&EPA  Methods for the Determination
       of Nonconventionai Pesticides in
       Municipal
       Volume If

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                            Acknowledgments
  This methods compendium was prepared under the direction of Thomas E. Fielding, Ph.D., and
 William A. Telliard of the Engineering and Analysis Division within EPA's Office of Water. This
document was prepared under EPA Contract No. 68-C9-0019 by the Environmental Services Division
of DynCorp Viar, Inc.  The methods contained in this compendium were developed by the U.S. EPA
              Environmental Monitoring Systems Laboratory in Cincinnati, Ohio.
                                  Disclaimer

     This methods compendium has been reviewed by the Engineering and Analysis Division,
   U.S. Environmental Protection Agency, and approved for publication. Mention of trade names
       or commercial products does not constitute endorsement or recommendation for use.

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                                                                            Introduction
    The Environmental Protection Agency (EPA) is promulgating effluent limitations guidelines and stan-
dards for the Pesticide Chemicals Industry at 40 CFR Part 455 to control the discharge of pollutants,
including certain pesticide active ingredients, into the  waters of the United States.  This compendium of
test procedures (methods) supports this rulemaking. The purpose of publishing these methods in a
compendium is to create a single reference for analysts seeking to  measure infrequently determined  active
ingredients.
    The proposed rule for the Pesticide Chemicals Manufacturing Subcategory (57 FR 12560) referenced
the original version of Volume I (EPA 821 RR-92-002) of this compendium of methods.  That volume has
since been revised and is referenced in the rule as EPA-821-R-93-010-A.  Volume II contains methods that
are not included in Volume I but that were incorporated by reference in the proposed rule.
    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  These methods were withdrawn as  a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 perfor-
mance 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 Engineer-
ing 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 one industry method. 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 EAD, 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 II contains 13 of the 500-series methods  and one 200-series  method developed by
EMSL-Ci since the mid-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 I 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

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    Questions about the methods in Volume II—Method 200.9 and the 500-series methods—should be
directed to:

                     U.S. EPA
                     Office of Research and Development
                     Environmental Monitoring Systems Laboratory
                     Chemistry Research Division
                     26 W. Dr. Martin Luther King Jr. Drive
                     Cincinnati, OH 45268

    Questions, as well as requests for Volume I, may also be directed to the following address:

                     Sample Control Center
                     Operated by DynCorp Viar, Inc.
                     P.O. Box 1407
                     Alexandria, VA  22313

                     (703) 557-5040

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	Contents

Method
No.          Title                                                                    Page

200.9         Determination of Trace Elements by Stabilized Temperature
              Graphite Furnace Atomic Absorption Spectrometry  	  1

505.          Analysis of Organohalide Pesticides and Commercial
              Polychlorinated Biphenyl (PCB) Products in Water
              by Microextraction and Gas Chromatography	29

506.          Determination of Phthalate and Adipate Esters in Drinking Water
              by Liquid-Liquid Extraction or Liquid-Solid Extraction
              and Gas Chromatography with Photoionization Detection	61

507.          The Determination of Nitrogen- and Phosphorus-Containing
              Pesticides in Water by Gas Chromatography
              with a Nitrogen-Phosphorus Detector	83

508.          Determination of Chlorinated Pesticides in Water
              by Gas Chromatography with an Electron Capture Detector	  109

515.1         Determination of Chlorinated Acids in Water by Gas Chromatography
              with an Electron Capture Detector	  135

515.2         Determination of Chlorinated Acids in Water
              Using Liquid-Solid Extraction and Gas Chromatography
              with an Electron Capture Detector	  165

525.1         Determination of Organic Compounds in Drinking Water
              by Liquids-Solid Extraction and Capillary Column
              Gas Chromatography/Mass Spectrometry	  195

              Appendix:  Detection Limits for Precision and Accuracy
              for the Analysis of Pesticide Compounds by EPA Method 525.1  	230

531.1         Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates
              in Water by Direct Aqueous Injection HPLC
              with Post-Column Derivatization	235

547           Determination of Glyphosate In Drinking Water By Direct-Aqueous-Injection
              HPLC, Post-Column Derivatization,  and Fluorescence Detection	255

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Contents	

Method
No.           Title                                                                    Page


548           Determination of Endothall in Drinking Water by Aqueous
              Derivatization, Liquid-Solid Extraction, and Gas Chromatography
              with Electron-Capture Detection  	271

              Appendix:  Preparation of Endothall-Pentafluorophenylhydrazine	286

548.1         Determination of Endothall in Drinking Water by Ion-Exchange Extraction,Acidic
              Methanol Methylation and Gas Chromatography/Mass Spectrometry	291

553.           Determination of Benzidines and Nitrogen-Containing Pesticides in Water
              by Liquid-Liquid Extraction or Liquid-Solid Extraction and Reverse Phase
              High Performance Liquid Chromatography/Particle Beam/Mass Spectrometry ...  319

555.           Determination of Chlorinated Acids in Water by High Performance Liquid
              Chromatography with a Photodiode Array Ultraviolet Detector	355
IV

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                                                               Cross-Reference
      Analytes shown in bold italic type are regulated under the Pesticide Chemicals Manufacturing
Rule found at 40 CFR Part 455.
                                                       Applicable Method(s)
Analytes                      CAS No.            Volume I               Volume II

Acenaphthylene  	  208-96-8	525.1
Acephote  	30560-19-1	1656, 1657	  -
Adfluorfen	50594-66-6	  -	575.7, 575.2, 555
Alachlor	15972-60-8	645, 1656	505, 507, 525.7
Aldicarb (Temik)	  116-06-3	-	   537.7
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, 525.7
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	633, 1656	  507, 525.7
Baythroid (Cyfluthrin)	68359-37-5	  1660  	  -
Bendiocarb	22781-23-3	  639  	  -
Benefin (Benfluralin)	   1861-40-1	627, 1656	  -
Benfluralin (Benefin)	   1861-40-1	627, 1656	  -

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Cross-Reference
                                                       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[b]fluoranthene	  205-99-2	525.1
Benzo[?,/u]perylene	  191-24-2	525.1
Benzo[Jt]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
(8-BHC	  319-85-7	617, 1656  	525.1
Y-BHC	  58-89-9	617, 1656  	525.1
6-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
Bolstur (Sulprofos)	35400-43-2	622, 1657	  -
Bromacil 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, 630.1'	  ~
Carbaryl  	  63-25-2	632	  531.1, 553
Carbendazim	10605-21-7	631  	   -
Carbofuran	   1563-66-2	632	   531.1
    1 Carbam-S was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Carbam-S.
VI

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                                                        Cross-Reference
                                                        Applicable Method(s)
Analyte                       CAS No.            Volume I               Volume II


Carbophenothion  	  786-19-6	617,  1656   	  -
Carboxin  	5234-68-4	507, 525.1
CDN	  97-00-7	   646   	  -
Chloramben  	  133-90-4	515.1, 555
Chlordane	  57-74-9	617,  1656   	505, 508
a-Chlordane  	5103-71-9	   1656   	  505, 508, 525.1
7-Chlordane  	5103-74-2	   1656   	  505, 508, 525.1
Chlorfevinphos	  470-90-6	   1657   	  -
Chlorobenzilate  	  510-15-6	608.1,  1656   	508, 525.1
2-Chlorobiphenyl  	2051-60-7	525.1
Chloroneb  	   2675-77-6	   608.1,  1656	  508, 525.1
o-Chlorophenyl thiourea	5344-82-1	  553
Chloropicrin	  76-06-2	   618   	  -
Chloropropylate	5836-10-2	608.1,  1656   	  -
Chlorothalonil	   1897-45-6	   608.2,  1656	  508, 525.1
Chlorpropham	  101-21-3	   632   	507, 525.1
Chlorpyrifos methyl  	5598-13-0	622,  1657   	  -
Chlorpyrifos	   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   	  --
Cyanatine  	21725-46-2	629	  SOT2
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, 515.2, 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, 630.1,31659	  --
2,4-DB   	  94-82-6	615,  1658	515.1, 515.2, 555
   2 Cyanazine was not specifically listed in this method, but data from EMSL-Ci and from industry indicate that
this method is applicable.

   3 Dazomet was not explicitly listed in these methods, but these methods are applicable to dithiocarbamate
pesticides such as Dazomet.

                                                                                        vii

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Cross-Reference  (Cont.>
                                                       Applicable Method(s)
Analyte                       CAS No.            Volume I               Volume II
DBCP	  96-12-8	  1656  	  -
DCPA (Dacthal)	   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-fl-butyl phthalate	  84-74-2	506, 525.1
Di-«-octyl phthalate	  117-84-0	 506
Diallate   	2303-16-4	  1656  	  -
Diaunon  	  333-41-5	   614, 622, 1657	  507, 525.1
Dibenz[a,/z]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-DichIorobiphenyl	16605-91-7	525.1
Dichlorophen	  97-23-4	604.1  	  --
Dichlorprop Salts & Esters	120-36-5	615, 1658	515.1, 515.2, 555
Dichlorvos 	62-73-7	622, 1657	  507, 525.7
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	515.1, 515.2, 555
Dioxathion	  78-34-2	   614.1, 1657	  -
Diphenamid  	  957-51-7	  645  	507, 525.1
Diphenylamine	  122-39-4	620  	  -
Disulfoton 	  298-04-4	   614, 622, 1657	  507, 525.1
VIII

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                                                        Cross-Reference  (com.)
                                                       Applicable Method(s)
Analyte                       CAS No.            Volume I               Volume II
Disulfoton sulfone	2497-06-5	507, 525.1
Disulfoton sulfoxide  	2497-07-6	507, 525.1
Diuron  	  330-54-1	632	553
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	   72-20-5	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	 -
Ethion	  563-12-2	  614, 614.1, 1657	 -
Ethoproprophos (Ethoprop)  . . 13194-48-4	
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Cross-Reference  (com.
                                                      Applicable Method(s)
Analyte                      CAS No.            Volume I               Volume II


6-HCH	  319-86-8	   508
a-HCH  	  319-84-6	   508
Heptachlor	  76-44-8	617, 1656	505, 508, 525.1
Heptachlor epoxide	1024-57-3	617, 1656 	  505, 508, 525.1
2,2',3,3',4,4',6-Heptachloro-
    biphenyl	52663-71-5	525.1
Hexachlorobenzene	  118-74-1	  505, 508, 525.1
2,2',4,4',5,6'-HexachIoro-
    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-Hydroxycarbofuran	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 	  -
Isopropalin (Paarlan)	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 (T-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./
Metham (Vapam)	  137-42-8	  630, 630.1	~
Methamidophos	10265-92-6	  7557	 -
Methiocarb	2032-65-7	632 	531.1

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                                                       Cross-Reference  (com.)
                                                       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, 525.7
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  	  -
Nabam   	  142-59-6	  630,  630.1	  -
Nabonate	  138-93-2	   630.1	  -
Noted  	  300-76-5	
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Cross-Reference (cont.)
                                                       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	  - . .  515.1, 515.2, 525.1, 555
Permethrin5  	52645-53-1 . .  .  608.2, 1656, 1660	  508, 525.1
cis-Permethrin6 	61949-76-6	1656, 1660	  508, 525.1
trans-Permethrin6	52645-53-1	1656, 1660	  505, 525.1
Perthane	 72-56-0	617, 1656  	  --
Phenanthrene	 85-01-8	525.1
Phenothrin (Sumithrin)	26002-80-2	  1660  	  -
o-PhenylphenoI  	 90-43-7	642  	  -
Phorate	 298-02-2	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 (Promitol)	   1610-18-0	619	  507, 525.7
Prometryn  	   7287-19-6	619	  507, 525.7
Pronamide	23950-58-5	  633.1	  507, 525.7
Propachlor	   1918-16-7	   608.1, 1656	  508, 525.1
Propanil	 709-98-8	   632.1, 1656	  -
Propazine	 139-40-2	679, 1656	  507, 525.7
Propham   	 122-42-9	632  	  -
Propoxur  	 114-26-1	632  	  --
Pydrin (Fenvalerate)	51630-58-1	  1660	  -
   5 Detected as cis-Permethrin and trans-Permethrin.

   6 Regulated as Permethrin.
XII

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                                                        Cross-Reference  (com.)
                                                       Applicable Method(s)
Analyte                       CAS No.            Volume I              Volume II


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
Rubigan (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
Simazine  	  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 (Tetrachlorvinphos)  .  2224S-79-97	622, 1657	  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, 525.7
Temik (Aldicarb)	  116-06-3	-	  531.1
2,2',4,4'-Tetrachlorobiphenyl  .  . 2437-79-8	525.1
Tetrachlorvinphos (Stirofos)  .  2224S-79-97	622, 1657	  507, 525.1
TEPP  	  107-49-3	  1657  	  -
Terbacil	   5902-51-2	633, 1656	  507, 525.7
Terbufos (Counter)	13071-79-9	   614.1, 1657	  507, 525.1
Terbuthylazine (Gardoprim)  . .   5915-41-3	619, 1656	  -
Terbutryn	  886-50-0	619	  507, 525.7
Tetramethrin	7696-12-0	  1660  	  -
Thallium  	7440-28-0	200.9
Thiabendazole	  148-79-8	641  	  -
   1 CAS number in Table 7 of proposed rule is incorrect.
                                                                                       XIII

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Cross-Reference  (com.)
                                                      Applicable Method (s)
Ana/yte                      CAS No.           Volume I              Volume II


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

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                       Method 200.9
     Determination of Trace Elements by
Stabilized Temperature Graphite Furnace
        Atomic Absorption Spectrometry
                    Revision 1.2 - EPA EMSL-Ci
                                  April 1991
  John T. Creed, Theodore D. Martin, Larry B. Lobring and James W. O'Dell
                           Inorganic Chemistry Branch
                           Chemistry Research Division

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                                    Method 200.9
 Determination of Trace Elements by Stabilized Temperature  Graphite
                  Furnace Atomic Absorption Spectrometry


 1.    SCOPE AND A PPLICA TION
1.1   This method provides procedures for the determination of dissolved and total recoverable
      elements  in ground water, surface water, drinking water and wastewater. This method is also
      applicable to total recoverable elements in sediment, sludges, biological tissues, and solid
      waste samples.
1.2   Dissolved elements are determined after suitable filtration and acid preservation. Acid diges-
      tion procedures are required prior to the determination of total recoverable elements.  Appro-
      priate digestion procedures for biological tissues should be utilized prior to sample analysis.
1.3   This method is applicable to the determination of the following elements by stabilized tempera-
      ture graphite furnace atomic absorption spectrometry (STGFAA).
Element
Aluminum
Antimony
Arsenic
Beryllium
Cadmium
Chromium
Cobalt
Copper
Iron
Lead
Manganese
Nickel
Selenium
Silver
Thallium
Tin
Zinc

(Al)
(Sb)
(As)
(Be)
(Cd)
(Cr)
(Co)
(Cu)
(Fe)
(Pb)
(Mn)
(Ni)
(Se)
(Ag)
(Tl)
(Sn)
(Zn)
CAS No.
7429-90-5
7440-36-0
7440-38-2
7440-41-7
7440-43-9
7440-47-3
7440-48-4
7440-50-8
7439-89-6
7439-92-1
7439-96-5
7440-02-0
7782-49-2
7440-22-4
7440-28-0
7440-31-5
7440-66-6
      NOTE:  Method detection limit and instrumental operating conditions for the applicable
      elements are listed in Table 2.  These are intended as a guide to instrumental detection
      limits typical of a system optimized for the element employing commercial instrumen-
      tation. However, actual method detection  limits and linear working ranges will be
      dependent on the sample matrix, instrumentation and selected operating conditions.

1.4   The sensitivity and limited linear dynamic  range (LDR) of GFAA often implies the need to
      dilute a sample prior to  the analysis. The  actual magnitude of the dilution as well as the
      cleanliness of the labware used to perform the dilution can dramatically influence the quality of
      the analytical results.  Therefore, samples  types requiring large dilutions should be analyzed by

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Method 200.9
       an alternative analytical method which has a larger LDR or which is inherently less sensitive
       than GFAA.
1.5    This method should be used by analysts experienced in the use of GFAA.

2.     SUMMARY OF METHOD

2.1    This method describes the determination of applicable  elements by stabilized temperature
       platform graphite furnace atomic absorption (STPGFAA).  In STPGFAA the sample (and the
       matrix modifier,  if required) is first pipetted onto the platform or a device which provides
       delayed atomization.  The sample is then dried at a relatively low temperature  (~ 120°C) to
       avoid spattering.  Once dried,  the sample is normally pretreated  in a char or ashing step which
       is designed to minimize the  interference effects caused by the concomitant sample matrix.
       After the char step the furnace is allowed to cool prior to atomization.  The atomization cycle
       is characterized by rapid heating of the furnace to a temperature where the metal (analyte) is
       atomized from the pyrolytic graphite surface.  The resulting atomic cloud absorbs the element
       specific atomic emission produced by a hollow cathode lamp (HCL) or a electrodeless dis-
       charge lamp (EDL).   Because  the resulting absorbance usually has a nonspecific component
       associated  with the actual analyte absorbance, an instrumental background correction device is
       necessary to subtract from the total signal the component which is nonspecific  to the analyte.
       In the absence of interferences, the background corrected absorbance is directly related to the
       concentration of the analyte.  Interferences relating to STPGFAA (Sect. 4) must be recognized
       and corrected.  Instrumental drift as well as suppressions or enhancements of instrument
       response caused by the sample matrix must be corrected for by the method of standard ad-
       dition (Sect.  11.5).

3.     DEFINITIONS

3.1    Dissolved: Material that will pass through a 0.45-/xm  membrane filter assembly, prior to
       sample acidification.
3.2    Total Recoverable: The concentration of analyte determined on an unfiltered sample following
       treatment with hot dilute mineral acid.
3.3    Instrument Detection Limit (IDL):  The concentration equivalent of an analyte  signal equal to
       three times the standard deviation of the calibration blank signal  at the selected absorbance
       line.
3.4    Method Detection Limit (MDL):  The minimum concentration of an analyte that can be
       identified,  measured and reported with  99% confidence that the analyte concentration is greater
       than zero.
3.5    Linear Dynamic Range (LDR): The concentration range over which the analytical working
       curve remains linear.
3.6    Laboratory Reagent Blank (LRB):  An  aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware, equipment, and reagents that are used with sam-
       ples.  The LRB is used to determine if method analytes or other interferences are present in
       the laboratory environment, reagents or apparatus.

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                                                                                   Method 200.9
3.7   Calibration Blank: A volume of ASTM type I water acidified such that the acid(s) concentra-
       tion is identical to the acid(s) concentration associated with the calibration standards.
3.8   Stock Standard Solution:  A concentrated solution containing one analyte  prepared in the
       laboratory using a assayed reference compound or purchased from a reputable commercial
       source.
3.9   Calibration Standard (CAL): A solution prepared from the stock standard solution which is
       used to calibrate the instrument response with respect to analyte  concentration.
3.10  Laboratory Fortified Blank (LFB):  An aliquot of reagent water to which a known quantity of
       each method analyte is added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to determine whether the method is within accepted control limits.
3.11  Laboratory Fortified Sample Matrix (LFM):  An aliquot of an environmental  sample  to which
       a known quantity of each method analyte is added in the laboratory.  The LFM  is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.
3.12  Quality Control Sample (QCS):  A solution containing a known concentration of each method
       analyte derived from externally prepared test materials. The QCS  is obtained from a source
       external to the laboratory and is used to check laboratory performance.
3.13  Matrix Modifier: A substance added to the graphite furnace along with the sample in order to
       minimize the  interference effects  by selective volatilization of either analyte or matrix compo-
       nents.
3.14  Standard Addition:  The addition of a known amount of analyte to  the sample in order to
       determine the relative response of the detector to an analyte within the sample matrix. The
       relative response is then used to assess the sample analyte concentration.

4.     INTERFERENCES

4.1   Several interference sources may cause inaccuracies in the determination  of trace elements by
       GFAA.  These interferences can  be classified into three major subdivisions, namely spectral,
       non-spectral and memory.
       4.1.1   Spectral: Interferences resulting from the absorbance of light by a molecule and/or an
               atom which is not the  analyte of interest.  Spectral interferences  caused by an element
               only  occur if there is a spectral overlap between the wavelength  of the interfering
               element and the analyte  of interest.  Fortunately, this type of interference is relatively
               uncommon in STPGFAA because of the narrow  atomic  line widths associated with
               STPGFAA.  In addition, the use of appropriate furnace  temperature programs and
               high  spectral purity lamps as light sources  can minimize the possibility  of this type of
               interference.  However,  molecular absorbances can span over several hundred nano-
               meters producing broadband  spectral interferences.  This type of interference is far
               more common in STPGFAA.  The use of matrix modifiers, selective volatilization and
               background correctors are all attempts to eliminate unwanted non-specific absorbance.
               The non-specific component of the total absorbance can vary  considerably from
               sample type to sample type.  Therefore, the effectiveness of a particular background

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Method 200.9
               correction device may vary depending on the actual analyte wavelength used as well
               as the nature and magnitude of the interference.

