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

-------
                            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.

-------
                                                                            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 These methods were withdrawn as a part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.

-------
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

-------
	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

-------
Contents (com.)
Appendix
Methods EV-024 and EV-025:
Analytical Procedures for Determining Total Tin and Triorganotin in Wastewater  	791
IV

-------
                                                              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	  -

-------
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

-------
                                                        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

-------
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

-------
                                                       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

-------
 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

-------
                                                       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

-------
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

-------
                                                       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

-------
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 400C 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 4C
               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 400C for 4 hours or
      perform a Soxhlet extraction with methylene chloride.
5.4   Water bath:  Heated, capable of temperature control (2C).  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 400C 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
               4C 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,j8natySflHB&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 4C 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 65C, 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 80C.  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.     "CarcinogensWorking 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

-------

-------
                                 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

-------
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 400C 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

-------
                                                                                 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
               4C 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

-------
 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 400C for 30 minutes or perform a
       Soxhlet extraction with methylene chloride.
 5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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 400C 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 675C 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 130C 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 4C 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

-------
                                                                                   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

-------
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 4C 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

-------
                                                                                  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 65C, 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 80C.  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

-------
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

-------
                                                                                  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

-------
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.     "CarcinogensWorking 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 200C.
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 400C 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 4C
               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
              (2C). 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 400C 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 675C and store in dark in
               glass containers with glass stoppers or foil-lined screw-caps.  Before use, activate each
               batch overnight at 130C in foil-covered glass container.
       6.1.4   Silica gel: Activate  approximately 100 g of silica gel at 200C  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  400C 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 4C 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 4C 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 85C. 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  85C 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.    "CarcinogensWorking 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 200C. 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 200C.  Detector:  electron capture.

   * Column temperature is  220C.
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 400C 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
              4C 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 400C 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 (2C).  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 400C
       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 675C 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 130C 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 4C 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

-------
                                                                                     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

-------
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 4C from the time of  collection until extraction.
66

-------
                                                                                   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  85C, 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

-------
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

-------
                                                                                   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 85C. 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

-------
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

-------
                                                                                  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

-------
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

-------
                                                                                 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

-------
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

-------
                                                                            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 200C.  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 212C.
                                                                                    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

-------
            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

-------
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 400C 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
               4C 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

-------
                                                                                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
              (2C). 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 400C 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

-------
Method 614.1
       6.1.2   Silica gel:  Woelm 70-230 mesh.  Activate approximately 100 g of silica gel at 200C
               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 400C 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 4C 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 4C 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

-------
                                                                                    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 85C) 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

-------
                                                                               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.    "CarcinogensWorking 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

-------
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 200C for 2 minutes,
then  programmed at 5/min to 240C 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 180C  for 2 minutes, then programmed at 8/min to 250C 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 400C 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 90C:  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
               4C 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.
                                                                                           99

-------
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 400C for 30 minutes or perform a
       Soxhlet extraction with methylene chloride.
5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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

-------
                                                                                    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
      400C  for a minimum of 4 hours to remove phthalates and other interfering organic substan-
      ces.  Alternatively, heat  16 hours at 450 to 500C 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 130C.
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 4C.
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 130C.
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 4C
              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:
                                                                                            707

-------
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.
 702

-------
                                                                                    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
                                                                                            103

-------
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 4C 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 65C), 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

-------
                                                                                   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 (4C) 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

-------
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

-------
                                                                                  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
                                                                                          707

-------
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.
 108

-------
                                                                                 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.     "CarcinogensWorking 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).
                                                                                        109

-------
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 185C, except for MCPP, MCPA,
dichlorprop and dinoseb, where the. column temperature was held at 140C for 6 minutes and then
programmed to 200C 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 185C.

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 150C at 10/min.
 110

-------
                                                                                  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 400C 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 4C
              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 400C for 4 hours or
       perform a Soxhlet extraction with methylene chloride.
5.4    Water bath: Heated,  capable of temperature control  (2C).  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 400C 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 130C.  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
               4C 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 4C 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 65C, 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

-------
                                                                                   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

-------
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

-------
                                                                                 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

-------
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 240C at 8C/min, injector temperature is 280C and
detector is 300C. 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 240C at 4C/min, injector temperature is 280C and
detector is 300C. A flame ionization detector is used.
130

-------
                                                                          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

-------

-------
              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

-------
                                                                                     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 400C 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

-------
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
               4C 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 400C for 30 minutes or perform a
      Soxhlet extraction with methylene chloride.
5.4   Water bath: Heated, with concentric ring cover, capable of temperature control (2C).  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 400C 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 675C 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 130C 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

