Mercaptobenzothiazole 521
641 Thiabendazole 539
642 Biphenyl and Ortho-Phenylphenol 553
643 Bentazon 567
644 Picloram 581
645 Certain Amine Pesticides and Lethane 597
646 Dinitro Aromatic Pesticides 615
1656 Organohalide Pesticides 631
1657 Organophosphorus Pesticides 671
1658 Phenoxy-Acid Herbicides 707
1659 Dazomet 741
1660 Pyrethrins and Pyrethroids 757
1661 Bromoxynil 775
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Contents (com.)
Appendix
Methods EV-024 and EV-025:
Analytical Procedures for Determining Total Tin and Triorganotin in Wastewater 791
IV
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Cross-Reference
Analytes shown in bold italic type are regulated under the Pesticide Chemicals Manufacturing
Rule found at 40 CFR Part 455.
Applicable Method(s)
Analytes CAS No. Volume I Volume II
Acenaphthylene 208-96-8 525.1
Acephote 30560-19-1 1656, 1657 -
Acifluorfen 50594-66-6 ~ 515.1, 515.2, 555
Alachlor 15972-60-8 645, 1656 505, 507, 525.1
Aldicarb (Temk) 116-06-3 ~ 531.1
Aldicarb sulfoxide 1646-87-3 '. 531.1
Aldicarb sulfone 1646-88-4 531.1
Aldrin 309-00-2 617, 1656 505, 508, 525.1
Allethrin (Pynamin) 584-79-2 1660 -
Aluminum 7429-90-5 200.9
Ametryn 834-12-8 619 507, 525.7
Aminocarb 2032-59-9 632 -
Amobam 3566-10-7 630,630.1 -
Anthracene 120-12-7 525.1
Antimony 7440-36-0 200.9
AOP - 630 --
Aroclor 1232 11141-16-5 505, 508
Aroclor 1221 11104-28-2 505, 508
Aroclor 1260 11096-82-5 505, 508
Aroclor 1242 53469-21-9 505, 508
Aroclor 1254 11097-69-1 505, 508
Aroclor 1248 12672-29-6 505, 508
Aroclor 1016 12674-11-2 505, 508
Arsenic 7440-38-2 200.9
Aspon 3244-90-4 622.1 --
Atraton 1610-17-9 619 507, 525.1
Atrazine 1912-24-9 619, 1656 505, 507, 525.1
Azinphos ethyl 2642-71-9 1657 -
Azinphos methyl (Guthion) 86-50-0 614, 622, 1657 -
Barban 101-27-9 632 -
Basalin (Fluchloralin) 33245-39-5 646 -
Baygon 114-26-1 531.1
Bayleton (Triadimefon) 43121-43-3 533, 1656 507, 525. /
Baythroid (Cyfluthrin) 68359-37-5 1660 -
Bendiocarb 22781-23-3 639 -
Benefin (Benfluralin) 1861-40-1 627, 1656 -
Benfluralin (Benefin) 1861-40-1 627, 1656 -
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Benomyl 17804-35-2 631 -
Bensulide 741-58-2 636 -
Bentazon (Basagran) 25057-89-0 643 515.1, 515.2, 555
Benzidine 92-87-5 553
Benzoylprop ethyl 33878-50-1 553
Benzo[a]pyrene 50-32-8 525.1
Benzo[fc]fluoranthene 205-99-2 525.1
Benzo[£,/u]perylene 191-24-2 525.1
Benzo[£]fluoranthene 207-08-9 525.1
Benz[a]anthracene 56-55-3 525.1
Beryllium 7440-41-7 200.9
a-BHC 319-84-6 617, 1656 525.1
0-BHC 319-85-7 617, 1656 525.1
7-BHC 58-89-9 617, 1656 525.1
5-BHC 319-86-8 617, 1656 525.1
Biphenyl 92-52-4 642 -
Bis(2-ethylhexyl)adipate 103-23-1 506, 525.1
Bis(2-ethylhexyl)phthalate .... 117-81-7 506, 525.1
Bolster (Sulprofos) 35400-43-2 622, 1657 -
Bromodl Salts & Esters 314-40-9 633, 1656 507, 525.1
Bromoxynil 1689-84-5 1661 -
Bromoxynil octanoate 1689-99-2 1656 -
Busan 40 51026-28-9 630, 630.1 -
Busan 85 128-03-0 630, 630.1 -
Butachlor 23184-66-9 645, 1656 507, 525.1
Butylate 2008-41-5 634 507, 525.1
Butylbenzyl phthalate 85-68-7 506, 525.1
Cadmium 7440-43-9 200.9
Caffeine 58-08-2 553
Captafol 2425-06-1 1656 ~
Captan 133-06-2 617, 1656 -
Carbam-S 128-04-1 630, 630.11 -
Carbaryl 63-25-2 632 531.1, 553
Carbendazim 10605-21-7 631 ~
Carbofuran 1563-66-2 632 531.1
1 Carbam-S was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Carbam-S.
VI
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Cross-Reference
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Carbophenothion 786-19-6 617, 1656 --
Carboxin 5234-68-4 507, 525.1
CDN 97-00-7 646 --
Chloramben 133-90-4 515.1, 555
Chlordane 57-74-9 617, 1656 505, 508
a-Chlordane 5103-71-9 1656 505, 508, 525.1
7-Chlordane 5103-74-2 1656 505, 508, 525.1
Chlorfevinphos 470-90-6 1657 -
Chlorobenzilate 510-15-6 608.1, 1656 508, 525.1
2-Chlorobiphenyl 2051-60-7 525.1
Chloroneb 2675-77-6 608.1, 1656 508, 525.1
o-Chlorophenyl thiourea 5344-82-1 553
Chloropicrin 76-06-2 618 -
Chloropropylate 5836-10-2 608.1, 1656 -
Chlorotholonil 1897-45-6 608.2, 1656 508, 525.1
Chlorpropham 101-21-3 632 507, 525.1
Chlorpyrifos methyl 5598-13-0 622, 1657 -
CMorpyrifos 2921-88-2 622, 1657 508
Chromium 7440-47-3 200.9
Chrysene 218-01-9 525.1
Cobalt 7440-48-4 200.9
Copper 7440-50-8 200.9
Coumaphos 56-72-4 622, 1657 -
Crotoxyphos 7700-17-6 1657 -
Cyarumne 21725-46-2 629 5072
Cycloate 1134-23-2 634 507, 525.1
Cycloprate 54460-46-7 616 -
Cyfluthrin (Baythroid) 68359-37-5 1660 -
2,4-D Salts & Esters 94-75-7 615, 1658 515.1, 575.2, 555
Dacthal (DCPA) 1861-32-1 608.2, 1656 . . 508, 515.1, 515.2, 525.1
Dalapon 75-99-0 615, 1658 515.1
Dazomet 533-74-4 630, 630.1/1659 -
2,4-DB 94-82-6 615, 1658 5/5.1, 515.2, 555
2 Cyanazine was not specifically listed in this method, but data from EMSL-Ci and from industry indicate that
this method is applicable.
3 Dazomet was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Dazomet.
vii
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Cross-Reference (com.)
Applicable MethodfsJ
Analyte CAS No. Volume I Volume II
DBCP 96-12-8 1656 -
DCPA (Docthol) 1861-32-1 608.2, 1656 . . 508, 515.1, 515.2, 525.1
4,4'-DDD 72-54-8 617, 1656 508, 525.1
4,4'-DDE 72-55-9 617, 1656 508, 525.1
4,4'-DDT 50-29-3 617, 1656 508, 525.1
Deet 134-62-3 633 ~
DBF 78-48-8 1657 -
Demeton 8065-48-3 614, 622, 1657 -
Di-n-butyl phthalate 84-74-2 506, 525.1
Di-n-octyl phthalate 117-84-0 506
Diallate 2303-16-4 1656 --
Diozinon 333-41-5 614, 622, 1657 507, 525.1
Dibenz[a,A]anthracene 53-70-3 525.1
Dibromochloropropane 96-12-8 608.1 ~
Dicamba 1918-00-9 615, 1658 515.1, 515.2, 555
Dichlofenthion 97-17-6 622.1, 1657 -
Dichlone 117-80-6 1656 -
Dichloran 99-30-9 608.2,617 -
3,3'-Dichlorobenzidine 91-94-1 553
3,5-Dichlorobenzoic acid 51-36-5 515.1, 515.2, 555
2,3-Dichlorobiphenyl 16605-91-7 525.1
Dichlorophen 97-23-4 604.1 ~
Dichhrprop Salts & Esters 120-36-5 615, 1658 515.1, 515.2, 555
DicMorvos 62-73-7 622, 1657 507, 525.1
Dicofol 115-32-2 617, 1656 -
Dicrotophos 141-66-2 1657 ~
Dieldrin 60-57-1 617, 1656 505, 508, 525.1
Diethyl phthalate 84-66-2 506, 525.1
Dimethoate 60-51-5 1657 -
3,3'-Dimethoxybenzidine 119-90-4 553
Dimethyl phthalate 131-11-3 506, 525.1
3,3'-Dimethylbenzidin 119-93-7 553
Dinocap 39300-45-3 646 -
Dinoseb 88-85-7 615, 1658 5/5.1, 515.2, 555
Dioxathion 78-34-2 614.1, 1657 -
Diphenamid 957-51-7 645 507, 525.1
Diphenylamine 122-39-4 620 -
Disulfoton 298-04-4 614, 622, 1657 507, 525.1
VIII
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Disulfoton sulfone 2497-06-5 507, 525.1
Disulfoton sulfoxide 2497-07-6 507, 525.1
Diuron 330-54-1 632 555
Endosulfan sulfate 1031-07-8 617, 1656 508, 525.1
Endosulfan I 959-98-8 617, 1656 508, 525.1
Endosulfan II 33213-65-9 617, 1656 508, 525.1
Endotholl Salts & Esters 145-73-3 - 548, 548.1
Endrin 72-20-8 617, 1656 505, 508, 525.1
Endrin aldehyde 7421-93-4 617, 1656 508, 525.1
Endrin ketone 53494-70-5 1656 -
EPN 2104-64-5 614.1, 1657 -
EPTC 759-94-4 634 507, 525.1
Ethalfluralin 55283-68-6 627, 1656 -
Etfuon 563-12-2 614, 614.1, 1657 -
Ethoproprophos (Ethoprop) . . 13194-48-4 622, 1657 507, 525.7
Ethylene dibromide 106-93-4 618 -
Ethylene thiourea 96-45-7 553
Etridiazole 2593-15-9 608.1, 1656 508, 525.1
EXD 502-55-6 630.1 -
Famphur 52-85-7 622.1, 1657 -
Fenamiphos 22224-92-6 507, 525.1
Fenarimol (Rubigan) 60168-88-9 633.1, 1656 507, 525.1
Fenitrothion 122-14-5 622.1 -
Fensulfothion 115-90-2 622, 1657 ~
Fenthion 55-38-9 622, 1657 ~
Fenuron 101-42-8 632 -
Fenuron-TCA 4482-55-7 632 -
Fenvalerate (Pydrin) 51630-58-1 1660 -
Ferbam 14484-64-1 630, 630.1 -
Fluchloralin (Basalin) 33245-39-5 646 -
Fluometuron 2164-17-2 632 -
Fluorene 86-73-7 525.1
Fluridone 59756-60-4 645 507, 525.1
Fonophos 944-22-9 622.1 -
Gardoprim (Terbuthylazine) . . 5915-41-3 619, 1656 -
Glyphosate 1071-83-6 - 547
Guthlon (Mnphos methyl) 56-50-0 614, 622, 1657 -
/3-HCH 319-85-7 508
7-HCH (Lindane) 58-89-9 505, 508, 525.1
IX
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
6-HCH 319-86-8 508
a-HCH 319-84-6 , 508
Heptachlor 76-44-8 617, 1656 505, 508, 525.1
Heptachlor epoxide 1024-57-3 617, 1656 505, 508, 525.1
2,2',3,3',4,4',6-Heptachloro-
biphenyl . 52663-71-5 525.1
Hexachlorobenzene 118-74-1 505, 508, 525.1
2,2',4,4',5,6'-Hexachloro-
biphenyl 6-145-22-4 525.1
Hexachlorocyclopentadiene 77-47-4 505, 525.1
Hexachlorophene 70-30-4 604.1 -
Hexazinone 51235-04-2 633 507, 525.1
3-Hydroxycarbofizran 16655-82-6 - 531.1
5-Hydroxydicamba 7600-50-2 515.1, 515.2, 555
Indeno[l,2,3,c,d]pyrene 193-39-5 525.1
Iron 7439-89-6 200.9
Isodrin 465-73-6 617, 1656 -
Isopropolin (Poarlan) 33820-53-0 627, 1656 -
Kepone 143-50-0 1656 ~
Kinoprene 42588-37-4 616 -
KN Methyl 137-41-7 630, 630.1 -
Lead 7439-92-1 200.9
Leptophos 21609-90-5 1657 -
Lethane 112-56-1 645 -
Lindane (7-HCH) 58-89-9 505, 508, 525.1
Linuron (Lorox) 330-55-2 632 553
Malathion 121-75-5 614, 1657 -
Mancozeb 8018-01-7 630 -
Maneb 12427-38-2 630 -
Manganese 7439-96-5 200.9
MBTS 120-78-5 637 -
MCPA 94-74-6 615, 1658 555
MCPP 7085-19-0 615, 1658 555
Mercaptobenzothiazole 149-30-4 640 —
Merphos 150-50-5 622, 1657 507, 525.1
Metham (Vapam) 137-42-8 630, 630.1 -
Methamidophos 10265-92-6 1657 -
Methiocarb 2032-65-7 632 531.1
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Cross-Reference (Cont.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Methomyl 16752-77-5 632 531.1
Methoprene 40596-69-8 616 -
Methoxychlor 72-43-5 608.2, 617, 1656 505, 508, 525.1
Methyl paraoxon 950-35-6 507, 525.1
Metolachlor 51218-45-2 507, 525.1
Metribuzin 21087-64-9 633, 1656 507, 525.1
Mevinphos 7786-34-7 622, 1657 507, 525.1
Mexacarbate 315-18-4 632 -
MGK 264 113-48-4 633.1 507, 525.1
MGK 326 136-45-8 633.1 -
Mirex 2385-85-5 617, 1656 -
Molinate 2212-67-1 634 507, 525.1
Monocrotophos 6923-22-4 1657 -
Monuron 150-68-5 632 553
Monuron-TCA 140-41-0 632 -
Nobom 142-59-6 630, 630.1 -
Nobonote 138-93-2 630.1 -
Noted 300-76-5 622, 1657 -
Napropamide 15299-99-7 632.1 507, 525.1
Neburon 555-37-3 632 ~
Niacide 8011-66-3 630 -
Nickel 7440-02-0 200.9
Nitrofen (TOK) 1836-75-5 1656 -
4-Nitrophenol 100-02-7 515.1, 555
cis-Nonochlor 5103-73-1 505
trons-Nonochlor 39765-80-5 505, 525.J
Norflurozon 27314-13-2 645, 1656 507, 525.1
2,2',3,3',4,5',6,6'-Octachloro-
biphenyl 40186-71-8 525.1
Organotin (as Tin=7440-31-5) . . . 0-1924 EV-024/025 200.P
Oryzalin 19044-88-3 638 -
Oxamyl 23135-22-0 632 531.1
Paarlan (Isopropalin) 33820-53-0 627, 1656 -
Parathion ethyl 56-38-2 614, 1657 -
Parathion methyl J. . 298-00-0 614, 622, 1657 -
PCB-1016 12674-11-2 617, 1656 -
4 CAS number in Table 7 of proposed rule is for Bis(tributyltin) dodecenyl succinate and is therefore incorrect.
XI
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Cross-Reference
Applicable Method(s)
Analyte CAS No. Volume I Volume II
PCB-1221 11104-28-2 617, 1656 -
PCB-1232 11141-16-5 617, 1656 -
PCB-1242 53469-21-9 617, 1656 -
PCB-1248 12672-29-6 617, 1656 -
PCB-1254 11097-69-1 617, 1656 -
PCB-1260 11096-82-5 617, 1656 -
PCNB 82-68-8 608.1, 617, 1656 -
Pebulate 1114-71-2 634 507, 525.1
Pendimetholin (Prowl) 40487-42-1 1656 -
2,2,3',4,6-Pentachlorobiphenyl . 60233-25-2 525.1
Pentachlorophenol (PCP) 87-86-5 - . . 5/5./, 5/5.2, 525./, 555
Permethrin5 52645-53-1 . . . 608.2, 1656, 1660 508, 525.1
ds-Permethrin6 61949-76-6 1656, 1660 505, 525./
trans-Permethrin7 52645-53-1 1656, 1660 508, 525.1
Perthane 72-56-0 617, 1656 -
Phenanthrene 85-01-8 525.1
Phenothrin (Sumithrin) 26002-80-2 1660 -
o-Phenylphenol 90-43-7 642 -
Phorate 295-02-2 622, 1657 -
Phosmet 732-11-6 622.1, 1657 ~
Phosphamidon 13171-21-6 1657 -
Phosphoramide 680-31-9 1657 -
Picloram 1918-02-1 644 515.1, 515.2, 555
Polyram 9006-42-2 630 -
Profluralin 26399-36-0 627 -
Prometon (PromUol) 1610-18-0 619 507, 525./
Prometryn 7287-19-6 619 507, 525./
Pronamide 23950-58-5 633.1 507, 525./
Propachlor 1918-16-7 608.1, 1656 505, 525./
Propanil 709-P5-5 632.1, 1656 -
Propazine 139-40-2 619, 1656 507, 525./
Propham 122-42-9 632 -
Propoxur 114-26-1 632 -
Detected as cis-Permethrin and trans-Permethrin.
Regulated as Permethrin.
XII
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Cross-Reference (com.)
Applicable Method(s)
Analyte CAS No. Volume I Volume II
Pydrin (Fenvolerote) 51630-58-1 1660 -
Pynamin (Allethrin) 584-79-2 1660 ~
Pyrene 129-00-0 525.1
Pyrethrin I 121-21-1 1660 -
Pyrethrin II 121-29-9 1660 -
Resmethrin 10453-86-8 616, 1660 -
Ronnel 299-84-3 622, 1657 -
Rotenone 83-79-4 635 553
Rubigon (Fenarimol) 60168-88-9 633.1, 1656 507, 525.1
Secbumeton 26259-45-0 619 -
Selenium 7782-49-2 200.9
Siduron 1982-49-6 632 553
Silver 7440-22-4 200.9
Silvex (2,4,5-TP) 93-72-1 615,1658 515.1, 515.2, 555
Simcaine 122-34-9 619, 1656 505, 507, 525.1
Simetryn 1014-70-6 619 507, 525.1
Sodium dimethyldithiocarbamate . 128-04-1 630, 630.1 -
Stirofos (Tetrachlorrinphos) . 2224S-79-97 522, 1*57 507, 525.1
Strobane 8001-50-1 617, 1656 -
Sulfotepp 3689-24-5 1657 -
Sulprofos (Bolster) 35400-43-2 622, 1657 -
Sumithrin (Phenothrin) 26002-80-2 1660 -
Swep 1918-18-9 632 -
2,4,5-T 93-76-5 615, 1658 515.1, 515.2, 555
TCMTB 21564-17-0 637 -
Tebuthiuron (Spike) 34014-18-1 - 507, 525.1
Tentik (Aldicarb) 116-06-3 ~ 531.1
2,2',4,4'-Tetrachlorobiphenyl . . 2437-79-8 525.1
Tetracfdorvinphos (Stirofos) . 2224S-79-97 622, 1657 507, 525.1
TEPP 107-49-3 1657 -
Terbacil 5902-51-2 633, 1656 507, 525.1
Terbufos (Counter) 13071-79-9 614.1, 1657 507, 525.1
Terbuthylazine (Gardoprim) . . 5915-41-3 619, 1656 ~
Terbutryn 886-50-0 619 507, 525.1
Tetramethrin 7696-12-0 1660 -
Thallium 7440-28-0 200.9
7 CAS number in Table 7 of proposed rule is incorrect.
xiii
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Cross-Reference
(cont.)
Applicable Methodfs)
Ana/yte CAS No. Volume I Volume If
Thiabendazole 148-79-8 641 -
Thionazin 297-97-2 622.1 -
Thiram 137-26-8 630, 630.1 -
Tin 7440-31-5 200.9
TOK (Nitrofen) 1836-75-5 1656 ~
Tokuthion 34643-46-4 622, 1657 ~
Toxaphene 8001-35-2 617, 1656 505, 508, 525.1
2,4,5-TP (Silvex) 93-72-1 615, 1658 515.1, 515.2, 555
Triadimefon (Bayleton) 43121-43-3 633, 1656 507, 525.1
2,4,5-Trichlorobiphenyl 15862-07-4 525.1
Trichlorofon 52-68-6 1657 -
Trichloronate 327-98-0 622, 1657 -
Tricresylphosphate 78-30-8 1657 -
Tricyclazole 41814-78-2 633 507, 525.1
Trifluralin 1582-09-8 617, 627, 1656 508, 525.1
Trimethylphosphate 512-56-1 1657 -
Trithion methyl 953-17-3 1657 -
Vacor 53558-25-1 632.1 -
Vapam (Metham) 137-42-8 630, 630.1 -
Vernolate 1929-77-7 634 507, 525.1
ZAC ~ 630 -
Zinc 7440-66-6 200.9
Zineb 12122-67-7 630, 630.1 -
Ziram 137-30-4 630, 630.1 -
XIV
-------
Method 604.1
The Determination of
Hexachlorophene and
D/chlorophen in Municipal and
Industrial Wastewaters
-------
-------
Method 604.1
The Determination of Hexachlorophene and Dichlorophen in
Municipal and Industrial Wastewater Method
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain phenolic pesticides. The following parameters
can be determined by this method:
Parameters CAS No.
Dichlorophen 97-23-4
Hexachlorophene 70-30-4
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the determi-
nation 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 alternative test proce-
dures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for each compound 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 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 using the proce-
dure described in Section 8.2.
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. This method describes analytical conditions for a second liquid chromatographic
column that can be used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is adjusted to pH 4 to 4.5 and extracted
with methylene chloride using a separatory funnel. The methylene chloride extract is dried
and exchanged to methanol during concentration to a volume of 5 mL or less. Liquid chro-
matographic conditions are described which permit the separation and measurement of the
compounds in the extract by HPLC using an ultraviolet detector (UVD).1
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
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.
-------
Method 604.1
3.1.1 Glassware must be scrupulously cleaned.2 Clean all glassware as soon as possible
after use by 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 minutes. Do not heat volumetric
ware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be sub-
stituted 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 in-
verted 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 being sampled. The
acid/base extraction cleanup described in Section 10 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 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 aware-
ness 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 identified3"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-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Aluminum foil may he substituted for PTFE 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 col-
lection 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, fol-
lowed by repeated rinsings with distilled water to minimize the potential for contami-
nation of the sample. An integrating flow meter is required to collect flow-propor-
tional composites.
-------
Method 604.1
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-0250 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 1000-mL (Kontes K-570001-1000 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 equi-
valent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 /* Spherisorb-ODS, 250 mm long by 4.6 mm
or equivalent. This column was used to develop the method performance statements
in Section 14. Alternative columns may be used in accordance with provisions de-
scribed in Section 12.1.
5.6.3 Column 2: Reversed-phase column, 5 n Lichrosorb RP-2, 250 mm long by 4.6 mm
or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm. This detector has proven effec-
tive in the analysis of wastewaters for the parameters listed hi the scope 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.
-------
Method 604.1
6.2 Methylene chloride, methanol, acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous. Heat in a muffle furnace at 400°C overnight.
6.4 Sodium phosphate, monobasic: ACS, crystal.
6.5 IN sodium hydroxide: Dissolve 4.0 grams of NaOH (ACS) in 100 mL of distilled water.
6.6 Phosphoric acid (85%).
6.7 Stock standard solutions (1.00 /ig//*L): 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 mate-
rial. Dissolve the material in distilled-in-glass quality methanol 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 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.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. 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.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid chromatographic system can be calibrated using either the external standard tech-
nique (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 50/50 methanol/water.
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 20 to 50 /nL of each calibration standard, tabulate peak height or
area responses against the mass injected. The results can be used to prepare a calibra-
tion 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.
-------
$04.1
7.2.3 Thefaofliiflg £alfflM9ri Wv^W^lifclfatrdh fiicttS mtist be
shift by jfte^ftSl^elfiei^fWM for
any compound varies from the predicted response by more than ±loWr,3thte3iest must
be repeated using a fresh calibration standard. Alternatively, a new calibration curve
or calibration factor must be prepared for that parameters 'v'••"••*»' T^.^—
7.3 Internal standard calibration procedure: To usththjs;appfpacjfej the,j8naty§SflHB&isdlBel one or
more internal standards similar in analytical behavior,to: Jhe^ieoojppunds MitfltStfi&uiThe
analyst must further demonstrate that the measurement of-the internal standard1 IS ;JlQt affected
by method or matrix interferences. Due to these limitations, no internal standard applicable to
all samples can be suggested. b -r-v -^
7.3.1 Prepare calibration standards at a minimum of three concentration levels fbr^each
parameter of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with 50/50 methanol/water. One of the
standards should be a representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of concen-
trations expected in the sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 20 to 50 pL 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:
Equation 1
RF = _
where
As = Response for the parameter to be measured
Au = Response for the internal standard
C^ = Concentration of the internal standard, in \iglL
C = Concentration of the parameter to be measured, in pg/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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A/A^ 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 compound
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.
-------
Method 604.1
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 interferences 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.
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 measure-
ments. 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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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
-------
Method 604.1
8.3.1 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 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 de-
fined 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.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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. When doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques, such as liquid chromatography with a dissimilar
column, 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
-------
Method 604,1
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-L separatory runnel.
10.2 The analyst may solvent-wash the sample at basic pH as described in Sections 10.2.1 and
10.2.2 to remove potential method interferences. 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 with IN sodium hydroxide.
10.2.2 Add 60 mL of methylene chloride to the separatory funnel and extract the sample by
shaking the funnel for 2 minutes with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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. Discard the methylene chloride extract. Perform a second and third extrac-
tion in the same manner.
10.3 Add 50 g of NaH2PO4 to the sample in the separatory funnel and shake to dissolve the solid.
The sample pH should be between 4.0 and 4.5. If necessary, adjust the pH with phosphoric
acid or sodium hydroxide. Add 200 mL of methylene chloride to the separatory funnel and
extract the sample by shaking the funnel for 2 minutes with periodic venting to release excess
pressure. Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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 separations. Col-
lect the methylene chloride extract in a 1-L Erlenmeyer flask .
10.4 Add a second 200-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.
10.5 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
1000-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.
10.6 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.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.7 Add one or two 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 approximately 60 minutes. At the proper rate of
distillation, the balls of the column will actively chatter but the chambers will not flood with
10
-------
Method 604.1
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 minutes.
10.8 Increase the temperature of the hot water bath to about 80°C. Momentarily remove the
Snyder column, add 15 mL of methanol and a new boiling chip, and attach a micro-Snyder.
Pour about 1 mL of methanol into the top of the micro-Snyder column and concentrate the
solvent extract as before. Elapsed time of concentration should be 5 to 10 minutes. 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 minutes.
10.9 Remove the micro-Snyder column and adjust the volume to 2.5 mL with methanol. Transfer
the liquid to a 5-mL volumetric flask and dilute to the mark with reagent water. Mix thor-
oughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. If the sample extract requires no
further cleanup, proceed with liquid chromatographic analysis. If the sample requires addi-
tional cleanup, proceed to Section 11.
10.10 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5 mL.
11. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The cleanup
procedure recommended in this method, namely the acid/base extraction described in Sec-
tion 10, has been used for the analysis of various clean waters and industrial effluents. If
particular Circumstances demand the use of additional cleanup, the analyst must demonstrate
that the recovery of each compound of interest 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 estimated retention times and method detection limits that can be
achieved by this method. An example of the separation achieved by Column 1 is shown in
Figure 1. Examples of the separation achieved by Column 2 are shown in Figure 2. Other
columns, chromatographic conditions, or detectors may be used if the requirements of Sec-
tion 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard
until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 20 to 50 jiL of the sample extract by completely filling the sample valve loop. Record
the resulting peak sizes 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a sug-
-------
Method GQ4.1
gested window size; 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.
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:
Equation 2
(A)(Vt)
Concentration, \iglL =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in /jL
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:
Equation 3
Concentration, pg/L = -
where
As - Response for parameter to be measured
Ais = Response for the internal standard
Is = Amount of internal standard added to each extract, in fig
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
12
-------
Method 604. 1
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.8 The MDL
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle's Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two diffe-
rent wastewaters were spiked and analyzed. The standard deviation of the percent recovery is
also included in Table 2.'
13
-------
Method 604.1
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, 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, August 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.
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. Glaser, J. A. et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
14
-------
Method 604.1
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time fmin) Method Detection
Parameter Column 1 \ Column 2 Limit (ug/L)
Dichlorophen 4.2 8.2 1.0
Hexachlorophene 9.7 14.4 1.2
Column 1 conditions: Spherisorb-ODS, 5 //, 250 mm long by 4.6 mm; 1 mL/min flow; 65/35
acetonitrile/water, 0.05% H3P04. A UV detector was used with this column to determine the
MDL.
Column 2 conditions: Lichrosorb RP-2, 5 //, 250 mm long by 4.6 mm; 1 mL/min flow; 50/50
acetonitrile/water, 0.5 acetic acid.
Table 2. Single-Laboratory Accuracy and Precision8
Spike Mean Standard
Sample Background Level Recovery Deviation Number of
Parameter Type6 (ug/LF (ug/L) (%) (%) Replicates
Dichlorophen 1 ND 10 58 12.4 7
1 ND 50 107 3.9 7
Hexachlorophene 1 ND 10 82 2.7 7
1 ND 50 102 5.8 7
(a) Column 1 conditions were used.
(b) 1 = POTW secondary effluent
(c) ND = Not detected
15
-------
Method 604.1
Hexachlorophene
~i 1 1 1 1 1 1 1—
2.0 4.0 6.0 8.0
n 1—
10.0
—i 1 1 1 1 r
12.0 14.0 16.0
—I 1 1
18.0 20.0
A52-OOZ-1A
Retention Time (minutes)
Figure 1. HPLC-UV Chromatogram of 10 ng Each of Hexachlorophene and
Dichlorophen (Column 1)
16
-------
Method 604.1
Hexachlorophene
2.0 4.0
i
6.0
8.0 10.0
\ r
12.0
i I
14.0
16.0
i i i
18.0 20.0
Retention Time (minutes)
52-002-2A
Figure 2. HPLC-UV Chromatogram of 250 ng Each of Hexachlorophene (Column 2)
17
-------
-------
Method 608.1
The Determination of
Organochlorine Pesticides in
Municipal and Industrial
Wastewater
-------
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Method 608.1
The Determination of Organochlorine Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
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.
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 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 alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 125) 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 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-series methods. 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 ap-
propriate cleanup procedures. Under gas chromatography, the analyst is allowed the latitude
to select chromatographic conditions appropriate for the simultaneous measurement of com-
binations 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 chromatographic
column for chlorobenzilate 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.
21
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Method 608.1
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is 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 chromatographic conditions are
described which permit the separation and measurement of the compounds in the extract by
electron capture (EC) gas chromatography.1
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 large 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.
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.
22
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Method 608.1
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 aware-
ness 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.5"7
4.2 The following parameters covered by this method have been tentatively classified as known or
suspected human or mammalian carcinogens: chlorobenzilate, dibromochloropropane, and
PCNB. Primary standards of these toxic compounds should be prepared in a hood.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 con-
tamination 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 by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 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.
23
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Method 608.1
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 minutes or perform a
Soxhlet extraction 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 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 1.5% SP-2250/1.95%
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 by 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 spec-
trometer, 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. Condition heating in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances. Alter-
natively, heat 16 hours at 450 to 500 °C in a shallow tray or perform a Soxhlet extraction with
methylene chloride for 48 hours.
6.5 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.6 Stock standard solutions (1.00 /*g//*L): Stock standard solutions may be prepared from pure
standard materials or purchased as certified solutions.
24
-------
Method 608.1
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 vol-
ume 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. Commer-
cially 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.
7.2.2 Using injections of 1 to 5 juL 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 injec-
ted, 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 ana-
lyst 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.
25
-------
Method 608.1
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 volumet-
ric 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 /LcL 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in
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, AJA-a 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 the lauric acid value is suggested. This procedure8 determines the
adsorption from hexane solution of lauric acid, in milligram, per gram 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.
26
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Method 608.1
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 measure-
ments. 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 pipette, 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
hi 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
hi 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 be-
fore 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 + 3s
Lower Control Limit (LCL) = R - 3s
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.
27
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Method 608.1
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 de-
fined 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.9
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-L
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
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.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices10 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 tune of collection until extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of extrac-
tion.
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-L separatory funnel.
28
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Method 608.1
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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 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 methylene chloride to complete the quantitative transfer.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 minutes. When the ap-
parent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes.
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 re-
quires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
29
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Method 608.1
11. CLEANUP AND SEPARA TION
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 pro-
cedure, 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% (v/v) ethyl ether in hexane (Fra-
ction 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% (v/v) ethyl ether
in hexane (Fraction 2), into a second K-D flask. Perform a third elution, using
200 mL of 50% (v/v) ethyl ether in hexane (Fraction 3), into a separate K-D flask.
The elution patterns for four of the pesticides are shown in Table 3.
11.2.4 Concentrate the eluates by standard K-D techniques (Section 10.6), substituting hex-
ane 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 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 demon-
strated 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.
30
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Method 608.1
12.4 Inject 1 to 5 /*L of the sample extract using the solvent-flush technique." Record the volume
injected to the nearest 0.05 /xL, 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 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.
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:
Equation 2
Concentration, ug/L =
(W.)
where
A = Amount of material injected, in ng
Vf = Volume of extract injected, in yL
Vt = Volume of total extract, in \>L
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:
Equation 3
Concentration, pglL = —
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
Va = Volume of water extracted, in L
31
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Method 608.1
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 out-
side 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 seconds 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.12
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.13
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 ng 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 ±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 re-analysis. These may include the use of alternative packed or capillary GC columns
or additional cleanup (Section 11).
32
-------
Method 60S. 1
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
concentrations listed in Table 1 were estimated from the response of an electron capture
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-/*L injection from a 10-mL
final extract of a 1-L sample.
15.2 In a single laboratory (West Coast 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.
-------
Method 608.1
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. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, 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, August 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, 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. McNair, H.M. and Bonelli, E.J., "Basic Chromatography," Consolidated Printing, Berkeley,
California, p. 52, 1969.
34
-------
Method 608.1
References
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).
35
-------
Method 608.1
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
Column * Column 2 Estimated
Temperature
Parameter (°C)
Retention Time Retention Time MDL
(min) (mini (ug/L)
Dibromochloropropane 100 3.1 -- 0.04
Etridiazole 140 1.3 -- 0.04
Chloroneb 150 2.0 -- 0.04
Propachlor 150 3.8 -- 1.0
PCNB 160 2.4 - 0.06
Chloropropylate 215 3.6 8.4 0.2
Chlorobenzilate 215 3.8 10.7 0.2
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/1.95% SP-2401
packed in a glass column 1.8m long by 2 mm ID 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 MOL.
Column 2 conditions: Ultrabond 20M (100/120 mesh) packed in a glass column 1.8 m long by
2 mm ID with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature is 200°C.
36
-------
Method 608.1
Table 2. Single-Operator Accuracy and Precision
Parameter
Chlorobenzilate
Chloroneb
Chloropropylate
Dibromochloropropane
Etridiazole
PCNB
Propachlor
Sample
Type
MW
MW
MW
MW
IW
IW
MW
MW
MW
MW
IW
IW
MW
MW
MW
MW
IW
MW
Background
(ug/U
NO
ND
NO
ND
0.84
110
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
21.3
ND
Spike
(ug/U
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
Mean
Recovery
(%)
74
97
92
93
53
97
78
96
83
70
61
55
144
91
100
91
87
83
Standard
Deviation
(%)
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
ND = Not detected
MW= Municipal wastewater
IW = Industrial wastewater, pesticide manufacturing
* For duplicate analyses, range is listed.
37
-------
Method 608.1
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
Etridiazole 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
38
-------
Method 608.2
The Determination of Certain
Organochlorine
Pesticides in Municipal and
Industrial Wastewater
-------
-------
Method 608.2
The Determination of Certain Organochlorine Pesticides in Municipal
and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain organochlorine pesticides in industrial and
municipal wastewater. The following parameters may be determined by this method:
Parameter Storet No. CAS No.
Chlorothalonil - 1897-45-6
DCPA 39770 1861-32-1
Dichloran - 99-30-9
Methoxychlor 39480 72-43-5
Permethrin - 5264553-1
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the electron capture detector equal to
5 times the detector background noise assuming a 10.0-mL final extract volume of a 1-L
reagent water sample and a gas chromatographic (GC) injection volume of 5 fiL. The EDL
for a specific wastewater may be different depending on the nature of interferences in the
sample matrix.
1.3 This is a GC method applicable to the determination of the compounds listed above in munici-
pal and industrial discharges. When this method is used to analyze unfamiliar samples for any
or all of the compounds listed above, compound identifications should be supported by at least
one additional qualitative technique. Section 13 provides gas chromatograph/mass spectro-
meter (GC/MS) conditions appropriate for the qualitative confirmation of compound iden-
tifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Organochlorine pesticides are removed from the sample matrix by extraction with methylene
chloride. The extract is dried, exchanged into hexane, and analyzed by gas chromatography.
Column chromatography is used as necessary to eliminate interferences which may be en-
countered. Measurement of the pesticides is accomplished with an electron capture detector.
2.2 Confirmatory analysis by gas chromatography/mass spectrometry is recommended (Section 13)
when a new or undefined sample type is being analyzed if the concentration is adequate for
such determination.
41
-------
Method 608.2
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analy-
sis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 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 hi a muffle furnace at 400 °C for 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits 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 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 aware-
ness 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 identified2"4 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to
air dry, then muffle the glass bottles at 400°C for 1 hour. After cooling, rinse the cap liners
with hexane, seal the bottles with aluminum foil, and store in a dust-free environment.
5.1.1 Automatic sampler (optional): Must incorporate glass sample containers for the col-
lection 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,
-------
Method 608.2
a minimum length of compressible silicone rubber tubing may be used. Before use,
however, the compressible tubing should be thoroughly rinsed with methanol, fol-
lowed by repeated rinsings with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to con-
centrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak
areas.
5.3.1.1 Column 1: 180 cm long by 2 mm ID, glass, packed with 1.5% OV-17/
1.95% OV-210 on Chromosorb W-HP (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 180 cm long by 2 mm ID, glass, packed with 4% SE-30/
6% SP-2401 on Supelcoport (100/120 mesh) or equivalent. Guidelines for
the use of alternative column packings are provided in Section 10.3.1.
5.3.1.3 Detector: Electron capture. This detector has proven effective in the
analysis of wastewaters for the parameters listed in Section 1.1 and was
used to develop the method performance statements in Section 12. Guide-
lines for the use of alternative detectors are provided in Section 10.3.1.
5.4 Chromatographic column: 400 mm long by 19 mm ID Chromaflex, equipped with coarse-
fritted bottom plate and PTFE stopcock. (Kontes K-420540-0224 or equivalent).
Chromatographic column: 300 mm long by 10 mm ID, equipped with coarse-fritted bottom
plate and PTFE stopcock (Kontes K-430540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control
(±2°C). The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes, or per-
form a Soxhlet extraction overnight with methylene chloride.
43
-------
Method 608.2
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, ethanol and methylene chloride: Demonstrated to be free of ana-
lytes.
6.1.2 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 perox-
ides are provided with the test strips. After cleanup, 20 mL ethyl alcohol preservative
must be added to each liter of ether.
6.1.3 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in
glass containers with glass stoppers or foil-lined screw-caps. Before use, activate each
batch overnight at 130°C in foil-covered glass container.
6.1.4 Silica gel: Activate approximately 100 g of silica gel at 200°C for 16 hours in a tared
500-mL Erlenmeyer flask with ground-glass stopper. Allow to cool to room tempera-
ture, and determine the weight of activated silica gel. Deactivate by adding 3% by
weight of distilled water. Restopper the flask, and shake on a wrist-action shaker for
at least 1 hour. Allow to equilibrate for 3 or more hours at room temperature.
6.1.5 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.
6.1.6 Sodium hydroxide (NaOH) solution (ION): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.7 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.8 Sulfuric acid (H2SO4) solution (1 +1): Add a measured volume of concentrated H2SO4
to an equal volume of reagent water.
6.2 Standard stock solutions (1.00 fig/fiL): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in hexane or other suitable solvent 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 96% or greater, the weight can be used
without correction to calculate the concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
44
-------
Method 608.2
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 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 extraction. If the samples will not be extracted within 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.
7.3 All samples must be extracted within 7 days of collection, and analyzed within 40 days of
extraction.6
8. CAL/BRA TION AND STANDARDIZA TION
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is needed.
One calibration solution should contain each analyte at a concentration approaching
but greater than the EDL (Table 1) for that compound; the other two solutions should
contain analytes at concentrations that bracket the range expected in samples. For
example, if the detection limit for a particular analyte is 0.2 /ig/L, and a sample
expected to contain approximately 5 /xg/L is analyzed, standard solutions should be
prepared at concentrations representing 0.3 /xg/L, 5 /ig/L, and 10 /xg/L of the ana-
lytes.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock
solution to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3.2 and tabulate peak height or area response versus the mass
of analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor)
is a constant over the working range (< 10% relative standard deviation), linearity
through the origin can be assumed and the average ratio or calibration factor can be
used in place of a calibration curve.
8.1.4 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
analyte varies from the predicted response by more than +10%, the test must be re-
peated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve.
8.2 Florisil standardization.
8.2.1 Florisil from different batches or sources may vary in absorptive capacity. To stan-
dardize the amount of Florisil which may be used in the cleanup procedure (Sec-
tion 10.2.2), use of the lauric acid value7 is suggested. The referenced procedure
determines the adsorption from hexane solution of lauric acid in milligrams per gram
of Florisil. The amount of Florisil to be used for each column is calculated by divi-
ding this factor into 110 and multiplying by 20 g.
45
-------
Method 608.2
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A labora-
tory reagent blank is a 1-L aliquot of reagent water. If the reagent blank contains a
reportable level of any analyte, immediately check the entire analytical system to
locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.3, prepare a laboratory control standard concen-
trate that contains each analyte of interest at a concentration of 2 jig/mL in
acetone or other suitable solvent.8
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For
each analyte in the laboratory control standard, calculate the percent recov-
ery (P^ with the equation:
Equation 1
100S.
P, =
where
St = Analytical results from the laboratory control standard, in pg/L
Tt = Known concentration of the spike, in pgIL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of most
of the analytes.
46
-------
Method 608.2
9.3.2 For each analyte in each duplicate pair, calculate the relative range (RR,) with the
equation:
Equation 2
RR. =
X,
where
Rt = Absolute difference between the duplicate measurements Xt and X2, in pg/L
f; = Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sample
by shaking the funnel for 2 minutes with periodic venting to release vapor pres-
sure. 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 com-
plete the phase separation. The optimum technique depends on the sample, but may
include stirring, filtration of the emulsion through glass wool, or centrirugation.
Collect the extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and
complete the extraction procedure a second time, combining the extracts in the Erlen-
meyer flask.
10.1 A Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect the
extract in a 500-mL K-D flask equipped with a 10-mL concentrator tube. Rinse the
Erlenmeyer flask and column with 20 to 30 mL of methylene chloride to complete the
quantitative transfer.
47
-------
Method 608.2
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top.
Place the K-D apparatus on a hot water bath (60 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 in steam. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the
proper rate of distillation, the balls of the column will actively chatter but the cham-
bers will not flood. When the apparent volume of liquid reaches about 3 mL, remove
the K-D apparatus and allow it to drain and cool for at least 10 minutes.
10.1.6 Increase the temperature of the hot water bath to about 80 to 85°C. Momentarily
remove the Snyder column, add 50 mL of hexane and a new boiling chip, and reat-
tach 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 minutes. When the apparent volume of liquid reaches about 3 mL,
remove the K-D apparatus, and allow it to drain at least 10 minutes while cooling.
Remove the Snyder column, rinse the flask and the 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 2 days, they should be transferred to PTFE-sealed screw-cap bottles. If
the sample extract requires no cleanup, proceed with gas chromatographic analysis.
10.1.7 If the sample requires cleanup, the extract obtained must be divided into two frac-
tions. One of the fractions is eluted through Florisil for the analysis of dicloran and
DCPA. The other fraction is eluted through silica gel for the analysis of chloro-
thalonil, methoxychlor, and the permethrins.
10.1.8 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.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. The single-operator precision and accu-
racy data in Table 2 were gathered using the recommended cleanup procedures. 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 is no less than that recorded in Table 2.
10.2.2 Florisil column cleanup.
10.2.2.1 Add a weighed amount of Florisil, about 21 g, to a chromatographic
column. The exact weight should be determined by calibration.7 Tap the
column to settle the Florisil. Add a 1 to 2 cm layer of sodium sulfate
above the Florisil. Rinse the Florisil and sodium sulfate by adding 60 mL
of hexane to the column. Just prior to exposure of the sodium sulfate to
48
-------
Method 608.2
air, stop the draining of the hexane by closing the stopcock on the column.
Discard the eluate.
10.2.2.2 Quantitatively, add the fraction of extract chosen for the analysis of dichlo-
ran and DCPA to the column. Drain the column into the flask, stopping
just prior to exposure of the sodium sulfate layer.
10.2.2.3 Elute the column with 200 mL of 6% ethyl ether in hexane (Fraction 1)
using a drip rate of about 5 mL/min. Remove and discard. Perform a
second elution using 200 mL of 15% ethyl ether in hexane (Fraction 2),
collecting the eluant in a 500-mL K-D flask equipped with a 10-mL con-
centrator tube.
10.2.2.4 Concentrate the eluate by standard K-D techniques (Section 10.1.5), substi-
tuting hexane for methylene chloride, and using the water bath at about
85 °C. Adjust the final volumes to 10 mL with hexane. Analyze by gas
chromatography.
10.2.3 Silica gel column cleanup.
10.2.3.1 Prepare silica gel columns using a glass column 300 mm long by
10 mm ID. Rinse column with hexane. Add approximately 50 mL of
hexane to the empty column. Add 3.5 g of 3% deactivated silica gel
(Section 6.1.4). Pack by rotating slowly to release air bubbles. Top with
1.5 cm of NajSO,,. Drain hexane to the top of the Na^O,,.
10.2.3.2 Add the fraction of extract chosen for the analysis of chlorothalonil, meth-
oxychlor, and the permethrins to the column. Open the stopcock and
allow it to drain to the surface of the sodium sulfate. Elute with the fol-
lowing solutions:
Fraction 1: 25 mL of hexane
Fraction 2: 25 mL of 6% (v/v) MeCl2 in hexane
Fraction 3: 25 mL of 50% MeCl2 in hexane
10.2.3.3 Collect Fraction 3 in a 500-mL K-D flask equipped with a 10-mL con-
centrator tube, and add 50 mL of hexane. Concentrate on an 85°C water
bath to 10.0 mL as described in Section 10.1.5.
10.2.4 The elution profiles obtained in these studies are listed in Tables 3 and 4 for the
convenience of the analyst. The analyst must determine the elution profiles and
demonstrate that the recovery of each compound of interest is no less than that re-
ported in Table 2 before the analysis of any samples utilizing these cleanup proce-
dures.
10.2.5 Proceed with gas chromatography.
10.3 Gas chromatographic analysis.
10.3.1 Recommended columns and detector for the gas chromatographic system are described
in Section 5.3.1. Table 1 summarizes the recommended operating conditions for the
gas chromatograph. Included in this table are estimated retention times and detection
limits that can be achieved by this method. Examples of the separations achieved by
Column 1 are shown in Figures 1 and 2. Other packed columns, chromatographic
49
-------
Method 608.2
conditions, or detectors may be used if data quality comparable to Table 2 are
achieved. 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 data quality comparable to Table 2 are achieved.
10.3.2 Inject 2 to 5 /*L of the sample extract using the solvent-flush technique.9 Record the
volume injected to the nearest 0.05 /iL, the total extract volume, the fraction of total
extract utilized in each cleanup scheme, and the resulting peak size in area or peak
height units.
10.3.3 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
the day. Three times the standard deviation of a retention time for a compound can
be used to calculate a suggested window size; however, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per
liter with the equation:
Equation 3
(A)(Vt)(Vt)
C =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \>L
Vt = Volume of total extract, in fjL
Vs = Volume of water extracted, in mL
Vc = Volume of final extract after cleanup, in \tL
Vf = Volume of extract utilized for cleanup scheme, in \>L
11.2 Report the results for the unknown samples in /ig/L. Round off the results to the nearest
0.1 jig/L or two significant figures.
12. METHOD PERFORMANCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in
Table I.10 The detection limits were calculated from the minimum detectable response of the
EC detector equal to 5 times the background noise, assuming a 10.0-mL final extract volume
of a 1-L sample and a GC injection of 5 /xL.
50
-------
Method 608.2
12.2 Single-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 using spiked industrial wastewater samples. The results of these studies
are presented in Table 2.
13. GC/MS CONFIRMATION
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 scans per peak 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.
13.2 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. The calculation.
of tailing factors is illustrated in Method 625."
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS
system must be checked to see that all DFTPP performance criteria are achieved.12
13.4 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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
13.4.1 The molecular ion and other 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 ±10%. For example, if
the relative abundance of an ion is 30% in the mass spectrum of the stan-
dard, the allowable limits for the relative abundance of that ion in the mass
spectrum for the sample would be 20 to 40%.
13.4.2 The retention time of the compound in the sample must be within 6 sec-
onds of the same compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by
GC/MS only on the basis of retention time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
51
-------
Method 608.2
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, PA, P. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, PA, p.76 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897. Unpub-
lished report available from U.S. Environmental Protection Agency, Environmental Moni-
toring and Support Laboratory, Cincinnati, Ohio.
7. Mills, P.A., "Variation of Floricil Activity: Simple Method for Measuring Adsorbent Capa-
city and Its Use in Standardizing Florisil Columns," Journal of the Association of Official
Analytical Chemists, 51, 19 (1968).
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, March 1979.
9. Burke, J.A., "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,"
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
10. "Evaluation of Ten Pesticide Methods," Contract Report #68-03-1760, Task No. 11, U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio.
11. "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater," EPA-600/
4-82-057. U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Analytical Chemistry, 46, 1912 (1975).
52
-------
Method 608.2
Table 1. Gas Chromatography of Organochlorine Pesticides
Parameter
Chlorothalonil
DCPA
Dicloran
Methoxychlor
cis-Permethrin***
trans-Permethrin * * *
Retention
Column 1*
3.
4.
2.
22.
18.
20.
40
19
23
35
52
02
Time (minj
Column 2**
4
5
2
10
16
17
.69
.44
.62
.85
.04
.53
Estimated
Detect/on
Limit
(ug/U
0.001
0.003
0.002
0.04
0.2
0.2
Column 1: 180 cm long by 2 mm ID, glass, packed with 1.5% OV-17/1.95% 0V 210 on
Chromosorb W-HP (100/120 mesh) or equivalent; 5% methane/95% Argon carrier gas at
30 mL/min flow rate. Column temperature is 200°C. Detector: electron capture.
Column 2: 180 cm long by 2 mm ID, glass, packed with 4% SE-30/6% SP-2401 on Supel-
coport (100/120 mesh) or equivalent; 5% methane/95% Argon carrier gas at 60 mL/min flow
rate. Column temperature is 200°C. Detector: electron capture.
* Column temperature is 220°C.
Table 2. Single-Laboratory Accuracy and Precision
Parameter
Chlorothalonil
DCPA
Dicloran
Methoxychlor
cis-Permethrin
trans-Permethrin
1 = Low-level industrial effluent
2 = High-level industrial effluent
Metric
Type*
1
2
1
2
1
2
1
2
1
2
1
2
Spike
Range
ftig/U
37.8
2,300
16
10,540
37.5
21,200
24.5
2,600
6.3
317
5.7
297
Number of
Replicates
7
7
7
7
7
7
7
7
7
7
7
7
Average
Percent
Recovery
84.1
94.9
77.6
89.5
98.6
90.8
102.4
102.2
99.5
77.5
78.8
88.9
Standard
Deviation
(%)
16.4
22.5
25.7
11.0
8.4
20.3
12.4
10.2
18.8
10.6
16.1
19.6
53
-------
Method 608.2
Table 3. Elution Profiles for Florisil Cleanup
Percent Recovery By Fraction*
Parameter 1 j 2
DCPA 0 99.3 0
Dicloran 0 96.3 0
Eluting solvent composition for each fraction given in Section 10.2.2.3.
Table 4. Elution Profiles for Silica Gel* Cleanup
Percent Recovery By Fraction**
Parameter
3
Chlorothalonil 0 0 93.8
Methoxychlor 0 0 93.8
cis-Permethrin 0 0 107.2
trans-Permethrin 0 0 92.5
* 3% deactivated
** Eluting solvent composition for each fraction given in Sections 10.2.3.2 and 10.2.3.3.
54
-------
Method 608.2
Dicloran
Chlorothalonil
DCPA
Methoxychlor
\
\ II I 1 I I I I 1 I r I 1 I I I I I I 1 I I
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0
Retention Time (minutes)
Figure 1. Gas Chromatogram of Chlorothalonil, DCPA, Dicloran,
and Methoxychlor in a Wastewater Extract (Column 1)
A52-002-14A
55
-------
Method 608.2
trans-Permethrin
cis-Permethrin A /
I IIII 1IiIIIIIII\ II 1I
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time (minutes)
AS2-002-13A
Figure 2. Gas Chromatogram of Permethrin Sample (Column 1)
56
-------
Method 614
The Determination of
Organophosphorus Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 614
The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
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
Demeton 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 shall be considered a major
modification subject to application and approval of alternative 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).
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.
59
-------
Method 614
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene chloride
in hexane using a separatory runnel. 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.1 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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimi-
nated 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 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 aware-
ness file of OSHA regulations regarding the safe handling of the chemicals specified in this
60
-------
Method 614
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 identified3 5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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 con-
tamination 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: 125-mL, 1000-mL, and 2000-mL, with TFE-fluorocarbon stop-
cock, ground-glass or TFE stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 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 equiva-
lent).
5.2.8 Pipette, disposable: 140 mm long by 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 minutes or perform a Soxh-
let extraction with methylene chloride.
61
-------
Method 614
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 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.
5.6.1 Column 1: 180 cm long by 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 perfor-
mance 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 by 4 mm ID glass, packed with 1.5% OV-17/1.95% QF-1
on Gas Chrom Q (100/120 mesh) or equivalent.
5.6.3 Detector: Phosphorus-specific; flame photometric detector (FPD, with 526 nm filter)
or thermionic bead detector in the nitrogen mode. These detectors have proven effec-
tive 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 provi-
sions 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 indi-
cated 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 Acetonitrile, hexane-sarurated: Mix pesticide-quality acetonitrile with an excess of hexane
until equilibrium is established.
6.5 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500 °C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.6 Sodium chloride solution, saturated: Prepare saturated solution of NaCl in reagent water and
extract with hexane to remove impurities.
6.7 Alumina: Woelm, neutral; deactivate by pipetting 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 hours on a mechanical shaker.
62
-------
Method 614
6.8 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.9 Stock standard solutions (1.00 ju,g//*L): 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 6 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 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 cor-
respond 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 /iL 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 injec-
ted, 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
63
-------
Method 614
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 ana-
lyst 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 volumet-
ric 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 pL 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:
Equation 1
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in fug/L
Cs = Concentration of the parameter to be measured, in pg/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, A/A^ 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 the lauric acid value is suggested. This procedure6 determines the
adsorption from hexane solution of lauric acid, in milligrams, per gram of Florisil. The
amount of Florisil to be used for each column is calculated by dividing this factor into 1 10 and
multiplying by 20 g.
64
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Method 614
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 capa-
ability and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. 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 pipette, 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 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 poten-
tial 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.
65
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Method 614
8.3.1 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 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 ali-
quots 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.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 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-L
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
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.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
66
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Method 614
9.3 All samples must be extracted within 7 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-L separately funnel.
10.2 Add 60 mL 15% (v/v) methylene chloride in hexane to the sample bottle, seal, and shake
30 seconds to rinse the inner walls. Transfer the solvent to the separatory funnel and extract
the sample by shaking the funnel for 2 minutes with periodic venting to release excess pres-
sure. 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 op-
timum 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 Erlen-
meyer 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 one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 PTFE-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.
67
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Method 614
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring fee water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 pro-
cedure, 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. The applicability of this procedure to organo-
phosphorus pesticides is 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 minute with 30-mL portions of hexanesaturated
acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-L separatory funnel and add
650 mL of reagent water and 40 mL of saturated sodium chloride solution. Mix
thoroughly for 30 to 45 seconds. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 seconds.
11.2.3 Combine the hexane extracts in a 1-L separatory funnel and wash with two 100-mL
portions of reagent water. Discard the water layer and pour the hexane layer through
a dry big 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.
11.2.5 Analyze by gas chromatography unless a need for further cleanup is indicated.
11.3 Florisil column cleanup: The following Florisil column cleanup procedure has been demon-
strated to be applicable to the seven organophosphorus pesticides listed in Table 3. It should
also be applicable to the cleanup of extracts for ethion.
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.
68
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Method 614
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% (v/v) ethyl ether in hexane (Fra-
ction 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% (v/v) ethyl ether
in hexane (Fraction 2) into a second K-D flask. Perform a third elution using 200 mL
of 50% (v/v) ethyl ether in hexane (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 hex-
ane. Analyze by gas chromatography.
11.4 Removal of sulfur:9 Elemental sulfur will elute in Fraction 1 of the Florisil cleanup proce-
dure. 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.
11.4.1 Add one or two boiling chips to the 10-mL hexane solution contained in a concentra-
tor tube. Attach a micro-Snyder column and concentrate the extract to about 0.2 mL
in a hot water bath at 85°C. Remove the micro K-D from the bath, cool, and adjust
the volume to 0.5 mL with hexane.
11.4.2 Plug a disposable pipette with a small quantity of glass wool. Add enough alumina to
produce a 3-cm column after settling. Top the alumina with a 0.5-cm layer of an-
hydrous sodium sulfate.
11.4.3 Quantitatively transfer the concentrated extract to the alumina microcolumn using a
lOO-pL syringe. Rinse the ampule with 200 pL of hexane and add to the microcol-
umn.
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. Inclu-
ded in this table are estimated retention-times and method detection lunits 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.
69
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Method 614
12.4 Inject 1 to 5 ^L of the sample extract using the solvent-flush technique.10 Record the volume
injected to the nearest 0.05 juL, 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 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.
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:
Equation 2
Concentration, uglL =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \)L
Vt = Volume of total extract, in \iL
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:
Equation 3
Concentration, fig/L = —
where
As = Response for parameter to be measured
Ais = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
70
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Method 614
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 seconds 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."
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
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 ng 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).
71
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Method 614
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.13 The MDL
concentrations listed in Table 1 were obtained using reagent water.14
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
72
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Method 614
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, 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, Phila-
delphia, 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, August 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, 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 in 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,
15, 1426 (1981).
73
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Method 614
References
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.
74
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Method 614
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time
(min)
Parameter
Diazinon
Disulfoton
Demeton
Parathion
methyl
Malathion
Parathion ethyl
Ethion
Azinphos
methyl
Column 1
1.8
1.9
2.3
2.5
2.9
3.1
6.8
14.5
Column 2
1.8
2.1
2.1
3.7
3.9
4.5
9.1
29.9
Method
Detection
(ug/L)
0.
ND
ND
0.
ND
0.
ND
ND
012
012
012
ND = Not determined
Column 1 conditions: Gas-Chrom Q (100/120 mesh) coated with 3% OV-1 packed in a glass
column 1.8m long by 4 mm ID 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 glass column 1.8 m long by 4 mm ID with nitrogen carrier gas at a flow rate of
70 mL/min. Column temperature, isothermal at 212°C.
75
-------
Method 6)4
Table 2. Single-Operator Accuracy and Precision
Parameter
Diazinon
Parathion methyl
Parathion ethyl
Average
Percent
Recovery
94
95
102
Standard
Deviation
(%)
5.2
3.2
4.1
Spike
Range
(t*g/U
0.04-40
0.06-60
0.07-70
Number of
Analyses
27
27
27
Matrix
Types
4
4
4
Table 3. Florisil Fractionation Patterns and Acetonitrile Partition Applicability
Parameter
Demeton
Disulfoton
Diazinon
Malathion
Parathion ethyl
Parathion methyl
Azinphos methyl
Ethion
Percent Recovery by Fraction Acetonitrile
No. 1 No. 2
100
100
100
5
100
100
ND ND
Partition
No. 3 No. 4 Applicability
ND
ND
Yes
95 Yes
Yes
Yes
20 80 ND
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
76
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Method 614.1
The Determination of
Organophosphorus Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 614.1
The Determination of Organophosphorus
Pesticides in Municipal and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain organophosphorus pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter STORET No. CAS No.
Dioxathion 78-34-2
EPN 2104-64-5
Ethion 39398 563-12-2
Terbufos 13071-79-9
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the nitrogen/phosphorus detector equal to
5 times the gas chromatographic (GC) background noise assuming a 1.0-mL final extract
volume of a 1-L reagent water sample and an injection of 5 /xL. The EDL for a specific
wastewater may be different depending on the nature of interferences in the sample matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications
should be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confir-
mation of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Organophosphorus pesticides are removed from the sample matrix by extraction with 15%
methylene chloride in hexane. The extract is dried, exchanged into hexane, and analyzed by
gas chromatography. Column chromatography is used as necessary to eliminate interferences
which may be encountered. Measurement of the pesticides is accomplished with a nitrogen/
phosphorus-specific detector.
2.2 Confirmatory analysis by GC/MS is recommended when a new or undefined sample type is
being analyzed if the concentration is adequate for such determination.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of gas chromatograms. All of these
79
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Method 614.1
materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 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 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits 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 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 aware-
ness 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 identified2^ for the information of the analyst.
5. APPARA TUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to
air dry, then muffle at 400°C for 1 hour. After cooling, rinse the cap liners with hexane, seal
the bottles, and store in a dust-free environment.
5.1.1 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 con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
80
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Method 614.1
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to con-
centrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak
areas.
5.3.1.1 Column 1: 180 cm long by 2 mm ID, glass, packed with 3% OV-225 on
Supelcoport (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 120 cm long by 2 mm ID, PyrexR glass, packed with
1.5% OV-17/1.95 % QF-1 on Gas Chrom Q, 80/100 mesh or equivalent.
5.3.1.3 Column 1 was used to develop the accuracy and precision statements in
Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.4 Detector: nitrogen/phosphorus. 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 12. Guidelines
for the use of alternative detectors are provided in Section 10.3.1.
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-
fitted bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fitted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control
(±2°C). The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, and methylene chloride: Demonstrated to be free of analytes.
81
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Method 614.1
6.1.2 Silica gel: Woelm 70-230 mesh. Activate approximately 100 g of silica gel at 200°C
for 6 hours in a tared 500-mL Erlenmeyer flask with ground-glass stopper. Allow to
cool to room temperature, reweigh, and determine the weight of activated silica gel.
Deactivate by adding 3% by weight of distilled water. Restopper the flask, and shake
on a wrist-action shaker for at least 1 hour. Allow to equilibrate for 3 or more hours
at room temperature.
6.1.3 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.
6.1.4 Sodium hydroxide (NaOH) solution (ION): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.6 Sulfuric acid (H2SO4) solution (1 + 1): Add measured volume of concentrated H2SO4 to
equal volume of reagent water.
6.2 Standard stock solutions (1.00 /xg//xL): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure
material. Dissolve the material in hexane 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 96% or greater, the weight can be used without correction to
calculate the concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
7.3 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 within 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.
7.4 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction.6
82
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Method 614.1
8. CALIBRA TION AND STANDARDIZA TION
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is
needed. One calibration solution should contain each analyte at a concentration
approaching but greater than the estimated detection limit (Table 1) for that com-
pound; the other two solutions should contain analytes at concentrations that bracket
the range expected in samples. For example, if the detection limit for a particular
analyte is 0.2 /xg/L, and a sample expected to contain approximately 5 /*g/L is ana-
lyzed, solutions of standards should be prepared at concentrations of 0.3 /ig/L,
5 Mg/L, and 10 /ig/L for the particular analyte.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock
solution to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3.2 and tabulate peak height or area responses versus the
mass of analyte injected. The results can be used to prepare a calibration curve for
each compound. Alternatively, if the ratio of response to concentration (calibration
factor) is a constant over the working range (< 10% relative standard deviation), line-
arity through the origin can be assumed and the average ratio or calibration factor can
be used in place of a calibration curve.
8.1.4 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
analyte varies from the predicted response by more than ± 10%, the test must be re-
peated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve.
9. QUALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is a 1-L aliquot of reagent water. If the reagent blank
contains a reportable level of any analyte, immediately check the entire analytical system to
locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every 10 samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.2, prepare a laboratory control standard con-
centrate that contains each analyte of interest at a concentration of 2 jig/ml
in acetone or other suitable solvent.7
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water.
83
-------
Method 614.1
9.2.1 .3 Analyze the laboratory control standard as described in Section 10. For
each analyte in the laboratory control standard, calculate the percent recov-
ery (Pj) with the equation:
Equation 1
1005..
P, =
where
S( = Analytical results from the laboratory control standard, in \iglL
Tt = Known concentration of the spike, in uglL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of most
of the analytes.
9.3.2 For each analyte in each duplicate pair, calculate the relative range7 (RR,) with the
equation:
RR.
where
Rf = Absolute difference between the duplicate measurements X1 and X2, in pg/L
Xt = Average concentration found
, in fig/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
84
-------
Method 614.1
10.1.2 Add 60 mL of 15% methylene chloride/hexane to the sample bottle and shake for
30 seconds to rinse the walls. Transfer the solvent to the separatory funnel and
extract the sample by shaking the funnel for 2 minutes with periodic venting to release
vapor 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 on the sample, but
may include stirring, filtration of the emulsion through glass wool, or centrifugation.
Collect the extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of 15% methylene chloride/hexane to the sample
bottle and complete the extraction procedure a second time, combining the extracts in
the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-mL K-D flask equipped with a 10-mL concentrator tube. Rinse the Erlenmeyer
flask and column with 20 to 30 mL of methylene chloride to complete the quantitative
transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of 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 in steam. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the
proper rate of distillation, the balls of the column will actively chatter but the cham-
bers 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 minutes. Remove the
Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation. If the
extract requires cleanup, proceed to Section 10.2 (cleanup and separation). If cleanup
has been performed or if the extract does not require cleanup, proceed with Sec-
tion 10.1.6.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of hexane to the
top. Place this micro K-D apparatus on a steaming-water bath (80 to 85°C) so that
the concentrator tube is partially immersed in the hot water. Adjust the vertical
position of the apparatus and water temperature as required to complete the concen-
tration in 5 to 10 minutes. At the proper rate of distillation, the balls will actively
chatter but the chambers will not flood. When the apparent volume of liquid reaches
0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min-
utes. Remove the micro-Snyder column and rinse its lower joint into the concentrator
tube with a small volume of hexane. Adjust the final volume to 1.0 mL or to a
volume suitable for cleanup or gas chromatography, and stopper the concentrator
tube; store refrigerated if further processing will not be performed immediately. If
85
-------
Method 614.1
the extracts will be stored longer than 2 days, they should be transferred to PTFE-
sealed screw-cap bottles. Proceed with gas chromatographic analysis.
10.1.7 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.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. The silica gel procedure allows for a
select fractionation of the compounds and will eliminate non-polar materials. The
single-operator precision and accuracy data in Table 2 were gathered using the recom-
mended cleanup procedures. If particular circumstances demand the use of an alter-
native cleanup procedure, the analyst must determine the elution profile and demon-
strate that the recovery of each compound of interest is no less than that recorded in
Table 2.
10.2.2 Prepare silica gel columns using a glass column 200 mm long by 10 mm ID. Rinse
column with hexane. Add approximately 50 mL of hexane to the empty column.
Add 3.5 grams of 3% deactivated silica gel. Pack by rotating slowly to release air
bubbles. Top with 1.5 cm of NajSO^ Drain hexane to the top of NajSC^ layer.
10.2.3 Just prior to exposure of the sodium sulfate layer to the air, transfer the sample ex-
tract onto the column using an additional 2 mL of hexane to complete the transfer.
10.2.4 Just prior to exposure of the sodium sulfate layer to the air, add 30 ml, of 6% methy-
lene chloride/hexane and continue the elution of the column, collecting the eluate in a
500-mL K-D flask equipped with a 10-mL concentration tube. Elution of the column
should be at a rate of about 2 mL per minute. Add 50 mL of hexane to the flask and
concentrate the collected fraction by the standard technique prescribed in Sections
10.1.5 and 10.1.6.
10.2.5 Continue the elution of the column according to the scheme outlined in Table 3. The
elution of the compounds may vary with different sample matrices.
10.2.6 Analyze the fractions by gas chromatography.
10.3 Gas chromatographic analysis.
10.3.1 Recommended columns and detector for the gas chromatography system are described
in Section 5.3.1. Table 1 summarizes the recommended operating conditions for the
gas chromatograph. Included in this table are estimated retention times and detection
limits that can be achieved by this method. Examples of the separations achieved are
shown in Figures 1 and 2. Other packed columns, chromatographic conditions, or
detectors may be used if data quality comparable to Table 2 are achieved. Capillary
(open-tubular) columns may also be used if the relative standard deviations of respon-
ses for replicate injections are demonstrated to be less than 6% and data quality
comparable to Table 2 are achieved.
10.3.2 Inject 2 to 5 ^tL of the sample extract using the solvent-flush technique.8 Record the
volume injected to the nearest 0.05 /iL, the total extract volume, and the resulting
peak size in area or peak height units.
86
-------
Method 614.1
10.3.3 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
the day. Three times the standard deviation of a retention time for a compound can
be used to calculate a suggested window size; however, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the ex-
tract and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per
liter with the equation:
Equation 3
Concentration, pg/L =
where
A = Amount of material injected, in ng
V. = Volume of extract injected, in \tL
Vt = Volume of total extract, in /jL
V = Volume of water extracted, in mL
11.2 Report the results for the unknown samples in microgram per Liter. Round off the results to
the nearest 0.1 ^g/L or two significant figures.
12. METHOD PERFORMANCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in
Table I.9 The detection limits were calculated from the minimum detectable response of the
N/'P detector equal to 5 times the GC background noise, assuming a 1.0-mL final extract
volume of a 1-L sample and a GC injection of 5 juL.
12.2 Single laboratory accuracy and precision studies were conducted by ESE,6 using spiked rele-
vant industrial wastewater samples. The results of these studies are presented in Table 2.
13. GC/MS CONFIRMATION
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 compounds
of interest. 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 scans per peak utilizing a 70 V (nominal) electron
87
-------
Method 614.1
energy m 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.
13.2 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. The calculation
of tailing factors is illustrated in Method 625.10
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS
system must be checked to see that all DFTPP performance criteria are achieved.11
13.4 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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
13.4.1 The molecular ion and other 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 ±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%.
13.4.2 The retention-time of the compound in the sample must be within 6 seconds of the
same compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by GC/MS only
on the basis of retention-time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
88
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Method 614.1
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, PA, p. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897. Unpub-
lished report available from U.S. Environmental Protection Agency, Environmental Monitor-
ing and Support Laboratory, Cincinnati, Ohio.
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. Burke, J.A., "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,"
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965T)
9. "Evaluation of Ten Pesticide Methods," U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio 45268.
10. "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater," EPA-600/
4-82-057, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268.
11. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Analytical Chemistry, 46, 1912 (1975).
89
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Method 614.1
Table 1. Gas Chromatography of Organophosphorus Pesticides
Retention Time (min) Detection Limit
Parameter Column 1 I Column 2 fffff/U
Terbufos 1.41 1.9 .004
Dioxathion 2.3 2.3 .01
Ethion 8.3 6.4 0.1
EPN 13.3 8.3 0.2
Column 1: 180 cm long by 2 mm ID, glass, packed with 3% OV-225 on 100/120 Supelcoport;
nitrogen carrier gas at a flow rate of 50 mL/min. Column temperature is 200°C for 2 minutes,
then programmed at 5°/min to 240°C and held for 5 minutes.
Column 2: 120 cm long by 2 mm ID, Pyrex" glass, packed with 1.5% OC-17/1.95% QF-1 on
80/100 mesh Gas Chrom Q or equivalent; nitrogen carrier gas at a flow rate of 30 mL/min.
Column temperature is 180°C for 2 minutes, then programmed at 8°/min to 250°C and held for 4
minutes.
Table 2. Single-Laboratory Accuracy and Precision
Matrix Spike Range Number of Average Percent Standard
Parameter Type* (fjg/U Replicates Recovery Deviation (%)
Dioxathion 1 1,978.0 7 94.3 19.9
1 19.8 7 99.0 27.5
EPN 1 1,293.0 7 96.1 6.1
Ethion 1 1,788.0 7 89.2 4.5
Terbufos 1 15.1 7 101.0 12.4
1 1,508.0 7 95.0 3.4
1 = Combined industrial wastewaters
90
-------
Method 614.1
Table 3. Silica Gel Cleanup of Organophosphorus Pesticides
Percent Recoveries
Silica Gel Fraction*
1
2
3
4
Total Percent Recoveries 93.0 87.8 101 102
Fraction 1 = 30 ml 6% MeCI2 in hexane
Fraction 2 = 30 mL 15% MeCI2 in hexane
Fraction 3 = 30 ml 50% MeCI2 in hexane
Fraction 4 = 30 ml 100% MeCI2
Terbufos
0
0
93.0
0
| Dioxathion |
0
0
35.1
52.7
Ethion |
0.8
1.9
94.9
3.0
EPN
0
0
46.4
56.0
91
-------
Method 614.1
Terbufus
Dioxathion
Ethion
EPN
iiiiiiinii i i i
4.0 6.0 8.0 10.0 12.0 14.0 16.0
Retention Time (minutes)
A5a-002-37A
Figure 1. Gas Chromatogram of Organophosphorous Pesticides (Column 1)
92
-------
Method 614.1
Terbufos
Ethion
Dioxathion
EPN
T I i
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
Retention Time (minutes)
A52-002-38A
Figure 2. Gas Chromatogram of Organophosphorous Pesticides (Column 2)
-------
-------
Method 615
The Determination of
Chlorinated Herbicides in
Municipal and Industrial
Wastewater
-------
-------
Method 615
The Determination of Chlorinated Herbicides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain chlorinated herbicides. The following parent
acids can be determined by this method:
Parameter STORET No. CAS No.
2,4-D 39736 94-75-7
Dalapon
2,4-DB
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
2,4,5-T
2,4,5-TP
-
-
-
-
-
-
-
39740
39760
75-99-0
94-82-6
1918-00-9
1 20-36-5
88-85-7
94-74-6
7085-19-0
93-76-5
93-72-1
1.2 This method is also applicable to the determination of salts and esters of these compounds.
These include, but are not limited to: the isobutyl and isooctyl esters of 2,4-D; the isobutyl
and isooctyl esters of 2,4-DB; 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.
1.3 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 shall be considered a major
modification subject to application and approval of alternative 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 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.
1.5 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 alternative gas chromatographic
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 qualita-
tive confirmation of compound identifications.
97
-------
Method 615
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is acidified. The acid herbicides and their
esters and salts are extracted with ethyl ether using a separatory funnel. The derivatives are
hydrolyzed with potassium hydroxide and extraneous organic material is removed by a solvent
wash. After acidification, the acids are extracted and converted to their methyl esters using
diazomethane as the derivatizing agent. Excess reagent is removed, and the esters are deter-
mined by electron capture (EC) gas chromatography.1
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 dilute acid, tap and reagent
water. Drain dry, and heat in an oven or muffle furnace at 400°C for 15 to 30 min-
utes. Do not heat volumetric ware. Thermally stable materials, such as PCBs, may
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 The acid forms of the herbicides are strong organic acids, which react readily with alkaline
substances and can be lost during analysis. Glassware and glass wool must be acid-rinsed with
(1+9) hydrochloric acid and the sodium sulfate must be acidified with sulfuric acid prior to
use to avoid this possibility.
3.3 Organic acids and phenols, especially chlorinated compounds, cause the most direct inter-
ference 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. 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.
98
-------
Method 615
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 aware-
ness 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 identified3"5 for the information of the analyst.
4.2 Diazomethane 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, and avoid the use of ground-glass joints, sleeve bearings,
and glass stirrers: EXPLOSION may result.
4.2.6 Do not store near 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, and boiling chips.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 con-
tamination 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 Separately funnels: 60-mL and 2000-mL, with TFE-fluorocarbon stopcocks, ground-
glass or TFE stoppers.
5.2.2 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.
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Method 615
5.2.3 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.4 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.5 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or equi-
valent).
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-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a
Soxhlet extraction 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 Diazomethane generator: assemble from two test tubes 150 mm long by 20 mm ID, two
Neoprene rubber stoppers, and a source of nitrogen. The generator assembly is shown in
Figure 1.
5.7 Glass wool: Acid-washed (Supelco 2-0383 or equivalent).
5.8 Gas chromatograph: Analytical system complete with 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 1: 180 cm long by 4 mm ID glass, packed with 1.5% SP-2250/1.95%
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 by 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 by 2 mm ID glass, packed with 0.1 % SP-1000 on Car-
bopak C (80/100 mesh) or equivalent.