               Spectral interferences are also caused by the emission from black body radiation
               produced during the atomization furnace cycle.  This black body emission reaches the
               photomultiplier tube producing erroneous results.  The magnitude of this  interference
               can be minimized by proper furnace tube alignment and  monochromator design. In
               addition, atomization temperatures which adequately volatilize the analyte of interest
               without producing unnecessary black body radiation can  help reduce unwanted back-
               ground emission produced during atomization.
       NOTE:  A spectral interference may be manifested by extremely high backgrounds (1.0
       abs*) which may exceed the capability of the background corrector and/or it may be
       manifested as a non-analyte element which may cause a direct spectral overlap with the
       analyte of interest.  If a spectral interference is suspected, the analyst  is advised to:

                  1.      Dilute the sample if the analyte absorbance is large enough to sacrifice
                         some of the sensitivity.  This dilution may dramatically reduce a molecular
                         background or reduce it to the point where the background correction
                         device is capable of adequately removing the remaining nonspecific com-
                         ponent.  If the non-specific component is produced by a spectral overlap
                         with an interfering element, the change in absorbance caused by dilution of
                         the sample should decrease in a linear fashion, provided the undiluted and
                         diluted sample are both within the linear range of the interfering element.

                  2.      If dilution is not acceptable because of the relatively low analyte absor-
                         bance readings or the dilution produces a linear decrease in the non-
                         specific absorbance, the analyst is advised to investigate another analyte
                         wavelength  which may eliminate the suspected spectral interference(s).

                  3.      If dilution and alternative spectral lines are not acceptable,  the analyst is
                         advised to attempt to selectively volatilize the analyte or the non-specific
                         component thereby eliminating the unwanted interference (s) by atomizing
                         the analyte in an interference-free environment.

                  4.      If none of the above advice is  applicable and the spectral interference
                         persists, an alternative analytical method which is not based on the same
                         type of physical/chemical principle may be necessary to evaluate the actual
                         analyte concentration.
    This background level  is given as a guide and is not intended to serve as an absolute value which
may be applied in all situations.

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                                                                                    Method 200.9
       4.1.2  Non-spectral:  Interferences caused by sample components which inhibit the formation
               of free atomic analyte atoms during the atomization cycle.  The use of a delayed
               atomization device which provides stabilized temperatures is required, because these
               devices provide an environment which is more conducive to the formation of free
               analyte atoms and thereby minimize this type of interference.  This type of interfer-
               ence can be detected by analyzing a sample plus a laboratory fortified sample matrix
               early within any analysis set.  From this data, immediately  calculate the percent
               recovery (Sect. 10.4.2).  If the percent recovery is outside the laboratory determined
               control limits (Sect. 10.3.3) a  potential problem  should be suspected.  If the result
               indicates a potential matrix effect, the analyst is  advised to:
               1.         Perform the method of standard addition* (see Sect. 11.5); if the "percent
                          recovery" from the method of standard addition is drastically different
                          from the percent recovery  from LFM, then lab contamination  or another
                          lab related problem should be suspected and corrected.

       NOTE: If contamination is suspected,  analyze the LFB and calculate a percent recov-
       ery.

               2.         If the two recoveries are approximately equal* and the response from the
                          standard addition is dramatically different than that which would be cal-
                          culated from the  calibration curve,  the sample should be suspected of a
                          matrix induced interference and analyzed by the method of standard ad-
                          dition (Sect. 11.5).
       4.1.3  Memory interferences resulting from analyzing a sample containing a high concentra-
               tion of an element (typically a  high  atomization temperature element) which cannot be
               removed quantitatively in one  complete set of furnace steps. The analyte which re-
               mains in the furnace can produce  false positive signals on subsequent sample(s).
               Therefore, the analyst should establish the analyte concentration which can  be injected
               into the furnace and adequately removed in one complete set of furnace cycles.  This
               concentration represents the  maximum concentration of analyte within a sample which
               will not cause a memory  interference on the subsequent sample(s).  If this con-
               centration is exceeded,  the sample should be diluted and a blank should be analyzed
               (to assure the memory affect has been eliminated) before  reanalyzing the diluted
               sample.

       NOTE: Multiple clean out furnace cycles  may be necessary in order to fully utilize the
       LDRfor certain elements.
* The limitations listed in Sect. 11.5 must be met in order to apply these recommendations.

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Method 200.9
       4.1.4   Specific Element Interferences
               Antimony:   Antimony suffers from an interference produced by K2SO4.5 In the ab-
                 sence of hydrogen in the char cycle (1300°C*), K2SO4 produces a relatively high
                 (1.2 abs) background absorbance which can produce a false signal even with Zee-
                 man background correction.  However, this background level can be dramatically
                 reduced (0.1 abs) by the use of a hydrogen/argon gas mixture in the char step.
                 This reduction in background is  strongly influenced by the temperature of the char
                 step.
               Aluminum:   The Pd may have elevated levels of Al which will cause elevated blank
                 absorbances.
               Arsenic:   The HC1 present from the digestion procedure can influence the sensitivity
                 for As.  A 1 % HC1 solution with Pd used as a modifier results in a 40% loss in
                 sensitivity relative to the analyte in a 1 % HNO3 solution.  The use of Pd/Mg/H2 as
                 a modifier reduces this suppression to about 10%.
               Cadmium:   The HC' present from the digestion procedure can influence the  sen-
                 sitivity for Cd. A  1 % HC1 solution with Pd used as a modifier results in a 70%
                 loss in sensitivity relative to the  analyte in a 1 %  HNO3 solution.  The use of
                 Pd/Mg/H2 as a modifier reduces this suppression to less than 10%.
               Copper:   Pd lines at 324.27 nm and  325.16 nm may produce an interference  on the
                 Cu line at 324.8 nm.5
               Lead:     The HC1 present from the digestion procedure can influence the sensitivity
                 for Pb.  A 1 % HC1 solution with Pd used as a modifier results in a 70% loss in
                 sensitivity relative to the analyte response in a 1 % HNO3 solution.  The use of
                 Pd/Mg/H2 as a modifier reduces this suppression to less than 10%.
               Selenium:    Iron has been shown to suppress Se response with continuum source
                 background correction.5  In addition, the use of hydrogen as a purge gas during the
                 dry and char steps can cause a suppression in Se response if not purged from the
                 furnace prior to atomization.
               Silver:      The Pd used in the modifier preparation may have elevated levels of Ag
                 which will cause elevated blank  absorbances.

5.     SAFETY
5.1    The toxicity or carcinogenicity of reagents used in this method has not been fully established.
       Each chemical should be regarded as a potential health hazard, and exposure to these com-
       pounds should be as low as  reasonably achievable. Each laboratory is responsible for main-
       taining a current awareness file of OSHA regulations regarding the safe handling of the
    *The  actual furnace temperature may vary from instrument to  instrument.   Therefore, the actual
furnace temperature should be determined on an individual bases.
8

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                                                                                  Method 200.9
       chemicals specified in this method.''2  A reference file of material data handling sheets should
       also be available to all personnel involved in the chemical analysis.
5.2    The graphite tube during atomization emits intense UV radiation.  Suitable precautions should
       be taken to protect personnel from such a hazard.
5.3    The use of argon/hydrogen gas mixture during the dry and char steps may evolve a consider-
       able amount of HC1  gas.  Therefore, adequate ventilation is required.

6.     APPARATUS AND EQUIPMENT

6.1    Graphite Furnace Atomic Absorbance Spectrophotometer
       6.1.1   The GFAA spectrometer must be capable of programmed heating of the graphite tube
               and the associated delayed atomization device.  The instrument should be equipped
               with an adequate background correction device capable of removing undesirable non-
               specific absorbance over the spectral region of interest.  The capability to record
               relatively fast (<  1 sec) transient signals and evaluate data on a peak area basis is
               preferred.  In addition, a recirculating refrigeration bath is recommended for im-
               proved  reproduc-ibility of furnace temperatures.  The data shown in the tables were
               obtained using the stabilized temperature platform and Zeeman background correction.
       6.1.2   Single element hollow cathode lamps or single element electrodeless discharge lamps
               along with the associated power supplies.
       6.1.3   Argon gas supply (high-purity grade, 99.99%).
       6.1.4   A 5% hydrogen in argon gas mix and the necessary hardware to use this gas mixture
               during specific furnace cycles.
       6.1.5   Autosampler: Although not specifically required, the use of an autosampler is highly
               recommended.
6.2    Graphite Furnace Operating Conditions: A guide to experimental conditions for the applicable
       elements are shown in Table 2.
6.3    Sample Processing Equipment
       6.3.1   Balance:  Analytical,  capable of accurately  weighing to 0.1 mg.
       6.3.2   Hot Plate: Corning PC100 or equivalent.
       6.3.3   Centrifuge:   Steel cabinet with guard bowl, electric timer and brake.
       6.3.4   Drying Oven capable of ± 3°C temperature control.
6.4    Labware:  The determination of trace level elements requires  a consideration of potential
       sources of contamination and analyte losses. Potential contamination sources include improp-
       erly cleaned laboratory apparatus and general contamination within the laboratory environment
       from dust, etc.  A clean laboratory work area designated for trace element sample handling
       must be used. Sample containers can introduce positive and  negative errors in the determina-
       tion of trace elements by contributing contaminants through surface desorption or leaching
       and/or depleting element concentrations through adsorption processes.  All reusable labware
       (glass, quartz, polyethylene, Teflon etc.), including  the sample container, should be cleaned
       prior to use. Labware should be soaked overnight and thoroughly washed with laboratory-
       grade detergent and water, rinsed with water, and soaked for four hours in a mixture of dilute

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Method 200.9
      nitric and hydrochloric acid (1 +2 + 9), followed by rinsing with ASTM type I water and oven
      drying.

      NOTE: Chromic acid must not be used for cleaning glassware.

      6.4.1  Glassware:  Volumetric flasks and graduated cylinders.
      6.4.2  Assorted calibrated pipettes.
      6.4.3  Conical Phillips beakers, 250-mL with 50-mm watch glasses. Griffin beakers, 250-
              mL with 75-mm  watch glasses.
      6.4.4  Storage bottles:  Narrow mouth bottles, Teflon FEP (fluorinated ethylene propylene)
              with Tefzel ETFE (ethylene tetrafluorethylene) screw closure, 125-mL and 250-mL
              capacities.
      6.4.5  Wash bottle:  One piece  stem, Teflon FEP bottle with Tefzel ETFE screw closure,
              125-mL capacity.

7.    REAGENTS AND CONSUMABLE MATERIALS

7.1   Reagents:  Reagents may  contain elemental impurities which might affect analytical data.
      Because of the high sensitivity of GFAA, high-purity reagents should be used whenever
      possible.  All acids used for this method must be ultra high-purity grade.  Suitable acids are
      available from a number of manufacturers or may be prepared by sub-boiling distillation.
      7.1.1  Nitric acid, concentrated (sp.gr.  1.41) (CASRN 7697-37-2).
      7.1.2  Nitric acid (1 + 1):  Add  500 mL cone, nitric acid to 400 rnL of ASTM type I water
              and dilute to 1 L.
      7.1.3  Nitric acid (1 +9):  Add  100 mL cone, to 400 mL of ASTM type I water and dilute to
              1 L.
      7.1.4  Hydrochloric acid, concentrated (sp.gr. 1.19) (CASRN 7647-01-0).
      7.1.5  Hydrochloric acid (I +4): Add 200 mL cone, hydrochloric acid to 400 mL ASTM
              type I water and  dilute to 1000 mL.
      7.1.6  Tartaric acid.  ACS reagent grade (CASRN 87-69-4).
      7.1.7  Matrix Modifier, dissolve 300 mg Palladium (Pd) powder  in concentrated HNO3 (1 ml
              of HNO3, adding 10 mL of concentrated HC1 if necessary).  Dissolve 200 mg of
              Mg(NO,)2 in ASTM type 1 water. Pour the two solution together and dilute to 100
              mL with ASTM type 1 water.

      NOTE: It is recommended that the matrix modifier be analyzed separately in order to
      assess the contribution of the modifier to the overall laboratory blank.

      7.1.8  Ammonium hydroxide, concentrated  (sp.gr. 0.902) (CAS no. 1336-21-6).
7.2   Water: For all sample preparation and dilutions, ASTM type I water (ASTM D1193) is
      required.  Suitable water  may be  prepared by passing distilled water through a  mixed bed of
      anion and cation exchange resins.

10

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                                                                                Method 200.9
7.3   Standard Stock Solution:  May be purchased from a reputable commercial source or prepared
      from ultra high-purity grade chemicals or metal (99.99-99.999% pure).  All salts should be
      dried for 1 h at 105°C, unless otherwise specified.  (CAUTION:  Many metal salts are
      extremely toxic if inhaled or swallowed.  Wash hands thoroughly after handling).  The stock
      solution should be stored in Teflon bottles. The following procedures may be used for pre-
      paring standard stock solutions:

      NOTE: Some metals, particularly those which form surface oxides, require cleaning
      prior to being weighed.  This may be achieved by pickling the surface of the metal in
      acid. An amount in excess  of the desired weight should be pickled repeatedly,  rinsed
      with water, dried and weighed until the desired weight is achieved.

      7.3.1  Aluminum solution, stock,  1 mL  = 1000 /*g Al:  Pickle aluminum metal in warm
              (1 +1) HC1 to an exact weight of 0.100 g.  Dissolve in 10 mL cone. HC1 and 2 mL
              cone, nitric acid, heating to effect solution. Continue heating until volume is reduced
              to 4 mL. Cool and add  4 mL ASTM type I water.   Heat until the volume is reduced
              to 2 mL. Cool and dilute to 100 mL with ASTM type I water.
      7.3.2  Antimony solution, stock, 1 mL = 1000 pig Sb:  Dissolve 0.100 g antimony powder
              in 2 mL (1 + 1) nitric acid and 0.5 mL cone, hydrochloric acid, heating to effect
              solution. Cool, add 20 ASTM type I  water and 0.15g tartaric acid. Warm the solu-
              tion to dissolve the white precipitate.  Cool and dilute to 100 mL with ATSM type I
              water.
      7.3.3  Arsenic solution, stock,  1 mL =  1000 ^g As:  Dissolve 0.1320 g As2O3 in a mixture
              of 50 mL ASTM type I water and 1 mL cone,  ammonium hydroxide.  Heat gently to
              dissolve. Cool and acidify the solution with 2  mL cone,  nitric acid.  Dilute to  100
              mL with ASTM type I water.
      7.3.4  Beryllium solution, stock 1 mL = 500 fig Be:  Dissolve 1.965 g BeSO4.4H2o (DO
              NOT DRY) in 50 mL ASTM Type I Water. Add 2 mL cone, nitric acid.  Dilute to
              200 mL with ASTM  type I water.
      7.3.5  Cadmium solution, stock, 1 mL = 1000 fig Cd:  Pickle Cd metal in (1+9) nitric acid
              to an exact  weight  of 0.100 g. Dissolve in 5 mL  (1 + 1) nitric acid, heating to effect
              solution. Cool and dilute to 100 mL with ASTM type I water.
      7.3.6  Chromium solution, stock, 1 mL = 1000 fig Cr: Dissolve 0.1923g CrO3 in a solu-
              tion mixture of 10  mL ASTM type I water and 1 mL cone, nitric acid. Dilute  to  100
              mL with ASTM type I water.
      7.3.7  Cobalt solution, stock 1  mL  = 1000 /ig Co: Pickle Co metal in (1+9) nitric acid to
              an exact weight of 0.100 g.  Dissolve in 5 mL (1 + 1) nitric acid, heating to effect
              solution. Cool and dilute to 100 mL with ASTM type I water.
      7.3.8  Copper solution, stock,  1 mL = 1000 /xg Cu:  Pickle Cu metal in (1 +9) nitric acid to
              an exact weight of 0.100 g.  Dissolve in 5 mL (1 +1) nitric acid, heating to effect
              solution. Cool and dilute to 100 mL with ASTM type I water.

-------
Method 200.9
       7.3.9   Iron solution, stock, 1 mL  = 1000 fig Fe: Pickle Fe metal in (1+9) hydrochloric
               acid to an exact weight of 0.100 g.  Dissolve in 10 mL (1 +1) hydrochloric acid,
               heating to effect solution.  Cool and dilute to 100 mL with ASTM type I water.
       7.3.10 Lead solution, stock, 1 mL =  lOOOAigPb: Dissolve 0.1599 g PbNO3 in 5 mL(l + l)
               nitric acid.  Dilute to 100 mL with ASTM type I water.
       7.3.11  Manganese solution, stock, 1 mL = 1000 pig Mn:  Pickle manganese flake in (1 +9)
               nitric acid to an exact weight of 0.100 g.  Dissolve in 5 mL (1 +1) nitric acid, heating
               to effect solution.  Cool and dilute to 100 mL with ASTM type I water.
       7.3.12 Nickel solution, stock,  1 mL = 1000 /xg Ni: Dissolve 0.100 g nickel power in 5 mL
               cone, nitric acid, heating to effect solution. Cool and dilute to 100 mL with ASTM
               type I water.
       7.3.13 Selenium solution, stock, 1 mL = 1000 itg Se:  Dissolve 0.1405 g SeO2 in 20 mL
               ASTM type I water. Dilute to 100 mL with ASTM type I water.
       7.3.14 Silver solution, stock, 1 mL =  1000 /*g Ag: Dissolve 0.100 g silver metal in  5 mL
               (1 + 1) nitric  acid, heating to effect solution.  Cool and dilute to 100 mL with ASTM
               type I water.  Store in amber container.
       7.3.15 Thallium solution, stock 1 mL = 500 /xg  Tl: Dissolve 0.1303 g T1NO3 in a solution
               mixture of 10 mL ASTM type I water and 2 cone, nitric acid. Dilute to 200 mL with
               ASTM type I water.
       7.3.16 Tin solution, stock, 1 mL  = 1000 ng Sn:  Dissolve 0.100 g Sn shot in 20 mL (1 + 1)
               hydrochloric acid, heating to effect solution.  Cool and dilute to  100 mL with (1 +1)
               hydrochloric acid.
       7.3.17 Zinc solution, stock, 1  mL  = 1000 ng Zn:  Pickle zinc metal in (1+9) nitric acid to
               an exact weight of 0.100 g.  Dissolve in 5 mL (1 +1) nitric acid, heating to effect
               solution. Cool and dilute to 100 mL with ASTM type I water.
7.4    Preparation of Calibration Standards:  Fresh calibration standards (CAL Solution) should be
       prepared every two weeks or as needed.  Dilute each of the stock standard solutions to  levels
       appropriate to the operating range of the instrument using the appropriate acid diluent (see
       note).  The element concentrations in each CAL solution should be sufficiently high to produce
       good measurement precision and to  accurately define the slope of the response curve.  The
       instrument calibration should be initially verified using a quality control sample (Sect. 7.6).

       NOTE:  The appropriate acid diluent for dissolved elements in water samples is 1 %
       HNO,.  For total recoverable elements in waters the appropriate acid diluent is 2%
       HNO, and I % HCI.  Finally, the appropriate add diluent for total recoverable ele-
       ments in solid samples is 2% HNO3 and 2% HCI.  The reason for these different
       diluents is to match the types of acids and the acid concentrations of the samples with
       the acid present in the standards and blanks.

7.5    Blanks:  Two types of blanks are required for this method.  A calibration blank is used to
       establish the analytical calibration curve and the laboratory reagent blank (LRB) is used to
 12

-------
                                                                                  Method 200.9
       assess possible contamination from the sample preparation procedure and to assess spectral
       background. All diluent acids should be made from concentrated acids (Sects. 7.1.1, 7.1.4)
       and ASTM type I water.
       7.5.1   Calibration blank:  Consists of the appropriate acid diluent (Sect. 7.4 note)
               (HCl/HNO3) in ASTM type I water.
       7.5.2   Laboratory reagent blank (preparation blank) must contain all the reagents in the same
               volumes as used in processing the samples. The preparation blank must be carried
               through the entire sample digestion and preparation scheme.
7.6    Quality Control Sample: Quality control samples are available from various sources.  Dilute
       (with the appropriate acid (HC1/HNO3) blank solution) an appropriate aliquot of analyte such
       that the resulting solution will result in an absorbance of approximately 0.1.
7.7    Laboratory Fortified Blank: To an aliquot of laboratory reagent blank, add an aliquot of the
       stock standard to provide a final concentration which will produce an absorbance of approxi-
       mately 0.1 for the analyte.  The fortified blank must be carried through the entire sample
       digestion and preparation scheme.

8.     SAMPLE  COLLECTION,  PRESERVATION AND STORAGE

8.1    Prior to sample collection, consideration should be given to the type of data required so that
       appropriate preservation and pretreatment steps can be taken.  Filtration,  acid preservation etc.
       should be performed at the time of sample collection or as soon thereafter as practically
       possible.
8.2    For the determination of dissolved elements, the sample should be filtered through a 0.45-/mi
       membrane filter.   Use a portion of the sample to rinse the filter assembly, discard and then
       collect the required volume of filtrate. Acidify the filtrate with (1 +1) nitric acid immediately
       following filtration to a pH of less than two.
8.3    For the determination of total recoverable elements in aqueous samples, acidify with (1 + 1)
       nitric acid  at the time of collection to  a pH of less than two.  The sample should not be filtered
       prior to analysis.

       NOTE:  Samples  that cannot be add preserved at the time of collection because of
       sampling limitations or transport restrictions, should be acidified with nitric acid to pH
       < 2 upon receipt in the laboratory (normally, 3 mL of (1 + 1) nitric acid per liter of
       sample is sufficient for most ambient and drinking water samples). Following acidifica-
       tion, the sample should be held for a minimum of 16 h before withdrawing an aliquot
      for sample processing.