-------
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 4C 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

-------
                                                                                     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

-------
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  4C 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

-------
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 85C, 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 85C.  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 200C. 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 200C.
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: 200C
 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: 200C
                             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: 200C
                            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: 160C
                   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: 160C
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: 160C
             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: 160C.
     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: 160C.
                  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: 200C
    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: 200C
            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

-------
Method 618
               water and detergent and thorough rinsing with tap and reagent water.  Drain dry, and
               heat in an oven or muffle furnace at 400C 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 105C 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 105C 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

-------
                                                                                     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
              4C and protect from light. Frequently check stock standard solutions for signs of

                                                                                            777

-------
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

-------
                                                                                    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

-------
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

-------
                                                                                  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 4C 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

-------
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
176

-------
                                                                                  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

-------
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

-------
                                                                                 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

-------
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 135C. 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 95C.
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

-------
                                                                            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

-------

-------
            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

-------
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 400C  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.
186

-------
                                                                                  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
              4C 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.
                                                                                          187

-------
Method 619
5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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 400C
       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 675C 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 130C 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.

 188

-------
                                                                                      Method 619
       6.6.2  Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
               Store at 4C 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-
                                                                                             189

-------
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.
190

-------
                                                                                     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
                                                                                            191

-------
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 4C 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

-------
                                                                                   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 65C, 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 80C.  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.
                                                                                          193

-------
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.
194

-------
                                                                                    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
                                                                                           195

-------
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

-------
                                                                                  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.
                                                                                         757

-------
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.     "CarcinogensWorking 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

-------
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 200C. 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 155C.
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

-------
                                                                             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

-------
              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

-------
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 400C 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
               4C 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 400C for 4 hours or
       extract in a Soxhlet extractor with methylene chloride.
5.4    Water bath: Heated, capable of temperature control  2C. 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 400C overnight.
6.4    Silica gel:  Davison Grade 923, 100-200 mesh; activated by heating for 24 hours at 150C.
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
               4C  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 40C, 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 65C, 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 80C 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.
214

-------
                                                                                 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

-------
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

-------
                                                                                 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.     "CarcinogensWorking 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

-------
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 80C for 4 minutes, programmed from 80 to 300C at 8C/min, and held at
300C 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 80C for 4 minutes, programmed from 80 to 250C at 80C/min, and held at
250C 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

-------
                                                                  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

-------

-------
              Method 622
         The Determination of
Organophosphorus Pesticides in
       Municipal and Industrial
                 Wastewater

-------

-------
                                  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).

                                                                                    223

-------
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 400C 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

-------
                                                                                   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
               4C 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.

                                                                                          225

-------
Method 622
5.3    Boiling chips:  Approximately 10/40 mesh. Heat at 400C for 30 minutes or perform Soxhlet
       extraction with methylene chloride.
5.4    Water bath:  Heated, with concentric ring cover, capable of temperature control (+2C).  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 400C
       for a minimum of 4 hours to remove phthalates and other interfering organic substances.  Al-
       ternatively, heat 16 hours at 450 to 500C 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.
 225

-------
                                                                                      Method 622
       6.5.2  Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
               Store at 4C 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-
                                                                                             227

-------
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.
225

-------
                                                                                     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.
                                                                                            225

-------
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 4C 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.
230

-------
                                                                                  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 65C, 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 80C.  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

-------
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

-------
                                                                                  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

-------
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

-------
                                                                                 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.     "CarcinogensWorking 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

-------
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 150C, hold for 1 minute, then program at 25C/min to 220C
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 170C, hold 2 minutes, then program at 20C/min
to 220C 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 170C, hold for 7  minutes, then program at 10C/min to  250C
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 100C, then program immediately at 25C/min to 200C and hold. An
electrolytic detector in the halogen mode was used with this column to estimate the MDL.
236

-------
                                                                          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

-------
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 400C 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

-------
                                                                                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
              4C 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 400C for 4 hours  or
      extract in a Soxhlet with methylene chloride.
5.4   Water bath:   Heated, capable of temperature control (2C).  The bath should be used in a
      hood.
                                                                                         247

-------
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 400C overnight.
6.4   Florisil:  PR grade (60/100 mesh). Purchase activated at 675C 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
       170C.   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

-------
                                                                                   Method 622.1
       6.7.2   Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. Store at
               4C 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

-------
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 4C 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 65C, 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.     "CarcinogensWorking 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 300C at 8C/min with a 4 minute hold at each extreme,
injector temperature is 250C and detector is 300C.  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 300C at 8C/min with a 4 minute hold at each extreme,
injector temperature is 250C and detector is 300C.