5.8.4 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 spec-
trometer, 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 interferant 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 hi glass if necessary. Must be free of peroxides as indi-
cated by EM Quant test strips (available from Scientific Products Co., Cat. No. PI 126-8, and
700
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Method 615
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. Condition heating in a shallow tray at
400°C for a minimum of 4 hours to remove phthalates and other interfering organic substan-
ces. Alternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet
extraction with methylene chloride for 48 hours. Acidify by slurry ing 100 g sodium sulfate
with enough ethyl ether to just cover the solid. Add 0.1 mL concentrated sulfuric acid 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 acid (1+9): Add one volume of concentrated acid (ACS) to 9 volumes reagent
water.
6.6 Potassium hydroxide solution: 37% 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 H2SO4 (sp. gr. 1.84) to 50 mL of reagent
water.
6.8 Sulfuric acid solution (1 + 3): Slowly add 25 mL H2SO4 (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: Af-methyl-Af-nitroso-p-toluenesulfonarnide, 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 /ig//xL): 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 g 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 96% or greater, the weight can be used without cor-
rection to calculate the concentration of the stock standard. Commercially prepared
stock standards can be used at any concentration if they are certified by the manufac-
turer or by an independent source.
6.12.2 Transfer the stock standard solutions into PTFE-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 1 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:
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Method 615
7.2.1 For each parameter of interest, prepare working standards of the free acids at a mini-
mum 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 represen-
tative of a concentration near, but above, the method detection limit. The other con-
centrations 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 fiL of each calibration
standard, tabulate peak height or area responses against the mass of free acid repre-
sented by the injection. 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), can 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 preparation 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 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 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 measure-
ments. 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.
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Method 615
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 pipette, 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 be-
fore 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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 14.3.
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-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each
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Method 615
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.
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. Com-
posite 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 7 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-L separately 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 seconds to rinse the
walls. Transfer the solvent to the separatory funnel and extract the sample by shaking the
funnel for 2 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, cen-
trifugation, 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 one or two 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 apparatus on a hot water bath (60 to 65°C), such that the bottom of the flask is
bathed in the water vapor. Although the ethyl ether will evaporate in about 15 minutes, con-
104
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Method 615
tinue heating for a total of 60 minutes, beginning from the time the flask is placed on the
water bath. Remove the apparatus and let stand at room temperature for at least 10 minutes.
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 1 minute with 20-mL portions of ethyl ether. Discard
the organic phase. The free acids remain in the aqueous phase.
10.6 Acidify the contents of the separatory funnel to pH 2 by adding 2 mL of cold (4°C) sulfuric
acid (1 + 3). Test with pH indicator paper. Add 20 mL ethyl ether and shake vigorously for
2 minutes. Drain the aqueous layer into the 250-mL Erlenmeyer flask, 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 in the 125-mL flask. Allow the extract to remain in contact with the sodium sulfate
for approximately 2 hours.
10.7 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.
10.8 Pour the combined extract through a funnel plugged with acid-washed glass wool, and collect
the extract in the K-D in concentrator. 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 one to two 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 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 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 liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least
10 minutes.
10.10Remove 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 concentrator tube is partially immersed in the hot water. Adjust the
vertical position of the apparatus and the water temperature as required to complete con-
centration in 5 to 10 minutes. When the apparent volume of liquid reaches 0.5 mL, remove
the micro K-D from the bath and allow it 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 volume to 1.0 mL with ethyl ether.
7 /. ESTERIFICA TION OF ACIDS
11.1 Assemble the diazomethane generator (see Figure 1) in a hood using two test tubes 150 mm
long by 20 mm ID. Use neoprene rubber stoppers with holes drilled in them to accommodate
705
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Method 615
glass delivery tubes. The exit tube must be drawn to a point to bubble diazomethane 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 carbitol,
1.5 mL of 37% aqueous KOH, and 0.1 to 0.2 g Diazald to the second test tube. Immediately
place the exit tube into the concentrator tube containing the sample extract. Apply nitrogen
flow (10 mL/min) to bubble diazomethane through the extract for 10 minutes or until the
yellow color of diazomethane persists.
11.3 Remove the concentrator tube and seal it with a neoprene or PTFE stopper. Store at room
temperature in a hood for 20 minutes.
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 if
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 mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
12. CLEANUP AND SEPARATION
12.1 No cleanup procedures were required to analyze the wastewaters described in 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 gas chromatograph. Inclu-
ded in 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 3. 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.
13.2 Calibrate the system daily as described in Section 7.
13.3 Inject 1 to 5 ^tL of the sample extract using the solvent-flush technique.8 Record the volume
injected to the nearest 0.05 /*L, 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.
13.4 The width of the retention-time window used to make identifications should be based upon
measurements of actual retention-tune 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.
106
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Method 615
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:
Equation 1
Concentration, pglL =
where
A = Amount of material injected, in ng
V, = Volume of extract injected, in pL
Vt - Volume of total extract, in /iL
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 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 out-
side of the control limits in Section 8.3, data for the affected parameters must be labeled as
suspect.
15. GC/MS CONFIRMATION
15.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative com-
pound 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 acid 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 seconds 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 chromato-
graphic 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. Chromatographic tailing factors of less than 5.0 must be achieved.9
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Method 615
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.10
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 ng 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
± 10%. For example, if the relative abundance of an ion is 30% in the mass spec-
trum 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.
15.4.3 Compounds that have very similar mass spectra can be explicitly identified by GC/MS
only on the basis of retention tune data.
15.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
15.6 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.
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 99% confidence that the value is above zero.11 The MDL
concentrations listed in Table 1 were obtained from reagent water with an electron capture
detector.1
16.2 In a single laboratory (West Coast 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.
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Method 615
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.
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, Phila-
delphia, 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, August 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.
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,
15, 1426 (1981).
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Method 615
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
fas methyl ester)
Dicamba
2,4-D
2,4,5-TP
2,4,5-T
2,4-DB
Dalapon
MCPP
MCPA
Dichlorprop
Dinoseb
Retention Time
Column 1 |
1.2
2.0
2.7
3.4
4.1
—
3.4
4.1
4.8
11.2
| Column 2 | Column 3
1.0 —
1.6 —
2.0 —
2.4 —
— —
— 5.0
— —
— —
— —
— —
Method Detection
Limit (ug/L)
0.27
1.20
0.17
0.20
0.91
5.80
192.00
249.00
0.65
0.07
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/1.95% SP-2401
packed in a glass column 1.8 m long by 4 mm ID 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 minutes 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 glass
column 1.8 m long by 4 mm ID 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 glass
column 1.8 m long by 2 mm ID with nitrogen carrier gas at a flow rate of 25 mL/min. Column
temperature: programmed at injection from 100 to 150°C at 10°/min.
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Method 615
Table 2. Single-Operator Accuracy and Precision*
Parameter
2,4-D
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
(tig/L)
10.9
10.1
200.0
23.4
23.4
468.0
10.3
10.4
208.0
1.2
1.1
22.2
10.7
10.7
213.0
0.5
102.0
2020.0
2020.0
21400.0
2080.0
2100.0
20440.0
1.1
1.3
25.5
1.0
1.3
25.0
Mean Recovery
(%)
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
* All results based upon seven replicate analyses.
DW = Reagent water
MW = Municipal water
-------
Method 615
Glass Tubing
Nitrogen •
Rubber Stopper
o«
O
N '
' f
1 I
\>
b
V^v
0
°0
°0
D
Tubel
Tube 2
Figure 1. Diazomethane Generator
A52-002-17A
112
-------
Method 615
I I
2.0
I
4.0
I I
6.0
I
8.0
I I
10.0
Retention Time (minutes)
AS2-002-16A
Figure 2. Gas Chromatogram of Methyl Esters of Chlorinated Herbicides on
Column 1 (for conditions, see Table 1)
113
-------
Method 615
>MCPA
Dinoseb
I
2.0
I
4.0
I I I
6.0
I
8.0
I I I
10.0
I \
12.0
I I I
14.0
16.0
Retention Time (minutes)
A 52-002-1SA
Figure 3. Gas Chromatogram of Methyl Esters of Chlorinated Herbicides
on Column 1 (for conditions, see Table 1)
114
-------
Method 616
The Determination of Certain
Carbon-, Hydrogen-, and
Oxygen-Containing Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 616
The Determination of Certain Carbon-, Hydrogen-, and
Oxygen-Containing Pesticides in Municipal and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain carbon-, hydrogen-, and oxygen-containing
pesticides. The following parameters can be determined by this method:
Parameter CAS No.
Cycloprate 54460-46-7
Kinoprene 42588-37-4
Methoprene 40596-69-8
Resmethrin 10453-86-8
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial 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 alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each parameter is listed in
Table 2. 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 cer-
tain other 600-series methods. Thus, a single sample may be extracted to measure the com-
pounds included in the scope of the methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply ap-
propriate cleanup procedures.
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 L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
117
-------
Method 616
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by flame ionization detector/gas chromatography (GC/FID).1
2.2 This method provides a silica gel column cleanup procedure to aid in the elimination of inter-
ferences 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 4 hours. Do not heat volumetric
ware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substi-
tuted 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 in-
verted 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 MDL listed in Table 2.
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 aware-
ness 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 identified3'5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is
118
-------
Method 616
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 col-
lection 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, fol-
lowed by repeated rinsings with distilled water to minimize the potential for contami-
nation of the sample. An integrating flow meter is required to collect flow-propor-
tional composites.
5.2 Glassware (all specifications are suggested; catalog numbers are included for illustration only).
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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.
A ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250-mL (Kontes K-570001-0250 or equivalent).
Attach to concentrator or 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 equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Graduated cylinder: 1000-mL.
5.2.10 Erlenmeyer flask: 250-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, 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 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method perfor-
mance statements in Section 15. Alternative columns may be used in accordance with
the provisions described in Section 12.1.
119
-------
Method 616
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 10% OV-210 on Supel-
coport (100/120 mesh) or equivalent.
5.6.3 Detector: Flame ionization detector (FID). This detector has proven effective in the
analysis of wastewaters for the compounds listed in the scope and was used to develop
the method performance statements in Section 15.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferant is not observed at
the method detection limit of each parameter of interest.
6.2 Methylene chloride, methanol, petroleum ether, ethyl ether, methyl t-butyl ether, distilled-in-
glass quality or equivalent. Ethyl ether must be free of peroxides as indicated by EK Quant
Test Strips (available from Scientific Products Co., Catalog No. PI 126-8 and other suppliers).
Procedures recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davison Grade 923 (100/120 mesh). Purchase activated. To prepare for use,
place in a wide-mouth jar and heat overnight at 120 to 130°C. Seal tightly with PTFE or
aluminum- foil-lined screw-cap and cool to room temperature.
6.5 Sodium phosphate: Monobasic, monohydrate.
6.6 Sodium phosphate: Dibasic.
6.7 Stock standard solutions (1.00 /ig/jtL): 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 distilled-in-glass quality methyl t-butyl 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 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.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check standard solutions for signs of degrada-
tion or evaporation, especially just prior to preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after 6 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 2.
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.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumet-
720
-------
Method 616
ric flask and diluting to volume with methyl t-butyl ether. One of the external stan-
dards should be at a concentration near, but above, the method detection limit. The
other concentrations should correspond to the range of concentrations expected in the
sample concentrations or should define the working range of the detector.
7.2.2 Using injection of 1 to 5 pL 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, the ratio of the response to the mass injec-
ted, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volumet-
ric flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with methyl t-butyl ether. One of the stan-
dards should be at a concentration near, but above, the method detection limit. The
other concentrations expected in the sample concentrates or should define the working
range of the detector.
7.3.2 Using injections of 1 to 5 /*L 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:
Equation 1
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
Cis = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in pg/L
121
-------
Method 616
7.3.3 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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve or
response ratios, A,/A-a against RF. The working calibration curve or RF must be
verified on each working shift by the measurement of one or more calibration stan-
dards. If the response for any compound 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 interferences 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.
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 measure-
ments. 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 methyl t-butyl
ether, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore R and s calculations are performed.
122
-------
Method 616
8.2.4 Using the appropriate data from Table 3, determine the recovery and single operator
precision expected for the method, and compare these results to the values measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL): R - 3s
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 de-
fined 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. 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.
723
-------
Method 616
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. Com-
posite 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 Adjust the pH of the sample to 6.8 by addition of 2 g each of monobasic and dibasic sodium
phosphate per liter of sample.
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-L separatory funnel. Add 2 g each of monobasic
sodium phosphate and dibasic sodium phosphate to the sample to adjust the pH to 6.8.
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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
250-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two 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 minutes. At the proper rate of distillation,
the balls of the column will actively chatter but the chambers will not flood with condensed
124
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Method 616
solvent. When the apparent volume of liquid reaches 3 to 4 mL, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride. Adjust the sample extract volume to 5 mL with
methylene chloride.
10.8 Stopper the concentrator tube and store refrigerated if further processing will not be performed
immediately. If the extract is to be stored longer than 2 days, transfer the extract to a screw-
capped vial with a PTFE-lined cap. If the sample extract requires no further cleanup, proceed
to Section 10.9. If the sample requires cleanup, proceed to Section 11.
10.9 Add one or two boiling chips and attach a two-ball micro-Snyder column to the concentrator
tube. Prewet the micro-Snyder column with methylene chloride and concentrate the solvent
extract to 1 mL as before.
10.10 Add 20 mL of methyl t-butyl ether to the concentrator tube and reconcentrate the solvent
extract as before. When an apparent volume of 0.5 mL is reached, or the solution stops
boiling, remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.11 Remove the micro-Sayder column and adjust the volume of the extract to 1.0 mL with methyl
t-butyl ether. Transfer the extract to an appropriate container for subsequent GC analysis.
10.12Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 /. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is no less than 85%.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to
the four C, H, and O pesticides listed in Table 1.
11.2.1 Deactivate silica gel by mixing 100 mL of acetone, 1.2 mL of distilled water, and
20 g of silica gel thoroughly for 30 minutes in a 250-mL beaker. Transfer the slurry
to a chromatographic column (silica gel is retained with a plug of glass wool). Allow
the solvent to elute from the column until the silica gel is almost exposed to the air.
Wash the column sequentially with 10 mL of acetone, two 10-mL portions of methy-
lene chloride, and three 10-mL portions of petroleum ether. Use a column flow rate
of 2 to 2.5 mL/min throughout the wash and elution profiles. Add an additional
50 mL of petroleum ether to the head of the column.
11.2.2 Quantitatively add the methylene chloride extract from Section 10.8 to the head of the
column. Allow the solvent to elute from the column until the silica gel is almost
exposed to the air. Elute the column with 25 mL of petroleum ether. Discard this
fraction.
125
-------
Method 616
11.2.3 Elute the column with 50 mL of 6% ethyl ether in petroleum ether (Fraction 1) and
collect eluate in a K-D apparatus. Repeat process with 50 mL of 15% ethyl ether in
petroleum ether (Fraction 2), add 100 mL of 50% ethyl ether in petroleum ether
(Fraction 3). Collect each fraction in a separate K-D apparatus. The elution patterns
for the C, H, and O pesticides are shown in Table 1. Concentrate each fraction to
1 mL as described in Sections 10.9, 10.10, and 10.11. Proceed with gas chromato-
graphic analysis.
11.2.4 The above-mentioned fractions can be combined before concentration at the discretion
of the analyst.
12. GAS CHROMATOGRAPHY
12.1 Table 2 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded in this table are estimated retention times and method detection limits that can be achieved
by this method. Examples of the separations achieved by Columns 1 and 2 are shown in
Figures 1 and 2. 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 gas chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 ^L of the sample extract using the solvent flush technique. Record the volume
injected to the nearest 0.05 /*L, and the resulting peak sizes 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
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:
726
-------
Method 616
Equation 2
Concentration,
•where
A = Amount of material injected, in ng
Vf = Volume of extract injected, in \iL
Vt = Volume of total extract, in \iL
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:
Equation 3
Concentration, ng/L =
(Aa)(RF)(Vo)
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative iden-
tifications 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 seconds 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. When using a fused-silica capillary column, the column outlet
should be threaded through the interface to within a few millimeters of the entrance to the
source ionization chamber. 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.
727
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Method 616
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.10
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 DFTPP performance criteria are achieved.9
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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above 10% relative abundance in
the mass be present in the mass spectrum of the sample with agreement to .+ 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 spec-
trum 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.11 The MDL
concentrations listed in Table 2.1 Similar results were obtained using reagent water were
achieved using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained after silica gel cleanup. Seven repli-
cates of each of two different wastewaters were spiked and analyzed. The standard deviation
of the percent recovery is also included in Table 3.1
128
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Method 616
References
1. "Development of Methods for Pesticides in Wastewater," Report for EPA Contract 68-03-2956
(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, Phila-
delphia, 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, August, 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, 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. 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).
10. McNair, H.M., and Bonelli, E.J., "Basic Chromatography", Consolidated Printing, Berkeley,
California, p. 52 (1969).
11. Glaser, J.A. et al., "Trace Analysis for Wastewaters", Environmental Science and Technology,
15, 1426 (1981)
129
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Method 616
Table 1. Elution Characteristics of the C, H, And 0 Compounds from
6% Deactivated Silica Gel
Recovery in Specified Fraction'*
Parameter
Cycloprate
Kinoprene
Methoprene
Resmethrin
F1
97
100
ND
65
F2
ND
ND
101
27
F3
ND
ND
<1
ND
Total
97
100
101
92
(a) Elution solvents are 50 mL each of the following:
F1 = 6% ethyl ether in petroleum ether
F2 = 15% ethyl ether in petroleum ether
F3 = 50% ethyl ether
(b) ND = Not detected
Table 2. Chromatographic Conditions and Method Detection Limits
Retention Time (min)
Parameter Column 1
MDL
Column 2 (ug/L)
Cycloprate 3.6 3.9 21
Kinoprene 4.4 5.5 18
Methoprene 5.5 6.5 22
Resmethrin 8.4 8.9 36
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is programmed from 180 to 240°C at 8°C/min, injector temperature is 280°C and
detector is 300°C. A flame ionization detector is used.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 10% OV-210 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is programmed from 180 to 240°C at 4°C/min, injector temperature is 280°C and
detector is 300°C. A flame ionization detector is used.
130
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Method 616
Table 3. Single-Laboratory Accuracy and Precision3
Parameter
Cycloprate
Kinoprene
Methoprene
Resmethrin
Sample
Type"
1
1
1
1
1
1
1
1
Background
(ug/Lf
ND
ND
ND
ND
ND
ND
ND
ND
Spike
(ug/L)
100
1000
100
1000
100
1000
100
1000
Mean
Recovery
(%)
84
94
89
92
93
90
86
91
Standard
Deviation
(%)
14
4
6
6
13
4
8
• 3
Number of
Replicates
7
7
7
7
7
7
7
7
(a) Column 1 conditions were used.
(b) 1 = Columbus POTW secondary effluent
(c) ND = Not detected
131
-------
Method 616
Methoprene
Cycloprate
/
Resmethrin
~~l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
0 3.8 4.8 5.8 6.8 7.8 8.8 9.8 10.8 11.8 12.8
Retention Time (minutes)
A52-002-19A
Figure 1. GC-FID Chromatogram of 200 ng Each of C, H, and O Compounds
(Column 1)
132
-------
Method 616
Resmethrin
Methoprene
Kinoprene
\
Cycloprate
I I I I 1 l I I I I I I I I I I I I I I
0 3.8 4.8 5.8 6.8 7.8 8.8 9.8 10.8 11.8 12.8
Retention Time (minutes)
A52-002-1BA
Figure 2. GC-FID Chromatogram of 200 ng Each of C, H, and O Compounds
(Column 2)
133
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Method 617
The Determination of
Organohalide Pesticides and
PCBs in Municipal and Industrial
Wastewater
-------
-------
Method 617
The Determination of Organohalide Pesticides and PCBs in Municipal
and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1
This method covers the determination of certain Organohalide pesticides and PCBs. The
following parameters can be determined by this method:
Parameter
Aldrin
a-BHC
/3-BHC
5-BHC
•y-BHC
Captan
Carbophenothion
Chlordane
4,4'-DDD
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
Perthane
Strobane
Toxaphene
Trifluralin
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1 248
PCB-1254
PCB-1 260
Storet 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
39500
39504
39508
CAS No.
309-00-2
319-84-6
319-85-7
319-86-8
58-89-9
133-06-2
786-19-6
57-74-9
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
1 582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
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
137
-------
Method 617
modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative 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 clean-
up procedures. Under gas chromatography, the analyst is allowed the latitude to select chro-
matographic 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 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 L, is 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.
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 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
138
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Method 617
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimi-
nated 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 large 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 re-
quired to eliminate background phthalate contamination.3'4 The interferences from phthalate
esters can be avoided by using a microcoulometric or electrolytic conductivity detector.
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. 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 aware-
ness 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, hepta-
chlor, PCNB, PCBs, and toxaphene. Primary standards of these toxic materials should be
prepared in a hood.
139
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Method 617
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 con-
tamination 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 Separately funnel: 125-mL, 1000-mL, and 2000-mL, with TFE-fluorocarbon stop-
cock, ground-glass or TFE stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 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 minutes or perform a
Soxhlet extraction 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 strip-chart recorder. A data system is recommended for measuring peak areas.
140
-------
Method 617
5.6.1 Column 1: 180 cm long by 4 mm ID glass, packed with 1.5% SP-2250/1.95% 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 by 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 spec-
trometer, 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 interferant 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 indi-
cated 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 Acetonitrile, hexane-saturated: Mix pesticide-quality acetonitrile with an excess of hexane
until equilibrium is established.
6.5 Sodium sulfate: ACS, granular, anhydrous. Heat in a shallow tray at 400°C for a minimum
of 4 hours to remove phthalates and other interfering organic substances. Alternatively, heat
16 hours at 450 to 500 °C in a shallow tray or Soxhlet extract with methylene chloride for
48 hours.
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 H2SO4 (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 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.11 Stock standard solutions (1.00 /ig/^L): 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 in pesticide-quality isooctane and dilute to vol-
ume 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
141
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Method 617
used without correction to calculate the concentration of the stock standard. Commer-
cially-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 6 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 pL 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 in-
jected, 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 volu-
metric 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
142
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Method 617
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 detec-
tor.
7.3.2 Using injections of 1 to 5 jiL 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
Ais = Response for the internal standard
Cu = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in
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/AU 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 the lauric acid value is suggested. This procedure8 determines the
adsorption from hexane solution of lauric acid, in milligrams, per gram 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. Rec-
ommended procedures for calibration, separation and measurement of PCBs is discussed in
detail in the previous edition of this method.1 Illustrated methods for the calibration and
measurement of chlordane and strobane/toxaphene are available elsewhere.9
143
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Method 617
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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. 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 pipette, 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 + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts10 that are useful in observing trends in performance.
144
-------
Method 617
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 ali-
quots 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
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-L
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
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.
9. SAMPLE COLLECT/ON, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices11 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 within 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.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction.
145
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Method 617
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-L separatory funnel.
10.2 Add 60 mL 15% (v/v) methylene chloride in hexane to the sample bottle, seal, and shake 30
seconds to rinse the inner walls. Transfer the solvent to the separatory funnel and extract the
sample by shaking the funnel for 2 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, 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 Erlen-
meyer 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 one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 PTFE-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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
746
-------
Method 617
11. CLEANUP AND SEPARA TION
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 minute with 30-mL portions of hexane-satu-
rated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-L separatory runnel and add
650 mL of reagent water and 40 mL of saturated sodium chloride solution. Mix
thoroughly for 30 to 45 seconds. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 seconds.
11.2.3 Combine the hexane extracts in a 1-L 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.
11.2.5 Analyze by gas chromatography unless a need for further cleanup is indicated.
11.3 Florisil column cleanup: The following Florisil column cleanup procedure has been demon-
strated to be applicable to most of the organochlorine pesticides and PCBs listed in Table 3. It
should also be applicable to the cleanup of extracts for PCNB, strobane, and trifluralin.
11.3.1 Add a weight of Florisil (nominally 20 g), predetermined by calibration (Sections 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% (v/v) ethyl ether in hexane (Fra-
ction 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% (v/v) ethyl ether
747
-------
Method 617
in hexane (Fraction 2), into a second K-D flask. Perform a third elution using
200 mL of 50% (v/v) ethyl ether in hexane (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 Pipette 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.
11.4.2 Agitate the contents of the vial for 15 to 30 seconds.
11.4.3 Place the vial in an upright position on a reciprocal laboratory shaker and shake for
up to 2 hours.
11.4.4 If the mercury appears shiny after this treatment, analyze the extract by gas chromato-
graphy. 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. Inclu-
ded in this table are estimated retention tunes 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 /*L of the sample extract using the solvent-flush technique.12 Record the volume
injected to the nearest 0.05 fiL, 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 PCBs1 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.
148
-------
Method 617
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:
Equation 2
Concentration, ng/L =
(v,xvf)
where
A = Amount of material injected, in ng
V. = Volume of extract injected, in \tL
Vt = Volume of total extract, in \tL
V = 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:
Equation 3
Concentration, pg/L =
where:
As = Response for parameter to be measured.
Au = Response for the internal standard.
Is = Amount of internal standard added to each extract, in
V = 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.
149
-------
Method 617
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 five scans per peak but not to exceed seven 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.13
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 ng 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
± 10%. For example, if the relative abundance of an ion is 30% in the mass spec-
trum 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).
750
-------
Method 617
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.'5 The MDL
concentrations 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
151
-------
Method 617
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, 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, Phila-
delphia, 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., and 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, August 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. "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 Sup-
port Laboratory — Cincinnati, Ohio, 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.
752
-------
Method 617
References
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,
15, 1426 (1981).
16. McGrath, T. P., "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.
753
-------
Method 617
Table 1. Gas Chromotagraphy of Pesticides and PCBs
Parameter
Aldrin
ff-BHC
0-BHC
tf-BHC
y-BHC
Captan
Carbophenothion
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dichloran
Dicofol
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epox-
ide
Isodrin
Methoxychlor
Mi rex
PCNB
Trifluralin
Retention
Column 1
2.40
1.35
1.90
2.15
1.70
6.22
10.9
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
Time (min)
| Column 2
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
8.19
9.30
3.35
5.00
4.83
26.60
15.50
2.01
1.35
Method
Detection
i ifnif
L till 1 1
(ug/L)
0.009
0.004
ND
ND
0.002
ND
ND
0.012
0.004
0.032
ND
ND
0.011
0.11
0.17
ND
ND
ND
0.004
0.003
ND
0.176
0.015
0.002
0.013
* For multipeak materials, see Figures 2 through 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 in
a glass column 1.8 m long by 4 mm ID 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 glass
column 1.8 m long by 4 mm ID with 95% argon/5% methane carrier gas at a flow rate of
60 mL/min. Column temperature: isothermal at 200°C.
154
-------
Method 617
Table 2. Single-Operator Accuracy and Precision for Tap Water
Parameter
Aldrin
(J-BHC
K-BHC
4,4'-DDD
4,4'-DDE
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
(ug/U
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
155
-------
Method 617
Table 3. Distribution and Recovery of Chlorinated Pesticides and PCBs Using
Florisil Column Chromatography
Percent Recovery by Fraction
Parameter No. 1 \ No. 2 \ No. 3
Aldrin 100
o-BHC 100
0-BHC 97
-------
Method 617
Column: 1.5%SP-2250+
1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
o *
m
1 o
SI
*o 5
_i ^:
u
V-'-J
I I T 1 I I I I
0 4.0 8.0 12.0 16.0
Retention Time (minutes)
A52-002-38A
Figure 1. Gas Chromatogram of Pesticides
757
-------
Method 617
Column: 1.5% SP-2250+
1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: ElectronCapture
1 I I I \ I I
4.0 8.0 12.0
16.0
Retention Time (minutes)
A52-002-40A
Figure 2. Gas Chromatogram of Chlordane
158
-------
Method 617
Column: 1.5%SP-2250+
1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
0 2.0 6.0 10.0 14.0 18.0 22.0 26.0
Retention Time (minutes)
A52-002-41A
Figure 3. Gas Chromatogram of Toxaphene
159
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Temperature: 160°C
Detector: Electron Capture
—l 1 1 1 1 1 1
0 2.0 6.0 10.0 14.0
—I 1 1—
18.0 22.0
Retention Time (minutes)
A52-002-42A
Figure 4. Gas Chromatogram of PCB-1016
160
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Temperature: 160°C
Detector: Electron Capture
II I I I I I I I I I
2.0 6.0 10.0 14.0 18.0 22.0
Retention Time (minutes)
A52-002-43A
Figure 5. Gas Chromatogram of PCB-1221
161
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Temperature: 160°C
Detector: Electron Capture
i r~
14.0
~I T
22.0
0 2.0
6.0
10.0
18.0
22.0
Retention Time (minutes)
A52-002-44A
Figure 6. Gas Chromatogram of PCB-1232
162
-------
Met hod 617
Column: 1.5% SP-2250 + 1.95% SP-2401
on Supelcoport
Temperature: 160°C.
Detector: Electron Capture
I I
0 2.0
I I
6.0
I I I
10.0 14.0
I I
18.0
22.0
Retention Time (minutes)
A52-002-45A
Figure 7. Gas Chromatogram of PCB-1242
763
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on
Supelcoport
Temperature: 160°C.
Detector: Electron Capture
I I I I I I I T I 1 I 1 I
0 2.0 6.0 10.0 14.0 18.0 22.0 26.0
Retention Time (minutes)
A52-002-46A
Figure 8. Gas Chromatogram of PCB-1248
164
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
I I I I I I I T I I I
0 2.0 6.0 10.0 14.0 18.0 22.0
Retention Time (minutes)
A52-002-47A
Figure 9. Gas Chromatogram of PCB-1254
165
-------
Method 617
Column: 1.5% SP-2250+ 1.95% SP-2401 on Supelcoport
Temperature: 200°C
Detector: Electron Capture
0 2.0
i i
6.0
i i
10.0
14.0
18.0
22.0
26.0
Retention Time (minutes)
A52-002-48A
Figure 10. Gas Chromatogram of PCB-1260
766
-------
Method 618
The Determination of Volatile
Pesticides in Municipal and
Industrial Wastewater
-------
-------
Method 618
The Determination of Volatile Pesticides in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain volatile pesticides. The following parameters
can be determined by this method:
Parameter CAS No.
Chloropicrin 76-06-2
Ethylene dibromide 106-93-4
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges.
1.3 The method detection limit (MDL, defined in Section 15) for each compound 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 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.
1.5 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. 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, 20 mL, is extracted with cyclohexane. The cyclohexane
extract is analyzed with no additional treatment. Gas chromatographic conditions are de-
scribed which permit the separation of the compounds in the extract and their measurement by
an electron capture 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.1 Clean all glassware as soon as possible after
use by thoroughly rinsing with the last solvent used in it. Follow by washing with hot
169
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Method 618
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 minutes. Do not heat volu-
metric ware. Thorough rinsing with acetone may be substituted for the heating.
After drying and cooling, seal and store glassware in a clean environment to prevent
any accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
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 being sampled. Some
samples may require a cleanup approach 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 should be treated as a potential health hazard.
Chloropicrin produces severe sensory irritation in upper respiratory passages. It has strong
lacrimatory properties and produces increased sensitivity after frequent exposures. Taken
orally, chloropicrin causes severe nausea, vomiting, colic, and diarrhea. Chloropicrin is a
potent skin irritant. Ethylene dibromide liquid on the skin causes blisters if evaporation is
delayed. Inhalation of ethylene dibromide causes delayed pulmonary lesions. Prolonged
exposure may also result in liver and kidney injury. Exposure to these chemicals must be
reduced to the lowest possible level by whatever means available. The laboratory is respon-
sible 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 identified2"4 for the information of
the analyst.
5. APPARATUS AND MATERIALS
5.1 Sampling equipment, for discrete sampling.
5.1.1 Vial: 25-mL capacity or larger, equipped with a screw-cap with hole in center (Pierce
No. 13075 or equivalent). Detergent wash, rinse with tap and distilled water, and dry
at 105°C before use.
5.1.2 Septum: PTFE-faced silicone (Pierce No. 12722 or equivalent). Detergent wash,
rinse with tap and distilled water, and dry at 105°C before use.
5.2 Glassware (all specifications are suggested).
5.2.1 Centrifuge tube: 40-mL, with screw-cap lined with PTFE.
5.2.2 Pipette: 4-mL graduated.
5.2.3 Graduated cylinder: 25-mL.
5.2.4 Volumetric flask: 10-mL, ground-glass stoppered.
170
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Method 618
5.3 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.4 Gas chromatograph: Analytical system complete with 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.
5.4.1 Column 1: 180 cm long by 2 mm ID glass, packed with 1 % SP-1000 on Carbopak B
(60/80 mesh) or equivalent. This column was used to develop the method perfor-
mance statements in Section 15. Alternative columns may be used in accordance with
the provisions described in Section 11.1.
5.4.2 Column 2: 180 cm long by 2 mm ID glass, packed with 30% OV-17 on Gas
Chrom Q (100/120 mesh) or equivalent.
5.4.3 Detector: electron capture. This detector has proven effective in the analysis of
wastewaters for the compounds listed in the scope and was used to develop the meth-
od performance statements in Section 15. Alternative detectors, including a mass
spectrometer, may be used in accordance with the provisions described in Sec-
tion 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferant is not observed at
the method detection limit of each compound of interest.
6.2 Cyclohexane: Pesticide-quality or equivalent. Because of the frequent occurrence of con-
taminants in solvents, interfering with electron capture several lots of solvent, or a different
solvent, e.g., hexane, heptane, or isooctane, may have to be analyzed to find a suitable extrac-
tion solvent.
6.3 Sodium hydroxide: 6N in distilled water.
6.4 Sulfuric acid: 6N in distilled water.
6.5 Stock standard solutions (20 mg/ml): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions. Prepare stock solutions in cyclohexane
using assayed liquids.
6.5.1 Place about 9.5 mL of pesticide-quality cyclohexane in a 10-mL volumetric flask.
Allow the flask to stand, unstoppered, for about 5 minutes or until all cyclohexane-
wetted surfaces have dried. Weigh the flask to the nearest 0.1 mg. Using a 250-^iL
syringe, immediately add 121 /^L of chloropicrin (d420 = 1.66) and/or 92 /*L of
ethylene dibromide (d420 = 2.18). The liquid must fall directly into the cyclohexane
without contacting the neck of the flask. Reweigh, dilute to volume, stopper, and mix
by inverting the flask several times. Calculate the concentration in milligrams per
milliliter (mg/mL) from the net gain in weight. Larger volumes can be used 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.
6.5.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check stock standard solutions for signs of
777
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Method 618
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.5.3 Stock standard solutions must be replaced after 6 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 compound 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 cyclohexane. 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 pL 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 in-
jected, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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 specified; however, bromoform has been shown to be satisfactory in some
cases.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with cyclohexane. One of the standards
should be representative of a concentration near, but above, the method detection lim-
it. The other concentrations should correspond to the range of concentrations ex-
pected in the sample concentrates or should define the working range of the detector.
772
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Method 618
7.3.2 Using injections of 1 to 5 jtL 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
Ais = Response for the internal standard
Ca = Concentration of the internal standard, in \iglL
Cs = Concentration of the parameter to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A^A^ 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 compound
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 interferences 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.
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 measure-
ments. 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.
773
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Method 618
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 methanol such
that a 4-fjLL aliquot of the check sample concentrate in 20 mL of water gives the
selected concentration.
8.2.2 Using a 10-//L syringe, add 4 /*L of the check sample concentrate to each of a mini-
mum of four 20-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 ana-
lyzed 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 be-
fore 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 measured
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 compound being measured.
8.3.1 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 in Section 8.2.3. The UCL and LCL can be used to
construct control charts5 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 de-
fined 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.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 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 compound does not fall
174
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Method 618
within the control limits for method performance, the results reported for that compound 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
20-mL 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. 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 having a total volume of at least 25 mL.
Fill the sample bottle just to overflowing in such a manner that no air bubbles pass through the
sample as the bottle is being filled. Seal the bottle so that no air bubbles are entrapped in it.
Store the sample in an inverted position and maintain the hermetic seal on the sample bottle
until the time of analysis.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
10. SAMPLE EXTRACTION
10.1 Measure 20 mL of sample by pouring the sample into a 40-mL centrifuge tube equipped with a
PTFE-lined screw-cap to a predetermined 20-mL mark. Adjust pH of sample to 6 to 8 by
addition of 6N sodium hydroxide or 6N sulfuric acid. Measure 4.0 mL of extraction solvent
with a 4-mL graduated pipette and add to the centrifuge tube.
10.2 Shake the tube vigorously for 1 minute. Allow the layers to separate for at least 10 minutes.
Centrifuge, if necessary, to facilitate phase separation.
10.3 Withdraw an aliquot of the solvent layer and proceed with gas chromatographic analysis.
7 7. CLEANUP AND SEPARA TION
11.1 Cleanup procedures are not generally necessary. 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 is no less than 85%.
775
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Method 618
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. Examples of the separations achieved by Columns 1 and 2 are
shown in Figures 1 and 2 respectively. 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 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 in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 /*L of the sample extract using the solvent-flush technique.6 Record the volume
injected to the nearest 0.05 jiL, 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 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.
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:
Equation 2
Concentration, ng/L =
where
A = Amount of material injected, in ng
Vi = Volume of extract injected, in yL
Vt = Volume of total extract, in \>L
Vs = Volume of water extracted, in mL
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Method 618
13.1.2 If the internal standard calibration procedure is used, calculate the concentration in the
sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, ug/L = s.—f
(AU)(RF)(V0)
where
As = Response for parameter to be measured
AJJ = Response for the internal standard
Is = Amount of internal standard added to each extract, in ^g
Vo = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 seconds 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.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 decafluorotriphenyl phosphine (DFTPP) performance
criteria are achieved.7
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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
777
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Method 618
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
± 10%. For example, if the relative abundance of an ion is 30% in the mass spec-
trum 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 alternative 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.10 The MDL
concentrations listed in Table 1 were obtained using reagent water.8 Similar results were
achieved using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented hi Table 2 were obtained. Seven replicates each of two different
wastewaters were spiked and analyzed. The relative standard deviations of the percent recov-
ery of these measurements are also included in Table 2.
778
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Method 618
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, PA, p. 679, 1980.
2. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. "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.
6. Burke, J.A., "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,"
Journal of the Association of Official Analytical Chemists, 48 1037 (1965).
7. Eichelberger, J.W., Harris, L. E., and Budde, W. L., "Reference Compound to Calibrate
Ion Abundance Measurement in Gas Chromatography-Mass Spectrometry," Analytical Chem-
istry, 47, 995 (1975).
8. "Development of Methods for Pesticides in Wastewaters," Report from Battelle's Columbus
Laboratories for EPA Contract 68-03-2956 (in preparation).
9. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, 52 (1969).
10. Glaser, J.A. et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
179
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Method 618
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
Parameter
Chloropicrin
Ethylene Dibromide
Retention Time (mm)
Column 1
5.60
9.90
Column 2
2.03
3.15
Method Detection Limits
(u/U
0.8
0.2
Column 1 Conditions: Carbopak B (60/80 mesh) coated with 1% SP-1000 packed in a glass
column 1.8 m long by 2 mm ID with nitrogen carrier gas at a flow rate of 30 mL/minutes. Column
temperature, isothermal at 135°C. An electron capture detector was used with this column to
determine the MDL.
Column 2 Conditions: Gas Chrom Q (100/120 mesh) coated with 30% OV-17 packed in glass
column a 1.8 m long by 2 mm ID with helium carrier gas at a flow rate of 25 mL/minutes. Column
temperature, isothermal at 95°C.
Table 2. Single-Laboratory Accuracy and Precision3
Parameter
Chloropicrin
Ethylene Dibromide
Sample
Type*
1
2
1
2
Background
(ug/Lf
ND
ND
ND
ND
Spike
Level
ffig/U
5
50
5
50
Mean
Recovery
Standard
Deviation
98
98
69
108
12
3.3
6.9
4.8
No. of
Replicates
7
7
7
7
(a) Column 1 conditions were used.
(b) 1 = Low background relevant industrial effluent
2 = High background relevant industrial effluent
(c) ND = Not detected
180
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Method 618
Chloropicrin
Ethylene
Dibromide
i i i i i I i i I i i i i i i i i I i i
0 1.1 2.2 3.3 4.4 5.5 6.6 7.7 8.8 9.9 11.0
Retention Time (minutes)
A52-QOZ-20A
Figure 1. GC-ECD Chromatogram of 200 ng Chloropicrin and Ethylene
Dibromide in Cyclohexane (Column 1)
181
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Method 619
The Determination of Triazine
Pesticides in Municipal and
Industrial Wastewater
-------
-------
Method 619
The Determination of Triazine Pesticides in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain triazine pesticides. The following parameters
can be determined by this method:
Parameter STORET No. CAS No.
Ametryn — 834-12-8
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 — 86-50-0
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 shall be considered a major
modification subject to application and approval of alternative 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 600-series methods. 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).
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 tech-
nique. 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 pro-
785
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Method 619
vides 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 L, is 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 chromatographic conditions are de-
scribed which permit the separation and measurement of the compounds in the extract by gas
chromatography with a thermionic bead detector in the nitrogen mode.1'2
2.2 Method 619 represents an editorial revision of a previously promulgated 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.4 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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 hi 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 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.
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Method 619
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 aware-
ness 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 identified5"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-L 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 meth-
ylene 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 con-
tamination 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 by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fritted disc at
bottom and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderaa-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 400CC for 30 minutes or perform a
Soxhlet extraction with methylene chloride.
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Method 619
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 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 5% Carbowax 20M-TPA on
Supelcoport (80/100 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 by 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 de-
scribed in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferant 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 indi-
cated 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. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500 °C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.5 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in the dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.6 Stock standard solutions (1.00 ^g//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 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.
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Method 619
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 6 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 concentrations should cor-
respond 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 jtL 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 injec-
ted, 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 volumet-
ric 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 concentrations should correspond to the range of concentra-
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Method 619
tions expected in the sample concentrates, or should define the working range of the
detector.
7.3.2 Using injections of 1 to 5 jiL 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:
Equation 1
RF = _
where
As - Response for the parameter to be measured
Ais = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in
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, \/A-a 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 the lauric acid value is suggested. This procedure8 determines the
adsorption from hexane solution of lauric acid, in milligrams, per gram 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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory is required to maintain performance records to define the quality of data that is generated.
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Method 619
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 measure-
ments. 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 pipette, 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 be-
fore 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 in Sec-
tion 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 + 3s
Lower Control Limit (1CL) = R - 3s
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 ali-
quots 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
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Method 619
through the requirement for continuing quality control in Section 8.4. The accuracy
statements should be updated regularly.9
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-L
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
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.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices10 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 7 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-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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,
732
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Method 619
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 extrac-
tion 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 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 methylene chloride to complete the quantitative transfer.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 minutes. When the ap-
parent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes.
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 oftriazines 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 trans-
ferred 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
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Method 619
11. CLEANUP AND SEPARA TION
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% (v/v) ethyl ether in hexane (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 chromatography column. Elute the column into the flask,
using 200 mL of 15% (v/v) ethyl ether in hexane (Fraction 2). Perform a third
elution using 200 mL of 50% (v/v) ethyl ether in hexane (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 hex-
ane 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.
72. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded in this table are estimated retention times and method detection limits that can be achieved
by this 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 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 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.
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Method 619
12.4 Inject 1 to 5 /iL of the sample extract using the solvent-flush technique." Record the volume
injected to the nearest 0.05 /xL, 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 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.
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:
Equation 2
Concentration, ng/L = ''
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in fuL
Vt = Volume of total extract, in uL
Vs = Volume of water extracted, in mL
1 3.1 .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:
Equation 3
Concentration, pg/L = —
where
As = Response for parameter to be measured
A^ = Response for the internal standard
ls = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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Method 619
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 seconds 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.12
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.13
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 ng 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
±10%. For example, if the relative abundance of an ion is 30% in the mass spec-
trum 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).
736
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Method 619
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
concentrations listed in Table 1 were estimated from the response of the thermionic bead
nitrogen detector to each compound. The estimate is based upon the amount of material
required to yield a signal 5 times the GC background noise, assuming a 5-p.L injection from a
10-mL final extract of a 1-L sample.
15.2 In a single laboratory (either West Cost Technical Services, Inc., or Midwest Research Insti-
tute), using effluents from pesticide manufacturers and publicly owned treatment works
(POTW), the average recoveries presented in Table 2 were obtained after Florisil cleanup.1-2
The standard deviations of the percent recoveries of these measurements are also included in
Table 2.
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Method 619
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.
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. PB#82 132507, National Technical Information Service, Springfield,
Virginia.
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, September 1978.
4. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, 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, August 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, Pennsylvania, 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, March 1979.
10. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
198
-------
Method 619
References
fcont.)
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).
199
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Method 619
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
Prometon
Atraton
Propazine
Terbuthylazine
Secbumeton
Atrazine
Prometryn
Terbutryn
Simazine
Ametryn
Simetryn
ND = Not determined
Column 1 conditions: Supelcoport {80/100 mesh) coated with 5% Carbowax 20M-TPA packed in
a glass column 1.8 m long by 2 mm ID 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 1.0% Carbowax 20 M packed in
a glass column 1.8m long by 4 mm ID with helium carrier gas at 80 mL/min flow rate. Column
temperature, isothermal at 155°C.
Retention
Column 1
6.9
—
9.2
10.2
—
12.4
13.8
15.4
16.3
17.7
23.0
Time (min)
Column 2
4.9
6.3
6.7
7.3
8.3
9.4
10.3
—
12.7
14.0
—
Method
Detection Limit
(ijg/U
0.03
ND
0.03
0.03
ND
0.05
0.06
0.05
0.06
0.06
0.07
200
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Method 619
Table 2. Single-Laboratory Accuracy and Precision
Parameter
Ametryn
Atrazine
Prometon
Prometryn
Propazine
Simatryn
Simazine
Terbuthylazine
Terbutryn
Sample
Type*
3
3
3
1
1
2
3
3
1
3
3
3
1
3
3
3
1
2
Spike
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
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 =s Industrial process water
2 = Industrial effluent
3 =80% Industrial process water/20% industrial effluent
Table 3. Florisil Fractionation Patterns
Parameter
Propazine
Terbuthylazine
Atrazine
Ametryn
Prometryn
Simazine
Atraton
Secbumeton
Prometon
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
Percent Recovery by Fraction
No. 1 No. 2
0 90
0 30
0 20
No. 3 No. 4
10
70
80
100
100
100
100
100
100
201
-------
Method 619
fPrometon
/Propazine
j yTerbuthylazine
^Atrazine
Prometryn
/ /Simazine
'Terbutryn
/Ametryn
/Simetryn
I
0
i
5.0
I I I I I T I 1
10.0
15.0 20.0
25.0
Retention Time (minutes)
A52-002-49A
Figure 1. Gas Chromatogram of Triazine Pesticides on Column 1
For Conditions, See Table 1
202
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Method 620
The Determination of
Diphenylamine in Municipal and
Industrial Wastewater
-------
-------
Method 620
The Determination of Diphenylamine in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of diphenylamine, CAS. No. 122-39-4.
1.2 This is a gas chromatographic (GC) method applicable to the determination of diphenylamine
in municipal and industrial discharges.
1.3 The method detection limit (MDL, defined in Section 15) for diphenylamine 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 similar to those of other 600-
series methods. Thus, a single sample may be extracted to measure the compounds included
in the scope of the 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.
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 L, is extracted with methylene chloride using
a continuous extractor. The methylene chloride extract is dried and concentrated to 5.0 mL.
Gas chromatographic conditions are described which permit the separation and measurement of
the compounds in the extract by alkali flame detector (AFD) gas chromatography.1
2.2 This method provides an optional silica gel column cleanup procedure 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
205
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Method 620
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 minutes. Thermally
stable materials, such as PCBs, may 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 being 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 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 aware-
ness 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.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE/PIFE. Aluminum foil may be substituted for PTFE
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 meth-
ylene 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,
205
-------
Method 620
followed by repeated rinsings with reagent water to minimize the potential for con-
tamination 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 Continuous extractor: 2000-mL, available from Paxton Woods Glass Shop, Cincin-
nati, Ohio, or equivalent.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K570001-0500 or equivalent).
Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K503000-0121 or equi-
valent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K569001-0219 or equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 5-mL with glass stopper.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, 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 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method perfor-
mance statements in Section 15. Guidelines for the use of alternative columns are
provided in Section 12.1.
5.6.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP 1000 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali-flame detector (AFD), sometimes referred to as a nitrogen-phos-
phorous detector (NPD) or a thermionic-specific detector (TSD). This detector has
proven effective in the analysis of wastewaters for the compounds listed in the scope
and was used to develop the method performance statements in Section 15. Alter-
native detectors, including a mass spectrometer, may be used in accordance with the
provisions described in Section 12.1.
207
-------
Method 620
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, acetone, methanol, petroleum ether, ethyl ether, toluene (distilled-in-glass
quality or equivalent). Ethyl ether must be free of peroxides as indicated by EM Quant test
strips (available from Scientific Products Co., Catalog No. PI 126-8, and other suppliers).
Procedures recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davison Grade 923, 100-200 mesh; activated by heating for 24 hours at 150°C.
6.5 6N sulftiric acid: Slowly add 16.7 mL of concentrated H2SO4 (94%) to about 50 mL of
reagent water. Dilute to 100 mL with reagent water.
6.6 6N sodium hydroxide: Dissolve 24.0 grams of sodium hydroxide in 100 mL of reagent water.
6.7 Stock standard solutions (1.00 /*g//iL): 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 distilled-in-glass quality methanol 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 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can 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 PTFE-sealed screw-cap bottles. 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.
6.7.3 Stock standard solutions must be replaced after 6 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 compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with toluene. One of the external standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations found in real
samples or should define the working range of the detector.
208
-------
Method 620
7.2.2 Using injections of 2 to 5 /xL 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 in-
jected, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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 ana-
lyst 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, although carbazole has been used successfully in some instances.
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 volumet-
ric flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with toluene. One of the standards should be
at a concentration near, but above, the method detection limit. The other concentra-
tions 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 /*L 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:
Equation 1
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in pg/L
Cs = Concentration of the parameter to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of re-
sponse ratios, A/A^ against RF.
205
-------
Method 62O
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 compound
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 interferences 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.
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 measure-
ments. 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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must review potential
problem areas and repeat the test.
270
-------
Method 620
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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
R and s. Alternatively, the analyst must use four wastewater datapoints gathered
through the requirement for continuing quality control in Section 8.4. The accuracy
statements6 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
211
-------
Method 620
9.2 The samples must be iced or refrigerated at 40°C, from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid imme-
diately after sampling.
10. SAMPLE EXTRACTION
10.1 Assemble continuous extraction apparatus by placing five to ten carborundum chips into the
500-mL round-bottom flask and attaching to the extraction flask.
10.2 Add 400 mL methylene chloride to the extraction flask. Some methylene chloride should
displace into the round-bottom flask.
10.3 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into the extraction flask and add sufficient distilled water to
fill the extraction flask (2 L total volume aqueous phase).
10.4 Check the pH of the sample with wide-range pH paper and adjust to 6 to 8 with 6N sodium
hydroxide or 6N sulfuric acid.
10.5 Connect the stirring apparatus to the extraction flask without the frit touching the sample.
Heat methylene chloride in round-bottom flask to continuous reflux and continue heating for
30 minutes to 1 hour until frit is thoroughly wetted with methylene chloride.
10.6 Lower frit until it just touches the sample and start the stirring apparatus rotating.
10.7 Continuously extract sample for 18 to 24 hours.
10.8 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.
10.9 Pour the extract from the round-bottom flask through a drying column containing about 10 cm
of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the flask
and column with 20 to 30 ml of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.10 Add one to two 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 minutes. At the proper rate of distillation,
the balls of the column will actively chatter but the chambers will not flood with condensed
solvent. When the apparent volume of liquid reaches approximately 4 mL, remove the K-D
apparatus and allow it to drain and cool for at least 10 minutes.
10.11 Remove the Snyder column and flask and adjust the volume of the extract to 5.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further processing
will not be performed immediately. If the extract is to be stored longer than two days, trans-
fer the extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with solvent exchange to toluene and gas chromatographic analysis as
212
-------
Method 620
described in Sections 11.5 and 12 respectively. If the sample requires cleanup, proceed to
Section 11.
10.12Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
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 clean waters
and industrial 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 is no less than 85%.
11.2 Stir 20 g of silica gel in 100 mL of acetone and 1.2 mL of reagent water for 30 minutes on a
stirring plate. Transfer the slurry to a chromatographic column (silica gel may be retained
with a plug of glass wool). Wash the column with 20 mL of methylene chloride and then with
30 mL of petroleum ether. Use a column flow rate of 2 to 2.5 mL/min throughout the wash
and elution profiles. Add an additional 50 mL of petroleum ether to the head of the column.
11.3 Add the extract from Section 10.11 to the head of the column. Allow the solvent to elute
from the column until the Florisil is almost exposed to the air. Elute the column with 50 mL
of 6% ethyl ether in petroleum ether. Discard this fraction.
11.4 Elute the column with 100 mL of 15% ethyl ether in petroleum ether and collect in a K-D
apparatus.
11.5 Add 2.5 mL of toluene to the fraction. Concentrate the fraction to approximately 4 mL with
the water bath at 75 to 80°C as described in Section 10.10. Transfer the sample to a 5-mL
volumetric flask and dilute to 5 mL with toluene. Proceed with gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded 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 and Column 2 are
shown in Figures 1 and 2. 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 demon-
strated to be less than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 \tL of the sample extract using the solvent flush technique.8 Record the volume
injected to the nearest 0.05 /iL and the resulting peak sizes 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
213
-------
Method 62O
suggested window size; 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.
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:
Equation 2
(A)(Vt)
Concentration, ng/L =
(V,XV,)
where
A = Amount of material injected, in ng
Vf = Volume of extract injected, in \>L
Vt = Volume of total extract, in \>L
Vs = Volume of water extracted, in mL
13.1.2 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:
Equation 3
Concentration, ftg/L = —
where
As = Response for parameter to be measured
A.a = Response for the internal standard
I5 = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
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Method 620
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 seconds 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. When using a fused-silica capillary column, the column
outlet should be threaded through the interface to within a few millimeter of the entrance to the
source ionization chamber. 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. The calculation
of tailing factors is illustrated in Method 625.
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 DFTPP performance criteria are achieved.9
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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
14.4.1 The molecular ion and all other 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 ±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 30 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. The MDL
275
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Method 620
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two
different wastewaters were spiked and analyzed. The standard deviation of the percent recov-
ery is also included in Table 2.1
276
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Method 620
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 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 Publications,
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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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. 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).
217
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Method 620
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (min) Method Detection Limit
Parameter Column 1 | Column 2 (fjg/L)
Diphenylamine 18.1 19.3 1.6
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is held at 80°C for 4 minutes, programmed from 80 to 300°C at 8°C/min, and held at
300°C for 4 minutes.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-1000 packed in a glass
column 1.8 m long by 2 m ID with helium carrier gas at a flow rate of 30 mL/min Column tempera-
ture is held at 80°C for 4 minutes, programmed from 80 to 250°C at 80°C/min, and held at
250°C for 4 minutes.
Table 2. Single-Laboratory Accuracy and Precision8
Relative
Average Standard Spike Number
Percent Deviation Level of Matrix
Parameter Recovery (%) (vg/U Analyses Type"
Diphenylamine 120 25 5.0 7 1
89 11 50.0 7 1
(a) Column 1 conditions were used.
(b) 1 = Columbus secondary POTW effluent.
218
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Method 620
29.0
31.0
33.0 35.0
Retention Time (minutes)
A52-002-SOA
Figure 1. GC-AFD Chromatogram of 100 ng of Diphenylamine (Column 1)
219
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Method 622
The Determination of
Organophosphorus Pesticides in
Municipal and Industrial
Wastewater
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Method 622
The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
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
Bolstar — 35400-43-2
Chlorpyrifos — 2921-88-2
Chlorpyrifos methyl — 5598-13-0
Coumaphos 81293 56-72-4
Demeton 39560 8065-48-3
Diazinon 39570 333-41-5
Dichlorvos — 62-73-7
Disulfoton 39010 298-04-4
Ethoprop — 13194-48-4
Fensulfothion — 115-90-2
Fenthion 39016 55-38-9
Merphos 39019 150-50-5
Mevinphos — 7786-34-7
Naled — 300-76-5
Parathion methyl 39600 298-00-0
Phorate 39023 298-02-2
Ronnel 39357 299-84-3
Stirofos — 961-11-5
Tokuthion — 34643-46-4
Trichloronate — 327-98-0
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 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 seve-
ral others in the 600-series methods. 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).
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Method 622
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.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is 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 chromatographic 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.1
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 special cleanup approaches or selective GC detectors to achieve the MDL
listed in Table 1. Use of a flame photometric detector in the phosphorus mode will minimize
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 conductivity or microcoulo-
224
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Method 622
metric) 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 aware-
ness 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 identified3 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-L 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 meth-
ylene 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 con-
tamination 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 by 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.
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Method 622
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform Soxhlet
extraction 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 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.
5.6.1 Columns: These columns were used to develop the method performance statements in
Section 15. Alternate columns may be used in accordance with the provisions de-
scribed in Section 12.1.
5.6.1.1 Column 1: 180 cm long by 2 mm ID glass, packed with 5% SP-2401 on
Supelcoport (100/120 mesh) or equivalent.
5.6.1.2 Column 2: 180 cm long by 2 mm ID glass, packed with 3% SP-2401 on
Supelcoport (100/120 mesh) or equivalent.
5.6.1.3 Column 3: 50 cm long by W OD PTFE, 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 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. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.5 Stock standard solutions (1.00 f*g//iL): 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 stock standards may be used at any
concentration if they are certified by the manufacturer or by an independent source.
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Method 622
6.5.2 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 6 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 concentrations should cor-
respond 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 /xL 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 in-
jected, 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 volumet-
ric 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 concentrations should correspond to the range of concentra-
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Method 622
tions expected in the sample concentrates, or should define the working range of the
detector.
7.3.2 Using injections of 1 to 5 /xL 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:
Equation 1
RF = _
where
As = Response for the parameter to be measured
Ais = Response for the internal standard
Ca = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in fig/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, A/A^ 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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. Each time such modifications to the method are made, the analyst is required
to repeat the procedure in Section 8.2.
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Method 622
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 pipette, 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 be-
fore 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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.
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Method 622
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-L
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
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.
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. Com-
posite 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 7 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-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion procedure a second time, combining the extracts in the Erlenmeyer flask. Perform a third
extraction in the same manner.
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Method 622
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 methylene chloride to complete the quantitative transfer.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 minutes. When the ap-
parent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes.
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 re-
quires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
11. CLEANUP AND SEPARA TION
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 tunes and method detection limits that can be
231
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Method 622
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 in the sample, GC analysis for
dichlorvos and naled must be performed using Column 3. Examples of the separations
achieved are shown in Figures 1 through 4. Other packed columns, chromatographic con-
ditions, 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 /xL of the sample extract using the solvent-flush technique.8 Record the volume
injected to the nearest O.OS /*L, 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 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, 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:
Equation 2
Concentration, pg/L =
where
A = Amount of material injected, in ng
V. = Volume of extract injected, in \>L
Vt = Volume of total extract, in \>L
Vs = Volume of water extracted, in mL
232
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Method 622
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:
Equation 3
Concentration, uglL =
(Ais)(RF)(Va)
where
As = Response for parameter to be measured
Ais = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 seconds 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.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 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 chromatographic conditions. It is
recommended that at least 25 ng 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
233
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Method 622
to ± 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." The MDL
concentrations listed in Table 1 were estimated from the response of the detector to each com-
pound. The estimate is based upon the amount of material required to yield a signal 5 times
the GC background noise, assuming a 5-fiL injection from a 10-mL final extract of a 1-L
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.1 The standard deviations of the percent recoveries of these measure-
ments are also included in Table 2.
234
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Method 622
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.
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, Phila-
delphia, Pennsylvania, 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, August 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 Sup-
port Laboratory: Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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,
15, 1426 (1981).
235
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Method 622
Table 1. Chromatographic Conditions and Estimated Method Detection Limits
GC Retention Time Estimated MDL
Parameter Column (mm) fag/L)
Demeton 1a 1.16 0.25
2.53
Phorate 1a 1.43 0.15
Disulfoton 1a 2.10 0.20
Trichloronate 1a 2.94 0.15
Fenthion 1a 3.12 0.10
Tokuthion 1a 3.40 0.5
Bolster 1a 4.23 0.15
Fensulfothion 1a 6.41 1.5
Azinphos methyl 1a 6.80 1.5
Coumaphos 1a 11.6 1.5
Dichlorvos 1b 0.8 0.1
Mevinphos 1b 2.41 0.3
Stirofos 1b 8.52 5.0
Ethoprop 2 3.02 0.25
Parathion methyl 2 3.37 0.3
Ronnel 2 5.57 0.3
Chlorpyrifos methyl 2 5.72 0.3
Chlorpyrifos 2 6.16 0.3
Merphos 2 7.45 0.25
Oiazinon 2 7.73 0.6
Dichlorvos 3 1.50 0.1
Naled 3 3.28 0.1
Stirofos 3 5.51 5.0
Column la conditions: Supelcoport (100/120 mesh) coated with 5% SP-2401 packed in a glass
column 180 cm long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature, programmed: Initial 150°C, hold for 1 minute, 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 1b conditions: Same as Column 1a, except nitrogen carrier gas at a flow rate of
30 mL/min. Temperature, programmed: Initial 170°C, hold 2 minutes, 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 glass
column 180 cm long by 2 mm ID with helium carrier gas at a flow rate of 25 mL/min. Column
temperature, programmed, initial 170°C, hold for 7 minutes, then program at 10°C/min to 250°C
and hold. A thermionic bead detector was used with this column to estimate the MDL.
Column 3 Conditions: Gas Chrom Q (100/120 mesh) coated with 15% SE-54 packed in a PTFE
column 50 cm long by Va" OD 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 MDL.
236
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Method 622
Table 2. Single-Operator Accuracy and Precision
Parameter
Azinphos methyl
Bolstar
Chlorpyrifos
Coumaphos
Demeton
Diazinon
Dichlorvos
Disulfoton
Ethoprop
Fensulfothion
Fenthion
Merphos
Mevinphos
Naled
Parathion methyl
Phorate
Ronnel
Stirofos
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 (%)
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 Range
frg/U
21-250
4.9-46
1.0-50.5
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
Number of
Analyses
17
17
18
17
17
7
16
17
18
17
. 17
18
16
16
21
17
18
16
17
3
Types
3
3
3
3
3
1
3
3
3
3
3
3
3
3
3
3
3
3
3
1
237
-------
Method 622
8.0
9.0
10.0 11.0 12.0
Retention Time (minutes)
A52-002-53A
Figure 1. Gas Chromatogram of Organophosphorus Pesticides on Column 1a
(for conditions, see Table 1)
238
-------
Method 622
Dichlorvos
\
Mevinphos
/
Stirofos
—i r~
o 1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
Retention Time (minutes)
A52-002-52A
Figure 2. Gas Chromatogram of Organophosphorous Pesticides on Column 1b
(for conditions, see Table 1)
239
-------
Method 622
Merphos
Diazinon
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Retention Time (minutes)
A52-002-S4A
Figure 3. Gas Chromatogram of Organophosphorus Pesticides on Column 2
for conditions, see Table 1)
240
-------
Method 622
Dichlorvos
rStirofos
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Retention Time (minutes)
A52-002-5SA
Figure 4. Gas Chromatogram of Organophosphorus Pesticides on
Column 3 (for conditions, see Table 1)
241
-------
-------
Method 622.1
The Determination of
Thiophosphate Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 622.1
The Determination of Thiophosphate Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain thiophosphate pesticides. The following
parameters can be determined by this method:
Parameter CAS No.
Aspon 3244-90-4
Dichlofenthion 97-17-6
Famphur 52-85-7
Fenitrothion 122-14-5
Fonophos 944-22-9
Phosmet 732-11-6
Thionazin 297-97-2
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial 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 alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each parameter is listed in
Table 2. 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
certain other 600-series methods. Thus, a single sample may be extracted to measure the
compounds included in the scope of the methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply ap-
propriate cleanup procedures.
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.
245
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Method 622.1
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory runnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by alkali flame detector gas chromatography (GC/AFD).1
2.2 This method provides a Florisil column cleanup procedure to aid in the elimination of inter-
ferences that 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 thoroughly rinsing with tap and reagent water.
Drain dry and heat in an oven or muffle furnace at 400°C for 15 to 30 minutes. Do
not heat volumetric glassware. Some thermally stable materials, such as PCBs, may
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 overcome many of these interferences, but unique
samples may require additional cleanup approaches to achieve the MDLs listed in Table 2.
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 aware-
ness 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 identified3"5 for the information of the analyst.
246
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Method 8#. f
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-quart or 1-L volume, fitted
with screw-caps lined with PTFE. Foil may be substituted for PTFE 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 should be thoroughly rinsed with methanol,
followed by repeated rinsing with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-propor-
tional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory runnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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. A
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 equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or
extract in a Soxhlet with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±2°C). The bath should be used in a
hood.
247
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Method 622.1
5.5 Balance: 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 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 by 2 mm ID glass, packed with 3% SP-2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop the method perfor-
mance 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 by 2 mm ID glass, packed with 3% SP-2100 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phos-
phorous detector (NPD) or a thermionic-specific detector (TSD). This detector has
proven effective in the analysis of wastewaters for the compounds listed in the scope
and was used to develop the method performance statements in Section 15.
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, petroleum ether, anhydrous ethyl ether, and acetone: Dis-
tilled-in-glass quality or equivalent. Ethyl ether must be free of peroxides as indicated by EM
Quant Test Strips (available from Scientific Products Co., Catalog No. P 1126-8 and other
suppliers). Procedures recommended for removal of peroxides are provided with the test
strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in a brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFE or aluminum-foil-lined screw-cap and cool to room tempera-
ture.
6.5 6N sodium hydroxide.
6.6 6N sulfuric acid.
6.7 Stock standard solutions (1.00 /*g//*L): 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 mate-
rial. Dissolve the material in distilled-in-glass 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 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.
248
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Method 622.1
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check standard solutions for signs of degrada-
tion or evaporation, especially just prior to preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after 6 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 2.
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:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumet-
ric flask and diluting to volume with ethyl ether. One of the external standards should
be at a concentration near, but above, the method detection limit. The other con-
centrations 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 /*L 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, the ratio of the response to the mass injec-
ted, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volumet-
ric flask. To each calibration standard, add a known constant amount of one or more
internal standards, and dilute to volume with ethyl ether. One of the standards should
be at a concentration near, but above, the method detection limit. The other con-
centrations should correspond to the range of concentrations expected in the sample
concentrates or should define the working range of the detector.
249
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Method §22.1
7.3.2 Using injections of 1 to 5 /*L 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in \nglL
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A^A^ 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 compound
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 interferences 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.
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 measure-
ments. 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.
250
-------
Method 622.1
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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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%.1
251
-------
Method 622.1
8.5 Before processing any samples, the analyst should demonstrate though the analysis of a 1-L
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. 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid im-
mediately after sampling.
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid.
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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 concentration devices or techniques may be used in place of
the K-D if the requirements of Section 8.2 are met.
252
-------
Method 622.1
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.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a
two-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
methylene chloride and concentrate the solvent extract as before. When an apparent volume of
0.5 mL is reached, or the solution stops boiling, remove the K-D apparatus and allow it to
drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 3 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with the gas chromatographic analysis in Section 12. 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5 mL.
71. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is no less than that reported in Table 3.
11.2 The following Florisil column cleanup procedure has been demonstrated to be applicable to the
seven thiophosphate pesticides listed in Table 1.
11.2.1 Add 20 g of Florisil to 100 mL of ethyl ether and 400 /*L of reagent water in a
250-mL Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry
to a chromatographic column (Florisil may be retained with a plug of glass wool).
Allow the solvent to elute from the column until the Florisil is almost exposed to the
253
-------
air. Wash the column with 25 mL of petroleum ether. Use a column flow of 2 to
2.5 mL/min throughout the wash and elution profiles. Add an additional 50 mL of
petroleum ether to the head of the column.
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the Florisil is almost exposed to the
air. Elute the column with 50 mL of 6% ethyl ether in petroleum ether. Discard this
fraction.
11.2.3 Elute the column with 50 mL of 15% ethyl ether in petroleum ether (Fraction 1)
and collect eluate in a K-D apparatus. Repeat process with 50 mL of 50% ethyl
ether in petroleum ether (Fraction 2), 50 mL of 100% ethyl ether (Fraction 3),
50 mL 6% acetone in ethyl ether (Fraction 4), and 100 mL 15% acetone in ethyl
ether (Fraction 5), collecting each fraction in a separate K-D apparatus. The elution
patterns for the thiophosphates are shown in Table 1. Concentrate each fraction to
1 mL as described in Sections 10.6 and 10.7. Proceed with gas chromatographic
analysis.
11.2.4 The above-mentioned fractions can be combined before concentration at the discretion
of the analyst.
12. GAS CHROMATOGRAPHY
12.1 Table 2 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. Examples of the separations achieved by Column 1 and Column 2
are shown in Figures 1 and 2. Other packed columns, chromatographic conditions, or detec-
tors 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 gas chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 pL of the sample extract using the solvent flush technique.8 Record the volume
injected to the nearest 0.05 jiL, and record the resulting peak sizes 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-tune 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 can be used to calculate a
suggested window size; 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.
254
-------
Method 622.1
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:
Equation 2
Concentration, p.g/L =
where
A = Amount of material injected, in ng
Vf = Volume of extract injected, in pL
Vt = Volume of total extract, in \>L
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:
Equation 3
Concentration, fj.g/L = —
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
Vo = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 seconds per scan utilizing a 70 V (nominal) electron
energy in the electron impact ionization mode. A GC-to-MS interface constructed of all glass
255
-------
Method 622.1
or glass-lined materials is recommended. When using a fused-silica capillary column, the
column outlet should be threaded through the interface to within a few millimeters of the
entrance to the source ionization chamber. A computer system should be interfaced to the
mass spectrometer that allows the continuous acquisition 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.10
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 DFTPP performance criteria are achieved.9
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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
14.4.1 The molecular ion and all other 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 ± 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 alternative 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.11 The MDL
concentrations listed in Table 2 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained after Florisil cleanup. Seven replicates
of each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 3.1
256
-------
Method 622 1
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 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-6007
4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory - Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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. 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).
10. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52 (1969).
11. Glaser, J.A. et al., "Trace Analysis for Wastewaters", Environmental Science and Technology,
15, 1426 (1981).
257
-------
Method 622.1
F1 F
2 F3
94
92
84
F4
2
51
6
2
F5
55
93
F6
6
37
F7
103
69
Total
96
92
109
106
90
106
95
Table 1. Elution Orders and Recoveries of Thiophosphates from Florisil
Recovery in Specified Fraction (%)"
Compound
Aspon
Dichlofenthion
Famphur
Fenitrothion
Fonophos
Phosmet
Thionazine
(a) Results of single determination with 100 pg of each compound. Elution solvents were 50 mL
each of the following:
F1 = 2% methylene chloride in petroleum ether
F2 = 6% ethyl ether in petroleum ether
F3 = 15% ethyl ether in petroleum ether
F4 = 50% ethyl ether in petroleum ether
F5 = 100% ethyl ether
F6 = 6% acetone in ethyl ether
F7 = 15% acetone in ethyl ether
Table 2. Chromatographic Conditions and Estimated Method Detection Limits
Retention Time (mini
- — T - MDL
Parameter Column 1 Column 2
Thionazin 18.3 25.0 1
Fonophos 20.5 27.8 0.7
DichJofenthion 21.4 29.4 0.7
Aspon 22.6 30.2 0.6
Fenitrothion 23.1 30.8 2
Famphur 28.1 34.8 19
Phosmet 30.0 36.2 1
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is programmed from 80 to 300°C at 8°C/min with a 4 minute hold at each extreme,
injector temperature is 250°C and detector is 300°C. Alkali flame detector at bead voltage of 16
volts.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a glass
column 1.8 m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/minute. Column
temperature is programmed from 80 to 300°C at 8°C/min with a 4 minute hold at each extreme,
injector temperature is 250°C and detector is 300°C.
258
-------
Method 622.1
Table 3. Single-Laboratory Accuracy and Precision3
Relative
Mean Standard
Parameter
Aspon
Dichlofenthion
Famphur
Fenitrothion
Fonophos
Phosmet
Thionizin
(a) Column 1 conditions were used.
(b) 1 = Low-level relevant industrial effluent
2 = Municipal sewage influent
(c) ND = Not detected
Sample
Type"
1
2
1
2
1
2
1
2
1
2
1
2
1
2
Background
frg/LJe
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Spike
(ug/U
50
500
50
500
50
500
50
500
50
500
50
500
50
500
Recovery
(%)
83
87
83
84
86
86
82
83
84
86
85
87
84
89
Deviation /I
(%) A
7
3
7
4
6
4
7
4
7
4
5
5
7
5
fumbi
teplic
7
7
7
7
7
7
7
7
7
7
7
7
7
7
259
-------
Method 622.1
Thionazin
\
V
Aspon
Fenitrothion
Famphur
Phosmet
/i i i i i i i i i i i i i \ \ \ i i i
' 17.0 19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0
Retention Time (minutes)
A52-002-56
Figure 1. GC-AFD Chromatogram of 100 ng Each of Seven Thiophosphates (Column 1)
260
-------
Method 622.1
Fonofos
Aspon
Famphur
Phosmet
' I i i i i r i i i i i i i i i i i i i i
0 25.5 27.0 28.5 30.0 31.5 33.0 34.5 36.0 37.5 39.0
Retention Time (minutes)
A52-002-57A
Figure 2. GC-FID Chromatogram of 100 ng Each of Seven Thiophosphates (Column 2)
261
-------
-------
Method 627
The Determination of
Dinitroaniline Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 627
The Determination of Dinitroaniline Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain dinitroaniline pesticides. The following
parameters can be determined by this method:
Parameter STORET No. CAS No.
Benfluralin 39002 1861-40-1
Ethalfluralin — 55283-68-6
Isopropalin — 33820-53-0
Profluralin — 26399-36-0
Trifluralin 39030 1582-09-8
1.2 This method fails to distinguish between benfluralin, ethalfluralin, and trifluralin. When more
than one of these materials may be present in a sample, the results are reported as trifluralin.
1.3 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 shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR
136.4 and 136.5.
1.4 The method detection limits (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.
1.5 The sample extraction and concentration steps in this method are essentially the same as
several other the 600-series methods. 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).
1.6 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.
1.7 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 chromatographic
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 ehromatograph/mass spectro-
meter (GC/MS) criteria appropriate for the qualitative confirmation of compound identifications.
265
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Method 627
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is 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 chromatographic conditions are
described which permit the separation and measurement of the compounds in the extract by
electron capture (EC) gas chromatography.1
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 hi 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 materials during laboratory operations. Cross-contami-
nation 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.
266
-------
Method 627
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 aware-
ness 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 identified5"7 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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 con-
tamination 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 by 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 minutes or perform a Soxh-
let extraction 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.
257
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Method 627
5.6 Gas chromatograph: Analytical system complete with 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 1.5% OV-17/1.95% OV-210
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 by 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. Condition by heat in a shallow tray at 400°C for
a minimum of 4 hours to remove phthalates and other interfering organic substances. Alterna-
tively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction with
methylene chloride for 48 hours.
6.4 Stock standard solutions (1.00 fig//*L): 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.
6.4.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
268
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Method 627
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 cali-
brated 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 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. 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 ^L 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 injec-
ted, 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 volumet-
ric 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.
269
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Method 627
7.3.2 Using injections of 1 to 5 pL 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:
Equation 1
RF
where
As = Response for the parameter to be measured
Ais = Response for the internal standard
Cis = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in \iglL
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, A/A^ 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 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 measure-
ments. 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.
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Method 627
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 pipette, 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 be-
fore 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.
8.3.1 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 in Section 8.2.3. The UCL and LCL can be used to
construct control charts8 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 ali-
quots 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
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-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each
271
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Method 627
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.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices9 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 7 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-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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 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 methylene chloride to complete the quantitative transfer.
272
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Method 627
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 minutes. When the ap-
parent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes.
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 re-
quires cleanup, proceed to Section 11.
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 proce-
dure is no less than 85%.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded 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 conditions, or detectors 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 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 tri-
fluralin if more than one of these materials may be present in a sample.
273
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Method 627
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 /*L, 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 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.
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:
Equation 2
(A)( V )
Concentration, ug/L = '
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in uL
Vt = Volume of total extract, in pL
Vs = Volume of water extracted, in mL
13.1.2 If the internal standard calibration procedure was used, calculate the concentration hi
the sample using the response factor (RF) determined in Section 7.3.2 as follows:
Equation 3
Concentration, ug/L = —
where
As = Response for parameter to be measured
AX = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
274
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Method 627
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 charac-
terized beyond the capabilities provided in this method.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls out-
side 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 seconds 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.11
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
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 ng 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
±10%. For example, if the relative abundance of an ion is 30% in the mass spec-
trum 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.
275
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Method 627
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.13 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 reagent water and effluents
from pesticide manufacturers and 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.
276
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Method 627
References
1. "Pesticide Methods Evaluation," Letter Report #5 for EPA Contract No. 68-03-2697. Avail-
able from U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
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, Phila-
delphia, Pennsylvania, 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, August 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, March 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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,
15, 1426, 1981.
277
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Method 627
Table 1. Gas Chromatography and Method Detection Limits of Dinitroanilines
Parameter
Trifluralin
Benfluralin
Ethalfluralin
Profluralin
Isopropalin
Retention
Column 1
1.6
1.6
1.6
2.3
6.4
Time (min)
Column 2
1
2.2
2.3
2.3
3.4
6.3
Method
Detection Limit
M1
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 glass column 1.8 m long by 2 mm ID 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 glass column 1.8 m long by
2 mm ID with nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature: held at
160°C for 2 minutes, then programmed to 200°C at 10°C/min.