8.4    Solid samples usually require preservation prior to analysis other than storage at 4°C.

9.     CALIBRATION AND  STANDARDIZATION

9.1    Calibration:  Demonstration and documentation of acceptable initial calibration is required
       before any samples are analyzed and is required periodically throughout sample analysis as
                                                                                            13

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Method 200.9
       dictated by results of continuing calibration checks.  After initial calibration is successful, a
       calibration check is required at the beginning and end of each period during which analyses are
       performed
       9.1.1   Initiate proper operating configuration of instrument and data system.  Allow a period
               of not less than 30 min for the instrument to warm up if an EDL is to be used.
       9.1.2   Instrument stability must  be demonstrated by analyzing a standard solution of a con-
               centration 20 times the IDL a minimum of five times with the resulting relative stan-
               dard deviation of absorbance signals less than 5%.
       9.1.3   Initial calibration.  The instrument must be calibrated for the analyte to be determined
               using the calibration blank (Sect. 7.5.1) and calibration standards prepared at three or
               more concentration levels within the linear dynamic range  of the analyte.
9.2    Instrument Performance:   Check the performance of the instrument and verify the calibration
       using data gathered from analyses of calibration blanks, calibration standards and the quality
       control sample.
       9.2.1   After the calibration has been established, it must be initially verified for the analyte
               by analyzing the QCS (Sect. 7.6).  If measurements exceed ±  10% of the established
               QCS value, the analysis should be terminated, the source of the problem identified
               and corrected, the instrument recalibrated, and the  new calibration must be verified
               before continuing analyses.
       9.2.2   To verify that the instrument is properly calibrated on a continuing basis, analyze the
               calibration blank and an intermediate concentration calibration standard as surrogate
               samples after every ten analyses.  The results of the analyses of the standard will
               indicate whether the calibration remains valid.  If the indicated concentration of any
               analyte deviates  from the true concentration by more than  10%, the instrument must
               be recalibrated and the response of the QCS checked as in Sect. 9.2.1. After the QCS
               sample has met specifications, the previous ten samples must be reanalyzed in groups
               of five with an intermediate concentration calibration standard analyzed after every
               fifth sample.  If the intermediate concentration calibration standard is found to deviate
               by more than 10%, the analyst is instructed to identify the source of instrumental
               drift.

       NOTE: If the sample matrix is responsible for the calibration drift and/or the sample
       matrix is affecting analyte response, it may be necessary to perform standard additions
       in order to assess an analyte concentration (Sect. 11.5).
 10.   QUALITY CONTROL (QC)
 10.1  Formal Quality Control:  The minimum requirements of this QC program consist of an initial
       demonstration of laboratory capability, and the analysis of laboratory reagent blanks and
       fortified blanks and samples as a continuing check on performance.  The laboratory is required
       to maintain performance  records that define the quality of the data thus generated.
 14

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                                                                                   Method 200.9
10.2  Initial Demonstration of Performance
       10.2.1 The initial demonstration of performance is used to characterize instrument perfor-
               mance (MDLs and linear calibration ranges) for analyses conducted by this method.
       10.2.2 Method  detection limits (MDL):  The method detection limit should be established for
               the analyte, using reagent water (blank) fortified at a concentration of two to five
               times  the estimated detection limit3.  To determine MDL values, take seven  replicate
               aliquots  of the fortified reagent water and process through the entire  analytical meth-
               od.  Perform all calculations defined in the method and report the concentration values
               in the appropriate units.  Calculate the MDL as follows:
                                       MDL = (0  x (S)
                  where:
                   t = Student's t value for a 99 %  confidence level and a
                       standard deviation estimate with  n-l degrees of freedom
                       [t  = 3.14/or seven  replicates],
                   S = standard deviation of the replicate analyses.

                 Method detection limits should be determined every six months or whenever a
                 significant change in background or instrument response is expected.
       10.2.3 Linear calibration ranges:  Linear calibration ranges are metal dependent.  The upper
               limit of the linear calibration range should be established by determining the signal
               responses from a minimum of four different concentration standards, one of which is
               close to the upper limit of the linear  range.  The linear calibration range which may
               be used for the analysis of samples should be judged by the analyst from the resulting
               data.  Linear calibration ranges should be determined every six months or whenever a
               significant change in instrument response maybe expected.
10.3  Assessing Laboratory Performance:  Reagent and Fortified Blanks
       10.3.1  Laboratory  reagent blank (LRB):  The laboratory must analyze at least one LRB
               (Sect. 7.5.2) with each set of  samples. Reagent blank data are used to assess contami-
               nation from the laboratory environment and to characterize spectral background from
               the reagents used in  sample processing. If an analyte value in the reagent blank
               exceeds its determined MDL,  then laboratory or reagent contamination should be
               suspected.   Any determined source of contamination should be corrected and the
               samples reanalyzed.
       10.3.2 Laboratory  fortified blank (LFB):  The laboratory must analyze at least one LFB
               (Sect. 7.7) with each set of samples.   Calculate accuracy as percent recovery (Sect.
               10.4.2).  If the recovery of any analyte falls outside the control limits (Sect. 10.3.3),
               that analyte is judged out of control,  and the source of the problem should be identi-
               fied  and resolved before continuing analyses.
       10.3.3 Until sufficient data (usually a minimum of 20 to 30 analyses) become available, a
               laboratory should assess  laboratory performance against recovery limits of 80-120%.
               When sufficient internal performance data become available, develop control limits
                                                                                             15

-------
Method 200.9
               from the percent mean recovery (x) and the standard deviation (S) of the mean recov-
               ery.  These data are used to establish upper and lower control limits as follows:

                            Upper Control Limit = x + 3S
                            Lower Control Limit = x  — 3S

               After each 5-10 new recovery measurements, new control limits should be calculated
               using only the most recent 20 to 30 data points.

       NOTE:  Antimony and Aluminum do manifest relatively  low percent recoveries (see
       Table I A, NBS River Sediment  1645).

10.4  Assessing Analyte Recovery: Laboratory Fortified Sample Matrix
       10.4.1  The laboratory must fortify a minimum of 10% of the samples or one fortified sample
               per set, whichever is greater.  Ideally for  solid  samples, the concentration added
               should be approximately equal to 0.1  abs  units  after the solution has been diluted.  In
               other words  if the sample (after dilution) results in an absorbance of 0.05, ideally the
               laboratory fortified sample will result in an absorbance of 0.150 (after dilution).  Over
               time, samples from all routine sample sources should be fortified.
       10.4.2  Calculate the percent recovery for the analyte, corrected for background concentra-
               tions measured in the unfortified sample, and compare these values  to the control
               limits established in Sect. 10.3.3 for the analyses  of LFBs. Fortified recovery calcu-
               lations are not required if the fortified concentration is less than  10% of the sample
               background concentration. Percent recovery may be calculated in units appropriate to
               the matrix, using the following equation:
                                      R  =     _ ^1x100
                                               S
                      where:
                       R  = percent recovery.
                      Cs  = fortified sample concentration.
                       C  = sample background concentration.
                       S  = concentration equivalent of the fortified sample.
       10.4.3 If the recovery of the analyte on the fortified sample falls outside the designated
               range, and the laboratory performance on the LFB for the analyte is shown to be in
               control (Sect. 10.3) the recovery problem encountered with the fortified sample is
               judged to be matrix related (Sect. 4), not system related. The data obtained for that
               analyte should be verified with the methods of standard  additions (Sect.  11.5).
10.5  Quality Control Samples (QCS):  Each quarter, the laboratory should analyze one or more
       QCS (if available).  If criteria provided with the QCS are not met, corrective action should be
       taken and documented.

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                                                                                  Method 200.9
11.  PROCEDURE

11.1  Sample Preparation: Dissolved Elements
      11.1.1 For the determination of dissolved elements in drinking water, wastewater, ground
              and surface waters, take a 100-mL (± 1 mL) aliquot of the filtered acid preserved
              sample, and add 1 mL of concentrated nitric acid.  The sample is now ready for
              analysis.  Allowance should be made in the calculations for the appropriate dilution
              factors.
      NOTE: If a precipitate is formed during acidification, transport or storage, the sample
      aliquot must be treated using the procedure in Sect. 11.2.1 prior to analysis.

11.2 Sample Preparation: Total Recoverable Elements.
      11.2.1 For the determination of total recoverable elements in water or waste water, take a
              100-mL (±1 mL) aliquot from a well mixed, acid preserved sample and transfer it to
              a 250-mL Griffin beaker.  Add 1 mL of concentrated HNO3 .and 0.5 mL of con-
              centrated HC1.  Heat the sample on a hot plate at 85 °C until the volume has been
              reduced to approximately 20 mL,  ensuring that the sample does not boil.  (A spare
              beaker containing 20 mL of water can be used as a gauge.)

      NOTE: For proper heating adjust the temperature control of the hotplate such that an
      uncovered beaker containing 50 mL of water located in the center of the hot plate can
      be maintained at approximately but no higher than 85 °C.  Evaporation time for 100 mL
      of sample at 85 °C is approximately 2 h with the rate of evaporation rapidly increasing
      as the sample volume approaches 20 mL.

              Cover the beaker with a watch glass and reflux for 30 min. Slight boiling may occur
              but vigorous boiling should be avoided. Allow to cool and quantitatively transfer to
              either a 50-mL volumetric or a 50-mL class A stoppered graduated cylinder.  Dilute
              to volume with ASTM type I water and mix.  Centrifuge the sample or allow to stand
              overnight to separate insoluble material.  The sample is now ready for analysis.  Prior
              to the analysis of samples the calibration standards must be analyzed and the calibra-
              tion verified using a QC sample (Sect. 9).  Once the  calibration has been verified, the
              instrument is ready for sample analysis. Because the effects of various matrices on
              the stability of diluted samples cannot be characterized, samples should be  analyzed as
              soon as possible after preparation.
      11.2.2 For the determination of total recoverable elements in solid samples (sludge, soils,
              and sediments), mix the sample thoroughly to achieve homogeneity and weigh accu-
              rately a 1.0 ± 0.01 g portion of the sample.  Transfer to a 250-mL Phillips beaker.
              Add 4 mL  (1 +1) nitric acid and  10 mL (1 +4) HC1.  Cover with a watch glass. Heat
              the sample on a hot plate and gently reflux for 30 min. Very slight boiling may
              occur, however, vigorous boiling must be avoided to prevent the loss of the HC1
              azeotrope.
                                                                                           77

-------
Method 200.9
       NOTE:  For proper heating adjust the temperature control of the hot plate such that an
       uncovered Griffin beaker containing 50 mL of water located in the center of the hot
       plate can be maintained at a temperature approximately but no higher than 85 °C.

               Allow the sample to cool and quantitatively transfer to either 100-mL (± 1 mL)
               volumetric flask or a 100-mL class A stoppered graduate cylinder. Dilute to volume
               with ASTM type I water and mix.  Centrifuge the sample or allow to stand overnight
               to separate insoluble material. The sample is now ready for analysis. Prior to  the
               analysis of samples the calibration standards must be analyzed and the calibration
               verified using a QC sample (Sect.  9).  Once the calibration has been  verified, the
               instrument is ready for sample analysis.  Because the effects of various matrices on
               the stability of diluted samples cannot be characterized,  samples should be analyzed as
               soon as  possible after preparation.

       NOTE:  Determine the percent solids in the sample for use in calculations and for
       reporting data on a dry  weight basis.

       11.2.3 Appropriate digestion procedures for biological tissues should be utilized prior to
               sample analysis.
11.3  For every new or unusual matrix, it is highly recommended that an inductively coupled plasma
       atomic emission  spectrometer be used to screen for high element concentrations.  Information
       gained from this may be used to prevent potential damage of the instrument and better estimate
       which elements may require analysis by graphite furnace.
11.4  Samples having concentrations higher than the established linear  dynamic range should be
       diluted into range and reanalyzed.  If methods of standard additions are  required,  follow the
       instructions in Sect. 11.5.
11.5  Standard Additions: If methods of standard addition are required, the following procedure is
       recommended.
       11.5.1  The standard addition technique4 involves preparing new standards in the sample
               matrix by adding known amounts of standard to one or more aliquots of the processed
               sample solution. This technique compensates for a sample constituent that  enhances
               or depresses the analyte signal thus producing a different slope from  that of the
               calibration standards.  It will not correct for additive interference which causes  a
               baseline shift.  The simplest version of this technique is the single-addition method.
               The procedure is as follows.  Two identical aliquots of the sample solution, each of
               volume  Vx, are taken.  To the first (labeled A) is added a small volume Vs, of a
               standard analyte solution of concentration Cs.  To the second (labeled B)  is added the
               same volume V,. of the solvent.  The analytical signals of A and B are measured and
               corrected for nonanalyte signals. The unknown sample  concentration cx is  calculated:
 18

-------
                                                                                   Method 200.9
               where SA and SB are the analytical signals (corrected for the blank) of solutions A and
               B, respectively.  Vs and cs should be chosen so that SA is roughly twice SB on the
               average.  It is best if Vs is made  much less than Vs, and thus cs is much greater than
               cx, to avoid excess dilution of the sample matrix. If a separation or concentration step
               is used, the additions are best made first and carried through the entire procedure.
               For the results from this technique to be valid, the following limitations must be  taken
               into consideration:
                  1.      The analytical curve must be linear.
                  2.      The chemical form of the analyte added must respond the same as the
                         analyte in the sample.
                  3.      The interference effect must be constant over the working range of con-
                         cern.
                  4.      The signal must be corrected for any additive interference.

 12.   CALCULATIONS

 12.1  Do not report element concentrations  below the determined MDL.
 12.2  For aqueous samples prepared by total recoverable procedure (Sect. 11.2.1), multiply solution
       concentrations by the appropriate dilution factor.  Round the data to the tenths place and report
       the data in /^g/L with up to three significant figures.
 12.3  For solid samples prepared by total recoverable procedure (Sect. 11.2.2)  round the solution
       concentration (/xg/L in the analysis  solution) to the tenths place and multiply by the dilution
       factor.  Data should be reported to  a tenth mg/kg up to three significant figures taking into
       account the percent solids if the data are reported on a dry weight* basis.
 12.4  If additional dilutions were  performed, the appropriate dilution factor must be applied to
       sample values.
 12.5  The QC data obtained during the analyses provide an  indication of the quality of the sample
       data and should be provided with the  sample results.

 13.   PRECISION AND ACCURACY

 13.1  Instrument operating conditions used for single laboratory testing of the method and MDLs are
       listed in Table 2.
 13.2  Data obtained from single laboratory testing of the method are summarized  in Table 1A-C for
       three solid samples consisting of SRM 1645 River Sediment, EPA Hazardous Soil and EPA
       Electroplating Sludge.  Samples were prepared using the procedure described in Sect. 11.2.2.
       For each matrix, five replicates were  analyzed and an average of the replicates used for
       determining the sample background concentration.  Two further pairs of duplicates were
       fortified at different concentration levels.  The sample background concentration, mean spike
    The dry weight should be determined on a separate sample aliquot if the sample is available.  The
dry weight can be determined by transferring a uniform 1-g aliquot to an evaporating dish and drying the
sample to a constant weight at 103-105 °C.

                                                                                             19

-------
Method 200.9
       percent recovery, the standard deviation of the average percent recovery and the relative
       percent difference between the duplicate fortified determinations are listed in Table 1A-C.  In
       addition, Table 1D-F contains a single laboratory testing of the method in aqueous media
       including drinking water, pond water and  well water. Samples were prepared using the
       procedure described in  Sect. 11.2.1.  For each aqueous matrix, five replicates were analyzed
       and an average of the replicates used for determining the sample background concentration.
       Four samples were fortified at the levels reported in Table ID-IF.  A percent relative standard
       deviation is reported  in Table ID-IF for the fortified samples.  An average percent recovery
       is also reported in Tables 1D-F.
20

-------
                                                                             Method 200.9
                                    References

1.    "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
      and Health Administration, OSHA 2206, revised January, 1976.

2.    "Proposed OSHA Safety and Health Standards, Laboratories," Occupational Safety and Health
      Administration, Federal Register, July 24,  1986.

3.    Code of Federal Regulations 40, Ch. 1, Pt. 136, Appendix B.

4.    Winefordner, J.D.,  "Trace Analysis: Spectroscopic Methods for Elements, "Chemical Analy-
      sis, Vol. 46, pp. 41-42.

5.    Waltz, B,, G. Schlemmar and J.R. Mudakavi, JAAS. 1988, 3, 695.

-------
Nj
No
Table  1A.  Precision and Recovery Data For NBS River  Sediment 1645
Certified
Value +
22600
(51)
(66)
10.2
29600
109
785
1.5
-
—
A verage
Sed. Cone.
(mg/kgj
6810
25.8
69.2
10.8
32800
132
893
0.7
1.7
439
% RSD
4.6
8.2
3.4
3.7
1.6
4.8
5.1
20.4
3.1
4.4
Average
Percent
Recovery
(20 mg/kgr S(r)
*
74.9
69.8
115.3
*
99.1
*
96.0
101.8
-
-
8.3
19.0
2.6
-
14.2
-
15.9
3.8
-
Average
Percent
Recovery
RPD (100 mg/kgr S(r)
-
9.5
12.0
4.0
-
0
-
45.2
9.7
-
*
99.0
89.2
1 10.7
#
111.5
103.2
105.4
93.5
_
.
1.5
4.3
0.7
-
3.6
26.4
4.0
1.9
_
RPD
_
2.7
7.3
1.7
-
2.6
4.7
10.7
5.6
_
            Solid Sample
            Aluminum
            Antimony
            Arsenic
            Cadmium
            Chromium
            Copper
            Manganese
            Selenium
            Silver
            Tin
             % RSD Percent relative standard deviation (n  = 5)
             S(r)     Standard deviation of average percent recovery
             RPD    Relative  percent difference between duplicate recovery determinations
             *       Fortified concentration < 10% of sample concentration
                    Not determined
             *       Values in parenthesis are noncertified
             *       Fortified concentration
                                                                                                                                     o
                                                                                                                                     Q.
                                                                                                                                     No
                                                                                                                                     O
                                                                                                                                     O
                                                                                                                                     CO

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Table 1B.  Precision and Recovery Data For EPA Hazardous Soil
Average
Average Percent
Sed. Cone. Recovery
Solid Sample (mg/kg) % RSD (20 mg/kgf
Aluminum 6410 3.3 *
Antimony 4.6 14.7 61.4
Arsenic 8.7 4.6 109.8
Cadmium 1.8 10.3 115.4
Chromium 84.0 4.2 95.5
Copper 127 4.3 108.0
Manganese 453 6.0 *
Selenium 0.6 7.5 95.0
Silver 0.9 18.5 100.1
Tin 18.4 3.7
% RSD Percent relative standard deviation (n = 5)
S(r) Standard deviation of average percent recovery
S(r)
-
2.7
2.1
0.8
33.8
15.2
-
8.4
3.8
-


Average
Percent
Recovery
RPD (100 mg/kgy S(r)
-
7.4
3.5
1.4
17.9
2.6
-
24.1
10.2
-


*
60.9
103.7
99.0
120.8
117.7
99.2
96.9
93.5
-


-
1.7
1.5
4.3
6.6
5.4
13.9
3.3
1.3
-


RPD
-
7.1
3.6
12.1
8.9
5.7
1.6
9.7
3.8
-


RPD Relative percent difference between duplicate recovery determinations
* Fortified concentration < 1 0% of sample concentration
Not determined
" Fortified concentration










                                                                                                      I
                                                                                                      KJ
                                                                                                      o
                                                                                                      p

                                                                                                      io

-------
Table  1C.  Precision and Recovery Data For EPA Electroplating Sludge 286
               Average
              Sed. Cone.
Solid Sample   (mg/kg)
Aluminum      6590
Antimony          7.7
Arsenic           33.7
Cadmium        119
Chromium      8070
Copper          887
Manganese      320
Selenium          0.8
Silver             6.5
Tin              21.8
% BSD  Percent relative standard deviation (n = 5)
S(r)     Standard deviation of average percent recovery
RPD    Relative percent difference between duplicate recovery determinations
*       Fortified concentration  <10% of sample concentration
        Not determined
"        Fortified concentration


%RSD
2.7
3.9
2.7
1.3
4.5
1.6
1.6
6.7
2.3
3.2
Average
Percent
Recovery
(20 mg/kgr
*
68.6
87.6
81.9
*
|*
*
99.4
102.8
-


S(r)
-
2.3
2.6
7.9
-
-
-
0.8
2.5
-


RPD
-
5.7
1.7
3.0
-
-
-
2.3
5.3
-
Average
Percent
Recovery
(100 mg/kgr
#
60.7
100.2
112.5
*
99.5
101.0
96.8
92.3
-


S(r)
-
3.1
1.5
3.9
-
21.9
6.4
0.7
1.9
-


RPD
-
12.8
3.1
4.7
-
6.0
4.0
1.9
5.4
-
o
a
                                                                                                                     §
                                                                                                                     to

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Method 200.9
Table 1 D. Precision and


Average
Cone.
Element (vg/U
Ag
Al
As3
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb3
Se3
Sn3
Tl
<0.5
550
3.2
0.05
<0.05
<0.7
0.75
2.98
773
751
2.11
1.24
<0.8
<0.6
<1.7
<0.7
Recovery Data for Pond Water

Fortified Cone.
% RSD (fjg/L)1
1.25
T.2
4.1 10
36.4 2.5
0.5
10
8.7 2.5
11.2 10
5.7
2.2
6.8 20
20.5 25
* 25
25
50
75.0 50
< Sample concentration less than the established method
*
1
2
3
Not determined on sample concentrations less than the
% RSD at
Fortified
Cone.2
3.7
-
0.8
14.0
4.5
2.8
1.8
2.9
-
-
1.6
1.8
0.4
1.6
3.3
5.2
detection limit.
method.
Average
Percent
Recovery
107.5
.
100.5
90.0
99.1
97.3
98.5
101.9
-
-
105.6
101.6
115.2
97.8
117.5
101.0


Fortified sample concentration based on 100 ml sample volumes.
RSD are reported on 50
Electrodeless discharge
ml sample volumes.
lamps were used.