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

-------
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 400C 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
               4C 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 400C 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 (2C).  The
       bath should be used in a hood.
5.5    Balance:  Analytical, capable of accurately weighing to the nearest 0.0001 g.
                                                                                          257

-------
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 400C for
       a minimum of 4 hours to remove phthalates and other interfering organic substances. Alterna-
       tively, heat 16 hours at 450 to  500C 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 4C 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

-------
                                                                                     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

-------
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.
270

-------
                                                                                     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

-------
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 4C 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

-------
                                                                                  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 65C, 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 80C. 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

-------
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

-------
                                                                                  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

-------
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

-------
                                                                                  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.     "CarcinogensWorking 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

-------
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 190C.

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
160C for 2 minutes, then programmed to 200C at 10C/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

-------
                                                                     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

-------

-------
              Method  629
The Determination of Cyanazine
     in Municipal and Industrial
                 Wastewater

-------

-------
                                   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

-------
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 400C 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
               4C  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-
284

-------
                                                                                   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 400C for 30 minutes or perform a
       Soxhlet extraction with methylene chloride.
5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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.
                                                                                          285

-------
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 400C
       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 675C 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  130C 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 4C 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

-------
                                                                                     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
                                                                                             287

-------
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.
255

-------
                                                                                     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.
                                                                                            289

-------
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 4C 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 65C, 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


290

-------
                                                                                  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  80C, 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)
                                                                                         291

-------
       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


292

-------
                                                                                  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.
                                                                                         293

-------
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.     "CarcinogensWorking 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).
294

-------
	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
                                                                                 235

-------
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)
296

-------
          Method 630
        The Determination
       of Dithiocarbamate
Pesticides in Municipal and
    Industrial Wastewater

-------

-------
                                  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.
                                                                                    299

-------
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 200C for 15
              minutes.  Rinse the CS2 trap with methanol three times between each use and follow
              by heating for 15 minutes at 200C.  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

-------
                                                                                   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
               4C 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

-------
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:  SnCl22H2O, 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 4C 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

-------
                                                                                    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.

                                                                                            303

-------
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 4C from the time of collection until extraction.
9.3    All samples must be analyzed within 7 days of collection.
304

-------
                                                                                   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

-------
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

-------
                                                                                 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.     "CarcinogensWorking 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

-------
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

-------
                                                          Method 630
                                                             A52-002-60A
Figure 1.   Dithiocarbamate Hydrolysis Apparatus
                                                                309

-------

-------
           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

-------
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 400C 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

-------
                                                                                 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 105C 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
               4C 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 (2C).  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

-------
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: SnCl22H20, 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 45C 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
               4C.  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

-------
                                                                                   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

-------
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

-------
                                                                                Method 630.1
9.2   The samples must be iced or refrigerated at 4C 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
      30C, 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 50C 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

-------
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

-------
                                                                                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.     "CarcinogensWorking 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 7C for 3 minutes, programmed at
    20C/min to 120C, and then held at 120C for 5 minutes. Column effluent is vented from
    the Hall detector after elution of CS2 from the column.  Injector temperature and detector
    temperatures are 200C.  The Hall detector is operated in the sulfur mode following manufac-
    turer's specifications.
322

-------
                                                                          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

-------
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

-------
            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

-------
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 400C 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
               4C 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 400C for 30 minutes or perform a
       Soxhlet extraction with methylene chloride.
5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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 400C
       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 4C 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

-------
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

-------
                                                                                   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-
                                                                                           333

-------
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 4C 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 65C, 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.
334

-------
                                                                                  Method 631
10.9 Increase the temperature of the hot water bath to 85 to 90C.  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.
                                                                                         335

-------
 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

336

-------
                                                                                   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.
                                                                                           337

-------
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.     "CarcinogensWorking 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).
338

-------
                                                                          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
                                                                                339

-------
Method 631
                                                  Carbindazim
                                               10
                                Retention Time (minutes)
                                                                             A52-002-62A
            Figure 1.   Liquid Chromatogram of Carbendazim on Column 1
                       (for conditions, see Table 1)
340

-------
             Method 632
         The Determination of
Carbamate and Urea Pesticides
    in Municipal and Industrial
                Wastewater

-------

-------
                                  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.
                                                                                   343

-------
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 400C 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.
344

-------
                                                                                   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
               4C 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 (2C).  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.
                                                                                          345