Table 2. Single-Operator Accuracy and Precision
Parameter
Benfluralin
Isopropalin
Profluralin
Trifluralin
Sample
Type
IW
DW
IW
DW
IW
DW
IW
Spike Range
(ug/L)
2.00
0.50
2.20
0.50
2.04
0.50
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
278
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Method 627
Trifluralin
Profluralin
Isopropalin
i i r T i i i I i
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Retention Time (minutes)
A52-002-58
Figure 1. Gas Chromatogram of Dinitroaniline Pesticides on Column 1
(for conditions, see Table 1)
279
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Method 629
The Determination of Cyanazine
in Municipal and Industrial
Wastewater
-------
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Method 629
The Determination of Cyanazine in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of cyanazine. The following parameter can be deter-
mined 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 deter-
mination 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 alternative 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 /xg/L.
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 liquid chromatography and in the interpretation of liquid chromatograms. Each analyst
must demonstrate the ability to generate acceptable results with this method using the proce-
dure described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for cyanazine, compound identifi-
cations should be supported by at least one additional qualitative technique.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is 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
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
283
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Method 629
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 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 aware-
ness 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 identified3"5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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 con-
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Method 629
lamination 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 by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 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 minutes or perform a
Soxhlet extraction 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 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 by 2.6 mm ID stainless steel packed with Spherisorb ODS
(10 ju,m) 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 state-
ments in Section 14. Alternative detectors may be used in accordance with the provi-
sions 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.
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Method 629
6.2 Acetone, hexane, methylene chloride: Pesticide-quality or equivalent.
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as indi-
cated 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 Methanol: HPLC/UV quality.
6.5 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500 °C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.7 Stock standard solution (1.00 /xg//xL): 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-fluorocarbon-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 6 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 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 /ng/L of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
286
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Method 629
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 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 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 demon-
strate 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 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 /tg/L 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
Cis = Concentration of the internal standard, in
C = Concentration of the parameter to be measured, in \t,glL
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/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
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Method 629
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 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 the lauric acid value is suggested. This procedure6 determines the
adsorption from hexane solution of lauric acid, in milligrams per gram 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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. 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. 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 pipette, 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 be-
fore R and s calculations are performed.
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Method 629
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 performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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 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 labora-
tory 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-L
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
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 wavelengths, must be used. When-
ever possible, the laboratory should perform analysis of quality control materials and par-
ticipate in relevant performance evaluation studies.
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Method 629
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices8 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 7 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-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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-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.
10.6 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-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 minutes. At the proper rate of distillation, the balls of
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Method 629
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 minutes.
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. Pour about 1 mL of solvent into the top of the Snyder column and con-
centrate the solvent extract as before. Elapsed time of concentration should be 5 to 10 min-
utes. 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 minutes.
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 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 extracts will be stored longer than 2
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.
10.10Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
11. CLEANUP AND SEPARA TION
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 demonstrated 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 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 clo-
sing 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% (v/v) ethyl ether in hexane (Fraction 1) and with 200 mL of
15% (v/v) ethyl ether in hexane (Fraction 2) using a drip rate of about 5 mL/min.
These fractions may be discarded. Place a 500-mL K-D flask and clean concentrator
tube under the chromatography column. Elute the column with 200 mL of 50% (v/v)
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629
ethyl ether in hexane (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 ug/L 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 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.
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:
Equation 2
Concentration, ug/L =
where
A = Amount of cyanazine injected, in nanograms.
V.= Volume of extract injected, in ug/L
Vt = Volume of total extract, in ug/L
Vs = Volume of water extracted, in mL
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Method 629
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:
Equation 3
Concentration, ng/L =
(A^RPtW)
where:
As = Response for cyanazine
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = 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 cyanazine 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
concentration listed in Table 1 was estimated from the response of a 254 nm UV detector to
the compound. The estimate is based upon the amount of material required to yield a signal 5
times the HPLC background noise, assuming a 10-fig injection from a 10-mL final extract of a
1-L sample.
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.1 The standard deviations of the percent recoveries of these measure-
ments are also included in Table 2.
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Method 629
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 Labora-
tory, Cincinnati, Ohio.
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, Phila-
delphia, Pennsylvania, 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, August 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, 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 ah, "Trace Analysis for Wastewaters," Environmental Science & Technology,
15, 1426 (1981).
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Method 629
Table 1. Chromatographic Conditions and Estimated Detection Limit
Retention Time Estimated MDL
Parameter (min) fag/U
Cyanazine 10.0 6
Column conditions: Spherisorb ODS (10 ism) packed in a stainless steel column 25 cm long by
2.6 mm ID with a mobile phase flow rate of 1.0 mL/min. Mobile phase: Linear gradient from 50%
Solvent B to 100% Solvent B in 2 min, where Solvent A is 25% methanol in water and Solvent B is
50% methanol in water.
Table 2. Single-Operator Accuracy and Precision
Average Standard
Spike No. of Percent Deviation
Parameter Sample Type (pg/U Replicates Recovery (%)
Cyanazine DW 121 7 100.0 8.9
MW 60.8 7 85.5 3.9
PW 10,100 3 94.3
IW 10,100 2 78.0
DW - Reagent water
MW= Municipal wastewater
PW = Process water, pesticide manufacturing
IW = Industrial wastewater, pesticide manufacturing
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Method 629
Cyanazine
5.0
10.0
15.0
Retention Time (minutes)
A52-002-58A
Figure 1. Liquid Chromatogram of Cyanazine in Process Water
Extract on Column 1 (for conditions, see Table 1)
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Method 630
The Determination
of Dithiocarbamate
Pesticides in Municipal and
Industrial Wastewater
-------
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Method 630
The Determination of Dithiocarbamate Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of dithiocarbamate pesticides. The following parameters
can be determined by this method:
Parameter CAS No.
Amoban 3566-10-7
AOP
Busan40 51026-28-9
Busan 85 128-03-0
Ferbam 14484-64-1
KN Methyl 137-41-7
Mancozeb 8018-01-7
Maneb 12427-38-1
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 dithiocarbamates. The compounds
above are reduced to carbon disulflde 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 hi
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 alternative test procedures under 40 CFR 136.4 and 136.5.
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.
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Method 630
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is digested with acid to yield carbon
disulfide by hydrolysis of the dithiocarbamate moiety. The evolved C£, is purged from
the sample and absorbed by a color reagent. The absorbance of the solution is measured
at 380 and 435 nm using a UV-visible spectrophotometer.1
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 necessary. Clean the H2S scrubber between each use with
0.1N HC1 in methanol, rinse three times with methanol, and bake at 200°C for 15
minutes. Rinse the CS2 trap with methanol three times between each use and follow
by heating for 15 minutes at 200°C. Should it become difficult to force the color
reagent through the glass frit of the CS2 trap, clean in the same manner as the H2S
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 is not addressed in this method. If correction for background carbon disulfide is
required, the CSj 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 interferences 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 H2S 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. 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 aware-
ness 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 identified3"5 for the information of the analyst.
300
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Method 630
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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 con-
tamination of the sample. An integrating flow meter is required to collect flow-
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 com-
ponents.
5.2.1 Hot plate with magnetic stirrer.
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 g. The preparation
of calibration standards for some dithiocarbamates (e.g., metham) requires the use of a balance
capable of weighing 10 jig.
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 minutes immediately before use.
6.2 Acetonitrile, diethanolamine, methanol: ACS grade.
6.3 Ethanol: 95%.
307
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6.4 Cupric acetate: Monohydrate, ACS grade.
6.5 Hydrochloric acid: Concentrated.
6.6 Hydrochloric acid, 0.1N in methanol: Slowly add 8.3 mL concentrated HC1 to methanol and
dilute to 100 mL.
6.7 Sodium hydroxide, 4N: Dissolve 16 g ACS grade NaOH pellets in reagent water and dilute to
100 mL.
6.8 Stannous chloride: SnCl2«2H2O, ACS grade.
6.9 Zinc acetate solution, 20%: Dissolve 20 g ACS grade Zn(C2R3OJ 2H2O in reagent water and
dilute to 100 mL.
6.10 Color reagent: Add 0.012 g cupric acetate monohydrate to 25 g diethanolamine. Mix thor-
oughly 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 concentrated hydro-
chloric acid. Prepare fresh daily.
6.12 Stock standard solutions (1.00 ng/fJiL): 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 1-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 6 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 is
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.
302
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Method 63O
7.3 Spike the water in each flask with an accurately known weight of dithiocarbamate standard.
Use a series of weights equivalent to 5 to 200 /ig of CS^. Follow the procedure outlined
Section 10.
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 /xg ziram (4 cm cell), and the 380 nm curve is used for calibration below 30 jig
ziram. The choice of which curve to use is left to the discretion of the analyst. It is recom-
mended 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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 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.
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 deter-
mine 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 be-
fore 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.
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Method 630
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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 11.3.
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-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each
tune 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. Whenever possible, the laboratory should perform analy-
sis 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. Com-
posite 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 7 days of collection.
304
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Method 630
10. SAMPLE ANAL YSIS
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-L
hydrolysis flask. Rinse the bottle four times with 100-mL aliquots of reagent water, adding
the washes to the hydrolysis flask. Bring the volume in the hydrolysis flask to approximately
1500 mL with reagent water.
10.3 Place 5.0 mL of color reagent into the CS2 trap (second gas-washing bottle). Place 9 mL of
zinc acetate solution into the H2S scrubber (first gas washing bottle). Add 2 mL of ethanol to
the H2S scrubber. Place a magnetic stirring bar in the hydrolysis flask and place the flask on
the hotplate/magnetic stirrer (ambient at this time). Assemble the apparatus providing ade-
quate 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
CS2 trap.) Add 30 mL of decomposition reagent to the flask.
NOTE: The analyst must ensure that the samplepH 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 CS2 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 pipette bulb. Ensure
quantitative 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 minutes
but not more than 2 hours 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 Section 7.4.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the liquid to a 1000-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.
305
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Method 630
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 out-
side of the control limits in Section 8.3, data for the affected parameters must be labeled as
suspect.
12. METHOD PERFORMANCE
12.1 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.8 The MDL
concentrations listed in Table 1 were determined using wastewater, and are expressed in con-
centration units of the spiked materials.1
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 I.1 All recoveries are based on calibrations
using the specific dithiocarbamate instead of ziram.
306
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Methfts/ $30
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, Phila-
delphia, 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, August 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.
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. Glaser, J.A. et al., "Trace Analysis for Wastewaters," Environmental Science & Technology,
15, 1426 (1981).
307
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Method 63O
Table 1. Method Performance
Parameter
Maneb
Metham
Ziram
Method
Detection
Limit
frg/U
15.3
3.7
1.9
Sample
Type*
1
2
3
4
5
Number of
Replicates
7
7
7
8
8
Spike
(pg/L)
31.5
20.1
250.0
32.2
1050.0
Mean
Recovery
(%)
97.1
94.5
65.2
100.0
96.2
Standard
Deviation
(%)
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 40% industrial and 60% municipal wastewater
5 = 7% industrial process water, 7% industrial wastewater, 86% municipal wastewater
308
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Method 630
A52-002-60A
Figure 1. Dithiocarbamate Hydrolysis Apparatus
309
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Method 630.1
The Determination of
Dithiocarbamates Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 630.1
The Determination of Dithiocarbamates Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain dithiocarbamates pesticides after conversion to
carbon disulfide. The following parameters can be determined by this method:
Parameter CAS No.
Amobam 3566-10-7
Busan40 51026-28-9
Busan 85 128-03-0
EXD 502-55-6"
Ferbam 14484-64-1
KN Methyl 137-41-7
Metham 137-42-8
Nabam 142-59-6
Nabonate 138-93-2
Sodium dimethyldithiocarbamate 128-04-1
Thiram 137-26-8
Zineb 12122-67-7
Ziram 137-30-4
1.2 The compounds are decomposed to form carbon disulfide (CSj) and the total dithiocarbamate
concentration is measured from the amount of CS2 produced by acid hydrolysis. Unless the
sample can be otherwise characterized, all results are reported as ziram.
1.3 This is a total-residue gas chromatographic (GC) method applicable to the determination of the
compounds listed above in municipal and industrial 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 alternative test procedures under
40 CFR 136.4 and 136.5.
1.4 The method detection limits (MDLs, defined in Section 14) for the parameters listed in Sec-
tion 1.1 are listed in Table 1. The MDLs for a specific 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 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.
373
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Method 630.1
2. SUMMARY OF METHOD*
2.1 A measured 5-mL volume of sample is digested with acid to yield CS2 by hydrolysis of the
dithiocarbamate moiety. The evolved CS2 is extracted from water into hexane. Gas chromato-
graphic conditions are described which permit the separation and measurement of CS2 in the
extract by gas chromatography with a Hall detector in the sulfur mode.
2.2 This method provides a cleanup procedure involving purging of any indigenous CS2 from the
sample at pH 12 to 13. This procedure is performed using a vortex evaporator.
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, drain dry, and heat in an oven or muffle furnace at 400°C for 15 to
30 minutes. Do not heat volumetric ware. Some thermally stable materials, such as
PCBs, may 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 Carbon disulfide may be a direct interferent in wastewaters. This method includes procedures
to purge CS2 from the wastewater prior to acid hydrolysis of the sample. A vortex evaporator
is used for CS2 removal.
3.3 Additional matrix interferences may be caused by contaminants that are coextracted from the
sample and from other CS2 generating compounds. The extent of matrix interferences will
vary considerably from source to source, depending upon the nature of the sample.
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 aware-
ness 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 identified3"5 for the information of the analyst.
4.2 Nabam (ethylene bis (dithiocarbamate)) has been identified as having substantial evidence of
carcinogenicity and should be handled according to OSHA regulations.
314
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Method 630.1
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Sample containers: 40-mL screw-cap vials (Pierce No. 13075 or equivalent): each
equipped with a polytetrafluoroethylene (PTFE)-faced silicone septum (Pierce No.
12722 or equivalent). Prior to use, wash vials and septa with detergent and rinse with
tap and distilled water. Allow the vials and septa to air dry at room temperature,
place in a 105°C oven for 1 hour, then remove and allow to cool in an area known to
be free of organics.
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 con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Glassware.
5.2.1 Centrifuge tube: 15-mL, conical, with PTFE-lined screw-cap.
5.2.2 Volumetric flask: 250-mL with glass stopper.
5.2.3 Bottles: 100- to 200-mL capacity with PTFE-lined screw-caps.
5.3 Vortex Evaporator: Buchler 3-2200, equipped with sample block to hold 36 15-mL conical-
bottom centrifuge tubes and appropriate vacuum cover.
5.4 Water bath: Heated, 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 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.
5.6.1 Column: 180 cm by 2 mm ID glass, packed with 0.1% SP-1000 on Carbopack C
(80/100 mesh) or equivalent. This column was used to develop the method perfor-
mance statements in Section 14. Alternative columns may be used in accordance with
the provisions described in Section 11.1.
5.6.2 Detector: Hall detector operated in the sulfur mode. This detector has proven effec-
tive in the analysis of wastewaters for the compounds listed in the scope and was used
to develop the method performance statements in Section 14.
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 Hexane: Distilled-in-glass quality or equivalent.
315
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Method 630
6.3 Sulfuric acid, 12N: Slowly add 100 mL concentrated sulfuric acid to 200 mL reagent water.
6.4 Sodium phosphate, tribasic, dodeca-hydrate: Baker reagent grade or equivalent.
6.5 Tribasic sodium phosphate, 0.1M: Dissolve 38 g of tribasic sodium phosphate in reagent
water and dilute to 1000 mL with reagent water.
6.6 Stannous chloride: SnCl2«2H20, ACS grade.
6.7 Stannous chloride reagent: Dissolve 1.5 g stannous chloride in 100 mL 12N sulfuric acid.
Prepare fresh daily.
6.8 Sodium chloride: Heated at 45°C for 8 hours.
6.9 Stock standard solutions (0.1 /ig//xL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.9.1 Prepare dithiocarbamate spiking solutions by accurately weighing about 0.025 g of
pure material. Dissolve the material in 0.1M Na3PO4 and dilute to volume in a
250-mL volumetric flask. Larger volumes can be used 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.
6.9.2 (0.1 Mg//tL) Prepare CS2 stock standard solution by adding 7.9 /iL of CS2 to hexane
and diluting to volume in a 100-mL volumetric flask.
6.9.3 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
4°C. Frequently check standard solutions for signs of degradation or evaporation.
6.9.4 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Use ziram as the standard for total dithiocarbamates when a mixture of dithiocarbamates is
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 Establish gas chromatographic operating parameters equivalent to those indicated in Table 1.
The gas chromatographic system can be calibrated using the external standard technique
(Section 7.3).
7.3 External standard calibration procedure.
7.3.1 Prepare calibration standards at a minimum of three concentration levels by adding
volumes of the C$2 stock standard to a volumetric flask and diluting to volume with
hexane. One of the external standards should be at 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 /*L 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 CS2. Alternatively, the ratio of the response to the mass injected, defined as
316
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Method 630.1
the calibration factor (CF), can be calculated 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.
7.3.3 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 CS2
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 calibra-
tion factor must be prepared.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration standards
through the procedure to validate the absence of interferences 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.
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 measure-
ments. 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.
8.2.2 Add a known amount of an individual dithiocarbamate standard to a minimum of four
5-mL aliquots of 0.1M tribasic sodium phosphate. 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 be-
fore 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must review potential
problem areas and repeat the test.
317
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Method 630.1
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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 state-
ments 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound in all
samples processed as part of the same set must be qualified as described in Section 12.3. 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 5-mL
aliquot of 0.1M tribasic sodium phosphate 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 processes 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
318
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Method 630.1
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Add 15.2 g of tribasic sodium phosphate per 40 mL of sample to the sample to adjust pH to
12 to 13 at time of collection.
10. SAMPLE CLEANUP AND EXTRACT/ON
10.1 Place 5 mL of sample in a 15-mL conical centrifuge tube.
10.2 Add 0.75 g of NaCl and shake tube to dissolve salt.
10.3 Add 2 mL of MTBE and process in a vortex evaporator for 10 minutes with the temperature at
30°C, a vacuum of 30 inches Hg, and the vortex speed control set at 4.5.
10.4 Repeat step in Section 10.3 twice.
10.5 Add 0.75 mL of hexane and 2.5 mL of SnCl2 reagent to the aqueous layer. Cap tube tightly
and invert in a water bath at 50°C for 30 minutes.
10.6 Remove tube from water bath and let cool inverted to room temperature.
10.7 Shake tube for 1 minute without venting. Analyze the hexane layer by GC with a Hall detec-
tor in the sulfur mode. If CS2 levels are outside of the GC calibration range, the sample can
be diluted a known amount with hexane and reanalyzed.
11. GAS CHROMA TOGRAPHY
11.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded in this table are estimated retention time and MDLs that can be achieved by this method.
An example of the chromatography achieved from 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 stan-
dard deviations of responses for replicate injections are demonstrated to be less than 6% and
the requirements of Section 8.2 are met.
11.2 Calibrate the gas chromatographic system daily as described in Section 7.
11.3 Inject 1 to 5 /tL of the sample extract using the solvent flush technique.8 Record the volume
injected to the nearest 0.05 pL, and the resulting peak sizes in area or peak height units. An
automated system that consistently injects a constant volume of extract may also be used.
11.4 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 can be used to calculate a
suggested window size; however, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
11.5 If the response for the peak exceeds the working range of the system, dilute the extract with
hexane and reanalyze.
11.6 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
319
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Method 630.1
12. CALCULATIONS
12.1 Determine the concentration of carbon disulfide in the sample.
12.1.1 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 of dithiocarbamate in the sample can be calculated as
follows:
Equation 1
Concentration, pg/L =
where
A = Amount of C52 injected, in ng
V( = Volume of extract injected, in
Vt = Volume of total extract, in \>L
Vs = Volume of water extracted, in mL
Mc = Molecular weight of dithiocarbamate
C = Theoretical number of moles of CS2 formed per mole of dithicoabamate
12.2 Determine the concentration of total dithiocarbamates in the sample as ziram. When a specific
dithiocarbamate is being measured, quantitate in terms of the selected pesticide.
12.3 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.
12.4 For samples processed as part of a set where the laboratory spiked sample recovery falls out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
13. METHOD PERFORMANCE
13.1 The 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 obtained using spiked reagent water samples.1
13.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 /ig/L to 1000 /*g/L.
13.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries of the parameters listed in Section 1.1 presented in Table 2 were obtained.
Seven replicates of the wastewater were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 2.1
320
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Method 630.1
References
1. "Determination of Pesticides and Priority Pollutants in Industrial and Municipal Wastewaters,"
EPA Contract Report 68-03-1760, Work Assignment 4 (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, Phila-
delphia, Pennsylvania, 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, August 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, Com-
mittee 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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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. Glaser, J.A. et al., "Trace Analysis for Wastewaters", Environmental Science and Technology,
15, 1426 (1981).
327
-------
Method 630.1
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time MDL
Parameter (min)1 (vg/LJ
Amobam 1.3 1.1
Busan 40 1.3 4.4
Busan 85 1.3 1.3
EXD 1.3 5.2
Ferbam 1.3 2.9
KN Methyl 1.3 2.7
Metham 1.3 3.1
Nabam 1.3 1.6
Nabonate 1.3 0.9
NaDMDTC 1.3 2.8
Thiram 1.3 2.2
Zineb 1.3 4.1
Ziram 1.3 4.6
1. Retention time of CS2 under the following conditions: Carbopack C (80/100 mesh) coated with
0.1 % Sp-1000 packed in a glass column 180 cm long by 2 mm ID with helium carrier gas at a
flow rate of 25 mL/min. Column temperature held at 7°C for 3 minutes, programmed at
20°C/min to 120°C, and then held at 120°C for 5 minutes. Column effluent is vented from
the Hall detector after elution of CS2 from the column. Injector temperature and detector
temperatures are 200°C. The Hall detector is operated in the sulfur mode following manufac-
turer's specifications.
322
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Method 630.1
Table 2. Single-Laboratory Accuracy and Precision
Parameter
Amobam
Busan 40
Busan 85
EXD
Ferbam
KN Methyl
Methan
Nabam
Nabonate
Na DMDTC
Thiram
Zineb
Ziram
Sample
Type'
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Background
(ug/L)
4.6
4.6
6.6
6.6
5.9
5.9
4.5
4.5
5.2
5.2
5.4
5.4
6.2
6.2
4.8
4.8
6.1
6.1
5.4
5.4
4.5
4.5
5.2
5.2
5.7
5.7
Spike
(ug/L)
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
50
500
Mean
Recovery
(%)
90
93
110
100
110
100
71
76
94
110
90
89
110
84
62
65
66
56
110
110
89
82
87
86
100
95
Standard
Deviation
7.8
8.7
7.2
6.1
5.5
2.0
7.5
2.4
4.8
1.8
6.1
2.5
5.2
5.9
6.6
13
11
12
2.5
4.2
2.9
3.4
3.4
9.4
12
19
Number of
Replicates
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
(a) 1 = Wastewater from a manufacturer of a dithiocarbamate diluted 1000:1 with Columbus
POTW secondary effluent.
323
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Method 630.1
i i i i i i i i i i i
1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
Retention Time (minutes)
A52-002-61A
Figure 1. GC-HALL Chromatogram of 0.1 ng of CS
324
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Method 631
The Determination of Benomyl
and Carbendazim in Municipal
and Industrial Wastewater
-------
-------
Method 631
The Determination of Benomyl and Carbendazim in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
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 Benomyl cannot be determined directly by this method. Benomyl is hydrolyzed to carben-
dazim, and both compounds are measured and reported as carbendazim.
1.3 This is a high-performance liquid chromatographic (HPLC) 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 proce-
dures 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 /ig/L. The
MDL for a specific wastewater may differ from those listed, depending upon the nature of in-
terferences 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 using the proce-
dure described in Section 8.2.
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.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is acidified if necessary to hydrolyze
benomyl to carbendazim. The total carbendazim 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.1'2
327
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Method 631 __
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
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.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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimi-
nated 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 toxieity 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 aware-
ness 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 identified4"6 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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
325
-------
Method 631
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 con-
tamination 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 by 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 minutes or perform a
Soxhlet extraction 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 by 4 mm ID stainless steel, packed with /i Bondapak C18 (10 ^)
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 Sec-
tion 14. Alternative detectors may be used in accordance with the provisions de-
scribed 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.
329
-------
Method 631
6.3 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500 °C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
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 H2SO4 (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.
6.7 Stock standard solution (1.00 /ig/juL): 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. Commer-
cially 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.
6.7.3 The stock standard solution must be replaced after 6 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 repre-
sentative 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 pL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
330
-------
Method 631
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 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.
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 standard applicable to all samples can be
suggested.
7.3. 1 Prepare calibration standards at a minimum of three concentration levels of carben-
dazim 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 /iL 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:
Equation 1
where
As = Response for the parameter to be measured
Ab = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in fig/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, A/A^ against RF.
331
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Method 631
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 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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. 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 of either benomyl
or carbendazim in methanol, 1000 times more concentrated than the selected con-
centrations.
8.2.2 Using a pipette, 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 be-
fore 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
332
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Method 631
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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 parame-
ter 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-L
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
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 wavelengths may be used. When-
ever 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 practices8 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
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Method 631
posite 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 7 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 hours.
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 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, cen-
trifugation, 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-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 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 one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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Method 631
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 minutes. When the apparent vol-
ume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at
least 10 minutes.
10.10Remove 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 recom-
mended 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
2 days, they should be transferred to TFE-fluorocarbon-sealed screw-cap vials. Proceed with
HPLC analysis.
7 7. CLEANUP AND SEPARA TION
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 proce-
dure 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 /xL of the sample extract. Record the volume injected to the nearest 0.05 /iL, 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 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.
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Method 631
13. CALCULATIONS
13.1 Determine the concentration of carbendazim 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:
Equation 2
Concentration, \LglL =
where
A = Amount of material injected, in ng
V( = Volume of extract injected, in \iL
Vt = Volume of total extract, in \>L
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:
Equation 3
CW.)
Concentration, pg/L =
where
As = Response for parameter to be measured
Ab = Response for the internal standard
Is = Amount of internal standard added to each extract, in
Vo = Volume of water extracted, in L
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 chloride.1
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Method 631
14.2 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. All results were obtained using the same experimental scale de-
scribed in Section 14.1.
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Method 63;
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.
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, Virginia.
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, Phila-
delphia, Pennsylvania, 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, August 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, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
9. Glaser, J.A. et al., "Trace Analysis for Wastewaters," Environmental Science & Technology,
15, 1426(1981).
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Method 631
Table 1. Chromatographic Conditions and Method Detection Limits
.^*
Retention Time Method Detection Limit (fjg/U
Parameter (min)
Benomyl (as carbendazim) - 25.0
Carbendazim 8.1 8.7
Column conditions: // Bondapak C18 (10jum) packed in a stainless steel column 30 cm long by
4 mm ID with a mobile phase flow rate of 2.0 mL/min at ambient temperature.
Mobile phase: methanol/water (1 +1).
Table 2. Single-Operator Accuracy and Precision
Number Average Standard
Sample of Spike Percent Deviation
Parameter Type Replicates (fjg/L) Recovery (%)
Benomyl (as carbendazim) DW 7 51.5 70 15.5
MW 7 51.5 78 8.8
MW 7 103 99 6.4
Carbendazim DW 7 50 106 5.5
MW 7 50 117 18.5
MW 7 100 108 11.3
DW = Reagent water
MW = Municipal wastewater
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Method 631
Carbindazim
10
Retention Time (minutes)
A52-002-62A
Figure 1. Liquid Chromatogram of Carbendazim on Column 1
(for conditions, see Table 1)
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Method 632
The Determination of
Carbamate and Urea Pesticides
in Municipal and Industrial
Wastewater
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Method 632
The Determination of Carbamate and Urea Pesticides in Municipal
and Industrial Wastewater
1. SCOPE AND APPLICA TION
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
Neburon — 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 fenuron 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 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 alternative 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 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 use
of liquid chromatography and in the interpretation of liquid chromatograms. Each analyst
must demonstrate the ability to generate acceptable results with this method using the proce-
dure described in Section 8.2.
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Method 632
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 L, is extracted with methylene chloride using
a separatory runnel. 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 hi liquid
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.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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimi-
nated 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 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.
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Method 632
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 aware-
ness 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 identified4* for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grap-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene 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 con-
tamination 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 by 19 mm ID with coarse-
fritted disc.
5.2.3 Chromatographic column: 400 mm long by 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.
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Method 632
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 by 4 mm ID stainless steel packed with /* Bondapak Clg (10
/mi) or equivalent. This column was used to develop the method performance state-
ments in Section 14. Alternative columns may be used in accordance with the provi-
sions 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 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 (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. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.5 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use activate each batch at
least 16 hours at 130°C in a foil-covered glass container.
6.6 Acetic acid: Glacial.
6.7 Stock standard solutions (1.00 /ig//xL): 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 material. 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 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.
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Method 632
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.
6.7.3 Stock standard solutions must be replaced after 6 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 /tL 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 in-
jected, 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 volumet-
ric 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
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Method 632
standards should be representative of a concentration near, but above, the method
detection limit. The other concentrations should correspond to the range of con-
centrations expected in the sample concentrates, or should define the working range of
the detector.
7.3.2 Using injections of 10 pL 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:
Equation 1
RF = _
where
As = Response for the parameter to be measured
A.K = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in fig/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, A/A,, 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 the lauric acid value is suggested. This procedure7 determines the
adsorption from hexane solution of the lauric acid, in milligrams per gram 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 capa-
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Method 632
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. 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 pipette, 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 most of the carbamate and
urea pesticides. Similar results should be expected from reagent water for all com-
pounds listed in the 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 + 3s
Lower Control Limit (LCL) = R - 3s
where R and s are calculated as in Section 8.2.3. The UCL and LCL can be used to
construct control charts8 that are useful in observing trends in performance.
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Method 632
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 de-
fined 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
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-L
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
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 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 Crap-samples must be collected in glass containers. Conventional sampling practices9 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 7 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-L separatory funnel.
350
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Method 632
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. Transfer the solvent to the separatory funnel and extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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
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 SO 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 con-
centrated extract to a 10-mL volumetric flask, quantitatively 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 2 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
11. CLEANUP AND SEPARA TION
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
357
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Method 632
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
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.
11.2.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration (Sections 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 volu-
metric 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% (v/v) ethyl ether in hexane (Fraction 1) using a drip rate of
about 5 mL/min. Place a 500-mL round-bottom flask under the chromatography
column. Elute the column again, using 200 mL of 6% (v/v) acetone in hexane (Fra-
ction 2), into a second flask. Perform a third elution using 200 mL of 15% (v/v)
acetone in hexane (Fraction 3), and a final elution with 200 mL of 50% (v/v) acetone
in hexane (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 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. 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 jaL of the sample extract. Record the volume injected to the nearest 0.05 /xL, 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 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.
352
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Method 632
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:
Equation 2
n C*)(V,)
Concentration, (iglL = —
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \iL
V, = Volume of total extract, in pL
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:
Equation 3
Concentration, \LglL =
(^XJWXV,)
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls out-
side of the control limits in Section 8.3, data for the affected parameters must be labeled as
suspect.
353
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Method 632
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.10 The MDL
concentrations listed in Table 1 were obtained using reagent water or river water.2-11
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.
354
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Method 632
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, Virginia.
2. Farrington, D.S., Hopkins, R.G. and Ruzicka, J.H.A. "Determination of Residues of Substitu-
ted Phenylurea Herbicides in Grain, Soil, and River Water by Use of Liquid Chromato-
graphy," 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, Phila-
delphia, 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, August 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.
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. Glaser, J.A. et al., "Trace Analysis for Wastewaters," Environmental Science & Technology,
15, 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.
355
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Method 632
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
Mexacarbate
Propoxur
Monuron
Carbaryl
Propham
Diuron
Linuron
Methiocarb
Chlorpropham
Barban
Neburon
Propoxur
Methomyl
Carbaryl
Diuron
Linuron
Propoxur
Carbofuran
Fluorometuron
Oxamyl
Mobile
Phase*
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
C
C
C
D
Retention
Time
fmin)
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
3.2
UV Wavelength
(nmj
254
280
254
280
254
254
254
254
254
254
254
280
254
280
254
254
280
280
254
254
Method Detection
Limit
0.52
0.11
0.003
0.02
0.07
0.009
0.009
0.02
0.03
0.05
0.012
0.11
8.9
0.02
0.009
0.009
0.11
3.2
11.1
9.2
'Mobile Phase:
A = Methanol/1 % acetic acid, programmed linearly from 5 to 95% methanol at a flow rate
of 2.0 mL/min and at ambient temperature.
B = Acetonitrile/water, programmed linearly from 10 to 100% acetonitrile in 30 minutes 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: fj Bondapak C18 (10 //m) packed in a stainless steel column 30 cm long by 4 mm ID, with
a Whatmann Co. PELL ODS (30-38 fjm) guard column 7 cm long by 4 mm ID.
356
-------
Method 632
Table 2. Single-Operator Accuracy and Precision
Parameter
Fluorometuron
Propoxur
Oxamyl
Methomyl
Diuron
Linuron
Carbofuran
Barban
Carbaryl
Chlorpropham
Methiocarb
Mexacarbate
Monuron
Neburon
Propham
* Sample Type
Sample
Type*
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
5
5
5
5
5
Spike
(vg/U
50
50
1724
550
2200
550
0.5
100
53
1080
100
30660
100
1960
10
500
10
400
0.05
10
4000
10
210
0.05
37
148
0.3
0.1
0.2
0.2
4.0
0.05
0.05
0.3
No. of
Analyses
1
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
3 = Industrial
4 = Industrial
wastewater
process water, pesticide manufacturing
wastewater,
pesticide manufacturing
5 = River Water
557
-------
Method 631
Table 3. Florisil Fractionation Patterns
Percent Recovery by Fraction
Parameter
Diuron
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
Fractions = 200 mL of 15% acetone in hexane
Fraction 4 = 200 mL of 50% acetone in hexane
358
-------
Method 632
Diuron
Methomyl
Linuron
5.0
10.0
15.0
20.0
Retention Time (minutes)
AS2-00243A
Figure 1. Liquid Chromatogram of Diuron, Linuron and Methomyl on Column 1
(for conditions, see Table 1)
359
-------
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Method 632.1
The Determination of
Carbamate and Amide
Pesticides in Municipal and
Industrial Wastewater
-------
-------
Method 632.1
The Determination of Carbamate and Amide Pesticides
in Municipal and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain carbamate/amide pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Napropamide 15299-99-7
Propanil 709-98-8
Vacor 53558-25-1
1.2 The estimated detection limits (EDLs) for the parameters above are listed in Table 1. The
EDL was calculated from the minimum detectable response being equal to five times the
background noise using a 10-mL final extract volume of a 1-L sample and an injection volume
of 100 fjiL. The EDL for a specific wastewater may be different depending on the nature of
interferences in the sample matrix.
1.3 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compounds listed above in municipal and industrial discharges. When this
method is used to analyze unfamiliar samples for any or all of the compounds above, com-
pound identification should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second HPLC column that can be used to confirm
measurements made with the primary column.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 The carbamate/amide pesticides are removed from the sample matrix by extraction with methy-
lene chloride. The extract is dried, exchanged to HPLC mobile phase and analyzed by liquid
chromatography with ultraviolet (UV) detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample processing hardware may yield discrete ar-
tifacts and/or elevated baselines causing misinterpretation of liquid chromatograms. All of
these materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.' Clean all glassware as soon as possible
after use by rinsing with the last solvent used in it. This should be followed by
363
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Method 632.1
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 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that are coextracted from the
samples. 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. Unique samples may require cleanup approaches to achieve the detection limits
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 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 aware-
ness 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 identified2"4 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample Containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to
air dry, then muffle the bottles at 400°C for 1 hour. After cooling, rinse the bottle and cap
liners with hexane, seal the bottles, and store in a dust-free environment.
5.1.1 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 reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Rotary evaporator: With 24/40 joints and associated water bath and vacuum for operation at
reduced pressure (Servo Instruments VE-1000-B or equivalent).
5.3 High-performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
364
-------
Method 632.1
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Gradient pumping system.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 100-/zL loop.
5.3.3 Column 1: 250 mm long by 4.0 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 5/i, or equivalent.
5.3.4 Column 2: 250 mm long by 4.6 mm ID, packed with reverse phase Dupont Zorbax
ODS, 10 /i, or equivalent.
5.3.5 Ultraviolet detector, variable wavelength, capable of monitoring at 254 nm.
5.3.6 Strip-chart recorder compatible with detector, 250-mm. (A data system for measuring
peak areas is recommended.)
5.4 Boiling flask: 250-mL, flat-bottom, 24/40 joint.
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5,6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride for 2 hours.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Volumetric flasks: 5- and 10-mL, Class A.
5.6.6 Pasteur pipettes with bulbs.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, acetonitrile, hexane, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 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.
6.1.3 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.4 HPLC mobile phase, Column 1: Add 400 mL of acetonitrile to a 1-L volumetric
flask and dilute to volume with reagent water.
6.1.5 HPLC mobile phase, Column 2: Add 550 mL of acetonitrile to a 1-L volumetric
flask and dilute to volume with reagent water.
6.1.6 Sodium hydroxide solution (l.ON): Dissolve 40 g of NaOH in reagent water and
dilute to l.OOOmL.
6.1.7 Sodium chloride: ACS, crystals.
365
-------
Method 632.1
6.1.8 Sodium thiosulfate: ACS, granular.
6.1.9 Sulfuric acid solution (1 +1): Slowly add 50 mL of H2S04 (specific gravity 1.84) to
50 mL of reagent water.
6.2 Standard stock solutions (1.00 /xg/jiL): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality (9:1) acetonitrile/acetone and
dilute to volume in a 10-mL volumetric flask. Larger volumes can be used at the
convenience of the analyst. When compound purity is 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.
6.2.2 Transfer the stock standards to PTFE-sealed screw-cap bottles. Store at 4°C and
protect from light. Stock standards should be checked frequently for signs of degra-
dation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 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 within 48 hours of
collection, the sample should be adjusted to a pH of 2.0 to 4.0 with sulfuric acid, and add
35 mg of sodium thiosulfate per liter of sample for each part per million of free chlorine.
7.3 All samples must be extracted within 7 days and completely analyzed within 30 days of
extraction.
8. CALIBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in
Table 1. The chromatographic system can be calibrated using the external standard tech-
nique (Section 8.2).
8.2 External standard calibration procedure.
8.2.1 Prepare calibration standards at a minimum of three concentration levels of the ana-
lytes by adding volumes of the stock standard to a volumetric flask and diluting to
volume with HPLC mobile phase. One of the standards should be at a concentration
near, but greater than, the EDL, and the other concentrations should correspond to the
366
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Method 632.1
expected range of concentrations found in real samples or should define the working
range of the detector.
8.2.2 Using injections of 100 pL of each calibration standard, tabulate peak height or area
response against the mass injected. The results are used to prepare a calibration curve
for the analytes. Alternatively, if the ratio of response to amount injected (calibration
factor) is a constant over the working range (< 10% relative standard deviation,
RSD), linearity of the calibration curve can be assumed and the average ratio or
calibration factor can be used in place of a calibration curve.
8.2.3 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
analyte varies from the predicted response by more than ±10%, the test must be re-
peated using a fresh calibration standard. Alternatively, a new calibration curve or
factor must be prepared.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A labora-
tory reagent blank is an aliquot of reagent water. If the reagent blank contains a
reportable level of the analytes, immediately check the entire analytical system to
locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.2, prepare a laboratory control standard con-
centrate that contains the analytes at a concentration of 10 /*g/mL in aceto-
nitrile.6
9.2.1.2 Laboratory control standard: Using a pipette, add 1.0 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Cal-
culate the percent recovery (Pj) with the equation:
Equation 1
1005,
P, =
•where
S{ = Analytical results from the laboratory control standard, in
Tt = Known concentration of the spike, in pg/L
367
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Method 632.1
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both aliquots for at least 10% of all samples. To the extent
practical, the samples for duplication should contain reportable levels of the analytes.
9.3.2 Calculate the relative range6 (RRj) with the equation:
Equation 2
100/?.
RR =
where
R, = Absolute difference between the duplicate measurements X, and X^, in pg/L
f = Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 6.5 to 7.5
with sodium hydroxide or sulfuric acid by slow addition and thorough mixing. Add
200 g of sodium chloride, and mix to dissolve.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sample
by shaking the funnel for 2 minutes with periodic venting to release vapor pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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 on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and
complete the extraction procedure a second time, combining the extracts in the Erlen-
meyer flask.
368
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Method 632.1
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, collecting the
extract in a 250-mL flat-bottom boiling flask. Rinse the Erlenmeyer flask and column
with about 30 mL of methylene chloride to complete the transfer.
10.1.5 Concentrate the combined methylene chloride extracts to about 1 mL on a rotary
evaporator with bath temperature between 35 and 40°C. Add 15 mL of acetonitrile,
and reconcentrate to about 1 mL. Transfer the extract to a 10-mL volumetric flask.
Rinse the boiling flask with about 1 mL of acetonitrile, and transfer to the volumetric
flask. A 5-mL syringe is recommended for this operation. Rinse the boiling flask
further with a 1-mL portion of acetonitrile, and transfer to the volumetric flask.
10.1.6 Add exactly 5.0 mL of HPLC-grade water to the flask, and dilute to 10 mL with
acetonitrile. If the extracts will be stored longer than 2 days, they should be trans-
ferred to PTFE-sealed screw-cap bottles. If the sample extract requires no cleanup,
proceed with chromatographic analysis. If the sample requires cleanup, proceed to
Section 10.2.
10.1.7 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.
10.2 Cleanup and separation.
10.2.1 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 is no less than 85%.
10.2.2 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromato-
graph. Included in this table are the estimated retention times and estimated detection
limits that can be achieved by this method. An example of the separation achieved by
the primary column of the analytes is shown in Figures 1 and 2. Other columns,
chromatographic conditions, or detectors may be used if data quality comparable to
Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 juL of the sample extract. Monitor the column eluent at 254 nm. Record
the resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon meas-
urements 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 can be used to
calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the
sample with mobile phase and reanalyze.
369
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Method 632.1
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
cleanup is required.
77. CALCULATIONS
11.1 Determine the concentration of analytes in the sample.
11.1.1 Calculate the amount of analytes injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
Equation 3
Concentration, ng/L =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \iL
Vt = Volume of total extract, in (iL
Vs = Volume of water extracted, in mL
11.2 Report results in milligrams per liter without correction for recovery data. When duplicate
and spiked samples are analyzed, report all data obtained with the sample results.
72. METHOD PERFORMANCE
12.1 The EDLs and associated chromatographic conditions for the analytes are listed in Table I.7
The EDL is defined as the minimum response being equal to 5 times the background noise,
assuming a 10-mL final extract volume of a 1-L sample and an HPLC injection volume of
100 nL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc., in the designated matrix. The results of these studies are presented in
Table 2.
370
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Method 632.1
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, p. 679, 1986.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1986.
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, March 1979.
7. "Evaluation of Ten Pesticides," U.S. Environmental Protection Agency, Contract 68-03-1760,
Task No. 11, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio (in preparation.).
377
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Method 632 1
Table 1. Chromatographic Conditions and Estimated Detection Limits
Retention Time (minj
Parameter
Vacor (RH 787)
Propanil
Napropamide
Column 7
6.0
12.4
15.2
Column 2
3.8
6.9
9.5
Estimated Detection
Limit (ug/U
0.20
0.85
0.31
Column 1: 25 cm long by 4 mm ID, stainless steel, packed with Ultrasphere OOS (particle size
5 fj); mobile phase: 40% acetonitrile/HPLC water programmed to 65% acetonitrile/HPLC water
over 10 minutes at a flow rate of 1.0 mL/min at ambient temperature.
Column 2: 25 cm long by 4.6 mm ID, stainless steel, packed with Zorbax ODS (DuPont); mobile
phase: Isocratic elution with 55% acetonitrile/HPLC water at a flow rate of 1.0 mL/min for 6
minutes then linear flow gradient to 1.5 mL/min over 3 minutes at ambient temperature.
Table 2. Single-Laboratory Accuracy and Precision
Parameter
Napropamide
Propanil
Vacor (RH787)
Matrix
Type*
1
1
1
1
1
1
Spike
Range
(ug/L)
11.5
597.0
14.0
676.0
12.9
655.0
Number
of
Replicates
7
7
7
7
7
7
Average
Percent
Recovery
113.8
104.0
99.8
96.4
98.2
111.2
Standard
Deviation
15.7
16.0
12.4
7.6
17.5
5.2
1 = Spiked municipal wastewater
372
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Method 632.1
Napropamide
12.0 14.0 16.0
Retention Time (minutes)
A52-002-64A
Figure 1. HPLC Chromatogram of Carbamates/Amides on Column 1
373
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Method 633
The Determination of
Organonitrogen Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 633
The Determination of Organonitrogen Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
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 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 alternative 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 8.2.
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 confirmation of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is 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 mL or less. Gas chromatographic conditions are described
which permit the separation and measurement of the compounds in the extract by gas chroma-
tography with a thermionic bead detector.1
377
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Method 633
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 minutes. Do not
heat volumetric ware. Thermally stable materials, such as PCBs, may not be elimina-
ted 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 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 aware-
ness 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 identified3"5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L 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 meth-
ylene chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the col-
lection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
378
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Method 633
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 com-
posites.
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 by 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 minutes or performed
Soxhlet extraction 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 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.
5.6.1 Column 1: 180 cm long by 2 mm ID glass, packed with 3% SP-2250DB on Supel-
coport (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 by 2 mm ID glass, packed with 3% SP-2401 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 de-
scribed in Section 12.1. 1
375
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Method 633
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. Condition by heating in a shallow tray at 400°C
for a minimum of 4 hours to remove phthalates and other interfering organic substances. Al-
ternatively, heat 16 hours at 450 to 500°C in a shallow tray or perform a Soxhlet extraction
with methylene chloride for 48 hours.
6.4 Stock standard solutions (1.00 /ig/jiL): 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
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.
6.4.3 Stock standard solutions must be replaced after 6 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 acetone. 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 \iL of each calibration standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a calibration
380
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Method 633
curve for each parameter. Alternatively, the ratio of the response to the mass injec-
ted, 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 volu-
metric 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 detec-
tor.
7.3.2 Using injections of 1 to 5 /iL 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
AIS = Response for the internal standard
Cis = Concentration of the internal standard, in fig/L
C = Concentration of the parameter to be measured, in fig/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, AyA^ against RF.
381
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Method 633
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 capa-
bility and the analysis of spiked samples as a continuing check on performance. The labora-
tory 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 measure-
ments. Each tune 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 pipette, 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 be-
fore 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.
382
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Method 633
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 ali-
quots 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.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-L
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 reagent, 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.
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. Com-
posite 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.
383
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Method 633
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 7 days and completely analyzed within 40 days of
extraction.
1 0. SAMPLE EXTRA c TION
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-L separatory funnel.
10.2 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 seconds to rinse the
inner walls. 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, cen-
trifugation, 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 extrac-
tion 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 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 methylene chloride to complete the quantitative transfer.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
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 minutes. When the ap-
parent volume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and
cool for at least 10 minutes.
384
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Method 633
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 2 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5
mL.
71. CLEANUP AND SEPARA TION
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 proce-
dure is no less than 85 %.
72. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded 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 conditions, or detectors 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 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 ^L of the sample extract using the solvent-flush technique.8 Record the volume
injected to the nearest 0.05 ^iL, 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 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, cleanup
is required.
385
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Method 633
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1 . J If the external standard calibration procedure is used, calculate the amount of material
.ajected from the peak response using the calibration curve or calibration factor in
Jrction 7.2.2. The concentration in the sample can be calculated as follows:
Equation 2
Concentration,
where
A = Amount of material injected, in ng
V{ = Volume of extract injected, in yiL
Vt = Volume of total extract, in \tL
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:
Equation 3
Cor '^ration, pg/L =
(Ais)(RF)(V0)
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side 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 seconds per scan utilizing a 70 V (nominal) electron
386
-------
Method 633
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.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 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 chromatographic conditions. It is
recommended that at least 25 ng 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 ±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.. 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.11 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 measure-
ments are also included in Table 2.
387
-------
Method 633
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 Monitor-
ing and Support Laboratory, Cincinnati, Ohio.
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, Phila-
delphia, Pennsylvania, 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, August 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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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,
15, 1426 (1981).
388
-------
Method 633
Table 1. Chromatographic Conditions and Method Detection Limits
Method
Retention Detection
GC Time Limit
Parameter Column (min) (pg/U
Terbacil 1a 2.1 ND
Bromacil 1a 3.7 2.38
Hexazinone 1a 7.6 0.72
Tricyclazole 1b 3.5 ND
Metribuzin 2a 2.4 0.46
Triadimefon 2a 4.1 0.78
Deet 2b 4.6 3.39
ND = Not determined
Column 1a conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250DB packed in a glass
column 180 cm long by 2 mm ID with nitrogen carrier gas at a flow rate of 30 mL/min. Column
temperature, programmed: initial 210°C, hold for 1 minute, then program at 10 to 250°C and
hold. A thermionic bead detector in the nitrogen mode was used to calculate the MDL.
Column 1b conditions: Same as Column 1a, except column temperature isothermal at 240°C.
Column 2a conditions: Supelcoport (100/120 mesh) coated with 3% SP-2401 packed in a glass
column 180 cm long by 2 mm ID with nitrogen carrier gas at a flow rate of 30 mL/min. Column
temperature, programmed: initial I60°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 minute, then program at 12°C/min to 200°C.
389
-------
Method 633
Table 2. Single-Operator Accuracy and Precision
Parameter
Bromacil
Deet
Hexazinon
Metribuzin
Terbacil
Triadmefon
Tricyclazole
Sample
Type
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
MW
MW
DW
PW
IW
MW
MW
Spike
(V9/U
5.0
11.1
333.0
5.8
5.2
515.0
4.9
10.1
369.0
5.2
32.8
656.0
5.2
515.0
154.5
12.3
303.0
Number
of
Replicates
7
7
7
7
7
7
7
7
7
6
7
7
6
4
7
7
7
Mean
Recovery
(%)
92.2
89.0
95.0
99.1
92.6
94.2
86.6
92.2
94.0
98.2
106.7
101.0
126.0
71.8
70.4
69.0
98.0
Standard
Deviation
(%)
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 = Reagent water
MW = Municipal wastewater
PW = Process water, pesticide manufacturing
IW = Industrial wastewater, pesticide manufacturing
390
-------
Method 633
Terbacil
\
Hexazinone
\ '
n—i—i—i—i—i—i—i—i—i—i—i—i—i i i i
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Retention Time (minutes)
A52-002-66A
Figure 1. Gas Chromatogram of Organonitrogen Pesticides on Column 1
(for conditions, see Table 1)
391
-------
-------
Method 633.1
The Determination of Neutral
Nitrogen-Containing Pesticides
in Municipal and Industrial
Wastewaters
-------
-------
Method 633.1
The Determination of Neutral Nitrogen-Containing Pesticides
in Municipal and Industrial Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain neutral nitrogen containing pesticides. The
following parameters can be determined by this method:
Parameter CAS No.
Fenarimol 60168-88-9
MGK264-A 113-48-4
MGK 264-B 113-48-4
MGK326 136-45-8
Pronamide 23950-58-5
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial 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 alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for each compound is listed in
Table 2. 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 similar to those in other 600-
series methods. Thus, a single sample may be extracted to measure the compounds included
in the scope of the 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.
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.
395
-------
Method 633.1
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Gas
chromatographic conditions are described which permit the separation and measurement of the
compounds in the extract by alkali flame detector gas chromatography (GC/AFD).1
2.2 This method provides an optional Florisil column cleanup procedure 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 rinsing with the last solvent used in it. Follow by rinsing 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 minutes. Do not heat volumetric
ware. Some thermally stable materials, such as PCBs, may not be eliminated by this
treatment. Thorough rinsing with acetone and pesticide-quality hexane may be substi-
tuted 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 in-
verted 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 MDL listed in Table 2.
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 aware-
ness file of OSHA regulations regarding the safe handling of the chemicals specified in this
method. A reference file of materials 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 identified3"5 for the information of the analyst.
396
-------
Method 633.1
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Foil may be substituted for PTFE 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 collec-
tion 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, fol-
lowed by repeated rinsings with distilled water to minimize the potential for contami-
nation of the sample. An integrating flow meter is required to collect flow-propor-
tional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory runnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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. A
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 e-
quivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.2.11 Beaker: 250-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±20°C). The bath should be used in a
hood.
397
-------
Method 633.1
5.5 Balance: 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 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 by 2 mm ID glass, packed with 3% SP-2250 on Supelcoport
(100/120 mesh) or equivalent. This column was used to develop tho method perfor-
mance 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 by 2 mm ID glass, packed with 3% SP-2100 on Supelcoport
(100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phospho-
rous detector (NPD) or a thermionic-specific detector (TSD). This detector has
proven effective in the analysis of wastewaters for the compounds listed in the scope
and was used to develop the method performance statements in Section 15.
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, petroleum ether, ethyl ether, acetone: Distilled-in-glass quality
or equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips
(available from Scientific Products Co., Catalog No. PI 126-8 and other suppliers). Proce-
dures recommended for removal of peroxides are provided with the test strips.
6.3 6N sodium hydroxide: Dissolve 24.0 g NaOH in 100 mL of reagent water.
6.4 6N sulfuric acid: Slowly add 16.7 mL of concentrated H2SO4 (94%) to about 50 mL of reagent
water. Dilute to 100 mL with reagent water.
6.5 Sodium sulfate: (ACS), granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFE or aluminum-foil-lined screw-cap and cool to room tempera-
ture.
6.7 Stock standard solutions (1.00 /xg/ML): 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 mate-
rial. Dissolve the material in distilled-in-glass quality methanol 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 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.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check stock standard solutions for signs of
398
-------
Method 633.1
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solutions must be replaced after 6 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 2.
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.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volumet-
ric flask and diluting to volume with acetone. One of the external standards should be
at a concentration near, but above, the method detection limit. The other concentra-
tions should correspond to the range of concentrations expected in the sample con-
centrates or should define the working range of the detector.
7.2.2 Using injections of 1 to 5 /iL 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, the ratio of the response to the mass in-
jected, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric 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 at 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 /*L 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:
399
-------
Method 633.1
Equation 1
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
Cis = Concentration of the internal standard, in \aglL
Cs = Concentration of the parameter to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A/A^ 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 compound
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 interferences 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.
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 measure-
ments. 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.
400
-------
Method 633,1
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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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.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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
aliquot of reagent water that all glassware and reagent interferences are under control. Each
407
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Method 633.1
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 rele-
vant 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. Com-
posite 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 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid im-
mediately after sampling.
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-L separately funnel. Check the pH of the sample
with wide range pH paper and adjust to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid.
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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 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
402
-------
Method 633.1
and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder
column. Prewet the macro 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 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 and
allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a
two-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
methylene chloride and concentrate the solvent extract as before. When an apparent volume of
0.5 mL is reached, or the solution stops boiling, remove the K-D apparatus and allow it to
drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 2 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no fur-
ther cleanup, proceed with solvent exchange to acetone as described in Section 10.9. If the
sample requires cleanup, proceed to Section 11.
10.9 Add one or two clean boiling chips to the concentrator tube along with 10 mL of acetone.
Attach the two-ball macro Snyder column and prewet the column with about 1 mL of acetone.
Adjust the temperature of the water bath to 85 to 95°C. Concentrate the solvent extract as
before to an apparent volume of 0.5 mL and allow it to drain and cool for 10 minutes. Add a
second 10 mL of acetone to the concentrator tube and repeat the concentration procedure a
second time. Adjust the final volume of the extract to 1.0 mL with acetone.
10.10Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is no less than 85 %.
11.2 The following Florisil cleanup procedure has been demonstrated to be applicable to the four
neutral nitrogen pesticides listed in Table 1.
403
-------
Method 633.1
11.2.1 Slurry 20 g of Florisil in 100 mL of ethyl ether and 400 jtL of reagent water. Trans-
fer the slurry to a chromatographic column (Florisil may be retained with a plug of
glass wool). Allow the solvent to elute from the column until the Florisil is almost
exposed to the air. Wash the column with 25 mL of petroleum ether. Use a column
flow rate of 2 to 2.5 mL/min throughout the wash and elution profiles. Add an addi-
tional 50 mL of petroleum ether to the head of the column.
11.2.2 Quantitatively transfer the sample from Section 10.8 to the petroleum ether suspended
over the column. Allow the solvent to elute from the column until the Florisil is
almost exposed to the air. Elute the column with 50 mL of 50% ethyl ether in petro-
leum ether. Discard this fraction.
11.2.3 Elute the column with 50 mL of 100% ethyl ether (Fraction 1) and collect in a K-D
apparatus. Repeat procedure with 50 mL 6% acetone in ethyl ether (Fraction 2),
50 mL 15% acetone in ethyl ether (Fraction 3), 50 mL 50% acetone in ethyl ether
(Fraction 4), and 100 mL 100% acetone (Fraction 5), collecting each in a separate
K-D apparatus. The elution patterns for the neutral nitrogen compounds are shown in
Table 1. Concentrate each fraction to 1 mL as described in Section 10.6 and 10.7.
The fractions may be combined before concentration at the discretion of the analyst.
Solvent exchange Fraction 1 to acetone as described in Section 10.9 if the fractions
are not combined.
11.2.4 Proceed with gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 2 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. Examples of the separations achieved by Column 1 and Column 2
are shown in Figures 1 and 2. Other packed columns, chromatographic conditions, or detec-
tors 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 gas chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 1 to 5 fj,L of the sample extract using the solvent flush technique.8 Record the volume
injected to the nearest 0.05 /itL, and the resulting peak sizes 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
404
-------
Method 633.1
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:
Equation 2
Concentration, \iglL =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \>L
Vt = Volume of total extract, in \tL
V = 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 fol-
lows:
Equation 3
e-w.)
Concentration, pglL =
(Ais)(RF)(Vo)
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
405
-------
Method 633.1
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 seconds 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. When using a fused-silica capillary column, the column
outlet should be threaded through the interface to within a few mm of the entrance to the
source ionization chamber. 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.10
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 DFTPP performance criteria are achieved.9
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 50 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
14.4.1 The molecular ion and all other 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 ± 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 alternative 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." The MDL
concentrations listed in Table 2 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
406
-------
Method 633.1
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle's Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 3 were obtained after Florisil cleanup. Seven replicates
of each of two different wastewaters were spiked and analyzed. The standard deviation of the
percent recovery is also included in Table 3.'
407
-------
Method 633.1
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 1977.
4. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), OccupationafSafety
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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, 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. 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).
10. McNair, H.M., and Bonelli, E.J., Basic Chromatography, Consolidated Printing, Berkeley,
California, 52 (1969).
11. Glaser, J.A., et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
408
-------
Method 633.1
Table 1. Elution Characteristics of the Neutral Nitrogen Compounds on 2% Deac-
tivated Florisil
Elution in Specified Fraction'
Parameter
Fenarimol
MGK 264
MGK326
Pronamide X
(a) Elution solvents are 50 mL each of the following:
F1 = 100% ethyl ether
F2 = 6% acetone in ethyl ether
F3 = 15% acetone in ethyl ether
F4 = 50% acetone in ethyl ether
F5 = 100% acetone (100 ml)
F1
F2
F3
X X
X X
F4
F5
Table 2. Chromatographic Conditions and Method Detection Limits
Retention Time (min) MDL
Parameter Column 1 | Column 2 (ug/L)
Pronamide 19.9 22.0 4
MGK 326 21.9 23.8 6
MGK 264 23.0 and 25.5 and 2
23.5a 27.5a
Fenarimol 30.6 32.2 4
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is programmed from 80°C to 300°C at 8°C/min with a 4 minute hold at each ex-
treme, injector temperature is 250°C, and detector is 300°C. Alkali flame detector at bead voltage
of 16 V.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. All other
conditions as for Column 1.
(a) Two isomers of MGK 264 are resolved from each other.
409
-------
Method 633.1
Table 3. Single-Laboratory Accuracy and Precision3
Parameter
Fenarimol
MGK 264
MGK 326
Pronamide
Sample
Type"
1
2
1
2
1
2
1
2
Background
(ug/L)c
1.8
ND
ND
ND
ND
ND
ND
210
Spike
Level
(ug/L)
20
500
20
500
20
500
20
500
Mean
Recovery
98
96
96
74
108
95
102
86
Standard Number
Deviation of
(%) Replicates
4 7
4 7
23
4
7
4
5
3
7
7
7
7
7
7
(a) Column 1 conditions were used.
(b) 1 = Low-level relevant industrial effluent
2 = High-level relevant industrial effluent
(c) ND = Not detected
410
-------
Method 633.1
MGK 326
Pronamide
MGK 264
Fenarimol
/ i i i i r T i i i i i i i i i r i I i
0 ' 17.0 19.0 21.0 23.0 25.0 27.0 29.0 31.0 33.0 35.0
Retention Time (minutes)
A52-002-32A
Figure 1. GC-AFD Chromatogram of 100 ng Each of the Neutral Nitrogen
Compounds (Column 1)
411
-------
Method 633.1
Fenarimol
21.0
24.0
27.0
l I
30.0
33.0
Retention Time (minutes)
A52-002-33
Figure 2. GC-AFD Chromatogram of 200 ng Each of the Neutral Nitrogen
Compounds (Column 2)
412
-------
Method 634
The Determination of
Thiocarbate Pesticides in
Municipal and Industrial
Wastewaters
-------
-------
Method 634
The Determination of Thiocarbate Pesticides in Municipal and
Industrial Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain thiocarbamate pesticides. The following
parameters can be determined by this method:
Parameter CAS No.
Butylate 2008-41-5
Cycloate 1134-23-2
EPTC 759-94-4
Molinate 2212-67-1
Pebulate 1114-71-2
Vernolate 1929-77-7
1.2 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial 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 alternative test procedures under 40 CFR
136.4 and 136.5.
1.3 The 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 similar to other 600-series
methods. Thus, a single sample may be extracted to measure the compounds included in the
scope of the 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.
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.
415
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Method 634
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a continuous extractor. The methylene chloride extract is dried and concentrated to 5.0 mL.
Gas chromatographic conditions are described which permit the separation and measurement of
the compounds in the extract by alkali flame detector (AFD) gas chromatography.1
2.2 This method provides an optional silica gel column cleanup procedure 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 minutes. Thermally
stable materials, such as PCBs, may 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.
3.1.1 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 being 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 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 aware-
ness 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 identified3"5 for the information of the analyst.
416
-------
Method 634
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE 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 should 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 Continuous extractor: 2000-mL, Hirschberg-Wolf, (Paxton Woods Glass Shop #1029
or equivalent).
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A
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 equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 5-mL with glass stopper.
5.2.10 Volumetric flask: 10-mL with glass stopper.
5.2.11 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (+2°C). The bath should be used in a
hood.
417
-------
Method 634
5.5 Balance: 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 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: Glass, 180 cm long by 2 mm ID, packed with 3% SP-2250 on Supel-
coport (100/120 mesh) or equivalent. This column was used to develop the method
performance statements in Section 15. Guidelines for the use of alternative columns
are provided in Section 12.1.
5.6.2 Column 2: Glass, 180 cm long by 2 mm ID, packed with 3% SP-1000 on Supel-
coport (100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-phos-
phorus detector (NPD) or a thermionic-specific detector (TSD). This detector has
proven effective in the analysis of wastewaters for the compounds listed in the scope
and was used to develop the method performance statements in Section 15. Alter-
native 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 Methylene chloride, methanol, petroleum ether, ethyl ether, toluene: distilled-in-glass quality
or equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips
(available from Scientific Products Co., Catalog No. PI 126-8 and other suppliers). Proce-
dures recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davision Grade 923, 100/200 mesh; activated by heating for 24 hours at 150°C.
6.5 6N sodium hydroxide: Dissolve 24.0 grams NaOH in 100 mL distilled water.
6.6 6N sulfuric acid: Slowly add 16.6 mL concentrated H2SO4 to 50 mL distilled water and dilute
to 100 mL with distilled water.
6.7 Stock standard solutions (1.00 ^g//*L): 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 distilled-in-glass quality methanol 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 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can 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 PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check stock standard solutions for signs of
418
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Method 634
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solutions must be replaced after 6 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 compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with toluene. One of the external standards
should be at a concentration near, but above, the method detection limit. 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 /*L 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 injec-
ted, defined as the calibration factor (CF), can be calculated for each compound 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 compound 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, although carbazole has been used successfully in some instances.
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 volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with toluene. One of the standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations found in real
samples, or should define the working range of the detector.
419
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Method 634
7.3.2 Using injections of 2 to 5 /*L 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:
Equation 1
RF = _
where
As = Response for the parameter to be measured
Ais = Response for the internal standard
Cis = Concentration of the internal standard, in \LglL
Cs = Concentration of the parameter to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, AS/A^ 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 compound
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 interferences 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 are
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 measure-
ments. Each time such modifications to the method are made, the analyst is required
to repeat the procedure in Section 8.2
420
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Method 634
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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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
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.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 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 compound does not fall
421
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Method 634
within the control limits for method performance, the results reported for that compound 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 1-L
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. 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid imme-
diately after sampling.
7 0. SAMPLE EXTRA c TION
10.1 Assemble continuous extraction apparatus by placing 5 to 10 carborundum chips into the
500-mL round-bottom flask and attaching to the extraction flask.
10.2 Add 400 mL methylene chloride to the extraction flask. Some methylene chloride should
displace into the round-bottom flask.
10.3 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into the extraction flask and add sufficient distilled water to
fill extraction flask (2 L total volume aqueous phase).
10.4 Check the pH of the sample with wide range pH paper and adjust to 6 to 8 with 6N sodium
hydroxide or 6N sulfuric acid.
10.5 Connect the stirring apparatus to the extraction flask without the frit touching the sample.
Heat the methylene chloride in the round-bottom flask to continuous reflux and continue
heating for 30 minutes to 1 hour until frit is thoroughly wetted with methylene chloride.
422
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Method 634
10.6 Lower frit until it just touches the sample and start the stirring apparatus rotating.
10.7 Continuously extract sample for 18 to 24 hours.
10.8 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-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.
10.9 Pour the extract from the round-bottom flask through a drying column containing about 10 cm
of anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the flask
and column with 20 to 30 mL of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.10 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
10.11 Remove the Snyder column and flask and adjust the volume of the extract to 5.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further processing
will not be performed immediately. If the extract is to be stored longer than 2 days, transfer
the extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with solvent exchange to toluene as described in Section 10.12, and
then to gas chromatographic analysis as described in Section 12. If the sample requires
cleanup, proceed to Section 11.
10.12 Add 2.5 mL of toluene and one or two clean boiling chips to the extract in the 25-mL con-
centrator tube and attach a two ball micro-Snyder column. Place the K-D apparatus in a hot
water bath, 70 to 75°C. when the apparent volume of liquid reaches 2 to 2.5 mL. Remove
the K-D apparatus and allow it to drain and cool for at least 10 minutes. Transfer the sample
to a 5 mL volumetric flask and dilute to 5-mL with toluene. Proceed with gas chromato-
graphic analysis.
10.13Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 100-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is no less than 85 %.
423
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Method 634
11.2 Add 20 g of silica gel to a mixture of 100 mL of acetone and 1.2 mL of reagent water and stir
for 30 minutes on a stirring plate. Transfer the slurry to a chromatographic column (silica gel
may be retained with a plug of glass wool). Wash the column with 20 mL of methylene
chloride followed by 30 mL petroleum ether. Allow the solvent to elute from the column until
the silica gel is almost exposed to the air. Discard washings. Use a column flow rate of 2 to
2.5 mL/min throughout the wash and elution profiles. Add an additional 50 mL of petroleum
ether to the head of the column.
11.3 Add the extract from Section 10.12 to the petroleum ether suspended above column. Allow
the solvent to elute from the column until the silica gel is almost exposed to the air. Elute the
column with 25 mL of petroleum ether (Fraction 1). Discard this fraction.
11.4 Elute the column with 100 mL of 50% ethyl ether in petroleum ether and collect in a K-D
apparatus. Alternatively, separate fractions may be collected or combined at the discretion of
the analyst. The elution profile of these compounds from silica gel is given in Table 3.
11.5 Concentrate the fraction to less than 5-mL after addition of 2.5 mL toluene as described in
Section 10.12. Transfer sample to a 5-mL volumetric flask and dilute to 5 mL with toluene.
Proceed with gas chromatographic analysis.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph. Inclu-
ded 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 and Column 2 are
shown in Figures 1 and 2. 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 demon-
strated to be less than 6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 /xL of the sample extract using the solvent-flush technique.8 Record the volume
injected to the nearest 0.05 /*L, and the resulting peak sizes 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-tune variations of standards over the course of a day. Three
times the standard deviation of a retention-time for a compound can be used to calculate a
suggested window size; 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.
424
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Method 634
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:
Equation 2
Concentration, pg/L =
where
A = Amount of material injected, in ng
V( = Volume of extract injected, in \>L
Vt = Volume of total extract, in \tL
V = 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:
Equation 3
C-W.)
Concentration, uglL =
where
As = Response for parameter to be measured
Ais = Response for the internal standard
Is = Amount of internal standard added to each extract, in
Vo = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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.
425
-------
Method 634
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 seconds 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. When using a fused-silica capillary column, the column
outlet should be threaded through the interface to within a few millimeters of the entrance to
the source ionization chamber. A computer system should be interfaced to the mass spectro-
meter 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. The calculation
of tailing factors is illustrated in Method 625.
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 DFTPP performance criteria are achieved.9
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 50 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
14.4.1 The molecular ion and all other 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 +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 30 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 alternative 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. The MDL
concentrations listed in Table 1 were obtained using reagent water.1
15.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
426
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Method 634
15.3 In a single laboratory, Battelle's Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two diffe-
rent wastewaters were spiked and analyzed. The standard deviation of the percent recovery is
also included in Table 2.'
427
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Method 634
References
1. "Development of Methods for Pesticides in Wastewaters." EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 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, Com-
mittee 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, Pennsylvania, 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. 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).
428
-------
Method 634
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (minutes)
Parameter
EPIC
Butylate
Vernolate
Pebulate
Molinate
Cycloate
Column 1
12.8
13.5
14.2
14.5
16.6
17.5
Column 2
17.9
18.2
19.6
20.2
23.8
24.2
Method
Detection Limit
(ug/L)
0.9
0.6
1.1
0.8
0.6
1.6
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8 m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is held at 80°C for 4 minutes, programmed from 80 to 300°C at 8°C/min and held at
300°C for 4 minutes.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a glass
column 1.8m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is held at 80°C for 10 minutes, programmed from 80 to 250°C at 8°C/min and held
at 250°C for 10 minutes.
Table 2. Single Laboratory Accuracy and Precision3
Parameter
Butylate
Cycloate
EPIC
Molinate
Pebulate
Vernolate
Average
Percent
Recovery
80
95
93
95
100
100
87
93
97
98
93
96
Relative
Standard
Deviation
(%)
18
7.2
16
7.3
18
4.8
17
8.4
26
5.7
18
10
Spike
Level
(ug/L)
5.0
50
5.0
50
5.0
50
5.0
50
5.0
50
5.0
50
Number
of
Analyses
7
7
7
7
7
7
7
7
7
7
7
7
Matrix
Type2
1
1
1
1
1
1
1
1
1
1
1
1
(a) Column 1 conditions were used.
(b) 1 = Secondary POTW effluent
429
-------
Method 634
Table 3. Elution Characteristics of the Thiocarbamates from 6% Deactivated
Silica Gel
Appearance in Specified Fraction'
Parameter F1 \ F2 \ F3 \ F4
Butylate X X
Cycloate X
EPTC X
Molinate
Pebulate X
Vernolate X
Eluant composition by fraction:
(a) F1 = 25 mL petroleum ether
F2 = 50 mL 6% ethyl ether in petroleum ether
F3 = 50 ml 15% ethyl ether in petroleum ether
F4 = 50 mL 50% ethyl ether in petroleum ether
430
-------
Method 634
Molinate
Vernolate
EPIC
Butylate
'/• 1 1 1 1 1 1 1 1 I T
Cycloate
—1 T
19.0
—I 1
21.0
7.0
9.0
11.0
13.0
15.0
17.0
Retention Time (minutes)
A52-002-23A
Figure 1. GC-AFD Chromatogram of 200 ng of Each Thiocarbamate (Column 1)
431
-------
Method 634
\ i i i i i i i i I I i i i
16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0
Retention Time (minutes)
AS2-002-24A
Figure 2. GC-AFD Chromatogram of 200 ng of Each Thiocarbamate (Column 2)
432
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Method 635
The Determination of Rotenone
in Municipal and Industrial
Wastewaters
-------
-------
Method 635
The Determination of Rotenone in Municipal and Industrial
Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of rotenone pesticide. The following parameter can be
determined by this method:
Parameter CAS No.
Rotenone 83-79-4
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compound listed above in municipal and industrial 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 alternative test
procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for rotenone compound 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 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 using the proce-
dure described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second liquid chromatographic column that can be
used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory funnel. Liquid chromatographic conditions are described which permit the separa-
tion and measurement of the compounds in the extract by HPLC- UV.1
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 or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
435
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Method 635
ferences 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 thoroughly rinsing with tap and reagent water.
Drain dry, and heat in an oven or muffle furnace at 400°C for 15 to 30 minutes.
Some thermally stable materials, such as PCBs, may not be eliminated by this treat-
ment. 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 environ-
ment to prevent 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 being sampled. The
cleanup procedure in Section 11 can be used to overcome 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 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 aware-
ness 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 identified3"5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE 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 col-
lection 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,
436
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Method 635
however, the compressible tubing should be thoroughly rinsed with methanol, fol-
lowed by repeated rinsings with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-propor-
tional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock,
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit,
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290) or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250-mL (Kontes K-570001-0250 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 equi-
valent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 10-mL.
5.2.10 Erlenmeyer flask: 250-mL.
5.2.11 Graduated cylinder: 1000-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Normal-phase column, 5 \L Zorbax-CN, 250 mm long by 4.6 mm ID or
equivalent.
5.6.3 Column 2: Reversed-phase column, 5 /* Spherisorb-ODS, 250 mm long by 4.6 mm
ID or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm.
437
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Method 635
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, acetonitrile, acetone, hexane: Distilled-in-glass quality or
equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 IN sulfuric acid.
6.5 IN sodium hydroxide.
6.6 Silica gel, Davison grade 923, 100-200 mesh, dried for 12 hours at 150°C.
6.7 Stock standard solutions (1.00 fig/L): 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 distilled-in-glass quality methylene chloride for
analyses performed using Column 1 and methanol for analyses performed using
Column 2. 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 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 con-
centration if they are certified by the manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into PTFE-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.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with 50/50 hexane/methylene chloride for Column
1 standards and acetonitrile for Column 2 standards. One of the external standards
should be at a concentration near, but above, the method detection limit. 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 5 to 20 /*L of each calibration standard, tabulate peak height or
area responses against the mass injected. The results can be used to prepare a calibra-
438
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Method 635
tion curve for each compound. Alternatively, the ratio of the response to the mass
injected, defined as the calibration factor (CF), can be calculated for each compound
at each standard concentration. If the relative standard deviation of the calibration
factor is less than 10% over the working range, linearity through the origin can be
assumed and 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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with 50/50 hexane/methylene chloride
for Column 1 standards and acetonitrile for Column 2 standards. One of the stan-
dards should be at a concentration near, but above, the method detection limit. 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 5 to 20 /*L 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
Cis = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in \iglL
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 can be used for
calculations. Alternatively, the results can be used to pilot a calibration curve of
response ratios, AS/A^ against RF.
439
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Method 635
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 compound
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 interferences 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.
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 measure-
ments. 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 methylene
chloride, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 percentage recovery (R), and the standard deviation of the per-
centage 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 measured
440
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Method 635
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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 with this method. This ability is established as de-
scribed 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. When doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques, such as liquid chromatography with a dissimilar
column, 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
441
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Method 635
posite 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.
9.3 Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or IN sulfuric acid im-
mediately after sampling.
1 0. SAMPLE EXTRA c TION
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 7 with IN sodium hydroxide or IN H2SO4.
10.2 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 extract the sample by shaking the
funnel for 2 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, centrifuga-
tion, or other physical methods. Collect the methylene chloride extract in a 250-mL Erlen-
meyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the extrac-
tion procedure a second time, collecting the extract. Perform a third extraction in the same
manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
250-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with an additional
30 to 40 mL of methylene chloride.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-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 opera-
tion. Add one or two clean boiling chips and attach a two-ball micro-Snyder column to the
442
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Method 635
concentrator tube. Prewet the micro-Snyder column with methylene chloride and concentrate
the solvent extract as before. When an apparent volume of 0.5 mL is reached, or the solu-
tion stops boiling, remove the K-D apparatus and allow it to drain and cool for 10 minutes.
If analysis is being performed using Column 1 or if sample cleanup is required, proceed
with Section 10.9. If Column 2 is used and no sample cleanup is required, proceed with
Section 10.8.
10.8 Add 10 mL of acetonitrile to the concentrator tube along with one or two clean boiling chips.
Attach a two-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder
column with acetonitrile and concentrate the solvent extract as before. When an apparent
volume of 1 mL is reached, remove the K-D apparatus and allow it to drain and cool for
10 minutes. Transfer the liquid to a 10-mL volumetric flask and dilute to the mark with
acetonitrile. Mix thoroughly prior to analysis. Proceed with Section 12 using Column 2.