         25

-------
Method 200.9
Table 1E.
Element
Ag
Al
As
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb3
Se3
Sn3
Tl
< Sample
Precision and Recovery Data For Drinking Water
%RSD at
Average Fortified Fortified Average Percent
Cone. (pg/L) % RSD Cone. (fjg/U1 Cone.2
<0.5
163.6
0.5
<0.02
<0.05
<0.7
<0.1
2.6
9.1
0.9
0.8
<0.7
<0.8
<0.6
<1.7
<0.7
concentration less
* Not determined on sample
1 Fortified
2 RSD are
*
2.5
10.5
#
*
*
*
7.3
17.6
1.3
32.7
#
#
*
#
#
1.25
150
10
2.5
0.5
10
2.5
10
150
2.5
20
10
15
25
50
20
than the established method detection
5.6
6.4
0.6
9.4
6.3
3.9
3.1
1.2
5.9
0.7
4.3
4.0
14.7
1.5
0.4
2.8
limit.
Recovery
94.6
111.7
88.4
106.0
105.2
88.5
105.7
111.5
107.8
96.7
103.8
101.8
101.4
88.9
100.7
95.4

concentrations less than the method.
sample concentration based on 100
mL sample volumes


reported on 50 mL sample volumes.
3 Electrodeless discharge lamps were used.
26

-------
                                                                           Method 200.9
Table 1F.  Precision and Recovery Data For Well Water

Average
Cone. (vg/U
<0.5
14.4
0.9
<0.02
1.8
4.0
<0.1
35.9
441
3580
11.8

%RSD
*
26.7
14.2
#
11.9
2.9
#
1.2
6.6
2.7
3.2

Fortified Cone.
(tig/U1
1.25 '
150
10
2.5
0.5
10
2.5
10
-
-
20
% RSD at
Fortified
Cone.2
3.6
1.5
2.1
3.4
4.6
1.0
4.0
0.6
-
-
4.0

Average Percent
Recovery
108.3
97.1
101.6
103.7
109.3
95.8
102.6
90.2
-
-
105.7
  Element
  Ag
  Al
  As3
  Be
  Cd
  Co
  Cr
  Cu
  Fe
  Mn
  Ni
  Pb                <0.7         *            25           0.7             102.2
  Sb3               <0.8         *            25           1.2             114.3
  Se3               <0.6         *            25           1.2              95.9
  Sn3               <1.7         *            50           3.0             106.1
  Tl                <0.7         *            50           1.4              98.0

<   Sample concentration less than the established method detection limit.
*   Not determined on sample concentration less than the method detection limit.
1    Fortified sample concentration based on 100 mL sample volume.
2    RSD reported on 50 mL sample volume.
3    Electrodeless discharge lamps were used.
                                                                                   27

-------
Method 200.9
Table  2.  Recommended Graphite Furnace Operating Conditions and Recommended
Matrix Modifier1^3
Element
Ag
Al
As7
Be
Cd
Co
Cr
Cu
Fe
Mn
Ni
Pb
Sb7
Se7
Sn7
Tl
Zn
Temperature
Wave-length
328.1
309.3
193.7
234.9
228.8
242.5
357.9
324.8
248.3
279.5
232.0
283.3
217.6
196.0
286.3
276.8
213.9
Slit
0.7
0.7
0.7
0.7
0.7
0.2
0.7
0.7
0.2
0.2
0.2
0.7
0.7
2.0
0.7
0.7
0.7
Char
1000
1700
1300
1200
800
1400
1650
1300
1400
1400
1400
1250
1100
1000
1400s
1000
700
(Cf Atom.
1800
2600
2200
2500
1600
2500
26006
26006
2400
2200
2500
2000
2000
2000
2300
1600
1800
MDL4 fug/L)
0.59
7.89
0.5
0.02
0.05
0.7
0.1
0.7

0.3
0.6
0.7
0.8
0.6
1.7
0.7
0.3
    Matrix Modifier = 0.015 mg Pd + 0.01 mg Mg(NO3)2.
    A 5% H2 in Ar gas mix is used during the dry and char steps at 300 mL/min for all elements.
    A cool down step between the char and atomization is recommended.
    Obtained using a 20 /A. sample size and stop flow atomization.
    Actual char and atomization temperatures may vary from instrument to instrument and are best
    determined on an individual basis. The actual drying temperature may vary depending on the
    temperature of the water used to cool the furnace.
    A 7 second atomization is necessary to quantitatively remove the analyte from the graphite
    furnace.
    An electrodeless discharge lamp was  used for this element.
    An additional low temperature (approximately 200°C) prechar is recommended.
    Pd modifier was determined to have trace level contamination of this element.
28

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                           Method  505
   Analysis of Organohalide Pesticides and
Commercial Polychlorinated Biphenyl (PCB)
      Products in Water by Microextraction
                  and Gas Chromatography
                     Revision 2.0 - EPA EMSL-Ci
                T.W. Winfield - Method 505, Revision 1.0 (1986)
                T.W. Winfield - Method 505, Revision 2.0 (1989)

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                                 Method  505
 Analysis of Organohalide Pesticides and Commercial Polychlorinated
          Biphenyl (PCB) Products in Water by Microextraction
                           and Gas Chromatography


1.    SCOPE AND APPLICA TION
1.1   This method is applicable to the determination of the following analytes in finished drinking
      water, drinking water during intermediate stages of treatment,  and the raw source water:'"3
1.2
1.3
             Analyte                         CAS No.
             Alachlor                       15972-60-8
             Aldrin                           309-00-2
             Atrazine                        1912-24-9
             Chlordane                        57-74-9
             a-Chlordane                     5103-71-9
             7-Chlordane                     5103-74-2
             Dieldrin                          60-57-1
             Endrin                            72-20-8
             Heptachlor                        76-44-8
             Heptachlor Epoxide               1024-57-3
             Hexachlorobenzene                118-74-1
             Hexachlorocyclopentadiene          77-74-4
             Lindane                          58-89-9
             Methoxychlor                     72-43-5
             cis-Nonachlor                   5103-73-1
             trans-Nonachlor                39765-80-5
             Simazine                         122-34-9
             Toxaphene                      8001-35-2
             Aroclor1016                  12674-11-2
             Aroclor1221                   11104-28-2
             Aroclor1232                  11141-16-5
             Aroclor1242                  53469-21-9
             Aroclor1248                  12672-29-6
             Aroclor1254                  11097-69-1
             Aroclor1260                  11096-82-5


For compounds other than the above mentioned analytes or for other sample sources, the
analyst must demonstrate  the applicability of the method by collecting precision and accuracy
data on fortified samples (i.e., groundwater, tap water) and provide qualitative confirmation of
results by Gas Chromatography/Mass Spectrometry (GC/MS), or by GC analysis using
dissimilar columns.4^

Method detection limits (MDL)6 for the above organohalides and Aroclors have been ex-
perimentally determined (Sect. 13.1). Actual detection limits are highly dependent upon the
characteristics of the gas chromatographic system used (e.g. column type,  age, and proper
conditioning; detector condition; and injector mode and condition).
                                                                                  31

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Method 505
1.4    This method is restricted to use by or under the supervision of analysts experienced in the use
       of GC 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 Sect.
       II.
1.5    Analytes that are not separated chromatographically, i.e., analytes which have very similar
       retention times, cannot be individually identified and measured in the same calibration mixture
       or water sample unless an alternative technique for identification and quantitation is used (Sect.
       11.4).
1.6    When this method is used to analyze unfamiliar samples for any or all of the analytes above,
       analyte identifications should be confirmed by at least one additional qualitative technique.
1.7    Degradation of Endrin, caused by active sites in the injection port and GC columns, may
       occur.  This is not as much a problem with new  capillary columns as with packed columns.
       However, high boiling sample residue in capillary columns will create the same problem after
       injection of sample extracts.

2.     SUMMARY OF METHOD

2.1    Thirty-five mL of sample are extracted with 2 mL of hexane.  Two  /iL of the extract are then
       injected into a gas chromatograph equipped with a linearized electron capture detector for
       separation and analysis.  Aqueous calibration standards are extracted and  analyzed in an
       identical manner in order to compensate for possible extraction losses.
2.2    The extraction and analysis time is 30 to 50 min per sample depending upon the analytes and
       the analytical conditions chosen.  (See Sect. 6.9.)

3.     DEFINITIONS

3.1    Laboratory duplicates (LD1 and LD2): Two sample aliquots taken  in the analytical laboratory
       and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
       of the precision associated with laboratory procedures,  but not with  sample collection,
       preservation, or storage procedures.
3.2    Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the same  throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.3    Laboratory reagent blank (LRB): An aliquot of reagent water  that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples.  The LRB is used to determine if method
       analytes or other interferences are present  in the laboratory environment, the reagents, or the
       apparatus.
3.4    Field reagent blank (FRB):  Reagent water placed in a sample  container in the laboratory and
       treated as a  sample in all respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures.  The purpose of the  FRB  is to determine if method
       analytes or other interferences are present  in the field environment.
32

-------
                                                                                      Method 505
3.5   Laboratory performance check solution (LPC):  A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with respect to a defined set of method criteria.
3.6   Laboratory fortified blank (LFB):  An aliquot of reagent water to which known quantities of
       the method analytes are added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to determine whether the methodology is in control, and whether the
       laboratory is capable of making accurate and precise  measurements at the required method
       detection limit.
3.7   Laboratory fortified sample matrix (LFM):   An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results. The background concentrations of the analytes in the sample
       matrix must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.8   Stock standard solution:  A concentrated solution containing  a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
       an assayed  reference compound. Stock standard solutions are used to prepare primary dilution
       standards.
3.9   Primary dilution standard solution:  A solution of several  analytes prepared  in the laboratory
       from stock  standard solutions and diluted as  needed to prepare calibration solutions and other
       needed analyte solutions.
3.10  Calibration standard (CAL):  a solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards  and surrogate analytes. The CAL
       solutions are used to calibrate the instrument response with respect to analyte concentration.
3.11  Quality control sample (QCS):  a sample matrix containing method analytes or a solution of
       method analytes in a water miscible solvent which is  used  to fortify reagent water or
       environmental samples.  The QCS  is obtained from a source  external to the laboratory, and is
       used to check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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 Sect. 10.2.
       4.1.1   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 wih tap and reagent water.  Drain
               dry, and heat in an oven or muffle furnace at 400°C for 1 hr.  Do not heat volumetric
               ware.  Thermally stable materials might not be eliminated by this treatment.  Thor-
               ough 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.

                                                                                             33

-------
Method 505
       4.1.2   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. WARN-
               ING:  When a solvent is purified, stabilizers put into the solvent by the manufacturer
               are removed thus potentially making the solvent hazardous.  Also, when a solvent is
               purified, preservatives put into the solvent by the manufacturer are removed thus
               potentially reducing the shelf-life.
4.2    Interfering  contamination may occur when a sample containing low concentrations of analytes
       is analyzed immediately following a sample containing relatively high concentrations of
       analytes. Between-sample rinsing of the sample syringe and associated equipment with hexane
       can minimize sample cross contamination.  After analysis of a sample containing high
       concentrations of analytes, one or more injections of hexane should be made to ensure that
       accurate values are obtained for the next sample.
4.3    Matrix interferences may be caused by contaminants that are coextracted from the sample.
       Also, note that all the analytes listed in the scope and application section are not resolved from
       each  other on any one column, i.e., one analyte of interest may be an interferent for another
       analyte of interest. The extent of matrix  interferences will vary considerably from source to
       source, depending upon the water sampled.  Cleanup of sample extracts may be necessary.
       Positive identifications should be confirmed (Sect. 11.4).
4.4    It is important that samples and working standards be contained in the same solvent. The
       solvent for working standards must be the same as the final solvent used in sample
       preparation. If this is not the case, chromatographic comparability of standards to sample may
       be affected.
4.5    Caution must be  taken in the determination of endrin since it has  been reported that the
       splitless injector  may cause endrin degradation.7 The analyst should be alerted to this possible
       interference resulting in an erratic response for endrin.
4.6    Variable amounts of pesticides and commercial PCB products from aqueous solutions adhere to
       glass surfaces. It is recommended that sample transfers and glass surface contacts be
       minimized.
4.7    Aldrin, hexachlorocyclopentadiene and methoxychlor are rapidly  oxidized by chlorine.
       Dechlorination with sodium thiosulfate at time of collection will retard further oxidation of
       these compounds.
4.8    WARNING:  An interfering, erratic peak has been observed within the retention window of
       heptachlor during many analyses of reagent, tap, and groundwater. It appears to be related to
       dibutyl phthalate; however, the specific source has not yet been definitively determined.  The
       observed magnitude and character of this peak randomly varies in numerical value from
       successive injections made from the same vial.

5.     SAFETY

5.1    The toxicity and carcinogenicity of chemicals used in this method have not been precisely
       defined; each chemical should be  treated  as a potential health hazard, and exposure to  these
       chemicals should be minimized.  Each laboratory is responsible for maintaining awareness  of
       OSHA regulations regarding safe  handling of chemicals used in this method.  Additional
       references to laboratory safety are available for  the information of the analyst.8'"

34

-------
                                                                                   Method 505
5.2   The following organohalides have been tentatively classified as known or suspected human or
      mammalian carcinogens: aldrin, commercial PCB products, chlordane, dieldrin, heptachlor,
      hexachlorobenzene, and toxaphene. Pure standard materials and stock standard solutions of
      these compounds should be handled in a hood or glovebox.
5.3   WARNING: When a solvent is purified, stabilizers put into the solvent by the manufacturer
      are removed thus potentially making the solvent hazardous.

6.    APPARATUS AND EQUIPMENT

6.1   Sample Containers: 40-mL screw cap vials (Pierce #13075 or equivalent) each equipped with
      a size 24 cap with a flat, disc-like TFE facing backed with a polyethylene film/foam  extrusion
      (Fisher #02-883-3F 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
      the vials in a 400°C oven for one hour, then remove and allow to cool in an area known to be
      free of organics.
6.2   Vials:  auto sampler, screw cap with septa, 1.8 mL, Varian #96-000099-00 or equivalent or
      any other autosampler vials not requiring more than 1.8 mL sample volumes.
6.3   Auto Sampler:  Hewlett-Packard 7671 A, or equivalent.
6.4   Micro Syringes:  10 and 100 /nL.
6.5   Micro Syringe:  25 pL with a 2-inch by 0.006-inch needle; Hamilton 702N or equivalent.
6.6   Pipettes:  2.0 and 5.0 mL transfer.
6.7   Volumetric Flasks: 10 and 100 mL, glass  stoppered.
6.8   Standard Solution Storage Containers:   15-mL bottles with PTFE-lined screw caps.
6.9   Gas Chromatograph:  Analytical system complete with temperature programmable GC suitable
      and split/splitless injector for use with capillary columns and all required accessories including
      syringes, analytical columns, gases, a linearized electron capture detector and stripchart
      recorder.  A data  system is recommended for measuring peak areas.  Table 1  lists retention
      times observed for method analytes using the columns and analytical conditions described
      below.
      6.9.1  Three gas chromatographic columns are recommended.  Column 1 (Sect.  6.9.2)
              should be used as the primary  analytical column unless routinely occurring analytes
              are not adequately resolved.  Validation data presented in this method were  obtained
              using this column. Columns 2 and 3 are recommended for use as confirmatory
              columns when GC/MS confirmation is not available.  Alternative columns may be
              used in accordance with the provisions described  in Sect. 10.3.
      6.9.2  Column  1 (Primary Column):  0.32 mm ID  x  30 M long fused silica capillary with
              chemically bonded methyl polysiloxane phase (DB-1, 1.0 jtm film, or equivalent).
              Helium carrier gas flow is about 25 cm/sec linear velocity, measured at 180° with
              9  psi column head pressure.  The  oven temperature is programmed from 180°C to
              260°C at 4°C/min and held at 260°C until all expected compounds have eluted.
              Injector temperature:  200°C.  Splitless Mode: 0.5 min. Detector temperature:
              290°C.  Sample chromatograms for selected  pesticides are presented in Figures  1
                                                                                           35

-------
Method 505
              and 2. Chromatograms of the Aroclors, toxaphene, and technical chlordane are
              presented in Figures 3 through 11.
      6.9.3  Column 2 (alternative column 1): 0.32mm ID x 30 M long fused silica capillary
              with a 1:1 mixed phase of dimethyl silicone and polyethylene glycol (Durawax-DX3,
              0.25/*m film, or equivalent). Helium carrier gas flow  is about 25 cm/sec linear
              velocity and oven temperature is programmed from 100°C to 210°C at 8°C/min, and
              held at 210°C until all expected compounds have eluted.  Then the post temperature is
              programmed to 240°C at 8°C/min for 5 min.
      6.9.4  Column 3 (alternative column 2): 0.32mm ID x 25 M long fused silica capillary
              with chemically bonded 50:50 Methyl-Phenyl silicone (OV-17, 1.5/mi film thickness,
              or equivalent).  Helium carrier gas flow is about 40 cm/sec linear velocity and oven
              temperature is programmed from  100°C to 260°C at 4°C/min and held at 260°C until
              all expected compounds have eluted.

7.    REAGENTS AND CONSUMABLE MATERIALS

      WARNING:       When a solvent is purified stabilizers put into the solvent by the
      manufacturer are removed thus potentially making the solvent hazardous. Also, when a
      solvent is purified, preservatives put into the solvent by the manufacturer are removed
      thus potentially making the shelf-life short.

7.1   Reagents
      7.1.1  Hexane extraction solvent:  UV Grade, Burdick and Jackson #216 or equivalent.
      7.1.2  Methyl alcohol:  ACS Reagent Grade, demonstrated to be free of analytes.
      7.1.3  Sodium chloride, NaCl, ACS Reagent Grade: For pretreatment before use, pulverize
              a batch of NaCl and place in a muffle furnace at room temperature. Increase  the
              temperature to 400°C and hold for 30 min. Place in a bottle and cap.
      7.1.4  Sodium thiosulfate, Na2S2O3, ACS Reagent Grade:  For preparation of solution
              (0.04 g/mL),  mix 1 g of Na2S2O3 with reagent water and bring to 25-mL  volume in a
              volumetric flask.
7.2   Reagent Water:  Reagent water is defined as water free of interference when employed in the
      procedure described herein.
      7.2.1  A Millipore Super-Q Water System or its equivalent may be used to generate
              deionized reagent water.
      7.2.2  Test reagent water each day it is used by analyzing it according to Sect.  11.
7.3   Stock Standard Solutions:  These solutions may be  obtained as  certified solutions or prepared
      from pure standard materials using the following procedures:
      7.3.1  Prepare stock standard solutions (5000 /xg/mL) by accurately weighing about 0.0500 g
              of pure material.  Dissolve  the material in methanol 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 assayed to be 96%  or greater, the weight can be used
              without correction to calculate the concentration of the stock standard.  Commercially

36

-------
                                                                                     Method 505
               prepared stock standards can be used at any concentration if they are certified by the
               manufacturer or by an independent source.
       7.3.2  Transfer the stock standard solutions into Teflon-sealed screw-cap bottles.  Store at
               4°C and protect from light.  Stock standard solutions should be checked frequently for
               signs of degradation or evaporation, especially just prior to  preparing calibration
               standards from them.
       7.3.3  Stock standard solutions must be replaced after six months,  or sooner if comparison
               with check standards indicates a problem.
7.4   Primary Dilution Standard Solutions:  Use stock standard solutions to prepare primary dilution
       standard solutions that contain the analytes in methanol.  The primary dilution standards should
       be prepared at concentrations that can be easily diluted to prepare aqueous calibration
       standards (Sect. 9.1.1) that will bracket the working concentration range.  Store the primary
       dilution standard solutions with minimal headspace and check frequently for signs of
       deterioration or evaporation, especially just before preparing calibration standards.  The
       storage time described for stock standard solutions in Sect. 7.3.3 also applies to primary
       dilution standard solutions.