-------
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 400C
       for a minimum of 4 hours to remove phthalates and  other interfering organic substances. Al-
       ternatively, heat 16 hours at 450 to 500C 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 675C 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 130C 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.
 346

-------
                                                                                      Method 632
       6.7.2  Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap vials.
               Store at 4C 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
                                                                                             347

-------
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-
348

-------
                                                                                    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.
                                                                                            349

-------
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 4C 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

-------
                                                                                   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 50C water bath.
10.7 Concentrate the extract to approximately 5 mL in the rotary evaporator at a temperature of
      50C. 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 4C 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

-------
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

-------
                                                                                   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

-------
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

-------
                                                                                 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.     "CarcinogensWorking 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

-------
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

-------

-------
        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

-------
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 400C 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
                4C 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 400C 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 400C 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 4C 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 4C 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

-------
                                                                                    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

-------
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

-------
                                                                                 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 40C.  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

-------
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

-------
                                                                                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.    "CarcinogensWorking 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

-------
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

-------
                                                              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

-------

-------
           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

-------
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 400C 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 4C
378

-------
                                                                                   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 400C for 30  minutes or performed
      Soxhlet extraction with methylene chloride.
5.4   Water bath:  Heated, with concentric ring cover, capable of temperature control (2C).  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

-------
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 400C
       for a minimum  of 4 hours to remove phthalates and other interfering organic substances.  Al-
       ternatively, heat 16 hours at 450 to 500C 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 4C 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

-------
                                                                                     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

-------
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

-------
                                                                                    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

-------
Method 633
9.2    The samples must be iced or refrigerated at 4C 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 65C, 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 70C.  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

-------
                                                                                   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

-------
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.     "CarcinogensWorking 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 210C, hold for 1  minute, then program at 10 to 250C 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 240C.

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 I60C, programmed at injection at 10C/min to 230C.

Column 2b conditions: Same as Column 2a, except temperature programmed: initial 130C, hold
for 1 minute, then program at 12C/min to 200C.
                                                                                  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
                                                    \    '
         niiiiiiiiiiiii    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 400C 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 4C
              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 400C for 4 hours  or
       perform a Soxhlet extraction with methylene chloride.
5.4    Water bath: Heated, capable of temperature control (20C).  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 400C overnight.
6.6    Florisil: PR grade (60/100 mesh). Purchase activated at  675C and store in brown glass
       bottle.  To prepare for  use, place 150 g in a wide-mouth jar and heat overnight at 160 to
       170C.  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
               4C 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

-------
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 4C 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 65C, 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 95C.  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.     "CarcinogensWorking 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 80C to 300C at 8C/min with a 4 minute hold at each ex-
treme, injector temperature is 250C, and detector is 300C. 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

-------
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 400C 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
              4C 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 400C for 4 hours or
      extract in a Soxhlet extractor with methylene chloride.
5.4   Water bath:  Heated, capable of temperature control (+2C).  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 400C overnight.
6.4    Silica gel:  Davision Grade 923, 100/200 mesh;  activated by  heating for 24 hours at 150C.
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
               4C and protect from light.  Frequently check stock standard solutions for signs of
418

-------
                                                                                     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

-------
 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

-------
                                                                                    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

-------
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 4C 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

-------
                                                                                   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 75C. 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

-------
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

-------
                                                                                  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

-------
                                                                                     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

-------
 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.     "CarcinogensWorking 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 80C for 4 minutes, programmed from 80 to 300C at 8C/min and held at
300C 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 80C for 10 minutes,  programmed from 80 to 250C at 8C/min and held
at 250C 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

-------
             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

-------
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 400C 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 4C
               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

-------
                                                                                  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 400C for 4 hours or
      extract in a Soxhlet extractor with methylene chloride.
5.4   Water bath:  Heated, capable of temperature control (2C). 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

-------
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 400C 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 150C.
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
               4C  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

-------
                                                                                     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

-------
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

-------
                                                                                    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

-------
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 4C 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 65C, 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

-------
                                                                                   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

-------
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

-------
                                                                                   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

-------
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.     "CarcinogensWorking 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

-------
                                                                          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

-------
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

-------
                                                                      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

-------

-------
             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

-------
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 400C 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
               4C 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

-------
                                                                                  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 400C for 4 hours or
      extract in a Soxhlet extractor with methylene  chloride.
5.4   Water bath:  Heated, capable of temperature control (+2C).  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 400C overnight.
6.4   Sodium phosphate, monobasic: ACS,  crystal.
                                                                                         455

-------
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
       170C. 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
               4C  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 calibrati