10.9 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further processing
will not be performed immediately. If the extract is to be stored longer than two days,
transfer the extract to a screw-capped vial with a PTFE-lined cap. If the sample extract
requires no further cleanup, proceed with the liquid chromatographic analysis in Section 12
using Column 1. 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 clean waters
and industrial effluents. If particular circumstances demand the use of additional cleanup, the
analyst must demonstrate that the recovery of each compound of interest is no less than 85 %.
11.2 Slurry 10 g of silica gel in 50 mL of acetone to which has been added 600 ^L of reagent
water. Transfer the slurry to a chromatographic column (silica gel is retained with a plug of
glass wool). Wash the column with 100 mL of methylene chloride. Use a column flow rate
of 2 to 2.5 mL/min throughout the wash and elution profiles.
11.3 Add the extract from Section 10.9 to the head of the column. Allow the solvent to elute from
the column until the silica gel is almost exposed to the air. Elute the column with 50 mL of
methylene chloride. Discard this fraction.
11.4 Elute the column with 60 mL of 6% acetone in methylene chloride. Collect this fraction in
a K-D apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6
and 10.7. If Column 1 is being used, proceed with Section 11.5. If Column 2 is being used,
proceed with Section 11.7.
11.5 Add 5 mL of hexane to the concentrate along with one or two clean boiling chips. Attach a
three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column
with hexane and concentrate the solvent extract to an apparent volume of 1 mL. Allow the
K-D apparatus to drain and cool for 10 minutes.
443
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Method 635
11.6 Transfer the liquid to a 10-mL volumetric flask and dilute to the mark with hexane. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid chromato-
graphic analysis using Column 1.
11.7 Add 10 mL of acetonitrile to the concentrate along with one or two clean boiling chips.
Attach a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder
column with acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL.
Allow the K-D apparatus to drain and cool for 10 minutes.
11.8 Transfer the liquid to a 10-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid chromato-
graphic analysis using Column 2.
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 Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may
be used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 /iL of the sample extract by completely filling the sample valve loop. Record
the resulting peak sizes 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
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:
444
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Method 635
Equation 2
Concentration, ng/L =
where
A = Amount of material injected, in ng
V( = Volume of extract injected, in \)L
Vt - Volume of total extract, in \tL
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:
Equation 3
Concentration, pglL =
(Ais)(RF)(V0)
where
As = Response for parameter to be measured
Ais = Response for the internal standard
ls = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds 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 about zero. The MDL
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two
different wastewaters were spiked and analyzed. The standard deviation of the percent recov-
ery is also included in Table 2.1
445
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Method 635
References
1. "Development of Methods for Pesticides in Wastewaters," Report for EPA Contract
68-03-2956 (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, Phila-
delphia, Pennsylvania, 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, August 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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J. A. et al. "Trace Analysis for Wastewaters," Environmental Science and Technology,
15, 1426 (1981).
446
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Method 635
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (min) Method Detection
Limit
Parameter Column 1
Column 2
(ug/L)
Rotenone 8.6 8.0 1.6
Column 1 conditions: Zorbax-CN, 5 p, 250 mm long by 4.6 mm ID; 1 mL/min flow; 30/70
methylene cnloride/hexane.
Column 2 conditions: Spherisorb-ODS, 5 //, 250 mm long by 4.6 mm ID; 1 mL/min flow;
60/40 acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision3
Average Standard
Percent Deviation Spike Level Number of Matrix
Parameter Recovery Percent (ug/L) Analyses Type*
Rotenone 85 8 5.5 7 1
88 3 109 71
(a) Column 1 conditions were used.
(b) 1 = Pesticide manufacturing wastewater
447
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Method 635
Rotenone
r
o
i—
1.2
1 1—
4.8
—I—
6.0
2.4
1 1 1 1 1 1 1 \ 1
7.2
8.4
9.0
10.0 12.0
Retention Time (minutes)
52-002-25A
Figure 1. HPLC-UV Chromatogram of Standard Solution Representing 5 ug/L
of Rotenone in Water (Column 1)
448
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Method 635
Rotenone
IX
i i i i i i i i I i i i i i i i i i i
1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
A52-002-26
Figure 2. HPLC-UV Chromatogram of Standard Solution Representing 200 ug/L
of Rotenone in Water (Column 2)
449
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Method 636
The Determination of Bensulide
in Municipal and Industrial
Wastewater
-------
-------
Method 636
The Determination of Bensulide in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of bensulide pesticide. The following parameter can be
determined by this method:
Parameters CAS No.
Bensulide 741-58-2
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compound listed above in municipal and industrial 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 alternative test
procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) for bensulide compound 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 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 using the proce-
dure described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second liquid chromatographic column that can be
used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and exchanged to acetonitrile
during concentration to a volume of 2 mL or less. Liquid chromatographic conditions are
described which permit the separation and measurement of the compounds in the extract by
HPLC-UV.1
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 or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
453
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Method 636
ferences 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 minutes. Some
thermally stable materials, such as PCBs, may 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 miniimize 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 being sampled. The cleanup
procedure in Section 11 can be used to overcome 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 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 aware-
ness 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 identified3 5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Borosilicate or flint glass, 1-L or 1-quart volume, fitted with
screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE if the
sample is not corrosive. 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 should be thoroughly rinsed with methanol,
followed by repeated rinsings with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
454
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Method 636
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
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 equi-
valent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 ji Spherisorb-ODS, 250 mm long by 4.6 mm
ID or equivalent.
5.6.3 Column 2: Reversed-phase column, 5 //, Lichrosorb RP-2, 250 mm long by 4.6 mm
ID or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 270 mm.
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, acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Sodium phosphate, monobasic: ACS, crystal.
455
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Method 636
6.5 IN sulfuric acid: Slowly add 2.8 ml. of concentrated II2SO4 (94%) to about 50 mL of
distilled water. Dilute to KX) mL with distilled water.
6.6 IN sodium hydroxide: Dissolve 4.0 grams of NaOH in KM) mL of distilled water.
6.7 Florisil: PR grade (60/1 (X) mesh). Purchase activated at 675 °C and store in brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFL7 or aluminum foil-lined screw-cap and cool to room tempera-
ture.
6.8 Stock standard solutions (1.00 /*g//xL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.8.1 Prepare stock standard solutions by accurately weighing about O.OIOO g of pure
material. Dissolve the material in distilled-in-glass quality methane] 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 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can be used at any concentration if they are certified
by the manufacturer or by an independent source.
6.8.2 Transfer the stock standard solutions into PTFE-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.
6.8.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with acetonitrile. One of the external standards
should be at a concentration near, but above, the method detection limit. 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 (jiL 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, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation of the calibration factor is
less than 10% over the working range, linearity through the origin can be assumed
and the average calibration factor can be used in place of a calibration curve.
456
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Method 636
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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with acetonitrile. One of the standards
should be at a concentration near, but above, the method detection limit. 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 jtL 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:
Equation 1
where
As = Response for the compound to be measured
A^ = Response for the internal standard
Cis = Concentration of the intermalstandard, in ug/L
Cs = Concentration of the compound to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A^/A.^ 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 compound
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 interferences from the
reagents.
457
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Method 636
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 measure-
ments. 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 methanol 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample of 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 deter-
mine 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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.
458
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Method 636
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 de-
fined 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 gather through
the requirement for continuing quality control in Section 8.4. The accuracy state-
ments should be updated with this method. This ability is established as described
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 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 compound does not fall
within the control limits for method performance, the results reported for that compound in
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 though the analysis of a 1-L
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. When doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as liquid chromatography with a dissimilar
column, 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
9.3 Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or IN sulfuric acid imme-
diately after sampling.
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 7 with IN sodium hydroxide or IN H2SO4.
459
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Method 636
10.2 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 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, cen-
trifugation, 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 extrac-
tion procedure a second time, collecting the extract. Perform a third extraction in the same
manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball macro-
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 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 and
allow it to drain and cool for at least ten minutes. If the sample extract requires no further
cleanup, proceed with solvent exchange to acetonitrile and chromatographic analysis as de-
scribed in Sections 11.5 and 12 respectively. If the sample requires cleanup, proceed to
Section 10.7.
10.7 Remove the macro-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 ope-
ration. Add one or two clean boiling chips and attach a two-ball micro-Snyder column to the
concentrator tube. Prewet the micro-Snyder column with methylene chloride and concentrate
the solvent extract as before. When an apparent volume of 0.5 mL is reached, or the solution
stops boiling remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 2 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap.
460
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Method 636
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
71. CLEANUP AND SEPARA TION
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 clean waters
and industrial effluents. If particular circumstances demand the use of additional cleanup, the
analyst must demonstrate that the recovery of each compound of interest is no less than 76%.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column (Florisil may be retained with a plug
of glass wool). Wash the column with 100 mL of methylene chloride. Use a column flow
rate of 2 to 2.5 mL/min throughout the wash and elution profiles.
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from
the column until the Florisil is almost exposed to the air. Elute the column with 50 mL of
methylene chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction in a
K-D apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and
10.7.
11.5 Add 15 mL of acetonitrile to the concentrate along with one or two clean boiling chips. At-
tach a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder
column with acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL.
Allow the K-D apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid chroma-
tographic analysis.
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 Column 1 and Column 2
are 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 liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
the sample extracts until immediately before injections into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 /*L of the sample extract into the sample valve loop. Record the resulting peak
sizes in area or peak heights units.
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
461
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Method 636
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
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 mate-
rial 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:
Equation 2
Concentration, pg/L =
where
A = Amount of material injected, in nanograms
V. = Volume of extract injected, in ng/L
Vt = Volume of total extract, in pg/L
V = 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:
Equation 3
Concentration, pg/L =
where
As = Response for parameter to be measured
Ais = Response for the internal standard
Is = Amount of internal standard added to each extract, in fj,g
V = Volume of water extracted, in liters
13.2 Report results in microgram 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 out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
452
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Method 636
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.8 The MDL
concentrations listed in Table 1 were obtained using reagent water.8 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two
different wastewaters were spiked and analyzed. The standard deviation of the percent
recovery is also included in Table 2.'
463
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Method 636
References
I. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Pennsylvania, 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, August 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, March 1979.
7. ASTM Annual Book of Standards, Part 31, 03370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J. A. et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
464
-------
Method 636
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time fminj
Parameter
Bensulide
Column 1
14.1
Column 2
7.2
Method Detection Limit
(ug/L)
1.6
Column 1 conditions: Spherisorb ODS, 5 fj, 250 mm long by 4.6 mm ID; 1 mL/min flow; 55/45
acetonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5 //, 250 mm long by 4.6 mm ID; 1 mL/min flow; 60/40
acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision8
Parameter
Bensulide
Average
Percent
Recovery
86
76
Standard
Deviation
(%)
18
18
Spike
Level
(ug/L)
25
250
Number
of
Analyses
7
7
Matrix
Type*
1
1
(a) Column 1 conditions were used.
(b) 1 = Relevant industrial wastewater
465
-------
Method 636
Bensulide
\
i i i i i i i i i l i i i i i i i i i i
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
A52-002-27A
Figure 1. HPLC-UV Chromatogram of 60 ng of Bensulide (Column 1)
466
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Method 637
The Determination of MBTS
and TCMTB in Municipal and
Industrial Wastewaters
-------
-------
Method 637
The Determination of MBTS and TCMTB in Municipal and Industrial
Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of MBTS and TCMTB pesticides. The following
parameters can be determined by this method.
Parameter CAS No.
MBTS 120-78-5
TCMTB 21564-17-0
1.2 This is a liquid chromatographic (LC) method applicable to the determination of the com-
pounds listed above in municipal and industrial 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 alternative test procedures under 40 CFR .
136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) 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 in
certain other 600-series methods. Thus, a single sample may be extracted to measure the
compounds included in the scope of the methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply ap-
propriate cleanup procedures.
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 using the proce-
dure 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 liquid chromatographic
column that can be used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and concentrated to 5.0 mL.
Liquid chromatographic conditions are described which permit the separation and measurement
of the compounds in the extract by high-performance liquid chromatography with ultraviolet
detection.1
469
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Method 637
2.2 This method provides a silica gel column cleanup procedure 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 liquid
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 minutes. Do not heat volu-
metric ware. Some thermally stable materials, such as PCBs, may 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 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 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 aware-
ness 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 identified3"5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Foil may be substituted for PTFE if the sample is
not corrosive. If amber bottles are not available, protect samples from light. The
470
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Method 637
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 col-
lection 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, fol-
lowed by repeated rinsings with distilled water to minimize the potential for con-
tamination 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 PTFE stopcock.
5.2.2 Drying column: Chromatographic column, 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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. A
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 PTFE-lined screw-cap.
5.2.8 Erlenmeyer flask: 250-mL.
5.2.9 Graduated cylinder 1000-mL.
5.2.10 Volumetric flask: 5-mL, 10-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph suitable for on-
column injection and all required accessories including syringes, analytical columns, detector,
and strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: 5 p Dupont Zorbax-CN, 250 mm long by 4.6 mm ID or equivalent. This
column was used to develop the method performance statements in Section 14. Al-
ternative columns may be used in accordance with the provisions described in Sec-
tion 12.1.
5.6.2 Column 2: 5 ju. Dupont Zorbax Silica, 250 mm long by 4.6 mm ID or equivalent.
471
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Method 637
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, ethyl ether, and hexane: Distilled-in-glass quality of equi-
valent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips (available
from Scientific Products Co., Catalog No. PI 126-8 and other suppliers). Procedures recom-
mended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 40°C overnight.
6.4 Silica gel: Davison Grade 923, 100-120 mesh, dried for 12 hours at 150°C.
6.5 IN sodium hydroxide: Dissolve 4.0 g of NaOH (ACS) in 100 mL of distilled water.
6.6 IN sulruric acid: Slowly add 2.8 mL of concentrated H2SO4 (94%) to about 50 mL of dis-
tilled water. Dilute to 100 mL with distilled water
6.7 Sodium phosphate: Monobasic, ACS grade.
6.8 Sodium phosphate: Dibasic, ACS grade.
6.9 Stock standard solutions (1.00 jig/^L): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in distilled-in-glass quality methylene chloride 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 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can 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 PTFE-sealed screw-cap bottles. Store at
4°C and protect from light. Frequently check standard solutions for signs of degrada-
tion or evaporation, especially just prior to preparing calibration standards from them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with methylene chloride. One of the external
standards should be at 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.
472
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Method 637
7.2.2 Using injections of 5 to 20 fiL of each calibration standard, tabulate peak height or
area responses against the mass injected. The results can be used to prepare a calibra-
tion curve for each compound. Alternatively, the ratio of the response to the mass
injected, defined as the calibration factor (CF), can be calculated for each compound
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 compound 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 similar in analytical behavior to the compounds of interest. The ana-
lyst 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with methylene chloride. One of the
standards should be at 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 5 to 20 /xL 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:
Equation 1
RF = -
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in fig/L
Cs = Concentration of the parameter to be measured, in
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of re-
sponse ratios, AS/A^ against RF.
473
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Method 637
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 compound
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 interferences 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.
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 measure-
ments. 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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-operator
precision expected for the method, and compare these results to the values measured
in Section 8.2.3. If the data are not comparable, the analyst must review potential
problem areas and repeat the test.
474
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Method 637
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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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.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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 though the analysis of a 1-L
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. 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
475
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Method 637
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 6 to 8 with IN sodium hydroxide or IN sulfuric acid.
Dissolve 5 g of monobasic sodium phosphate and 5 g of dibasic sodium phosphate in the
sample.
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion 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 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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder
column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top.
Place the K-D apparatus on a hot water bath, 60 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 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 and
allow it to drain and cool for at least 10 minutes. If the sample extract requires no cleanup,
proceed with Section 10.7. If the sample extract requires cleanup, proceed to Section 11.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride. Adjust the volume of the extract to 5.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further processing
476
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Method 637
will not be performed immediately. If the extract is to be stored longer than 2 days, transfer
the extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with the liquid chromatographic analysis in Section 12. 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5
mL.
17. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is no less than the recovery values reported in Table 2.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to
the pesticides listed in Table 1.
11.2.1 Add 10 g of silica gel to 100 mL of ethyl ether and 600 /*L of reagent water in a
250-mL Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry to a
chromatographic column (silica gel may be retained with a plug of glass wool). Al-
low the solvent to elute from the column until the silica gel is almost exposed to the
air. Wash the column with 100 mL of 50% hexane in methylene chloride as before
and discard. Use a column flow of 2 to 2.5 mL/min throughout the wash and elution
profiles.
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the silica gel is almost exposed to the
air. Elute the column with 50 mL of 50% hexane in methylene chloride. Discard
this fraction.
11.2.3 Elute the column with 50 mL of methylene chloride (Fraction 1) and collect eluate in
a K-D apparatus. Repeat process with 50 mL of 6% ethyl ether in methylene chloride
(Fraction 2). The TCMTB elutes in Fraction 1 and the MBTS elutes in Fraction 2.
Concentrate each fraction to 5.0 mL as described in Sections 10.6 and 10.7. Proceed
with liquid chromatographic analysis.
11.2.4 The above-mentioned fractions can be combined before concentration at the discretion
of the analyst.
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. Examples of the separations achieved by Column 1 and Column 2
are shown in Figures 1,2, and 3. Other columns, chromatographic conditions, or detectors
may be used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
477
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Method 637
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 /iL of the sample extract by completely filling the sample value loop. Record
the resulting peak sizes in areas of 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 variations of standards over the course of a day. Three
times the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
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:
Equation 2
Concentration, uglL =
where
A = Amount of material injected, in ng
V. = Volume of extract injected, in \iL
Vt = Volume of total extract, in \iL
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:
Equation 3
Concentration, ng/L = -
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
478
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Method 637
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 out-
side of the control limits in Section 8.3, data for the affected compounds 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.8 The MDL
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x -MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained after silica gel cleanup. Seven repli-
cates of each of two different wastewaters were spiked and analyzed. The standard deviation
of the percent recovery is also included in Table 2.'
479
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Method 637
References
1. "Development of Methods for Pesticides in Wastewaters," Report for EPA Contract
68-03-2956 (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, Phila-
delphia, 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, August 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.
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. Glaser, J. A., et ah, "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
480
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Method 637
Table 1. Chromatographic Conditions and Estimated Detection Limits
Retention Time (min)
Parameter
MBTS
TCMTB
Column 1
Column 2
6.6 6.3
9.3 7.9
MDL
fug/I
0.5
1.0
Column 1 conditions: Dupont Zorbax-CN, 5 fj, 250 mm long by 4.6 mm ID; 1 mL/min flow; 15/85
methylene chloride/hexane.
Column 2 conditions: Dupont Zorbax silica, 5 fj, 250 mm long by 4.6 mm ID; 1 mL/min flow;
90/9.5/0.5 hexane/methylene chloride/methanol.
Table 2. Single Laboratory Accuracy and Precision3
Sample Background Spike Mean Standard Number of
7 Replicates
7
7
7
7
(a) Column 1 conditions were used.
(b) 1 = Municipal sewage effluent
(c) ND = Not detected
Parameter
MBTS
TCMTB
Type*
1
1
1
1
(ug/Uc
ND
ND
ND
ND
(ug/U Recovery (%) j
5 35
100 69
5 69
100 90
Devic
23
6
20
2
481
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Method 637
/MBTS TCMTB
\ I I I I I I I I I \ I I I I I I I I I
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
A52-002-34A
Figure 1. HPLC-UV Chromatogram of 10 ng Each of MBTS and
TCMTB (Column 1)
482
-------
Method 637
±
MBTS
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
AS2-002-35A
Figure 2. HPLC-UV Chromatogram of 100 ng of MBTS (Column 2)
483
-------
Method 637
TCMTB
iiiiiiiir i riiir T \ i Ti
0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
A52-002-36A
Figure 3. HPLC-UV Chromatogram of 100 ng of TCMTB (Column 2)
484
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Method 638
The Determination of Oryzalln
in Municipal and Industrial
Wastewaters
-------
-------
Method 638
The Determination of Oryzalin in Municipal and Industrial
Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of oryzalin pesticide. The following parameter can be
determined by this method:
Parameters CAS No.
Oryzalin 19044-88-3
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compound listed above in municipal and industrial 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 alternative test
procedures under 40 CFR 136.4 and 136.5.
1.3 The estimated method detection limit (MDL, defined in Section 15) for oryzalin 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 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 using the proce-
dure described in Section 8.2.
1.5 When this method is used to analyze unfamiliar samples for the compound above, compound
identifications should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second liquid chromatographic column that can be
used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separatory funnel. The methylene chloride extract is dried and exchanged to acetonitrile
during concentration to a volume of 2 mL or less. Liquid chromatographic conditions are
described which permit the separation and measurement of the compounds in the extract by
HPLC-UV.1
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 or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
ferences under the conditions of the analysis by running laboratory reagent blanks as described
in Section 8.5.
487
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Method 638
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 minutes. Some
thermally stable materials, such as PCBs, may not be eliminated by this treatment.
Thorough rinsing with acetone and pesticide-quality hexane may be substituted for
heating. Volumetric wave should not be heated in a muffle furnace. 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 being sampled. The
cleanup procedure in Section 11 can be used to overcome 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 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 aware-
ness 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 identified3"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-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Aluminum foil may be substituted for PTFE 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 should be thoroughly rinsed with methanol,
followed by repeated rinsings with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
488
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Method 638
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separatory funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 25-mL, graduated (Kontes K-570050-2525 or
equivalent). Calibration must be checked at the volumes employed in the test. A
ground-glass stopper is used to prevent evaporation of extracts.
5.2.5 Evaporative flask, Kuderna-Danish: 250-mL (Kontes K-570001-0250 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 equi-
valent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.2.9 Volumetric flask: 2-mL with glass stopper.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 /* Spherisorb-ODS, 250 mm long by
4.6 mm ID or equivalent.
5.6.3 Column 2: Reversed-phase column, 5 /* Lichrosorb RP-2, 250 mm long by
4.6 mm ID or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 nm.
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, acetone, acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated hi a muffle furnace at 400°C overnight.
489
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Method 638
6.4 Sodium hydroxide, IN: Prepared by adding 4 g of sodium hydroxide in distilled water and
diluting to 100 mL.
6.5 Sulfuric acid, IN: Prepared by diluting 2.8 mL of concentrated sulfuric acid to distilled water
and diluting to 100 mL.
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFE-or aluminum-foil-lined screw-cap and cool to room tempera-
ture.
6.7 Stock standard solutions (1.00 ptg/jDiL): 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 0.010 g of pure mate-
rial. Dissolve the material in distilled-in-glass quality acetonitrile and dilute to vol-
ume in a 10-mL volumetric flask. Larger volumes can be used 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.
6.7.2 Transfer the stock standard solutions into PTFE-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.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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:
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with acetonitrile. One of the external standards
should be at a concentration near, but above, the method detection limit. 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 /*L 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, the ratio of the response to the mass injec-
ted, defined as the calibration factor (CF), can be calculated for each compound at
each standard concentration. If the relative standard deviation of the calibration factor
is less than 10% over the working range, linearity through the origin can be assumed
and the average calibration factor can be used in place of a calibration curve.
490
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Method 638
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 compound 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 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with acetonitrile. One of the standards
should be at a concentration near, but above, the method detection limit. 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 /xL 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:
Equation 1
op _ ^
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
C^ = Concentration of the internal standard, in
Cs = Concentration of the parameter to be measured, in pg/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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A/Ajs 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 compound
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 interferences from the
reagents.
491
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Method 638
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 measure-
ments. 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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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.
492
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Method 638
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 de-
fined 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 with this method. This ability is established as de-
scribed 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. When doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as liquid chromatography with a dissimilar
column, 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite samples should be collected in 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.
9.3 Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or IN sulfuric acid im-
mediately after sampling.
9.4 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction.
493
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Method 638
10. SAMPLE EXTRA c TION
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 7 with IN sodium hydroxide or IN H2SO4.
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion procedure a second time, collecting the extract. Perform a third extraction in the same
manner and combine the extracts.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 25-mL concentrator tube to a
250-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two 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 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 and
allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-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 opera-
tion. Add one or two clean boiling chips and attach a two-ball micro-Snyder column to the
concentrator tube. Prewet the micro-Snyder column with methylene chloride and concentrate
the solvent extract as before. When an apparent volume of 0.5 mL is reached, or the solution
stops boiling, remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 2 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
494
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Method 638
further cleanup, proceed with solvent exchange to acetonitrile as described beginning with
Section 11.5. 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 transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest 5
mL.
71. CLEANUP AND SEPARA TION
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 clean waters
and industrial effluents. If particular circumstances demand the use of additional cleanup, the
analyst must demonstrate that the recovery of each compound of interest is no less than 85%.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column (Florisil is retained with a plug of
glass wool). Wash the column with 100 mL of methylene chloride. Use a column flow rate
of 2 to 2.5 mL/min throughout the wash and elution profiles.
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from
the column until the Florisil is almost exposed to the air. Elute the column with 50 mL of
methylene chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction in a
K-D apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6 and
10.7.
11.5 Add 15 mL of acetonitrile to the concentrate along with one or two clean boiling chips.
Attach a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder
column with acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL.
Allow the K-D apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid chroma-
tographic analysis.
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 Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may
be used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument.
12.4 Inject 2 to 5 //L of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes in area or peak height units.
495
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Method 638
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 for a compound can be used to calculate a
suggested window size; 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.
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:
Equation 2
Concentration, ng/L =
where
A = Amount of material injected, in ng
Vi = Volume of extract injected, in (jL
Vt = Volume of total extract, in fiL
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:
Equation 3
Concentration, fuglL = —
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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.
496
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Method 638
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls out-
side of the control limits in Section 8.3, data for the affected compounds must be labeled as
suspect.
14. METHOD PERFORMANCE
14.1 The method detection limit (MDL)8 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 in Table 1 were obtained using reagent water.1 Similar results
were achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two dif-
ferent wastewaters were spiked and analyzed. The standard deviation of the percent recovery
is also included in Table 2.'
497
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Method 638
References
1. "Devek oment of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(in prepj/ation).
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, Phila-
delphia, Pennsylvania, 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, August 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.
7. ASTM Annual Book of Sttiv' r 3, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Te^ag and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, S. A. et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
498
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Method 638
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time fmin)
Parameter
Oryzalin
Column 7
6.2
Column 2
10.7
MDL
fug/Li
0.5
Column 1 conditions: Spherisorb-ODS, 5 fj, 250 mm long by 4.6 mm ID; 1 mL/min flow; 40/60
acetonitrile/water. A UV detector was used with this column to determine the MDL.
Column 2 conditions: Lichrosorb RP-2, 5 //, 250 mm long by 4.6 mm ID; 1 mL/min flow;
50/50 acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision3
Spike
Sample Background Level
Parameter Type" fug/L) (ug/L)
Oryzalin 1 4 10
2 40 200
Mean
Recovery
(%)
106
100
Standard
Deviation
(%)
6
10
Number •
of Replicates
7
7
(a) Column 1 conditions were used.
(b) 1 = Relevant industrial wastewater diluted 1000:1 with municipal sewage effluent
2 = Relevant industrial wastewater diluted 100:1 with municipal sewage effluent
499
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Method 638
Oryzalin
I 1 I I 1 I
2.0 4.0 6.0
I I I I 1 I I I I
8.0 10.0 12.0 14.0 16.0
Retention Time (minutes)
52-002-28A
Figure 1. HPLC-UV Chromatogram of 10 ng of Oryzalin (Column 1)
500
-------
Method 638
Oryzalin
/
\\ \ r
iiii
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0
Retention Time (minutes)
A52-002-29A
Figure 2. HPLC-UV Chromatogram of 250 ng of Oryzalin (Column 2)
501
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Method 639
The Determination of
Bendiocarb in Municipal and
Industrial Wastewaters
-------
-------
Method 639
The Determination of Bendiocarb in Municipal and Industrial
Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of bendiocarb pesticide. The following parameter can
be determined by this method:
Parameter CAS No.
Bendiocarb 22781-23-3
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compound listed above in municipal and industrial discharges as provided
under 40 CFR 136.1. When this method is used to analyze unfamiliar samples for the com-
pound above, compound identifications should be supported by at least one additional qualita-
tive technique. This method describes analytical conditions for a second liquid chromato-
graphic column that can be used to confirm measurements made with the primary column.
1.3 The method detection limit (MDL, defined in Section 15) for bendiocarb 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 Any modification of this method beyond those expressly permitted shall be considered a major
modification subject to application and approval of alternative test procedures under 40 CFR
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 liquid chromatography and in the interpretation of liquid chromatograms. Each analyst
must demonstrate the ability to generate acceptable results with this method using the proce-
dure described in Section 8.2.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separately funnel. The methylene chloride extract is dried and exchanged to acetonitrile
during concentration to a volume of 2 mL or less. Liquid chromatographic conditions are
described which permit the separation and measurement of the compounds in the extract by
HPLC-UV.1
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 or elevated baselines in liquid
chromatograms. All of these materials must be routinely demonstrated to be free from inter-
505
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Method 639
ferences 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 rinsing with the last solvent used in it. This should be followed by
detergent washing with hot water and rinses with tap 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 treat-
ment. Solvent rinses with acetone and pesticide-quality hexane may be substituted for
the heating. 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 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 being sampled. 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 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 aware-
ness 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 identified3 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 glass, 1-L or 1-quart volume, fitted with screw-caps lined
with PTFE. Foil may be substituted for PTFE 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 should be thoroughly rinsed with methanol,
followed by repeated rinsings with distilled water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
506
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Method 639
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for illustration
only.)
5.2.1 Separately funnel: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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. A
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 equi-
valent).
5.2.8 Vials: Amber glass, 5- to 10-mL capacity with PTFE-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
extract in a Soxhlet extractor with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph and all required
accessories including syringes, analytical columns, detector, and strip-chart recorder. A data
system is recommended for measuring peak areas.
5.6.1 Pump: Isocratic pumping system, constant flow.
5.6.2 Column 1: Reversed-phase column, 5 /* Spherisorb-ODS, 250 mm long by
4.6 mm ID or equivalent. f
5.6.3 Column 2: Reversed-phase column, 5 p Lichrosorb RP-2, 250 mm long by
4.6 mm ID or equivalent.
5.6.4 Detector: Ultraviolet absorbance detector, 254 run.
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 acetonitrile: Distilled-in-glass quality or equivalent.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Sodium hydroxide, IN: Prepare by adding 4 g of sodium hydroxide to distilled water and
diluting to 100 mL.
507
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Method 639
6.5 Sulfuri© aeid, IN: Prepare by adding 2.8 mL of concentrated sulfuric acid to distilled water
and diluting to 100 mL.
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in a brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFE- or aluminum-foil-lined cap and cool to room temperature.
6.7 Stock standard solutions (1.00 ^g//iL): 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 mate-
rial. Dissolve the material in distilled-in-glass quality methanol 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 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.
6.7.2 Transfer the stock standard solution into PTFE-sealed screw-cap bottles. Store at 4°C
and protect from light. Stock standard solution should be checked frequently for signs
of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Stock standard solution must be replaced after 6 months, or sooner if comparison with
quality control check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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:
7.2.1 For the compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with acetonitrile. One of the external standards
should be at a concentration near, but above, the method detection limit. 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 ftL 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 bendiocarb. Alternatively, the ratio of the response to the mass injected,
defined as the calibration factor (CF), can be calculated at each standard concentra-
tion. If the relative standard deviation of the calibration factor is less than 10% over
the working range, linearity through the origin can be assumed and the average cali-
bration 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
bendiocarb varies from the predicted response by more than ±10%, the test must be
508
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Method 639
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 one or
more internal standards similar in analytical behavior to bendiocarb. 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 bendio-
carb 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 stan-
dards, and dilute to volume with acetonitrile. One of the standards should be at a
concentration near, but above, the method detection limit. 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 /*L of each calibration standard, tabulate the peak height or
area responses against the concentration for bendiocarb and internal standard. Cal-
culate response factors (RF) as follows:
Equation 1
RF = -
where
As = Response for the parameter to be measured
AK = Response for the internal standard
C^ = Concentration of the internal standard, in fig/L
Cs = Concentration of the parameter to be measured, in pg/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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A/A^ against 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 bendiocarb
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 interferences from the
reagents.
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Method 639
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 measure-
ments. 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 methanol 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore 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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R — 3s
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.
510
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Method 639
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 de-
fined 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 with this method. This ability is established as de-
scribed 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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 1-L
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. When doubt exists over the identification of a peak on
the chromatogram, confirmatory techniques such as liquid chromatography with a dissimilar
column 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 practices7 should
be followed; however, the bottle must not be prerinsed with sample before collection. Com-
posite 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.
9.3 Adjust the pH of the sample to 6 to 8 with IN sodium hydroxide or IN sulfuric acid imme-
diately after sampling.
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 7 with IN sodium hydroxide or IN H2SO4.
577
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Method 639
10.2 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 extract the sample by shaking
the funnel for 2 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, cen-
trifugation, 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 extrac-
tion procedure a second time, collecting the extract. Perform a third extraction in the same
manner and collect the extract.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube to a
250-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a macro-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 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 and allow it to
drain and cool for at least 10 minutes.
10.7 Remove the macro-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 opera-
tion. Add one or two clean boiling chips and attach a two-ball micro-Snyder column to the
concentrator tube. Prewet the micro-Snyder column with methylene chloride and concentrate
the solvent extract as before. When an apparent volume of 0.5 mL is reached, or the solution
stops boiling, remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 2 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with solvent exchange to acetonitrile as described beginning in
Section 11.5. If the sample requires cleanup, proceed to Section 11.
5/2
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Method 639
10.9 Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
7 7. CLEANUP AND SEPARA TION
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 clean waters
and industrial effluents. If particular circumstances demand the use of additional cleanup, the
analyst must demonstrate that the recovery of each compound of interest is no less than 65 %.
11.2 Slurry 10 g of Florisil in 100 mL of methylene chloride which has been saturated with reagent
water. Transfer the slurry to a chromatographic column. Wash the column with 100 mL of
methylene chloride. Use a column flow rate of 2 to 2.5 mL/min throughout the wash and
elution profiles.
11.3 Add the extract from Section 10.8 to the head of the column. Allow the solvent to elute from
the column until the Florisil is almost exposed to the air. Elute the column with 50 mL of
methylene chloride. Discard this fraction.
11.4 Elute the column with 50 mL of 5% acetone in methylene chloride. Collect this fraction
in a K-D apparatus. Concentrate the column fraction to 1 mL as described in Sections 10.6
and 10.7.
11.5 Add 10 mL of acetonitrile to the concentrate along with one or two clean boiling chips.
Attach a three-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder
column with acetonitrile and concentrate the solvent extract to an apparent volume of 1 mL.
Allow the K-D apparatus to drain and cool for 10 minutes.
11.6 Transfer the liquid to a 2-mL volumetric flask and dilute to the mark with acetonitrile. Mix
thoroughly prior to analysis. If the extracts will not be analyzed immediately, they should be
transferred to PTFE-sealed screw-cap vials and refrigerated. Proceed with the liquid chroma-
tographic analysis.
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 Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may
be used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If an internal standard approach is being used, the analyst must not add the internal standard to
sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 2 to 5 juL of the sample extract by completely filling the sample valve loop. Record the
resulting peak sizes 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 variations of standards over the course of a day. Three
513
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Method 639
times the standard deviation of the retention time for a compound can be used to calculate a
suggested window size; 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.
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:
Equation 2
Concentration, \iglL =
where
A = Amount of material injected, in ng
Vt - Volume of extract injected, in jjL
Vt - Volume of total extract, in \tL
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:
Equation 3
Concentration, pg/L = —
where
As = Response for parameter to be measured
A^ = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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.
514
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Method 639
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls out-
side of the control limits in Section 8.3, data for the affected compounds 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.8 The MDL
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 10 x MDL to 1000 x MDL.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained. Seven replicates of each of two diffe-
rent wastewaters were spiked and analyzed. The standard deviation of the percent recovery is
also included in Table 2.1
515
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Method 639
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 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 Sup-
port Laboratory - Cincinnati, Ohio, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J. A. et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
516
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Method 639
Table 1 . Chromatographic Conditions and Method Detection Limits
Parameter Retention Time (min) Method Detection Limits
Column 1 Column 2
Bendiocarb 9.3 6.0 1.8
Column 1 conditions: Spherisorb-ODS, 5 //, 250 mm long by 4.6 mm ID; 1 mL/min flow; 40/60
acetonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5 //, 250 mm long by 4.6 mm ID; 1 mL/min flow; 50/50
acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision3
Parameter Average Percent Relative Standard Spike Level No. of Matrix
Recovery Deviation (%) fag/LJ Analyses Type*
Bendiocarb 65 35.6 8 7 1
70 5.7 80 7 1
(a) Column 1 conditions were used.
(b) 1 = Relevant industrial wastewater
517
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Method 639
Bendiocarb
0
I l I
2.0
I
4.0
1 I I I I I
6.0 8.0 10.0
n i i i i
12.0
14.0
16.0
Retention Time (minutes)
A52-002-22A
Figure 1. HPLC-UV Chromatogram of 10 ng of Bendiocarb (Column 1)
518
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Method 639
\L
• Bendiocarb
2.0
\
4.0
I\\ I I I I I I I I I
6.0
8.0
10.0
12.0
14.0
16.0
Retention Time (minutes)
52-002-03
Figure 2. HPLC-UV Chromatogram of 600 ng of Bendiocarb (Column 2)
519
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Method 640
The Determination of
Mercaptobenzothiazole in
Municipal and Industrial
Wastewaters
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Method 640
The Determination of Mercaptobenzothiazole in Municipal and
Industrial Wastewaters
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of mercaptobenzothiazole. The following parameter can
be determined by this method:
Parameter CAS No.
Mercaptobenzothiazole 149-30-4
1.2 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compound listed above in municipal and industrial 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 alternative test
procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 14) 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 in
certain other 600-series methods. Thus, a single sample may be extracted to measure the
compounds included in the scope of the methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as necessary, in order to apply ap-
propriate cleanup procedures.
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 using the proce-
dure 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 liquid chromatographic
column that can be used to confirm measurements made with the primary column.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride using
a separately funnel. The methylene chloride extract is dried and concentrated to 1.0 mL. Li-
quid chromatographic conditions are described which permit the separation and measurement
of the compounds in the extract by high-performance liquid chromatography with ultraviolet
detection.
2.2 This method provides a silica gel column cleanup procedure to aid in the elimination of inter-
ferences which may be encountered.
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Method 640
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 baseline in liquid
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 minutes. Do not
heat volumetric ware. Some thermally stable materials, such as PCBs, may 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 con-
taminants. 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 MDL listed in Table 2.
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 aware-
ness 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 identified3 5 for the information of the analyst.
5. APPARA TUS AND MA TERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume, fitted
with screw-caps lined with PTFE. Foil may be substituted for PTFE 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 col-
lection of a minimum of 250 mL. Sample containers must be kept refrigerated at 4°C
524
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Method 640
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, fol-
lowed by repeated rinsings with distilled water to minimize the potential for con-
tamination 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 PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at the
top and PTFE stopcock (Kontes K-420290 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. A
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 equi-
valent).
5.2.8 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
5.2.10 Graduated cylinder: 1000-mL.
5.2.11 Volumetric flask: 5-mL, 10-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat at 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, 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: Analytical system complete with liquid chromatograph suitable for on-
column injection and all required accessories including syringes, analytical columns, detectors,
and strip-chart recorder. A data system is recommended for measuring peak areas.
5.6.1 Column 1: Spherisorb-ODS, 5jt , 250 mm long by 4.6 mm ID or equivalent. This
column was used to develop the method performance statements in Section 14. Al-
ternative columns may be used in accordance with the provisions described in Sec-
tion 12.1.
5.6.2 Column 2: Lichrosorb RP-2, 5/x, 250 mm long by 4.6 mm ID or equivalent.
525
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Method 640
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, acetonitrile, ethyl ether, and acetone: Distilled-in-glass quality
or equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant test strips
(available from Scientific Products Co., Catalog No. PI 126-8 and other suppliers). Proce-
dures recommended for removal of peroxides are provided with the test strips.
6.3 Sodium sulfate: ACS, granular, anhydrous; heated in a muffle furnace at 400°C overnight.
6.4 Silica gel: Davison Grade 923, 100-200 mesh, dried for 12 hours at 150°C.
6.5 IN sodium hydroxide: Dissolve 4 g of sodium hydroxide in 100 mL of distilled water.
6.6 IN sulfuric acid: Slowly add 2.8 mL of concentrated H2SO4 (94%) to about 50 mL of
distilled water. Dilute to 100 mL with distilled water.
6.7 Sodium phosphate: monobasic, ACS grade.
6.8 Sodium phosphate: dibasic, ACS grade.
6.9 Stock standard solutions (1.00 /ig/piL): Stock standard solutions can be prepared from pure
standard materials or purchased as certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in distilled-in-glass quality methanol 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 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.
6.9.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
40°C and protect from light. Frequently check standard solutions for signs of degra-
dation or evaporation, especially just prior to preparing calibration standards from
them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. CALIBRATION
7.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
The liquid 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.
7.2.1 For each compound of interest, prepare calibration standards at a minimum of three
concentration levels by adding volumes of one or more stock standards to a volu-
metric flask and diluting to volume with methanol. One of the external standards
526
-------
Method 640
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sam-
ple concentrates or should define the working range of the detector.
7.2.2 Using injection of 5 to 20 /*L of each calibration standard, tabulate peak height or
area responses against the mass injected. The results can be used to prepare a calibra-
tion curve for each compound. Alternatively, the ratio of the response to the mass
injected, defined as the calibration factor (CF), can be calculated for each compound
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 compound 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 similar in analytical behavior to the compounds of interest. The ana-
lyst 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
compound of interest by adding volumes of one or more stock standards to a volu-
metric flask. To each calibration standard, add a known constant amount of one or
more internal standards, and dilute to volume with methanol. One of the standards
should be at a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the sam-
ple concentrates or should define the working range of the detector.
7.3.2 Using injections of 5 to 20 pL 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:
Equation 1
RF = _
where
As = Response for the parameter to be measured
A^ = Response for the internal standard
CK = Concentration of the internal standard, in \iglL
C = Concentration of the parameter to be measured, in
527
-------
Method 640
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 can be used for
calculations. Alternatively, the results can be used to plot a calibration curve of
response ratios, A^A^ 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 compound
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 inter-
ferences from there agents.
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 measure-
ments. 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 methanol, 1000
times more concentrated than the selected concentrations.
8.2.2 Using a pipette, 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 be-
fore R and S calculations are performed.
528
-------
Method 640
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 measured
in Section 8.2.3. If the data are not comparable, the analyst must 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 + 3s
Lower Control Limit (LCL) = R - 3s
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 de-
fined 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.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 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 compound does not fall
within the control limits for method performance, the results reported for that compound 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 though the analysis of a 1-L
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. 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.
523
-------
Method 640
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. Com-
posite 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 Adjust the pH of the sample to 6 to 8 with sodium hydroxide or sulfuric acid immediately after
sampling.
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-L separatory funnel. Check the pH of the sample
with wide range pH paper and adjust to 6 to 8 with IN sodium hydroxide or IN sulfuric acid.
Dissolve 5 g of monobasic sodium phosphate and 5 g of dibasic sodium phosphate in the
sample.
10.2 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 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, cen-
trifugation, 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 extrac-
tion 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 25-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 methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse the column with 30 to 40 mL
of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball Snyder
column. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top.
Place the K-D apparatus on a hot water bath, 60 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 minutes. At the proper rate of distillation,
530
-------
Method 640
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 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride. Add one or two clean boiling chips and attach a
two-ball micro-Snyder column to the concentrator tube. Prewet the micro-Snyder column with
methylene chloride and concentrate the solvent extract as before. When an apparent volume of
0.5 mL is reached, or the solution stops boiling, remove the K-D apparatus and allow it to
drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with methy-
lene chloride. Stopper the concentrator tube and store refrigerated if further processing will
not be performed immediately. If the extract is to be stored longer than 2 days, transfer the
extract to a screw-capped vial with a PTFE-lined cap. If the sample extract requires no
further cleanup, proceed with Section 10.9. If the sample requires cleanup, proceed to Sec-
tion 11.
10.9 Add one or two clean boiling chips to the concentrator tube along with 10-mL of methanol.
Attach a two-ball micro-Snyder column and prewet the micro-Snyder column with about 1 mL
of methanol. Concentrate the solvent extract as before to an apparent volume of 2 mL and
allow it to drain and cool for 10 minutes. Transfer the solvent extract to a 5 mL volumetric
flask and dilute to the mark with methanol. Proceed with the liquid chromatographic analysis
in Section 12.
10.10Determine the original sample volume by refilling the sample bottle to the mark and transfer-
ring the water to a 1000-mL graduated cylinder. Record the sample volume to the nearest
5mL.
11. CLEANUP AND SEPARA TION
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 clean waters
and industrial 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 is not less than 85 %.
11.2 The following silica gel column cleanup procedure has been demonstrated to be applicable to
mercaptobenzothiazole.
11.2.1 Add 10 g of silica gel to 100 mL of ethyl ether and 600 pL of reagent water in a
250-mL Erlenmeyer flask. Shake vigorously for 15 minutes. Transfer the slurry to a
chromatographic column (silica gel may be retained with a plug of glass wool). Allow
the solvent to elute from the column until the silica gel is almost exposed to the air.
Wash the column with 100 mL of methylene chloride. Use a column flow of 2 to
2.5 mL/min throughout the wash and elution profiles.
11.2.2 Quantitatively add the sample extract from Section 10.8 to the head of the column.
Allow the solvent to elute from the column until the silica gel is almost exposed to the
air. Elute the column with 50 mL of methylene chloride. Discard this fraction.
531
-------
Method 640
11.2.3 Elute the column with 50 mL of 6% acetone in methylene chloride and collect eluate
in a K-D apparatus. Concentrate this fraction to 1 mL as described in Sections 10.6
and 10.7. Exchange solvent with methanol as described in Section 10.9 and proceed
with liquid chromatographic analysis.
12. Liaum 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. Examples of the separations achieved by Column 1 and Column 2
are shown in Figures 1 and 2. Other columns, chromatographic conditions, or detectors may
be used if the requirements of Section 8.2 are met.
12.2 Calibrate the liquid chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal standard
to the sample extracts until immediately before injection into the instrument. Mix thoroughly.
12.4 Inject 5 to 20 /*L of the sample extract. Record the resulting peak sizes 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 variations of standards over the course of a day. Three
tunes the standard deviation of a retention time for a compound can be used to calculate a
suggested window size; 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.
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:
Equation 2
Concentration, iiglL =
(V,XV,)
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in pL
Vt = Volume of total extract, in ftL
V = Volume of water extracted, in mL
532
-------
Method 640
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:
Equation 3
(A WM
Concentration,
(Ais)(RF)(V0)
where
As = Response for parameter to be measured
A.a = Response for the internal standard
Is = Amount of internal standard added to each extract, in
V = Volume of water extracted, in L
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 out-
side of the control limits in Section 8.3, data for the affected compounds 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.8 The MDL
concentrations listed in Table 1 were obtained using reagent water.1 Similar results were
achieved using representative wastewaters.
14.2 This method has been tested for linearity of recovery from spiked reagent water and has been
demonstrated to be applicable over the concentration range from 5 to 1000 /*g/L.
14.3 In a single laboratory, Battelle Columbus Laboratories, using spiked wastewater samples, the
average recoveries presented in Table 2 were obtained after silica gel cleanup. Seven repli-
cates of each of two different wastewaters were spiked and analyzed. The standard deviation
of the percent recovery is also included in Table 2.1
533
-------
Method 640
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report 68-03-2956
(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, Phila-
delphia, Pennsylvania, 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, August 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, March 1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
8. Glaser, J.A., et al., "Trace Analysis for Wastewaters," Environmental Science and Tech-
nology, 15, 1426 (1981).
534
-------
Method 640
Table 1. Chromatographic Conditions and Method Detection Limits
Retention Time (min) MDL
Parameter Column 1 Column 2 (ug/L)
Mercaptobenzothiazole 8.4 9.5 1.7
Column 1 conditions: Spherisorb-ODS, 5//, 250mm long by 4.6 mm ID; 1 mL/min flow; 50/50
acetonitrile/water.
Column 2 conditions: Lichrosorb RP-2, 5/j, 250mm long by 4.6 mm ID; 1 mL/min flow; 10/90
acetonitrile/water.
Table 2. Single-Laboratory Accuracy and Precision3
Average Standard Number
Sample Background Spike Recovery Deviation of
Parameter Type" (ug/L)c (ug/L) (%) (%) Replicates
Mercaptobenzothiazole 1 ND 5 79 5 7
1 ND 10 87 4 7
(a) Column 1 conditions were used.
(b) 1 = Municipal sewage effluent
(c) ND = Not detected
535
-------
Method 640
Mercaptobenzothiazole
/
i i
0 1.5
i i i
i i i i
4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0
Retention Time (minutes)
A52-002-30A
Figure 1. HPLC-UV Chromatogram of 10 ng of Mercaptobenzothiazole (Column 1)
536
-------
Method 640
Mercaptobenzothiazole
/
I I I I IIIIIIIIIIIIT T 1
2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time (minutes)
A52-002-31A
Figure 2. HPLC-UV Chromatogram of 10 ng of Mercaptobenzothiazole (Column 2)
537
-------
-------
Method 641
The Determination of
Thiabendazole in Municipal and
Industrial Wastewater
-------
-------
Method 641
The Determination of Thiabendazole in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of thiabendazole in municipal and industrial wastewater.
Parameter CAS No.
Thiabendazole 148-79-8
1.2 The estimated detection limit (EDL) for thiabendazole is listed in Table 1. The EDL was cal-
culated from the minimum detectable response being equal to 5 times the background noise
using a 100-/iL injection. The EDL for a specific wastewater may be different depending on
the nature of interferences in the sample matrix.
1.3 This is a liquid chromatographic method applicable to the determination of thiabendazole in
municipal and industrial discharges. When this method is used to analyze unfamiliar samples
for thiabendazole, compound identification should be supported by at least one additional
qualitative technique.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of chromatograms. Each analyst
must demonstrate the ability to generate acceptable results with this method using the proce-
dure described in Sections 9.2 and 9.3.
2. SUMMARY OF METHOD
2.1 Thiabendazole is analyzed in the sample matrix after solubilization with acid and filtration to
remove particulate matter. Chromatographic conditions are described which permit the separa-
tion and accurate measurement of thiabendazole by direct aqueous injection and HPLC with
fluorescence detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of the chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 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 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
541
-------
Method 641
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Matrix interferences may be caused by fluorescing contaminants that coelute with thiaben-
dazole. 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. Matrix interferences caused by the presence of particulate matter are removed by
filtration. Unique samples may require additional cleanup approaches to achieve the detection
limit 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 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 aware-
ness 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 identified2"4 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sampling equipment for discrete sampling.
5.1.1 Vial: 25-mL capacity or larger, equipped with a screw-cap with hole in center (Pierce
13074 or equivalent). Detergent wash, rinse with tap and distilled water, and dry at
105°C before use.
5.1.2 Vial: 3.5-mL, equipped with a screw-cap with hole in center (Pierce 13019 or equi-
valent). Wash vial and cap as in Section 5.1.1.
5.1.3 Septum: PTFE-faced silicone (Pierce 12722 or equivalent). Detergent wash and dry
at 105°C for 1 hour before use.
5.1.4 Septum: PTFE-faced silicone (Pierce 12712 or equivalent). Detergent wash and dry
at 105° for 1 hour before use.
5.2 Syringe: Glass, 5-mL with Leur tip.
5.3 Syringe-filter holder: Stainless steel with Leur connection (Rainin 38 to 101 or equivalent).
5.4 Filters: 13 mm, Nylon 66, 0.45-/* pore (Rainin 38 to 112 or equivalent).
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 High-performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
542
-------
Method 641
5.6.1 Isocratic pumping system, constant flow.
5.6.2 Injector valve (Rheodyne 7125 or equivalent) with 100-/iL loop.
5.6.3 Column: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 10 p.
5.6.4 Fluorescence detector, for excitation at 300 nm and emission at 360 nm (Perkin Elmer
650 to IS or equivalent). Fluorometer should have dispersive optics for excitation
and utilize either filter or dispersive optics at the emission detector.
5.6.5 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not observed at
the EDL of each parameter of interest.
6.2 Sodium hydroxide solution (ION): Dissolve 40 grams of 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 H2SO4 (specific gravity 1.84) to 50 mL of
reagent water.
6.5 HPLC buffer (pH 8.2): Add 8 mL of triethanolamine (Eastman 1599) and 1 mL of glacial
acetic acid (ACS) to 1 L of reagent water.
6.6 High-purity methanol: HPLC quality, distilled in glass.
6.7 Stock standard solution (1.0 /*g//iL): Stock standard solutions are prepared from pure standard
material or purchased as a certified solution.
6.7.1 Prepare the stock standard solution by accurately weighing about O.OlOOg of pure
material. Dissolve the material in pesticide-quality methanol, dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the
analyst. When compound purity is certified at 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can be used at any concentration if they are certified
by the manufacturer or by an independent source.
6.7.2 Transfer the stock standard to a PTFE-sealed screw-cap bottle. Store at 4°C and
protect from light. The stock standard should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards.
6.7.3 The stock standard must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
543
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Method 641
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
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 analyzed within 48 hours of
collection, the sample should be adjusted to a pH range of 1.0 to 3.0 with sodium hydroxide
or sulfuric acid, and 35 mg of sodium thiosulfate per liter of sample for each part per million
of free chlorine should be added.
7.3 All samples must be analyzed within 30 days of collection.6
8. CALIBRA TION AND STANDARDIZA TION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of thiabendazole by
adding volumes of the stock standard to a volumetric flask and diluting to volume with HPLC
mobile phase. One of the standards should be at a concentration near, but greater than, the
EDL, 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.
8.3 Using injections of 100 juL of each calibration standard, tabulate peak height or area responses
against the mass injected. The results are used to prepare a calibration curve for thiaben-
dazole. Alternatively, if the ratio of response to amount injected (calibration factor) is a
constant over the working range (< 10% relative standard deviation), linearity of the calibra-
tion curve can be assumed and the average ratio or calibration factor can be used in place of a
calibration curve.
8.4 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 thiabendazole 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 factor must be prepared.
8.5 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.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank as described in Section 10 each time a set of sam-
ples is extracted. A laboratory reagent blank is an aliquot of reagent water. If the
reagent blank contains a reportable level of thiabendazole, immediately check the
entire analytical system to locate and correct for possible interferences and repeat the
test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
544
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Method 641
9.2.1.1 Laboratory control standard concentrate: From the stock standard pre-
pared as described in Section 6.7, prepare a laboratory control standard
concentrate that contains thiabendazole at a concentration of 2 ng/mL in
methanol or other suitable solvent.7
9.2.1.2 Laboratory control standard: using a pipette or microliter syringe, add
50.0 (jL of the laboratory control standard concentrate to a 10-mL aliquot
of reagent water contained in a 10-mL volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Cal-
culate the percent recovery (Pj) with the equation:
Equation 1
1005.
P. =
where
Sj = Analytical results from the laboratory control standard, in \iglL
T(. = Known concentration of the spike, in figlL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample vials for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of thia-
bendazole.
9.3.2 Calculate the relative range (RRj) with the equation:
Equation 2
where
Rf= Absolute difference between the duplicate measurements Xl and X^, in
X.= Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
545
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Method 641
10. PROCEDURE
10.1 Sample preparation.
10.1.1 Adjust the pH of the sample to pH 1 to 3 with sulfuric acid solution.
10.1.2 Assemble the syringe-filtration assembly by attaching the filter holder (with filter) to a
5-mL glass syringe equipped with a Leur tip.
10.1.3 Remove the barrel from the syringe and pour a 4- to 5-mL aliquot of the acidifield
sample into the syringe, allowing room for reinsertion of the syringe barrel.
10.1.4 Filter a portion of the sample through 0.45-/H filter using a syringe-filter holder. The
first few milliliters should be discarded. Collect the filtrate in a 4-mL vial equipped
with a PTFE-sealed screw-cap.
10.1.5 The syringe and filter holder should be rinsed with acetone or methanol and then
HPLC-grade water between samples.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. Use
of fluorescent detectors, however, often obviates the necessity for cleanup of relatively
clean sample matrices. If particular circumstances demand the use of an alternative
cleanup procedure, the analyst must determine the elution profile and demonstrate that
recovery is no less than 85%.
10.3 Liquid chromatographic analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromato-
graph. Included in this table are the estimated retention time and estimated detection
limit that can be achieved by this method. An example chromatogram achieved by
this column is shown in Figure 1. Figure 2 is a chromatogram of thiabendazole in a
POTW wastewater sample. Other columns, chromatographic conditions, or detectors
may be used if data quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.4 Inject 100 jiL of the filtered aqueous sample. Monitor the column eluent at excitation wave-
length 300 nm (5-nm slit width) and emission wavelength 360 nm (10-nm slit width). Record
the resulting peak size in area or peak height units.
10.5 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 stan-
dard deviation of a retention time for a compound can be used to calculate a suggested window
size; however, the experience of the analyst should weigh heavily in the interpretation of chro-
matograms.
10.6 If the response for the peak exceeds the working range of the system, dilute the sample with
mobile phase and reanalyze.
10.7 If the measurement of the peak response is prevented by the presence of interferences, further
cleanup is required.
546
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Method 641
11. CALCULATIONS
11.1 Determine the concentration of thiabendazole in the sample.
11.1.1 Calculate the amount of thiabendazole injected from the peak response using the
calibration curve or calibration factor in Section 8.2.2. The concentration in the
sample can be calculated from the following equation:
Equation 3
Concentration, fj.g/L =
*
(V,)
where
A = Amount of the thiabendazole injected, in ng
Vt = Volume of sample injected, in \>L
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.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for thiabendazole are listed in Table I.8
The EDL is defined as the minimum response being equal to 5 times the background noise,
using a 100-jiL injection. -•**'
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 using a spiked POTW sample. The results of these studies are presented in
Table 2.
547
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Method 641
References
1. ASTM Annual Book of Standards, Part 31, "Standard Practice for Preparation of Sample
Containers and for Preservation," American Society for Testing and Materials, Philadelphia,
Pennsylvania, p. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Vol. 11.01, D3370, "Standard Practice for Sampling
Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report 68-03-2897, unpublished
report available from U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio.
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, March 1979.
8. "Evaluation of Ten Pesticides Methods," U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio.
548
-------
Method 641
Table 1. Liquid Chromatography of Thiabendazole*
Retention Time Estimated Detection
Compound (min) Limit (ug/L)
Thiabendazole 4.3 1.7
*HPLC conditions: 10/y reverse-phase Ultrasphere ODS; Column, 250 mm long by 4.6 mm ID;
isocratic 70% methanol/30% buffer; flow rate 1 mL/min.
Table 2. Single-Operator Accuracy and Precision*
Average
Spike Con- Number of Percent Standard
Parameter centration (ug/L) Replicates Recovery Deviation (%)
Thiabendazole 12.5 7 100 9.5
625 7 92.8 4.5
*POTW effluent was used in this study.
549
-------
Method 641
Thiabendazole
I I I I I
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
AS2-002-67A
Figure 1. HPLC of Thiabendazole
550
-------
Method 641
I—I—I—I—I—I—I 1—I—I I
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
A52-O02-68A
Figure 2. Chromatogram of Thiabendazole in Wastewater Sample
551
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-------
Method 642
The Determination of Biphenyl
and Ortho-Phenylphenol in
Municipal and Industrial
Wastewater
-------
-------
Method 642
The Determination of Biphenyl and Ortho-Phenylphenol in Municipal
and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of biphenyl and o-phenylphenol in municipal and
industrial waste water.
Parameter CAS No.
Biphenyl 92-52-4
o-Phenylphenol 90-43-7
1.2 The estimated detection limits (EDL) for the parameters above are listed in Table 1. The
EDLs were calculated from the minimum detectable response being equal to 5 times the
background noise using a 2-mL final extract volume of a 1-L sample and an injection volume
of 50 juL. The EDL for a specific wastewater may be different depending on the nature of
interferences in the sample matrix.
1.3 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of the compounds listed above in municipal and industrial discharges. When this
method is used to analyze unfamiliar samples for any or all of the compounds above, com-
pound identification should be supported by at least one additional qualitative technique. This
method describes analytical conditions for a second HPLC column that can be used to confirm
measurements made with the primary column.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 The fungicides are removed from the sample matrix by extraction with methylene chloride.
The extract is dried, exchanged to acetonitrile or methanol, and analyzed by liquid chroma-
tography with ultraviolet (UV) detection.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analy-
sis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 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
555
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Method 642
should then be drained dry and heated in a muffle furnace at 400°C for 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that are coextracted from the
samples. 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 sam-
pled. While general cleanup procedures are provided as part of this method, unique samples
may require additional cleanup approaches to achieve the detection limits 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 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 aware-
ness 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 identified2^ for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to
air dry, then muffle the bottles at 400°C for 1 hour. After cooling, rinse the bottles and cap
liners with hexane, seal the bottles, and store in a dust-free environment.
5.1.1 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 reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent) and two-ball micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
556
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Method 642
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to concen-
trator tube with springs.
5.3 High-performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Gradient pumping system, constant flow.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 50-/*L loop.
5.3.3 Column 1: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Perkin Elmer HC-ODS Sil-X 10 n, or equivalent.
5.3.4 Column 2: 250 mm long by 4.6 mm ID, packed with reverse-phase Dupont Zorbax
ODS, 6 to 7 /a, or equivalent.
5.3.5 Ultraviolet detector, capable of monitoring at 254 run.
5.3.6 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-
fritted bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnels: 2-L, 500-mL, and 250-mL, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride for 2 hours.
5.6.4 Water bath: Heated with concentric ring cover, capable of temperature control
(±2°C). The bath should be used in a hood.
5.6.5 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, acetonitrile, methanol, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferant is not
observed at the method detection limit of each parameter of interest. The water is
held at 90°C. Store in clean, narrow-mouth bottles with PTFE-lined septa and screw-
caps.
6.1.3 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
557
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Method 642
6.1.4 HPLC Mobile Phase 1: Add 400 mL of acetonitrile to a 1-L volumetric flask and
dilute to volume with reagent water.
6.1.5 HPLC Mobile Phase 2: Add 500 mL of methanol to a 1-L volumetric flask and dilute
to volume with reagent water.
6.2 Standard stock solutions (1.00 fig//xL): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in pesticide-quality methanol or acetonitrile, dilute to
volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience
of the analyst. When compound purity is certified at 96% or greater, the weight can
be used without correction to calculate the concentration of the stock standard. Com-
mercially prepared stock standards can be used at any concentration if they are cer-
tified by the manufacturer or by an independent source.
6.2.2 Transfer the stock standards to PTFE-sealed screw-cap bottles. Store at 4°C and
protect from light. Stock standards should be checked frequently for signs of degra-
dation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
7.3 All samples must be extracted and analyzed as soon as possible after sampling, since preser-
vation studies6 have shown that these compounds undergo almost complete decomposition
within 7 days.
8. CALIBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of the analytes by
adding volumes of the stock standard to a volumetric flask and diluting to volume with HPLC
mobile phase (40% acetonitrile in water or 50% methanol in water). One of the standards
should be at a concentration near, but greater than, the EDL, 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.
8.3 Using injections of 50 pL of each calibration standard, tabulate peak height or area responses
against the mass injected. The results are used to prepare a calibration curve for the analytes.
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over
the working range (< 10% relative standard deviation), linearity of the calibration curve can
555
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Method 642
be assumed and the average ratio or calibration factor can be used in place of a calibration
curve.
8.4 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 analyte varies
from the predicted response by more than ±10%, the test must be repeated using a fresh cali-
bration standard. Alternatively, a new calibration curve or factor must be prepared.
8.5 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.
9. QUALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is an aliquot of reagent water. If the reagent blank
contains a reportable level of the analytes, immediately check the entire analytical system to
locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.2, prepare a laboratory control standard con-
centrate that contains the analytes at a concentration of 2 fig/mL in metha-
nol or acetonitrile.
9.2.1.2 Laboratory control standard: Using a pipette, add 1.0 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water con-
tained in a 1000-mL volumetric flask.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Cal-
culate the percent recovery (Pj) with the equation:
P, =
where
5 = Analytical results from the laboratory control standard, in
Tt = Known concentration of the spike, in pg/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.7
559
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Method 642
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section. 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of the
analytes.
9.3.2 Calculate the relative range7 (RR,) with the equation:
Equation 2
RR =
*,
where
Rt = Absolute difference between the duplicate measurements X} and X2, in
X(. = Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sample
by shaking the funnel for 2 minutes with periodic venting to release vapor pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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 on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and com-
plete the extraction procedure a second time, combining the extracts in the Erlenmeyer
flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, collecting the
extract in a 500-mL K-D flask equipped with a 10-mL concentrator tube. Rinse the
Erlenmeyer flask and column with about 30 mL of methylene chloride to complete the
transfer.
560
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Method 642
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of 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 in steam. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the
proper rate of distillation, the balls of the column will actively chatter but the cham-
bers will not flood. When the apparent volume of liquid reaches 5 mL, remove the
K-D apparatus and allow it to drain and cool for at least 10 minutes. If the extract
requires cleanup, proceed to Section 10.2. If the extract does not require cleanup,
proceed with Sections 10.1.6 and 10.1.7.
10.1.6 Add 50 mL of methanol or acetonitrile and a clean boiling chip to the flask and repeat
the concentration as described above. When the apparent volume of the liquid reaches
1 mL, remove the K-D apparatus and allow it to drain and cool for at least 10 min-
utes. Remove the Snyder column and rinse the flask and its lower joint into the con-
centrator tube with 1 to 2 mL of methanol or acetonitrile. A 5-mL syringe is recom-
mended for this operation.
10.1.7 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of methanol or
acetonitrile to the top. Place the micro K-D apparatus on a hot water bath (80 to
85°C) so that the concentrator tube is partially immersed in the hot water. Adjust the
vertical position of the apparatus and water temperature as required to complete the
concentration in 5 to 10 minutes. At the proper rate of distillation, the balls will
actively chatter but the chambers will not flood. When the apparent volume of liquid
reaches 0.5 mL, remove the K-D apparatus and allow it to drain and cool for at least
10 minutes. Remove the micro-Snyder column and rinse its lower joint into the
concentrator tube with a small volume of methanol or acetonitrile. Adjust the volume
to 1.0 mL with methanol or acetonitrile. Add 1.0 mL of reagent water to the extract
if methanol or 1.5 mL of reagent water to the extract if acetonitrile (Table 1).
NOTE: At high concentrations (approximately 1,000 mg/L or greater) ofbiphenyl in
the extract, low recoveries may be obtained due to insolubility in the acetonitrile.
Larger volumes of acetonitrile or acetone may be required to dissolve all the biphenyl
and to prevent precipitation.
10.1.8 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.
10.2 Cleanup and separation.
10.2.1 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 is no less than 85 %.
561
-------
Method 642
10.2.2 Prior to HPLC analysis, the composition of the extracts must be as specified under
chromatographic conditions in Table 1 and described in Sections 10.1.6 and 10.1.7.
10.2.3 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromato-
graph. Included in this table are the estimated retention times and estimated detection
limits that can be achieved by this method. An example of the separation achieved by
Column 1 of the analytes in a POTW extract is shown in Figure 1. Other columns,
chromatographic conditions,8 or detectors may be used if data quality comparable to
Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 50 /xL of the sample extract. Monitor the column eluent at 254 nm. Record
the resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon meas-
urements 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 can be used to
calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the
sample with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
cleanup is required.
77. CALCULATIONS
11.1 Determine the concentration of analytes in the sample.
11.1.1 Calculate the amount of analytes injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
Equation 3
Concentration, pg/L =
where:
A = Amount of analytes injected, in ng
V. = Volume of extract injected, in
Vt = Volume of total extract, in
Vs = Volume of water extracted, in mL
562
-------
Method 642
12. METHOD PERFORMANCE
12.1 The EDLs and associated chromatographic conditions for the analytes are listed in Table 1.
The EDL is defined as the minimum response being equal to 5 times the background noise,
assuming a 2-mL final extract volume of a 1-L sample and an HPLC injection volume of
50 fiL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 in the designated matrices. The results of these studies are presented in
Table 2.
563
-------
Method 642
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, p. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report No. 68-03-2897. Un-
published report available from U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
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, March 1979.
8. Beernaert, H., "Determination of Biphenyl and Ortho-Phenylphenol in Citrus Fruits by Gas
Chromatography," Journal of Chromatography, 77: 331-8, 1973.
9. "Evaluation of Ten Pesticide Methods" U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio.
564
-------
Method 642
Table 1. Chromatographic Conditions and Estimated Detection Limits
Parameter
o-Phenylphenol
Biphenyl
Retention Time (mini
Column 1
Column 2
7.7 11.3
18.8 16.5
Estimated
Detection
Limit
.01
.04
Column 1: Reverse-phase column, 4.6 mm ID by 250 mm long; 10/y, Perkin-Elmer HC-ODS Sil-X
or equivalent; isocratic elution for 5 minutes using 40% acetonitrile in water, then linear gradient
elution to 100% acetonitrile over 25 minutes; flow rate of 0.5 mL/min.
Column 2: Reverse-phase column, 4.6 mm ID by 250 mm long; 6 to 7 p, Pupont Zorbax ODS or
equivalent; isocratic elution for 3 minutes using 50% methanol in water, then linear gradient to
80-percent methanol over 10 minutes; flow rate 1.0 mL/min.
Table 2. Single-Operator Accuracy and Precision'
Parameter
o-Phenylphenol
Biphenyl
Spike Range
fpg/U
2.5
6,500
2.4
6,300
Number of Average Percent
Replicates Recovery* *
7
7
7
7
102.3
94.1
86.3
100.7
Standard
Deviation
(%)
36.3
6.3
16.2
9.9
* POTW effluent was used in this study.
** No cleanup was employed in validation studies.
565
-------
Method 642
I \ i i i i i in i i T i r i r^ i r it
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention Time (minutes)
A52-002-69A
Figure 1. Liquid Chromatogram of Wastewater Extract Fortified With
o-Phenylphenol and Biphenyl (Column 1)
566
-------
Method 643
The Determination of Bentazon
in Municipal and Industrial
Wastewater
-------
-------
Method 643
The Determination of Bentazon in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of bentazon in municipal and industrial wastewater.
Parameter CAS No.
Bentazon (Basagran) 25057-89-0
1.2 The estimated detection limit (EDL) for bentazon is listed in Table 1. The EDL was calcu-
lated from the minimum detectable response being equal to 5 times the background noise using
a 5-mL final extract volume of a 1-L sample and an injection volume of 100 /xL. The EDL
for a specific wastewater may be different depending on the nature of interferences in the
sample matrix.
1.3 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of bentazon in municipal and industrial discharges. When this method is used to
analyze unfamiliar samples for bentazon, compound identification should be supported by at
least one additional qualitative technique. This method describes analytical conditions for a
second HPLC column that can be used to confirm measurements made with the primary
column.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 Bentazon is removed from an acidified sample matrix by extraction with methylene chloride.
The extract is discarded after back extraction with aqueous base. HPLC conditions are de-
scribed which permit the separation and measurement of bentazon in the aqueous extract.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the analy-
sis by running laboratory reagent blanks as described in Section 9.1
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 Clean all glassware as soon as possible
after use by rinsing with the last solvent used in it. This should be followed by
569
-------
detergent washing with hot walei and rinses with tap water and reagent water It
should then be drained diy and healed in a inutile furnace at 400"C lor 15 to 30
minutes Solvent rinses with acetone and pesticide-quality hexane may be substituted
r >r the healing Volumetric ware should not be heated in a muffle furnace. Alter
u. 'ing and cooling, glasswaie should be sealed and stored in a clean environment to
prevent any accumulation ol dust or other contaminants. Store the glassware inverted
or capped witli aluminum foil
3.2 Matrix interferences may be caused by UV active contaminants that are coextracted from the
samples. The extent of matrix inteiteieiices will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality being
sampled. Unique samples may require additional cleanup approaches to achieve the detection
limit listed in Table I.
4. SAFETY
4.1 The toxicity or carcmogemcity 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 aware-
ness 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-1' for the information of the analyst.
5. APPARATUS AND EQUIPS .
5.1 Sample containers: Narro\v-mouth glass bottles, I-Lor I -quart volume, equipped with poly-
tetrafluoroethylene (PTI;E)-lined screw-caps. Wide-mouth glass bottles, l-quart volume,
equipped with PTFH-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to
air dry, then muffle the bottles at 4()()°C for I hour. After cooling, rinse the bottles and cap
liners with hexane, seal the bottles, and store in a dust-free environment.
5.1.1 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 reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 High-performance liquid chromatography (HPLC) apparatus: Analytical system complete with
liquid chromatograph and all required accessories including syringes, analytical columns, and
mobile phases. The system must be compatible with the specified detectors and strip-chart
recorder. A data system is recommended for measuring peak areas.
570
-------
Method 643
5.2.1 Gradient pumping system, constant flow
5.2.2 Injector valve (Khcodyne 7125 or equivalent) with 100-^L loop.
5.2.3 Column I • 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ullrasphcre ODS, 10 /x, or equivalent
5.2.4 Column 2: 300 mm long hy 4.0 mm ID, packed with reverse phase /x Bondapak C18,
!()//, (Waters Associates), or equivalent.
5.2.5 Ultraviolet detector, variable wavelength, capable of monitoring at 340 run.
5.2.6 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.3 Miscellaneous.
5.3.1 Balance' analytical, capable of accurately weighing to the nearest 0.0001 g.
5.3.2 Separatory funnels: 2-L, and 250-mL, equipped with PTFE stopcocks.
5.3.3 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.3.4 Pasteur pipettes with bulbs.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, methanol, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 Reagent water: Reagent water is defined as a water in which an interferant is not
observed at the method detection limit of each parameter of interest.
6.1.3 Sodium hydroxide solution (0.IN): Dissolve 0.4 g of NaOH in reagent water and
dilute to 100 mL.
6.1.4 Sodium chloride: ACS, crystals.
6.1.5 Sodium thiosulfate: ACS, granular.
6.1.6 Sulfuric acid solution (I +1): Slowly add 50 mL of H2SO4 (specific gravity 1.84) to
50 mL of reagent water.
6.1.7 Sodium hydroxide solution (6N): Dissolve 24 g of NaOH in reagent water and dilute
to 100 mL.
6.1.8 Acetate buffer solution: Dissolve 0.41 g of anhydrous sodium acetate (ACS) and
1.5 mL of glacial acetic acid (ACS) in 100 mL of reagent water.
6.1.9 Glacial acetic acid: ACS.
6.1.10 HPLC mobile phase buffer (pH 4.7, 0.062 M acetate): Dissolve 0.87 g of anhydrous
sodium acetate (ACS) and 3.0 mL of glacial acetic acid (ACS) in 1 L of reagent
water.
6.2 Standard stock solution (1.00 /xg//xL): This solution may be purchased as a certified solution
or prepared from a pure standard material using the following procedures.
577
-------
Method 643
6.2.1 Prepare the stock standard solution by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality methanol, dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the
analyst. When compound purity is certified at 96% or greater, the weight can be
used without correction to calculate the concentration of the stock standard. Commer-
cially prepared stock standards can be used at any concentration if they are certified
by the manufacturer or by an independent source.
6.2.2 Transfer the stock standards to a PTFE-sealed screw-cap bottles. Store at 4°C and
protect from light. Stock standards should be checked frequently for signs of degra-
dation or evaporation, especially just prior to preparing calibration standards from
them.
6.2.3 Stock standards must be replaced after 6 months, or when comparison with quality
control check samples indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
If the samples will not be extracted within 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 and add 35 mg of
sodium thiosulfate per liter of sample for each part per million of free chlorine.
7.3 All samples must be extracted within 7 days and completely analyzed within 30 days of
extraction.6
8. CALIBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of bentazon
by adding volumes of the stock standard to a volumetric flask and diluting to volume with
HPLC mobile phase (35% methanol in HPLC mobile phase buffer or 40% methanol in HPLC
mobile phase buffer). One of the standards should be at a concentration near, but greater
than, the EDL, 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.
8.3 Using injections of 100 /xL of each calibration standard, tabulate peak height or area response
against the mass injected. The results are used to prepare a calibration curve for the analytes.
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over
the working range (< 10% relative standard deviation, RSD), linearity of the calibration curve
can be assumed and the average ratio or calibration factor can be used in place of a calibration
curve.
8.4 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 bentazon varies
572
-------
Method 643
from the predicted response by more than ± 10%, the test must be repeated using a fresh
calibration standard. Alternatively, a new calibration curve or factor must be prepared.
8.5 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.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A labora-
tory reagent blank is an aliquot of reagent water. If the reagent blank contains a
reportable level of bentazon, immediately check the entire analytical system to locate
and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.2, prepare a laboratory control standard con-
centrate that contains bentazon at a concentration of 2 pg/nL in methanol.
9.2.1.2 Laboratory control standard: Using a pipette or microliter syringe, add
50.0 jtL of the laboratory control standard concentrate to a 1-L aliquot of
reagent water.7
9.2.1.3 Analyze the laboratory control standard as described in Section 10. Cal-
culate the percent recovery (Pj) with the equation:
Equation 1
100 S,
p =
where
S. = Analytical results from the laboratory control standard, in
Ti = Known concentration of the spike, in \nglL
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.7
573
-------
Method 643
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both samples for at least 10% of all samples. To the extent
->ractical, the samples for duplication should contain reportable levels of bentazon.
9.3.2 calculate the relative range7 (RR,) with the equation:
Equation 2
IOOR.