8.     SAMPLE COLLECTION, PRESERVATION, AND  STORAGE

8.1    Sample Collection
       8.1.1   Collect all samples in 40-mL bottles into which  3 mg of sodium thiosulfate crystals
               have been added to the empty bottles just prior to shipping to the sampling site.
               Alternately, 75 /xL of freshly prepared sodium thiosulfate solution (0.04 g/mL) may
               be added to empty 40-mL bottles just prior to sample collection.
       8.1.2  When sampling from a water tap, open the tap and allow the system to flush until the
               water  temperature has stabilized (usually about 10 min). Adjust the flow to about 500
               mL/min and collect samples from the flowing stream.
       8.1.3  When sampling from a well, fill a wide-mouth bottle or beaker with sample,  and
               carefully fill 40-mL sample bottles.
8.2   Sample Preservation
       8.2.1   The samples must be chilled to 4°C at the time  of collection and  maintained at that
               temperature until the analyst is prepared for the  extraction process.  Field samples that
               will not be received at the laboratory on the day of collection must be  packaged for
               shipment with sufficient ice to insure that they will be maintained at 4°C until arrival
               at the  laboratory.
8.3   Sample Storage
       8.3.1   Store samples and extracts at 4°C until extraction and analysis.
       8.3.2  Extract all samples as soon as possible after collection.  Results of holding time studies
               suggest that all analytes  with the possible exception of heptachlor were adequately
               stable for 14 days when stored under these conditions.  In general, heptachlor showed
               inconsistent results. If heptachlor is to be determined, samples should be extracted
               within 7  days of collection.  Analyte stability may be affected by the matrix;
                                                                                             37

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Method SOS
               therefore, the analyst should verify that the preservation technique is applicable to the
               samples under study.

9.     CALIBRA TION AND STANDARDIZA TION

9.1    Establish GC operating parameters equivalent to those indicated in Sect. 6.9.

       WARNING:       Endrin is easily degraded in the injection port if the injection port
       or front of the column is dirty.   This is the result of buildup of high boiling residue
      from sample injection.  Check for degradation problems by injecting a mid-level
       standard containing only endrin.  Look for the degradation products ofendrin (endrin
       ketone and endrin aldehyde).  If degradation ofendrin exceeds 20%, take corrective
       action before proceeding with calibration.  Calculate percent breakdown as follows:

            Total endrin degradation peak area  (endrin aldehyde +  endrin ketone)
             Total endrin peak area (endrin  + endrin aldehyde  + endrine ketone)
9.2    At least three calibration standards are needed; five are recommended. One should contain
       analytes at a concentration near but greater than the method detection limit for each compound;
       the other two should be at concentrations that bracket the range expected in samples.  For
       example, if the MDL is 0.01 /*g/L, and a sample expected to contain approximately 0.10 ng/L
       is to be analyzed, aqueous standards should be prepared at concentrations of 0.02 /ig/L, 0.10
       Mg/L, and 0.20 /zg/L.
       9.2.1   To prepare a calibration standard (CAL), add an appropriate volume of a secondary
               dilution  standard to a 35-mL aliquot of reagent water in a 40-mL bottle. Do not add
               less than 20 fj.L of an alcoholic standard to the reagent water. Use a 25-/iL micro
               syringe and rapidly inject the alcoholic standard into the middle point of the water
               volume.  Remove the needle as  quickly as possible after injection. Mix by inverting
               and shaking the capped bottle several times.  Aqueous standards must be prepared
               fresh daily.
       9.2.2   Starting with the standard  of lowest concentration, prepare,  extract, and analyze each
               calibration standard beginning with Sect.  11.2 and tabulate peak height  or area
               response versus the concentration in the standard.  The results are to be used to
               prepare  a calibration curve for each compound by plotting the peak height  or area
               response versus the concentration. Alternatively, if the ratio of concentration to
               response (calibration factor)  is a constant over the working range (20%  RSD or less),
               linearity  to the origin can  be assumed and the average ratio or calibration factor can
               be used  in place of a calibration curve.
       9.2.3   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 an
               analyte varies  from the predicted response by more than ±20%,  the test must be
               repeated using a fresh calibration standard.  If the results  still do not agree, generate a
               new calibration curve  or use a single point calibration standard as described in Sect.
               9.2.4.

38

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                                                                                    Method 505
       9.2.4   Single point calibration is an acceptable alternative to a calibration curve.  Prepare
               single point standards from the secondary dilution standard solutions. The single
               point calibration standard should be prepared at a concentration that produces a
               response close (±20% or less) to that of the unknowns. Do not use less than 20 jtL
               of the secondary dilution standard solution to produce a single point calibration
               standard in reagent water.
9.3    Instrument Performance: Check the performance of the entire analytical system daily using
       data gathered from analyses of laboratory reagent blanks (LRB), (CAL), laboratory duplicate
       samples (LD1 and LD2), and the laboratory performance check solution (LPC) (Sect. 10.6).
       9.3.1   Significant peak tailing in excess of that shown for the target compounds in the
               method chromatograms (Figures 1-11) must be corrected.  Tailing problems are
               generally traceable to active sites on the GC column, improper column installation, or
               operation of the detector.
       9.3.2   Check the precision between replicate analyses.  Poor precision is generally traceable
               to pneumatic leaks, especially  at the injection port.  If the GC system is apparently
               performing acceptably but with decreased sensitivity, it may be necessary  to generate
               a new curve or set of calibration factors to verify the decreased responses before
               searching for the source of the problem.
       9.3.3   Observed relative  area responses of endrin (Sect. 4.5) must meet  the following general
               criteria:
               9.3.3.1   The breakdown of  endrin into its aldo and keto forms  must be  adequately
                         consistent during a period in which a series  of analyses is made.
                         Equivalent  relative amounts of breakdown should be demonstrated in the
                         LRB, LPC, LFB, CAL and QCS.  Consistent breakdown resulting in these
                         analyses would suggest that the breakdown occurred in the instrument
                         system and that the methodology is  in control.
               9.3.3.2   Analyses of laboratory fortified matrix (LFM) samples must also be
                         adequately consistent after corrections for potential background
                         concentrations are made.

10.    QUALITY CONTROL

10.1   Minimum quality control (QC)  requirements are initial demonstration of laboratory capability,
       analysis of laboratory reagent blanks (LRB), laboratory fortified blanks (LFB), laboratory
       fortified sample matrix (LFM), and quality  control samples  (QCS).
10.2   Laboratory Reagent Blanks. Before processing any samples, the analyst must demonstrate that
       all glassware and reagent interferences  are under control. Each time a set of samples is
       extracted or reagents are changed, an LRB must be analyzed. If within the retention time
       window of any analyte the  LRB produces a peak that would prevent the determination of that
       analyte, determine the  source of contamination and eliminate the interference before processing
       samples.
                                                                                            39

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Method 505
10.3  Initial Demonstration of Capability
       10.3.1  Select a representative concentration (about 10 times MDL or at the regulatory
               Maximum Contaminant Level, whichever is lower) for each analyte.  Prepare a
               primary dilution standard solution (in methanol) containing each analyte at 1000 times
               selected concentration.  With a syringe, add 35 /zL of the concentrate to each of at
               least four 35-mL aliquots of reagent water, and analyze each aliquot according to
               procedures beginning in Sect.  11.
       10.3.2  For each analyte the recovery value should for at least three out of four consecutively
               analyzed samples fall in the range of R±30% (or within R±3SR if broader) using the
               values for  R and SR for reagent water in Table 2.  For those compounds that meet the
               acceptance criteria, performance is  considered acceptable and  sample analysis may
               begin.  For those compounds that fail these criteria, initial demonstration procedures
               should be  repeated.
       10.3.3  The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it. It is expected that as laboratory personnel gain experience with this
               method the quality of data will improve beyond those required here.
10.4  The analyst is permitted to modify GC columns, GC conditions, or detectors to improve
       separations or lower analytical costs.  Each time such method modifications are made, the
       analyst must repeat the procedures in Sect. 10.3.
10.5  Assessing Laboratory Performance: Laboratory Fortified Blank (LFB)
       10.5.1  The laboratory must analyze at least one laboratory fortified blank (LFB) per sample
               set (all samples extracted within a 24-h period).  If the sample set contains more than
               20 samples, analyze one LFB for every 20 samples.  The fortifying concentration of
               each analyte in the LFB sample should be 10 times MDL or the MCL, whichever is
               less. Calculate accuracy as percent recovery (X,).  If the recovery of any analyte falls
               outside the control limits (see Sect.  10.5.2), that analyte is judged out of control, and
               the source of the problem should be identified and resolved before  continuing
               analyses.
       10.5.2  Until sufficient data become available from within their own laboratory, usually a
               minimum  of results from 20 to 30 analyses, the laboratory may assess laboratory
               performance against the control limits in Sect. 10.3.2 that are derived from the data in
               Table 2.  When sufficient internal performance data becomes  available, develop
               control limits from the mean percent recovery (X) and standard deviation (S) of the
               percent recovery.  These data are used to establish upper and  lower control limits as
               follows:

                               Upper Control  Limit   =  X + 3S
                               Lower Control Limit  =  X -  3S

               After each five to ten new recovery measurements, new control limits should be
               calculated  using only the most recent 20-30 data points.
40

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                                                                                    Method 505
       10.5.3 It is recommended that the laboratory periodically determine and document its
               detection limit capabilities for analytes of interest.

       CAUTION:        No attempts to establish low detection limits should be made before
       instrument optimization and adequate conditioning of both the column and the GC
       system.  Conditioning includes the processing ofLFB and LFM samples containing
       moderate concentration levels of these analytes.

       10.5.4 At least  each quarter the laboratory should analyze quality control samples (QCS) (if
               available).  If criteria provided with the QCS are not met, corrective action should be
               taken and documented.
10.6  Assessing Analyte Recovery: Laboratory Fortified Sample Matrix (LFM)
       10.6.1  The laboratory must add a known concentration to a minimum of 10% of the routine
               samples  or one LFM per set, whichever is greater.  The fortified concentration should
               not be less than the background concentration of the sample selected for fortification.
               Ideally the LFM concentration should be the same as that used for the LFB (Sect.
               10.5). Periodically, samples from all routine sample sources should be fortified.
       10.6.2 Calculate the percent recovery (R;) for each analyte, corrected for background
               concentrations measured  in the unfortified sample, and compare these values to the
               control limits  established in Sect.  10.5.2 from the analyses of LFBs.
       10.6.3 If the recovery of any such analyte falls outside the designated range, and the
               laboratory performance for that analyte is shown to be in control (Sect. 10.5), the
               recovery problem encountered with the dosed sample is judged to be matrix related,
               not system related.  The result for that analyte in the unfortified sample is labeled
               suspect/matrix to inform  the data user that the results are suspect due to matrix
               effects.
10.7  The laboratory may adopt additional quality control 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.  For example, field or  laboratory duplicates may be analyzed to assess
       the precision of the environmental measurements or field reagent blanks may be used to assess
       contamination of samples under site conditions, transportation and storage.

11.    PROCEDURE

11.1   Sample Preparation
       11.1.1  Remove  samples from storage and allow them to equilibrate to room temperature.
       11.1.2  Remove  the container caps.  Withdraw and discard a 5-mL volume using a  10-mL
               graduated cylinder.  Replace the container caps and weigh the containers with contents
               to the nearest  0.1 g and record these  weights for subsequent sample volume
               determinations (Sect. 11.3).
11.2  Extraction and  Analysis
       11.2.1  Remove  the container cap of each sample, and add 6 g NaCl (Sect.  7.1.3) to the
               sample bottle. Using a transfer or automatic dispensing pipet, add 2.0 mL of hexane.

                                                                                            41

-------
Method 505
               Recap and shake vigorously by hand for 1 min. Invert the bottle and allow the water
               and hexane phases to separate.
       11.2.2 Remove the cap and carefully transfer approximately 0.5 mL of hexane layer into an
               autosampler vial using a disposable glass pipet.
       11.2.3 Transfer the remaining hexane phase, being careful not to include any of the water
               phase, into a second autosampler vial. Reserve this second vial at 4°C for an
               immediate reanalysis if necessary.
       11.2.4 Transfer the first sample vial to an autosampler set up to inject 1-2 /*L portions into
               the gas chromatograph for analysis (See Sect. 6.9 for GC conditions).  Alternately,
               1-2 mL portions of samples, blanks, and standards may be manually injected,
               although an autosampler is strongly recommended.
11.3  Determination of Sample Volume in Bottles Not Calibrated
       11.3.1  Discard the remaining sample/hexane mixture from the sample bottle.  Shake off the
               remaining few drops using short, brisk wrist movements.
       11.3.2 Reweigh the empty container with original cap and calculate the net weight of sample
               by difference  to the nearest 0.1 g (Sect. 11.1.2 minus  Sect. 11.3.2). This net weight
               (in grams) is equivalent to the  volume (in mL) of water extracted (Sect. 12.3).  By
               alternately using 40-mL bottles precalibrated at 35-mL levels,  the gravimetric steps
               can be omitted, thus increasing the speed and ease of this extraction process.
11.4  Identification of Analytes
       11.4.1  Identify a sample component by comparison of its retention time to the retention time
               of a reference chromatogram.  If the retention time of an unknown compound
               corresponds, within  limits, to the retention time of a standard  compound, then
               identifiction is considered positive.
       11.4.2 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.
       11.4.3 Identification  requires expert judgement when sample components are not resolved
               chromatographically. When peaks obviously  represent more than one sample
               component (i.e.,  broadened peak with shoulder(s) or valley between two or more
               maxima), or any time doubt exists  over the identification of a  peak on a
               chromatogram, appropriate alternative techniques to help confirm peak identification
               need be employed.   For example, more positive identification may be made by the use
               of an alternative detector which operates on a chemical/physical principle different
               from that originally used, e.g., mass spectrometry, or the use of a second
               chromatography column.  Suggested alternative columns are described in Sect. 6.9.

 12.   CALCULATIONS

 12.1  Identify the organohalides in the sample chromatogram by comparing the retention time of the
       suspect peak to  retention times generated by the calibration standards and the laboratory
42

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                                                                                      Method 505
       fortified blanks.  Identify the multicomponent compounds using all peaks that are characteristic
       of the specific compound from chromatograms generated with individual standards.  Select the
       most sensitive and reproducible peaks to obtain a sum for calculation purposes (See Table 1).
12.2  Use the single point calibration (Sect. 9.2.4) or use the calibration curve or calibration factor
       (Sect. 9.2.3) to directly calculate the uncorrected concentration (Ci) of each analyte in the
       sample (e.g., calibration factor  x response).

12.3  Calculate the sample volume (Vs) as equal to the net sample weight:
                   Vs  = Gross weight (Sect.  11.1.2) - bottle tare (Sect. 11.3.2)
12.4  Calculate the corrected sample concentration as:
                                                         35(c)
                                  Concentration,  uglL =  	-
12.5  Results should be reported with an appropriate number of significant figures.  Experience
       indicates that three significant figures may be used for concentrations above 99 fig/L, two
       significant figures for concentrations between 1-99 /*g/L, and 1 significant figure for lower
       concentrations.

13.   ACCURACY AND PRECISION

13.1   Single laboratory (EMSL-Cincinnati) accuracy  and precision at several concentrations in
       reagent, ground, and tap water matrices are presented in Table 2." These results were
       obtained from data generated with a DB-1 column.
13.2  This method has been tested by 10 laboratories using reagent water and groundwater fortified
       at three concentration levels. Single operator precision, overall precision, and method accuracy
       were found to be directly related to the concentration of the analyte and virtually independent
       of the sample matrix. Linear equations  to describe the relationships are presented in Table  3.12
                                                                                             43

-------
Method 505
                                     References
\.    Glaze, W.W., Lin, C.C., Optimization of Liquid-Liquid Extraction Methods for Analysis of
      Organics in Water, EPA-600/S4-83-052, January 1984.

2.    Henderson, J.E., Peyton, G.R. and Glaze, W.H. (1976).  In "Identification and Analysis of
      Organic Pollutants in Water" (L.H. Keith ed.), pp.  105-111.  Ann Arbor Sci. Publ., Ann
      Arbor, Michigan.

3.    Richard, J.J., Junk, G.A., "Liquid Extraction for Rapid Determination of Halomethanes in
      Water," Journal AWWA, 69, 62, January 1977.

4.    "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.

5.    Budde, W.L., Eichelberger, J.W., "Organic Analyses Using Gas Chromatography-Mass
      Spectrometry," Ann Arbor Science, Ann Arbor, Michigan 1979.

6.    Glaser, J.A. et al., "Trace Analyses for Wastewaters," Environmental Science and
      Technology, 15, 1426(1981).

7.    Bellar, T.A., Stemmer, P., Lichtenberg,  J.J., "Evaluation of Capillary Systems for the
      Analysis of Environmental Extracts," EPA-600/S4-84-004, March 1984.

8.    "Carcinogens-Working with Carcinogens," Department of Health, Education, and Welfare,
      Public Health Service, Center for Disease Control,  National Institute of Occupational Safety
      and Health, Publication No. 77-206, August, 1977.

9.    "OSHA Safety and Health Standards, General Industry," (29CF/?1910), Occupational Safety
      and Health Administration, OSHA 2206, (Revised,  January 1976).

10.   "Safety in  Academic Chemistry Laboratories," American Chemical Society Publication,
      Committee on Chemical Safety, 3rd Edition, 1979.

11.   Winfield, T., et al.  "Analysis of Organohalide Pesticides and Commercial PCB  Products in
      Drinking Water by Microextraction and Gas Chromatography." In preparation.

12.   Multilaboratory Method Validation Study #40, conducted by the Quality  Assurance Branch,
      EMSL-Ci. Report in progress.
 44

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                                                                               Method SOS
Table 1.    Retention Times for Method Analytes
Analyte
Hexachlorocyclopentadiene
Simazine
Atrazine
Hexachlorobenzene
Lindane
Alachlor
Heptachlor
Aldrin
Heptachlor Epoxide
7-Chlordane
a-Chlordane
trans-Nonachlor
Dieldrin
Endrin
cis-Nonachlor
Methoxychlor
                                                    Retention Time' (min)
Primary
5.5
10.9
11.2
11.9
12.3
15.1
15.9
17.6
19.0
19.9
20.9
21.3
22.1
23.2
24.3
30.0
I Confirm 1
6.8
25.7
22.6
13.4
18.4
19.7
17.5
18.4
24.6
25.9
26.6
24.8
45.1
33.3
39.0
58.5
| Confirm 2
5.2
19.9
19.6
15.6
18.7
21.1
20.0
21.4
24.6
26.0
26.6
26.3
27.8
29.2
30.4
36.4
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Chlordane
Toxaphene
Primary*

13.6, 14.8, 15.2, 16.2, 17.7
7.7, 9.0, 15.9, 19.1, 24.7
11.2, 14.7, 13.6, 15.2, 17.7
11.2, 13.6, 14.7, 15.2, 17.7, 19.8
14.8, 16.2, 17.1, 17.7, 19.8, 22.0
19.1, 21.9, 23.4, 24.9, 26.7
23.4, 24.9, 26.7, 28.2, 29.9, 32.6
15.1, 15.9, 20.1, 20.9, 21.3
21.7, 22.5, 26.7, 27.2
    Columns and analytical conditions are described in Sect. 6.9.2, 6.9.3, and 6.9.4.
    Column and conditions described in Sect. 6.9.2.  More than one peak listed does not implicate
    the total number of peaks characteristic of the multi-component analyte. Listed peaks indicate
    only the ones chosen for summation in the quantification.
                                                                                      45

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Method 505
Table 2. Single Laboratory Ace
(MDLs) for Analytes from Reage
Concen-
MDLb tration*
Analyte (ug/L) (ug/U
Aldrin 0.075 0.15
Alachlor 0.225 0.50
Aldrm 0.007 0.05
Atrazme 2.4 5.0
20.0
a-Chlordane 0.006 0.06
0.35
7-Chlordane 0.01 2 0.06
0.35
Chlordane 0.14 0.17
3.4
Dieldrin 0.012 0.10
3.6
Endrin 0.063 0.10
3.6
Heptachlor 0.003 0.032
1.2
Heptachlor 0.004 0.04
Epoxide 1.4
Hexachloro- 0.002 0.003
benzene 0.09
Hexachlorocyclo- 0.13 0.15
pentadiene 0.35
Lindane 0.003 0.03
1.2
Methoxychlor 0.96 2.10
7.03
cis-Nonachlor 0.027 0.06
0.45
trans-Nonachlor 0.011 0.06
0.35
Simazine 6.8 25
60
Toxaphene 1.0 10
80
Aroclor1016 0.08 1.0
Aroclor1221 15.0 180
Aroclor 1232 0.48 3.9
Aroclor 1242 0.31 4.7
uracy, Precision and Method Detection Limits
nt Water, Groundwater, and Tap Water3
Accuracy and Standard Deviation Data
Reagent Water
Ff
86
102
106
85
95
95
86
95
86
NA
NA
87
114
119
99
77
80
100
115
104
103
73
73
91
111
100
98
110
82
95
86
99
65
NA
NA
NA
NA
NA
NA
SFf
9.5
13.4
20.0
16.2
5.2
3.5
17.0
0.4
18.5
8.0
3.6
17.1
9.1
29.8
6.5
10.2
7.4
15.6
6.6
13.5
4.4
5.1
11.7
6.5
5.0
21.0
10.9
15.2
21.3
9.6
21.8
8.3
3.6
12.6
15.3
6.6
8.3
13.5
6.0
Ground Water
R
100
-
86
95
86
83
94
86
95
-
-
67
94
94
100
37
71
90
103
91
101
87
69
88
109
-
-
101
93
83
94
97
59
-
-
-
-
-
-
sa
11.0
-
16.3
7.3
9.1
4.4
10.2
5.3
14.5
-
-
10.1
8.6
20.2
11.3
6.8
9.8
14.2
6.9
12.0
4.4
5.1
4.8
7.7
3.4
-
--
7.2
18.3
7.1
17.2
9.2
18.0
-
-
-
-
-
-
Tap Water
R
69
-
-
108
91
85
91
83
91
105
95
92
81
106
85
200
106
112
81
100
88
191
109
103
93
-
-
93
87
73
86
102
67
110
114
97
92
86
96
S*
9.0
-
-
10.9
3.1
7.1
2.4
14.7
6.0
12.4
9.6
15.7
14.0
14.0
12.4
22.6
16.8
7.5
5.9
15.6
15.2
18.5
14.3
8.1
18.4
-
-
14.3
5.4
4.1
5.1
13.4
6.2
9.5
13.5
7.5
9.6
7.3
7.4
 46

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                                                                              Method 505
Table  2.   Single Laboratory Accuracy, Precision and Method Detection Limits
(MDLs) for Analytes from Reagent Water, Groundwater, and Tap Water (cont.)