RR =
X
where
/?. = Absolute difference between the duplicate measurements Xl and X2, in
Xt = Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water mer.,^ , on the side of the sample bottle for later determination of
sample volume. Po- tf , entire sample into a 2-L separatory funnel. Check the pH
of the sample wit i wide-range pH paper and adjust to within the range of 2.5 to 3.5
with sulfuric acid. Add 200 g of sodium chloride and mix to dissolve.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sample
by shaking the funnel for 2 minutes with periodic venting to release vapor pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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 on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL separatory funnel.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and
complete the extraction procedure a second time, combining the extracts in the
250-mL separatory funnel.
10.1.4 Perform a third extraction in the same manner. Add 2 mL of 0.1 M NaOH in reagent
water to the 250-mL separatory funnel, and extract by shaking the funnel for 2 min-
utes with periodic venting to release vapor pressure. Allow the organic layer to
separate from the water phase for a minimum of 10 minutes. Drain the methylene
574
-------
Method 643
chloride into a 250-mL Erlenmeyer flask. Transfer the aqueous layer with a Pasteur
pipette to a 5-mL volumetric flask.
10.1.5 Add the methylene chloride back to the 250-mL separatory funnel, and extract with an
additional 2 mL of 0.1M NaOH. Combine the extracts in the 5-mL volumetric flask.
10.1.6 Add two drops of glacial acetic acid to the volumetric flask, and dilute to volume with
acetate buffer solution (Section 6.1.7).
10.1.7 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.
10.2 Cleanup and separation.
10.2.1 The cleanup procedure recommended in this method involves the back extraction of a
methylene chloride extract with aqueous base, and has been used for the analysis of
various clean waters and industrial effluents. If additional cleanup is required, a 1-L
sample is adjusted to pH 12 with 6N NaOH and extracted with three 60-mL aliquots
of methylene chloride in a 2-L separatory funnel. The methylene chloride extracts are
discarded and the aqueous sample adjusted to pH range of 2.5 to 3.5 with 1:1 sulfuric
acid solution for re-extraction as in Section 10.1.1. If additional cleanup is required,
or if particular circumstances demand the use of an alternative cleanup procedure, the
analyst must determine the elution profile and demonstrate that the recovery for each
compound of interest is no less than 85 %.
10.3 Liqur jhromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromato-
graph. Included in this table are the estimated retention times and estimated detection
limit that can be achieved by this method. An example of the separation achieved by
Column 2 is shown in Figure 1. Other columns, chromatographic conditions, or
detectors may be used if data quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 ^L of the sample extract. Monitor the column eluent at 340 nm. Record
the resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon meas-
urements 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 can be used to
calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the sam-
ple with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
additional cleanup is required.
7 7. CALCULA TIONS
11.1 Determine the concentration of bentazon in the sample.
575
-------
Method 643
11.1.1 Calculate the amount of bentazon injected from the peak response using the calibration
curve. The concentration in the sample can be calculated from the equation:
Equation 3
Concentration, uglL =
where
A = Amount of material injected, in ng
V1 = Volume of extract injected, in yL
V, = Volume of total extract, in \>L
Vs = Volume of water extracted, in mL
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.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for bentazon are listed in Table I.8 The
EDL is defined as the minimum response being equal to 5 times the background noise, as-
suming a 5-mL final extract volume of a 1-L sample and an HPLC injection volume of
100 nL.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 in the designated matrices. The results of these studies are presented in
Table 2.
576
-------
Method 643
References
1. ASTM Annual Book of Standards, Vol. 11.02, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, 1986.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Vol. 11.01, D3370, "Standard Practice for Sampling
Water," American Society for Testing and Materials, Philadelphia, Pennsylvania, 1986.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report No. 68-03-2897 (in
preparation). Unpublished report available from U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
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. "Evaluation of Ten Pesticide Methods," U.S. Environmental Protection Agency Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio.
577
-------
Method 643
Table 1. Liquid Chromatography of Bentazon
Retention Time (min) Estimated Detection Limit
Pan meter Column 1 j Column 2 (ug/L)
Ben-zon 7.9 4.3 1.1
HPLC Column 1: Reverse-phase column, 250 mm long by 4.6 mm ID, stainless steel, packed with
10 fj Ultrasphere ODS or equivalent. Isocratic elution with 35% methanol/65% buffer; flow rate
2.0 mL/min.
HPLC Column 2: Reverse-phase column, 300 mm long by 4 mm ID, stainless steel, packed with a
// Bondapak C18, 10//, Waters Associates or equivalent. Linear gradient elution of 40% metha-
nol/60% buffer to 52% methanol/48% buffer over 9 minutes; flow rate 1 mL/min.
Table 2. Single-Laboratory Accuracy and Precision
Average
Matrix No. of Percent Standard
Parameter Type* Range (ug/L) Replicates Recovery Deviation (%)
Bentazon 1 125 7 85.1 4.8
2 20,400 7 88.4 8.4
1 = 50% industrial efflue.n * F0% POTW effluent
2 = 100% industrial effl'=vit
578
-------
Method 643
Bentazon
I I I I I I \
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
A52-002-70A
Figure 1. HPLC of Bentazon (Column 2)
579
-------
-------
Method 644
The Determination of Picloram
in Municipal and Industrial
Wastewater
-------
-------
Method 644
The Determination of Plcloram in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of picloram in municipal and industrial wastewater.
Parameter CAS No.
Picloram 1918-02-1
1.2 The estimated detection limit (EDL) for picloram is listed in Table 1.. The EDL was cal-
culated from the minimum detectable response being equal to 5 times the background noise
using a 100-/xL injection. The EDL for a specific wastewater may be different depending on
the nature of interferences in the sample matrix.
1.3 This is a high-performance liquid chromatographic (HPLC) method applicable to the deter-
mination of picloram in municipal and industrial discharges. When this method is used to
analyze unfamiliar samples for picloram, compound identification should be supported by at
least one additional qualitative technique.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of liquid chromatographs and in the interpretation of liquid chromatograms.
2. SUMMARY OF METHOD
2.1 Picloram is removed from the acidified sample matrix by extraction with methylene chloride.
The extract is dried, exchanged to HPLC mobile phase, and analyzed by HPLC with ultra-
violet (UV) detection. An alkaline back-extraction is used as necessary to eliminate interferen-
ces which may be encountered.
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of liquid chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.1 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 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the heating. Volumetric ware should not be heated in a muffle furnace. After
583
-------
Method 644
drying and cooling, glassware should be sealed and stored in a clean environment to
prevent any accumulation of dust or other contaminants. Store the glassware inverted
or capped with aluminum foil.
3.2 Matrix interferences may be caused by UV-active contaminants that coelute with picloram.
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. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limit 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 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 aware-
ness 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 identified2"4 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and reagent water. Allow the bottles and cap liners to
air dry, then muffle at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with
hexane, seal the bottles, and store in a dust-free environment.
5.1.1 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 reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to con-
centrator tube with springs.
584
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Method 644
5.3 High-performance liquid chromatography (HPLC) apparatus: Analytical system complete
with liquid chromatograph and all required accessories including syringes, analytical columns,
and mobile phases. The system must be compatible with the specified detector and strip-chart
recorder. A data system is recommended for measuring peak areas.
5.3.1 Isocratic pumping system, constant flow.
5.3.2 Injector valve (Rheodyne 7125 or equivalent) with 100-/*L loop.
5.3.3 Column: 250 mm long by 4.6 mm ID, stainless steel, packed with reverse-phase
Ultrasphere ODS, 10 M.
5.3.4 Ultraviolet detector, variable wavelength, capable of monitoring at 225 nm.
5.3.5 Strip-chart recorder compatible with detector, 250 mm. (A data system for measuring
peak areas is recommended.)
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-
fritted bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g,
5.6.2 Separatory funnels: 2-L, 500-mL, and 250-mL, equipped with PTFE stopcocks.
5.6.3 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride for 2 hours.
5.6.4 Water bath: Heated with concentric ring cover, capable of temperature control
(±2°C). The bath should be used in a hood.
5.6.5 Pasteur pipettes and bulbs.
5.6.6 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, methanol, and methylene chloride: Demonstrated to be free of
analytes and interferences.
6.1.2 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.
6.1.3 Sodium hydroxide (NaOH) solution (0.3N): Dissolve 12 g NaOH in reagent water
and dilute to 1000 mL.
6.1.4 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.5 Sodium chloride: ACS, crystals.
6.1.6 Sulfuric acid (H2SO4) solution (1 +1): Add a measured volume of concentrated H2SO4
to an equal volume of reagent water.
585
-------
Method 644
6.1.7 HPLC buffer (pH 2, 0.1M phosphate): Dissolve 5.83 g of KH2P04 (ACS) and
3.9 mL of 85% phosphoric acid (ACS) in 1 L of reagent water.
6.1.8 HPLC mobile phase: Add 570 mL of HPLC buffer solution to a 1-L volumetric flask
and dilute to volume with methanol.
6.2 Standard stock solutions (1.00 /^g/^L): These solutions may be purchased as a certified
solution or prepared from the pure standard material using the following procedures.
6.2.1 Prepare the stock standard solution by accurately weighing about 0.0100 g of pure
material. Dissolve the material in pesticide-quality methanol and dilute to volume in a
10-mL volumetric flask. Larger volumes can be used at the convenience of the ana-
lyst. When 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.
6.2.2 Transfer the stock standard to a PTFE-sealed screw-cap bottle. Store at 4°C and
protect from light. The stock standard should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards
from it.
6.2.3 The stock standard must be replaced after 6 months, or when comparison with a
quality control check sample indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until analysis.
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 analyzed within 48 hours of
collection, the sample should be adjusted to a pH range of 1.0 to 3.0 with sodium hydroxide
or sulfuric acid.
7.3 All samples must be extracted within 7 days of collection and analyzed within 30 days of
extraction.6
8. CALIBRATION
8.1 Establish liquid chromatographic operating parameters equivalent to those indicated in Table 1.
8.2 Prepare calibration standards at a minimum of three concentration levels of picloram by adding
volumes of the stock standard to a volumetric flask and diluting to volume with HPLC mobile
phase. One of the standards should be at a concentration near, but greater than, the EDL, 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.
8.3 Using injections of 100 /xL of each calibration standard, tabulate peak height or area response
against the mass injected. The results are used to prepare a calibration curve for picloram.
586
-------
Method 644
Alternatively, if the ratio of response to amount injected (calibration factor) is a constant over
the working range (< 10% relative standard deviation), linearity of the calibration curve can
be assumed and the average ratio or calibration factor can be used in place of a calibration
curve.
8.4 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 picloram varies
from the predicted response by ±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve or factor must be prepared.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1 .1 Analyze a laboratory reagent blank each time a set of samples is extracted. A labora-
tory reagent blank is an aliquot of reagent water. If the reagent blank contains a
reportable level of picloram, immediately check the entire analytical system to locate
and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1 .1 Laboratory control standard concentrate: From the stock standard pre-
pared as described in Section 6.3, prepare a laboratory control standard
concentrate that contains picloram at a concentration of 2 /ig/mL in metha-
nol or other suitable solvent.7
9.2.1 .2 Laboratory control standard: Using a pipette add 1.0 mL of the laboratory
control standard concentrate to a 1-L aliquot of reagent water.
9.2.1 .3 Analyze the laboratory control standard as described in Section 10. Cal-
culate the percent recovery (P) with the equation:
Equation 1
1005.
P = _ 1
' T,
where
Sf = Analytical results from the laboratory control standard, in fj.g/L
T{ = Known concentration of the spike, in pg/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.7
587
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Method 644
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of pic-
loram.
9.3.2 Calculate the relative range7 (RRj) with the equation:
Equation 2
RR= i
where
Rf= Absolute difference between the duplicate measurements X, and X2, in
IX + X,
1 .
2 .
, in (Jig/L
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 1.5 to 2.5
with sulfuric acid. Add 200 g of sodium chloride and mix to dissolve.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sam-
ple by shaking the funnel for 2 minutes with periodic venting to release vapor pres-
sure. 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 com-
plete the phase separation. The optimum technique depends on the sample, but may
include stirring, filtration of the emulsion through glass wool, or centrifugation.
Collect the extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and
complete the extraction procedure a second time, combining the extracts in the Erlen-
meyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate. If the extract
requires cleanup, collect the extract in a 500-mL separatory runnel and proceed to
Section 10.2 (cleanup and separation). If the extract does not require cleanup, collect
588
-------
Method 644
the extract in a 500-mL K-D flask equipped with a 10-mL concentrator tube and
proceed with Sections 10.1.5 and 10.1.6.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of 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 in steam. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the
proper rate of distillation, the balls of the column will actively chatter but the cham-
bers will not flood. When the apparent volume of liquid reaches 5 mL, remove the
K-D apparatus and allow it to drain and cool for at least 10 minutes. Add 50 mL of
methanol and a clean boiling chip to the flask and repeat the concentration as de-
scribed above. When the apparent volume of the liquid reaches 1 mL, remove the
K-D apparatus and allow it to drain and cool for at least 10 minutes. Remove the
Snyder column and rinse the flask and its lower joint into the concentrator tube with
1 to 2 mL of methanol. A 5-mL syringe is recommended for this operation.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of methanol to the
top. Place the micro K-D apparatus on a hot water bath (80 to 85°C) so that the
concentrator tube is partially immersed in the hot water. Adjust the vertical position
of the apparatus and water temperature as required to complete the concentration in
5 to 10 minutes. At the proper rate of distillation, the balls will actively chatter but
the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL,
remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
Remove the micro-Snyder column and rinse its lower joint into the concentrator tube
with a small volume of methanol. Quantitatively transfer the extract to a 25-mL
volumetric flask by means of a Pasteur pipette or other suitable device. Rinse the
concentrator tube with about 0.5 mL of methanol and add to the volumetric flask.
Adjust the final volume to 25 mL or to a volume suitable for liquid chromatography
with HPLC mobile phase. Store refrigerated if further processing will not be per-
formed immediately. Proceed with liquid chromatographic analysis.
10.1.7 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.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters 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 is no less than that
recorded in Table 2.
10.2.2 Collect the dried extracts from Section 10.1.4 in a 500-mL separatory funnel. Add
10 mL of 0.3N NaOH and extract by shaking the funnel for 2 minutes with periodic
venting to release excess pressure. Allow a 10-minute separation time. Drain the
589
-------
Method 644
methylene chloride and discard. Allow 2 minutes for the aqueous layer to drain from
the walls, and collect it in a 25-mL volumetric flask.
10.2.3 Adjust the pH of the aqueous extract to 1.5 to 2.5 with sulfuric acid solution, and
dilute to volume with HPLC mobile phase.
10.2.4 Proceed with liquid chromatography as described in Section 10.3.
10.3 Liquid chromatography analysis.
10.3.1 Table 1 summarizes the recommended operating conditions for the liquid chromato-
graph. Included in this table are the estimated retention time and estimated detection
limit that can be achieved by this method. An example of the separation achieved by
this column is shown in Figure 1. Figure 2 is a chromatogram of picloram in a
POTW wastewater sample. Other columns, chromatographic conditions, or detectors
may be used if data quality comparable to Table 2 is achieved.
10.3.2 Calibrate the system daily as described in Section 8.
10.3.3 Inject 100 /^L of the sample extract. Monitor the column eluent at 225 ran. Record
the resulting peak size in area or peak height units.
10.3.4 The retention-time window used to make identifications should be based upon meas-
urements 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 can be used to
calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
10.3.5 If the response for the peak exceeds the working range of the system, dilute the
sample with mobile phase and reanalyze.
10.3.6 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
11. CALCULATIONS
11.1 Determine the concentration of picloram in the sample.
11.1.1 Calculate the amount of picloram injected from the peak response using the calibration
curve or calibration factor in Section 8.2.2. The concentration in the sample can be
calculated from the equation:
Equation 3
Concentration, pg/L =
where
A = Amount of material injected, in ng
Vt = Volume of extract injected, in \tL
Vt = Volume of total extract, in \iL
Vs = Volume of water extracted, in mL
590
-------
Method 644
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.
12. METHOD PERFORMANCE
12.1 The EDL and associated chromatographic conditions for picloram are listed in Table I.8 The
EDL is defined as the minimum response being equal to 5 times the background noise, using a
100-/iL injection.
12.2 Single-operator accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 in the designated matrix. The results of these studies are presented in
Table 2.
591
-------
Method 644
References
i. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia. Pennsylvania, p 679, 1980.
2 "Carcinogen-;—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report 68-03-2897 Unpublished
report, available from the U.S. Environmental Protection Agency, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
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, March 1979.
8. "Evaluation of Ten Pesticide Methods," U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio,
532
-------
Method 644
Table 1. Liquid Chromatography of Picloram*
Retention Time Estimated Detect/on
Compound (min) Limit (fjg/U
Picloram 4.0 0.3
*HPLC conditions: Reverse-phase 10// Ultrasphere ODS, 4.6 mm ID by 250 mm long; isocratic
elution; flow rate 1 mL/min. Mobile Phase: 57% (v/v) HPLC buffer solution in methanol.
Table 2. Single-Operator Accuracy and Precision
Spike Average Standard
Matrix Range Number of Percent Deviation
Parameter Type* fag/D Replicates Recovery (%)
Picloram 1 25 7 93.9 9.1
1 778 7 79.0 7.7
' 1 = Municipal effluent
593
-------
Method 644
r
Picloram
I 1 I I I I
0 1.0 2.0 3.0 4.0 5.0 6.0
Retention Time (minutes)
AS2-002-71A
Figure 1. HPLC of Picloram
594
-------
Method 644
i
Picloram
I T T I 1 I I
0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Retention Time (minutes)
A52-002-72A
Figure 2. HPLC of Picloram in Wastewater Extract
595
-------
-------
Method 645
The Determination of Certain
Arnine Pesticides and Lethane
in Municipal and Industrial
Wastewater
-------
-------
Method 645
The Determination of Certain Amine Pesticides and Lethane in
Municipal and Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain amine pesticides and lethane in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Alachlor 15972-60-8
Butachlor 23184-66-9
Diphenamid 957-51-7 .
Fluridone 59756-60-4
Lethane 112-56-1
Norflurazon 27314-13-2
1.2 The estimated detection limit (EDL) for each parameter is listed in Tables 1 and 2. The EDL
was calculated from the minimum detectable response of the nitrogen/phosphorous detector
equal to 5 times the gas chromatographic (GC) background noise assuming a 10 mL final
extract volume of a 1-L reagent water sample and a GC injection of 5 /iL. The EDL for a
specific wastewater may be different depending on the nature of interferences in the sample
matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications
should be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confir-
mation of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 The amine pesticides and lethane are removed from the sample matrix by extraction with
methylene chloride. The extract is dried, exchanged into hexane, and analyzed by gas chro-
matography. Column chromatography is used as necessary to eliminate interferences which
may be encountered. Measurement of the pesticides is accomplished with a nitrogen/
phosphorous specific detector.
2.2 Confirmatory analysis by gas chromatography/mass spectrometry is recommended (Section 13)
when a new or undefined sample type is being analyzed if the concentration is adequate for
such determination.
555
-------
Method 645
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.' 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 15 to 30
minutes. 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 the
glassware inverted or capped with aluminum foil.
3.2 Interferences co-extracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits listed in Tables 1 and 2.
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 aware-
ness 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 identified2"1 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to
air dry, then muffle at 400°C for 1 hour. After cooling, rinse the bottles and cap liners with
hexane, seal the bottles, and store in a dust-free environment.
5.1.1 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
600
-------
Method 645
use, however, the compressible tubing should be thoroughly rinsed with methanol,
followed by repeated rinsings with reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to con-
centrator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak
areas.
5.3.1.1 Column 1: 180 cm long by 2 mm ID, glass, packed with 10% OV-11 on
Gas Chrom W-HP (100/120 mesh) or equivalent.
5.3.1.2 Column 2: 180 cm long by 2 mm ID, PyrexR glass, packed with 3%
Dexsil 300 on Chromasorb W-HP (80/100 mesh) or equivalent.
5.3.1.3 Column 3: 180 cm long by 2mm ID Glass, packed with 3% SP-2100 on
Supelcoport (100/120 mesh) or equivalent.
5.3.1.4 Column 1 was used to develop the accuracy and precision statements in
Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.5 Detector: Nitrogen/phosphorous. This detector has proven effective in the
analysis of wastewaters for the parameters listed in Section 1.1 and was
used to develop the method performance statements in Section 12. Guide-
lines for the use of alternate detectors are provided in Section 10.3.
5.4 Chromatographic column: 300 mm long by 10 mm ID Chromaflex, equipped with coarse-
fritted bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous.
5.6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: Heated with concentric ring cover, capable of temperature control
(±2°C). The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
5.6.5 Boiling chips: Approximately 10/40 mesh. Heat to 400°C for 30 minutes or perform
a Soxhlet extraction with methylene chloride.
601
-------
Method 645
6. REAGENTS AND CONSUMABLE MATERIALS
6.1 Reagents.
'- \
6.1.1 Acetone, hexane, and methylene chloride: demonstrated to be free of ari&lytes.
6.1.2 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in glass
containers with glass stoppers or foil-lined screw-caps. Before use, activate each
batch overnight at 200°C in foil-covered glass containers. To prepare for use, place
the amount necessary for the number of columns to be run in a 500-mL reagent bottle
and add 2% by weight of reagent water. Seal and mix thoroughly by shaking or
rolling for 10 minutes. Allow to stand for at least 2 hours prior to use. The mixture
must be homogeneous. Keep the bottle tightly sealed to ensure proper activity.
6.1.3 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.
6.1.4 Sodium hydroxide (NaOH) solution (ION): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.6 Sulfuric acid (H2SO4) solution (1 + 1): Add a measured volume of concentrated H2SO4
to an equal volume of reagent water.
6.1.7 Sodium thiosulfate: ACS, granular.
6.2 Standard stock solutions (1.00 /*g//*L): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in hexane or other suitable solvent 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 96% or greater, the weight can be used
without correction to calculate the concentration of the standard stock.
6.2,2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
7.3 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 within 48 hours of
602
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Method 645
collection, the sample should be adjusted to a pH range of 6.0 to 8.0 with sodium hydroxide
or sulfuric acid.
7.4 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction6.
5. CALIBRATION AND STANDARDIZATION
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is needed.
One calibration solution should contain each analyte at a concentration approaching
but greater than the estimated detection limit (Tables 1 and 2) for that compound; the
other two solutions should contain analytes at concentrations that bracket the range
expected in samples. For example, if the detection limit for a particular analyte is
0.2 /ig/L, and a sample expected to contain approximately 5 fig/L is analyzed, solu-
tions of standards should be prepared at concentrations representing 0.3 jig/L, 5jtg/L,
and 10 jtg/L for the particular analyte.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock
solution to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3 and tabulate peak height or area response versus the mass of
analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor)
is a constant over the working range (< 10% relative standard deviation), linearity
through the origin can be assumed and the average ratio or calibration factor can be
used in place of a calibration curve.
8.1.4 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
analyte varies from the predicted response by more than ±10%, the test must be re-
peated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve.
9. QUALITY CONTROL
9.1 Monitoring for interferences.
9.1.1 Analyze a laboratory reagent blank each time a set of samples is extracted. A labora-
tory reagent blank is a 1-L aliquot of reagent water. If the reagent blank contains a
\ reportable level of any analyte, immediately check the entire analytical system to
x locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
603
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Method 645
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.3, prepare a laboratory control standard con-
centrate that contains each analyte of interest at a concentration of 2 /ig/mL
in acetone or other suitable solvent.
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water.
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For
each analyte in the laboratory control standard, calculate the percent recov-
ery (PJ) with the equation:
Equation 1
1005
P =
T,
where
Sf = Analytical results from the laboratory control standard, in pg/L
Tt = Known concentration of the spike, in
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Sect. 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of most
of the analytes.
9.3.2 For each analyte in each duplicate pair, calculate the relative range7 (RRj) with the
equation:
Equation 2
100/J
RR =
where
/?(. = Absolute difference between the duplicate measurements Xl and X2, in ng/L
Xt = Average concentration found
X.-KX,
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
604
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Method 645
10. PROCEDURE
10.1 Sample extraction.
10.1.1 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 of the sample with wide-range pH paper and adjust to within the range of 5 to 9
with sodium hydroxide or sulfuric acid.
10.1.2 Add 60 mL of methylene chloride to the sample bottle and shake for 30 seconds to
rinse the walls. Transfer the solvent to the separatory funnel and extract the sample
by shaking the funnel for 2 minutes with periodic venting to release vapor pressure.
Allow the organic layer to separate from the water phase for a minimum of 10 min-
utes. 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 on the sample, but may include
stirring, filtration of the emulsion through glass wool, or centrifugation. Collect the
extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of methylene chloride to the sample bottle and
complete the extraction procedure a second time, combining the extracts in the Erlen-
meyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-mL K-D flask equipped with a 10 mL concentrator tube. Rinse the Erlenmeyer
flask and column with 20 to 30 mL of methylene chloride to complete the quantitative
transfer.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of 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 in steam. Adjust the vertical position of the apparatus and the water
temperature as required to complete the concentration in 15 to 20 minutes. At the
proper rate of distillation, the balls of the column will actively chatter but the cham-
bers 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 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.
10.1.6 For Florisil column cleanup or gas chromatography, the extract must be in hexane
solution. To exchange the solvent to hexane, add one or two fresh boiling chips to
the flask and ampule containing the extract, add 50 mL of hexane, and reattach the
Snyder column. Pour about 1 mL of hexane into the top of the Snyder column, and
concentrate the extract at 85 to 95°C in the hot water bath as above. When the
apparent volume of liquid reaches 1 mL, remove the K-D apparatus from the water
bath and allow it to drain and cool for at least 10 minutes.
10.1.7 Remove the Snyder column, rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of hexane. A 5-mL syringe is recommended for this operation.
605
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Method 645
Dilute to 10 mL with hexane for analysis by gas chromatography (Section 10.3) if
cleanup is not required. If the extract requires cleanup, proceed to Section 10.2. If
the extracts will be stored longer than 2 days, they should be transferred to PTFE-
sealed screw-cap bottles. Proceed with gas chromatographic analysis.
10.1.8 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.
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
alachlor, butachlor, diphenamid, and lethane in various clean waters and municipal
effluents. The use of Florisil as the cleanup material for fluridone and norflurazon
has been demonstrated to yield recoveries of less than 50%, and is not recommended
as a cleanup material for these compounds. Use of specific detectors may obviate the
necessity for cleanup of relatively clean sample matrices. 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 is no
less than 85%.
10.2.2 Place the necessary amount of deactivated Florisil into a 20 mm ID chromatographic
column and tap the column to settle the Florisil. Add 1 to 2 cm of anhydrous sodium
sulfate to the top of the Florisil.
10.2.3 Pre-elute the column with 50 to 60 mL of hexane. Discard the eluate and, just prior
to exposure of the sodium sulfate layer to the air, transfer the sample extract onto the
column by decantation. Complete the transfer by rinsing with two additional 2-mL
volumes of hexane. Alternatively, a measured aliquot of the extract may be taken for
cleanup.
10.2.4 Just prior to exposure of the sodium sulfate layer to the air, elute the column with
100 mL hexane. Discard the eluate and repeat the elution with 200 mL of 6% (v/v)
acetone in hexane. Collect the eluate in a 500-mL K-D flask equipped with a 10-mL
concentrator tube (Fraction 1). All elutions should be carried out using a flow rate of
about 5 mL/min.
10.2.5 Perform a second elution with 200 mL of 15% acetone in hexane (Fraction 2).
Collect each fraction in a separate K-D apparatus. The elution pattern for these
compounds is shown in Table 3.
10.2.6 Determine, from Table 3, the fractions of interest and concentrate by standard K-D
technique, as indicated in Section 10.1.5, using hexane in place of methylene
chloride, to a volume of 10 mL.
10.2.7 Analyze the fractions by gas chromatography.
10.3 Gas chromatography analysis.
10.3.1 Recommended columns and detectors and operating conditions for the gas chroma-
tography system are described in Section 5.3. Tables 1 and 2 summarize the recom-
mended operating conditions for the gas chromatograph. Included in these tables are
retention times and estimated detection limits that can be achieved by this method.
606
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Method 645
Examples of the separations achieved are shown in Figures 1 through 3. Other
packed columns, chromatographic conditions, or detectors may be used if data quality
comparable to Table 4 is achieved. Capillary (open-tubular) columns may also be
used if the relative standard deviations of responses for replicate injections are demon-
strated to be less than 6% and data quality comparable to Table 4 is achieved.
10.3.2 Inject 2 to 5 ^L of the sample extract using the solvent-flush technique.8 Record the
volume injected to the nearest 0.05 ^L, the total extract volume, and the resulting
peak size in area or peak height units.
10.3.3 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
the day. Three times the standard deviation of a retention time for a compound can
be used to calculate a suggested window size; however, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
77. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per
liter with the equation:
Equation 3
Concentration,
(V,XVf)
where
A = Amount of material injected, in ng
Vf = Volume of extract injected, in t>L
Vt = Volume of total extract, in \iL
Vs = Volume of water extracted, in mL
11.2 Report the results for the unknown samples in micrograms per liter. Round off the results to
the nearest 0.1 /*g/L or two significant figures.
72. METHOD PERFORMANCE
12.1 Estimated detection limits (EDL) and associated chromatographic conditions are listed in
Table I.9 The detection limits were calculated from the minimum detectable response of the
EC detector equal to 5 times the GC background noise, assuming a 10-mL final extract volume
of a 1-L sample and a GC injection of 5 /*L.
12.2 Single-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering,6 using spiked samples. The results of these studies are presented in Table 2.
607
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Method 645
13. GC/MS CONFIRMATION
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative
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 compounds
of interest. 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 scans per peak 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.
13.2 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. The calculation
of tailing factors is illustrated in Method 625.10
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS
system must be checked to see that all DFTPP performance criteria are achieved."
13.4 To confirm an identification of a compound, the background-corrected mass spectrum of the
compound must be obf^ined 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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
13.4.1 The molecular ion and other 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 +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%.
13.4.2 The retention time of the compound in the sample must be within 6 seconds of the
same compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by GC/MS only
on the basis of retention time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
608
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Method 645
References
1. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, p. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897. Un-
published report available from U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
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, March 1979.
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. "Evaluation of Ten Pesticide Methods," U.S. Environmental Protection Agency, Contract No.
68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio (in preparation).
10. "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater" EPA-600/
4-82-057, U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory - Cincinnati, Ohio.
11. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Analytical Chemistry, 46, 1912 (1975).
609
-------
Method 645
Table 1. Gas Chromatography and Detection Limits of Certain Amines and Leth-
ane
Retention Time fmin)
Parameter
Alachlor
Butachlor
Diphenamide
Fluridone
Lethane
Norflurazon
Column 1
6.9
10.5
10.8
2.2
2.0
18.4
Column 2
2.45
Column 3
2.1
Estimated
Detection Limit
(ug/L)
0.2
0.3
0.2
0.5
0.1
0.02
Column 1: Glass, 180 cm long by 2 mm ID, packed with 10% OV-11 on Gas Chrom W-HP,
100/120 mesh; nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature is held at
225°C for 4 minutes after injection and then programmed to 275°C at 4°/min and held for
8 minutes.
Column 2: Glass, 180 cm long by 2 mm ID, packed with 3% Dexsil 300 on Chromasorb W-HP,
80/100 mesh; nitrogen carrier gas at a flow rate of 30 mL/min. Column temperature at 300°C
isothermal.
Column 3: Glass, 180 cm long by 2 mm ID, packed with 3% SP-2100 on Supelcoport, 100/120
mesh; nitrogen carrier gas at a flow rate of 40 mL/min. Column temperature at 275°C isothermal.
Table 2. Single-Laboratory Accuracy and Precision
Parameter
Alachlorn
Butachlor
Diphenamid
Fluridone**
Lethane
Norlurazone**
Matrix Type*
1
1
1
1
2
3
1
1
1
1
1
3
Spike Range
(ug/L)
255
996
286
1,420
9.3
740
20.8
998
167
576
243
1,048
No. of
Replicates
7
7
7
7
7
7
7
7
7
7
7
7
Average
Percent
Recovery
113
104
93.1
92.8
100
98.8
92.0
88.4
93.3
97.6
89.5
102
Relative
Standard
Deviation (%)
9.0
13.3
8.2
4.3
14.2
7.0
11.5
11.4
19.9
29.4
7.4
6.1
1 = Manufacturing effluent wastewaters.
2 = Manufacturing effluent wastewater +
3 = Manufacturing effluent wastewater +
POTW effluent at a ratio of 1:200.
POTW effluent at a ration of 1:1.
610
-------
Method 645
Table 3. Florisil* Cleanup Recoveries
Solvent Frac- Average Percent Recoveries Lethane
lion** Alachlor Butachlor Diphenamid
1 103 95 106
2 ND ND 96 ND
2% deactivated.
1 =6% acetone/hexane
2 = 15% acetone/hexane
611
-------
Method 645
. Lethane (2.0)*
\
-Butachlor(10.5)*
Alachlor(6.9)* i /Diphenamid(10.8)*
Norflurazon(18.4)*
'Retention Time in parentheses
Figure 1. Gas Chromatogram of Amines/Lethane (Column 2)
A52-002-73A
612
-------
Method 645
Fluridone
Retention Time (minutes)
Figure 2. Gas Chromatogram of Fluridone (Column 2)
A52-002-74A
613
-------
Method 645
Fluridone
2.0
4.0
Retention Time (minutes)
A52-002-75A
Figure 3. Gas Chromatogram of Fluridone (Column 3)
674
-------
Method 646
The Determination of Dinitro
Aromatic Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 646
The Determination of Dinitro Aromatic Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of certain dinitro aromatic pesticides in municipal and
industrial wastewater. The following parameters may be determined by this method.
Parameter CAS No.
Basalin (Fluchloralin) 33245-39-5
CDN 97-00-7
Dinocap 39300-45-3
1.2 The estimated detection limit (EDL) for each parameter is listed in Table 1. The EDL was
calculated from the minimum detectable response of the electron capture detector (ECD) equal
to 5 times the GC background noise assuming a 1.0 mL final extract volume of a 1-L reagent
water sample and a GC injection of 5 /iL. The EDL for a specific wastewater may be dif-
ferent depending on the nature of interferences in the sample matrix.
1.3 This is a gas chromatographic (GC) method applicable to the determination of the compounds
listed above in municipal and industrial discharges. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above, compound identifications
should be supported by at least one additional qualitative technique. Section 13 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the qualitative confir-
mation of compound identifications.
1.4 This method is restricted to use by or under the supervision of analysts experienced in the
operation of gas chromatographs and in the interpretation of chromatograms.
2. SUMMARY OF METHOD
2.1 Dinitroaromatic pesticides are removed from the sample matrix by extraction with 15%
methylene chloride in hexane. The extract is dried, exchanged into hexane, and analyzed by
gas chromatography (GC). Column chromatography is used as necessary to eliminate inter-
ferences which may be encountered. Measurement of the pesticides is accomplished with an
electron capture detector.
2.2 Confirmatory analysis by gas chromatography/mass spectrometry (GC/MS) is recommended
(Section 13) when a new or undefined sample type is being analyzed, if the concentration is
adequate for such determination.
617
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Method 646
3. INTERFERENCES
3.1 Solvent, reagents, glassware, and other sample-processing hardware may yield discrete arti-
facts and/or elevated baselines causing misinterpretation of gas chromatograms. All of these
materials must be demonstrated to be free from interferences under the conditions of the
analysis by running laboratory reagent blanks as described in Section 9.1.
3.1.1 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.
3.1.2 Glassware must be scrupulously cleaned.' 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 15 to 30
minutes. Solvent rinses with acetone and pesticide-quality hexane may be substituted
for the 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 the glassware inverted
or capped with aluminum foil.
3.2 Interferences coextracted from the samples will vary considerably from source to source,
depending on the diversity of the industrial complex or municipality being sampled. While
general cleanup procedures are provided as part of this method, unique samples may require
additional cleanup approaches to achieve the detection limits 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 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 aware-
ness 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 identified2 4 for the information of the analyst.
5. APPARATUS AND EQUIPMENT
5.1 Sample containers: Narrow-mouth glass bottles, 1-L or 1-quart volume, equipped with poly-
tetrafluoroethylene (PTFE)-lined screw-caps. Wide-mouth glass bottles, 1-quart volume,
equipped with PTFE-lined screw-caps may also be used. Prior to use, wash bottles and cap
liners with detergent and rinse with tap and distilled water. Allow the bottles and cap liners to
air dry, then muffle at 400°C for 1 hour. After cooling, rinse the cap liners with hexane, seal
the bottles, and store in a dust-free environment.
5.1.1 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
618
-------
Method 646
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 reagent water to minimize the potential for con-
tamination of the sample. An integrating flow meter is required to collect flow-
proportional composites.
5.2 Kuderna-Danish (K-D) glassware.
5.2.1 Synder column: Three-ball macro (Kontes K-503000-0121 or equivalent) and two-ball
micro (Kontes K-569001-0219 or equivalent).
5.2.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025 or equivalent) with
ground-glass stopper.
5.2.3 Evaporative flask: 500-mL (Kontes K-570001-0500 or equivalent). Attach to concen-
trator tube with springs.
5.3 Gas chromatography system.
5.3.1 The gas chromatograph must be equipped with a glass-lined injection port compatible
with the detector to be used. A data system is recommended for measuring peak
areas.
5.3.1.1 Chromatography column: 180 cm long by 4 mm ID, glass, packed with
1.5% OV-17/1.95% OV-210 on Supelcoport (100/120 mesh) or equiva-
lent. This column was used to develop the method performance statements
in Section 12. Guidelines for the use of alternative column packings are
provided in Section 10.3.1.
5.3.1.2 Detector: Electron capture. This detector has proven effective in the anal-
ysis of wastewaters for the parameters listed in the scope and was used to
develop the method performance statements in Section 12. Guidelines for
the use of alternative detectors are provided in Section 10.3.
5.4 Chromatographic column: 200 mm long by 10 mm ID Chromaflex, equipped with coarse-
fritted bottom plate and PTFE stopcock. (Kontes K-420540-0213 or equivalent).
5.5 Drying column: Approximately 400 mm long by 20 mm ID borosilicate glass, equipped with
coarse-fritted bottom plate.
5.6 Miscellaneous
5.6.1 Balance: analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6.2 Separatory funnel: 2-L, equipped with PTFE stopcock.
5.6.3 Water bath: heated, with concentric ring cover, capable of temperature control
(+2°C). The bath should be used in a hood.
5.6.4 Standard solution storage containers: 15-mL bottles with PTFE-lined screw-caps.
6. REAGENTS AND CONSUMABLE MA TERIALS
6.1 Reagents.
6.1.1 Acetone, hexane, and methylene chloride: Demonstrated to be free of analytes.
619
-------
Method 646
6.1.2 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in
glass containers with glass stoppers or foil-lined screw-caps. Before use, activate each
batch overnight at 200°C in foil-covered glass container.
6.1.3 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.
6.1.4 Sodium hydroxide (NaOH) solution (ION): Dissolve 40 g NaOH in reagent water and
dilute to 100 mL.
6.1.5 Sodium sulfate: Granular, anhydrous. Condition by heating at 400°C for 4 hours in
a shallow tray.
6.1.6 Sulfuric acid (H2SO4) solution (1 + 1): Add measured volume of concentrated H2SO4
to equal volume of reagent water.
6.2 Standard stock solutions (1.00 /^g/^L): These solutions may be purchased as certified solu-
tions or prepared from pure standard materials using the following procedures.
6.2.1 Prepare standard stock solutions by accurately weighing about 0.0100 g of pure mate-
rial. Dissolve the material in hexane 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 96% or greater, the weight can be used without correction to
calculate the concentration of the standard stock.
6.2.2 Store standard stock solutions at 4°C in 15-mL bottles equipped with PTFE-lined
screw-caps. Standard stock solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.2.3 Standard stock solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
7.1 Collect all samples in duplicate. Grab samples must be collected in glass-containers. Conven-
tional sampling practices5 should be followed, except that the bottle must not be prewashed
with sample before collection.
7.2 The samples must be iced or refrigerated at 4°C from the time of collection until extraction.
7.3 All samples must be extracted within 30 days of collection.6
8. CALIBRA TION AND STANDARDIZA TION
8.1 Calibration.
8.1.1 A set of at least three calibration solutions containing the method analytes is needed.
One calibration solution should contain each analyte at a concentration approaching
but greater than the estimated detection limit (Table 1) for that compound; the other
two solutions should contain analytes at concentrations that bracket the range expected
in samples. For example, if the detection limit for a particular analyte is 0.2 /ig/L,
620
-------
Method 646
and a sample expected to contain approximately 5 /zg/L is analyzed, standard solutions
should be prepared at concentrations of 0.3 Mg/L, 5/ig/L, and 10 ^tg/L.
8.1.2 To prepare a calibration solution, add an appropriate volume of a standard stock
solution to a volumetric flask and dilute to volume with hexane.
8.1.3 Starting with the standard of lowest concentration, analyze each calibration standard
according to Section 10.3 and tabulate peak height or area responses versus the mass
of analyte injected. The results can be used to prepare a calibration curve for each
compound. Alternatively, if the ratio of response to concentration (calibration factor)
is a constant over the working range (< 10% relative standard deviation), linearity
through the origin can be assumed and the average ratio or calibration factor can be
used in place of a calibration curve.
8.1.4 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
analyte varies from the predicted response by more than ±10%, the test must be
repeated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve.
8.2 Florisil standardization.
8.2.1 Florisil from different batches or sources may vary in absorptive capacity. To stan-
dardize the amount of Florisil which may be used in the cleanup procedure (Sec-
tion 10.2) use of the lauric acid value7 is suggested. The referenced procedure deter-
mines the adsorption from hexane solution of lauric acid, in milligrams per gram 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.
9. QUALITY CONTROL
9.1 Monitoring for interferences: Analyze a laboratory reagent blank each time a set of samples is
extracted. A laboratory reagent blank is a 1-L aliquot of reagent water. If the reagent blank
contains a reportable level of any analyte, immediately check the entire analytical system to
locate and correct for possible interferences and repeat the test.
9.2 Assessing accuracy.
9.2.1 After every ten samples, and preferably in the middle of each day, analyze a labora-
tory control standard. Calibration standards may not be used for accuracy assess-
ments and the laboratory control standard may not be used for calibration of the
analytical system.
9.2.1.1 Laboratory control standard concentrate: From stock standards prepared
as described in Section 6.3, prepare a laboratory control standard concen-
trate that contains each analyte of interest at a concentration of 2 /ig/mL in
acetone or other suitable solvent.8
9.2.1.2 Laboratory control standard: Using a pipette, add 1.00 mL of the labora-
tory control standard concentrate to a 1-L aliquot of reagent water.
627
-------
Method 646
9.2.1.3 Analyze the laboratory control standard as described in Section 10. For
each analyte in the laboratory control standard, calculate the percent recov-
ery (P,) with the equation:
Equation 1
100S
where
Sf = Analytical results from the laboratory control standard, in
r. = Known concentration of the spike, in ng/L
9.2.2 At least annually, the laboratory should participate in formal performance evaluation
studies, where solutions of unknown concentrations are analyzed and the performance
of all participants is compared.
9.3 Assessing precision.
9.3.1 Precision assessments for this method are based upon the analysis of field duplicates
(Section 7.1). Analyze both sample bottles for at least 10% of all samples. To the
extent practical, the samples for duplication should contain reportable levels of most
of the analytes.
9.3.2 For each analyte in each duplicate pair, calculate the relative range (RRJ with the
equation:
Equation 2
100/f
where
Rt = Absolute difference between the duplicate measurements X{ and X^ , in
Xt = Average concentration found
, in
9.3.3 Individual relative range measurements are pooled to determine average relative range
or to develop an expression of relative range as a function of concentration.
10. PROCEDURE
10.1 Sample extraction.
10.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel. Check the pH
of the sample with wide-range pH paper and adjust to within the range of 5 to 9 with
sodium hydroxide or sulfuric acid.
522
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Method 646
10.1.2 Add 60 mL of 15% methylene chloride/hexane to the sample bottle and shake for
30 seconds to rinse the walls. Transfer the solvent to the separatory funnel and
extract the sample by shaking the funnel for 2 minutes with periodic venting to release
vapor pressure. Allow the organic layer to separate from the water phase for a mini-
mum 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 on the sample, but
may include stirring, filtration of the emulsion through glass wool, or centrifugation.
Collect the extract in a 250-mL Erlenmeyer flask.
10.1.3 Add an additional 60-mL volume of 15% methylene chloride/hexane to the sample
bottle and complete the extraction procedure a second time, combining the extracts in
the Erlenmeyer flask.
10.1.4 Perform a third extraction in the same manner. Pour the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate, and collect it in a
500-mL K-D flask equipped with a 10-mL concentrator tube.
10.1.5 Add one or two clean boiling chips to the flask and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of hexane 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
in steam. Adjust the vertical position of the apparatus and the water temperature as
required to complete the concentration in 15 to 20 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 liquid reaches 1 mL, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes. Remove the Snyder column
and rinse the flask and its lower joint into the concentrator tube with 1 to 2 mL of
hexane. A 5-mL syringe is recommended for this operation. If the extract requires
cleanup proceed to Section 10.2 (cleanup and separation). If cleanup has been per-
formed or if the extract does not require cleanup, proceed with Section 10.1.6.
10.1.6 Add a clean boiling chip to the concentrator tube. Attach a two-ball micro-Snyder
column. Prewet the micro-Snyder column by adding about 0.5 mL of hexane to the
top. Place this micro K-D apparatus on a steaming-water bath (80 to 85°C) so that
the concentrator tube is partially immersed in the hot water. Adjust the vertical posi-
tion of the apparatus and water temperature as required to complete the concentration
in 5 to 10 minutes. At the proper rate of distillation, the balls will actively chatter but
the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL,
remove the K-D apparatus and allow it to drain and cool for at least 10 minutes.
Remove the micro-Snyder column and rinse its lower joint into the concentrator tube
with a small volume of hexane. Adjust the final volume to 1.0 mL, and stopper the
concentrator tube; store refrigerated if further processing will not be performed imme-
diately. If the extracts will be stored longer than 2 days, they should be transferred to
PTFE-sealed screw-cap bottles. Proceed with gas chromatographic analysis.
10.1.7 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.
623
-------
Method 646
10.2 Cleanup and separation.
10.2.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedures recommended in this method have been used for the analysis of
various clean waters and municipal effluents. The Florisil cleanup procedure allows
for a select fractionation of the compounds and will eliminate non-polar materials.
The single-operator precision and accuracy data in Table 2 were gathered using the
recommended cleanup procedures. 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 is no less than that recor-
ded in Table 2.
10.2.2 Prepare a slurry of 10 g of Florisil in methylene chloride. Use it to pack a 10-mm ID
chromatography column, gently tapping the column to settle the Florisil. Add a 1-cm
layer of anhydrous sodium sulfate to the top of the Florisil.
10.2.3 Just prior to exposure of the sodium sulfate layer to the air, transfer the sample
extract onto the column using an additional 2 mL of hexane to complete the transfer.
10.2.4 Just prior to exposure of the sodium sulfate layer to the air, add 30 mL of 50%
methylene chloride/hexane and continue the elution of the column. Elution of the
column should be at a rate of about 2 mL/min. Discard the eluate from this fraction.
10.2.5 Next, elute the column with 30 mL of methylene chloride, collecting the eluate in a
500-mL K-D flask equipped with a 10-mL concentrator tube. Add 50 mL of hexane
to the flask and concentrate the collected fraction by the standard technique prescribed
in Sections 10.1.5 and 10.1.6. This fraction should contain DCN and basalin.
10.2.6 Elute the column with 30 mL of 10% acetonelmethylene chloride collecting the eluate
in a 500-mL K-D flask equipped with a 10 mL concentrator tube. Add 50-mL of
hexane to the flask and concentrate the collected fraction by the standard technique
prescribed in Sections 10.1.5 and 10.1.6. This fraction should contain dinocap.
10.2.7 Analyze the fractions by gas chromatography.
10.3 Gas chromatography analysis.
10.3.1 Recommended columns and detectors for the gas chromatography system are de-
scribed in Section 5.3. Table 1 summarizes the recommended operating conditions
for the gas chromatograph. Included in this table are estimated retention times and
detection limits that can be achieved by this method. Examples of the separations
achieved are shown in Figures 1 and 2. Other packed columns, chromatographic
conditions, or detectors may be used if data quality comparable to Table 2 are
achieved. 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 data quality comparable to Table 2 are achieved.
10.3.2 Inject 2 to 5 juL of the sample extract using the solvent-flush technique.9 Record the
volume injected to the nearest 0.05 juL, the total extract volume, and the resulting
peak size in area or peak height units.
10.3.3 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
624
-------
Method 646
the day. Three times the standard deviation of a retention time for a compound can
be used to calculate a suggested window size; however, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
10.3.4 If the response for the peak exceeds the working range of the system, dilute the
extract and reanalyze.
10.3.5 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
77. CALCULATIONS
11.1 Determine the concentration (C) of individual compounds in the sample in micrograms per
liter with the equation:
Equation 3
Concentration, \iglL = —
where
A = Amount of material injected, in ng
Vi = Volume of extract injected, in \iL
Vt = Volume of total extract, in \tL
Vs = Volume of water extracted, in mL
11.3 Report the results for the unknown samples in micrograms per liter. Round off the results of
the nearest 0.1 /xg/L or two significant figures.
12. METHOD PERFORMANCE
12.1 Estimated detection limits and associated chromatographic conditions are listed in Table I.10
The detection limits were calculated from the minimum detectable response of the BCD equal
to 5 times the GC background noise, assuming a 1.0-mL final extract volume of a 1-L sample
and a GC injection of 5 /iL.
12.2 Single-laboratory accuracy and precision studies were conducted by Environmental Science and
Engineering, Inc.,6 using spiked wastewater samples. The results of these studies are presen-
ted in Table 2.
13. GC/MS CONFIRMATION
13.1 It is recommended that GC/MS techniques be judiciously employed to support qualitative iden-
tifications 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 seconds 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
625
-------
Method 646
that allows the continuous acquisition and storage on machine-readable media of all mass
spectra obtained throughout the duration of the chromatographic program.
13.2 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. The calculation
of tailing factors is illustrated in Method 625."
13.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS
system must be checked to see that all DFTPP performance criteria are achieved.12
13.4 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 ng of material be injected into the GC/MS. The criteria below
must be met for qualitative confirmation.
13.4.1 The molecular ion and other 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 ±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%.
13.4.2 The retention time of the compound in the sample must be within seven seconds of the
same compound in the standard solution.
13.4.3 Compounds that have similar mass spectra can be explicitly identified by GC/MS only
on the basis of retention time data.
13.5 Where available, chemical ionization mass spectra may be employed to aid in the qualitative
identification process.
13.6 Should these MS procedures fail to provide satisfactory results, additional steps may be taken
before reanalysis. These may include the use of alternative packed or capillary GC columns or
additional cleanup.
626
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Method 646
References
1. ASTM Annual Book of Standards, Part 31, 03694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials, Phila-
delphia, Pennsylvania, p. 679, 1980.
2. "Carcinogens—Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part 31, 03370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76, 1980.
6. Test procedures for Pesticides in Wastewaters, EPA Contract Report #68-03-2897 unpublished
report available from the U.S.Environmental Protection Agency, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
7. Mills, P.A., "Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capa-
city and Its Use in Standardizing Florisil Columns," Journal of the Association of Official
Analytical Chemists, 51, 19, 1968.
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. Burke, J.A., "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects,"
Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
10. "Evaluation of Ten Pesticide Methods," U.S. Environmental Protection Agency, Contract
No.68-03-1760, Task No. 11, U.S. Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
11. "Methods for Organic Chemical Analysis of Municipal and Industrial Wastewater," U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincin-
nati, Ohio.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L., Analytical Chemistry, 46, 1912, 1975.
627
-------
Method 646
Table 1. Chromatographic Conditions and Estimated Detection Limits
Retention Time Estimated Detection
Parameter (min) Limit (fjg/U
CDN 2.0 .0005
Basalin 6.4 .0005
Dinocap* 10-16 0.1
* Oven temperature 200°C isothermal.
Conditions: Glass column, 180 cm long by 4 mm ID, packed with 1.5% OV-17/1.95% OV-210 on
Supelcoport (100/120 mesh) or equivalent; 5% methane/95% argon carrier gas at 33 mL/min flow
rate. Oven temperature 160°C isothermal.
Table 2. Single-Laboratory Accuracy and Precision
Matrix
Type*
1
1
1
1
1
1
Spike
Range
(pg/U
10
121
10
99.2
10
161
Number of
Replicates
7
7
7
7
7
7
Average
Percent
Recovery
79.0
99.3
78.6
99.5
108.5
100.3
Standard
Deviation
{%)
7.0
10.1
7.6
6.1
4.5
4.4
Parameter
Basalin
CDN
Dinocap
* 1 = Publicly Owned Treatment Works (POTW) wastewater
628
-------
Method 646
-Dinocap-
1 I I 1 I 7 1 I I I I I I I I I I I
0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
Retention Time (minutes)
AS2-002-76A
Figure 1. Four-Peak Gas Chromatogram of Dinocap
629
-------
Method 646
CDN
Basalin
i i r i i i i i i r
0 2.0 4.0 6.0 8.0 10.0
Retention Time (minutes)
A52-002-77A
Figure 2. Gas Chromatogram of CDN and Basalin
630
-------
Method 1656
The Determination of
Organo-Halide Pesticides in
Municipal and Industrial
Wastewater
-------
-------
Method 1656
The Determination of Organo-Halide Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method is designed to meet the survey requirements of the Environmental Protection
Agency (EPA). It is used to determine (1) the organo-halide pesticides and polychlorinated
biphenyls (PCBs) associated with the Clean Water Act, the Resource Conservation and Recov-
ery Act, and the Comprehensive Environmental Response, Compensation and Liability Act;
and (2) other compounds amenable to extraction and analysis by wide-bore capillary column
gas chromatography (GC) with halogen-specific detectors.
1.2 The compounds listed in Table 1 may be determined in waters, soils, Sediments, and sludges
by this method. The method is a consolidation of several EPA wastewater methods. For
waters, the sample extraction and concentration steps are essentially the same as in these
methods. However, the extraction and concentration steps have been extended to other sample
matrices. The method may be applicable to other pesticides as well. The quality control
requirements in this method give the steps necessary to determine this applicability. Not all
compounds listed in Table 1 have corresponding calibration data in Table 3 and acceptance
criteria in Table 4. Calibration data for such analytes may be found in other EPA methods
(References 1 and 2).
1.3 This method is applicable to a large number of compounds. Calibrating the GC systems for
all compounds is time-consuming. If only a single compound or small number of compounds
is to be tested for, it is necessary to calibrate the GC systems and meet the performance speci-
fications in this method for these compounds only. In addition, the GC conditions can be
optimized for these compounds provided that all performance specifications in this method are
met.
1.4 When this method is applied to analysis of unfamiliar samples, compound identity must 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. Gas chromatography/mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.5 The detection limits of this method are usually dependent on the level of interferences rather
than instrumental limitations. The limits in Table 2 typify the minimum quantities that can be
detected with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that
uses this method must demonstrate the ability to generate acceptable results using the proce-
dure in Section 8.2.
633
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Method 1656
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is determined.
2.1.2 Samples containing low solids: If the solids content is 1% or less, a 1-L sample is
extracted with methylene chloride using continuous extraction techniques.
2.1.3 Samples containing less than 1 % solids.
2.1.3.1 Non-sludge samples: If the solids content is 1 to 30%, the sample is
diluted to 1% solids with reagent water, homogenized ultrasonically, and
extracted with methylene chloride using continuous extraction techniques.
If the solids content is greater than 30%, the sample is extracted with
methylene chloride:acetone using ultrasonic techniques.
2.1.3.2 Municipal sludge samples and other intractable sample types: If the solids
content is less than 30%, the sample is diluted to 1% solids and extracted
with methylene chloride using continuous extraction techniques. If the
solids content is greater than 30%, the sample is extracted with aceto-
nitrile and then methylene chloride using ultrasonic techniques. The ex-
tract is back-extracted with 2% (w/v) sodium sulfate in reagent water to
remove water-soluble interferences and residual acetonitrile.
2.2 Concentration and cleanup: The extract is dried over sodium sulfate, concentrated using a
Kuderaa-Danish evaporator, cleaned up (if necessary) using gel permeation chromatography
(GPC) and/or adsorption chromatography and/or solid-phase extraction, and then concentrated
to 1 mL. Sulfur is removed from the extract, if required.
2.3 Gas chromatography: A l-/xL aliquot of the extract is injected into the gas chromatograph
(GC). The compounds are separated on a wide-bore, fused-silica capillary column. The
analytes are detected by an electron capture, microcoulometric, or electrolytic conductivity
detector.
2.4 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention
times of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within
their respective windows.
2.5 Quantitative analysis is performed using an authentic standard to produce a calibration factor
or calibration curve, and using the calibration data to determine the concentration of a pollu-
tant in the extract. The concentration in the sample is calculated using the sample weight or
volume and the extract volume.
2.6 Quality is assured through reproducible calibration and testing of the extraction and GC
systems.
3. CONTAMINA TION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the anal-
634
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Method 1656
ysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450°C for
a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials, such as
PCBs, may not be eliminated by this treatment, and thorough rinsing with acetone and pes-
ticide-quality hexane may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interference by phthalate esters can pose a major problem in pesticide analysis when using the
electron capture detector. Phthalates usually appear in the chromatogram as large, late-eluting
peaks. Phthalates may be leached from common flexible plastic tubing and other plastic mate-
rials during the extraction and cleanup processes. Cross-contamination of clean glassware
routinely occurs when plastics are handled during extraction, especially when solvent-wetted
surfaces are handled. Interferences from phthalates can best be minimized by avoiding the use
of plastics in the laboratory, or by using a microcoulometric or electrolytic conductivity
detector.
3.5 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled. The cleanup procedures given in this method
can be used to overcome many of these interferences, but unique samples may require addi-
tional cleanup to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 3 through 5.
4.2 The following compounds covered by this method have been tentatively classified as known or
suspected human or mammalian carcinogens: 4,4'-DDD, 4,4'-DDT, the BHCs and the PCBs.
Primary standards of these compounds shall be prepared in a hood, and a NIOSH/MESA-
approved toxic gas respirator should be worn when high concentrations are handled.
4.3 Mercury vapor is highly toxic. If mercury is used for sulfur removal, all operations involving
mercury shall be performed in a hood.
4.4 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
The oven used for sample drying to determine percent moisture should be located in a hood so
that vapors from samples do not create a health hazard in the laboratory.
635
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Method 1656
5. APPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1.1.1 Liquid samples (waters, sludges, and similar materials that contain less
than 5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-
cap.
5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain >5% solids): Sample bottle, wide mouth, amber
glass, 500-mL minimum.
5.1.1.3 If amber bottles are not available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with
PTFE.
5.1.1.5 Cleaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent or
baked at 450°C for a minimum of 1 hour before use.
5.1.1.5.2 Liners are detergent-water washed, then rinsed with reagent
water and solvent, and baked at approximately 200°C for a
minimum of 1 hour prior to use.
5.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle-cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for determining percent moisture.
5.2.1 Oven, capable of maintaining a temperature ofllO°C(±5°C).
5.2.2 Desiccator.
5.2.3 Crucibles, porcelain.
5.2.4 Weighing pans, aluminum.
5.3 Extraction equipment.
5.3.1 Equipment for ultrasonic extraction.
636
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Method 1656
5.3.1.1 Sonic disrupter: 375 watt with pulsing capability and W or %" disrupter
horn (Ultrasonics, Inc, Model 375C, or equivalent).
5.3.1.2 Sonabox (or equivalent), for use with disrupter.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Continuous liquid-liquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L capacity (Hershberg-Wolf Ex-
tractor, Cal-Glass, Costa Mesa,California, 1000- or 2000-mL continuous
extractor, or equivalent).
5.3.2.2 Round-bottom flask, 500-mL, with heating mantle.
5.3.2.3 Condenser, Graham, to fit extractor.
5.3.2.4 pH meter, with combination glass electrode.
5.3.2.5 pH paper, wide range (Hydrion Papers, or equivalent).
5.2.3 Separatory funnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 Filtration apparatus.
5.3.4.1 Glass powder funnels: 125-to 250-mL.
5.3.4.2 Filter paper for above (Whitman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to 1 L.
5.3.5.2 400- to 500-mL.
5.3.6 Spatulas: Stainless steel or PTFE.
5.3.7 Drying column: 400 mm long x 15 to 20 mm ID Pyrex chromatographic column
equipped with coarse glass frit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for a mini-
mum of 1 hour.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1,1 Evaporation flask: 500-mL (Kontes K-570001-0500, or equivalent), at-
tached to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equi-
valent) with calibration verified. Ground-glass stopper (size 19/22 joint) is
used to prevent evaporation of extracts.
5.4.1.3 Snyder column: Three-ball macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450 °C for a minimum of
1 hour.
5.4.1.5.2 PTFE (optional): Extracted with methylene chloride.
637
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Method 1656
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control
(±2°C), installed in a fume hood.
5.4.3 Nitrogen evaporation device: Equipped with heated bath that can be maintained at 35
to 40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit
GC autosampler.
5.5 Balances.
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc., Colum-
bia, MO, Model GPC Autoprep 1002, or equivalent).
5.6.1.1 Column: 600 to 700 mm long x 25 mm ID, packed with 70 g of SX-3
Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
5.6.1.2 Syringe, 10-mL, with Luer fitting.
5.6.1.3 Syringe-filter holder, stainless steel, and glass fiber or PTFE filters (Ge-
Iman Acrodisc-CR, 1 to 5 /i, or equivalent).
5.6.1.4 UV detector: 254-nm, preparative or semi-prep flow cell: (Isco, Inc.,
Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 fiL micro-
prep flow cell, 2 mm path; Pharmacia UV-1, 3 mm flow cell; LDC Mil-
ton-Roy UV-3, monitor #1203; or equivalent).
5.6.2 Vacuum system and cartridges for solid-phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar (house vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack for holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, CIS Octadecyl, 10 g/60 mL (Ana-
lytichem International Cat. No. 607H060, or equivalent).
5.6.3 Chromatographic column: 400 mm long x 22 mm ID, with PTFE stopcock and
coarse frit (Kontes K-42054, or equivalent).
5.6.4 Sulfur removal tubes: 40- to 50-mL bottle or test tube with PTFE-lined screw-cap.
5.7 Centrifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes
at 5,000 rpm minimum.
5.7.2 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-mL, with screw-caps, to fit centrifuge.
5.7.3 Funnel, Buchner, 15 cm.
638
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Method 1656
5.7.3.1 Flask, filter, for use with Buchner funnel.
5.7.3.2 Filter paper, 15 cm (Whatman #41, or equivalent).
5.8 Miscellaneous glassware.
5.8.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.8.2 Syringes, glass, with Luerlok tip, 0.1-, 1.0- and 5.0-mL. Needles for syringes, 2",
22-gauge.
5.8.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.8.4 Scintillation vials, glass, 20- to 50-mL, with PTFE-lined screw-caps.
5.9 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a halide-specific detector at the end of each column, temper-
ature program with isothermal holds, data system capable of recording simultaneous signals
from the two detectors, and shall meet all of the performance specifications in Section 14.
5.9.1 GC columns: Bonded-phase, fused-silica capillary.
5.9.1.1 Primary: 30 m (±3 m) long x 0.5 mm (+0.05 mm) ID DB-608 (or e-
quivalent).
5.9.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.9.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or
tape, process GC data, compute peak areas, store calibration data including retention
times and calibration factors, identify GC peaks through retention times, compute
concentrations, and generate reports.
5.9.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.9.2.2 Calibration factors and calibration curves: The data system shall be used
to record and maintain lists of calibration factors, and multi-point cali-
bration curves (Section 7). Computations of relative standard deviation
(coefficient of variation) are used for testing calibration linearity. Statistics
on initial (Section 8.2) and ongoing (Section 14.6) performance shall be
computed and maintained.
5.9.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software rou-
tines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
5.9.3 Halide-specific detector: Electron capture or electrolytic conductivity (Micoulometric,
Hall, O.I., or equivalent), capable of detecting 8 pg of aldrin under the analysis con-
ditions given in Table 2.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
639
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Method 1656
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (ION): Dissolve 40 g NaOH in 100 mL reagent
water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL
H2SO4 (specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (w/v). Dissolve 37 g KOH in 100 mL reagent water.
6.3 Solution drying and back-extraction.
6.3.1 Sodium sulfate, reagent grade, granular anhydrous (Baker 3375, or equivalent), rinsed
with methylene chloride (20 mL/g), baked at 450°C for a minimum of 1 hour, cooled
in a desiccator, and stored in a pre-cleaned glass bottle with screw-cap which prevents
moisture from entering.
6.3.2 Sodium sulfate solution: 2% (w/v) in reagent water, pH-adjusted to 8.5 to 9.0 with
KOH or H2S04.
6.4 Solvents: Methylene chloride, hexane, ethyl ether, acetone, acetonitrile, isooctane, and meth-
anol; pesticide-quality; lot-certified to be free of interferences.
6.4.1 Ethyl ether must be shown to be free of peroxides before it is used, as indicated by
EM Laboratories Quant Test Strips (Scientific Products PI 126-8, or equivalent).
Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL of ethyl alcohol is added to each liter of ether as a preservative.
6.5 GPC calibration solution: Solution containing 300 mg/mL corn oil, 15 mg/mL bis (2-et-
hylhexyl) phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL
sulfur.
6.6 Sample cleanup.
6.6.1 Florisil: PR grade, 60/100 mesh, activated at 650 to 700°C, stored in the dark in
glass container with PTFE-lined screw-cap. Activate at 13Q°C for 16 hours minimum
immediately prior to use. Alternatively, 500-mg cartridges (J.T. Baker, or equivalent)
may be used.
6.6.2 Solid-phase extraction.
6.6.2.1 SPE cartridge calibration solution: 2,4,6-trichlorophenol, 0.1 ug/mL in
acetone.
6.6.2.2 SPE elution solvent: Methylene chloride:acetonitrile:hexane (50:3:47).
6.6.3 Alumina, neutral, Brockman Activity I, 80 to 200 mesh (Fisher Scientific Certified,
or equivalent). Heat for 16 hours at 400 to 450°C. Seal and cool to room temper-
ature. Add 7% (WAV) reagent water and mix for 10 to 12 hours. Keep bottle tightly
sealed.
6.6.4 Silicic acid, 100 mesh.
6.6.5 Sulfur removal: Mercury (triple-distilled), copper powder (bright, non-oxidized),
or TBA sodium sulfite. If mercury is used, observe the handling precautions in Section 4.
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Method 1656
6.7 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
6.8 High-solids reference matrix: Playground sand or similar material in which the compounds of
interest and interfering compounds are not detected by this method. May be prepared by
extraction with methylene chloride and/or baking at 450°C for 4 hours minimum.
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition.
If compound purity is 96% or greater, the weight may be used without correction to compute
the concentration of the standard. When not being used, standards are stored in the dark at
-20 to -10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the
level of the solution so that solvent evaporation loss can be detected. The vials are brought to
room temperature prior to use. Any precipitate is redissolved and solvent is added if solvent
loss has occurred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10 mg aldrin in a 10-mL ground-glass stoppered volumetric flask and fill
to the mark with isooctane. After the aldrin is completely dissolved, transfer the
solution to a 15-mL vial with PTFE-lined cap.
6.10.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.10.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.11 Secondary mixtures: Using stock solutions (Section 6.10), prepare mixtures at the levels
shown in Table 3 for calibration and calibration verification (Sections 7.3 and 14.5), for initial
and ongoing precision and recovery (Sections 8.2 and 14.6), and for spiking into the sample
matrix (Section 8.4).
6.12 Surrogate spiking solution: Prepare dibutyl chlorendate (DEC) at a concentration of 2 /ig/mL
in acetone.
NOTE: If DEC is not available, compounds such as tetrachloro-m-xylene or deca-
chlorobiphenyl may be used provided that the laboratory performs the tests described in
Section 8.2 using these compounds.
6.13 DDT and endrin decomposition solution: Prepare a solution containing endrin at a concen-
tration of 1 /xg/mL and DDT at a concentration of 2 /ig/mL.
6.14 Stability of solutions: All standard solutions (Sections 6.9 through 6.13) shall be analyzed
within 48 hours of preparation and on a monthly basis thereafter for signs of degradation.
Standards will remain acceptable if the peak area remains within ±15% of the area obtained in
the initial analysis of the standard.
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Method 1656
7. SETUP AND CALIBRATION
7.1 Configure the GC system as given in Section 5.9 and establish the operating conditions in
Table 2.
7.2 Attainment of method detection limit (MDL) and DDT/Endrin decomposition requirements:
Determine that each column/detector system meets the MDLs (Table 2), and the DDT and
Endrin decomposition test (Section 13.4).
7.3 Calibration.
7.3.1 Inj ection of calibration solutions.
7.3.1.1 Compounds with calibration data in Table 3: The compounds in each cali-
bration group in Table 3 were chosen so that each compound would be
separated from the others by approximately 1 minute on the primary col-
umn. The concentrations were chosen to bracket the working range of
either the BCD or the ELCD. However, because the response of the
ECLD is less for some compounds than that of the BCD, it may be neces-
sary to inject a larger volume of calibration solution when the ELCD is
used.
7.3.1.2 Compounds without calibration data in Table 3: Prepare calibration stan-
dards at a minimum of three concentration levels. One of these concen-
trations should be near, but above, the MDL (Table 2) and the other con-
centrations should define the working range of the detectors.
7.3.1.3 Set the automatic injector to inject a constant volume in the range of 0.5 to
5.0 pL of each calibration solution into the GC column/detector pairs, be-
ginning with the lowest level mixture and proceeding to the highest. For
each compound, compute and store, as a function of the concentration
injected, the retention time and peak area on each column/detector system
(primary and confirmatory). For the multi-componenent analytes (PCBs,
toxaphene), store the retention time and peak area for the five largest
peaks.
7.3.2 Retention time: The polar nature of some analytes causes the retention time to de-
crease as the quantity injected increases. To compensate this effect, the retention time
for compound identification is correlated with the analyte level.
7.3.2.1 If the difference between the maximum and minimum retention times for
any compound is less than 5 seconds over the calibration range, the reten-
tion time for that compound can be considered constant and an average
retention time may be used for compound identification.
7.3.2.2 Retention time calibration curve (retention time vs. amount): If the reten-
tion time for a compound in the lowest level standard is more than 5 sec-
onds greater than the retention time for the compound in the highest level
standard, a retention time calibration curve shall be used for identification
of that compound.
642
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Method 1656
7.3.3 Calibration factor (ratio of area to amount injected).
7.3.3.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
column/detector system.
7.3.3.2 Linearity: If the calibration factor for any compound is constant
(Cv < 20%) over the calibration range, an average calibration factor may
be used for that compound; otherwise, the complete calibration curve (area
vs. amount) for that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration
groups of compounds (Table 3), the GC systems are calibrated with combined solutions as a
final step. Not all of the compounds in these standards will be separated by the GC columns
used in this method. Retention times and calibration factors are verified for the compounds
that are resolved, and calibration factors are obtained for the unresolved peaks.
7.4.1 Analyze the combined QC standard on each column/detector pair.
7.4.1.1 For those compounds that exhibit a single, resolved GC peak, the retention
time shall be within ±5 seconds of the retention time of the peak in the
medium level calibration standard (Table 3), and the calibration factor
using the primary column shall be within ±20% of the calibration factor in
the medium level standard (Table 3).
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns (primary and confir-
matory), and also compute and store the calibration factors on both col-
umns. These results will be used for calibration verification (Section 13.2
and 13.5) and for precision and recovery studies (Sections 8.2 and 13.6).
7.5 Florisil calibration: The cleanup procedure in Section 11 utilizes Florisil column chroma-
tography. Florisil from different batches or sources may vary in adsorptive capacity. To
standardize the amount of Florisil that is used, the use of the lauric acid value (Reference 6)
is suggested. The referenced procedure determines the adsorption of lauric acid (in milligrams
per gram of Florisil) from hexane solution. The amount of Florisil to be used for each column
is calculated by dividing 110 by this ratio and multiplying by 20 g.
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program
(Reference 7). The minimum requirements of this program consist of an initial demonstration
of laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to samples
containing high solids with very little moisture (e.g., soils, compost), the high-solids reference
643
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Method 1656
matrix (Section 6.8) k substituted for reagent water (Section 6.7) in all performance tests, and
the high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of 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 analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If detection limits will be affected by the modification, the analyst is required to
repeat the demonstration of detection limits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to moni-
tor method performance. This test is described in Section 8.3. When results of these
spikes indicate atypical method performance for samples, the samples are diluted to
briag method performance within acceptable limits (Section 16).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 6.11) that the analysis system
is in control. These procedures are described in Sections 13.1, 13.5, and 13.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control limit for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
8.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four 1-L aliquots of reagent water spiked with the combined
QC standard (Section 6.11) according to the procedure in Section 10. Alternatively,
sets of four replicates of the individual calibration groups (Table 3) may be used. For
samples containing high solids, a set of four 30-g aliquots of the high-solids reference
matrix are used.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X)
and the coefficient of variation (Cv) of percent recovery (s) for each compound.
8.2.3 For each compound, compare s and X with the corresponding limits for initial preci-
sion and accuracy in Table 4. For coeluting compounds, use the coeluted compound
with the teast restrictive specification (largest Cv and widest range). If s and X for all
compound* meet the acceptance criteria, system performance is acceptable and anal-
ysis of blanks and samples may begin. If, however, any individual s exceeds the
precision limitor or any individual X falls outside the range for accuracy, system
performance is unacceptable for that compound. In this case, correct the problem and
repeat the test.
644
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Method 1656
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the DBC or other surrogate.
8.3.3 The surrogate recovery shall be 40 to 120%. If the recovery of the surrogate falls
outside of these limits, method performance is unacceptable for that sample, and the
sample is complex. Water samples are diluted, and smaller amounts of soils, sludges,
and sediments are reanalyzed per Section 16.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from
a given site type (e.g., influent to treatment, treated effluent, produced water, river sediment).
If only one sample from a given site type is analyzed, a separate aliquot of that sample shall be
spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows:
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in
the sample is being checked against a regulatory concentration limit, the
matrix spike shall be at that limit or at 1 to 5 times higher than the back-
ground concentration determined in Section 8.4.2, whichever concentration
is larger.
8.4.1.2 If the concentration of an analyte in the sample is not being checked
against a limit specific to that analyte, the matrix spike shall be at the
concentration of the combined QC standard (Table 3) or at 1 to 5 times
higher than the background concentration, whichever concentration is
larger.
8.4.1.3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Table 3).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyte. If necessary, prepare a standard solution appropriate to produce a level in
the sample 1 to 5 times the background concentration. Spike a second sample aliquot
with the standard solution and analyze it to determine the concentration after spiking
(A) of each analyte. Calculate the percent recovery (P) of each analyte:
Equation 1
p _ 100Q4-g)
T
where
T - True value of the spike
645
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Method 1656
8.4.3 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sam-
ple is complex and must be diluted and reanalyzed per Section 16.
8.4.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests
in Section 8.4.3, compute the average percent recovery (P) and the standard deviation
of the percent recovery (sp) for each compound (or coeluting compound group).
Express the accuracy assessment as a percent recovery interval from P - 2sp to
P + 2sp for each matrix. For example, if P=90% and sp= 10% for five analyses of
compost, the accuracy interval is expressed as 70 to 110%. Update the accuracy
assessment for each compound in each matrix on a regular basis (e.g., after each five
to ten new accuracy measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1-L reagent water blank or a 30-g high-solids reference
matrix blank with each sample batch (samples started through the extraction process
on the same 8-hour shift, to a maximum of 20 samples). Analyze the blank imme-
diately after analysis of the combined QC standard (Section 13.6) to demonstrate
freedom from contamination.
8.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound
is found in an aqueous blank at greater than 0.05 /*g/L, or in a high-solids reference
matrix blank at greater than 1 /ig/kg (assuming the same calibration factor as aldrin
for compounds not listed in Table 1), analysis of samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination at this
level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with
an analyte in the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery (sn)
for the new analyte, and measure the recovery and standard deviation of recovery for the other
analytes. The data for the new analyte is assumed to be valid if the precision and recovery
specifications for the other analytes are met; otherwise, the analytical problem is corrected and
the test is repeated. Establish a preliminary quality control limit of A + 2sn for the new
analyte and add the limit to Table 4.
8.7 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 13.5), and for initial (Section 8.2) and ongoing (Section 13.6)
precision and recovery should be identical, so that the most precise results will be obtained.
The GC instruments will provide the most reproducible results if dedicated to the settings and
conditions required for the analyses of the analytes given in this method.
646
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Method 1656
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes
of interest into samples may be required to assess the precision and accuracy of the sampling
and sample transporting techniques.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices (Reference 8),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples
which flow freely are collected in refrigerated bottles using automatic sampling equipment.
Solid samples are collected as grab samples using wide-mouth jars.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 9.0 using
sodium hydroxide or sulfuric acid solution. Record the volume of acid or base used. If resi-
dual chlorine is present in aqueous samples, add 80 mg sodium thiosul.fate per liter of water.
EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 9).
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRACTION AND CONCENTRATION
Samples containing 1 % solids or less are extracted directly using continuous liquid-liquid extraction
techniques (Section 10.2.1). Samples containing 1 to 30% solids are diluted to the 1% level with
reagent water and extracted using continuous liquid-liquid extraction techniques (Section 10.2.2).
Samples containing more than 30% solids are extracted using ultrasonic techniques (Section 10.2.5).
Figure 1 outlines the extraction and concentration steps.
10.1 Determination of percent solids .
10.1.1 Weigh 5 to 10 g of sample into a tared beaker. Record the weight to three significant
figures.
10.1 .2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a dessicator.
10.1 .3 Determine percent solids as follows:
Equation 2
% solids = Mis* of dry sample
weight of wet sample
10.2 Preparation of samples for extraction
10.2.1 Samples containing 1% solids or less: Extract the sample directly using continuous
liquid-liquid extraction techniques.
10.2.1 ,1 Measure 1.00 L (±0.01 L) of sample into a clean 1.5- to 2.0-L beaker.
10.2.1 .2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the
sample aliquot. Proceed to preparation of the QC aliquots for low solids
samples (Section 10.2.3).
647
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Method 1656
10.2.2 Samples containing 1 to 30% solids.
10.2.2.1 Mix sample thoroughly.
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1 % solids as fol-
lows:
Equation 3
sample weight = £_
% solids
10.2.2.3 Place the weight determined in Section 10.2.2.2 in a clean 1.5- to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior
to weighing.
10.2.2.4 Bring the volume of the sample aliquot(s) to 100 to 200 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into each
sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into
small pieces.
10.2.2.7 Place the %" horn on the ultrasonic probe approximately W below the
surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any
large pieces using the metal spatula or a stirring rod and repeat the sonica-
tion. Clean the probe with methylene chloride:acetone (1:1) between
samples to preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at
the same time, place two 1.0 L (±0.01 L) aliquots of reagent water in
clean 1.5- to 2.0-L beakers.
10.2.3.2 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into
one reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into the remaining reagent
water aliquot.
10.2.3.4 If a matrix spike is required, prepare an aliquot at the concentrations
specified in Section 8.4.
10.2.4 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Extract the samples
and QC aliquots per Section 10.3.
648
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Method 1656
10.2.5 Samples containing 30% solids or more.
10.2.5.1 Mix the sample thoroughly.
10.2.5.2 Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker. Discard all
sticks, rocks, leaves, and other foreign material prior to weighing.
10.2.5.3 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the
aliquot.
10.2.5.4 QC aliquot: For each sample or sample batch (to a maximum of 20) to be
extracted at the same time, place 30 g (±0.3 g) of the high-solids refer-
ence matrix in each of two clean 400- to 500-mL beakers.
10.2.5.5 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into
one aliquot of the high-solids reference matrix.
10.2.5.6 Spike the combined QC standard (Section 6.11) into the remaining high-
solids reference matrix aliquot. Extract the high-solids samples per Sec-
tion 10.4.
10.3 Continuous extraction of low-solids (aqueous) samples: Place 100 to 150 mL methylene
chloride in each continuous extractor and 200 to 300 mL in each distilling flask.
10.3.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass
containers with 50 to 100 mL methylene chloride and add to the respective extractors.
Include all solids in the extraction process.
10.3.2 Extraction: Adjust the pH of the waters in the extractors to 5 to 9 with NaOH or
H2SO4 while monitoring with a pH meter. Caution: Some samples require acidifica-
tion in a hood because of the potential for generating hydrogen sulfide.
10.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling.
When properly adjusted, one to two drops of methylene chloride per second will fall
from the condenser tip into the water. Test and adjust the pH of the waters during the
first 1 to 2 hours of extraction. Extract for 18 to 24 hours.
10.3.4 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 mL), and pour the contents through a prerinsed drying column containing 7 to
10 cm of anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of
methylene chloride and pour through the drying column. For extracts to be cleaned
up using GPC, collect the solution in a 500-mL K-D evaporator flask equipped with a
10-mL concentrator tube. Seal, label the pesticide and herbicide fractions, and con-
centrate per Sections 10.5 to 10.6.
10.4 Ultrasonic extraction of high solids samples: Procedures are provided for extraction of non-
municipal sludge (Section 10.4.1) and municipal sludge samples (Section 10.4.2).
10.4.1 Ultrasonic extraction of non-municipal sludge high-solids aliquots.
10.4.1.1 Add 60 to 70 g of powdered sodium sulfate to the sample and QC aliquots.
Mix each aliquot thoroughly. Some wet sludge samples may require more
than 70 g for complete removal of water. All water must be removed
prior to addition of organic solvent so that the extraction process is ef-
ficient.
649
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Method 1656
10.4.1.2 Add 100 mL (±10 mL) of acetone:methylene chloride (1:1) to each of the
aliquots and mix thoroughly.
10.4.1.3 Place the % " horn on the ultrasonic probe approximately Vi" below the
surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and
repeat the sonication.
10.4.1.4 Decant the extract through a prerinsed drying column containing 7 to
10 cm anhydrous sodium sulfate into a 500- to 1000-mL graduated cylin-
ders.
10.4.1.5 Repeat the extraction steps (Sections 10.4.1.3 to 10.4.1.4) twice more for
each sample and QC aliquot. On the final extraction, swirl the sample or
QC aliquot, pour into its respective drying column, and rinse with acetone:
methylene chloride. Record the total extract volume. If necessary, trans-
fer the extract to a centrifuge tube and centrifuge for 10 minutes to settle
fine particles.
10.4.2 Ultrasonic extraction of high-solids municipal sludge aliquots.
10.4.2.1 Add 100 mL (± 10 mL) of acetonitrile to each of the aliquots and mix
thoroughly.
10.4.2.2 Place the 3A" horn on the ultrasonic probe approximately W below the
surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and
repeat the sonication.
10.4.2.3 Decant the extract through filter paper into a 1000- to 2000-mL separatory
funnel.
10.4.2.4 Repeat the extraction and filtration steps (Sections 10.4.2.2 to 10.4.2.3)
using a second 100 mL (± 10 mL) of acetonitrile.
10.4.2.5 Repeat the extraction step (Section 10.4.2.3) using 100 mL (±10 mL) of
methylene chloride. On this final extraction, swirl the sample or QC
aliquot, pour into its respective filter paper, and rinse with methylene
chloride. Record the total extract volume.
10.4.2.6 For each extract, prepare 1.5 to 2 L of reagent water containing 2% sodi-
um sulfate. Adjust the pH of the water to 6.0 to 9.0 with NaOH or H2SO4.
10.4.2.7 Back-extract each extract three times sequentially with 500 mL of the
aqueous sodium sulfate solution, returning the bottom (organic) layer to the
separatory funnel the first two times while discarding the top (aqueous)
layer. On the final back extraction, filter each pesticide extract through a
prerinsed drying column containing 7 to 10 cm anhydrous sodium sulfate
into a 500- to 1000-mL graduated cylinder. Record the final extract vol-
ume.
650
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Method 1656
10.4.3 For extracts to be cleaned up using GPC, filter these extracts through Whatman #41
paper into a 500-mL K-D evaporator flask equipped with a 10-mL concentrator tube.
Rinse the graduated cylinder or centrifuge tube with 30 to 50 mL of methylene chlo-
ride and pour through filter to complete the transfer. Seal and label the K-D flasks.
Concentrate these fractions per Sections 10.5 through 10.8.
10.5 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL concen-
trator tubes. Add one to two clean boiling chips to the flask and attach a three-ball
macro Snyder column. Prewet the column by adding approximately 1 mL of methy-
lene chloride through the top. Place the K-D apparatus in a hot water bath so that the
entire lower rounded surface of the flask is bathed with steam. Adjust the vertical
position of the apparatus and the water temperature as required to complete the con-
centration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood.
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus
from the bath and allow the solvent to drain and cool for at least 10 minutes.
10.5.3 If the extract is to be cleaned up using GPC, remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chlo-
ride. A 5-mL syringe is recommended for this operation. Adjust the final volume to
10 mL and proceed to GPC cleanup in Section 11.
10.6 Hexane exchange: Extracts to be subjected to Florisil or silica gel cleanup and extracts that
have been cleaned up are exchanged into hexane.
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
chip, and reattach the Snyder column. Concentrate the extract as in Section 10.5
except use hexane to prewet the column. The elapsed time of the concentration
should be 5 to 10 minutes.
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the concentra-
tor tube with 1 to 2 mL of hexane. Adjust the final volume of extracts that have
not been cleaned up by GPC to 10 mL and those that have been cleaned up by GPC
to 5 mL (the difference accounts for the 50% loss in the GPC cleanup). Clean up the
extracts using the Florisil, silica gel, and/or sulfur removal procedures in Section 11.
7 7. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents,
ground water, drinking water). If particular circumstances require the use of a cleanup
procedure, the analyst may use any or all of the procedures below or any other appropriate
procedure. However, the analyst shall first repeat the tests in Section 8.2 to demonstrate that
the requirements of Section 8.2 can be met using the cleanup procedure(s) as an integral part
of the method. Figure 1 outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
interferents that cause GC column performance to degrade. It is used for all soil and
651
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Method 1656
sediment extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols.
11.1.3 The Florisil column (Section 11.4) allows for selected fractionation of the organo-
chlorine compounds and will also eliminate polar interferences.
11.1.4 Alumina column cleanup (Section 11.5) may also be used for cleanup of the organo-
chlorine compounds.
11.1.5 Elemental sulfur, which interferes with the electron capture gas chromatography of
some of the pesticides, is removed using GPC, mercury, or activated copper. Sulfur
removal (Section 11.6) is required when sulfur is known or suspected to be present.
11.2 Gel permeation chromatography (GPC).
11.2.1 Column packing.
11.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight
(12 hours minimum).
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 5.5 mL/min prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis (2-ethylhexyl) phthalate, pen-
tachlorophenol, perylene, and sulfur.
11.2.2.3 Set the "dump time" to allow greater than 85% removal of the corn oil and
greater than 85% collection of the phthalate.
11.2.2.4 Set the "collect time" to the peak minimum between perylene and sulfur.
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachlorophenol is greater
than 85%. If calibration is not verified, the system shall be recalibrated
using the calibration solution, and the previous 20 samples shall be re-
extracted and cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: GPC requires that the column not be overloaded. The column
specified in this method is designed to handle a maximum of 0.5 g of high molecular
weight material in a 5-mL extract. If the extract is known or expected to contain
more than 0.5 g, the extract is split into fractions for GPC and the fractions are
652
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Method 1656
combined after elution from the column. The solids content of the extract may be
obtained gravimetrically by evaporating the solvent from a 50-^iL aliquot.
11.2.3.1 Filter the extract or load through the filter holder to remove particulates.
Load the 5.0-mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange into hexane per Sections 10.5
and 10.6. Adjust the final volume to 5.0 mL.
11.3 Solid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with
the trap and gauge installed between the manifold and vacuum source.
11.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge washing: Pre-elute each cartridge prior to use sequentially with 10-mL
portions each of hexane, methanol, and water using vacuum for 30 seconds after each
eluant. Follow this pre-elution with 1 mL methylene chloride and three 10-mL por-
tions of the elution solvent (Section 6.6.2.2) using vacuum for 5 minutes after each
eluant. Tap the cartridge lightly while under vacuum to dry between eluants. The
three portions of elution solvent may be collected and used as a blank if desired.
Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum
for 30 seconds after each eluant.
11.3.3 Cartridge certification: Each cartridge lot must be certified to ensure recovery of the
compounds of interest and removal of 2,4,6-trichIorophenol.
11.3.3.1 To make the test mixture, add the trichlorophenol solution (Section
6.6.2.1) to the combined calibration standard (Section 6.11). Elute the
mixture using the procedure in Section 11.3.4.
11.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 fiL of the concentrated
eluant into the GC using the procedure in Section 13. The recovery of all
analytes (including the unresolved GC peaks) shall be within the ranges for
recovery specified in Table 4, and the peak for trichlorophenol shall not be
detectable; otherwise the SPE cartridge is not performing properly and the
cartridge lot shall be rejected.
11.3.4 Extract cleanup.
11.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack containing the 50-mL volumetric flasks (Section 5.6.2.4) in the vac-
uum manifold. Re-establish the vacuum at 5 to 10 psia.
653
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Method 1656
11.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE
cartridge. Apply vacuum for 5 minutes to dry the cartridge. Tap gently
to aid in drying.
11.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 min-
utes after each portion. Collect the eluants in the 50-mL volumetric flasks.
11.3.4.4 Release the vacuum and remove the 50-mL volumetric flasks.
11.3.4.5 Concentrate the eluted extracts to 1.0 mL using the nitrogen blow-down
apparatus.
11.4 Florisil column.
11.4.1 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5)
in a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm
of anhydrous sodium sulfate to the top.
11.4.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate layer to the air, stop the elution of the hexane by
closing the stopcock on the chromatographic column. Discard the eluate.
11.4.3 Transfer the concentrated extract (Section 10.6.2) onto the column. Complete the
transfer with two 1-mL hexane rinses.
11.4.4 Place a clean 500-mL K-D flask and concentrator tube under the column. Drain the
column into the flask until the sodium sulfate layer is nearly exposed. Elute Frac-
tion 1 with 200 mL of 6% (v/v) ethyl ether in hexane at a rate of approximately
5 mL/min. Remove the K-D flask. Elute Fraction 2 with 200 mL of 15% (v/v) ethyl
ether in hexane into a second K-D flask. Elute Fraction 3 with 200 mL of 50% (v/v)
ethyl ether in hexane.
11.4.5 Concentrate the fractions as in Section 10.6, except use hexane to prewet the column.
Readjust the final volume to 5 or 10 mL as in Section 10.6, depending on whether the
extract was subjected to GPC cleanup, and analyze by gas chromatography per the
procedure in Section 12.
11.5 Alumina column.
11.5.1 Reduce the volume of the extract to 0.5 mL and bring to 1.0 mL with acetone.
11.5.2 Add 3 g of activity III neutral alumina to a 10-mL chromatographic column. Tap the
column to settle the alumina.
11.5.3 Transfer the extract to the top of the column and collect the eluate in a clean 10-mL
concentrator tube. Rinse the extract container with 1 to 2 mL portions of hexane (to a
total volume of 9 mL) and add to the alumina column. Do not allow the column to
go dry.
11.5.4 Concentrate the extract to 1.0 mL if sulfur is to be removed, or adjust the final
volume to 5 or 10 mL as in Section 10.6, depending on whether the extract was
subjected to GPC cleanup, and analyze by gas chromatography per Section 13.
654
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Method 1656
11.6 Sulfur removal: Elemental sulfur will usually elute entirely in Fraction 1 of the Florisil
column cleanup.
11.6.1 Transfer the concentrated extract into a clean concentrator tube or PTFE-sealed vial.
Add 1 to 2 drops of mercury or 100 mg of activated copper powder and seal (Refer-
ence 10). If TBA sulfite is used, add 1 mL of the TEA sulfite reagent and 2 mL of
isopropanol.
11.6.2 Agitate the contents of the vial for 1 to 2 hours on a reciprocal shaker. If the mer-
cury or copper appears shiny, or if precipitated sodium sulfite crystals from the TBA
sulfite reagent are present, and if the color remains unchanged, all sulfur has been
removed; if not, repeat the addition and shaking.
11.6.2.1 If mercury or copper is used, centrifuge and filter the extract to remove all
residual mercury or copper. Dispose of the mercury waste properly.
Bring the final volume to 1.0 mL and analyze by gas chromatography per
the procedure in Section 13.
11.6.2.2 If TBA sulfite is used, add 5 mL of reagent water and shake for 1 to
2 minutes. Centrifuge and filter the extract to remove all precipitate.
Transfer the hexane (top) layer to a sample vial and adjust the final volume
to 5 or 10 mL as in Section 10.6, depending on whether the extract was
subjected to GPC cleanup, and analyze by gas chromatography per Seer
tion 12.
12. GAS CHROMATOGRAPHY
Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in
these tables are the retention times and minimum levels that can be achieved under these conditions.
Examples of the separations achieved by the primary and confirmatory columns are shown in Fig-
ure 2.
12.1 Calibrate the system as described in Section 7.
12.2 Set the auto-sampler to inject the same volume that was chosen for calibration (Section
7.3.1.3) for all standards and extracts of blanks and samples.
12.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection
after the last analyte is expected to elute and to return the column to the initial temperature.
13. SYSTEM AND LABORA TORY PERFORMANCE
13.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified for all pollutants and surrogates on both column/
detector systems. For these tests, analysis of the combined QC standard (Section 6.11) shall
be used to verify all performance criteria. Adjustment and/or recalibration (per Section 7)
shall be performed until all performance criteria are met. Only after all performance criteria
are met may samples, blanks, and precision and recovery standards be analyzed.
13.2 Retention times: The absolute retention times of the peak maxima shall be within ±10
seconds of the retention times in the initial calibration (Section 7.4.1).
655
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Method 1656
13.3 GC resolution: Resoktfk* ia acceptable if the valley height between two peaks (as measured
from the baseline) is less than 10% of the taller of the two peaks.
13.3.1 Primary column (D8-608): DDT and endrin aldehyde.
13.3.2 Confirmatory column (DB-1701): Alpha and gamma chlordane.
13.4 Decomposition of DDT and endrin.
13.4.1 Analyze a total of 2 ng DDT and 1 ng endrin on each column using the analytical
conditions specified in Table 2.
13.4.2 Measure the total area of all peaks in the chromatogram.
13.4.3 The area of peaks other than the sum of the areas of the DDT and endrin peaks shall
be less than 20% the sum of the areas of these two peaks. If the area is greater than
this sum, the system is not performing acceptably for DDT and endrin. In this case,
the GC system that failed shall be repaired and the performance tests (Sections 13.1 to
13.4) shall be repeated until the specification is met.
NOTE: DDT and endrin decomposition are usually caused by accumulations ofpar-
ticulates in the injector and in the front end of the column. Cleaning and silanizing the
injection port liner, and breaking off a short section of the front end of the column will
usually eliminate the decomposition problem.
13.5 Calibration verification: Calibration is verified for the combined QC standard only.
13.5.1 Inject the combined QC standard (Section 6.11)
13.5.2 Compute the percent recovery of each compound or coeluting compounds, based on
the calibration data (Section 7.4).
13.5.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing accuracy in Table 4. For co-
eluting compounds, use the coeluted compound with the least restrictive specification
(the widest range). If the recoveries for all compounds meet the acceptance criteria,
system performance is acceptable and analysis of blanks and samples may begin. If,
however, aay recovery falls outside the calibration verification range, system perfor-
mance is unacceptable for that compound. In this case, correct the problem and
repeat the test, or recalibrate (Section 7). If verification requirements are met, the
calibration is assumed to be valid for the multicomponent analytes (PCBs and toxa-
phene).
13.6 Ongoing precision and recovery.
13.6.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Sections 10.2.3.3 and 10.2.5.7).
13.6.2 Compute the percent recovery of each analyte and for coeluting compounds.
13.6.3 For each compound or coeluted compound, compare the percent recovery with the
limits for ongoing recovery in Table 4. For coeluted compounds, use the coeluted
compound with the least restrictive specification (widest range). If all analytes pass,
the extraction, concentration, and cleanup processes are in control and analysis of
656
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Method 1656
blanks and samples may proceed. If, however, any of the analytes fail, these proces-
ses are not in control. In this event, correct the problem, re-extract the sample batch,
and repeat the ongoing precision and recovery test.
13.6.4 Add results which pass the specifications in Section 13.6.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued labor-
atory performance. Develop a statement of laboratory data quality for each analyte by
calcualting the average percent recovery (R) and the standard deviation of percent
recovery sr. Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
For example, if R=95% and sr=5%, the accuracy is 85 to 105%.
14. QUALITATIVE DETERMINATION
14.1 Quantitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 13.2), and with data stored in the
retention-time and calibration libraries (Section 7.3.2 and 7.3.3.2). Identification is confirmed
when retention time and amounts agree per the criteria below.
14.2 For each compound on each column/detector system, establish a retention-time window of
3 RSD on either side of the average retention time in the calibration data (Section 7.3.2).
For compounds that have a retention-time curve (Section 7.3.2.2), establish this window as
the minimum -10 seconds and maximum +10 seconds. For the multi-component analytes, use
the retention times of the five largest peaks in the chromatogram from the calibration data
(Section 7.3.2).
14.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the
analysis of a sample or blank is within a window (as defined in Section 14.2) on the
primary column/detector system, it is considered tentatively identified. A tentatively
identified compound is confirmed when (1) the retention time for the compound on
the confirmatory column/detector system is within the retention-time window on that
system, and (2) the computed amounts (Section 15) on each system (primary and
confirmatory) agree within a factor of three.
14.2.2 Compounds requiring a retention-time calibration curve: If a peak from the analysis
of a sample or blank is within a window (as defined in Section 14.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention times on both systems (primary and
confirmatory) are within ±10 seconds of the retention times for the computed
amounts (Section 15), as determined by the retention-time calibration curve (Section
7.3.2.2), and (2) the computed amounts (Section 15) on each system (primary and
confirmatory) agree within a factor of 3.
15. QUANTITA TIVE DETERMINA TION
15.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
657
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Method 1656
1 5.2 Liquid samples: Compute the concentration in the sample using the following equation:
Equation 4
(V,)
where
Cs = Concentration in the sample, in
10 = Final extract total volume, in mL
Cex = Concentration in the extract, in
Vs = Sample extracted, in L
15.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
(C >
C. = 10-
lOOO(Ws)(solids)
where
Cs = Concentration in the sample, in pg/kg
10 = Final extract total volume, in mL
CK = Concentration in the extract, in (iglmL
1000 = Conversion factor, g to kg
Ws = Sample weight, in g
solids = Percent solids in Section 10.1.3 divided by 100
15.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1-fiL aliquot of the diluted extract is analyzed.
15.5 Two or more PCBs in a given sample are quantitated and reported as total PCS.
15.6 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at
which the concentration is in the calibration range.
16. ANALYSIS OF COMPLEX SAMPLES
16.1 Some samples may contain high levels (greater than 1000 ng/L) of the compounds of interest,
interfering compounds, and/or polymeric materials. Some samples may not concentrate to
10 mL (Section 10.6); others may overload the GC column and/or detector.
16.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), the SPE cart-
ridge (Section 11.3), by Florisil (Section 11.4) or Alumina (Section 11.5), and sulfur removal
(Section 11.6). If these techniques do not remove the interfering compounds, the extract is
diluted by a factor of 10 and reanalyzed (Section 15.4).
658
-------
Method 1656
16.3 Recovery of surrogate: In most samples, surrogate recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3.3, the sample shall be reextracted and reanalyzed. If the
surrogate recovery is still outside this range, (he extract is diluted by a factor of 10 and
reanalyzed (Section 15.4).
16.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those
from reagent water or from the high-solids reference matrix. If the matrix spike recovery is
outside the range specified in Table 4, the sample shall be diluted by a factor of 10, respiked,
and reanalyzed. If the matrix spike recovery is still outside the range, the method may not
apply to the sample being analyzed and the result may not be reported for regulatory compli-
ance purposes.
17. METHOD PERFORMANCE
17.1 Development of this method is detailed in References 11 and 12.
659
-------
Method 1656
References
1. "Guideline Establishing Test Procedures for the Analysis of Pollutants under the Clean Water
Act; Final Rule and Interim Final Rule and Proposed Rule," 40 CFR Part 136.
2. "Methods for the Determination of Organic Compounds in Drinking Water," U.S. Environ-
mental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinatti, Ohio:
EPA-600/4-88/039, December 1988.
3. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
4. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
6. Mills, P. A., "Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capa-
city and Its Use in Standardizing Florisil Columns," Journal of the Association of Official
Analytical Chemists, 51, 29: 1968.
7. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
8. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
9. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
10. Goerlitz, D.F., and Law, L.M., "Bulletin for Environmental Contamination and Toxicology":
6, 9, 1971.
11. "Consolidated GC Method for the Determination of ITD/RCRA Pesticides using Selective GC
Detectors," S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-
-01, Document R70, September 1986.
12. "Method Development and Validation, EPA Method 1618, Cleanup Procedures," Pesticide
Center, Department of Environmental Health, Colorado State University: November 1988 and
January 1989.
660
-------
Method 1656
Table 1 . Organo-Halide Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with
EPA EGO
089
102
103
104
105
434
433
441
091
431
094
093
092
432
478
090
095
096
097
098
099
435
100
101
437
439
Compound
Acephate
Alachlor
Aldrin
Atrazine
Benfluralin (Benefin)
a-BHC
/3-BHC
-y-BHC (Lindane)
5-BHC
Bromacil
Bromoxynil octanoate
Butachlor
Captafol
Captan
Carbophenothion (Trithion)
a-Chlordane (cis-Chlordane)
•y-Chlordane (trans-Chlordane)
Chlorobenzilate
Chloroneb (Terraneb)
Chloropropyiate (Acaralate)
Chlorothalonil
DBCP (Dibromochloropropane)
DCPA (Dacthal)
4,4'-DDD (IDE)
4,4'-DDE
4,4'-DDT
Diallate (Avadex)
Dichlone
Dicofol
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Endrin ketone
Ethalfluralin (Sonalan)
Etridiazole
Fenarimol (Rubigan)
Heptachlor
Heptachlor epoxide
Isodrin
Isopropalin (Paarlan)
Kepone
Halide-Specific Detector
CAS Registry
30560-19-1
15972-60-8
309-00-2
1912-24-9
1861-40-1
319-84-6
319-85-7
58-89-9
319-86-8
314-40-9
1689-99-2
23184-66-9
2425-06-1
133-06-2
786-19-6
5103-71-9
5103-74-2
510-15-6
2675-77-6
5836-10-2
1897-45-6
96-12-8
1861-32-1
72-54-8
72-55-9
50-29-3
2303-16-4
1 1 7-80-6
115-32-2
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
53494-70-5
55283-68-6
2593-15-9
60168-88-9
76-44-8
1024-57-3
465-73-6
33820-53-0
1 43-50-0
661
-------
Method 1656
Table 1. Organo-Halide Pesticides Determined by Large-Bore, Fused-Silica Cap-
illary Column Gas Chromatography with Halide-Specific Detector
(cont.)
EPA EGO
430
438
436
112
108
109
106
110
107
111
440
113
442
Compound
Methoxychlor
Metribuzin
Mirex
Nitrofen (TOK)
Norfluorazon
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
PCNB (pentachloronitrobenzene)
Pendamethalin (Prowl)
cis-Permethrin
trans-Permethrin
Perthane (Ethylan)
Propachlor
Propanil
Propazine
Simazine
Strobane
Terbacil
Terbuthylazine
Toxaphene
Triadimefon (Bayleton)
Trifluralin
CAS Registry
72-43-5
21087-64-9
2385-85-5
1836-75-5
27314-13-2
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
82-68-8
40487-42-1
61949-76-6
61949-77-7
72-56-0
1918-16-7
709-98-8
139-40-2
122-34-9
8001-50-1
5902-51-2
5915-41-3
8001-35-2
43121-43-3
1582-09-8
562
-------
Method 1656
Table 2.
Gas Chromatography of Organo-Halide Pesticides
Retention Time (min)1
EPA EGO
442
432
102
440
104
103
100
478
105
089
437
101
091
095
093
090
433
431
098
436
439
094
096
092
441
099
097
434
Compound
Acephate
Trifluralin
Ethalfluralin
Benfluralin
Diallate-A
Diallate-B
a-BHC
PCNB
Simazine
Atrazine
Terbuthylazine
7-BHC (Lindane)
/3-BHC
Heptachlor
Chlorothalonil
Dichlone
Terbacil
5-BHC
Alachlor
Propanil
Aldrin
DCPA
Metribuzin
Triadimefon
Isopropalin
Isodrin
Heptachlor epoxide
Pendamethalin
Bromacil
7-Chlordane
Butachlor
a-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Captan
Chlorobenzilate
Endrin
Nitrofen (TOK)
Kepone
4,4'-DDD
Endosulfan II
Bromoxynil octanoate
4,4'-DDT
Carbophenothion
Endrin aldehyde
Endosulfan sulfate
Captafol
DB-608
5.03
5.16
5.28
5.53
7.15
7.42
8.14
9.03
9.06
9.12
9.17
9.52
9.86
10.66
10.66
10.80
11.11
11.20
11.57
11.60
11.84
12.18
12.80
12.99
13.06
13.47
13.97
14.21
14.39
14.63
15.03
15.24
15.25
16.34
16.41
16.83
17.58
17.80
17.86
17.92
18.43
18.45
18.85
19.48
19.65
19.72
20.21
22.51
DB-1701
3
6.79
6.49
6.87
6.23
6.77
7.44
7.58
9.29
9.12
9.46
9.91
11.90
10.55
10.96
__3
12.63
12.98
11.06
14.10
11.46
12.09
11.68
13.57
13.37
11.12
12.56
13.46
__3
14.20
15.69
14.36
13.87
14.84
15.25
15.43
17.28
15.86
17.47
24.03
17.77
18.57
18.57
18.32
18.21
19.18
20.37
21.22
Method Detection
Limit1
fng/U
2000
50
5
20
45
32
6
6
400
500
300
11
7
5
15
__4
200
5
20
8
3
5
50
20
13
12
30
70
9
30
8
11
10
6
100
25
4
13
100
5
8
30
12
50
11
7
100
est (ECD)
est
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
est (ECD)
663
-------
Method 1656
Table 2. Gas Chromatography of Organo-Halide Pesticides (cont.
Retention Time fm/n)1
Method Detection
EPA EGD
438
430
435
106
109
112
108
110
107
111
113
Notes:
1.
2.
3.
4.
Compound
Norfluorazon
Mirex
Methoxychlor
Endrin ketone
Fenarimol
cis-Permethrin
trans-Permethrin
PCB-1242
PCB-1232
PCB-1016
PCB-1221
PCB-1248
PCB-1254
PCB-1260
Toxaphene
DB-608
20.68
22.75
22.80
23.00
24.53
25.00
25.62
15.44
15.73
16.94
17.28
19.17
16.60
17.37
18.11
19.46
19.69
DB-1701
22.01
19.79
20.68
21.79
23.79
23.59
23.92
14.64
15.36
16.53
18.70
19.92
16.60
17.52
17.92
18.73
19.00
fng/U
50 est (ECD)
4
30
8
20 est (ECD)
200 est (ECD)
200 est (ECD)
1 50 est
1 50 est
1 50 est
150 est
1 50 est
1 50 est
140
910
Columns: 30 m long x 0.53 mm ID; DB-608: 0.83 //; DB-1701: 1.0 ;/. Conditions sug-
gested to meet retention times shown: 150°C for 0.5 minutes, 150 to 270° at 5°C/min,
270°C until trans-permethrin elutes. Carrier gas flow rates approximately 7 mL/min.
40 CFR Part 136, Appendix B (49 FR 43234). MDLs were obtained by a single laboratory
with an electrolytic conductivity detector, except as noted. MDL's for soils (in ng/kg) are
estimated to be 30 to 100 times this level.
Does not elute from DB-1701 column at level tested.
Not recovered from water at the levels tested.
664
-------
Method 1656
Table 3. Concentrations of Calibration Solutions for Electron Capture Detector
and Suggested Calibration Groups
Concentration (ng/mL)
EPA EGD Compound1
Calibration group 1
103
434
432
097
098
437
Acephate
Alachlor
Atrazine
/3-BHC
Bromoxynil octanoate
Captafol
Diallate
Endosulfan sulfate
Endrin
Isodrin
Pendimethalin (Prowl)
trans-Permethrin
Calibration group 2
102
093
092
478
430
a-BHC
DCPA
4,4'-DDE
4,4'-DDT
Dichlone
Ethalfluralin
Fenarimol
Methoxychlor
Metribuzin
Calibration group 3
105
091
435
101
436
440
442
•y-BHC (Lindane)
7-Chlordane
Endrin ketone
Heptachlor epoxide
Isopropalin
Nitrofen (TDK)
PCNB
cis-Permethrin
Trifluralin
Calibration group 4
431
090
095
438
Benfluralin
Chlorobenzilate
Dieldrin
Endosulfan I
Mirex
Terbacil
Terbuthylazine
Triadimefon
Low
2000
20
1000
10
50
200
200
10
20
10
50
200
5.0
5.0
10
10
20
10
20
20
10
5
5
10
5
20
20
5
200
10
20
50
5
10
20
200
500
100
Medium |
10000
100
5000
50
250
1000
1000
50
' 100
50
250
1000
25
25
50
50
100
50
100
100
50
25
25
50
25
100
100
25
1000
50
100
500
20
50
100
1000
2500
500
High
40000
400
20000
200
1000
4000
4000
200
400
200
1000
4000
100
100
200
200
400
200
400
400
200
100
100
200
100
400
400
100
4000
200
400
5000
100
200
400
4000
10000
2000
665
-------
Method 1656
Table 3. Concentrations of Calibration Solutions for Electron Capture Detector
and Suggested Calibration Groups (cont.)
EPA EGD Compound1
Calibration group 5
a-Chlordane
433 Captan
Chlorothalonil
094 4,4'-DDD
Norfluorazon
Simazine
Calibration group 6
089 Aldrin
104 6-BHC
Bromacil
Butachlor
096 Endosulfan II
100 Heptachlor
439 Kepone
Concentration (ng/mL)
Low
10
100
20
20
100
800
20
5
100
50
10
10
100
Medium
50
500
100
100
500
4000
100
25
500
250
50
50
500
High
200
2000
400
400
2000
20000
400
100
2000
1000
200
200
2000
For compounds listed in Table 2 that are not listed in this table, determine appropriate
ranges for calibration standards.
666
-------
Method 1656
Table 4.
EGD
No.
089
102
103
105
104
434
433
441
091
431
094
093
092
432
478
090
095
096
097
098
099
435
100
101
437
439
Acceptance Criteria for Performance
Compounds
Compound
Acephate
Alachlor
Aldrin
Atrazine
Benfluralin
a-BHC
0-BHC
6-BHC
•y-BHC (Lindane)
Bromacil
Bromoxynil octanoate
Butachlor
Captafol
Captan
Carbophenothion
Chlordane-a
Chlordane-7
Chlorobenzilate
Chlorothalonil
DCPA
4,4'-DDD
4,4'-DDE
4,4'-DDT
Diallate
Dichlone
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Endrin ketone
Ethalfluralin
Fenarimol
Heptachlor
Heptachlor epoxide
Isodrin
Isopropalin
Kepone
Spike
Level
(ng/U
100000
1000
1000
50000
1000
250
500
250
250
5000
2500
2500
10000
5000
1000
500
250
5000
1000
250
1000
500
500
10000
1000
200
500
500
500
1000
1000
500
500
1000
500
250
500
1000
5000
Tests for Organo-Halide
Acceptance Criteria
Initial
Precision
and Accuracy
s
94
20
12
26
22
10
10
24
10
84
28
32
76
32
10
10
13
19
20
20
12
13
19
16
20
11
14
19
17
13
13
25
24
26
12
13
15
20
46
X
0-195
26-100
82-108
35-129
45-125
57-135
66-130
60-122
66-112
0-263
31-131
21-137
0-221
28-144
63-141
79-122
32-140
58-118
37-109
57-129
69-117
66-114
86-112
44-120
45-117
66-140
41-133
78-142
50-130
17-149
0-149
36-126
46-132
46-140
78-104
63-117
69-113
47-129
31-197
Calibration
Verifi-
cation*
(%)
6-194
80-120
79-113
74-126
78-122
69-108
85-102
79-103
75-119
16-184
72-128
68-132
24-176
49-114
79-102
73-102
79-113
54-129
80-120
80-120
77-109
81-121
77-118
70-124
79-110
48-115
78-119
76-119
70-109
5-117
86-117
68-135
76-124
74-126
80-114
79-117
71-126
80-120
47-134
Recovery/
Ongoing
Accuracy
R(%)
0-209
23-101
76-114
31-132
42-128
38-154
50-146
45-136
55-123
0-275
27-135
17-141
0-232
24-148
43-161
69-133
4-169
43-133
34-112
54-132
57-129
54-126
79-119
24-139
42-120
48-158
18-156
62-158
31-149
0-182
0-190
14-148
42-136
42-144
71-111
49-131
45-127
54-132
25-203
667
-------
Method 1656
Table 4. Acceptance Criteria for Performance Tests for Organo-Halide Com-
pounds (cont.)
Acceptance Criteria
EGD
No. Compound
430 Methoxychlor
Metribuzin
438 Mirex
436 Nitrofen (TOK)
Norfluorazon
112 PCB-1016
108 PCB-1221
109 PCB-1232
106 PCB-1242
110 PCB-1248
107 PCB-1254
111 PCB-1260
440 PCNB
Pendimethalin
cis-Permethrin
trans-Permethrin
Simazine
Terbacil
Terbuthylazine
113 Toxaphene
Triadimefon
442 Trifluralin
Spike
Level
(ng/L)
1000
500
1000
1000
5000
1000
250
2500
10000
10000
40000
10000
25000
5000
5000
500
Initial
Precision
and Accuracy
s X
19 50-136
24 54-140
23 25-155
22 15-139
20 71-143
20 82-112
11 49-129
24 32-118
30 45-153
20 59-131
20 16-100
82 0-217
20 32-104
20 82-112
54 32-104
12 32-148
Calibration
Verifi-
cation*
47-128
76-124
78-114
59-142
80-120
79-126
78-101
76-124
70-130
80-120
80-120
18-182
80-120
68-134
80-120
47-134
Recovery/
Ongoing
Accuracy
28-158
50-155
0-188
0-170
68-146
75-119
29-149
28-122
41-157
56-134
13-101
0-228
29-107
76-122
0-107
3-177
* Verified at the level of the median standard in Table 3.
668
-------
Method 1656
Percent Solids
< 30% Solids
> 30% Solids
Oil. To 1% Solids
CH2 CI2 LiqVLiq. Ext.
Concentrate
1
To Cleanup
ACN & CH2CI2 Sonic
H2O Back-Extract
Concentrate
I
To Cleanup
Extraction and Concentraction Steps
From Extraction
Gel Permeation
Solid Phase Ext.
Florisil
I
Remove Sulfur
GC/HSD
Cleanup and Analysis Steps
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
669
-------
Method 1656
(B)
uiiiiiiiiiiimii
(A)
1
56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Retention Time (minutes)
A52-002-87
Figure 2. Gas Chromatogram of Selected Organo-Chlorine Compounds
670
-------
Method 1657
The Determination of
Organo-Phosphorus Pesticides
in Municipal and Industrial
Wastewater
-------
-------
Method 1657
The Determination of Organo-Phosphorus Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method is designed to meet the survey requirements of the Environmental Protection
Agency (EPA). It is used to determine the (1) organo-phosphorus pesticides associated with
the Clean Water Act, the Resource Conservation and Recovery Act, and the Comprehensive
Environmental Response, Compensation and Liability Act; and (2) other compounds amenable
to extraction and analysis by automated, wide-bore capillary column gas chromatography (GC)
with a flame photometric detector.
1.2 The compounds listed in Table 1 may be determined in waters, soils, sediments, and sludges
by this method. The method is a consolidation of several EPA methods. For waters, the
sample extraction and concentration steps are essentially the same as in these methods. How-
ever, the extraction and concentration steps have been extended to other sample matrices. The
method may be applicable to other phosphorus containing pesticides. The quality assurance/
quality control requirements in this method give the steps necessary to determine this ap-
plicability. Not all compounds listed in Table 1 have corresponding calibration data in Table 3
and acceptance criteria in Table 4. Calibration data for such analytes may be found in other
EPA methods (References 1 and 2).
1.3 This method is applicable to a large number of compounds. Calibrating the GC systems for all
compounds is time-consuming. If only a single compound or small number of compounds is
to be tested for, it is necessary to calibrate the GC systems and meet the performance specifi-
cations in this method for these compounds only. In addition, the GC conditions can be opti-
mized for these compounds provided that all performance specifications in this method are
met.
1.4 When this method is applied to analysis of unfamiliar samples, compound identity must 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. Gas chromatography/mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.5 The detection limits of this method are usually dependent on the level of interferences rather
than instrumental limitations. The limits in Table 2 typify the minimum quantities that can be
detected with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that
uses this method must demonstrate the ability to generate acceptable results using the proce-
dure in Section 8.2.
673
-------
Method 1657
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is determined.
2.1.2 Aqueous samples containing less than or equal to 1% solids.
2.1.2.1 Samples containing water-insoluble compounds: A 1-L sample is extracted
with methylene chloride using continuous extraction techniques.
2.1.2.2 Samples containing highly water-soluble compounds such as methamido-
phos: Salt is added to a 1-L sample and the sample is extracted with an
azeotropic mixture of chloroform:acetone using continuous extraction
techniques.
2.1.3 Samples containing greater than 1% solids:
2.1.3.1 Non-sludge samples: If the solids content is 1 to 30%, the sample is
diluted to 1% solids with reagent water, homogenized ultrasonically, and
extracted as an aqueous sample. If the solids content is greater than 30%,
the sample is extracted with methylene chloride:acetone using ultrasonic
techniques.
2.1.3.2 Municipal sludge samples and other intractable sample types: If the solids
content is less than 30%, the sample is diluted to 1% solids and extracted
as an aqueous sample. If the solids content is greater than 30%, the sam-
ple is extracted with acetonitrile and then methylene chloride using ultra-
sonic techniques. The extract is back-extracted with 2% (w/v) sodium
sulfate in reagent water to remove water-soluble interferences and residual
acetonitrile.
2.2 Concentration and cleanup: Each extract is dried over sodium sulfate, concentrated using a
Kuderna-Danish evaporator, cleaned up (if necessary) using gel permeation chromatography
(GPC) and/or solid-phase extraction, and concentrated to 1 mL.
2.3 Gas chromatography: A fixed volume of the extract is injected into the gas chromatograph
(GC). The compounds are separated on a wide-bore, fused-silica capillary column and detec-
ted using a flame photometric detector.
2.4 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention
times of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within
their respective windows.
2.5 Quantitative analysis is performed by using an authentic standard to produce a calibration
factor or calibration curve, and using the calibration data to determine the concentration of a
pollutant in the extract. The concentration in the sample is calculated using the sample weight
or volume and the extract volume.
2.6 Quality is assured through reproducible calibration and testing of the extraction and GC
systems.
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3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the anal-
ysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at
450°C for a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials,
such as PCBs, may not be eliminated by this treatment and thorough rinsing with acetone and
pesticide-quality hexane may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled. The cleanup procedures given in this method
can be used to overcome many of these interferences, but unique samples may require addi-
tional cleanup to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 3 through 5.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
The oven used for sample drying to determine percent moisture should be located in a hood so
that vapors from samples do not create a health hazard in the laboratory.
5. APPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting the requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1.1.1 Liquid samples (waters, sludges and similar materials that contain less than
5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-cap.
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5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain greater than 5% solids): Sample bottle, wide-mouth,
amber glass, 500-mL minimum.
5.1.1.3 If amber bottles are not available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with
PTFE.
5.1.1.5 Cleaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent or
baked at 450°C for a minimum of 1 hour before use.
5.1.1.5.2 Liners are detergent water washed, then rinsed with reagent
water and solvent, and baked at approximately 200°C for a
minimum of 1 hour prior to use.
5.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample con-
tamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for determining percent moisture.
5.2.1 Oven, capable of maintaining a temperature ofllO°C(+5°C).
5.2.2 Desiccator.
5.2.3 Crucibles, porcelain.
5.2.4 Weighing pans, aluminum.
5.3 Extraction equipment.
5.3.1 Equipment for ultrasonic extraction.
5.3.1.1 Sonic disrupter: 375 watt with pulsing capability and 1A" or % " disrupter
horn (Ultrasonics, Inc, Model 375C, or equivalent).
5.3.1.2 Sonabox (or equivalent): For use with disrupter.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Continuous liquid-liquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L capacity (Hershberg-Wolf Ex-
tractor, Cal-Glass, Costa Mesa, California, 1000- or 2000-mL continuous
extractor, or equivalent).
5.3.2.2 Round-bottom flask: 500-mL, with heating mantle.
5.3.2.3 Condenser: Graham, to fit extractor.
5.3.2.4 pH meter: With combination glass electrode.
5.3.2.5 pH paper: Wide-range (Hydrion Papers, or equivalent).
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5.3.3 Separatory funnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 Filtration apparatus.
5.3.4.1 Glass powder funnels: 125-to 250-mL.
5.3.4.2 Filter paper for above (Whatman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to 1 L.
5.3.5.2 400- to 500-mL.
5.3.6 Spatulas: Stainless steel or PTFE.
5.3.7 Drying column: 400 mm long x 15 to 20 mm ID Pyrex chromatographic column
equipped with coarse glass frit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for a mini-
mum of 1 hour.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1.1 Evaporation flask: 500-mL (Kontes K-570001-0500, or equivalent), at-
tached to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equi-
valent) with calibration verified. Ground-glass stopper (size 19/22 joint) is
used to prevent evaporation of extracts.
5.4.1.3 Snyder column: Three-ball macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450°C for a minimum of
1 hour.
5.4.1.5.2 PTFE (optional): Extracted with methylene chloride.
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control
(±2°C), installed in a fume hood.
5.4.3 Nitrogen-evaporation device: Equipped with heated bath that can be maintained at
35 to 40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit
GC autosampler.
5.5 Balances.
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Colum-
bia, MO, Model GPC Autoprep 1002, or equivalent).
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Method 1657
5.6.1.1 Column: 600 to 700 mm long x 25 mm ID, packed with 70 g of SX-3
Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
5.6.1.2 Syringe: 10-mL, with Luer fitting.
5.6.1.3 Syringe-filter holder: Stainless steel. Glass fiber or PTFE filters (Gelman
Acrodisc-CR, 1 to 5 /i, or equivalent).
5.6.1.4 UV detectors: 254-nm, preparative or semi-prep flow cell (Isco, Inc.,
Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8 ^iL micro-
prep flow cell, 2 mm path; Pharmacia UV-1, 3 mm flow cell; LDC Mil-
ton-Roy UV-3, monitor #1203; or equivalent).
5.6.2 Vacuum system and cartridges for solid-phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar (house vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack: For holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, CIS Octadecyl, 10 g/60 mL (Ana-
lytichem International Cat. No. 607H060, or equivalent).
5.7 Centrifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes
at 5,000 rpm minimum.
5.7.2 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-mL, with screw-caps, to fit centrifuge.
5.7.4 Funnel: Buchner, 15 cm.
5.7.4.1 Flask: Filter, for use with Buchner funnel.
5.7.4.2 Filter paper: 15 cm (Whatman #41, or equivalent).
5.8 Miscellaneous glassware.
5.8.1 Pipettes: Glass, volumetric, 1-, 5-, and 10-mL.
5.8.2 Syringes: Glass, with Luerlok tip, 0.1 -, 1- and 5-mL. Needles for syringes, 2",
22-gauge.
5.8.3 Volumetric flasks: 10-, 25-, and 50-mL.
5.8.4 Scintillation vials: Glass, 20- to 50-mL, with PTFE-lined screw-caps.
5.9 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a flame photometric detector at the end of each column,
temperature program with isothermal holds, data system capable of recording simultaneous
signals from the two detectors, and shall meet all of the performance specifications in Sec-
tion 14.
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5.9.1 GC columns: Bonded-phase fused-silica capillary.
5.9.1.1 Primary: 30 m (±3 m) long x 0.5 mm (+0.05 mm) ID DB-1, or equi-
valent.
5.9.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.9.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or
tape, process GC data, compute peak areas, store calibration data including retention
times and calibration factors, identify GC peaks through retention times, compute
concentrations, and generate reports.
5.9.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.9.2.2 Calibration factors and calibration curves: The data system shall be used
to record and maintain lists of calibration factors and multi-point calibra-
tion curves (Section 7). Computations of relative standard deviation (co-
efficient of variation) are used for testing calibration linearity. Statistics
on initial (Section 8.2) and ongoing (Section 13.6) performance shall be
computed and maintained.
5.9.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software rou-
tines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
5.9.2.4 Flame photometric detector: Capable of detecting 11 pg of malathion
under the analysis conditions given in Table 2.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (ION): Dissolve 40 g NaOH in 100 mL reagent
water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2.2 Sulfuric acid (1 +1): Reagent grade, 6N in reagent water. Slowly add 50 mL H2SO4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (w/v); dissolve 37 g KOH in 100 mL reagent water.
6.3 Solution drying and back-extraction.
6.3.1 Sodium sulfate: Reagent grade, granular anhydrous (Baker 3375, or equivalent),
rinsed with methylene chloride (20 mL/g), baked at 450°C for a minimum of 1 hour,
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Method 1657
cooled in a desiccator, and stored in a pre-cleaned glass bottle with screw-cap which
prevents moisture from entering.
6.3.2 Sodium sulfate solution: 2% (w/v) in reagent water, pH adjusted to 8.5 to 9.0 with
KOH or H2SO4.
6.4 Solvents: Methylene chloride, hexane, acetone, acetonitrile, isooctane, and methanol; pes-
ticide-quality; lot-certified to be free of interferences.
6.5 GPC calibration solution: Solution containing 300 mg/mL corn oil, 15 mg/mL bis(2-ethyl-
hexyl)phthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
6.6 Sample cleanup.
6.6.1 Solid-phase extraction.
6.6.1.1 SPE cartridge calibration solution: 2,4,6-trichlorophenol, 0.1 /ng/mL in
acetone.
6.6.1.2 SPE elution solvent: methylene chloride: acetonitrile: hexane (50:3:47).
6.7 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
6.8 High-solids reference matrix: Playground sand or similar material in which the compounds of
interest and interfering compounds are not detected by this method. May be prepared by ex-
traction with methylene chloride and/or baking at 450°C for 4 hours minimum.
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity, con-
centration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at -20 to
-10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the level of
the solution so that solvent evaporation loss can be detected. The vials are brought to room
temperature prior to use. Any precipitate is redissolved and solvent is added if solvent loss
has occurred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10 mg of malathion in a 10-mL ground-glass stoppered volumetric flask
and fill to the mark with isooctane. After the malathion is completely dissolved,
transfer the solution to a 15-mL vial with PTFE-lined cap.
6.10.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.10.3 Stock standard solutions shall be replaced after 6 months, or sooner if comparison
with quality control check standards indicates a change in concentration.
6.11 Secondary mixtures: Using stock solutions (Section 6.10), prepare mixtures at the levels
shown in Table 3 for calibration and calibration verification (Sections 7.3 and 13.5), for initial
and ongoing precision and recovery (Sections 8.2 and 13.6), and for spiking into the sample
matrix (Section 8.4).
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Method 1657
6.12 Surrogate spiking solution: Prepare tributyl phosphate and triphenyl phosphate each at a con-
centration of 2 /ig/mL in acetone.
6.13 Stability of solutions: All standard solutions (Sections 6.9 to 6.12) shall be analyzed within 48
hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will
remain acceptable if the peak area remains within ±15% of the area obtained in the initial
analysis of the standard.
7. SETUP AND CALIBRATION
7.1 Configure the GC system as given in Section 5.9 and establish the operating conditions in
Table 2.
7.2 Attainment of method detection limit (MDL): Determine that each column/detector system
meets the MDL's in Table 2.
7.3 Calibration.
7.3.1 Injection of calibration solutions.
7.3.1.1 Compounds with calibration data in Table 3: The compounds in each
calibration group in Table 3 were chosen so that each compound would be
separated from the others by approximately 1 minute on the primary col-
umn. The concentrations were chosen to bracket the working range of the
FPD. However, because the response of some models of FPD are greater
than others, it may be necessary to inject a larger volume of calibration
solution for these detectors.
7.3.1.2 Compounds without calibration data in Table 3: Prepare calibration stan-
dards at a minimum of three concentration levels. One of these concentra-
tions should be near, but above, the MDL (Table 2) and the other con-
centrations should define the working range of the detectors.
7.3.1.3 Set the automatic injector to inject a constant volume in the range of 0.5 to
5.0 fj.L of each calibration solution into the GC column/detector pairs,
beginning with the lowest level mixture and proceeding to the highest. For
each compound, compute and store, as a function of the concentration
injected, the retention time and peak area on each column/detector system
(primary and confirmatory).
7.3.2 Retention time: The polar nature of some analytes causes the retention time to de-
crease as the quantity injected increases. To compensate this effect, the retention time
for compound identification is correlated with the analyte level.
7.3.2.1 If the difference between the maximum and minimum retention times for
any compound is less than 5 seconds over the calibration range, the reten-
tion time for that compound can be considered constant and an average
retention time may be used for compound identification.
7.3.2.2 Retention-time calibration curve (retention time vs. amount): If the reten-
tion time for a compound in the lowest level standard is more than 5 sec-
onds greater than the retention time for the compound in the highest level
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Method 1657
standard, a retention-time calibration curve shall be used for identification
of that compound.
7.3.3 Calibration factor (ratio of area to amount injected).
7.3.3.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
column/detector system.
7.3.3.2 Linearity: If the calibration factor for any compound is constant
(Cv < 20%) over the calibration range, an average calibration factor may
be used for that compound; otherwise, the complete calibration curve (area
vs. amount) for that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration
groups of compounds (Table 3), the GC systems are calibrated with combined solutions as a
final step. Not all of the compounds in these standards will be separated by the GC columns
used in this method. Retention-times and calibration factors are verified for the compounds
that are resolved, and calibration factors are obtained for the unresolved peaks. These com-
bined QC standards are prepared at the level the mid-range calibration standard (Table 3).
7.4.1 Analyze the combined QC standard on each column/detector pair.
7.4.1.1 For those compounds that exhibit a single, resolved GC peak, the retention
time shall be within +5 seconds of the retention time of the peak in the
medium level calibration standard (Section 7.3.1), and the calibration
factor using the primary column shall be within +20% of the calibration
factor in the medium level standard (Table 3).
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns (primary and confir-
matory), and also compute and store the calibration factors on both col-
umns. These results will be used for calibration verification (Section 13.2
and 13.5) and for precision and recovery studies (Sections 8.2 and 13.6).
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program
(Reference 6). The minimum requirements of this program consist of an initial demonstration
of laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to samples
containing high solids with very little moisture (e.g., soils, compost), the high-solids reference
matrix (Section 6.8) is substituted for the reagent water (Section 6.8) in all performance tests,
and the high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 8.2.
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Method 1657
8.1.2 The analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If detection limits will be affected by the modification, the analyst is required to
repeat the demonstration of detection limits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to mon-
itor method performance. This test is described in Section 8.3. When results of these
spikes indicate atypical method performance for samples, the samples are diluted to
bring method performance within acceptable limits (Section 16).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 7.4) that the analysis system is
in control. These procedures are described in Sections 13.1, 13.5, and 13.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control limit for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
8.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four 1-L aliquots of reagent water spiked with the combined
QC standard (Section 7.4) according to the procedure in Section 10. Alternatively,
sets of four replicates of the individual calibration groups (Section 7.3) may be used.
For samples containing high solids, sets of four 30 g aliquots of the high-solids
reference matrix are used.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X)
and the coefficient of variation (Cv) of percent recovery (s) for each compound.
8.2.3 For each compound, compare s and X with the corresponding limits for initial preci-
sion and accuracy in Table 4. For coeluting compounds, use the coeluted compound
with the least restrictive specification (largest Cv and widest range). If s and X for all
compounds meet the acceptance criteria, system performance is acceptable and analy-
sis of blanks and samples may begin. If, however, any individual s exceeds the
precision limit or any individual X falls outside the range for recovery, system perfor-
mance is unacceptable for that compound. In this case, correct the problem and
repeat the test.
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the surrogate compounds.
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Method 1657
8.3.3 The recovery of the surrogate compounds shall be within the limits of 40 to 120%. If
the recovery of any surrogate falls outside of these limits, method performance is
unacceptable for that sample, and the sample is complex. Water samples are diluted,
and smaller amounts of soils, sludges, and sediments are reanalyzed per Section 16.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from
a given site type (e.g., influent to treatment, treated effluent, produced water, river sediment).
If only one sample from a given site type is analyzed, a separate aliquot of that sample shall be
spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows.
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in
the sample is being checked against a regulatory concentration limit, the
matrix spike shall be at that limit or at 1 to 5 times higher than the back-
ground concentration determined in Section 8.4.2, whichever concentration
is larger.
8.4.1.2 If the concentration of an analyte in the sample is not being checked
against a limit specific to that analyte, the matrix spike shall be at the
concentration of the combined QC standard (Section 7.4) or at 1 to 5 times
higher than the background concentration, whichever concentration is
larger.
8.4.1.3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Section 7.4).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyte. If necessary, prepare a standard solution appropriate to produce a level in
the sample one to five times the background concentration. Spike a second sample
aliquot with the standard solution and analyze it to determine the concentration after
spiking (A) of each analyte. Calculate the percent recovery (P) of each analyte:
Equation 1
p = 100 Q4-B)
T
where
T = True value of the spike
8.4.3 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the
sample is complex and must be diluted and reanalyzed per Section 16.
8.4.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
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a given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests
in Section 8.4, compute the average percent recovery (P) and the standard deviation of
the percent recovery (sp) for each compound (or coeluting compound group). Express
the accuracy assessment as a percent recovery interval from P — 2sp to P + 2sp for
each matrix. For example, if P = 90% and sp = 10% for five analyses of compost,
the accuracy interval is expressed as 70 to 110%. Update the accuracy assessment for
each compound in each matrix on a regular basis (e.g., after each five to ten new
accuracy measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1 L reagent water blank or a 30 g high-solids reference
matrix blank with each sample batch (samples started through the extraction process
on the same 8 hour shift, to a maximum of 20 samples). Analyze the blank immedi-
ately after analysis of the combined QC standard (Section 13.6) to demonstrate free-
dom from contamination.
8.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound
is found in an aqueous blank at greater than 0.05 jtg/L, or in a high-solids reference
matrix blank at greater than 1 /ig/kg (assuming the same calibration factor as mala-
thion for compounds not listed in Table 1), analysis of samples is halted until the
source of contamination is eliminated and a blank shows no evidence of contamination
at this level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with
an analyte in the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery (sn)
for the analyte, and measure the recovery and standard deviation of recovery for the other
analytes. The data for the new analyte is assumed to be valid if the precision and recovery
specifications for the other analytes are met; otherwise, the analytical problem is corrected and
the test is repeated. Establish a preliminary quality control limit of A + 2sn for the new
analyte and add the limit to Table 4.
8.7 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 13.5), and for initial (Section 8.2) and ongoing (Section 13.6)
precision and recovery should be identical, so that the most precise results will be obtained.
The GC instruments will provide the most reproducible results if dedicated to the settings and
conditions required for the analyses of the analytes given in this method.
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes
of interest into samples may be required to assess the precision and accuracy of the sampling
and sample transporting techniques.
685
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Method 1657
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices (Reference 7),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples
which flow freely are collected in refrigerated bottles using automatic sampling equipment.
Solid samples are collected as grab samples using wide-mouth jars.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 9.0 using
odium hydroxide or sulfuric acid solution. Record the volume of acid or base used. If resi-
dual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water.
EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 8).
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRA c TION AND CONCENTRA T/ON
Samples containing 1 % solids or less are extracted directly using continuous liquid-liquid extraction
techniques (Section 10.2.1). Samples containing 1 to 30% solids are diluted to the 1% level with
reagent water (Section 10.2.2) and extracted using continuous liquid-liquid extraction techniques.
Samples containing greater than 30% solids are extracted using ultrasonic techniques (Section 10.2.5).
For highly water soluble compounds such as methamidophos, samples are salted and extracted using a
chloroform:acetone azeotrope (Section 10.2.6). Figure 1 outlines the extraction and concentration
steps.
10.1 Determination of percent solids.
10.1.1 Weigh 5 to 10 g of sample into a tared beaker. Record the weight to three significant
figures.
10.1.2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a desiccator.
10.1.3 Determine percent solids as follows:
Equation 2
% solids = ^ight of dry sample x m
weight of wet sample
10.2 Preparation of samples for extraction.
10.2.1 Aqueous samples containing 1% solids or less: Extract the sample directly using
continuous liquid-liquid extraction techniques.
10.2.1.1 Measure 1 L (+0.01 L) of sample into a clean 1.5- to 2-L beaker.
10.2.1.2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the
sample aliquot. Proceed to preparation of the QC aliquots for low-solids
samples (Section 10.2.3).
10.2.2 Samples containing 1 to 30% solids.
10.2.2.1 Mix sample thoroughly.
686
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Method 1657
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1 % solids as fol-
lows:
Equation 3
sample weight =
% solids
10.2.2.3 Place the weight determined in Section 10,2.2.2 in a clean 1.5-L to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior
to weighing.
10.2.2.4 Bring the volume of the sample aliquot(s) to 100 to 200 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the appropriate surrogate spiking solution (Section 6.12)
into each sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into
small pieces.
10.2.2.7 Place the %" horn on the ultrasonic probe approximately Vi" below the
surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any
large pieces using the metal spatula or a stirring rod and repeat the soni-
cation. Clean the probe with methylene chloride:acetone (1:1) between
samples to preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at
the same time, place two 1.0 L (±0.01 L) aliquots of reagent water in
clean 1.5- to 2.0-L beakers.
10.2.3.2 Blank: Spike 0.5 mL of the pesticide surrogate spiking solution (Sec-
tion 6.12) into one reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into a reagent water aliquot.
10.2.3.4 If a matrix spike is required, prepare an aliquot at the concentrations
specified in Section 8.4.
10.2.4 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Extract the samples
and QC aliquots per Section 10.3.
10.2.5 Samples containing 30% solids or greater.
10.2.5.1 Mix the sample thoroughly.
10.2.5.2 Weigh 30 g (±0.3 g) into a clean 400- to 500-mL beaker. Discard all
sticks, rocks, leaves, and other foreign material prior to weighing.
10.2.5.3 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the
sample aliquot.
687
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Method 1657
10.2.5.4 QC aliquots: For each sample or sample batch (to a maximum of 20) to
be extracted at the same time, place two 30 g (±0.3 g) aliquots of the
high-solids reference matrix in clean 400- to 500-mL beakers.
10.2.5.5 Blank: Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into
an aliquot of the high-solids reference matrix.
10.2.5.6 Spike the combined QC standard (Section 7.4) into a high-solids reference
matrix aliquot. Extract the high-solids samples per Section 10.4.
10.2.6 Samples containing methamidophos and other highly water-soluble compounds: Pre-
pare samples containing less than 30% solids per Sections 10.2.6.1 to 10.2.6.5;
prepare samples containing greater than 30% solids per Section 10.2.5.
10.2.6.1 Interferences: If interferences are expected, aqueous samples can be pre-
extracted with methylene chloride to remove these interferences. This
extract can be used for determination of insoluble or slightly soluble com-
pounds and the surrogates. Methamidophos is only sightly soluble in
methylene chloride and will not be in this extract unless carried by polar
species in the sample matrix. If compounds other than methamidophos are
not to be determined, the methylene chloride extract can be discarded.
10.2.6.2 Determine the percent solids and prepare a 1-L sample aliquot and the QC
aliquots per Sections 10.2.2 and 10.2.4 or 10.2.3 and 10.2.4, except do
not spike the surrogate into the sample aliquot if the methylene chloride
extract will be discarded (Section 10.2.6.1).
10.2.6.3 Extract the aliquots per Section 10.3 using methylene chloride to remove
interferences.
10.2.6.4 After extraction, remove the water and methylene chloride from the extrac-
tor. Decant the aqueous portion into a beaker and combine the remaining
methylene chloride with the extract in the distilling flask. If the methylene
chloride extract is to be used for determination of other analytes and the
surrogate, proceed to Section 10.5 with that extract.
10.2.6.5 Saturate the water sample with sodium chloride. Approximately 350 g will
be required.
10.2.6.6 If the methylene chloride extract was discarded, spike the surrogates into
the sample aliquot.
10.2.6.7 Extract the sample per Section 10.3 except use a chlorofornracetone azeo-
trope (2:1 v/v or 4:1 w/w) for the extraction.
NOTE: Note: As a result of the increased density of the water caused by saturation
with salt, the sample may sink to where the water enters the siphon tube of the con-
tinuous extractor. To prevent this from occurring, use a smaller volume of water (e.g.,
800 mL) in the extractor. Correct for this adjustment in the calculation of the con-
centration of the pollutants in the extract (Section 15).
688
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Method 1657
10.3 Continuous extraction of low-solids (aqueous) samples: Place 100 to 150 mL methylene
chloride in each continuous extractor and 200 to 300 mL in each distilling flask.
10.3.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass
containers with 50 to 100 mL methylene chloride and add to the respective extractors.
Include all solids in the extraction process.
10.3.2 Extraction: Adjust the pH of the waters in the extractors to 5 to 9 with NaOH or
H2SO4 while monitoring with a pH meter.
NOTE: Caution: Some samples require acidification in a hood because of the potential
for generating hydrogen sulfide.
10.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling.
When properly adjusted, one to two drops of methylene chloride per second will fall
from the condenser tip into the water. Test and adjust the pH of the waters during the
first 1 to 2 hours of extraction. Extract for 18 to 24 hours.
10.3.4 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 mL), and pour the contents through a prerinsed drying column containing 7 to
10 cm of anhydrous sodium sulfate. Rinse the distilling flask with 30 to 50 mL of
methylene chloride and pour through the drying column. For extracts to be cleaned
up using GPC, collect the solution in a 500-mL K-D evaporator flask equipped with a
10-mL concentrator tube. Seal, label, and concentrate per Sections 10.5 to 10.6.
10.4 Ultrasonic extraction of high-solids samples: Procedures are provided for extraction of non-
municipal sludge (Section 10.4.1) and municipal sludge samples (Section 10.4.2).
10.4.1 Ultrasonic extraction of non-municipal sludge high-solids aliquots.
10.4.1.1 Add 60 to 70 g of powdered sodium sulfate to the sample and QC aliquots.
Mix each aliquot thoroughly. Some wet sludge samples may require more
than 70 g for complete removal of water. All water must be removed
prior to addition of organic solvent so that the extraction process is ef-
ficient.
10.4.1.2 Add 100 mL (±10 mL) of acetone:methylene chloride (1:1) to each of the
aliquots and mix thoroughly.
10.4.1.3 Place the % " horn on the ultrasonic probe approximately W below the
surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and
repeat the sonication.
10.4.1.4 Decant the pesticide extracts through a prerinsed drying column containing
7 to 10 cm anhydrous sodium sulfate into 500- to 1000-mL graduated
cylinders.
10.4.1.5 Repeat the extraction steps (Sections 10.4.1.2 to 10.4.1.4) twice more for
each sample and QC aliquot. On the final extraction, swirl the sample or
QC aliquot, pour into its respective drying column, and rinse with acetone:
689
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Method 1657
methylene chloride. Record the total extract volume. If necessary, trans-
fer the extract to a centrifuge tube and centrifuge for 10 minutes to settle
fine particles.
10.4.2 Ultrasonic extraction of high solids municipal sludge aliquots.
10.4.2.1 Add 100 mL ( +10 mL) of acetonitrile to each of the aliquots and mix
thoroughly.
10.4.2.2 Place the %" horn on the ultrasonic probe approximately W below the
surface of the solvent but above the solids layer and pulse at 50% for
3 minutes at full power. If necessary, remove the probe from the solution
and break any large pieces using a metal spatula or a stirring rod and
repeat the sonication.
10.4.2.3 Decant the extract through filter paper into a 1000- to 2000-mL separatory
funnel.
10.4.2.4 Repeat the extraction and filtration steps (Sections 10.4.2.1 to 10.4.2.3)
using a second 100 mL (±10 mL) of acetonitrile.
10.4.2.5 Repeat the extraction step (Sections 10.4.2.1 and 10.4.2.2) using 100 mL
( + 10 mL) of methylene chloride. On this final extraction, swirl the sam-
ple or QC aliquot, pour into its respective filter paper, and rinse with
methylene chloride. Record the total extract volume.
10.4.2.6 For each extract, prepare 1.5 to 2 L of reagent water containing 2% so-
dium sulfate. Adjust the pH of the water to 6.0 to 9.0 with NaOH or
H2S04.
10.4.2.7 Back-extract each extract three times sequentially with 500 mL of the aque-
ous sodium sulfate solution, returning the bottom (organic) layer to the
separatory funnel the first two times while discarding the top (aqueous)
layer. On the final back-extraction, filter each pesticide extract through a
prerinsed drying column containing 7 to 10 cm anhydrous sodium sulfate
into a 500- to 1000-mL graduated cylinder. Record the final extract vol-
ume.
10.4.3 For extracts to be cleaned up using GPC, filter these extracts through Whatman #41
paper into a 500-mL K-D evaporator flask equipped with a 10-mL concentrator tube.
Rinse the graduated cylinder or centrifuge tube with 30 to 50 mL of methylene chlo-
ride and pour through filter to complete the transfer. Seal and label the K-D flask.
Concentrate these fractions per Sections 10.5 through 10.8.
10.5 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL concen-
trator tubes. Add one to two clean boiling chips to the flask and attach a three-ball
macro Snyder column. Prewet the column by adding approximately 1 mL of methy-
lene chloride through the top. Place the K-D apparatus in a hot water bath so that the
entire lower rounded surface of the flask is bathed with steam. Adjust the vertical
position of the apparatus and the water temperature as required to complete the con-
centration in 15 to 20 minutes. At the proper rate of distillation, the balls of the
column will actively chatter but the chambers will not flood.
690
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Method 1657
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus
from the bath and allow the solvent to drain and cool for at least 10 minutes.
10.5.3 If the extract is to be cleaned up using GPC, remove the Snyder column and rinse the
flask and its lower joint into the concentrator tube with 1 to 2 mL of methylene chlo-
ride. A 5-mL syringe is recommended for this operation. Adjust the final volume to
10 mL and proceed to GPC cleanup in Section 11.
10.6 Hexane exchange: Extracts that have been cleaned up are exchanged into hexane.
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
chip, and reattach the Snyder column. Concentrate the extract as in Section 10.5
except use hexane to prewet the column. The elapsed time of the concentration
should be 5 to 10 minutes.
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the concen-
trator tube with 1 to 2 mL of hexane. Adjust the final volume of extracts that have
not been cleaned up by GPC to 10 mL and those that have been cleaned up by GPC
to 5 mL (the difference accounts for the 50% loss in the GPC cleanup).
7 7. CLEANUP AND SEPARA TION
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents,
ground water, drinking water). If particular circumstances require the use of a cleanup
procedure, the analyst may use arjy or all of the procedures below or any other appropriate
procedure. However, the analyst shall first repeat the tests in Section 8.2 to demonstrate that
the requirements of Section 8.2 can be met using the cleanup procedure(s) as an integral part
of the method. Figure 1 outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
interferents that cause GC column performance to degrade. It is used for all soil and
sediment extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols. It is used for cleanup of organo-chlorine and organo-phosphate
extracts.
11.2 Gel permeation chromatography (GPC).
11.2.1 Column packing.
11.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight
(12 hours minimum).
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 5.5 mL/min prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
691
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Method 1657
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis(2-ethylhexyl) phthalate, pentachloro-
phenol, perylene, and sulfur.
11.2.2.3 Set the "dump time" to allow >85% removal of the corn oil and >85%
collection of the phthalate.
11.2.2.4 Set the "collect time" to the peak minimum between perylene and sulfur.
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachlorophenol is greater
than 85%. If calibration is not verified, the system shall be recalibrated
using the calibration solution, and the previous 20 samples shall be re-
extracted and cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: GPC requires that the column not be overloaded. The column
specified in this method is designed to handle a maximum of 0.5 g of high molecular
weight material in a 5 mL extract. If the extract is known or expected to contain
more than 0.5 g, the extract is split into fractions for GPC and the fractions are
combined after elution from the column. The solids content of the extract may be
obtained gravimetrically by evaporating the solvent from a 50-/uL aliquot.
11.2.3.1 Filter the extract or load through the filter holder to remove particulates.
Load the 5.0 mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0 mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange into hexane per Sections 10.5
and 10.6. Adjust the final volume to 5.0 mL.
11.3 Solid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with
the trap and gauge installed between the manifold and vacuum source.
11.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge washing: Pre-elute each cartridge prior to use sequentially with 10-mL
portions each of hexane, methanol, and water using vacuum for 30 seconds after each
eluant. Follow this pre-elution with 1 mL methylene chloride and three 10-mL
portions of the elution solvent (Section 6.6.2.2) using vacuum for 5 minutes after each
692
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Method 1657
eluant. Tap the cartridge lightly while under vacuum to dry between eluants. The
three portions of elution solvent may be collected and used as a blank if desired.
Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum
for 30 seconds after each eluant.
11.3.3 Cartridge certification: Each cartridge lot must be certified to ensure recovery of the
compounds of interest and removal of 2,4,6-trichlorophenol.
11.3.3.1 To make the test mixture, add the trichlorophenol solution (Section
6.6.2.1) to the combined calibration standard (Section 7.4). Elute the
mixture using the procedure in Section 11.3.4.
11.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 /zL of the concentrated
eluant into the GC using the procedure in Section 13. The recovery of all
analytes (including the unresolved GC peaks) shall be within the ranges for
recovery specified in Table 4, and the peak for trichlorophenol shall not be
detectable; otherwise the SPE cartridge is not performing properly and the
cartridge lot shall be rejected.
11.3.4 Extract cleanup.
11.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack containing the 50-mL volumetric flasks (Section 5.6.2.4) in the vac-
uum manifold. Reestablish the vacuum at 5 to 10 psia.
11.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE
cartridge. Apply vacuum for 5 minutes to dry the cartridge. Tap gently
to aid in drying.
11.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 min-
utes after each portion. Collect the eluants in the 50-mL volumetric flasks.
11.3.4.4 Release the vacuum and remove the 50-mL volumetric flasks.
11.3.4.5 Concentrate the eluted extracts to approximately 0.5 mL using the nitrogen
blow-down apparatus. Adjust the final volume to 1.0 mL (per Section
10.6) and proceed to Section 13 for GC analysis.
12. GAS CHROMATOGRAPHY
Table 2 summarizes the recommended operating conditions for the gas chromatograph. Included in
this table are the retention times and estimated detection limits that can be achieved under these
conditions. Examples of the separations achieved by the primary and confirmatory columns are
shown in Figure 2.
12.1 Calibrate the system as described in Section 7.
12.2 Set the autosampler to inject the same volume that was chosen for calibration (Section 7.3.1.3)
for all standards and extracts of blanks and samples.
12.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection
after the last analyte is expected to elute and to return the column to the initial temperature.
693
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Method 1657
13. SYSTEM AND LABORA TORY PERFORMANCE
13.1 At the beginning of each 8-hour shift during which analyses are performed, GC system perfor-
mance and calibration are verified for all pollutants and surrogates on both column/detector
systems. For these tests, analysis of the combined QC standard (Section 7.4) shall be used to
verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be per-
formed until all performance criteria are met. Only after all performance criteria are met may
samples, blanks, and precision and recovery standards be analyzed.
13.2 Retention times: The absolute retention times of the peak maxima shall be within ±10 sec-
onds of the retention times in the initial calibration (Section 7.4.1).
13.3 GC resolution: Resolution is acceptable if the valley height between two peaks (as measured
from the baseline) is less than 10% of the taller of the two peaks.
13.3.1 Primary column (DB-1): Malathion and ethyl parathion.
13.3.2 Confirmatory column (DB-1701): Terbufos and diazinon.
13.4 Calibration verification: Calibration is verified for the combined QC standard only.
13.4.1 Inject the combined QC standard (Section 7.4).
13.4.2 Compute the percent recovery of each compound or coeluting compounds, based on
the calibration data (Section 7.4).
13.4.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing accuracy in Table 4. For co-
eluting compounds, use the coeluted compound with the least restrictive specification
(the widest range). If the recoveries for all compounds meet the acceptance criteria,
system performance is acceptable and analysis of blanks and samples may begin. If,
however, any recovery falls outside the calibration verification range, system perfor-
mance is unacceptable for that compound. In this case, correct the problem and
repeat the test, or recalibrate (Section 7).
13.5 Ongoing precision and recovery.
13.5.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Sections 10.2.3.3 and 10.2.5.7).
13.5.2 Compute the percent recovery of each analyte and coeluting compounds.
13.5.3 For each compound or coeluted compounds, compare the percent recovery with the
limits for ongoing recovery in Table 4. For coeluted compounds, use the coeluted
compound with the least restrictive specification (widest range). If all analytes pass,
the extraction, concentration, and cleanup processes are in control and analysis of
blanks and samples may proceed. If, however, any of the analytes fail, these proces-
ses are not in control. In this event, correct the problem, re-extract the sample lot,
and repeat the ongoing precision and recovery test.
13.5.4 Add results which pass the specifications in Section 13.6.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued labora-
tory performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
694
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Method 7657
recovery sr. Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.
14. QUALITATIVE DETERMINATION
14.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 14.2), and with data stored in the
retention-time and calibration libraries (Sections 7.3.2 and 7.3.3.2). Identification is con-
firmed when retention time and amounts agree per the criteria below.
14.2 For each compound on each column/detector system, establish a retention-time window
±20 seconds on either side of the retention time in the calibration data (Section 7.3.2).
For compounds that have a retention-time curve (Section 7.3.2.2), establish this window as
the minimum -20 seconds and maximum +20 seconds.
14.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the
analysis of a sample or blank is within a window (as defined in Section 14.2) on the
primary column/detector system, it is considered tentatively identified. A tentatively
identified compound is confirmed when (1) the retention time for the compound on
the confirmatory column/detector system is within the retention-time window on that
system, and (2) the computed amounts (Section 16) on each system (primary and
confirmatory) agree within a factor of 3.
14.2.2 Compounds requiring a retention-time calibration curve: If a peak from the analysis
of a sample or blank is within a window (as defined in Section 14.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention times on both systems (primary and
confirmatory) are within ±30 seconds of the retention times for the computed
amounts (Section 15), as determined by the retention-time calibration curve (Section
7.3.2.2), and (2) the computed amounts (Section 15) on each system (primary and
confirmatory) agree within a factor of 3.
15. QUANTITATIVE DETERMINATION
1 5.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
1 5.2 Liquid samples: Compute the concentration in the sample using the following equation:
Equation 4
C - 10
where
Cs = Concentration in the sample, in
10 = Final extract total volume, in mL
Ca = Concentration in the extract, in
Vs = Sample extracted, in L
695
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Method 1657
15.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
(C }
c. = 10.