Analyte
Aroclor

Aroclor

Aroclor

1248

1254

1260

MDL"
(ug/L)
0.102

0.102

0.189

tration*
(ug/U
3.6
3.4
1.8
1.7
2.0
1.8
Rea,
IT
NA
-
NA
-
NA
NA
                                          Accuracy and Standard Deviation Data
Reagent
* \
NA
Water
SRd
11.5
Ground Water
R
S*
Tap Water
R
s*
                                              10.4
                                              20.7
                                                                         84
                                                                         85
                                                                         88
 9.9
11.8
19.8
    NA  =  Not applicable. A separate set of aqueous standards was not analyzed, and the
           response factor for reagent water was used to calculate a recovery for the tap water
           matrix.
    Data corrected for amount detected in blank and represent the mean of 5-8 samples.
    MDL=  method detection limit in sample in fjg/L; calculated by multiplying standard deviation
           (S) times the students' t value appropriate for a 99% confidence level and a standard
           deviation estimate with n-1 degrees of freedom.
    R   =  average  percent recovery.
    SR   =  Standard deviation about percent recovery.
    Refers to concentration levels used to generate R and SR data for the three types of water
    Matrices, not for MDL determinations.
    No analyses conducted.
                                                                                     47

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Method 505
Table 3.  Method Accuracy and Precision as Functions of Concentration
Reagent Water
Parameter
Atrazine
Simazine
Hexachlorobenzene
Lindane
Alachlor
Heptachlor
Aldrin
Heptachlor epoxide
Dieldrin
Endrin
Methoxychlor
Chlordane
Toxaphene
PCB-1016
PCS-1254
 Applicable
Cone. Range
    (pg/U
(3.06-45.90)
(12.55-50.20)
 (0.01-0.37)
 (0.04-1.39)
(0.50-37.50)
 (0.04-1.41)
 (0.04-1.42)
 (0.04-1.42)
 (0.10-7.53)
 (0.10-7.50)
(0.20-15.00)
(0.51-50.90)
(5.63-70.40)
(0.50-49.80)
(0.50-50.40)
                                      Accuracy as
                                      Recovery X
 1.122C + 0.97
0.892C + 1.446
 1.028C-0.00
 1.009C-0.00
 1.004C-0.08
 1.002C + 0.02
 1.066C + 0.00
 0.952C + 0.00
 1.027C+0.00
 0.958C + 0.01
 0.950C + 0.15
 1.037C + 0.06
 1.087C + 0.24
 0.856C + 0.31
 0.872C-0.01
Single Analyst
 Precision Sf
    (ng/U
O.OOOX + 1.21
-0.049X + 3.52
0.108X + 0.00
0.057X+0.01
0.077X+0.10
0.107X+0.01
0.031 X+0.02
0.032X+0.02
0.091 X+0.01
0.116X+0.01
0.115X+0.12
0.084X+0.06
 0.131X-0.31
0.106X+0.31
0.122X+0.11
   Overall
 Precision S
    (ug/U
0.045X + 2.23
0.209X + 1.23
0.227X+0.00
0.142X + 0.00
0.105X+0.16
0.211X+0.02
0.264X-0.00
0.129X+0.02
0.198X+0.02
0.136X+0.02
0.125X+ 0.20
0.125X+0.19
0.269X+0.69
0.147X+0.45
0.281 X+0.05
* The concentration range applicable to the multi-laboratory study from which the data was
generated.
48

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                                                                   Method 505
                                     Column:  Fused Silica Capillary
                                     Liquid Phase: DB-1
                                     Film Thickness: 1.0^m
                                     Column Dimensions: 0.32mm ID, 30 M Long
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Method 505
                                                   Column:  Fused Silica Capillary
                                                   Liquid Phase: DB-1
                                                   Film Thickness: t.O^m
                                                   Column Dimensions: 0.32mm ID,
                                                                      30 M Long
                    10       15       20      25

                                  Time (Minutes)
30
35
                                                                             52-015-17
         Figure 2. Extract of Reagent Water Spiked at 20 [ig/L with Atrazine,
                  60 |ig/Lwith Simazine, 0.45 (ig/Lwith cis-Nonachlor, and
                  0.35 ng/L with Hexachlorocyclopentadiene,  Heptachlor,
                  y-Chlordane, a-Chlordane, and trans-Nonachlor
50

-------
                                                                    Method 505
                                    Column: Fused Silica Capillary
                                    Liquid Phase: DB-1
                                    Film Thickness: 1.0(im
                                    Column Dimensions: 0.32mm ID, 30 M Long
i
4
T  I
 6  8
\    i   i    I   I    l   I    I    I   I    I   I    I   I    I   i    I    l
10 12 14   16  18  20 22  24 26  28 30  32  34  36  38 40  42 44
                           Time (Minutes)
                                                                       52-015-18
       Figure 3.  Hexane Spiked at 11.4 (ig/L with Aroclor 1016
                                                                          51

-------
Method 505
                                            Column: Fused Silica Capillary
                                            Liquid Phase: DB-1
                                            Film Thickness: 1.0|im
                                            Column Dimensions: 0.32mm ID, 30 M Long
        ~1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	I~
         4    68   10  12  14   16 18  20 22  24 26  28 30  32  34  36  38  40  42 44

                                    Time (Minutes)
               Figure 4.  Hexane Spiked at 171.4 H.Q/L with Aroclor 1221
52

-------
                                                             Method 505
                              Column: Fused Silica Capillary
                              Liquid Phase: DB-1
                              Film Thickness: LO^m
                              Column Dimensions: 0.32mm ID, 30 M Long

I
2
i
4
I i
6 8
l i i
10 12 14
i
16
i i
18 20
i i
22 24
i \
26 28
i 1
30 32
i
34
i r
36 38
i 1 I
40 42 44
                     Time (Minutes)
                                                                 52-015-20
Figure 5.  Hexane Spiked at 57.1 |j.g/L with Aroclor 1232
                                                                    53

-------
Method 505
                                            Column: Fused Silica Capillary
                                            Liquid Phase:  DB-1
                                            Film Thickness: LO^m
                                            Column Dimensions: 0.32mm ID, 30 M Long
            i—r
            6   8
n—i—i—i—i—i—i—i—i—i—i—i—i—\—i—i—i    r
10  12  14   16 18  20  22  24  26  28  30 32  34  36  38  40  42  44

                Time (Minutes)
               Figure 6. Hexane Spiked at 57.1 |j,g/L with Aroclor 1242
54

-------
                                                                    Method 505
                                    Column: Fused Silica Capillary
                                    Liquid Phase:  DB-1
                                    Film Thickness:  1.0^m
                                    Column Dimensions: 0.32mm ID, 30 M Long
I
4
\
8
i   i   rrrri   i    i   i    i   i    IIITTT
10 12 14   16  18  20  22  24 26  28 30  32 34  36 38  40  42 44
                           Time (Minutes)
                                                                       52-015-22
      Figure 7.  Hexane Spiked at 57.1 \ig/L with Aroclor 1248
                                                                          55

-------
Method 505
                                            Column: Fused Silica Capillary
                                            Liquid Phase: DB-1
                                            Film Thickness: LO^m
                                            Column Dimensions: 0.32mm ID, 30 M Long
     i    i   i   i    i   I    i   i    i   i    i   i    i   i   i    i   i    i   i    i   i    i
    2   4   6  8   10  12 14  16 18  20 22  24 26  28  30  32  34  36  38  40  42  44
                                   Time (Minutes)
              Figure 8.  Hexane Spiked at 42.9 ^L with Aroclor 1254
56

-------
                                                                       Method 505
                                        Column:  Fused Silica Capillary
                                        Liquid Phase: DB-1
                                        Film Thickness: 1.0|im
                                        Column Dimensions: 0.32mm ID, 30 M Long
Jl
      i
      4
I
8
i    r   r
10 12 14
I    I   I    I   I   i    i   i    i   i    i   i    i   i    i
16 18  20 22  24  26  28  30 32  34 36  38 40  42 44
                                Time (Minutes)
                                                                          52-015-24
            Figure 9.  Hexane Spiked at 34.3 ng/L with Aroclor 1260
                                                                              57

-------
Method 505
                                            Column: Fused Silica Capillary
                                            Liquid Phase:  DB-1
                                            Film Thickness: 1 .O^m
                                            Column Dimensions: 0.32mm ID, 30 M Long
        T
         4
T"
 8
n—i	r~
 10  12 14
16 18  20 22  24 26  28 30  32 34  36  38  40  42 44

      Time (Minutes)
                                                                               52-015-25
                Figure 10.  Hexane Spiked at 28.6 (ig/L with Chlordane
 58

-------
                                                                    Method 505
                                    Column: Fused Silica Capillary
                                    Ljquid Phase:  DB-1
                                    Film Thickness: LOfxm
                                    Column Dimensions: 0.32mm ID, 30 M Long
~
 4
T
8
iirriiiiiiiiiiiiii
10 12 14   16  18 20  22 24  26 28  30  32  34  36  38  40 42  44
                            Time (Minutes)
                                                                       52-015-26
        Figure 11.  Hexane Spiked at 57.1 |ig/L with Toxaphene
                                                                           59

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                 Method 506
  Determination of Phthalate and
Adipate Esters in Drinking Water
      by Liquid-Liquid Extraction
       or Liquid-Solid Extraction
       and Gas Chromatography
  with Photoionization Detection
                      EPA EMSL-Ci
                        July 1990
      F.K. Kawahara, J.W. Hodgeson, J.W. Eichelberger

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                                   Method 506
   Determination of Phthalate and Adipate Esters in Drinking Water
        by Liquid-Liquid Extraction or Liquid-Solid Extraction and
           Gas Chromatography with Photoionization Detection


 1.    SCOPE AND APPLICA TION

 1.1   This method describes a procedure for the determination of certain phthalate and adipate esters
      in drinking water by liquid/liquid or liquid/solid extraction. The following analytes can be
      determined by this method:

                      Parameter                       CAS No.
                      Bis (2-ethylhexyl) phthalate         117-81-7
                      Butylbenzyl phthalate               85-68-7
                      Di-n-butyl phthalate                 84-74-2
                      Diethyl phthalate                   84-66-2
                      Dimethyl phthalate                 131-11-3
                      Bis(2-ethylhexyl) adipate           103-23-1
                      Di-n-octyl phthalate                117-84-0

 1.2   This is a capillary column gas chromatographic (GC) method applicable to the determination of
      the compounds listed above in ground water and finished drinking water. 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. Method
      525 provides gas chromatograph/mass spectrometer (GC/MS) conditions appropriate for the
      qualitative and quantitative confirmation of results for all the analytes listed above, using the
      extract produced by this method.
 1.3   This method has been validated in a single laboratory and method detection limits (MDLs)
      have been determined for the analytes above (Table 2).'  Observed detection limits may vary
      among waters, depending upon the nature of interferences in the sample matrix and the
      specific instrumentation used.
 1.4   This method is restricted to use by or under the supervision of analysts experienced in  the use
      of GC and in the interpretation of gas chromatograms obtained by a computerized system.
      Each analyst must demonstrate the ability to generate acceptable results with this method using
      the procedure described in Sect. 10.

2.    SUMMARY OF METHOD

2.1   A measured volume of sample, approximately  1-L, is extracted with a ternary solvent consist-
      ing of methylene chloride, hexane and ethyl acetate using a glass separatory runnel.  The
      solvent extract is isolated, dried and concentrated to a volume of 5 mL or less.  The extract is
      further concentrated by gentle use of nitrogen gas blowing  to a volume of 1 mL or less. The
      analytes in the extract are separated by means of capillary column gas Chromatography using
      temperature programming and the phthalate and adipate esters are then measured with a
      photoionization detector.2^*  Alternatively, a measured volume of sample is extracted with a

                                                                                       63

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Method 506
       liquid-solid extraction (LSE) cartridge or disk. The LSE cartridge or disk is eluted with
       methylene chloride.  The eluant is then concentrated by use of a gentle nitrogen purge to a
       volume of 1 mL or less.

3.     DEFINITIONS

3.1    Laboratory reagent blank (LRB):  An aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples.  The LRB is used to determine if method
       analytes or other interferences are present in the laboratory environment,  the reagents, or the
       apparatus.
3.2    Field reagent blank (FRB):   Reagent water placed in a sample container in the laboratory and
       treated as a sample in all respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures. The purpose of the FRB is to determine if method
       analytes or other interferences are present in the field environment.
3.3    Laboratory fortified blank (LFB):  An aliquot of reagent water to which known quantities of
       the method analytes are added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to  determine whether the methodology is in control, and whether the labora-
       tory is capable of making accurate and precise measurements at the required method detection
       limit.
3.4    Laboratory fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory. The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample  matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the sample
       matrix must be determined  in a separate aliquot and the measured values  in the LFM corrected
       for background concentrations.
3.5    Stock standard solution:  A concentrated solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
       an assayed reference compound. Stock standard solutions are used to prepare primary dilution
       standards.
3.6    Primary dilution standard solution:  A solution of several analytes prepared in the laboratory
       from stock standard solutions and diluted as needed to prepare calibration solutions and other
       needed analyte  solutions.
3.7    Calibration standard  (CAL):  A solution prepared from the primary  dilution standard solution
       and stock standard solutions of the internal  standards and surrogate analytes. The CAL
       solutions are used to calibrate the instrument response with respect to analyte concentration.
3.8    Quality control sample (QCS): A sample matrix containing method analytes or a solution of
       method analytes in a water miscible solvent which is used to fortify reagent water or environ-
       mental samples.  The QCS is obtained from a source external to the laboratory, and is used to
       check laboratory performance with  externally prepared test materials.
 64

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                                                                                     Method 506
4.     INTERFERENCES

4.1    Method interferences may be caused by contaminants in water, solvents, reagents, glassware,
       and sampling processing hardware.  These lead to discrete artifacts and/or elevated baselines in
       gas chromatograms.  All of these materials must be routinely demonstrated to be free from
       interferences under the conditions of the analysis by running laboratory reagent blanks (Sect.
       10.2).
       4.1.1   Phthalate esters are contaminants in many  products found in the laboratory. It is
               particularly important to avoid the use of plastics because phthalates are commonly
               used as plasticizers and  are easily extracted from plastic materials.  Great care must
               be exercised to prevent  contamination.  Exhaustive clean up of reagents and glassware
               must be required to eliminate background  phthalate that is  not derived from the
               sample.
       4.1.2   Glassware must be scrupulously cleaned.  Clean all glassware as soon as possible after
               use by thoroughly rinsing with the last solvent used.  Follow by washing with hot
               water and detergent and thorough rinsing with tap and reagent water. Drain dry and
               heat in an oven or muffle furnace at 400°C for 1 hour. Do not heat volumetric
               glassware.  Thorough rinsing with acetone may be substituted for the heating.  After
               cooling, the glassware should be sealed with aluminum foil and stored in a clean envi-
               ronment to prevent accumulation of dust and other contaminants.
       4.1.3   The use of high purity reagents and solvents helps to minimize interference problems.
               Purification of solvents by distillation in an all glass system may be required.

       WARNING:      When a solvent is purified, stabilizers added by the manufacturer
       are removed thus potentially making the solvent hazardous.  Also,  when a solvent is
       purified, preservatives added by  the manufacturer are removed thus potentially reducing
       the shelf-life.

4.2    Matrix interferences may be caused  by contaminants that are co-extracted from the sample.
       The extent of matrix interferences will vary from source to source, dependent upon the nature
       and diversity of the  industrial complex or municipality being sampled.  Clean up procedures
       can be  used  to overcome many of these interferences.

5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
       ing the safe  handling of the chemicals  specified in this method.  A reference file of material
       safety data 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 (All  specifications are suggested,  catalog numbers are
       included for illustration only.)
                                                                                              65

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Method 506
6.    APPARA rus AND MA TERIALS

6.1    Sampling Equipment
      6.1.1  Grab Sample Bottle:  1-L or 1-qt amber glass, fitted with a screw cap lined with
              Teflon.  Foil may be substituted for Teflon if the sample is not corrosive. Protect
              samples from light if amber bottles are not available. The bottle and cap liner must
              be washed, rinsed with acetone or methylene chloride and dried before use in order to
              minimize contamination.  (Sect. 4.1.1.)
6.2   Glassware
      6.2.1  Separatory Funnel:  2-L with Teflon stopcock.
      6.2.2  Drying Column: Chromatographic column-300 mm long x 10 mm ID, with Teflon
              stopcock and coarse frit filter disc at bottom (Kontes K  - 420540-0213 or equivalent).
      6.2.3  Concentrator Tube:  Kuderna-Danish, 10 mL,  graduated (Kontes K-570050-1025) or
              equivalent), calibration must be checked at the volumes employed in the test. Tight
              ground glass stopper is used to prevent evaporation of extracts.
      6.2.4  Evaporative Flask:  Kuderna-Danish, 500 mL (Kontes K-57000-0500 or equivalent).
              Attach to concentrator tube with springs.
      6.2.5  Snyder Column: Kuderna-Danish, three-ball macro size (Kontes K-503000-0121 or
              equivalent).
      6.2.6  Snyder Column: Kuderna-Danish, 2 or 3 ball  micro size (Kontes 590025-0125 or
              equivalent).
      6.2.7  Vials:  10 to 15 mL, amber glass with Teflon-lined screw cap.
      6.2.8  Boiling Chips:  Approximately 10/40 mesh.  Heat to 400°C for 30 min. or extract
              with methylene chloride in a Soxhlet apparatus.
      6.2.9  Flask,  Erlenmeyer:  250 mL. (A.H. Thomas Co. #1590-033 or equivalent.)
      6.2.10 Chromatography column similar to 6.2.2.
      6.2.11 Pasteur Pipets (and Bulb): VWR Scientific 14672-200 or equivalent.
      6.2.12 Autosampler Vials:  Equipped with Teflon-lined septum and threaded or crimp top
              caps.
6.3   Water Bath:  Heated (with concentric ring  covers) capable of temperature control  (± 2°C).
      The water bath should be used in a ventilating hood.
6.4   Balance:  Analytical, capable of weighing accurately  to nearest 0.0001  gm.
6.5   Gas Chromatograph:  An analytical system complete  with temperature  programmable GC fitted
      with split-splitless injection mode system,  suitable for use with capillary columns and all
      required accessory syringes, analytical columns, gases, detector  and stripchart recorder. A
      data system for processing chromatographic data is recommended.  The gas chromatograph
      was interfaced with the  Nelson Analytical "760" System, IBM personal computer  display,
      personal computer AT,  IBM Model  M Keyboard  and Epson FX-85 select type recorder.
      6.5.1  Column, Fused Silica Capillary:  DB-5 or equivalent, 30 m long x 0.32 mm ID with
              a film  thickness of 0.25 micron.
66

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                                                                                    Method 506
       6.5.2   The alternate column, Fused Silica Capillary:  30 m long x  0.32 mm ID with a film
               thickness of 0.25 micron, DB-1 or equivalent.
       6.5.3   Detector:  A high temperature photoio.nization detector equipped for 10.0 electron
               volts and capable of operating from 250°C to 350°C is required. (Tracor Instru-
               ments, Inc., Model 703 or equivalent.)
       6.5.4   An automatic injector system is suggested, but was not used  for the development of
               this method.
6.6   Vacuum pump, 110 VAC, capable of maintaining a vacuum of 8-10 mm Hg.