1000 (Ws) (solids)
where
Cs = Concentration in the sample, in pg/kg
10 = Final extract total volume, in mL
Ca = Concentration in the extract, in pg/mL
1000 = Conversion factor, g to kg
Ws = Sample weight, in g
solids = Percent solids in Section 10.1.3 divided by 100
15.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1-/*L aliquot of the diluted extract is analyzed.
15.5 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at
which the concentration is in the calibration range.
16. ANALYSIS OF COMPLEX SAMPLES
16.1 Some samples may contain high levels (> 1000 ng/L) of the compounds of interest, interfering
compounds, and/or polymeric materials. Some samples may not concentrate to 10 mL (Se-
ction 10.6); others may overload the GC column and/or detector.
16.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), and the SPE
cartridge (Section 11.3). If these techniques do not remove the interfering compounds, the
extract is diluted by a factor of 10 and reanalyzed (Section 16.4).
16.3 Recovery of surrogates: In most samples, surrogate recoveries will be similar to those from
reagent water or from the high solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3, the sample shall be re-extracted and reanalyzed. If the surro-
gate recovery is still outside this range, the sample is diluted by a factor of 10 and reanalyzed
(Section 15.4).
16.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those
from reagent water or from the high solids reference matrix. If the matrix spike recovery is
outside the range specified in Table 4, the sample shall be diluted by a factor of 10, respiked,
and reanalyzed. If the matrix spike recovery is still outside the range, the method may not
apply to the sample being analyzed and the result may not be reported for regulatory compli-
ance purposes.
77. METHOD PERFORMANCE
17.1 Development of this method is detailed in References 9 and 10.
696
-------
Method 1657
References
1. "Guideline Establishing Test Procedures for the Analysis of Pollutants under the Clean Water
Act; Final Rule and Interim Final Rule and Proposed Rule," 40 CFR Part 136.
2. "Methods for the Determination of Organic Compounds in Drinking Water," U.S. Environ-
mental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinatti, Ohio:
EPA-600/4-88/039, December 1988.
3. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
4, "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
6. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
7. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
8. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
9. "Consolidated GC Method for the Determination of ITD/RCRA Pesticides using Selective GC
Detectors," S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-
01, Document R70, September 1986.
10. "Method Development and Validation, EPA Method 1618," Pesticide Center, Department of
Environmental Health, Colorado State University: November 1988, January 1989, and
March 1992.
697
-------
Method 1657
Table 1. Organo-Phosphorus Pesticides Determined by Large-Bore, Fused-Silica
Capillary Column Gas Chromatography with Flame Photometric Detector
EPA EGD Compound
468
453
461
469
443
479
471
460
450
455
449
452
458
467
463
446
454
447
464
474
475
456
444
470
459
448
457
465
473
477
476
472
466
445
451
462
Acephate
Azinphos ethyl
Azinphos methyl
Chlorfevinphos
Chlorpyrifos
Coumaphos
Crotoxyphos
DEF
Demeton
Diazinon
Dichlofenthion
Dichlorvos
Dicrotophos
Dimethoate
Dioxathion
Disulfoton
EPN
Ethion
Ethoprop
Famphur
Fensulfothion
Fenthion
Hexamethylphosphoramide
Leptophos
Malathion
Merphos
Methamidophos
Methyl Chlorpyrifos
Methyl parathion
Methyl trithion
Mevinphos
Monocrotophos
Naled
Parathion (ethyl)
Phorate
Phosmet
Phosphamidon
Ronnel
Sulfotepp
Sulprofos (Bolstar)
TEPP
Terbufos
Tetrachlorvinphos
Tokuthion
Trichlorfon
Trichloronate
Tricresylphosphate
Trimethylphosphate
CAS Registry
30560-19-1
2642-71-9
86-50-0
470-90-6
2921-88-2
56-72-4
7700-17-6
78-48-8
8065-48-3
333-41-5
97-17-6
62-73-7
141-66-2
60-51-5
78-34-2
298-04-4
2104-64-5
563-12-2
13194-48-4
52-85-7
115-90-2
55-38-9
680-31-9
21609-90-5
121-75-5
150-50-5
10265-92-6
5598-13-0
298-00-0
953-17-3
7786-34-7
6923-22-4
300-76-5
56-38-2
298-02-2
732-11-6
13171-21-6
299-84-3
3689-24-5
35400-43-2
107-49-3
13071-79-9
961-11-5
34643-46-4
52-68-6
327-98-0
78-30-8
512-56-1
698
-------
Method 1657
Table 2.
Gas Chromatography of Organo-Phosphorus Pesticides
Retention Time1 (min)
EPA EGO
450
444
445
471
459
455
470
477
457
449
452
472
473
458
460
456
475
447
448
469
461
479
466
454
463
446
465
467
Compound
Dichlorvos
Mevinphos
Acephate
Trichlorofon
Methamidophos
Demeton-A
Ethoprop
Naled
Dicrotophos
Monocrotpphos
Sulfotepp
Phorate
Dimethoate
Demeton-B
Dioxathion
Terbufos
Phosphamidon-E
Disulfoton
Diazinon
Tributyl phosphate (surr)
Phosphamidon-Z
Methyl parathion
Dichlorofenthipn
Methyl chlorpyrifos
Ronnel
Malathion
Fenthion
Parathion
Chlorpyrifos
Trichloronate
Chlorfevinphos
Crotoxyphos
Tokuthion
Tetrachlorvinphos
DEF
Merphos-B
Fensulfothion
Methyl trithion
Ethion
Sulprofos
Famphur
Phosmet
EPN
DB-1
6.56
11,85
12.60
12.69
15.10
17.70
18.49
18.92
19.33
19.62
20.04
20.12
20.59
21.40
22.24
22.97
23.70
23.89
24.03
24.50
25.88
25.98
26.11
26.29
27.33
28.87
29.14
29.29
29.48
30.44
32,05
32.65
33.30
33.40
34.05
35.16
36.58
36.62
37.61
38.10
38.24
41.24
41.94
| DB-1701
9.22
16.20
17.40
18.85
19.20
20.57
21.43
23.00
26.30
29.24
23.68
23.08
29.29
25.52
26.70
24.55
29.89
27.01
26.10
17.20
32.62
32.12
28.66
29.53
30.09
33.49
32.16
34.61
32.15
32.12
36.08
37.58
37.17
37.85
37.50
37.37
43.86
40.52
41.67
41.74
46.37
48.22
47.52
MDL2
(ng/U
4
74
500
1503
100
19
7
18
81
85
6
10
27
21
121
26
28
32
38
-
116
18
6
13
11
11
22
10
4
14
2
81
2
12
50
18
104
10
13
6
27
14
9
653
-------
Method 1657
Table 2. Gas Chromatography of Organo-Phosphorus Pesticides (cont.
Retention Time1 (min)
EPA EGD Compound
453 Azinphos methyl
474 Leptophos
468 Azinphos ethyl
Triphenyl phosphate (surr)
443 Coumaphos
Notes:
DB-1
43
44
45
47
48
.33
.32
.55
.68
.02
DB-1701
50.26
47.36
51.88
40.43
56.44
MDL2
(ng/U
9
14
22
24
2.
3.
Columns: 30 m long x 0.53 mm ID; DB-1: 1.5//; DB-1701: 1.0 p. Conditions suggested to
meet retention times shown: 110°C for 0.5 min, 110 to 250° at 3°C/min, 250°C until
coumaphos elutes. Carrier gas flow rate approximately 7 mL/min.
40 CFR Part 136, Appendix B (49 FR 43234).
Estimated. Detection limits for soils (in ng/kg) are estimated to be 30 to 100 times this level.
Table 3. Concentrations of Calibration Solutions
Concentration (ug/mL)
EPA EGD Compound
Calibration Group 1
453 Azinphos methyl
450 Dichlorvos
458 Disulfoton
447 Fenthion
Merphos-A
Merphos-B
Methyl trithion
Ronnel
Sulprofos
Calibration Group 2
461 Chlorfevinphos
469 Chlorpyrifos
471 Demeton-A
Demeton-B
Dichlofenthion
449 Dimethoate
446 Famphur
474 Leptophos
456 Methyl parathion
445 Trichlorofon
451 Tricresylphosphate
Low
0.1
0.5
0.2
0.2
0.2
0.2
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.5
0.2
0.2
0.5
1.0
Medium
0.5
2.5
1.0
1.0
1.0
1.0
2.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.5
2.5
1.0
1.0
2.5
5
High
2.0
10.0
4.0
4.0
4.0
4.0
10.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
2.0
10.0
4.0
4.0
10.0
20.0
700
-------
Method 1657
Table 3.
Concentrations of Calibration
Solutions (cont.
)
Concentration (ug/mU
EPA EGO
Calibration
468
479
454
444
459
448
465
473
477
472
Calibration
443
460
467
463
475
457
466
Compound
Group 3
Azinphos ethyl
Crotoxyphos
DEF
Fensulfothion
Methyl chlorpyrifos
Mevinphos
Naled
Parathion
Phosmet
Phosphamidon-E
Phosphamidon-Z
Sulfotepp
Terbufos
Group 4
Coumaphos
Diazinon
EPN
Ethion
Ethoprop
Malathion
Phorate
Tetrachlorvinphos
Trichloronate
Low
0.2
0.5
0.2
0.5
0.2
0.5
0.5
0.2
0.5
0.5
0.5
0.2
0.2
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Medium
1.0
2.5
1.0
2.5
1.0
2.5
2.5
1.0
2. 5
2.5
2.5
1.0
1.0
2.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
High
4.0
10.0
4.0
10.0
4.0
10.0
10.0
4.0
10.0
10.0
10.0
4.0
4.0
10.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
707
-------
Method 1657
Table 4.
EGD No.
468
453
461
469
443
479
471
460
450
455
449
452
458
467
463
446
454
447
464
474
475
456
444
470
459
448
457
465
473
477
Acceptance Criteria for
Compounds
Compound
Acephate
Azinphos ethyl
Azinphos methyl
Chlorfevinphos
Chlorpyrifos
Coumaphos
Crotoxyphos
DEF
Demeton
Diazinon
Dichlofenthion
Dichlorvos
Dicrotophos
Dimethoate
Dioxathion
Disulfoton
EPN
Ethion
Ethoprop
Famphur
Fensulfothion
Fenthion
Hexamethylphos-
phoramide
Leptophos
Malathion
Merphos-B
Methamidophos1
Methyl chlorpyrifos
Methyl parathion
Methyl trithion
Mevinphos
Monocrotophos
Naled
Parathion
Phorate
Phosmet
Phosphamidon-Z
Ronnel
Sulfotepp
Spike
Level
(ng/L)
50000
10
5
10
10
25
25
10
10
10
10
25
5
-
10
10
10
10
25
25
10
10
10
10
10000
10
10
25
25
25
10
10
25
25
10
10
Performance Tests for Organo-Phosphorus
Acceptance Criteria
Initial Precision
and Accuracy
' *
s x
25 32-122
10 71-117
10 52-112
11 56-132
10 61-112
10 78-104
46 28-116
31 45-107
23 33-101
10 70-110
10 75-115
18 52-106
not recovered
89 27-100
22 59-101
30 46-98
13 74-124
11 72-134
14 79-103
12 81-101
65 13-115
13 69-101
not recovered
10 85-105
10 75-109
10 68-102
33 66-132
10 88-108
15 72-112
20 21-137
23 24-100
not recovered
10 0-148
10 71-111
19 54-100
39 44-119
45 0-100
10 79-111
10 70-120
Calibration
Verification
(%)
68-132
77-127
83-119
83-114
80-119
82-120
68-136
68-132
64-123
86-114
80-110
77-103
78-122
73-127
79-121
70-118
81-108
70-118
84-108
81-113
42-139
73-137
70-130
85-112
82-108
72-118
70-128
81-114
89-114
78-122
73-135
1 9-206
77-114
79-110
70-118
61-159
81-102
78-113
75-115
Recovery/
Ongoing
Accuracy, R
(%)
28-126
59-129
37-127
37-151
48-125
72-110
6-138
42-110
16-118
60-120
65-125
39-119
22-100
49-1 1 1
33-111
62-136
47-149
73-109
76-106
0-141
61-109
80-110
66-118
59-111
63-135
83-113
61-123
0-166
7-107
0-176
61-121
43-109
25-138
0-100
71-119
58-132
702
-------
Method 1657
Table 4.
EGD No.
476
472
466
445
451
462
Notes:
1 . With
2. With
Acceptance Criteria for
Compounds (cont.)
Compound
Sulprofos
TEPP
Terbufos
Tetrachlorvinphos
Tokuthion
Trichlorofon2
Trichloronate
Tricresylphosphate
Trimethylphosphate
Spike
Level
(ng/U
10
Performance Tests for Organo-Phosphorus
Acceptance Criteria
Initial Precision
and Accuracy
(%)
s
10
X
75-100
not recovered
10
10
100
25
10
50
23
11
17
42
10
10
60-110
48-110
73-105
43-195
82-102
81-101
not recovered
Calibration
Verification
(%)
81-118
70-130
82-111
73-119
70-130
58-142
80-113
70-130
70-130
Recovery/
Ongoing
Accuracy, R
(%)
70-100
47-123
32-126
65-113
37-201
77-107
74-114
salt and azeotropic extraction
salt
703
-------
Method 1657
Percent Solids
< 30% Solids
> 30% Solids
Oil. To 1% Solids
ACN&CH2C12Sonic
CH2CI2 Liq./Liq. Ext.
H2O Back Extract
Methamidophos
"Normal" Compounds
Add NaCI
CHCI3:(CH3)2CO L7L Ext
Concentrate
I
To Cleanup
Extraction and Concentration Steps
From Extraction
I
Gel Permeation
Solid-Phase Ext.
GC/FPD
Cleanup and Analysis Steps
AS2-002-83A
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
704
-------
(BH
Method 1657
£ £
ID O
» 0
.K
(A)
§ 3.
i 1-
I
2
JV.
ll[Mn|mi|mi{IIM[mi|MII|IMI|nM|mi|MM(MII|mi|mijini|nil|lllt|IMI|mi|llll|m
78 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 3* 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 36
Retention Time (minutes)
A52-002-88
Figure 2. Gas Chromatogram of Selected Organo-Phosphorus Compounds
705
-------
-------
Method 1658
The Determination of
Phenoxy-Acid Herbicides in
Municipal and Industrial
Wastewater
-------
-------
Method 165S
The Determination of Phenoxy-Acid Herbicides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method is designed to meet the survey requirements of the Environmental Protection
Agency (EPA). It is used to determine (1) the phenoxy-acid herbicides and herbicide esters
associated with the Clean Water Act, the Resource Conservation and Recovery Act, and the
Comprehensive Environmental Response, Compensation and Liability Act; and (2) other
compounds amenable to extraction and analysis by automated, wide-bore capillary column gas
chromatography (GC) with electron capture or halogen-selective detectors.
1.2 The chemical compounds listed in Table 1 may be determined in waters, soils, sediments, and
sludges by this method. This method should be applicable to other herbicides. The quality
assurance/quality control requirements in this method give the steps necessary to determine this
applicability.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must 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. Gas chromatography mass spectrometry (GC/MS) can be used
to confirm compounds in extracts produced by this method when analyte levels are sufficient.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantity that can be
detected with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that
uses this method must demonstrate the ability to generate acceptable results using the proce-
dure in Section 8.2.
2. SUMMARY OF METHOD
2.1 Extraction.
2.1.1 The percent solids content of a sample is determined.
2.1.2 Samples containing low solids: If the solids content is less than or equal to 1 %, the
sample is extracted directly using continuous extraction techniques. The pH of a 1-L
sample raised to 12 to 13 to hydrolyze acid esters, and the sample is extracted with
methylene chloride to remove interferences. The pH is lowered to less than 2 and the
free acids are extracted with methylene chloride.
2.1.3 Samples containing greater than 1% solids.
2.1.3.1 Solids content 1 to 30%: The sample is diluted to 1 % solids with reagent
water, homogenized ultrasonically, and extracted as a low-solids sample
(Section 2.1.2).
709
-------
Method 1658
2.1.3.2 Solids content greater than 30%: The sample is placed in an extraction
bottle and approximately 1-L of basic (pH 12-13) water is added. The
bottle is tumbled for 18 hours. The water is removed and extracted as a
low-solids sample (Section 2.1.2).
2.2 Concentration and cleanup: The extract is dried over sodium sulfate, concentrated using a
Kuderna-Danish evaporator, cleaned up (if necessary) using gel permeation chromatography
(GPC) and concentrated to 5 or 10 mL (depending upon whether GPC was or was not used).
2.3 Derivatization and cleanup: The acids in the extract are derivatized to form the methyl esters.
The solution containing the methyl esters is cleaned up (if necessary) using solid-phase extrac-
tion (SPE) and/or adsorption chromatography and reconcentrated to 5 or 10 mL.
2.4 Gas chromatography: A 1-^L aliquot of the extract is injected into the gas chromatograph
(GC). The derivatized acids are separated on a wide-bore, fused-silica capillary column and
are detected by an electron capture, microcoulometric, or electrolytic conductivity detector.
2.5 Identification of a pollutant (qualitative analysis) is performed by comparing the GC retention
times of the compound on two dissimilar columns with the respective retention times of an
authentic standard. Compound identity is confirmed when the retention times agree within
their respective windows.
2.6 Quantitative analysis is performed by using an authentic standard to produce a calibration
factor or calibration curve, and using the calibration data to determine the concentration of a
pollutant in the extract. The concentration in the sample is calculated using the sample weight
or volume and the extract volume.
2.7 Quality is assured through reproducible calibration and testing of the extraction and GC .
systems.
3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the anal-
ysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.5.
3.2 Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450°C for
a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials, such as
PCBs, may not be eliminated by this treatment and thorough rinsing with acetone and pes-
ticide-quality hexane may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interference by phthalate esters can pose a major problem in herbicide analysis when using the
electron capture detector. Phthalates usually appear in the chromatogram as large, late-eluting
peaks. Phthalates may be leached from common flexible plastic tubing and other plastic mate-
rials during the extraction and clean-up processes. Cross-contamination of clean glassware
routinely occurs when plastics are handled during extraction, especially when solvent-wetted
surfaces are handled. Interferences from phthalates can best be minimized by avoiding the use
770
-------
Method 1658
of plastics in the laboratory, or by using a microcoulometric or electrolytic conductivity
detector.
3.5 The acid forms of the herbicides are strong acids that react readily with alkaline substances
and can be lost during analysis. Glassware, glass wool, and all other apparatuses should be
rinsed with dilute hydrochloric or sulfuric acid prior to use. Sodium sulfate and other reagents
that can be acidified should be acidified to preclude the herbicides from being adsorbed by
these reagents.
3.6 Organic acids and phenols cause the most direct interference with the herbicides. Alkaline
hydrolysis and subsequent extraction of the basic solution can remove many hydrocarbons and
esters that may interfere with the herbicide analysis.
3.7 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled. The cleanup procedures given in this method
can be used to overcome many of these interferences, but unique samples may require addi-
tional cleanup to achieve the minimum levels given in Table 2.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 1 through 3.
4.2 Primary standards of hazardous compounds shall be prepared in a hood, and a NIOSH/MESA
approved toxic gas respirator should be worn when high concentrations are handled.
4.3 Diazomethane is a toxic carcinogen which can decompose or explode under certain conditions.
Solutions decompose rapidly in the presence of solid materials such as copper powder, calcium
chloride, and boiling chips. The following operations may cause explosion: heating above
90°C; use of grinding surfaces such as ground-glass joints, sleeve bearings, and glass stirrers;
and storage near alkali metals. Diazomethane shall be used only behind a safety screen in a
well ventilated hood and should be pipetted with mechanical devices only.
4.4 Unknown samples may contain high concentrations of volatile toxic compounds. Sample con-
tainers should be opened in a hood and handled with gloves that will prevent exposure. The
oven used for sample drying to determine percent moisture should be located in a hood so that
vapors from samples do not create a health hazard in the laboratory.
5. APPARATUS AND MATERIALS
NO TE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting requirements of this method is the responsibility of the laboratory.
711
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Method 1658
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottles and caps.
5.1.1.1 Liquid samples (waters, sludges and similar materials that contain less
than 5% solids): Sample bottle, amber glass, 1-L or 1-quart, with screw-
cap.
5.1.1.2 Solid samples (soils, sediments, sludges, filter cake, compost, and similar
materials that contain more than 5% solids): Sample bottle, wide-mouth,
amber glass, 500-mL minimum.
5.1.1.3 If amber bottles are not available, samples shall be protected from light.
5.1.1.4 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with
PTFE.
5.1.1.5 Cleaning.
5.1.1.5.1 Bottles are detergent-water washed, then rinsed with solvent
rinsed or baked at 450°C for a minimum of 1 hour before use.
5.1.1.5.2 Liners are detergent-water washed, then rinsed with reagent
water and solvent, and baked at approximately 200°C for a
minimum of 1 hour prior to use.
5.1.2 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept
at 0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sam-
pler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing
may be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample con-
tamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for determining percent moisture.
5.2.1 Oven, capable of maintaining a temperature of 110°C (±5°C).
5.2.2 Dessicator.
5.2.3 Crucibles, porcelain.
5.2.4 Weighing pans, aluminum.
5.3 Extraction equipment.
5.3.1 Equipment for ultrasonic extraction.
5.3.1.1 Sonic disrupter: 375 watt with pulsing capability and W or % " disrupter
horn (Ultrasonics, Inc, Model 375C, or equivalent).
5.3.1.2 Sonabox (or equivalent), for use with disrupter.
5.3.2 Equipment for liquid-liquid extraction.
5.3.2.1 Continuous liquid-liquid extractor: PTFE or glass connecting joints and
stopcocks without lubrication, 1.5- to 2-L (Hershberg-Wolf Extractor,
Cal-Glass, Costa Mesa,California, 1000- or 2000-mL continuous extractor,
or equivalent).
772
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Method 1658
5.3.2.2 Round-bottom flask, 500-mL, with heating mantle.
5.3.2.3 Condenser, Graham, to fit extractor.
5.3.2.4 pH meter, with combination glass electrode.
5.3.2.5 pH paper, wide range (Hydrion Papers, or equivalent).
5.3.3 Separatory runnels: 250-, 500-, 1000-, and 2000-mL, with PTFE stopcocks.
5.3.4 Filtration apparatus.
5.3.4.1 Glass powder funnels: 125- to 250-mL.
5.3.4.2 Filter paper for above (Whatman 41, or equivalent).
5.3.5 Beakers.
5.3.5.1 1.5- to 2-L, calibrated to 1 L.
5.3.5.2 400- to 500-mL.
5.3.6 Spatulas: Stainless steel or PTFE.
5.3.7 Drying column: 400 mm long x 15 to 20 mm ID, Pyrex chromatographic column
equipped with coarse glass frit or glass wool plug.
5.3.7.1 Pyrex glass wool: Extracted with solvent or baked at 450°C for a mini-
mum of 1 hour.
5.3.8 TLCP extractor.
5.3.8.1 Rotary agitation apparatus: Capable of rotating the extraction vessel in an
end over end fashion at 30 rpm (±2 rpm) (Associated Design and Manu-
facturing Co., or equivalent).
5.3.8.2 Bottle, polyethylene or polypropylene, 1- to 4-L, with screw-cap with
PTFE-lined lid, to fit extractor.
5.4 Evaporation/concentration apparatus.
5.4.1 Kuderna-Danish (K-D) apparatus.
5.4.1.1 Evaporation flask: 500-mL (Kontes K-570001-0500, or equivalent), at-
tached to concentrator tube with springs (Kontes K-662750-0012).
5.4.1.2 Concentrator tube: 10-mL, graduated (Kontes K-570050-1025, or equiva-
lent) with calibration verified. Ground-glass stopper (size 19/22 joint) is
used to prevent evaporation of extracts.
5.4.1.3 Snyder column: Three-ball macro (Kontes K-503000-0232, or equivalent).
5.4.1.4 Snyder column: Two-ball micro (Kontes K-469002-0219, or equivalent).
5.4.1.5 Boiling chips.
5.4.1.5.1 Glass or silicon carbide: Approximately 10/40 mesh, extracted
with methylene chloride and baked at 450 °C for a minimum of
1 hour.
5.4.1.5.2 PTFE (optional): Extracted with methylene chloride.
5.4.2 Water bath: Heated, with concentric ring cover, capable of temperature control
(±2°C), installed in a fume hood.
713
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Method 1658
5.4.3 Nitrogen evaporation device: Equipped with heated bath that can be maintained at
35 to 40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit
GC auto-sampler.
5.5 Balances.
5.5.1 Analytical: Capable of weighing 0.1 mg.
5.5.2 Top loading: Capable of weighing 10 mg.
5.6 Apparatus for sample cleanup.
5.6.1 Automated gel permeation chromatograph (Analytical Biochemical Labs, Inc, Colum-
bia, MO, Model GPC Autoprep 1002, or equivalent).
5.6.1.1 Column: 600 to 700 mm long x 25 mm ID, packed with 70 g of SX-3
Bio-beads (Bio-Rad Laboratories, Richmond, CA, or equivalent).
5.6.1.2 Syringe, 10-mL, with Luer fitting.
5.6.1.3 Syringe-filter holder, stainless steel, and glass fiber or PTFE filters (Ge-
Iman Acrodisc-CR, 1 to 5 /*, or equivalent).
5.6.1.4 UV detectors: 254-nm, preparative or semi-prep flow cell: (Isco, Inc.,
Type 6; Schmadzu, 5 mm path length; Beckman-Altex 152W, 8-/*L micro-
prep flow cell, 2-mm path; Pharmacia UV-1, 3-mm flow cell; LDC Mil-
ton-Roy UV-3, monitor #1203; or equivalent).
5.6.2 Vacuum system and cartridges for solid phase extraction (SPE).
5.6.2.1 Vacuum system: Capable of achieving 0.1 bar (house vacuum, vacuum
pump, or water aspirator), with vacuum gauge.
5.6.2.2 VacElute Manifold (Analytichem International, or equivalent).
5.6.2.3 Vacuum trap: Made from 500-mL sidearm flask fitted with single-hole
rubber stopper and glass tubing.
5.6.2.4 Rack for holding 50-mL volumetric flasks in the manifold.
5.6.2.5 Column: Mega Bond Elut, Non-polar, CIS Octadecyl, 10 g/60 mL (Ana-
lytichem International Cat. No. 607H060, or equivalent).
5.6.3 Chromatographic column: 400 mm long x 22 mm ID, with PTFE stopcock and
coarse frit (Kontes K-42054, or equivalent).
5.7 Centrifuge apparatus.
5.7.1 Centrifuge: Capable of rotating 500-mL centrifuge bottles or 15-mL centrifuge tubes
at 5,000 rpm minimum.
5.7.2 Centrifuge bottles: 500-mL, with screw-caps, to fit centrifuge.
5.7.3 Centrifuge tubes: 12- to 15-mL, with screw-caps, to fit centrifuge.
5.7.4 Funnel, Buchner, 15 cm.
5.7.4.1 Flask, filter, for use with Buchner funnel.
5.7.4.2 Filter paper, 15 cm (Whatman #41, or equivalent).
774
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Method 1658
5.8 Derivatization apparatus: Diazald kit with clear seal joints for generation of diazomethane
(Aldrich Chemical Co. Z10,025-0, or equivalent).
5.9 Miscellaneous glassware.
5.9.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.9.2 Syringes, glass, with Luerlok tip, 0.1-, 1.0- and 5.0-mL. Needles for syringes, 2",
22-gauge.
5.9.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.9.4 Scintillation vials, glass, 20- to 50-mL, with PTFE-lined screw-caps.
5.10 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with an electron capture or halide-specific detector at the end of
each column, temperature program with isothermal holds, data system capable of recording
simultaneous signals from the two detectors, and shall meet all of the performance specifi-
cations in Section 14.
5.10.1 GC columns: Bonded-phase fused-silica capillary.
5.10.1.1 Primary: 30 m (±3 m) long x 0.5 mm (±0.05 mm) ID (DB-608, or e-
quivalent).
5.10.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.10.2 Data system shall collect and record GC data, store GC runs on magnetic disk or
tape, process GC data, compute peak areas, store calibration data including retention
times and calibration factors, identify GC peaks through retention times, compute
concentrations, and generate reports.
5.10.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.10.2.2 Calibration factors and calibration curves: The data system shall be used
to record and maintain lists of calibration factors, and multi-point calibra-
tion curves (Section 7). Computations of relative standard deviation (co-
efficient of variation) are used for testing calibration linearity. Statistics on
initial (Section 8.2) and ongoing (Section 14.6) performance shall be com-
puted and maintained.
5.10.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
5.10.3 Detectors.
5.10.3.1 Halide-specific: Electron capture or electrolytic conductivity (Micoulo-
metric, Hall, O.I., or equivalent), capable of detecting 100 pg of 2,4-D
under the analysis conditions given in Table 2.
715
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Method 1658
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide: Reagent grade.
6.2.1.1 Concentrated solution (ION): Dissolve 40 g NaOH in 100 mL reagent
water.
6.2.1.2 Dilute solution (0.1M): Dissolve 4 g NaOH in 1 L of reagent water.
6.2,2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H2SO4
(specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (w/v). Dissolve 37 g KOH in 100 mL reagent water.
6.3 Acidified sodium sulfate: Add 0.5 mL H2SO4 and 30 mL ethyl ether to 100 g sodium sulfate.
Mix thoroughly. Allow the ether to evaporate completely. Transfer the mixture to a clean
Container and store at 110°C (±5°C).
6.4 Solvents: Methylene chloride, hexane, ethyl ether, acetone, acetonitrile, isooctane, and metha-
nol; pesticide-quality; lot-certified to be free of interferences.
6.4.1 Ethyl ether must be shown to be free of peroxides before it is used, as indicated by
EM Laboratories Quant test strips (Scientific Products PI 126-8, or equivalent).
Procedures recommended for removal of peroxides are provided with the test strips.
After cleanup, 20 mL of ethyl alcohol is added to each liter of ether as a preservative.
6.6 GPC calibration solution: Solution containing 300 mg/mL corn oil, 15-mg/mL bis(2-ethyl-
hexyOphthalate, 1.4 mg/mL pentachlorophenol, 0.1 mg/mL perylene, and 0.5 mg/mL sulfur.
6.6 Sample cleanup.
6.6.1 Florisil: PR grade, 60/100 mesh, activated at 650 to 700°C, stored in the dark in
glass container with PTFE-lined screw-cap. Activate at 130°C for 16 hours minimum
immediately prior to use. Alternatively, 500-mg cartridges (J.T. Baker, or equi-
valent) may be used.
6.6.2 Solid-phase extraction.
6.6.2.1 SPE cartridge calibration solution: 2,4,6-trichlorophenol, 0.1 /xg/mL in
acetone.
6.6.2.2 SPE elution solvent: Methylene chloride: acetonitrile: hexane (50:3:47).
6.7 Derivatization: Diazald reagent (W-memyl-N-nitroso-p-toluenesulfonamide), fresh and high-
purity (Aldrich Chemical Co.).
6.8 Reference matrices.
6.8.1 Reagent water: Water in which the compounds of interest and interfering compounds
are not detected by this method.
6.8.2 High-solids reference matrix: Playground sand or similar material in which the com-
pounds of interest and interfering compounds are not detected by this method. May
be prepared by extraction with methylene chloride and/or baking at 450 °C for 4 hours
minimum.
776
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Method 1658
6.9 Standard solutions: Purchased as solutions or mixtures with certification to their purity, con-
centration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute the
concentration of the standard. When not being used, standards are stored in the dark at
-20 to -10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the
level of the solution so that solvent evaporation loss can be detected. The vials are brought to
room temperature prior to use. Any precipitate is redissolved and solvent is added if solvent
loss has occurred.
6.10 Preparation of stock solutions: Prepare in isooctane per the steps below. Observe the safety
precautions in Section 4.
6.10.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10 mg 2,4-D in a 10-mL ground- glass stoppered volumetric flask and fill
to the mark with isooctane. After the 2,4-D is completely dissolved, transfer the
solution to a 15-mL vial with PTFE-lined cap.
6.10.2 Stock standard solutions should be checked for signs of degradation prior to the
preparation of calibration or performance test standards.
6.10.3 Stock standard solutions shall be replaced after 6 months, or sooner if comparison
with quality control check standards indicates a change in concentration.
6.11 Secondary mixtures: Combine stock solutions (Section 6.10) into a secondary mixture at the
highest level required for required for calibration (Table 3). Derivatize the acids in this solu-
tion using the procedure in Section 12. After derivatization, prepare the solutions for calibra-
tion and calibration verification (Table 3), for initial and ongoing precision and recovery
(Sections 8.2 and 14.6), and for spiking into the sample matrix (Section 8.4).
6.12 Surrogate spiking solution: Prepare 2,4-dichlorophenylacetic acid at a concentration of
2 ng/mL in acetone.
6.13 Stability of solutions: All standard solutions (Sections 6.9 - 6.12) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards
will remain acceptable if the peak area remains within ±15% of the area obtained in the initial
analysis of the standard.
7. SETUP AND CALIBRATION
7.1 Configure the GC system as given in Section 5.10 and establish the operating conditions in
Table 2.
7.2 Attainment of method detection limit (MDL): Determine that the MDLs in Table 2 can be met
on each column/detector system.
7.3 Calibration: Inject the calibration solutions into each GC column/detector pair, beginning with
the lowest level mixture and proceeding to the highest. For each compound, compute and
store, as a function of the concentration injected, the retention time, and the peak area on each
column/detector system (primary and confirmatory).
7.3.1 Retention time: The polar nature of some analytes causes the retention time to de-
crease as the quantity injected increases. To compensate this effect, the retention time
for compound identification is correlated with the analyte level.
717
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Method 1658
7.3.1.1 If the difference between the maximum and minimum retention times for
any compound is less than 5 seconds over the calibration range, the reten-
tion time for that compound can be considered constant and an average
retention-time may be used for compound identification.
7.3.1.2 Retention time calibration curve (retention time vs. amount): If the reten-
tion time for a compound in the lowest level standard is more than 5 sec-
onds greater than the retention time for the compound in the highest level
standard, a retention time calibration curve shall be used for identification
of that compound
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each compound on each
column/detector system.
7.3.2.2 Linearity: If the calibration factor for any compound is constant
(Cv < 20%) over the calibration range, an average calibration factor may
be used for that compound; otherwise, the complete calibration curve (area
vs. amount) for that compound shall be used.
7.4 Combined QC standards: To preclude periodic analysis of all of the individual calibration
groups of compounds (Section 7.3.1), the GC systems are calibrated with combined solutions
as a final step. Not all of the compounds in these standards will be separated by the GC
columns used in this method. Retention times and calibration factors are verified for the
compounds that are resolved, and calibration factors are obtained for the unresolved peaks.
These combined QC standards are prepared at the level the mid-range calibration standard
(Table 3).
7.4.1 Analyze the combined QC standards on their respective column/detector pairs.
7.4.1.1 For those compounds that exhibit a single, resolved GC peak,the retention
time shall be within +5 seconds of the retention time of the peak in the
medium level calibration standard (Table 3), and the calibration factor
using the primary column shall be within ±20% of the calibration factor in
the medium level standard (Table 3).
7.4.1.2 For the peaks containing two or more compounds, compute and store the
retention times at the peak maxima on both columns (primary and confir-
matory), and also compute and store the calibration factors on both col-
umns. These results will be used for calibration verification (Section 14.2
and 14.5) and for precision and recovery studies (Sections 8.2 and 14.6).
7.5 Florisil calibration: The cleanup procedure in Section 11 utilizes Florisil column chromato-
graphy. Florisil from different batches or sources may vary in adsorptive capacity. To
standardize the amount of Florisil that is used, the use of the lauric acid value (Reference 4) is
suggested. The referenced procedure determines the adsorption of lauric acid (in milligrams
per gram Florisil) from hexane solution. The amount of Florisil to be used for each column is
calculated by dividing 110 by this ratio and multiplying by 20 g.
718
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Method 1658
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program
(Reference 5). The minimum requirements of this program consist of an initial demonstration
of laboratory capability, an ongoing analysis of standards and blanks as tests of continued
performance, and analysis of spiked samples to assess accuracy. Laboratory performance is
compared to established performance criteria to determine if the results of analyses meet the
performance characteristics of the method. If the method is to be applied routinely to
samples containing high solids with very little moisture (e.g., soils, compost), the high-solids
reference matrix (Section 6.8.2) is substituted for the reagent water (Section 6.8.1) in all
performance tests, and the high-solids method (Section 10) is used for these tests.
8.1.1 The analyst shall make an initial demonstration of 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 analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If detection limits will be affected by the modification, the analyst is required to
repeat the demonstration of detection limits (Section 7.2).
8.1.3 The laboratory shall spike all samples with at least one surrogate compound to moni-
tor method performance. This test is described in Section 8.3. When results of these
spikes indicate a typical method performance for samples, the samples are diluted to
bring method performance within acceptable limits (Section 17).
8.1.4 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the combined QC standard (Section 7.4) that the analysis system is
in control. These procedures are described in Sections 14.1, 14.5, and 14.6.
8.1.5 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.4.
8.1.6 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.5.
8.1.7 Other analytes may be determined by this method. The procedure for establishing a
preliminary quality control limit for a new analyte is given in Section 8.6.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
recovery, the analyst shall perform the following operations.
8.2.1 For analysis of samples containing low solids (aqueous samples), extract, concentrate,
and analyze one set of four 1-L aliquots of reagent water spiked with the combined
QC standard (Section 7.4) according to the procedure in Section 10. Alternatively,
sets of four replicates of the individual calibration groups (Section 7.3) may be used.
For samples containing high-solids, sets of four 30-g aliquots of the high-solids
reference matrix are used.
719
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Method 1658
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X)
and the coefficient of variation (Cv) of percent recovery (s) for each compound.
8.2.3 For each compound, compare s and X with the corresponding limits for initial preci-
sion and accuracy in Table 4. For coeluting compounds, use the coeluted compound
with the least restrictive specification (largest Cv and widest range). If s and X for all
compounds meet the acceptance criteria, system performance is acceptable and analy-
sis of blanks and samples may begin. If, however, any individual s exceeds the preci-
sion limit or any individual X falls outside the range for accuracy, system perfor-
mance is unacceptable for that compound. In this case, correct the problem and
repeat the test.
8.3 The laboratory shall spike all samples with at least one surrogate compound to assess method
performance on the sample matrix.
8.3.1 Analyze each sample according to the method beginning in Section 10.
8.3.2 Compute the percent recovery (P) of the surrogate compound(s).
8.3.3 The recovery of the surrogate compound shall be within the limits of 40 to 120%. If
the recovery of any surrogate falls outside of these limits, method performance is
unacceptable for that sample, and the sample is complex. Water samples are diluted,
and smaller amounts of soils, sludges, and sediments are reanalyzed per Section 17.
8.4 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from
a given site type (e.g., influent to treatment, treated effluent, produced water, river sediment).
If only one sample from a given site type is analyzed, that sample shall be spiked.
8.4.1 The concentration of the matrix spike shall be determined as follows.
8.4.1.1 If, as in compliance monitoring, the concentration of a specific analyte in
the sample is being checked against a regulatory concentration limit, the
matrix spike shall be at that limit or at 1 to 5 times higher than the back-
ground concentration determined in Section 8.4.2, whichever concentration
is larger.
8.4.1.2 If the concentration of an analyte in the sample is not being checked
against a limit specific to that analyte, the matrix spike shall be at the
concentration of the combined QC standard (Section 7.4) or at 1 to 5 times
higher than the background concentration, whichever concentration is
larger.
8.4.1.3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration or at the
concentration of the combined QC standard (Section 7.4).
8.4.2 Analyze one sample aliquot to determine the background concentration (B) of each
analyte. If necessary, prepare a standard solution appropriate to produce a level in
the sample one to five times the background concentration. Spike a second sample
720
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Method 1658
aliquot with the standard solution and analyze it to determine the concentration after
spiking (A) of each analyte. Calculate the percent recovery (P) of each analyte:
Equation 1
p _ 100(A-B)
T
where
T = True value of the spike
8.4.3 Compare the percent recovery for each analyte with the corresponding QC acceptance
criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the sam-
ple is complex and must be diluted and reanalyzed per Section 17.
8.4.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (water, soil, sludge, sediment) in which the analytes pass the tests
in Section 8.4.3, compute the average percent recovery (P) and the standard deviation
of the percent recovery (sp) for each compound (or coeluting compound group).
Express the accuracy assessment as a percent recovery interval from P - 2sp to
P + 2sp for each matrix. For example, if P = 90% and Sp = 10% for five analyses
of compost, the accuracy interval is expressed as 70 to 110%. Update the accuracy
assessment for each compound in each matrix on a regular basis (e.g., after each five
to ten new accuracy measurements).
8.5 Blanks: Reagent water and high-solids reference matrix blanks are analyzed to demonstrate
freedom from contamination.
8.5.1 Extract and concentrate a 1-L reagent water blank or a 30-g high-solids reference
matrix blank with each sample lot (samples started through the extraction process on
the same 8-hour shift, to a maximum of 20 samples). Analyze the blank immediately
after analysis of the combined QC standard (Section 14.6) to demonstrate freedom
from contamination.
8.5.2 If any of the compounds of interest (Table 1) or any potentially interfering compound
is found in an aqueous blank at greater than 0.05 /*g/L, or in a high-solids reference
matrix blank at greater than 1 jtg/kg (assuming the same calibration factor as 2,4-D
for compounds not listed in Table 1), analysis of samples is halted until the source of
contamination is eliminated and a blank shows no evidence of contamination at this
level.
8.6 Other analytes may be determined by this method. To establish a quality control limit for an
analyte, determine the precision and accuracy by analyzing four replicates of the analyte along
with the combined QC standard per the procedure in Section 8.2. If the analyte coelutes with
an analyte in the QC standard, prepare a new QC standard without the coeluting component(s).
Compute the average percent recovery (A) and the standard deviation of percent recovery ($„)
727
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Method 1658
for the analyte, and measure the recovery and standard deviation of recovery for the other
analytes. The data for the new analyte is assumed to be valid if the precision and recovery
specifications for the other analytes are met; otherwise, the analytical problem is corrected and
the test is repeated. Establish a preliminary quality control limit of A ± 2sn for the new ana-
lyte and add the limit to Table 4.
8.7 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 14.5), and for initial (Section 8.2) and ongoing (Section 14.6)
precision and recovery should be identical, so that the most precise results will be obtained.
The GC instruments will provide the most reproducible results if dedicated to the settings and
conditions required for the analyses of the analytes given in this method.
8.8 Depending on specific program requirements, field replicates and field spikes of the analytes
of interest into samples may be required to assess the precision and accuracy of the sampling
and sample transporting techniques.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices (Reference 6),
except that the bottle shall not be prerinsed with sample before collection. Aqueous samples
which flow freely are collected in refrigerated bottles using automatic sampling equipment.
Solid samples are collected as grab samples using wide-mouth jars.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH of less than 2 using
sulfuric acid solution. Record the volume of acid used. Caution: some samples require
acidification in a hood because of the potential for generating hydrogen sulfide.
9.3 If residual chlorine is present in aqueous samples, add 80 mg sodium thiosulfate per liter of
water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Refer-
ence 7).
9.4 Begin sample extraction within 7 days of collection, and analyze all extracts within 28 days of
extraction.
1 0. SAMPLE EXTRA c TION AND CONCENTRA TION
Samples containing 1 % solids or less are extracted directly using continuous liquid/liquid extraction
techniques (Section 10.2.1). Samples containing 1 to 30% solids are diluted to the 1% level with
reagent water (Section 10.2.2) and extracted using continuous liquid-liquid extraction techniques.
Samples containing greater than 30% solids are extracted by tumbling with water in a rotary agitation
apparatus. The aqueous phase is then extracted using continuous liquid-liquid extraction techniques.
Figure 1 outlines the extraction and concentration steps.
10.1 Determination of percent solids.
10.1.1 Weigh 5 to 10 g of sample into a tared beaker. Record the weight to three significant
figures.
10.1.2 Dry overnight (12 hours minimum) at 110°C (±5°C), and cool in a desiccator.
722
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Method 1658
10.1.3 Determine percent solids as follows:
Equation 2
solids = ^ight of dry sample x
weight of wet sample
10.2 Preparation of samples for extraction.
10.2.1 Samples containing 1% solids or less.
10.2.1.1 Measure 1.00 L (±0.01 L) of sample into a clean 1.5- to 2.0-L beaker.
10.2.1.2 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into the
sample aliquot.
10.2.1.3 Proceed to preparation of the QC aliquots for low-solids samples (Sec-
tion 10.2.3).
10.2.2 Samples containing 1 to 30% solids.
10.2.2.1 Mix sample thoroughly.
10.2.2.2 Using the percent solids found in Section 10.1.3, determine the weight of
sample required to produce 1 L of solution containing 1 % solids as fol-
lows:
Equation 3
1000
% solids
= grams
10.2.2.3 Place the weight determined in Section 10.2.2.2 in a clean 1.5- to 2.0-L
beaker. Discard all sticks, rocks, leaves, and other foreign material prior
to weighing.
10.2.2.4 Bring the volume of the sample aliquot(s) to 400 to 500 mL with reagent
water.
10.2.2.5 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into each
sample aliquot.
10.2.2.6 Using a clean metal spatula, break any solid portions of the sample into
small pieces.
10.2.2.7 Place the 3A " horn on the ultrasonic probe approximately W below the
surface of each sample aliquot and pulse at 50% for 3 minutes at full
power. If necessary, remove the probe from the solution and break any
large pieces using the metal spatula or a stirring rod and repeat the soni-
cation. Clean the probe with 5% aqueous sodium bicarbonate and then
723
-------
Method 1658
methylene chloride:acetone (1:1) between samples to prevent damage to the
horn and preclude cross-contamination.
10.2.2.8 Bring the sample volume to 1.0 L (±0.1 L) with reagent water.
10.2.3 Preparation of QC aliquots for samples containing low solids (less than 30%).
10.2.3.1 For each sample or sample batch (to a maximum of 20) to be extracted at
the same time, place two 1.0 L (±0.01 L) aliquots of reagent water in
clean 1.5- to 2.0-L beakers. Acidify to pH to less than 2 with HC1.
10.2.3.2 Blank spike 0.5 mL of the surrogate spiking solution (Section 6.12) into
one reagent water aliquot.
10.2.3.3 Spike the combined QC standard (Section 7.4) into a reagent water aliquot.
10.2.3.4 If a matrix spike is required, prepare an aliquot at the concentrations
specified in Section 8.4.
10.2.4 Hydrolysis of acid esters and flocculation of particulates.
10.2.4.1 While on a stirring plate, raise the pH of the sample and QC aliquots to
pH 12 to 13.
10.2.4.2 Stir and equilibrate all sample and QC solutions for 1 to 2 hours. Check
the pH after approximately 0.5 hour and adjust if necessary.
10.2.4.3 Add sufficient NaCl to saturate the solution. Approximately 350 g are
required. Stir to dissolve.
10.2.4.4 If the solution appears cloudy, add 2 g (±0.2 g) of CaCl2 and allow to
stand for approximately 10 minutes to flocculate particulates.
10.2.4.5 Pre-extract the samples and QC aliquots to remove interferents per Sec-
tion 10.3.
10.2.5 Samples containing 30% solids or greater (Reference 8).
10.2.5.1 Mix the sample thoroughly.
10.2.5.2 Weigh 30 g (±0.3 g) of sample into a clean tumbler bottle. Discard all
sticks, rocks, leaves, and other foreign material prior to weighing.
10.2.5.3 Add 1000 m (± 100 mL) of reagent water and adjust the pH to 12 to 13
using NaOH.
10.2.5.4 QC aliquots: For each sample or sample batch (to a maximum of 20) to
be extracted at the same time, place two 30 g (±0.3-g) aliquots of the
high-solids reference matrix in tumbler bottles. One aliquot will serve as
the blank.
10.2.5.5 Spike 0.5 mL of the surrogate spiking solution (Section 6.12) into each
aliquot.
10.2.5.6 To serve as the ongoing precision and recovery standard,spike 1.0 mL of
the combined QC standard (Section 7.4) into the remaining aliquot. Raise
the pH of the QC aliquots to 12 to 13.
10.2.5.7 Tightly cap the tumbler bottles and tumble for 2 to 4 hours.
724
-------
Method 1658
10.3 Pre-extraction to remove interferents: Place 100 to 150 mL methylene chloride in each conti-
nuous extractor and 200 to 300 mL in each distilling flask.
10.3.1 Pour the sample(s), blank, and standard aliquots into the extractors.
10.3.1.1 If a precipitate formed in the flocculation step (Section 10.2.4.4), or if the
sample contains other solids, pour the sample through filter paper into the
extractor.
10.3.1.2 Rinse the containers with 50 to 100 mL methylene chloride and add to the
respective extractors. For samples that were filtered, pour the rinse over
the residual sample in the filter funnel and drain into the respective extrac-
tor.
10.3.2 Verify that the pH of the water in the extractors is 12 to 13.
10.3.3 Begin the extraction by heating the flask until the methylene chloride is boiling.
When properly adjusted, one to two drops of methylene chloride per second will fall
from the condenser tip into the water. Test and adjust the pH of the waters during the
first 1 to 2 hours of extraction. Extract for 2 to 4 hours.
10.3.4 After extraction, remove the distilling flask, discard the methylene chloride, and add a
fresh charge of methylene chloride to the flask.
10.4 Extraction.
10.4.1 Adjust the pH of the water in the extractors to less than 2 with sulfuric acid.
10.4.2 Test and adjust the pH of the waters during the first 1 to 2 hours of the extraction.
Extract for 18 to 24 hours.
10.4.3 Remove the distilling flask, estimate and record the volume of extract (to the nearest
100 mL), and pour the contents through a prerinsed drying column containing 7 to
10 cm of acidified anhydrous sodium sulfate. Rinse the distilling flask with 30 to
50 mL of methylene chloride and pour through the drying column. Collect the solu-
tion in a 500-mL K-D evaporator flask equipped with a 10-mL concentrator tube.
Seal, label, and concentrate per Sections 10.5 and 10.6.
10.5 Macro concentration.
10.5.1 Concentrate the extracts in separate 500-mL K-D flasks equipped with 10-mL con-
centrator tubes. Add one or two clean, acid-rinsed boiling chips to the flask and
attach a three-ball macro Snyder column. Prewet the column by adding approxi-
mately 1 mL of methylene chloride through the top. Place the K-D apparatus in a hot
water bath so that the entire lower rounded surface of the flask is bathed with steam.
Adjust the vertical position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 minutes. At the proper rate of distillation, the
balls of the column will actively chatter but the chambers will not flood.
10.5.2 When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus
from the bath and allow the solvent to drain and cool for at least 10 minutes.
725
-------
V
Method 1658
10.5.3 For extracts to be cleaned upjiiing GPC, remove the Snyder column and rinse the
flask and its lower joint into the' concentrator tube with 1 to 2 mL of methylene
chloride. A 5-mL syringe is recommended for this operation. Adjust the final vol-
ume to 10 mL and proceed to GPC cleanup in Section 11.
10.5.4 For extracts to be cleaned up using the SPE cartridge, adjust the final volume to
5.0 mL for those that have been cleaned up using GPC, and to 10 mL for those that
have not. Proceed to SPE cleanup in Section 11.
10.6 Hexane exchange: Extracts containing acids to be derivatized, extracts to be subjected to
Florisil cleanup, and extracts that have been cleaned up are exchanged into hexane.
10.6.1 Remove the Snyder column, add approximately 50 mL of hexane and a clean boiling
chip, and reattach the Snyder column. Concentrate the extract as in Section 10.5,
except use hexane to prewet the column. The elapsed time of the concentration
should be 5 to 10 minutes.
10.6.2 Remove the Snyder column and rinse the flask and its lower joint into the con-
centrator tube with 1 to 2 mL of hexane.
10.6.2.1 For extracts containing acids to be esterified, adjust the final volume to
10 mL for those that have not been cleaned up by GPC, and to 5 mL for
those that have been cleaned up by GPC (the difference accounts for the
50% loss in the GPC cleanup). Proceed to Section 12 for esterification.
10.6.2.2 For extracts to be cleaned up using Florisil, adjust the final volume to 5 to
10 mL and proceed to Florisil cleanup in Section 11.
10.6.2.3 For extracts to be analyzed by GC (Section 13), adjust the final volume to
10 mL for those that have not been cleaned up by GPC, and to 5 mL for
those that have been cleaned up by GPC.
11. CLEANUP
11.1 Cleanup procedures may not be necessary for relatively clean samples (treated effluents,
ground water, drinking water). If particular circumstances require the use of a cleanup
procedure, the analyst may use any or all of the procedures below or any other appropriate
procedure. However, the analyst shall first repeat the tests in Section 8.2 to demonstrate that
the requirements of Section 8.2 can be met using the cleanup procedure(s) as an integral part
of the method. Figure 1 outlines the cleanup steps.
11.1.1 Gel permeation chromatography (Section 11.2) removes many high molecular weight
interferents that cause GC column performance to degrade. It is used for all soil and
sediment extracts and may be used for water extracts that are expected to contain high
molecular weight organic compounds (e.g., polymeric materials, humic acids).
11.1.2 The solid-phase extraction cartridge (Section 11.3) removes polar organic compounds
such as phenols.
11.1.3 The Florisil column (Section 11.4) allows for selected fractionation of the herbicides
and will also eliminate polar interferences.
726
-------
Method 1658
11.2 Gel permeation chromatography (GPC).
11.2.1 Column packing.
11.2.1.1 Place 70 to 75 g of SX-3 Bio-beads in a 400- to 500-mL beaker.
11.2.1.2 Cover the beads with methylene chloride and allow to swell overnight
(12 hours minimum).
11.2.1.3 Transfer the swelled beads to the column and pump solvent through the
column, from bottom to top, at 4.5 to 5.5 mL/min prior to connecting the
column to the detector.
11.2.1.4 After purging the column with solvent for 1 to 2 hours, adjust the column
head pressure to 7 to 10 psig, and purge for 4 to 5 hours to remove air.
Maintain a head pressure of 7 to 10 psig. Connect the column to the
detector.
11.2.2 Column calibration.
11.2.2.1 Load 5 mL of the calibration solution (Section 6.5) into the sample loop.
11.2.2.2 Inject the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis (2-ethylhexyl) phthalate, pentachloro-
phenol, perylene, and sulfur.
11.2.2.3 Set the "dump time" to allow >85% removal of the corn oil and >85%
collection of the phthalate.
11.2.2.4 Set the "collect time" to the peak minimum between perylene and sulfur.
11.2.2.5 Verify the calibration with the calibration solution after every 20 extracts.
Calibration is verified if the recovery of the pentachlorophenol is greater
than 85%. If calibration is not verified, the system shall be recalibrated
using the calibration solution, and the previous 20 samples shall be re-
extracted and cleaned up using the calibrated GPC system.
11.2.3 Extract cleanup: GPC requires that the column not be overloaded. The column
specified in this method is designed to handle a maximum of 0.5 gram of high molec-
ular weight material in a 5-mL extract. If the extract is known or expected to contain
more than 0.5 g, the extract is split into fractions for GPC and the fractions are
combined after elution from the column. The solids content of the extract may be
obtained gravimetrically by evaporating the solvent from a 50-jiL aliquot.
11.2.3.1 Filter the extract or load through the filter holder to remove particulates.
Load the 5.0-mL extract onto the column.
11.2.3.2 Elute the extract using the calibration data determined in Section 11.2.2.
Collect the eluate in a clean 400- to 500-mL beaker.
11.2.3.3 Rinse the sample loading tube thoroughly with methylene chloride between
extracts to prepare for the next sample.
11.2.3.4 If a particularly dirty extract is encountered, a 5.0-mL methylene chloride
blank shall be run through the system to check for carry-over.
11.2.3.5 Concentrate the extract and exchange to hexane per Section 10.6.
727
-------
Method 1658
11.3 Solid-phase extraction (SPE).
11.3.1 Setup.
11.3.1.1 Attach the Vac-elute manifold to a water aspirator or vacuum pump with
the trap and gauge installed between the manifold and vacuum source.
11.3.1.2 Place the SPE cartridges in the manifold, turn on the vacuum source, and
adjust the vacuum to 5 to 10 psia.
11.3.2 Cartridge washing: Pre-elute each cartridge prior to use sequentially with 10-mL
portions each of hexane, methanol, and water using vacuum for 30 seconds after each
eluant. Follow this pre-elution with 1 mL methylene chloride and three 10-mL
portions of the elution solvent (Section 6.6.2.2) using vacuum for 5 minutes after each
eluant. Tap the cartridge lightly while under vacuum to dry between eluants. The
three portions of elution solvent may be collected and used as a blank if desired.
Finally, elute the cartridge with 10 mL each of methanol and water, using the vacuum
for 30 seconds after each eluant.
11.3.3 Cartridge certification: Each cartridge lot must be certified to ensure recovery of the
compounds of interest and removal of 2,4,6-trichlorophenol.
11.3.3.1 To make the test mixture, add the trichlorophenol solution (Section
6.6.2.1) to the combined calibration standard (Section 7.4). Elute the
mixture using the procedure in Section 11.3.4.
11.3.3.2 Concentrate the eluant to 1.0 mL and inject 1.0 i*L of the concentrated
eluant into the GC using the procedure in Section 13. The recovery of all
analytes (including the unresolved GC peaks) shall be within the ranges for
recovery specified in Table 4, and the peak for trichlorophenol shall not be
detectable; otherwise the SPE cartridge is not performing properly and the
cartridge lot shall be rejected.
11.3.4 Extract cleanup.
11.3.4.1 After cartridge washing (Section 11.3.2), release the vacuum and place the
rack containing the 50-mL volumetric flasks (Section 5.6.2.4) in the vac-
uum manifold. Reestablish the vacuum at 5 to 10 psia.
11.3.4.2 Using a pipette or a 1-mL syringe, transfer 1.0 mL of extract to the SPE
cartridge. Apply vacuum for 5 minutes to dry the cartridge. Tap gently
to aid in drying.
11.3.4.3 Elute each cartridge into its volumetric flask sequentially with three 10-mL
portions of the elution solvent (Section 6.6.2.2), using vacuum for 5 min-
utes after each portion. Collect the eluants in the 50-mL volumetric flasks.
11.3.4.4 Release the vacuum and remove the 50-mL volumetric flasks.
11.3.4.5 Using the nitrogen blow-down apparatus, concentrate the eluted extracts to
1.0 mL, and proceed to Section 13 for GC analysis.
728
-------
Method 1658
11.4 Florisil column.
11.4.1 Place a weight of Florisil (nominally 20 g) predetermined by calibration (Section 7.5)
in a chromatographic column. Tap the column to settle the Florisil and add 1 to 2 cm
of anhydrous sodium sulfate to the top.
11.4.2 Add 60 mL of hexane to wet and rinse the sodium sulfate and Florisil. Just prior to
exposure of the sodium sulfate layer to the air, stop the elution of the hexane by
closing the stopcock on the chromatographic column. Discard the eluate.
11.4.3 Transfer the concentrated extract (Section 10.6.2) onto the column. Complete the
transfer with two 1-mL hexane rinses.
11.4.4 Place a clean 500-mL K-D flask and concentrator tube under the column. Drain the
column into the flask until the sodium sulfate layer is nearly exposed. Elute Frac-
tion 1 with 200 mL of 6 % (v/v) ethyl ether in hexane at a rate of approximately
5 mL/min. Remove the K-D flask. Elute Fraction 2 with 200 .mL of 15% (v/v) ethyl
ether in hexane into a second K-D flask. Elute Fraction 3 with 200 mL of 50% (v/v)
ethyl ether in hexane.
11.4.5 Concentrate the fractions as in Section 10.6, except use hexane to prewet the column.
Readjust the final volume to 5 or 10 mL as in Section 10.6, depending on whether the
extract was subjected to GPC cleanup, and analyze by gas chromatography per the
procedure in Section 13.
12. ESTERIFICATION
NOTE: Observe the safety precautions regarding diazomethane in Section 4.
12.1 Set up the diazomethane generation apparatus as given in the instructions in the Diazald kit.
12.2 Transfer 1 mL of the hexane solution containing the herbicides to a clean vial and add 0.5 mL
of methanol and 3 mL of ether.
12.3 Add 2 mL of diazomethane solution and let the sample stand for 10 minutes with occasional
swirling. The yellow color of diazomethane should persist throughout this period. If the
yellow color disappears, add 2 mL of diazomethane solution and allow to stand, with oc-
casional swirling, for another 10 minutes. Colored or complex samples will require at least
4 mL of diazomethane to ensure complete reaction of the herbicides. Continue adding dizao-
methane in 2-mL increments until the yellow color persists for the entire 10-minute period or
until 10 mL of diazomethane solution has been added.
12.4.3 Rinse the inside wall of the container with 0.2 to 0.5 mL of diethyl ether and add
10 to 20 mg of silicic acid to react excess diazomethane. Filter through Whatman #41
paper into a clean sample vial. If the solution is colored or cloudy, evaporate to near
dryness using the nitrogen blowdown apparatus, bring to 1.0 mL with hexane, and
proceed to Section 11.3 for SPE cleanup. If the solution is clear and colorless, eva-
porate to near dryness, bring to 1.0 mL with hexane and proceed to Section 13 for
GC analysis.
723
-------
Method 1658
13. GAS CHROMATOGRAPHY
NOTE: Table 2 summarizes the recommended operating conditions for the gas chroma-
tograph. Included in this table are the retention times and estimated detection limits
that can be achieved under these conditions. Examples of the separations achieved by
the primary and confirmatory columns are shown in Figures 2 and 3.
13.1 Calibrate the system as described in Section 7.
13.2 Set the injection volume on the auto-sampler to inject 1.0 /xL of all standards and extracts of
blanks and samples.
13.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection
after the last analyte is expected to elute and to return the column to the initial temperature.
14. SYSTEM AND LABORA TORY PERFORMANCE
14.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified for all pollutants and surrogates on both column/
detector systems. For these tests, analysis of the combined QC standard (Section 7.4) shall be
used to verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall
be performed until all performance criteria are met. Only after all performance criteria are
met may samples, blanks, and precision and recovery standards be analyzed.
14.2 Retention times: The absolute retention times of the peak maxima shall be within ±10 sec-
onds of the retention times in the initial calibration (Section 7.4.1).
14.3 GC resolution: Resolution is acceptable if the valley height between two peaks (as measured
from the baseline) is less than 10% of the taller of the two peaks.
14.3.1 Primary column (DB-608): Dicamba and MCPA.
14.3.2 Confirmatory column (DB-1701): MCPP and MCPA.
14.5 Calibration verification: Calibration is verified for the combined QC standard only.
14.5.1 Inject the combined QC standard (Section 7.4)
14.5.2 Compute the percent recovery of each compound or coeluting compounds, based on
the calibration data (Section 7.4).
14.5.3 For each compound or coeluted compounds, compare this calibration verification
recovery with the corresponding limits for ongoing recovery in Table 4. For coelu-
ting compounds, use the coeluted compound with the least restrictive specification (the
widest range). If the recoveries for all compounds meet the acceptance criteria,
system performance is acceptable and analysis of blanks and samples may begin. If,
however, any recovery falls outside the calibration verification range, system perfor-
mance is unacceptable for that compound. In this case, correct the problem and
repeat the test, or recalibrate (Section 7).
730
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Method 1658
14.6 Ongoing precision and recovery.
14.6.1 Analyze the extract of the precision and recovery standard extracted with each sample
lot (Sections 10.2.3.3 and 10.2.5.7).
14.6.2 Compute the percent recovery of each analyte and coeluting compounds.
14.6.3 For each compound or coeluted compounds, compare the percent recovery with the
limits for ongoing recovery in Table 4. For coeluted compounds, use the coeluted
compound with the least restrictive specification (widest range). If all analytes pass,
the extraction, concentration, and cleanup processes are in control and analysis of
blanks and samples may proceed. If, however, any of the analytes fail, these proces-
ses are not in control. In this event, correct the problem, re-extract the sample batch,
and repeat the on-going precision and recovery test.
14.6.4 Add results which pass the specifications in Section 14.6.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued labora-
tory performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery sr. Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.
/ 5. QUALITA TIVE DETERMINA TION
15.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 14.2), and with data stored in the
retention-time and calibration libraries (Sections 7.3.2 and 7.3.3.2). Identification is con-
firmed when retention time and amounts agree per the criteria below.
15.2 For each compound on each column/detector system, establish a retention-time window
±20 seconds on either side of the retention-time in the calibration data (Section 7.3.1). For
compounds that have a retention time curve (Section 7.3.1.2), establish this window as the
minimum -20 seconds and maximum +20 seconds.
15.2.1 Compounds not requiring a retention-time calibration curve: If a peak from the
analysis of a sample or blank is within a window (as defined in Section 15.2) on the
primary column/detector system, it is considered tentatively identified. A tentatively
identified compound is confirmed when (1) the retention time for the compound on
the confirmatory column/detector system is within the retention-time window on that
system, and (2) the computed amounts (Section 16) on each system (primary and
confirmatory) agree within a factor of 3.
15.2.2 Compounds requiring a retention-time calibration curve: If a peak from the analysis
of a sample or blank is within a window (as defined in Section 15.2) on the primary
column/detector system, it is considered tentatively identified. A tentatively identified
compound is confirmed when (1) the retention-times on both systems (primary and
confirmatory) are within ±30 seconds of the retention times for the computed
amounts (Section 16), as determined by the retention-time calibration curve (Section
7.3.1.2), and (2) the computed amounts (Section 16) on each system (primary and
confirmatory) agree within a factor of 3.
737
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Method 1658
7 6. QUANTITA TIVE DETERMINA TION
16.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.3.2).
16.2 Liquid samples: Compute the concentration in the sample using the following equation:
Equation 4
C - 10
(Vs)
where
Cs = Concentration in the sample, in pg/L
10 = Final extract total volume, in mL
CeK = Concentration in the extract, in
Vs = Sample extracted, in L
16.3 Solid samples: Compute the concentration in the solid phase of the sample using the following
equation:
Equation 5
(C. ^
C = 10.
1000 (Wt) (solids)
where
Cs = Concentration in the sample, in tuglkg
10 = Final extract total volume, in mL
CK = Concentration in the extract, in fig/mL
1000 = Conversion factor, g to kg
Ws = Sample weight, in g
solids = Percent solids in Section 10.1.3 divided by 100
16.4 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 1-/*L aliquot of the diluted extract is analyzed.
16.5 Report results for all pollutants found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at
which the concentration is in the calibration range.
77. ANALYSIS OF COMPLEX SAMPLES
17.1 Some samples may contain high levels (> 1000 ng/L) of the compounds of interest, interfering
compounds, and/or polymeric materials. Some samples may not concentrate to 10 mL (Sec-
tion 10.6); others may overload the GC column and/or detector.
732
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Method 1658
17.2 The analyst shall attempt to clean up all samples using GPC (Section 11.2), the SPE cartridge
(Section 11.3), and Florisil (Section 11.4). If these techniques do not remove the interfering
compounds, the extract is diluted by a factor of 10 and reanalyzed (Section 16.4).
17.3 Recovery of surrogates; in most samples, surrogate recoveries will be similar to those from
reagent water or from the high-solids reference matrix. If the surrogate recovery is outside the
range specified in Section 8.3, the sample shall be reextracted and reanalyzed. If the surrogate
recovery is still outside this range, the sample is diluted by a factor of 10 and reanalyzed
(Section 16.4).
17.4 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those
from reagent water or from the high-solids reference matrix. If the matrix spike recovery is
outside the range specified in Table 4, the sample shall be diluted by a factor of 10, respiked,
and reanalyzed. If the matrix spike recovery is still outside the range, the method does not
work on the sample being analyzed and the result may not be reported for regulatory compli-
ance purposes.
18. METHOD PERFORMANCE
18.1 Development of this method is detailed in References 9 and 10.
733
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Method 1658
References
1. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
2. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
3. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
4. Mills, P. A., "Variation of Florisil Activity: Simple Method for Measuring Adsorbent Capa-
city and Its Use in Standardizing Florisil Columns," Journal of the Association of Official *•
Analytical Chemists, 51, 29: 1968.
5. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
6. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
8. Jackson, Gary B. and Workman, Stephen M., "Analysis of Chlorophenoxy-Acid Herbicides in
Soil and Water," presented at the 14th Annual EPA Conference on the Analysis of Pollutants
in the Environment, Norfolk, Virginia: May 1991.
9. "Consolidated GC Method for the Determination of ITD/RCRA Pesticides using Selective GC
Detectors," S-CUBED, A Division of Maxwell Laboratories, Inc., La Jolla, CA: Ref. 32145-
01, Document R70, September 1986.
10. "Method Development and Validation, EPA Method 1618, Cleanup Procedures," Colorado
State University, Colorado Pesticide Center: November 1988 and January 1989.
734
-------
Method 1658
Table 1. Phenoxyacid Herbicides Determined by Large-Bore, Fused-Silica Capillary
Column Gas Chromatography with Halide-Specific Detector
EPA EGO Compound CAS Registry
481 2,4-D 94-75-7
480 Dinoseb 88-85-7
482 2,4,5-T 93-76-5
483 2,4,5-TP 93-72-1
Other phenoxyacid herbicides that can be analyzed by this method:
Compound CAS Registry
Dalgpon 75-99-0
2,4-DB (Butoxon) 94-82-6
Dicamba 1918-00-9
Dichlorprop 120-36-5
MCPA 94-74-6
MCPP 7085-19-0
Table 2. Gas Chromatography of Phenoxy-Acid Herbicides
Retention Time (minf Method Detection Limit1
EPA EGO Compound DB-608 \ DB-1701 (ng/L)
481 2.4-D 16,57 16.39 100
480 Dinoseb 20.75 23.55 50 (est)(ECD)
482 2,4,5-T 20.42 20.25 50
483 2,4,5-TP (Silvex) 18.65 18.66 40
Dalapon 3.52 3.63 100 (est)
2,4-DB (Butoxon) 21.94 21.87 50
Dicamba 13.51 12.97 110
Dichlorprop 15.21 15.19 40
MCPA 14.42 14.30 90
MCPP 13.51 13.49 56
2,4-DCPA (surrogate) 12.88 12.51
Notes:
1. Columns: 30 m long x 0.53 mm ID, i.e., DB-608: 0.83//; DB-1701: 1.0//. Conditions sug-
gested to meet retention times shown: 175 to 270°C at 5°C/min., 175 to 270C° @
5°C/min. Carrier gas flow rates approximately 7 mL/min.
2. 40 CFR Part 136, Appendix B (49 FR 43234). MDLs were obtained with an electrolytic
conductivity detector, except as noted. Detection limits for soils (in ng/kg) are estimated to be
30 to 100 times this level.
735
-------
Method 1658
Table 3. Concentrations of Calibration Solutions
EPA EGD
Compound
Electron Capture Detector
481
480
482
483
2,4-D
Dalapon
2,4-DB
2,4-DCPA (Surrogate)
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
Picloram
2,4,5-T
2,4,5-TP (Silvex)
Electrolytic Conductivity Detector
481
480
482
483
2,4-D
Dalapon
2,4-DB
2,4-DCPA (surrogate)
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
2,4,5-T
2,4,5-TP (Silvex)
Concentration (ng/mL)
Low
100
50
200
10
20
100
50
5,000
5,000
50
20
20
500
500
1,000
500
500
500
500
500
500
250
Medium
1,000
500
2,000
1,000
200
1,000
500
50,000
50,000
500
200
200
5,000
5,000
10,000
5,000
5,000
5,000
No Response
5,000
5,000
5,000
2,500
High
10,000
5,000
20,000
10,000
2,000
10,000
5,000
500,000
500,000
5,000
2,000
2,000
50,000
50,000
100,000
50,000
50,000
50,000
50,000
50,000
50,000
25,000
Table 4. Acceptance Criteria for Performance Tests for Phenoxy-Acid Compounds
Acceptance criteria
EGD No. Compound
481 2,4-D
480 Dinoseb
482 2,4,5-T
483 2,4,5-TP (Silvex)
Dalapon
2,4-DB (Butoxon)
Dicamba
Dichlorprop
MCPA
MCPP
Picloram
Electron capture detector
Spike
level1
(ug/U
to
5
2
2
5
20
2
10
500
500
5
Initial precision and
accuracy
f%i
f /
s
16
18
17
14
15
22
18
14
14
14
13
«*/
X
41-107
24-154
30-132
36-120
43-137
22-118
37-145
49-133
46-130
65-149
46-140
Calibration
verification
(%)
78-121
64-136
70-130
75-126
74-125
42-157
59-139
71-128
67-132
71-129
73-126
Recovery/
Ongoing
accuracy
R (%)
23-131
19-159
5-158
15-141
39-140
0-142
10-172
28-154
25-151
42-170
42-144
736
-------
Method 1658
Percent Solids
< 30 Percent
> 30 Percent
Oil. To 1% Solids
Spike Surrogate
Spike Surrogate
Hydrolyze Esters
Hydrolyze Esters
Tumble With Water
Extract With CH2CI2
Aqueous
Phase
Saturate With NaCI
pH<2
Extract With CH2CI2
Organic
Phase
Concentrate
Derivatize
_L
Florisil Cleanup
I
GC/ECD
A52-002-B2A
Figure 1. Extraction, Cleanup, Derivatization, and Analysis
737
-------
Method 1658
Dalapon (3.51) Silvex (18.66)
s /
Dicamba/MCPP (13.51)
Herb Sur (12.89)
CVI
in
2" So"
1 «
J> 5
" cvT
2,4,5-T (20.43)
2,4,-DB (21.94)
(31.70)
T
10
I
15
i
20
I
25
Retention Time (minutes)
n
30
A52-002-79A
Figure 2. Chromatogram of Herbicides DB-608 Column
738
-------
Method 1658
Dalapon(3.6)
Dicamba
/
Herb Surr (12.5)
\
(9'\
L , A i
/ Sih/ex(18.7)
/
in
Q.
2 ^
B- CD
x-x ° C-
• o Q
C,coQ •*
Q. ^
|<
o
II
V
CM"
^
L-
/
2, 4, 5-T (20.3)
/
L
/
2,4-DB(21.9)
(23.6)
1 1 1 1 1 ! 1 1 1 1 1 1 1
D.O 5.0 10.0 15.0 20.0 25.0 30.0
Retention Time (minutes)
A52-002-80A
Figure 3. Chromatogram of Herbicides (DB-1701 Column)
739
-------
-------
Method 1659
The Determination of Dazomet
in Municipal and Industrial
Wastewater
-------
Method 1659
The Determination of Dazomet in Municipal and Industrial
Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of dazomet (CAS 533-74-4) by base hydrolysis to
methyl isothiocyanate (MITC; CAS 556-61-6) and subsequent determination of MITC by wide
bore, fused-silica column gas chromatography (GC) with a nitrogen-phosphorus detector
(NPD).
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must 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. Gas chromatography mass spectrometry (GC/MS) can be used
to confirm dazomet in extracts produced by this method when the level is sufficient.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limit in Table 1 typifies the minimum quantity that can be
detected with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a gas
chromatograph and in the interpretation of gas chromatographic data. Each laboratory that
uses this method must demonstrate the ability to generate acceptable results using the proce-
dure in Section 8.2.
2. SUMMARY OF METHOD
2.1 A 50-mL sample is adjusted to pH 10 to 12 and allowed to stand for 3 hours to hydrolyze
dazomet to MITC. After hydrolysis, the sample is saturated with salt and extracted with
2.5 mL of ethyl acetate. Gas chromatographic conditions are described that permit the separa-
tion and measurement of MITC in the extract by wide-bore, fused-silica capillary column with
nitrogen-phosphorus detector (GC/NPD).
2.2 Identification of MITC (qualitative analysis) is performed by comparing the GC retention time
of the MITC on two dissimilar columns with the respective retention times of an authentic
standard. Compound identity is confirmed when the retention times agree within their respec-
tive windows.
2.3 Quantitative analysis is performed using an authentic standard of MITC to produce a calibra-
tion factor or calibration curve, and using the calibration data to determine the concentration of
MITC in the extract. The concentration in the sample is calculated using the sample volume,
the extract volume, and a factor to convert MITC to dazomet.
743
-------
Method 1659
2.4 Quality is assured through reproducible calibration and testing of the extraction and GC
systems.
3. CONTAMINA TION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the anal-
ysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with and baking at 450°C for a
minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials, such as
PCBs, may not be eliminated by this treatment and thorough rinsing with acetone and pes-
ticide-quality hexane may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 1 through 3.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
5. APPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting the requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottle: Amber glass, 1-L, with screw-cap. If amber bottles are not available,
samples shall be protected from light.
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
5.1.3 Cleaning.
744
-------
Method 1659
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent or baked at
450°C for a minimum of 1 hour.
5.1.3.2 Liners are detergent water washed, then rinsed with reagent water and
solvent, and baked at approximately 200°C for a minimum of 1 hour prior
to use.
5.1.4 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Extraction bottle: 4-oz with PTFE-lined screw-cap, cleaned by solvent rinse or baking at
450°C for a minimum of 1 hour.
5.3 pH meter, with combination glass electrode.
5.4 Sample vials: Amber glass, 1- to 5-mL with PTFE-lined screw- or crimp-cap, to fit GC
autosampler.
5.5 Balance: Analytical, capable of weighing 0.1 mg.
5.6 Miscellaneous glassware.
5.6.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.6.2 Pipettes, glass, Pasteur.
5.6.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.7 Gas chromatograph: Shall have splitless or on-column simultaneous automated injection into
separate capillary columns with a nitrogen-phosphorus detector at the end of each column,
temperature program with isothermal holds, data system capable of recording simultaneous
signals from the two detectors, and shall meet all of the performance specifications in Sec-
tion 12.
5.7.1 GC columns: Bonded-phase fused-silica capillary.
5.7.1.1 Primary: 30 m long (± 3 m) by 0.5 mm (± 0.05 mm) ID, DB-608 (or
equivalent).
5.7.1.2 Confirmatory: DB-1701, or equivalent, with same dimensions as primary
column.
5.7.2 Data system: Shall collect and record GC data, store GC runs on magnetic disk or
tape, process GC data, compute peak areas, store calibration data including retention
times and calibration factors, identify GC peaks through retention times, compute
concentrations, and generate reports.
5.7.2.1 Data acquisition: GC data shall be collected continuously throughout the
analysis and stored on a mass storage device.
5.7.2.2 Calibration factors and calibration curves: The data system shall be used
to record and maintain lists of calibration factors, and multi-point calibra-
745
-------
Method 1659
tion curves (Section 7). Computations of relative standard deviation (coe-
fficient of variation) are used for testing calibration linearity. Statistics on
initial (Section 8.2) and ongoing (Section 12.5) performance shall be com-
puted and maintained.
5.7.2.3 Data processing: The data system shall be used to search, locate, identify,
and quantify the compounds of interest in each GC analysis. Software
routines shall be employed to compute and record retention times and peak
areas. Displays of chromatograms and library comparisons are required to
verify results.
5.7.3 Nitrogen phosphorus detector: Thermionic bead or alkali flame detector, capable of
detecting 600 pg of MITC under the analysis conditions given in Table 1.
6. REAGENTS AND STANDARDS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting the requirements of this method is the responsibility of the laboratory.
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide (ION): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL
H2SO4 (specific gravity 1.84) to 50 mL reagent water.
6.2.3 Potassium hydroxide: 37% (w/v). Dissolve 37 g KOH in 100 mL reagent water.
6.3 Solvents: Methylene chloride, ethyl acetate, and acetone; pesticide-quality; lot-certified to be
free of interferences.
6.4 Reagent water: Water in which the compounds of interest and interfering compounds are not
detected by this method.
6.5 Salt: Sodium chloride, spread approximately 1 cm deep in a baking dish and baked at 450°C
for a minimum of 1 hour.
6.6 Standard solutions: Purchased as solutions or mixtures with certification to their purity, con-
centration, and authenticity, or prepared from materials of known purity and composition. If
compound purity is 96% or greater, the weight may be used without correction to compute
the concentration of the standard. When not being used, standards are stored in the dark at
-20 to -10°C in screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the
level of the solution so that solvent evaporation loss can be detected. The vials are brought to
room temperature prior to use.
6.7 Preparation of stock solutions: Prepare in ethyl acetate per the steps below. Observe the
safety precautions in Section 4.
746
-------
Method 1659
6.7.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10 mg MITC in a 10-mL ground-glass stoppered volumetric flask and fill
to the mark with ethyl acetate. After the MITC is completely dissolved, transfer the
solution to a 15-mL vial with PTFE-lined cap.
6.7.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.7.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.8 Secondary mixtures: Using stock solutions (Section 6.7), prepare mixtures for calibration and
calibration verification (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.8.1 Calibration solutions: Prepare MITC in ethyl acetate at concentrations of 0.2, 1.0,
and 5.0 /xg/mL. The midpoint solution (1.0 /ig/mL) is used for calibration verifica-
tion (Section 12.4).
6.8.2 Precision and recovery standard: Prepare MITC in acetone at a concentration of
25 /ig/mL.
6.8.3 Matrix spike solution: Prepare dazomet in acetone at a concentration of 25 /ig/mL.
6.9 Stability of solutions: All standard solutions (Sections 6.7 and 6.8) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards
will remain acceptable if the peak area remains within ±15% of the area obtained in the initial
analysis of the standard.
7. SETUP AND CALIBRATION
7.1 Configure the GC system as given in Section 5.7 and establish the operating conditions in
Table 1.
7.2 Attainment of minimum level: Determine that each column/detector system meets the mini-
mum level for MITC (Table 1).
7.3 Calibration.
7.3.1 Inject 3 pL of each calibration solution (Section 6.8.1) into each GC column/detector
pair, beginning with the lowest level mixture and proceeding to the highest. For each
compound, compute and store, as a function of the concentration injected, the reten-
tion time and peak area on each column/detector system (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for MITC on each column/
detector system.
7.3.2.2 Linearity: If the calibration factor is constant (Cv<20%) over the calibra-
tion range, an average calibration factor may be used; otherwise, the com-
plete calibration curve (area vs. amount) shall be used.
747
-------
Method 1659
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program.4
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to es-
tablished performance criteria to determine if the results of analyses meet the performance
characteristics of the method.
8.1.1 1 he analyst shall make an initial demonstration of 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 analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If detection the detection limit for dazomet will be affected by the modification, the
analyst is required to repeat demonstration of the detection limit (Section 7.2).
8.1.3 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.8.2) that the analy-
sis system is in control. These procedures are described in Sections 12.1, 12.4, and
12.5.
8.1.4 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.4.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations:
8.2.1 Extract, concentrate, and analyze one set of four 50-mL aliquots of reagent water
spiked with 0.1 mL of the precision and recovery standard (Section 6.8.2) according
to the procedure in Section 10.
8.2.2 Using results of the set of four analyses, compute the average percent recovery (X)
and the coefficient of variation (Cv) of percent recovery(s) for MITC.
8.2.3 Compare s and X with the corresponding limit for initial precision and recovery in
Table 1. If s and X meet the acceptance criteria, system performance is acceptable
and analysis of blanks and samples may begin. If, however, s exceeds the precision
limit or X falls outside the range for accuracy, system performance is unacceptable.
In this case, correct the problem and repeat the test.
8.3 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from
a given site type (e.g., influent to treatment, treated effluent, produced water). If only one
sample from a given site type is analyzed, a separate aliquot of that sample shall be spiked.
748
-------
Method 1659
8.3.1 The concentration of the matrix spike shall be determined as follows:
8.3.1 .1 If, as in compliance monitoring, the concentration of dazomet in the sam-
ple is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.3.2, whichever concentration is
larger.
8.3.1 .2 If the concentration is not being checked against a regulatory limit, the
matrix spike shall be at 50 /zg/L or at 1 to 5 times higher than the back-
ground concentration, whichever concentration is larger.
8.3.1 .3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
the larger of either 5 times the expected background concentration, or
50 jtg/L (the concentration produced by 0. 1 mL of the matrix spike solu-
tion spiked into a 50-mL sample).
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of dazo-
met. If necessary, prepare a standard solution appropriate to produce a level in the
sample 1 to 5 times the background concentration. Spike a second sample aliquot
with the standard solution and analyze it to determine the concentration after spiking
(A) with dazomet. Calculate the percent recovery (P):
Equation 1
p _
where
T = True value of the spike
8.3.3 Compare the percent recovery for dazomet with the corresponding QC acceptance
criteria in Table 1. If dazomet fails the acceptance criteria for recovery, the sample is
complex and must be diluted and reanalyzed per Section 15.
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (treated effluent, influent to treatment, produced water) in which
the recovery test (Section 8.3.3) is passed, compute the average percent recovery (P)
and the standard deviation of the percent recovery (sp). Express the accuracy as-
sessment as a percent recovery interval from P — 2sp to P + 2sp for each matrix.
For example, if P = 90% and sp = 10% for five analyses of wastewater, the ac-
curacy interval is expressed as 70 to 110%. Update the accuracy assessment in each
matrix on a regular basis (e.g., after each five to ten new accuracy measurements).
749
-------
Method 1659
8.4 Blanks: Reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Extract and concentrate a 50-mL reagent water blank with each sample batch (samples
started through the extraction process on the same 8 hour shift, to a maximum of 20
samples). Analyze the blank immediately after analysis of the precision and recovery
standard (Section 12.5) to demonstrate freedom from contamination.
8.4.2 If MITC or any potentially interfering compound is found in an aqueous blank at
greater than 2 pg/L (assuming the same calibration factor as MITC for interfering
compounds), analysis of samples is halted until the source of contamination is elimina-
ted and a blank shows no evidence of contamination at this level.
8.5 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 12.4), and for initial (Section 8.2) and ongoing (Section 12.5)
precision and recovery should be identical, so that the most precise results will be obtained.
The GC instrument will provide the most reproducible results if dedicated to the settings and
conditions required for the analyses of the analytes given in this method.
8.6 Depending on specific program requirements, field replicates and field spikes may be required
to assess the precision and accuracy of the sampling and sample transporting techniques.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices,5 except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow
freely are collected in refrigerated bottles using automatic sampling equipment.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH greater than 9.0 using
sodium hydroxide solution. Record the volume used. If residual chlorine is present in a-
queous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and
330.5 may be used to measure residual chlorine.6
9.3 Begin sample extraction within seven days of collection, and analyze all extracts within 40
days of extraction.
10. SAMPLE HYDROL YSIS AND EXTRACTION
10.1 Hydrolysis and preparation of QC aliquots.
10.1.1 Pour 50 mL of sample into a clean 4-oz bottle. If a matrix spike is to be prepared,
pour 50 mL into a second clean bottle.
10.1.2 For each sample or sample batch (to a maximum of 20) to be extracted at the same
time, place two 50-mL aliquots of reagent water in clean 4-oz bottles. One reagent
water aliquot serves as the blank.
10.1.3 Spike 0.1 mL of the precision and recovery standard (Section 6.8.2) into the remain-
ing reagent water aliquot.
10.1.4 Spike 0.1 mL of the matrix spike solution (Section 6.8.3) into the sample aliquot used
for the matrix spike.
750
-------
Method 1659
10.1.5 Test the pH of the sample and QC aliquots with a pH meter and adjust to 10 to 12
with potassium hydroxide solution. Cap and shake the bottles vigorously to mix.
Allow to stand.
10.1.6 Test and adjust the pH after 0.5 to 1 hour. Allow to stand for an additional 2 to 3
hours.
10.1.7 Extract the sample and QC aliquots per Section 10.2.
10.2 Extraction.
10.2.1 Add 20 g of clean NaCl (Section 6.5) and 2.5 mL of ethyl acetate to each sample and
QC aliquot and cap tightly.
10.2.2 Shake vigorously for 2 to 5 minutes. Allow the bottle to stand for 10 minutes for the
phases to separate.
10.2.3 Using a Pasteur pipette, transfer the organic phase to a GC autosampler vial. Meas-
ure its volume.
11. GAS CHROMATOGRAPHY
Table 1 summarizes the recommended operating conditions for the gas chromatograph. Included in
this table is the retention time for MITC achieved under these conditions. An example of the separa-
tion achieved by the primary column is shown in Figure 1.
11.1 Calibrate the system as described in Section 7.
11.2 Set the injection volume on the autosampler to inject 3.0 pL of all standards and extracts of
blanks and samples.
11.3 Set the data system or GC control to start the temperature program upon sample injection, and
begin data collection after the solvent peak elutes. Set the data system to stop data collection
after the last analyte is expected to elute and to return the column to the initial temperature.
12. SYSTEM AND LABORATORY PERFORMANCE
12.1 At the beginning of each 8-hour shift during which analyses are performed, GC system
performance and calibration are verified on both column/detector systems. For these tests,
analysis of the calibration verification standard (Section 6.8.1) shall be used to verify all
performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed until
all performance criteria are met. Only after all performance criteria are met may samples,
blanks, and precision and recovery standards be analyzed.
12.2 Retention times: The absolute retention time of the peak maxima shall be within ± 10 seconds
of the retention times in the initial calibration (Section 7.3.1).
12.3 GC resolution: Resolution is acceptable if the peak width at half-height is less than 10 sec-
onds.
12.4 Calibration verification.
12.4.1 Inject the calibration verification standard (Section 6.8.1).
12.4.2 Compute the concentration of MITC based on the calibration factor or calibration
curve (Section 7.3).
757
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Method 1659
12.4.3 Compare this concentration with the limits for calibration verification in Table 1. If
the recovery meets the acceptance criteria, system performance is acceptable and
analysis of blanks and samples may begin. If, however, the recovery falls outside the
calibration verification range, system performance is unacceptable. In this case,
correct the problem and repeat the test, or recalibrate (Section 7).
12.5 Ongoing precision and recovery.
12.5.1 Analyze the extract of the precision and recovery standard extracted with each sample
batch (Section 10.1.3).
12.5.2 Compute the percent recovery of MITC.
12.5.3 Compare the percent recovery with the limits for ongoing recovery in Table 1. If the
recovery meets the acceptance criteria, the extraction and concentration processes are
in control and analysis of blanks and samples may proceed. If, however, the recovery
falls outside the acceptable range, these processes are not in control. In this event,
correct the problem, re-extract the sample batch, and repeat the ongoing precision and
recovery test.
12.5.4 Add results which pass the specifications in Section 12.5.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued labora-
tory performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery sr. Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.
13. QUALITATIVE DETERMINATION
13.1 Qualitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention time and calibration libraries (Section 7.3.1). Identification is confirmed when
retention-time and amounts agree per the criteria below.
13.2 On each column/detector system, establish a retention-time window ±20 seconds on either
side of the retention-time in the calibration data (Section 7.3.1).
13.3 If the MITC peak from the analysis of a sample or blank is within a window (as defined in
Section 13.2) on the primary column/detector system, it is considered tentatively identified.
A tentatively identified compound is confirmed when (1) the retention time for the compound
on the confirmatory column/detector system is within the retention-time window on that
system, and (2) the computed amounts (Section 14) on each system (primary and confirmatory)
agree within a factor of 3.
14. QUANTITATIVE DETERMINATION
14.1 Using the GC data system, compute the concentration of the analyte detected in the extract (in
milligrams per milliliter) using the calibration factor or calibration curve (Section 7.3.2).
752
-------
Method 1659
14.2 Compute the concentration in the sample using the following equation:
Equation 2
(2.22)(V,)(CM)
' = K
where
Cs = Concentration in the sample, in ng/L
2.22 -» Converts MITC (MW 73.12) to dazomet (MW 162.27)
Ve = Extract total volume, in mL
CM = Concentration in the extract, in
Vs = Volume of sample extracted, in L
14.3 If the concentration of MITC exceeds the calibration range of the system, the extract is diluted
by a factor of 10, and a 3-/xL aliquot of the diluted extract is analyzed.
14.4 Report results for dazomet found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at
which the concentration is in the calibration range.
15. ANALYSIS OF COMPLEX SAMPLES
15.1 Some samples may contain high levels (> 1000 ng/L) of dazomet or of interfering compounds,
and/or polymeric materials. Some samples may form emulsions when extracted (Section
10.2); others may overload the GC column and/or detector. In these instances, the extract is
diluted by a factor of 10 and reanalyzed (Section 1 4.3).
15.2 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those
from reagent water. If the matrix spike recovery is outside the range specified in Table 1, the
sample is diluted by a factor of 10, respiked, and reanalyzed. If the matrix spike recovery is
still outside the range, the method may not work on the sample being analyzed and the result
may not be reported for regulatory compliance purposes.
16. METHOD PERFORMANCE
16.1 This method is based on industry Method 131.7
16.2 Development of this method is detailed in Reference 8.
753
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Method 1659
References
1. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
2. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
3. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
4. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
5. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
6. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
7. "Determination of Metham (Vapam) in Wastewater" (Method 131), Methods for Nonconven-
tional Pesticides Chemicals Analysis of Industrial and Municipal Wastewater. U.S. Environ-
mental Protection Agency Effluent Guidelines Division (WH-552), Washington, DC:
EPA 440/1-83/079-C, January 31, 1983.
8. "Narrative for SAS 1019," Pacific Analytical, Inc.: September 1989. Available from the
U.S. Environmental Protection Agency Sample Control Center, 300 N. Lee St., Alexandria,
VA 22314 (703-557-5040).
754
-------
Method 1659
Table 1. GC Data and Method Acceptance Criteria for Dazomet*
Acceptance Criterion Specification Note
Minimum Level 10//g/L 1
Method Detection Limit 3 //g/L 2
Calibration Verification {Section 12.4) 0.8-1.3 //g/mL 3
Initial Precision and Recovery (Section 8.2I
Precision [standard deviation (s)] 23 fjg/L
Recovery [mean (X)J 18-75 //g/L
Ongoing Precision and Recovery (Section 12.5) 15-78 //g/L
Matrix Spike Recovery (Section 8.3.3) 16-123%
MITC Retention-time 5
DB-608 2.17 minutes
DB-1701 3.80 minutes
*(3,5-dimethyl-2H-tetrahydro-1,3,5-thiadiazine-2-thipne) detected as methyl isothiocyanate (MITC).
Notes:
1. This is a minimum level at which the analytical system shall give recognizable signals and
acceptable calibration points.
2. Estimated; 40 CFR Part 136, Appendix B.
3. Test concentration 1.0 yt/g/mL
4. Test concentration 50 //g/L.
5. Columns: 30 mm long by 0.53 mm ID. DB-608: 0.83 x/. DB-1701: 1.0//. Conditions
suggested to meet retention times shown: 50°C for 1.Q minute, 50 to 200° at 10°C/min.
Carrier gas flow rates approximately 7 mL/min.
755
-------
Method 1659
(0.6)
Methyl isothiocyanate
(1.0)
CVI
VE- d
(2.8)
(5.4)
I I I I I T l I i
0.0 1.0 2.0 3.0 4.0
Retention Time (minutes)
i I i i i r
6.0 7.0 8.0
9.0
A52-002-81A
Figure 1. Chromatogram of Methyl Isothiocyanate
756
-------
Method 1660
The Determination of Pyrethrins
and Pyrethroids in Municipal
and Industrial Wastewater
-------
-------
Method 1660
The Determination of Pyrethrins and Pyrethroids in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICA TION
1.1 This method covers the determination of pyrethrins and pyrethroids in wastewater by extrac-
tion and high-performance liquid chromatography (HPLC) with an ultra-violet detector (UV).
The compounds in Table ! may be determined by this method.
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method lists a second UV
wavelength that can be used to confirm measurements made with the primary wavelength.
1.4 This method is specific to the determination of two pyrethrins and seven pyrethroids, but
should be applicable to other pyrethroids as well. The quality control requirements in this
method give the steps necessary to determine this applicability.
1.5 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limits in Table 2 typify the minimum quantity that can be
detected with no interferences present.
1.6 This method is for use by or under the supervision of analysts experienced in the use of a
high-performance liquid chromatograph and interpretation of liquid chromatographic data.
Each laboratory that uses this method must demonstrate the ability to generate acceptable
results using the procedure in Section 8.2.
2. SUMMARY OF METHOD
2.1 A 750-mL sample is saturated with salt and extracted by stirring with acetonitrile in a 1-L
volumetric flask. A small portion of the acetonitrile rises into the neck of the flask.1 The
extract is evaporated to a volume of 7.5 mL.
2.2 A 40-/tL aliquot of the extract is injected into the HPLC. Chromatographic conditions are
described that permit the separation and measurement of the pyrethrins and pyrethroids by
reverse-phase CIS column HPLC with a multiple-wavelength UV detector.
2.3 Identification of compound is performed by comparing the retention time of the compound
with that of an authentic standard. Compound identity is confirmed when the retention times
agree, and when the response at a second wavelength agrees with the response at the primary
wavelength.
2.4 Quantitative analysis is performed using an authentic standard of each compound to produce a
calibration factor or calibration curve, and using the calibration data to determine the con-
759
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Method 1660
centration of that compound in the extract. The concentration in the sample is calculated using
the sample and extract volumes.
2.5 Quality is assured through reproducible calibration and testing of the extraction and HPLC
systems.
3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the
analysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at
450 °C for a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials
may not be eliminated by this treatment and thorough rinsing with acetone and pesticide-
quality acetonitrile may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 2 through 4.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
5. APPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and pan numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottle: Amber glass, 1-L, with screw-cap. If amber bottles are not available,
samples shall be protected from light.
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
760
-------
Method 1660
5.1.3 Cleaning.
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent or baked at
450°C for a minimum of 1 hour before use.
5.1.3.2 Liners are detergent-water washed, then reagent water and solvent rinsed,
and baked at approximately 200°C for a minimum of 1 hour prior to use.
5.1.4 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept
at 0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sam-
pler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing
may be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for glassware cleaning.
5.2.1 Laboratory sink with overhead fume hood.
5.2.2 Kiln: Capable of reaching 450°C within 2 hours and holding 450°C within ± 10°C,
with temperature controller and safety switch (Cress Manufacturing Co, Sante Fe
Springs, CA, B31H or X31TS, or equivalent).
5.3 Equipment for sample extraction.
5.3.1 Laboratory fume hood.
5.3.2 Stirring plate: Thermolyne Cimarec 2 (Model 546725), or equivalent.
5.3.3 Stirring bar: PTFE coated, approximately 1 by 4 cm.
5.3.4 Extraction flask: 1000-mL volumetric flask cleaned by rinsing with solvent or baking
at 450°C for a minimum of 1 hour.
5.3.5 pH meter, with combination glass electrode.
5.4 Equipment for sample concentration.
5.4.1 Nitrogen evaporation device: Equipped with heated bath that can be maintained at 35
to 40°C (N-Evap, Organomation Associates, Inc., or equivalent).
5.4.2 Concentrator tube: 10- to 15-mL, graduated (Kontes K-570050-1025, or equivalent)
with calibration verified.
5.5 Sample vials: Amber glass, 10- to 15-mL with PTFE-lined screw- or crimp-cap, to fit HPLC
autosampler.
5.6 Balance: Analytical, capable of weighing 0.1 mg.
5.7 Miscellaneous glassware.
5.7.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.7.2 Pipettes, glass, Pasteur, 150 mm long by 5 mm ID (Fisher Scientific 13-678-6A, or
equivalent).
5.7.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL
761
-------
Method 1660
5.8 High-performance liquid chromatograph (HPLC): Analytical system complete with pumps,
sample injector, column oven, and multiple-wavelength ultra-violet (UV) detector.
5.8.1 Pumping system: Capable of isocratic operation and producing a linear gradient
from 70% water/30% acetonitrile to 100% acetonitrile in 25 minutes (Waters 600E,
or equivalent).
5.8.2 Sample injector: Capable of automated injection of up to 30 samples (Waters 700, or
equivalent).
5.8.3 Column oven: Capable of operation at room ambient to 50°C (Waters TCM, or
equivalent).
5.8.4 Column: Two 300 Angstrom CIS columns 150 mm long by 4.6 mm ID (Vydac 201
TP5415, or equivalent) connected in series and preceded by a 300 Angstrom CIS
guard column 30 mm long by 4.6 mm ID (Vydac 201 GCC54T, or equivalent),
operated at the conditions shown in Table 2.
5.8.5 Detector: UV operated at 235 and 245 nm (Waters 490E, or equivalent).
5.9 Data system.
5.9.1 Data acquisition: The data system shall collect and record LC peak areas and reten-
tion times on magnetic media.
5.9.2 Calibration: The data system shall be used to calculate and maintain lists of calibra-
tion factors (response divided by concentration) and multi-point calibration curves.
Computations of relative standard deviation (coefficient of variation) are used to test
calibration linearity.
5.9.3 Data processing: The data system shall be used to search, locate, identify, and quan-
tify the compounds of interest in each analysis. Displays of chromatograms are
required to verify results.
5.9.4 Statistics on initial (Section 8.2) and ongoing (Section 12.5) performance shall be
computed and maintained.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH adjustment.
6.2.1 Sodium hydroxide (ION): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H2SO4
(specific gravity 1.84) to 50 mL reagent water.
6.3 Solvents: Acetonitrile and acetone; pesticide-quality; lot-certified to be free of interferences.
6.4 Reagent water: HPLC grade water in which the compounds of interest and interfering com-
pounds are not detected by this method.
6.5 Salt: Sodium chloride, spread approximately 1 cm deep in a baking dish and baked at 450°C
for a minimum of 1 hour, cooled and stored in a precleaned glass bottle with PTFE-lined cap.
6.6 Standard solutions: Purchased as solutions or mixtures with certification to their purity,
concentration, and authenticity, or prepared from materials of known purity and composition.
762
-------
Method 1660
If compound purity is 96% or greater, the weight may be used without correction to compute
the concentration of the standard.
NOTE: The pyrethrins are normally available in a mixed standard consisting of the six
naturally occurring compounds (pyrethrin I and II, cinerin I and II, andjasmolin I and
II). The concentrations in this standard will be on the order of 10% each of pyrethrin I
and II. The concentration in the stock solution prepared from this mixed standard is to
be corrected for the exact concentration.
When not being used, standards are stored in the dark at -20 to -10°C in screw-capped
vials with PTFE-lined lids. A mark is placed on the vial at the level of the solution so
that solvent evaporation loss can be detected. The vials are brought to room tempera-
ture prior to use.
6.7 Preparation of stock solutions: Prepare in acetonitrile per the steps below. Observe the safety
precautions in Section 4.
6.7.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10-mg allethrin in a 10-mL ground-glass stoppered volumetric flask and
fill to the mark with acetonitrile. After the allethrin is completely dissolved, transfer
the solution to a 15-mL vial with PTFE-lined cap.
6.7.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.7.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.8 Secondary mixtures: Using stock solutions (Section 6.7), prepare mixtures for calibration and
calibration verification (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.8.1 Calibration solutions: Prepare two solutions in acetonitrile at the concentrations given
in Table 3. The midpoint solution is used for calibration verification (Section 12.4)
6.8.2 Precision and recovery standard and matrix spike solution: Prepare two solutions in
acetone at 7.5 times the concentration of the midpoint standard (Table 3).
6.9 Stability of solutions: All standard solutions (Sections 6.7 through 6.8) shall be analyzed
within 48 hours of preparation and on a monthly basis thereafter for signs of degradation.
Standards will remain acceptable if the peak area remains within ±15% of the area obtained in
the initial analysis of the standard.
7. SETUP AND CALIBRATION
7.1 Configure the HPLC system as given in Sections 5.8 through 5.9 and establish the operating
conditions in Table 2.
7.2 Attainment of minimum level: Determine that the minimum levels in Table 2 are met at each
wavelength.
753
-------
Method 1660
7.3 Calibration.
7.3.1 Inject 40 /iL of each calibration solution (Table 3) into the HPLC system, beginning
with the lowest level mixture and proceeding to the highest. For each compound,
compute and store, as a function of the concentration injected, the retention time and
the peak area at each wavelength (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range for each at each wavelength.
7.3.2.2 Linearity: If the calibration factor is constant (Cv<20%) over the calibra-
tion range, an average calibration factor may be used; otherwise, the
complete calibration curve (area vs. amount) shall be used.
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program.5
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to es-
tablished performance criteria to determine if the results of analyses meet the performance
characteristics of the method.
8.1.1 The analyst shall make an initial demonstration of 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 analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If detection limits will be affected by the modification, the analyst is required to
repeat demonstration of the detection limit (Section 7.2).
8.1.3 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.8.2) that the analy-
sis system is in control. These procedures are described in Sections 12.1, 12.4, and
12.5.
8.1.4 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.4.
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations:
8.2.1 Extract, concentrate, and analyze two sets of four 750-mL aliquots of reagent water
spiked with 1.0 mL of each solution of the precision and recovery standard (Sec-
tion 6.8.2) according to the procedure in Section 10.
764
-------
Method 1660
8.2.2 Using results of each set of four analyses, compute the average recovery (X) and the
standard deviation of recovery (s), in milligrams per liter, for the each compound.
8.2.3 Compare s and X with the corresponding limit for initial precision and recovery in
Table 4. If s and X meet the acceptance criteria, system performance is acceptable
and analysis of blanks and samples may begin. If, however, s exceeds the precision
limit or X falls outside the range for accuracy, system performance is unacceptable.
In this case, correct the problem and repeat the test.
8.3 Method accuracy: The laboratory shall spike (matrix spike) at least 10% of the samples from
a given site type (e.g., influent to treatment, treated effluent, produced water). If only one
sample from a given site type is analyzed, a separate aliquot of that sample shall be spiked.
8.3.1 The concentration of the matrix spike shall be determined as follows.
8.3.1 .1 If, as in compliance monitoring, the concentration of allethrin in the sam-
ple is being checked against a regulatory concentration limit, the matrix
spike shall be at that limit or at 1 to 5 times higher than the background
concentration determined in Section 8.3.2, whichever concentration is
larger.
8.3.1 .2 If the concentration is not being checked against a regulatory limit, the
matrix spike shall be at the level of the precision and recovery standard
(Section 6.8.2) or at 1 to 5 times higher than the background con-
centration, whichever concentration is larger.
8.3.1 .3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
at the level of the precision and recovery standard (Section 6.8.2) or at 1
to 5 times the expected background concentration, whichever is larger.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the
pyrethrins and pyrethroids. If necessary, prepare a standard solution appropriate to
produce a level in the sample 1 to 5 times the background concentration. Spike a
second sample aliquot with the standard solution and analyze it to determine the
concentration after spiking (A) of each analyte. Calculate the percent recovery (P):
Equation 1
p _
where
T = True value of the spike
8.3.3 Compare the percent recovery of each compound with the corresponding QC accep-
tance criteria in Table 4. If any analyte fails the acceptance criteria for recovery, the
sample is complex and must be diluted and reanalyzed per Section 15.
765
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Method 1660
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (water, sludge) in which the recovery test (Section 8.3.3) is
passed, compute the average percent recovery (P) and the standard deviation of the
percent recovery (sp). Express the accuracy assessment as a percent recovery interval
from P- 2sp to P + 2sp for each matrix. For example, if P = 90% and sp = 10% for
five analyses of wastewater, the accuracy interval is expressed as 70 to 110%. Up-
date the accuracy assessment in each matrix on a regular basis (e.g., after each five to
ten new accuracy measurements).
8.4 Blanks: Reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Extract and concentrate a 750-mL reagent water blank with each sample batch (sam-
ples started through the extraction process on the same 8 hour shift, to a maximum of
20 samples). Analyze the blank immediately after analysis of the precision and recov-
ery standard (Section 12.5) to demonstrate freedom from contamination.
8.4.2 If any compound or any potentially interfering compound is found in an aqueous
blank at greater than 20 /ig/L (assuming the same calibration factor as allethrin for
interfering compounds), analysis of samples is halted until the source of contamination
is eliminated and a blank shows no evidence of contamination at this level.
8.5 Other pyrethroids may be determined by this method. To establish a quality control limit for
another analyte, determine the precision and accuracy by analyzing four replicates of the
analyte along with the precision and recovery standard per the procedure in Section 8.2. If the
analyte coelutes with an analyte in the QC standard, prepare a new QC standard without the
coeluting component(s). Compute the average percent recovery (A) and the standard deviation
of percent recovery (sj for the analyte, and measure the recovery and standard deviation of
recovery for the other analytes. The data for the new analyte is assumed to be valid if the
precision and recovery specifications for the other analytes are met; otherwise, the analytical
problem is corrected and the test is repeated. Establish a preliminary quality control limit of
A + 2sn for the new analyte and add the limit to Table 4.
8.6 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 12.4), and for initial (Section 8.2) and ongoing (Section 12.5)
precision and recovery should be identical, so that the most precise results will be obtained.
The HPLC instrument will provide the most reproducible results if dedicated to the settings
and conditions required for the analyses of the analytes given in this method.
8.7 Depending on specific program requirements, field replicates and field spikes may be required
to assess the precision and accuracy of the sampling and sample transporting techniques.
9. SAMPLE COLLECT/ON, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices,6 except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow
freely are collected in refrigerated bottles using automatic sampling equipment.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH of 5.0 to 7.0 using
766
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Method 1660
sodium hydroxide or hydrochloric acid solution. Record the volume used. If residual chlorine
is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods
330.4 and 330.5 may be used to measure residual chlorine.7
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
10. SAMPLE EXTRACTION
10.1 Preparation of sample and QC aliquots.
10.1.1 Mix sample thoroughly.
10.1.2 Pour 750 mL of sample into a clean 1000-mL volumetric flask. If a matrix spike is to
be prepared, pour two 750-mL aliquots into clean flasks.
10.1.3 For each sample or sample batch (to a maximum of 20) to be.extracted at the same
time, place three 750-mL aliquots of reagent water (Section 6.4) in clean 1000-mL
volumetric flasks. One reagent water aliquot serves as the blank.
10.1.4 Spike 1.0 mL of each precision and recovery standard (Section 6.8.2) into the remain-
ing reagent water aliquots.
10.1.5 Spike the samples designated as the matrix spike at the level directed in Section 8.3.
10.1.6 Extract the sample and QC aliquots per Section 10.2.
10.2 Extraction.
10.2.1 Place each sample or QC aliquot on a stirring plate and add a clean PTFE-coated
stirring bar.
10.2.2 Add 230 g of clean NaCl (Section 6.5) to each sample and QC aliquot and stir 5 to 10
minutes to dissolve.
10.2.3 Extraction with acetonitrile.
10.2.3.1 Add 160 mL of acetonitrile to each sample and QC aliquot.
10.2.3.2 Begin stirring. Increase the rate of stirring until the vortex is drawn ap-
proximately one-half the depth of the water. Stir for 3 to 5 minutes.
10.2.3.3 Stop stirring and invert each flask a minimum of 3 times will holding the
stopper. Return the flask to the stirring plate.
10.2.3.4 Repeat steps 10.2.3.2 through 10.2.3.3 twice.
10.2.4 Allow the solutions to stand for approximately 5 minutes for the phases to separate.
If an acetonitrile layer does not appear, add acetonitrile in 5-mL increments, stirring
and settling between increments, until a 2- to 5-mL layer appears. If the acetonitrile
layer is more than 5 mL, add reagent water, stir, and settle until the acetonitrile vol-
ume is reduced to 2 to 5 mL.
10.2.5 Using a Pasteur pipette, transfer the organic phase to a clean K-D concentrator tube
(Section 5.4.2).
10.2.6 Add 5 mL of acetonitrile to the extraction flasks, stir, and allow to settle. Transfer
the organic phase to the respective concentrator tubes. Repeat the extraction a third
767
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Method 1660
time. If all of the extract will not fit into the concentrator tube, evaporate some of the
acetonitrile (Section 10.3), then add the remaining extract.
10.3 Concentration of extracts.
10.3.1 Place the concentrator tubes in the evaporation device (Section 5.4.1). Adjust the
height of the blow-down tubes to 1 to 3 cm above the surface of the liquid and gently
evaporate the acetonitrile until a volume of approximately 5 mL is reached.
10.3.2 Adjust the final extract volume to 7.5 mL and transfer to an HPLC autosampler vial.
7 7. HIGH-PERFORMANCE LIQUID CHROMA TOGRAPHY
Table 2 summarizes the recommended operating conditions for the HPLC system. Included in this
table are the retention times for the pyrethrins and pyrethroids achieved under these conditions. An
example of the separation achieved by the column system is shown in Figure 1. Pyrethrin I and II
are the major peaks in the naturally occurring pyrethrin standard. Pyrethrin II elutes prior to pyreth-
rin I. Jasmolin II and I will normally coelute with pyrethrin II and I respectively. Most HPLC
columns will resolve cinerin II and I, which are small peaks that elute after the respective pyrethrins.
Some HPLC columns may resolve all six of the naturally occurring pyrethrins.
11.1 Calibrate the system as described in Section 7.
11.2 Set the injection volume on the autosampler to inject 40 pL of all standards and extracts of
blanks and samples.
11.3 Set the data system or HPLC control to start the gradient upon sample injection, and begin
data collection after 10 minutes. Set the data system or HPLC control to stop data collection
after the last analyte is expected to elute and to return the gradient to the initial setting.
12. SYSTEM AND LABORA TORY PERFORMANCE
12.1 At the beginning of each 8 hour shift during which analyses are performed, HPLC system
performance and calibration are verified at both wavelengths. For these tests, analysis of the
calibration verification standard (Section 6.8.1) shall be used to verify all performance criteria.
Adjustment and/or recalibration (per Section 7) shall be performed until all performance
criteria are met. Only after all performance criteria are met may samples, blanks, and preci-
sion and recovery standards be analyzed.
12.2 Retention times.
12.2.1 The absolute retention time of sumithrin shall be no earlier than 23 minutes.
12.2.2 The absolute retention time of the peak maxima shall be within +15 seconds of the
average retention times in the initial calibration (Section 7.3.1).
12.3 GC resolution: Resolution is acceptable if the height of the valley between tetramethrin and
allethrin is less than 20% of the taller of the two peaks when chromatograms of the two
calibration verification solutions (Section 6.8.1) are superimposed.
12.4 Calibration verification.
12.4.1 Inject the two calibration verification standards (Section 6.8.1).
12.4.2 Compute the concentration of the pyrethrins and pyrethroids based on the calibration
factor or calibration curve (Section 7.3).
768
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Method 1660
12.4.3 Compare this concentration with the limits for calibration verification in Table 4. If
calibration is verified, system performance is acceptable and analysis of blanks and
samples may begin. If, however, the recovery falls outside the calibration verification
range, system performance is unacceptable. In this case, correct the problem and
repeat the test, or recalibrate (Section 7).
12.5 Ongoing precision and recovery.
12.5.1 Analyze the extract of the two precision and recovery standards extracted with each
sample batch (Section 10.1.3).
12.5.2 Compute the recovery of the compounds of interest in milligrams per liter.
12.5.3 Compare the recovery with the limits for ongoing recovery in Table 4. If the recov-
ery meets the acceptance criteria, the extraction and concentration processes are in
control and analysis of blanks and samples may proceed. If, however, the recovery
falls outside the acceptable range, these processes are not in control. In this event,
correct the problem, re-extract the sample batch, and repeat the ongoing precision and
recovery test.
12.5.4 Add results which pass the specifications in 12.5.3 to initial and previous ongoing
data. Update QC charts to form a graphic representation of continued laboratory
performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery sr. Express the accuracy as a recovery interval from R - 2sr to R + 2sr.
For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.
13. QUALITA TIVE DETERMINA TION
13.1 Quantitative determination is accomplished by comparison of data rom analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention-time and calibration libraries (Section 7.3.1). Identification is confirmed when
retention time and amounts agree per the criteria below.
13.2 Establish a retention-time window of ±20 seconds on either side of the mean retention-time in
the calibration data (Section 7.3.1).
13.3 If a peak from the analysis of a sample or blank is within a window (as defined in Section
13.2) at the primary wavelength (235 nm), it is considered tentatively identified. A tentatively
identified compound is confirmed is confirmed when (1) the retention time of the peak maxi-
mum at the confirmatory wavelength (245 nm) is within ±2 seconds of the retention-time of
the peak maximum at the primary wavelength, and (2) the computed amounts (Section 14) on
each system (primary and confirmatory) agree within a factor of 2.
14. QUANTITATIVE DETERMINATION
14.1 Using the HPLC data system, compute the concentration of the analyte detected in the extract
(in micrograms per milliliter) using the calibration factor or calibration curve (Section 7.3.2).
769
-------
Method 1660
14.2 Compute the concentration in the sample using the following equation:
Equation 2
where
C5 = Concentration in the sample, in pg/L
Ve - Extract total volume, in mL (nominally 7.5)
Ca = Concentration in the extract, in pg/L
Vs = Volume of sample extracted, in L (nominally 0.75)
14.3 If the concentration of any analyte exceeds the calibration range of the system, the extract is
diluted by a factor of 10, and a 40-/xL aliquot of the diluted extract is analyzed.
14.4 Report results for pyrethrins and pyrethroids found in all standards, blanks, and samples to
three significant figures. Results for samples that have been diluted are reported at the least
dilute level at which the concentration is in the calibration range.
15. ANALYSIS OF COMPLEX SAMPLES
15.1 Some samples may contain high levels (> 1000 ng/L) of the pyrethrins and pyrethroids or of
interfering compounds and/or polymeric materials. Some samples may form emulsions when
extracted (Section 10.2); others may overload the HPLC column and/or detector. In these
instances, the sample is diluted by a factor of 10 and re-extracted (Section 10), or the extract
is diluted by a factor of 10 and reanalyzed (Section 14.3).
15.2 Recovery of matrix spikes: In most samples, matrix spike recoveries will be similar to those
from reagent water. If the matrix spike recovery is outside the range specified in Table 4, the
sample is diluted by a factor of 10, respiked, and reanalyzed. If the matrix spike recovery is
still outside the range, the method does not work on the sample being analyzed and the result
may not be reported for regulatory compliance purposes.
16. METHOD PERFORMANCE
16.1 Development of this method is detailed in Reference 8.
770
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Method 1660
References
1. Leggett, Daniel F., Jenkins, T. F., and Miyares, P. H., Analytical Chemistry, pp 1355-1356:
July 1990.
2. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
3. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
5. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
6. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
7. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
8. "Narrative for SAS 1097," Analytical Technologies, Inc.: September 1991. Available from
the U.S. Environmental Protection Agency Sample Control Center, 300 N. Lee St., Alexan-
dria, VA 22314 (703-557-5040).
771
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Method 1660
Table 1. Pyrethrins and Pyrethroids Determined by High-Performance Liquid Chro-
matography with Ultra-Violet Absorption Detector
Compound CAS Registry
Allethrin (Pynamin) 584-79-2
Cyfluthrin (Bayttiroid) 68359-37-5
Fenvalerate (Pydrin) 51630-58-1
Cis-permethrin 61949-76-6
Trans-permethrin 61949-77-7
Pyrethrin I 121-21-1
Pyrethrin II 121-29-9
Resmethrin 10453-86-8
Sumithrin (phenothrin) 26002-80-2
Tetramethrin 7696-12-0
Table 2. High-Performance Liquid Chromatography of Pyrethrins and Pyrethroids
Estimated
Retention Time Minimum Level1 MDL2
Compound (min) iug/U (ug/L)
Pyrethrin II 17.48 3.3 1
Tetramethrin 18.98 5.0 2
Allethrin 19.27 5.0 2
Pyrethrin I 20.89 3.1 1
Cyfluthrin 21.84 5.0 2
Resmethrin 22.07 5.0 2
Fenvalerate 22.68 2.5 2
C/T-permethrin3 22.98 5.0 , 2
Sumithrin 23.47 5.0 1
C/T-permethrin3 23.56 5.0 2
1. This is a minimum level at which the analytical system shall give recognizable signals and
acceptable calibration points.
2. 40 CFR Part 136, Appendix B. Column system and conditions: Two 300 Angstrom C18
columns 150 mm long x 4.6 mm ID 300 Angstrom C18 connected in series preceded by a
300 Angstrom C18 guard column 30 mm long x 4.6 mm ID. Column temperature 30°C.
Solvent flow rate 1.5 mL/min. Gradient: linear from 70% water/30% acetonitrile at injection to
100% acetonitrile in 25 minutes.
3. Elution order of cis/trans isomers not known.
772
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Method 1660
Table 3. Concentration of Calibration Solutions
Compound
Calibration Solution 1
Cyfluthrin
Fenvalerate
Pyrethrin I
Pyrethrin II
Sumithrin
Tetramethrin
Calibration Solution 2
Allethrin
Resmethrin
C/T-permethrin*
C/T-permethrin*
Solution Concentration (ug/mL)
Low
0.50
0.25
0.31
0.33
0.50
0.50
0.50
0.50
0.50
0.50
Median
4.00
2.00
2.50
2.65
4.00
4.00
4.00
4.00
4.00
4.00
High
40.0
20.0
25.0
26.5
40.0
40.0
40.0
40.0
40.0
40.0
Table 4. Acceptance Criteria for Performance Tests for Pyrethrins and Pyrethroids
Acceptance Criteria
Spike
Level -
(U9/L)
40.0
40.0
20.0
40.0
40.0
26.5
25.0
40.0
40.0
40.0
Initial Precision
and Accuracy
(ug/L)
s
9.0
12.5
3.5
7.5
7.5
7.0
6.0
12.5
14.0
9.0
X
Calibration
Verification1
(ug/L)
Recovery/
Ongoing
Accuracy
R (ug/L)
16.0-52.0 3.5-4.6 15.0-53.0
11.0-61.0 3.0-5.2 9.4-63.0
12.0-26.0 1.6-2.4 6.2-32.0
23.0-53.0 3.0-4.6 22.0-54.0
23.0-53.0 3.0-4.6 21.0-54.0
8.6-32.0 2.2-2.8 7.7-33.0
11.0-33.0 2.0-3.5 10.0-34.0
4.3-51.0 2.3-5.2 2.5-52.0
4.6-57.0 3.5-4.7 2.5-59.0
17.0-53.0 1.5-6.1 15.0-55.0
Compound
Allethrin
Cyfluthrin
Fenvalerate
C/T-permethrin2
C/T-permethrin3
Pyrethrin I
Pyrethrin II
Resmethrin
Sumithrin
Tetramethrin
1. Verified at the level of the median standard in Table 3.
2. First of two permethrin peaks
3. Second of two permethrin peaks
773
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Method 1661
The Determination of
Bromoxynil in Municipal and
Industrial Wastewater
-------
-------
Method 1661
The Determination of Bromoxynil in
Municipal and Industrial Wastewater
1. SCOPE AND A PPLICA TION
1.1 This method covers the determination of bromoxynil in waste by direct aqueous injection high-
performance liquid chromatography (HPLC) with an ultraviolet detector (UV).
1.2 This method is designed to meet the monitoring requirements of the U.S. Environmental
Protection Agency under the Clean Water Act at 40 CFR Part 455. Any modification of this
method beyond those expressly permitted shall be considered a major modification subject to
application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.
1.3 When this method is applied to the analysis of unfamiliar samples, compound identity must be
supported by at least one additional qualitative technique. This method lists a second UV
wavelength that can be used to confirm measurements made with the primary wavelength.
1.4 The detection limit of this method is usually dependent on the level of interferences rather than
instrumental limitations. The limit in Table 1 typifies the minimum quantity that can be
detected with no interferences present.
1.5 This method is for use by or under the supervision of analysts experienced in the use of a
high-performance liquid chromatograph and interpretation of liquid chromatographic data.
Each laboratory that uses this method must demonstrate the ability to generate acceptable
results using the procedure in Section 8.2.
2. SUMMARY OF METHOD
2.1 A 40-jiL aliquot of sample is injected into the HPLC. Chromatographic conditions are de-
scribed that permit the separation and measurement of bromoxynil by reverse-phase CIS
column HPLC with a multiple-wavelength UV detector.
2.2 Identification of bromoxynil is performed by comparing the retention time of the chromato-
graph peak with that of an authentic standard. Compound identity is confirmed when the
retention times agree, and when the response at a second wavelength agrees with the response
at the primary wavelength.
2.3 Quantitative analysis is performed using an authentic standard of bromoxynil to produce a
calibration factor or calibration curve, and using the calibration data to determine the con-
centration of bromoxynil in the sample.
2.4 Quality is assured through reproducible calibration and testing of the HPLC system.
3. CONTAMINATION AND INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample-processing hardware may yield artifacts and/or
elevated baselines causing misinterpretation of chromatograms. All materials used in the
777
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Method 1661
analysis shall be demonstrated to be free from interferences under the conditions of analysis by
running method blanks as described in Section 8.4.
3.2 Glassware and, where possible, reagents are cleaned by rinsing with solvent and baking at
450°C for a minimum of 1 hour in a muffle furnace or kiln. Some thermally stable materials
may not be eliminated by this treatment and thorough rinsing with acetone and pesticide-
quality acetonitrile may be required.
3.3 Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be required.
3.4 Interferences coextracted from samples will vary considerably from source to source, depen-
ding on the diversity of the site being sampled.
4. SAFETY
4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been
precisely determined; however, each chemical compound should be treated as a potential health
hazard. Exposure to these compounds should be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations regar-
ding the safe handling of the chemicals specified in this method. A reference file of material
handling sheets should also be made available to all personnel involved in these analyses.
Additional information on laboratory safety can be found in References 1 to 3.
4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves that will prevent exposure.
5. A PPARA TUS AND MA TERIALS
NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only.
No endorsement is implied. Equivalent performance may be achieved using apparatus
and materials other than those specified here, but demonstration of equivalent perfor-
mance meeting requirements of this method is the responsibility of the laboratory.
5.1 Sampling equipment for discrete or composite sampling.
5.1.1 Sample bottle, amber glass, 40-mL minimum, with screw-cap. If amber bottles are
not available, samples shall be protected from light.
5.1.2 Bottle caps: Threaded to fit sample bottles. Caps shall be lined with PTFE.
5.1.3 Cleaning.
5.1.3.1 Bottles are detergent water washed, then rinsed with solvent rinsed or
baked at 450°C for a minimum of 1 hour before use.
5.1.3.2 Liners are detergent-water washed, then rinsed with reagent water and
solvent, and baked at approximately 200°C for a minimum of 1 hour prior
to use.
5.1.4 Compositing equipment: Automatic or manual compositing system incorporating glass
containers cleaned per bottle cleaning procedure above. Sample containers are kept at
778
-------
Method 1661
0 to 4°C during sampling. Glass or PTFE tubing only shall be used. If the sampler
uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used in the pump only. Before use, the tubing shall be thoroughly rinsed with
methanol, followed by repeated rinsings with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.
5.2 Equipment for glassware cleaning.
5.2.1 Laboratory sink with overhead fume hood.
5.2.2 Kiln: Capable of reaching 450°C within 2 hours and holding 450°C within ± 10°C,
with temperature controller and safety switch (Cress Manufacturing Co, Sante Fe
Springs, CA, B31H or X31TS, or equivalent).
5.3 pH meter, with combination glass electrode.
5.4 Sample vials: Amber glass, 10- to 15-mL with PTFE-lined screw- or crimp-cap, to fit HPLC
autosampler.
5.5 Balance: analytical, capable of weighing 0.1 mg.
5.6 Miscellaneous glassware.
5.6.1 Pipettes, glass, volumetric, 1.00-, 5.00-, and 10.0-mL.
5.6.2 Pipettes, glass, Pasteur, 150 mm long by 5 mm ID (Fisher Scientific 13-678-6A, or
equivalent).
5.6.3 Volumetric flasks, 10.0-, 25.0-, and 50.0-mL.
5.7 High-performance liquid chromatograph (HPLC): Analytical system complete with pumps,
sample injector, column oven, and multiple-wavelength ultra-violet (UV) detector.
5.7.1 Pumping system: Capable of isocratic operation (Waters 600E, or equivalent).
5.7.2 Sample injector: Capable of automated injection of up to 30 samples (Waters 700, or
equivalent).
5.7.3 Column oven: Capable of operation at room ambient to 50°C (Waters TCM, or equi-
valent).
5.7.4 Column: 150 mm long by 4.6 mm ID 300 Angstrom CIS column (Vydac 201
TP5415, or equivalent), operated at the conditions shown in Table 1.
5.7.5 Detector: UV operated at 255 and 280 nm (Waters 490E, or equivalent).
5.8 Data system.
5.8.1 Data acquisition: The data system shall collect and record LC peak areas and reten-
tion times on magnetic media.
5.8.2 Calibration: The data system shall be used to calculate and maintain lists of calibra-
tion factors (response divided by concentration) and multi-point calibration curves.
Computations of relative standard deviation (coefficient of variation) are used to test
calibration linearity.
5.8.3 Data processing: The data system shall be used to search, locate, identify, and quan-
tify the compounds of interest in each analysis. Displays of chromatograms are
required to verify results.
773
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Method 1661
5.8.4 Statistics on initial (Section 8.2) and ongoing (Section 12.5) performance shall be
computed and maintained.
6. REAGENTS AND STANDARDS
6.1 Sample preservation: Sodium thiosulfate (ACS), granular.
6.2 pH Adjustment.
6.2.1 Sodium hydroxide (ION): Dissolve 40 g NaOH in 100 mL reagent water.
6.2.2 Sulfuric acid (1 + 1): Reagent grade, 6N in reagent water. Slowly add 50 mL H2SO4
(specific gravity 1.84) to 50 mL reagent water.
6.3 Solvents: Methanol; pesticide-quality, lot-certified to be free of interferences.
6.4 Reagent water: HPLC grade water in which the compounds of interest and interfering com-
pounds are not detected by this method.
6.5 Standard: Purchased as a solution with certification as to purity, concentration, and authen-
ticity, or prepared from materials of known purity and composition. If compound purity is
96% or greater, the weight may be used without correction to compute the concentration of the
standard. When not being used, standards are stored in the dark at - 20°C to -10°C in
screw-capped vials with PTFE-lined lids. A mark is placed on the vial at the level of the
solution so that solvent evaporation loss can be detected. The vials are brought to room
temperature prior to use.
6.6 Preparation of stock solutions: Prepare in water per the steps below. Observe the safety
precautions in Section 4.
6.6.1 Dissolve an appropriate amount of assayed reference material in solvent. For exam-
ple, weigh 10 mg bromoxynil in a 100-mL ground-glass stoppered volumetric flask
and fill to the mark with water. After the bromoxynil is completely dissolved, trans-
fer the solution to a 150-mL vial with PTFE-lined cap.
6.6.2 Stock solutions should be checked for signs of degradation prior to the preparation of
calibration or performance test standards.
6.6.3 Stock solutions shall be replaced after 6 months, or sooner if comparison with quality
control check standards indicates a change in concentration.
6.7 Secondary mixtures: Using stock solutions (Section 6.6), prepare mixtures for calibration and
calibration verification (Sections 7.3 and 12.4), for initial and ongoing precision and recovery
(Sections 8.2 and 12.5), and for spiking into the sample matrix (Section 8.3).
6.7.1 Calibration solutions: Prepare in water at the concentrations given in Table 2. The
low level solution is used for calibration verification (Section 12.4)
6.7.2 Precision and recovery standard and matrix spike solution: Prepare in water at a
concentration of 10 /xg/mL.
6.8 Stability of solutions: All standard solutions (Sections 6.6 through 6.7) shall be analyzed
within 48 hours of preparation and on a monthly basis thereafter for signs of degradation.
Standards will remain acceptable if the peak area remains within +15% of the area obtained in
the initial analysis of the standard.
780
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Method 1661
7. SETUP AND CALIBRATION
7.1 Configure the HPLC system described in Sections 5.7 through 5.8 and establish the operating
conditions in Table 1.
7.2 Attainment of minimum level: Determine that the minimum level in Table 1 is met at each
wavelength.
7.3 Calibration.
7.3.1 Inject 40 /iL of each calibration solution (Table 2) into the HPLC system, beginning
with the lowest concentration and proceeding to the highest. Compute and store, as a
function of the concentration injected, the retention time and the peak area of Bromox-
ynil each wavelength (primary and confirmatory).
7.3.2 Calibration factor (ratio of area to amount injected).
7.3.2.1 Compute the coefficient of variation (relative standard deviation) of the
calibration factor over the calibration range at each wavelength.
7.3.2.2 Linearity: If the calibration factor is constant (Cv< 15%) over the calibra-
tion range, an average calibration factor may be used; otherwise, the
complete calibration curve (area vs. amount) shall be used.
8. QUALITY CONTROL
8.1 Each laboratory that uses this method is required to operate a formal quality control program.4
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, an ongoing analysis of standards and blanks as tests of continued performance, and
analysis of spiked samples to assess accuracy. Laboratory performance is compared to es-
tablished performance criteria to determine if the results of analyses meet the performance
characteristics of the method.
8.1.1 The analyst shall make an initial demonstration of 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 analyst is permitted to modify this method to improve separations or lower the
costs of measurements, provided all performance requirements are met. Each time a
modification is made to the method or a cleanup procedure is added, the analyst is
required to repeat the procedure in Section 8.2 to demonstrate method performance.
If minimum level will be affected by the modification, the analyst is required to repeat
demonstration of the minimum level (Section 7.2).
8.1.3 The laboratory shall, on an ongoing basis, demonstrate through calibration verification
and the analysis of the precision and recovery standard (Section 6.7.2) that the analy-
sis system is in control. These procedures are described in Sections 12.1, 12.4, and
12.5.
8.1.4 The laboratory shall maintain records to define the quality of data that is generated.
Development of accuracy statements is described in Section 8.3.
8.1.5 Analyses of blanks are required to demonstrate freedom from contamination. The
procedures and criteria for analysis of a blank are described in Section 8.4.
781
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Method 1661
8.2 Initial precision and recovery: To establish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following operations.
8.2.1 Analyze one set of four 10-mL aliquots of reagent water spiked with 100 /xL of the
precision and recovery standard (Section 6.7.2) according to the procedure in Sec-
tions 10 and 11.
8.2.2 Using the results of the set of four analyses, compute the average recovery (X.) and
the standard deviation of recovery (s) for bromoxynil.
8.2.3 Compare s and X with the corresponding limit for initial precision and recovery in
Table 1. If s and X meet the acceptance criteria, system performance is acceptable
and analysis of blanks and samples may begin. If, however, s exceeds the precision
limit or X falls outside the range for accuracy, system performance is unacceptable.
In this case, correct the problem and repeat the test.
8.3 Method accuracy: The aboratory shall spike (matrix spike) at least 10% of the samples from a
given site type (e.g., influent to treatment, treated effluent, produced water). If only one
sample from a given site type is analyzed, a separate aliquot of that sample shall be spiked.
8.3.1 The concentration of the matrix spike shall be determined as follows.
8.3.1.1 If, as in compliance monitoring, the concentration of bromoxynil in the
sample is being checked against a regulatory concentration limit, the ma-
trix spike shall be at that limit or at 1 to 5 times higher than the back-
ground concentration determined in Section 8.3.2, whichever concentration
is larger.
8.3.1.2 If the concentration is not being checked against a regulatory limit, the
matrix spike shall be at the level of the precision and recovery standard
(Section 6.7.2) or at 1 to 5 times higher than the background concentra-
tion, whichever concentration is larger.
8.3.1.3 If it is impractical to determine the background concentration before spi-
king (e.g., maximum holding times will be exceeded), the matrix spike
concentration shall be the regulatory concentration limit, if any; otherwise,
at the the level of the precision and recovery standard (Section 6.7.2) or
at 1 to 5 times the expected background concentration concentration,
whichever is larger.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of bromox-
ynil. If necessary, prepare a standard solution appropriate to produce a level in the
sample 1 to 5 times the background concentration. Spike a second sample aliquot
with the standard solution and analyze it to determine the concentration after spiking
(A) of each analyte. Calculate the percent recovery (P):
Equation 1
p _ IQO(A-B)
T
where
T = True value of the spike
752
-------
Method 1661
8.3.3 Compare the percent recovery of bromoxynil with the corresponding QC acceptance
criteria in Table 1. If it fails the acceptance criteria for recovery, the sample may be
complex and must be diluted and reanalyzed per Section 15.
8.3.4 As part of the QC program for the laboratory, method accuracy for samples shall be
assessed and records shall be maintained. After the analysis of five spiked samples of
a given matrix type (e.g., influent to treatment, treated effluent,produced water) in
which the the recovery test (Section 8.3.3) is passed, compute the average percent
recovery (P) and the standard deviation of the percent recovery (sp). Express the
accuracy assessment as a percent recovery interval from P — 2sp to P + 2sp for each
matrix. For example, if P = 90% and sp = 10% for five analyses of waste water, the
accuracy interval is expressed as 70 to 110%. Update the accuracy assessment in
each matrix on a regular basis (e.g., after each 5 to 10 new accuracy measurements).
8.4 Blanks: reagent water blanks are analyzed to demonstrate freedom from contamination.
8.4.1 Analyze a reagent water blank with each sample batch (samples started through the
extraction process on the same 8-hour shift, to a maximum of 20 samples). Analyze
the blank immediately after analysis of the precision and recovery standard (Section
12.5) to demonstrate freedom from contamination.
8.4.2 If any compound or any potentially interfering compound is found in an aqueous
blank at greater than 100 pg/L (assuming the same calibration factor as bromoxynil
for interfering compounds), analysis of samples is halted until the source of con-
tamination is eliminated and a blank shows no evidence of contamination at this level.
8.5 The specifications contained in this method can be met if the apparatus used is calibrated
properly, then maintained in a calibrated state. The standards used for calibration (Section 7),
calibration verification (Section 12.4), and for initial (Section 8.2) and ongoing (Section 12.5)
precision and recovery should be identical, so that the most precise results will be obtained.
The HPLC instrument will provide the most reproducible results if dedicated to the settings
and conditions required for the analyses of the analytes given in this method.
8.6 Depending on specific program requirements, field replicates and field spikes may be required
to assess the precision and accuracy of the sampling and sample transporting techniques.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Collect samples in glass containers following conventional sampling practices,5 except that the
bottle shall not be prerinsed with sample before collection. Aqueous samples which flow
freely are collected in refrigerated bottles using automatic sampling equipment.
9.2 Maintain samples at 0 to 4°C from the time of collection until extraction. If the samples will
not be extracted within 72 hours of collection, adjust the sample to a pH of 3.0 to 7.0 using
sodium hydroxide or hydrochloric acid solution. Record the volume used. If residual chlorine
is present in aqueous samples, add 80 mg sodium thiosulfate per liter of water. EPA Methods
330.4 and 330.5 may be used to measure residual chlorine.6
9.3 Begin sample extraction within 7 days of collection, and analyze all extracts within 40 days of
extraction.
783
-------
Method 1661
10. PREPARATION OF SAMPLE AND QC ALIQUOTS
10.1 Mix sample thoroughly.
10.2 Pour approximately 10 mL of sample into a clean HPLC autcsarnpler vial. If a matrix spike is
to be prepared, pour 10.0 mL into a second clean vial.
10.3 For each sample or sample batch (to a maximum of 20) to be analyzed in the same 8-hour
shift, place two 10.0-mL aliquots of reagent water (Section 6.4) in clean auto-sampler vials.
One reagent water aliquot serves as the blank.
10.4 Spike 100 fiL of the precision and recovery standard (Section 6.7.2) into the remaining reagent
water aliquot.
10.5 Spike the sample designated as the matrix spike at the level directed in Section 8.3.
10.6 Analyze the sample and QC aliquots per Section 11.
11. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
Table 1 summarizes the recommended operating conditions for the HPLC system. Included
in this Table is the retention times for bromoxynil achieved under these conditions. An
example of the separation achieved by the column system is shown in Figure 1.
11.1 Calibrate the system as described in Section 7.
11.2 Set the injection volume on the auto-sampler to inject 40 pL of ail standards, blanks, and
samples.
11.3 Set the data system or HPLC control to begin data collection upon injection and to stop data
collection after bromoxynil is expected to elute.
12. SYSTEM AND LABORA TORY PERFORMANCE
12.1 At the beginning of each 8-hour shift during which analyses are performed, HPLC system
performance and calibration are verified at both wavelengths. For these tests, analysis of the
calibration verification standard (Section 6.7.1) shall be used to verify all performance criteria.
Adjustment and/or recalibration (per Section 7) shall be performed until all performance
criteria are met. Only after all performance criteria are met may samples, blanks, and preci-
sion and recovery standards be analyzed.
12.2 Retention times.
12.2.1 The absolute retention time of bromoxynil shall be no less than 2.5 minutes.
12.2.2 The absolute retention time of the bromoxynil peak maximum shall be within
±15 seconds of the average of the retention times in the initial calibration (Section
7.3.1).
12.3 GC resolution: Resolution is acceptable if the peak width at half-height of bromoxynil is less
than 15 seconds.
12.4 Calibration verification.
12.4.1 Inject the calibration verification standard (Section 6.7.1).
12.4.2 Compute the concentration of bromoxynil based on the calibration factor or calibration
curve (Section 7.3).
784
-------
Method 1661
12.4.3 Compare this concentration with the limits for calibration verification in Table 1. If
calibration is verified, system performance is acceptable and analysis of blanks and
samples may begin. If, however, the recovery falls outside the calibration verification
range, system performance is unacceptable. In this case, correct the problem and
repeat the test, or recalibrate (Section 7).
12.5 Ongoing precision and recovery.
12.5.1 Analyze the precision and recovery standard prepared with each sample batch (Se-
ction 10.3).
12.5.2 Compute the recovery of bromoxynil.
12.5.3 Compare the recovery with the limit for ongoing recovery in Table 1. If the recovery
meets the acceptance criteria, the analytical process is in control and analysis of
blanks and samples may proceed. If, however, the recovery falls outside the accep-
table range, these processes are not in control. In this event,'correct the problem and
repeat the ongoing precision and recovery test.
12.5.4 Add results which pass the specifications in Section 12.5.3 to initial and previous
ongoing data. Update QC charts to form a graphic representation of continued labora-
tory performance. Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of percent
recovery (sr). Express the accuracy as a recovery interval from R — 2sr to R + 2sr.
For example, if R = 95% and sr = 5%, the accuracy is 85 to 105%.
13. QUALITATIVE DETERMINATION
13.1 Quantitative determination is accomplished by comparison of data from analysis of a sample or
blank with data from analysis of the shift standard (Section 12.1), and with data stored in the
retention-time and calibration libraries (Section 7.3.1). Identification is confirmed when reten-
tion-time and amounts agree per the criteria below.
13.2 Establish a retention-time window +15 seconds on either side of the mean retention-time in
the calibration data (Section 7.3.1).
13.3 If a peak from the analysis of a sample or blank is within this window (as defined in Section
13.2) at the primary wavelength (280 nm), it is considered tentatively identified. A tentatively
identified compound is confirmed when (1) the retention time of the peak maximum at the
confirmatory wavelength (255 nm) is within +2 seconds of the retention-time of the peak
maximum at the primary wavelength, and (2) the computed amounts (Section 14) on each
system (primary and confirmatory) agree within a factor of 2.
14. QUANTITATIVE DETERMINATION
14.1 Using the HPLC data system, compute the concentration of the analyte detected in the sample
(in milgram per liter) using the calibration factor or calibration curve (Section 7.3.2).
14.2 If the concentration of any analyte exceeds the calibration range of the system, the sample is
diluted by a factor of 10, and a 40-/*L aliquot of the diluted extract is analyzed.
785
-------
Method 1661
14.3 Report results for bromoxynil found in all standards, blanks, and samples to three significant
figures. Results for samples that have been diluted are reported at the least dilute level at
which the concentration is in the calibration range.
15. ANALYSIS OF COMPLEX SAMPLES
15.1 Some samples may contain high levels (> 1000 /*g/L) of bromoxynil or of interfering com-
pounds and/or polymeric materials. Some samples may overload the HPLC column and/or
detector. In these instances, the sample is diluted by a factor of 10 and reanalyzed (Section
14.2).
15.2 Recovery of matrix spikes: in most samples, matrix spike recoveries will be similar to those
from reagent water. If the matrix spike recovery is outside the range specified in Table 1, the
sample is diluted by a factor of 10, respiked, and reanalyzed. If the matrix spike recovery is
still outside the range, the method does not work on the sample being analyzed and the result
may not be reported for regulatory compliance purposes.
16. METHOD PERFORMANCE
16.1 Development of this method is detailed in Reference 7.
786
-------
Method 1661
References
1. "Carcinogens—Working with Carcinogens." Department of Health, Education, and Welfare;
Public Health Service; Center for Disease Control; National Institute for Occupational Health
and Safety: Publication 77-206, August 1977.
2. "OSHA Safety and Health Standards, General Industry" (29 CFR 1910). Occupational Safety
and Health Administration: January 1976.
3. "Safety in Academic Chemistry Laboratories," American Chemical Society Committee on
Chemical Safety: 1979.
4. "Handbook of Quality Control in Wastewater Laboratories," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-79-019, March 1979.
5. "Standard Practice for Sampling Water" (ASTM Annual Book of Standards), American Society
for Testing and Materials, Philadelphia, Pennsylvania: 76, 1980.
6. "Methods 330.4 and 330.5 for Total Residual Chlorine," U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH: EPA-600/
4-70-020, March 1979.
7. "Narrative for SAS 1019," Pacific Analytical, Inc.: September 1989. Available from the
U.S. Environmental Protection Agency Sample Control Center, 300 N. Lee St., Alexandria,
VA 22314 (703-557-5040).
787
-------
Method 1661
Table 1. HPLC Data and Method Acceptance
Acceptance Criteria
Minimum Level
Method Detection Limit
Calibration Verification (Section 12.4)
Initial Precision and Recovery (Section 8.2)
Precision [standard deviation (s)]
Recovery [mean (X)]
Ongoing Precision and Recovery (Section 12.5)
Matrix Spike Recovery (Section 8.3.3)
Bromoxynil Retention Time
Criteria for Bromoxynil
Specifica tion
100/yg/L
20 A/g/L
86-128//g/L
28 /ig/L
74-130//g/L
72-132/yg/L
68-129%
2.62 minutes
*
Note
1
2
3
3
4
*(3,5-dibromo-4-hydroxybenzonitrile; CAS 1689-84-5)
Notes:
1. This is a minimum level at which the analytical system shall give recognizable signals and
acceptable calibration points.
2. Estimated; 40 CFR Part 136, Appendix B.
3. Test concentration 100 jug/L.
4. Column and conditions: 300 Angstrom C18 column 150 mm long by 4.6 mm ID. Column
temperature 30°C. Solvent flow rate 0,5 mL/min. Isocratic at 50% methanol in water.
Table 2. Concentration of Bromoxynil Calibration Solutions
Level
Low
Median
High
Concentration (pg/U
100
600
3,000
788
-------
Method 1661
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Retention Time (minutes)
A52-002-86A
Figure 1. Chromatogram of Bromoxynil
789
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-------
APPENDIX
Methods EV-024
and EV-025
Analytical Procedures for
Determining Total Tin and
Triorganotin in Wastewater
Provided by
ATOCHEM North America
-------
-------
Addendum to Methods EV-024 and EV-025:
Quality Control
1. SCOPE AND APPLICA TION
1.1 Methods EV-024 and EV-025 are employed by the pesticides manufacturing industry to
measure tin and organotin in wastewater. Data collected with these methods were used to
support the Pesticide Chemicals Manufacturing Rule proposed at 57 PR 12560. As written,
the methods contain little quality control (QC). The QC given below must be used when
compliance monitoring data are collected using these methods.
1.2 This QC is patterned after the QC in the 40 CFR Part 136 Appendix A methods promulgated
at 49 FR 43234 (October 26, 1984).
2. CALIBRATION AND CALIBRATION VERIFICATION
2.1 For calibration, Section 2.7 of Method EV-024 requires repetitive injections until the "nu-
mbers are reproducible and close in range." For QC purposes, this requirement is interpreted
to mean that the relative standard deviation of a triplicate injection must be less than 10%.
2.2 Calibration is verified after every tenth sample using a single injection of the calibration
standard. If the recovery is not in the range of 90 to 110%, the instrument must be recali-
brated and the ten (or fewer) samples analyzed prior to the failed calibration must be reana-
lyzed.
3. INITIAL PRECISION AND RECOVERY (IPR)
3.1 Dissolve the organotin of interest in a water-miscible solvent and spike four aliquots of reagent
water to produce a concentration of 50 fig/L.
3.2 Extract and analyze the four aliquots. The average recovery of organotin must be in the range
of 89 to 120% and the standard deviation must be less than 10%. If either of these criteria are
not met, the analytical system is not in control. Correct the problem and repeat the test.
4. ONGOING PRECISION AND RECOVERY (OPR)
4.1 With each batch of samples analyzed on the same 8-hour shift (to a maximum of 10 samples),
spike and analyze a single ongoing precision and recovery (OPR) sample—also known as a
"laboratory control sample" (LCS)—in the same way as the IPR.
4.2 The recovery must be in the range of 75 to 125%. If this criterion is not met, the analytical
system is not in control. Correct the problem, and re-extract and reanalyze the batch of
samples with a new OPR sample.
5. BLANKS
5.1 With each batch of samples analyzed on the same 8-hour shift (to a maximum of 10 samples).
analyze a blank. The batch is the same batch used for the OPR/LCS test.
793
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Appendix: IND-01
5.2 If tin is detected in the blank at a concentration of 5 /ig/L or greater, the analytical system is
not in control. Correct the problem, and re-extract and reanalyze the batch of samples.
6. MA T/J/X SPIKE/MA TRIX SPIKE DUPLICA TE (MS/MSD)
6.1 With each set of samples from the same sample stream analyzed on the same 8-hour shift (to a
maximum of 10 samples), spike and analyze an MS/MSD at a concentration in the range of
1-5 times the background concentration found in the sample.
6.2 The recovery must be in the range of 75 to 125% and the relative percent difference must be
less than 20%. If these criteria are not met, the analytical system is not in control. Correct
the problem, and re-extract and re-analyze the batch of samples.
794
-------
Methods EV-024 and EV-025
Analytical Procedures for Determining Total Tin
and Triorganotin in Wastewater
EV-024
1. SAFETY
Wear rubber gloves and glasses with side shields. Follow standard laboratory safety procedures.
Any special safety notes are included in the procedure body.
2. PROCEDURE
2.1 Prepare the AA & HGA 500 with the appropriate instrumental operating conditions and
keyboard entries:
2.1.1 AA2380
2.1.1.1 Turn power on.
2.1.1.2 Open H2O drain exit valve and H2O inlet valve (% open).
2.1.1.3 Install Sn hollow cathode element and regular, uncoated tube.
2.1.1.4 Check to see that all control knobs are in extreme counter-clockwise posi-
tion.
2.1.1.5 Using lamp control knob, set lamp/energy to 30 mA.
2.1.1.6 Set slit nm to 0.7 ALT and wavelength to 286.3.
2.1.1.7 Adjust wavelength and lamp alignment (by turning signal to set up posi-
tion, turning gain control knob clockwise until about 35 registers on lamp/
energy display, then adjusting beat and lamp for maximum gain).
2.1.1.8 Position control knobs as follows:
Signal - cone
Mode - PkHT
Recorder - TCI
BG Correction - AA KN
2.1.1.9 Set integration time for 7 seconds.
2.1.1.10 Turn on inert gas supply (argon).
2.1.2 HGA 500
2.1.2.1 Turn power on.
2.1.2.2 Program keyboard with following entries.
795
-------
Appendix: IND-01
1 ;
Temp °C 100
Ramp Time (s) 15
Hold Time (s) 15
Rec. x
Read
Mini Flow, Argon x
mL/min 50
Step
? 3 \ 4
700 2500 50
1500
15 7 5
XXX
X
X
50
Stop flow x
2.2 Secure appropriate amount (usually 25 to 150 mL depending on source of sample) directly into
previously cleaned (see comments) 500-mL screw-top bottle.
2.3 Add 10 mL hydrobromic acid to each sample. Shake well then let stand for 10 minutes.
2.4 Add 50 mL (or more, also dependent upon source of sample) 0.05% tropolone in toluene
(T-T) solution. Shake for 10 minutes. Allow to separate into two layers.
2.5 If separation is sufficient (i.e., little emulsion), run 5 /tL on HGA-500 AA 2380 after standar-
dization. (See Step 6); if separation unsatisfactory, add 10 mL more of T-T solution, shake 10
minutes and let stand to separate into two layers.
2.6 To standardize:
2.6.1 Inject 5 ptL of 0.05 ppm standard inorganic Sn into the graphite furnace with an
Eppendorf pipette, equipped with a clean, disposable tip.
2.6.1 Engage program cycle and record peak height.
2.7 Repeat standard until numbers are reproducible and close in range and average.
2.8 Check cleanliness of furnace by engaging program cycle without sample until baseline returns.
2.9 Obtain 5 /iL directly from upper phase of each sample from Step 5 using the Eppendorf pipette
technique with a clean tip for each sample and run through cycle at least three times and
average results.
2.10 Run 5 JJ.L of blank (T-T solution) three times and average.
3. CALCULATIONS
Real conc. x ml T-T solution
Total Tin ppm =
sample volume
Conc - Average ABS normalized to 5 \>L x factor
Example:
1. Normalize all average to 5 jiL;
e.g., 25 (j.L sample average = 30.4 -f- 5 = 6.08
2. Determine factor of standard.
796
-------
Appendix: EV-024 and EV-025
Concentration of standard in ppm
Average ABS (normalize to 5 pL)
Example:
Avg ABS of 0.5 ppm was .115
et)0.5/.115 = 4.348 = Factor
Example:
Avg ABS of sample is 0.146. Dilution factor is lOx. Sample volume is 150 mL.
T-T solution volume is 50 mL. (Use previous example as factor for standard).
(4.38)(50)(0.146)(10;t) ,, ,., „ t , „•
-— —- = 2.116 ppm Total Tin
150 mL ^
4. COMMENTS
4.1 Mark original level of sample in sample bottle so that volume may be determined.
4.2 Amount of sample extract introduced into the graphite furnace may vary depending upon
actual concentration.
4.3 Suitable signals for analysis are obtained in the 10X expansion mode.
4.4 All sample bottles and glassware used must be scrupulously cleaned with concentrated HCI, by
soaking for several hours or overnight, and then rinsed several times with distilled water.
4.5 Label bottles with magic marker for sample name, date sample discharged, sample volume,
and T-T volume.
737
-------
Appendix: IND-01
EV-025
1. SAFETY
1.1 Wear rubber gloves and glasses with side shields. Follow standard laboratory safety proce-
dures. Any special safety notes are included in the procedure body.
2. PROCEDURE
2.1 Prepare A A and total tin samples as specified an EV-024..
* - ~ " ?
2.2 Using clean dram vials, extract 2 mL from top phase of total tin sample.
2.3 Add equal amount of prepared 3% NaOH solution to sample in vial. Label vial with sample
name using indelible marker.
2.4 Shake for 10 minutes.
2.5 Inject 5 mL on HGA 500, AA 2380 after standardization into graphite furnace with an Eppen-
dorf pipette, equipped with a clean disposable tip.
2.6 Engage program cycle and record peak height.
2.7 Repeat until numbers are close in range and reproducible, and average.
3. CALCULATIONS
Calculations are the same as in ppm total tin.
(Factof)(T-T Volume%Avg ABS)(Dilutiori)
Sample Volume
Example:
0.5 ppm standard with Avg. 0.115 ABS
(0'5) = 4.348 = Factor
ppm Tri Tin: Avg ABS of sample is 0.123. Dilution factor is none, but 25 jiL was used.
Sample volume is 150 mL and T-T solution volume is 50 mL.
(4.348X50X0.123) * 5 =
150 ~~"
798
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