7.     REAGENTS AND CONSUMABLE MATERIALS

7.1    Reagent Water: Reagent water is defined as water in which an  interfering substance is not
       observed at the MDL of the parameters of interest.  Reagent water used to generate data in
       this method was distilled water obtained from the Millipore L/A-7044 system comprised of
       prefiltration, organic adsorption, deionization and Millipore filtration  columnar units.
7.2   Acetone, hexane, methylene chloride, ethyl acetate, ethyl ether and iso-octane:  Pesticide
       quality or equivalent to distillation in glass quality.
7.3   Sodium Sulfate:  (ACS) Granular, anhydrous.  Several levels of purification may be required
       in order to reduce background phthalate levels towards acceptance: 1) Heat 4 h at 400°C  in a
       shallow tray, 2) Soxhlet extract with methylene chloride for 48 h.
7.4   Florisil:  PR grade (60/100 mesh).  To prepare for use, place 100 g of Florisil into a 500-mL
       beaker  and heat for approximately 16 h at 40°C.  After heating  transfer to a 500-mL reagent
       bottle.  Tightly seal and cool to room temperature.  When cool, add 3 mL of reagent water.
       Mix thoroughly by shaking or rolling for 10 min. and  let it stand for  at  least 2 h.  Store in the
       dark in glass containers with ground glass stoppers or  foil-lined screw caps.
7.5   Sodium Chloride:   (ACS) Granular.  Heat 4 h  at 400°C in a shallow  tray.  When cool, keep in
       tightly sealed bottle.
7.6   Ethyl Ether:  (ACS) reagent grade.
7.7   Sodium Thiosulfate (Na2S2O3): (ACS) reagent grade.
7.8   Alumina: Neutral activity  Super I,  W200 series (ICN  Life Sciences Group,  No. 404583): To
       prepare for use, place 100  g of alumina into a  500-mL beaker and heat for approximately  16 h
       at 400°C. After heating transfer to a 500-mL  reagent  bottle.  Tightly seal and cool to room
       temperature. When cool, add 3 mL of reagent water.   Mix thoroughly by shaking or rolling
       for 10 min.  and let it stand for at least 2 h.  Keep the bottle sealed tightly.
7.9   Liquid-solid extraction (LSE) cartridges:  Cartridges are inert non-leaching plastic, for exam-
       ple polypropylene, or glass, and must not contain plasticizers, such as phthalate esters or
       adipates,  that leach into methylene chloride. The cartridges are packed with about 1 gram of
       silica, or other inert inorganic support, whose  surface  is modified by  chemically bonded
       octadecyl (C,8) groups.  The packing must have a narrow size distribution and must not leach
       organic compounds into methylene chloride. One liter of water should pass through the
       cartridge  in about  2 hrs with  the assistance of a slight vacuum of about 13 cm (5 in.) of
       mercury.  The extraction time should not vary unreasonably among LSE cartridges.
                                                                                            67

-------
Method 506
7.10  Liquid-solid extraction disks, C-18, 47 mm:  Disks are manufactured with Teflon and should
       contain very little contamination.
7.11  Helium carrier gas, as contaminant free as possible.
7.12  Stock standard solutions (1.00 ng/fiL):  Stock standard solutions can be prepared from pure
       standard materials or purchased as certified solutions.
       7.12.1  Prepare stock standard solutions by accurately weighing about 0.0100 g of pure
               material. Dissolve the material in isooctane and dilute to volume in a 10-mL volumet-
               ric flask. Larger  volumes can be used at the convenience of the analyst.   When
               compound purity is assayed to be 96% or greater, the weight can be used without
               correction to calculate the concentration of the stock standard. Commercially pre-
               pared stock standards can be used at any concentration if they are certified by the
               manufacturer or by an independent source.
       7.12.2 Transfer the stock standard solutions into Teflon-sealed screw-cap bottles. Store at
               4°C and protect from light.  Stock standard solutions should be checked frequently for
               signs of degradation or evaporation, especially just prior to preparing  calibration
               standards from them.
       7.12.3 Stock standard solutions must be  replaced after six months, or sooner if comparison
               with check standards indicates a problem.  Butylbenzyl phthalate is especially vul-
               nerable to autoxidation.
7.2   Laboratory control sample concentrate:  See Sect. 10.3.1.

8.     SAMPLE COLLECTION,  PRESERVATION.  AND STORAGE

8.1    Grab samples must be collected in glass containers.  Conventional sampling practices should
       be followed; however, the bottle must not be prerinsed with sample before collection.8"9
8.2   Sample Preservation and Storage
       8.2.1   For sample dechlorination, add 60 mg sodium thiosulfate to the sample bottle at the
               sampling site or in the laboratory before shipping to the sampling site.
       8.2.2  After the sample is collected in a bottle containing preservative(s), seal the bottle and
               shake vigorously for 1 min.
       8.2.3  The samples must be iced or refrigerated at 4°C free from light from the time of
               collection until  extraction. Limited holding studies have indicated that the analytes
               thus stored are stable up to 14 days or longer.  Analyte stability may be affected by
               the matrix; therefore, the analyst should verify that the preservation technique is ap-
               plicable to the particular samples under study.
8.3   Extract Storage: Extracts  should be stored at 4°C in absence of light.  A  14-day maximum
       extract storage time is recommended. The analyst should verify appropriate extract holding
       times applicable to the samples under study.
68

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                                                                                    Method 506
9.     CALIBRATION

9.1    Establish gas chromatograph operating conditions equivalent to those given in Table 1.  The
       gas chromatographic system can be calibrated using the external standard technique (Sect.
       9.2).
       9.1.1   Performance of the detector should be checked daily by a specified procedure given in
               the gas chromatograph operator's manual.  If the response is weak, the ultraviolet
               lamp is removed carefully following disconnection of the power supply. It is cleaned
               and then placed into its original position with the aid of a leak detector.
9.2    External standard calibration procedure:
       9.2.1   Prepare calibration standards at a minimum of three concentration levels for  each
               analyte of interest by adding volumes of one or more stock standards to a volumetric
               flask and diluting to volume with n-hexane.  One of the external standards should be
               at a concentration near, but above, the MDL (Table 2) and the other concentrations
               should correspond to the expected range of concentrations found in real samples or
               should define the working range of the detector.
       9.2.2   Using injection of 1 to 2 /xL, analyze each calibration standard according to  Sect.  11.5
               and tabulate peak height or area responses against the mass injected.  The  results can
               be used to prepare a calibration curve for each compound.  Alternatively, if  the ratio
               of response to amount injected (calibration factor) is a constant over the working
               range (< 10% relative standard deviation, RSD), linearity through the origin can be
               assumed and the average ratio or calibration factor can be used in place of a cali-
               bration curve.

10.   QUALITY CONTROL

10.1   Minimum quality control (QC) requirements are initial  demonstration of laboratory capability,
       analysis of laboratory  reagent blanks, laboratory fortified samples, laboratory fortified blanks,
       and QC samples.  Additional quality control practices are recommended.
10.2  Laboratory reagent blanks:  Before processing any samples, the analyst must demonstrate that
       all glassware and reagent interferences are under control.  Each time a set of samples is
       extracted or reagents are changed, a LRB must be analyzed.  If within the retention time
       window of any analyte of interest the LRB produces a peak that would prevent the  determina-
       tion of that analyte using a known standard, determine the source  of contamination and  elimi-
       nate the interference before processing samples.
10.3  Initial Demonstration of Capability.
       10.3.1 Select a representative spike concentration, about 10  times EDL or at the regulatory
               Maximum Contaminant Level (MCL), (whichever is lower) for each analyte. Prepare
               a laboratory control sample concentrate (in methanol) containing each analyte at 1000
               times selected concentration. With a syringe, add 1 mL of the concentrate to each of
               seven 1-L aliquots of reagent water, and analyze each aliquot according to procedures
               in Sect. 11.1  or 11.2, and 11.3 and  11.4.
       10.3.2 For each analyte, the mean recovery value should fall in  the range of R ± 30% (or
               within R  + 3Sr if broader) using the values for R and Sr  for reagent water in Table 3

                                                                                             69

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Method 506
               or Table 4.  For those compounds that meet the acceptance criteria, performance is
               considered acceptable and sample analysis may begin.  For those compounds that fail
               these criteria, initial demonstration procedures should be repeated.
       10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it. It is expected that, as laboratory personnel gain experience with
               this method, the quality of data will  improve beyond those required here.
10.4  The analyst is permitted to modify GC columns, GC detectors, GC conditions, concentration
       techniques, internal standards or surrogate compounds.  Each time such method modifications
       are made, the analyst must repeat the procedures  in Sect.  10.3.
10.5  Assessing laboratory performance:  Laboratory Fortified Blank
       10.5.1 The laboratory must analyze at least one laboratory fortified  blank (LFB) sample per
               sample set (all samples extracted within  a 24-hr period).  The spiking concentration
               of each  analyte in  the LFB should be 10 times MDL or the MCL, whichever is less.
               Calculate accuracy as percent recovery, R.  If the recovery  of any analyte falls out-
               side the control limits (see Sect. 10.5.2), that analyte is judged out of control, and the
               source  of the problem should be identified and resolved before continuing analyses.
       10.5.2 Until sufficient internal data become available, usually a minimum of results from 20
               to 30 analyses,  the laboratory should assess laboratory performance against the control
               limits in Sect.  10.3.2 that are derived from the control limits developed during the
               initial demonstration  of capability (10.3).  When sufficient internal performance data
               becomes available, develop control limits from the mean percent recovery, R, and
               standard deviation, Sr, of the percent recovery.  These data are used to establish
               upper and lower control limits as follows:

                         Upper Control Limit =  R + 3Sr
                         Lower Control  Limit =  R — 3Sr

               After each five to ten new recovery  measurements, new control limits should be
               calculated using only the most recent 20-30 data points.
10.6  Assessing Analyte Recovery:  Laboratory Fortified Sample Matrix
       10.6.1 The laboratory must  fortify each analyte to a minimum of 10% of the routine samples
               or one fortified sample per set,  whichever is greater.  The fortified concentration
               should not  be less than the background concentration of the sample selected for
               fortifying.  Ideally, this concentration should be the same as that used for the labo-
               ratory fortified blank  (Sect.  10.5).  Over time, samples from all  routine sample
               sources should be fortified.
       10.6.2 Calculate the accuracy as percent recovery,  R,  for each analyte, corrected for back-
               ground  concentrations measured in the unfortified sample, and compare these values
               to the control limits established in Sect. 10.5.2 from the analyses of LFBs.
       10.6.3 If the recovery  of any such analyte falls outside the designated range, and the labora-
               tory performance for that analyte is  shown to be in control (Sect. 10.5), the recovery
               problem encountered with the dosed sample is judged to be matrix related, not system
 70

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                                                                                    Method 506
               related.  The result for that analyte in the unspiked sample is labeled suspect/matrix to
               inform the data user that the results are suspect due to matrix  effects.
10.7 Quality Control Samples (QCS):  Each quarter, the laboratory should analyze one or more
      QCS (if available). If criteria provided with the QCS are not met, corrective action should be
      taken and documented.
10.8 The laboratory may adopt additional quality control 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.   For example, field or laboratory duplicates may be analyzed to assess
      the precision of the environmental measurements.

11.  PROCEDURE

11.1  Liquid-Liquid Extraction
      11.1.1  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 containing 50 g
               of NaCl.
      11.1.2  Add a mixture of 40 ml CH2Cl2  5 mL hexane, and 5 ml ethyl acetate to the sample
               bottle. Seal, and shake gently 5 seconds to rinse the inner walls of the bottle. Trans-
               fer the solvent  to the separatory funnel.  Extract the sample by shaking the funnel for
               2 min with initial and periodic venting to release  excess pressure. Allow the organic
               layer to separate for a minimum of 10 min from the water phase. 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.  Collect
               the solvent extract in a 250-mL Erlenmeyer flask.
      11.1.3  Add a  second 50-mL volume of above mixture to the sample bottle  and repeat the
               extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
               Perform  a third extraction in the same manner. Then extract with 40-mL of hexane,
               which extract (top phase) is added to the total.
      11.1.4  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 concentrator, provided the concentration factor attained in
               Sect. 11.1.6-11.1.8 is achieved without loss of analytes.
      11.1.5  Pour the combined extract through a drying column (Sect. 6.2.2) containing about  10
               cm of prerinsed anhydrous sodium sulfate, and collect the extract in the K-D concen-
              trator.  Rinse the  Erlenmeyer flask and column with 20 to 30 mL of methylene chlo-
               ride to complete the quantitative transfer.
      11.1.6  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 I mL of methylene
              chloride to the  top.  Place the K-D apparatus on a hot water bath (60 to 65°C) so that
              the concentrator tube is partially immersed in the hot water, and the entire lower
               rounded surface of the flask is bathed with hot vapor.  Adjust the vertical position of
              the apparatus and the  water temperature as required to  complete the concentration in

                                                                                            77

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Method 506
               40 min.  At the proper rate of distillation the balls of the column will actively chatter
               but the chambers will not flood with condensed solvent.  When the apparent volume
               of liquid  reaches approximately 7 mL, remove the K-D apparatus and allow it to drain
               and cool  for at least 10 min.
       11.1.7 Increase the temperature of the hot water bath to about 85°C.  Remove the Snyder
               column, rinse the column and the 500-mL evaporative flask with 1-2 mL of methy-
               lene chloride.  Replace with a micro column and evaporative flask. Concentrate the
               extract as in Sect. 11.1.6 to 0.5-1 mL.  The elapsed time of concentration should be
               approximately 15 min.
       11.1.8 Remove the micro Snyder column and rinse the column by flushing with hexane using
               a 5-mL syringe.  Concentrate to a volume of 1 ml by purging the liquid surface with
               a gentle flow  of nitrogen.  Transfer the extract to an autosampler vial with a Pasteur
               pipet.  Seal the vial with a threaded or crimp top cap.  Store in refrigerator if further
               processing will not be performed.   If the sample extract requires no further cleanup,
               proceed with gas chromatographic analysis (Sect.  11.5).  If the sample requires
               further cleanup, proceed to Sect.  11.4.
       11.1.9 Determine the original sample volume by refilling the sample bottle to the mark and
               transferring the liquid to a 1000-mL graduated cylinder.  Record the sample volume
               to the nearest 5 mL.
11.2  Liquid-Solid Extraction
       11.2.1 This method is applicable to a wide range of organic compounds that are efficiently
               partitioned from the water sample onto a C)8 organic phase chemically bonded to a
               solid inorganic matrix, and are sufficiently volatile and thermally stable for gas chro-
               matography.10 Particulate bound organic matter will not be partitioned, and more than
               trace levels of particulates in the water may disrupt the partitioning process.  Single
               laboratory accuracy and precision data have been determined at a single concentration
               for the analytes listed in 1.1 fortified into reagent water and raw source water.
       11.2.2 Set up the extraction  apparatus shown in Figure 1A.  The reservoir is not required,
               but recommended for convenient operation. Water drains from the reservoir through
               the LSE cartridge and into a syringe needle which is inserted through a rubber stopper
               into the suction flask. A  slight vacuum of 13  cm (5 in.) of mercury  is used during all
               operations with the apparatus.  With this extraction apparatus,  sample elution requires
               approximately 2 hours. Acceptable new cartridge and extraction disk technology have
               recently become  available, which allow significantly faster elution rates.
       11.2.3 Mark the water meniscus on the side of the sample bottle  (approximately 1 liter) for
               later determination of sample volume. Pour the water sample  into the 2-L separatory
               funnel with the stopcock closed.
       11.2.4 Flush each cartridge with two 10 mL aliquots  of methylene chloride, followed by two
               10 mL aliquots of methanol, letting the cartridge drain dry after each flush.  These
               solvent flushes may be accomplished by  adding the solvents directly to the solvent
               reservoir in Figure 1 A.  Add  10-mL of reagent water to the solvent reservoir, but
               before the reagent water level drops below the top edge of the packing in the LSE
               cartridge, open the stopcock of the separatory funnel and begin adding sample water
72

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                                                                                   Method 506
              to the solvent reservoir.  Close the stopcock when an adequate amount of sample is in
              the reservoir.
       11.2.5 Periodically open the stopcock and drain a portion of the sample water into the solvent
              reservoir.  The water sample will drain into the cartridge, and from the exit into the
              suction flask. Maintain the packing material  in the cartridge immersed in water at all
              times.   After all of the sample has passed through the LSE cartridge, wash the separa-
              tory funnel and cartridge with  10 mL of reagent water, and draw air through the
              cartridge for 10 min.
       11.2.6 Transfer the  125-mL solvent reservoir and LSE cartridge (from Figure 1A) to the
              elution  apparatus (Figure IB).  The same  125 mL solvent reservoir is  used for both
              apparatus.  Wash the 2-liter separatory funnel with 5 mL of methylene chloride and
              collect the washings. Close the stopcock on the 100-mL separatory  funnel of the
              elution  apparatus, add the washings to the reservoir and enough additional methylene
              chloride to bring the volume back up to 5 mL and elute the LSE cartridge.  Elute the
              LSE cartridge with an additional 5 mL of methylene chloride (10-mL total).  A small
              amount of nitrogen positive pressure may be used to elute the cartridge. Small
              amounts of residual water from the LSE cartridge will form an immiscible layer with
              the methylene chloride in the 100-mL  separatory  funnel.  Open the stopcock and
              allow the methylene  chloride to pass through the drying column packed with anhy-
              drous sodium sulfate (1-in) and into the collection vial.  Do not allow  the water layer
              to enter the drying column.  Remove the 100 mL separatory funnel and wash the
              drying column with 2 mL of methylene chloride.  Add this to the extract.  Concen-
              trate the extract to 1  mL under a gentle stream  of nitrogen. The extract is now  ready
              for gas  chromatography (Sect.  11.4) or additional cleanup (Sect.  11.3).
11.3  Sample Extraction
       11.3.1 Preparation of disks.
              11.3.1.1  Insert the disk into the 47 mm filter apparatus. Wash the disk with 5 mL
                         methylene chloride (MeQ2) by adding the MeCl2 to the disk, pulling about
                         half through the disk and allowing it to soak the disk for about a minute,
                         then pulling the remaining MeCl2 through the disk. With the vacuum on,
                        pull air through the disk for a minute.
              11.3.1.2  Pre-wet the disk with 5 mL methanol (MeOH) by adding the MeOH  to the
                        disk, pulling about half through the disk and allowing it to  soak for about a
                         minute, then pulling most of the remaining MeOH through. A layer of
                         MeOH must be left on the  surface of the disk, which shouldn't be allowed
                         to go dry from this point until  the end of the sample extraction. THIS IS
                         A CRITICAL STEP FOR A UNIFORM FLOW AND GOOD RECOV-
                         ERY.
              11.3.1.3  Rinse the disk with 5  mL reagent water by adding the water to the disk
                         and pulling most through, again leaving a layer on the surface of the disk.
       11.3.2 Add 5  mL MeOH per liter of water sample.  Mix well.
       11.3.3 Add the water sample to the  reservoir and turn  on the vacuum to begin the filtration.
              Full aspirator vacuum may be used. Particulate-free water may filter in as little as 10

                                                                                            73

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Method 506
               minutes or less.  Filter the entire sample, draining as much water from the sample
               container as possible.
       11.3.4 Remove the filtration top from the vacuum flask, but don't disassemble the reservoir
               and fritted base.  Empty the water from the flask and insert a suitable sample tube to
               contain the eluant.  The only constraint on  the sample tube is that it fit around the
               drip tip of the fritted base. Reassemble the apparatus.
               Add 5 mL of acetonitrile (CH,CN) to rinse the sample bottle. Allow the CH3CN to
               settle to the bottom of the bottle and transfer to the disk with a dispo-pipet, rinsing the
               sides of the glass filtration reservoir in the process.  Pull about half of the CH3CN
               through the disk, release the vacuum, and allow the disk to soak for a  minute.  Pull
               the remaining CH3CN through the disk.
               Repeat the above step twice, using MeCl2 instead of CH3CN. Pour the combined
               eluates thru a small funnel with filter paper containing 3 grams of anhydrous sodium
               sulfate.  Rinse the test tube and sodium sulfate with two 5  mL portions of MeCl2.
               Collect the filtrate in  a concentrator tube.
       11.3.5 With the concentrator  tube in a 28°C heating block, evaporate the eluate with a
               stream of N: to 0.5 mL.
11.4  Extract Cleanup:  Cleanup procedures may not  be necessary  for a relatively clean sample
       matrix, such as most drinking waters. If particular  circumstances demand the use of a
       cleanup procedure, the analyst may use either procedure below or any other appropriate
       procedure.  However, the analyst first must  demonstrate that the requirements  of Sect.  10.3
       and 10.5 can be met using the method as revised to incorporate the cleanup  procedure.
       11.4.1 Florisil column cleanup for phthalate esters:
               11.4.1.1  Place  10 g of Florisil (Sect. 7.4) into a chromatographic column.  Tap the
                         column to settle the  Florisil and add 1  cm of anhydrous  sodium sulfate to
                         the top.
               11.4.1.2 Preelute the column with 40 mL of hexane.  Discard the eluate and just
                         prior to exposure of the sodium sulfate  layer to the air, quantitatively
                         transfer the sample extract (Sect. 11.1.8  or 11.2.6) onto the column, using
                         an additional 2  mL of hexane to complete the transfer.  Just prior to ex-
                         posure of the sodium sulfate layer to the air, add 40  mL of hexane and
                         continue the elution  of the column. Discard this hexane eluate.
               11.4.1.3 Next,  elute the  column with 100 mL of 20% ethyl ether in  hexane (V/V)
                         into a 500-mL K-D flask equipped with a 10-mL concentrator tube.  Elute
                         the column at a rate of about 2 mL/min for all  fractions. Concentrate the
                         collected fraction as in Sect. 11.1.  No solvent  exchange is necessary. .
                         Adjust the  volume of the cleaned extract to 1 mL in  the concentrator tube
                         and analyze by  gas chromatography.
       11.4.2 Alumina column  cleanup for phthalate  esters:
               11.4.2.1  Place  10 g of alumina into a chromatographic column.  Tap the column to
                         settle  the alumina and add I cm of anhydrous sodium sulfate to the top.
74

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                                                                                   Method 506
              11.4.2.2  Preelute the column with 40 mL of hexane.  The rate for all elutions
                         should be about 2 mL/min.  Discard the eluate and just prior to exposure
                         of the sodium sulfate layer to the air, quantitatively transfer the sample
                         extract (Sect.  11.1.8 or 11.2.6) onto the column, using an additional 2 mL
                         of hexane to complete the transfer. Just prior to exposure of the sodium
                         sulfate layer to the air, add 35 mL of hexane and continue the elution of
                         the column.  Discard this hexane eluate.
              11.4.2.3  Next, elute the column with 140 mL of 20% ethyl ether in hexane (V/V)
                         into a 500-mL K-D flask equipped with a 10-mL concentrator tube. Con-
                         centrate  the collected fraction as in Sect. 11.1.  No solvent exchange is
                         necessary. Adjust the volume of the cleaned extract to 1 mL in the con-
                         centrator tube and analyze by gas chromatography.
11.5 Gas Chromatography
      11.5.1 Table 1 summarizes the recommended operating conditions for the gas chromato-
              graph.  Included are retention data for the primary and confirmation columns.  Other
              capillary (open-tubular) columns, chromatographic conditions, or detectors may be
              used if the requirements of Sect.  10 are met.
      11.5.2 Calibrate the system daily as described in Sect. 9.
      11.5.3 Inject 1 to  2 pL of the sample extract or standard into the gas chromatograph.  Small-
              er (1.0 jiL) volumes may be injected if automatic devices are employed.   For opti-
              mum reproducibility, an auto injector is recommended.
      11.5.4 Identify the analytes in the sample by comparing the retention times of the peaks in
              the sample chromatogram with those of the peaks in standard chromatograms.  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.
      11.5.5 If the response for a peak exceeds the working range of the system, dilute the extract
              and reanalyze
      11.5.6 If the measurement  of the peak response is prevented by the presence of interferences,
              further cleanup is required.
      11.5.7 The calibration curves should be linear over the range of concentrations  in Tables
              2-5.

12.  CALCULATIONS

12.1 Calculate the amount of material injected from the peak response using the calibration curve or
      calibration factor determined in Sect. 9.2.2.  The concentration in the sample can be calculated
      from Equation 1.
                                                                                            75

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Method 506
                                          Equation 1
                                 Concentration (ug/L) =
                             where:
                             A = Amount of material injected (ng).
                             V = Volume of extract injected
                             Vt = Volume of total extract
                             V = Volume of water extracted (mL).
12.2  Report results in ^g/L without correction for recovery data.  All QC data obtained should be
       reported with the sample results.

13.   METHOD PERFORMANCE
       Single laboratory accuracy and precision data were obtained by replicate liquid-liquid extrac-
       tion analyses of reagent water fortified at two sets of concentrations of method analytes. The
       data are given in Tables 2 and 3.  Accuracy and precision data by liquid-solid extraction of
       reagent water fortified at a single concentration are given in Table 4.  Finally, method valida-
       tion data obtained by the analyses  of fortified tap water and raw source water are given in
       Tables 5-7.
 76

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                                                                                 Method 506
                                     References

1.     Glaser, J.V., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analysis for
       Waste Waters." Environ. Sci. Technol. 15. 1426,  1981,

2.     "Determination of Phthalates in Industrial and Municipal Wastewaters," EPA-600/4-81-063,
       U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
       Cincinnati, Ohio 45268, October 1981.

3.     Giam, C.S., Chan, H.S. and Nef, G.S. "Sensitive Method for Determination of Phthalate
       Ester Plasticizers  in Open-Ocean Biota Samples," Anal. Chem.. 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," U.S. National Bureau of Standards, 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, D3694-78. "Standard Practices for Preparation of
       Sample Containers and for Preservation of Organic Constituents," American Society for
       Testing and Materials, Philadelphia.

9.     ASTM Annual Book of Standards, Part 31, D3370. "Standard Practices for Sampling Water,"
       American Society for Testing and Materials, Philadelphia.

10.    J.W. Eichelberger, T.D. Behymer and W.L. Budde, "Determination of Organic Compounds in
       Drinking Water By Liquid-Solid Extraction and Capillary Column Gas  Chromatography/Mass
       Spectrometry", EPA Method 525, in Methods for the  Determination of Organic Compounds in
       Drinking Water. EPA-600/4-88/039, Environmental Monitoring Systems Laboratory, U.S.
       Environmental Protection Agency, Cincinnati, Ohio 45268,  December 1988,  pp. 325-356.
                                                                                         77

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Method 506
Table 1.  Retention Data and Chromatographic Conditions
                                                        Retention Time (min)
Parameter
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butylbenzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
Column
  17.23
  20.29
  27.57
  34.19
  34.85
  37.51
  41.77
Column 2
  17.89
  21.13
  28.67
  35.34
  36.76
  39.58
  44.44
Column 1:  DB-5, fused silica capillary, 30 m  x 0.32 mm I.D., 0.25 micron film thickness, Helium
linear velocity = 30 cm/s.

Column 2:  DB-1, fused silica capillary, 30 m  x 0.32 mm I.D., 0.25 micron film thickness, Helium
linear velocity = 30 cm/s.

Chromatographic Conditions:
    Injector temperature = 295 °C
    Detector temperature = 295°C
    Program - 1 min hold at 60°C,
    6°C/min to 260°C, 10 min hold.
    Splitless injection with 45 s delay
Table 2.  Accuracy, Precision, and Method Detection Limit Data from Six Liquid-
Liquid Extraction Analyses of Fortified Reagent Water
Analyte
Dimethyl  phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate


True Cone.
(ug/U
2.02
1.51
2.62
6.00
6.03
5.62
17.18
Mean
Meas.
Cone.
(ug/U
1.42
1.16
1.78
3.27
3.94
2.92
7.96


Std. Dev.
(ug/L)
0.38
0.28
0.41
0.89
1.44
0.75
2.14
Mean
Accuracy
(% of True
Cone.)
70.3
76.8
67.9
54.5
65.3
52.0
46.3


MDL
(ug/L)
1.14
0.84
1.23
2.67
11.82
2.25
6.42
78

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                                                                            Method 506
Table  3.  Accuracy and Precision Data from Seven Liquid-liquid Extraction Analyses
of Fortified Reagent Water

                                                Mean Accuracy     Relative Standard
                          True Concentration  <% of True Concen-      Deviation
Analyte                         (ug/U              tration)               (%)
Dimethyl phthalate                  15                  73                   16
Diethyl phthalate                    15                  71                  16
Di-n-butyl phthalate                 15                  68                   15
Butyl benzyl phthalate               15                  71                   15
Bis(2-ethylhexyl) adipate             30                  69                   18
Bis(2-ethylhexyl) phthalate           30                  67                   21
Di-n-octyl phthalate                 30                  62                   23
Table 4.  Accuracy And Precision Data from Six Liquid-Solid Extraction Analyses of
Fortified Reagent Water

                                                Mean Accuracy     Relative Standard
                          True Concentration  (% of True Concen-       Deviation
Analyte                         (ug/U              tration)               (%)
Dimethyl phthalate                  15                  74                   11
Diethyl phthalate                   15                  85                   10
Di-n-butyl phthalate                 15                  74                   11
Butyl benzyl phthalate               15                  72                   14
Bis(2-ethylhexyl) adipate             30                  84                   11
Bis(2-ethylhexyl) phthalate           30                  101                   13
Di-n-octyl phthalate                 30                  85                   13
Table 5.  Accuracy and Precision Data from Six Liquid-liquid Extraction Analyses of
Fortified Tap Water

                                                Mean Accuracy     Relative Standard
                          True Concentration  (% of True Concen-       Deviation
Analyte                         (ug/U              tration)               (%)
Dimethyl phthalate                   5                   103                  10.0
Diethyl phthalate                    5                   106                  10.0
Di-n-butyl phthalate                  5                   94                    6.8
Butyl benzyl phthalate                5                   93                    9.1
Bis(2-ethylhexyl) adipate              5                   87                   12.0
Bis(2-ethylhexyl) phthalate            5                   93                    4.9
Di-n-octyl phthalate                  5                   72                   26.0
                                                                                    79

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Method 506
Table  6.  Accuracy and Precision Data from Six Liquid-Liquid Extraction Analyses
of Fortified Raw  Source Water
                                                Mean Accuracy    Relative Standard
                          True Concentration  (% of True Concen-       Deviation
Analyte                         (ug/L)               tration)               (%)
Dimethyl phthalate                  5                   59                   51
Diethyl phthalate                    5                   78                  45
Di-n-butyl phthalate                 5                   99                  29
Butyl benzyl phthalate               5                   72                  23
Bis(2-ethylhexyl) adipate             5                   115                 32
Bis(2-ethylhexyl) phthalate           5                   91                  35
Di-n-octyl phthalate                 5                   54                  24
Table 7.  Accuracy and Precision Data From Six Liquid-Solid Extraction Analyses of
Fortified Raw Source Water

                                                Mean Accuracy    Relative Standard
                          True Concentration  (% of True Concen-       Deviation
Analyte                         (ug/L)               tration)               (%)
Dimethyl phthalate                  5                   110                  20
Diethyl phthalate                   5                   111                  32
Di-n-butyl phthalate                 5                   95                  30
Butyl benzyl phthalate               5                   82                  20
Bis(2-ethylhexyl) adipate             5                   65                  24
Bis(2-ethylhexyl) phthalate           5                   60                  21
Di-n-octyl phthalate                 5                   53                  15
80

-------
                                                                      Method 506
                    2 Liter
                    Separately Funnel
                                       H
                                                      \7
                          125mL
                          Solvent
                          Reservoir

                          Ground Glass
                          Stopper 14/35

                          LSE Cartridge
                    125mL
                    Solvent
                    Reservoir
                    Ground Glass
                    Stopper 14/35

                    LSE Cartridge

                    Rubber Stopper


                    No. 18-20 Luer-lok
                    Syringe Needle
H
                          100mL
                          Separately
                          Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
                                                                  10 mL
                                                                  Graduated
                                                                  Vial
                                                                          52-015-31
A. Extraction Apparatus
            B. Elution Apparatus
                                Figure 1
                                                                               81

-------
  Method 506
 o>
 w
 a
 w
 a>
 cc
                                            Time (Minutes)
                                                                                        52-015-32
                 Peaks obtained by injecting 5 ng for the 1st, 2nd, 4th and 5th

                 compounds, 10 ng for the 6th, 7th and 8th compounds, and 2.5 ng for

                 the 3rd compound.  (Table 1)
                                           Figure 2
82

-------
                             Method  507
        The Determination of Nitrogen- and
Phosphorus-Containing Pesticides in Water
              by Gas Chromatography with
            a Nitrogen-Phosphorus Detector
                      Revision 2.0 - EPA EMSL-Ci
      T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey
                            Method 1, Revision 1.0 (1987)
                  R.L. Graves - Method 507, Revision 2.0 (1989)

-------

-------
                                Method 507
 Determination of Nitrogen- and Phosphorus-Containing Pesticides in
 Water by Gas Chromatography with a Nitrogen-Phosphorus Detector
1.    SCOPE AND APPLICA TION
1.1
This is a gas chromatographic (GC) method applicable to the determination of certain
nitrogen- and phosphorus-containing pesticides in ground water and finished drinking
water." The following compounds can be determined using this method:
                   Analyte
                   Alachlor
                   Ametryn
                   Atraton
                   Atrazine
                   Bromacil
                   Butachlor
                   Butylate
                   Carboxin
                   Chlorpropham
                   Cycloate
                   Diazinon'*
                   Dichlorvos
                   Diphenarnid
                   Disulfoton"
                   Disulfoton sulfone*
                   Disulfoton sulfoxide'*
                   EPTC
                   Ethoprop
                   Fenamiphos
                   Fenarimol
                   Fluridone
                   Hexazinone
                   Merphos*
                   Methyl paraoxon
                   Metolachlor
                   Metribuzin
                   Mevinphos
                   MGK 264
                   Molinate
                   Napropamide
                   Norflurazon
                   Pebulate
                   Prometon
                   Prometryn
                   Pronamide3*
                   Propazine
                   Simazine
                   Simetryn
                   Stirofos
                                    CAS No.
                                  15972-60-8
                                    834-12-8
                                    1610-17-9
                                    1912-24-9
                                    314-40-9
                                  23184-66-9
                                    2008-41-5
                                    5234-68-4
                                    101-21-3
                                    1134-23-2
                                    333-41-5
                                      62-73-7
                                    957-51-7
                                    298-04-4
                                    2497-06-5
                                    2497-07-6
                                    759-94-4
                                  13194-48-4
                                  22224-92-6
                                  60168-88-9
                                  59756-60-4
                                  51235-04-2
                                    150-50-5
                                    950-35-6
                                  51218-45-2
                                  21087-64-9
                                    7786-34-7
                                    113-48-4
                                    2212-67-1
                                  15299-99-7
                                  27314-13-2
                                    1114-71-2
                                    1610-18-0
                                    7287-19-6
                                  23950-58-5
                                    139-40-2
                                    122-34-9
                                    1014-70-6
                                  22248-79-9
                                                                               85

-------
Method 507
                      Analyte                         CAS No.
                      Tebuthiuron                     34014-18-1
                      Terbacil                          5902-51-2
                      Terbufos3*                      13071-79-9
                      Terbutryn                         886-50-0
                      Triadimefon                     43121-43-3
                      Tricyclazole                     41814-78-2
                      Vernolate                        1929-77-7

            a    Compound exhibits aqueous instability.  Samples for which this com-
                pound is an analyte of interest must be extracted immediately (Sec-
                tions 11.1 through 11.3).

            *   These compounds are only qualitatively identified in the National
                Pesticides Survey (NPS) Program. These compounds are not quanti-
                tated because control over precision has not been accomplished.

1.2   This method has been validated in a single laboratory and estimated detection limits (EDLs)
      have been determined for the analytes above (Sect.  13). Observed detection limits may vary
      among waters, depending upon the nature of interferences in the sample matrix and the
      specific instrumentation used.
1.3   This method is restricted to use by or under the supervision of analysts experienced in the use
      of GC 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 Sect.
       10.3.
1.4   Analytes that are not separated chromatographically, i.e., analytes which have very similar
      retention times, cannot be individually identified and measured in  the same calibration mixture
      or water sample unless an alternative technique for identification and quantitation exist (Section
       11.5).
1.5   When this method is used to analyze unfamiliar samples for any or all of the analytes  above,
      analyte identifications should be confirmed by at least one additional qualitative technique.

2.    SUMMARY OF METHOD

2.1   A measured volume  of sample of approximately 1 L is  extracted with methylene chloride by
      shaking in a separatory  funnel or mechanical tumbling  in a bottle. The methylene  chloride
      extract is isolated, dried and concentrated to a volume of 5 mL during a  solvent  exchange to
      methyl tertbutyl ether (MTBE).  Chromatographic conditions are described which permit the
      separation and measurement of the analytes in the extract by Capillary Column GC with a
       nitrogen-phosphorus detector (NPD).

3.    DEFINITIONS

3.1    Internal standard:  A pure analyte(s) added to a solution in known amount(s) and used to
       measure the relative responses of other method analytes and  surrogates that are components of
       the same  solution. The internal standard must be an analyte that is not a sample component.
86

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                                                                                     Method 507
3.2    Surrogate analyte:  A pure analyte(s), which is extremely unlikely to be found in any sample,
       and which is added to a sample aliquot in known amount(s) before extraction and is measured
       with the same procedures used to measure other sample components. The purpose of a surro-
       gate analyte is to monitor method performance with each sample.
3.3    Laboratory duplicates (LD1 and LD2):  Two sample aliquots taken in the analytical laboratory
       and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
       of the precision associated with laboratory procedures, but  not with sample collection, preser-
       vation, or storage procedures.
3.4    Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated  exactly  the same  throughout field and laboratory
       procedures.  Analyses of FD1  and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents,  internal standards,
       and surrogates that are  used with other samples.  The LRB is used to determine if method
       analytes or other interferences  are present in the laboratory environment, the reagents,  or the
       apparatus.
3.6    Field reagent blank (FRB):  Reagent water placed in a sample container in  the laboratory and
       treated as a sample in all respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures. The purpose of the FRB is to determine if method
       analytes or other interferences  are present in the field environment.
3.7    Laboratory performance check solution (LPC): A solution of method analytes,  surrogate
       compounds, and internal standards used to evaluate the performance of the  instrument system
       with respect to a defined set  of method criteria.
3.8    Laboratory fortified blank (LFB): An aliquot of reagent  water to which known quantities of
       the method analytes are added  in the laboratory.  The LFB is analyzed  exactly like a sample,
       and its purpose is to determine whether the methodology is in control, and  whether the labora-
       tory  is capable of making accurate and precise measurements at the required method detection
       limit.
3.9    Laboratory fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results. The background concentrations of the analytes in the sample
       matrix must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock standard solution: A concentrated solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
       an assayed reference compound. Stock standard solutions are used to prepare primary dilution
       standards.
3.11  Primary dilution standard solution:  A solution of several analytes prepared in the laboratory
       from stock standard solutions and diluted  as needed to prepare calibration solutions and other
       needed analyte solutions.
                                                                                             87

-------
Method 5O7
3.12  Calibration standard (CAL): A solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards and surrogate analytes.  The CAL
       solutions are used to calibrate the instrument response  with respect to analyte concentration.
3.13  Quality control sample (QCS):  A sample matrix containing method analytes or a solution of
       method analytes in a water miscible  solvent which is used to fortify reagent water or environ-
       mental samples.  The QCS is obtained from a source external to the laboratory, and is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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 Sect.  10.2.
       4.1.1   Glassware must be scrupulously cleaned.2  Clean all glassware as soon as possible
               after use  by thoroughly rinsing with the last solvent  used in it. Follow by  washing
               with hot water and detergent and thorough rinsing with tap and reagent water.  Drain
               dry, and  heat in an oven or muffle furnace at  400°C for 1 hour.  Do not heat volu-
               metric ware. Thermally stable materials might not be eliminated by this treatment.
               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.
       4.1.2   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.

       WARNING:        When a solvent is purified, stabilizers added by the manufacturer
       may be removed thus potentially making the solvent hazardous.  Also, when a solvent is
       purified, preservatives added by the manufacturer are removed thus potentially reducing
       the  shelf-life,

4.2   Interfering  contamination may occur when a sample containing low concentrations of  analytes
       is analyzed immediately following a sample containing relatively high concentrations of
       analytes. Between-sample rinsing of the sample syringe and associated equipment with MTBE
       can minimize sample cross contamination. After analysis of a sample containing  high concen-
       trations  of analytes, one or more injections of MTBE should be made to ensure that accurate
       values are obtained for the next sample.
4.3   Matrix interferences may be caused  by  contaminants that are coextracted from the sample.
       Also, note  that all the analytes listed in the scope and application section are not resolved from
       each other  on  any one column,  i.e., one analyte of interest may be an  interferant for another
       analyte of interest.  The extent of matrix interferences will vary considerably from  source to
       source, depending upon the water sampled.  Further processing of sample extracts may be
       necessary.  Positive identifications should be confirmed (Sect. 11.5).
88

-------
                                                                                   Method 507
4.4    It is important that samples and working standards be contained in the same solvent. The
       solvent for working standards must be the same as the final solvent used in sample prepara-
       tion. If this is not the case, chromatographic comparability of standards to sample may be
       affected.

5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level.  The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
       ing the safe handling of the chemicals specified in this method.  A reference file of material
       safety data 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.
5.2    WARNING: When a solvent is purified, stabilizers added by the manufacturer may be
       removed thus potentially making the solvent hazardous.

6.     APPARATUS AND EQUIPMENT
(All specifications are suggested.  Catalog numbers are included for illustration only.)
6.1    Sample bottle:  Borosilicate, 1-L volume with graduations (Wheaton Media/Lab bottle 219820
       or equivalent), fitted with screw caps lined with TFE-fluorocarbon.  Protect samples from
       light.The  container must be washed and dried as described in Sect. 4.1.1 before use to mini-
       mize contamination.  Cap liners are cut to fit from sheets (Pierce Catalog No. 012736 or
       equivalent) and extracted with methanol overnight prior to use.
6.2    Glassware
       6.2.1   Separatory funnel:   2000-mL, with TFE-fluorocarbon stopcock, ground glass or
              TFE-fluorocarbon stopper.
       6.2.2  Tumbler bottle:  1.7-L (Wheaton Roller Culture Vessel or equivalent), with TFE-
              fluorocarbon lined screw cap. Cap liners are cut to fit from sheets (Pierce Catalog
              No.  012736) and extracted with methanol overnight prior to use.
       6.2.3  Flask,  Erlenmeyer:  500-mL.
       6.2.4  Concentrator tube, Kuderna-Danish (K-D):  10- or 25-mL, graduated (Kontes
              K-570050-2525 or K-570050-1025 or equivalent).  Calibration must be checked at the
              volumes employed in the test.  Ground glass stoppers are used to prevent evaporation
              of extracts.
       6.2.5  Evaporative flask, K-D: 500-mL (Kontes K-570001-0500 or equivalent).  Attach to
              concentrator tube with springs.
       6.2.6  Snyder column, K-D:  Three-ball macro (Kontes K-503000-0121 or equivalent).
       6.2.7  Snyder column, K-D:  Two-ball micro (Kontes K-569001-0219 or equivalent).
       6.2.8  Vials:  Glass, 5- to  10-mL capacity with TFE-fluorocarbon lined screw cap.
                                                                                           89

-------
Method 507
6.3   Separator}' funnel shaker (Optional):  Capable of holding 2-L separatory funnels and shaking
      them with rocking motion to achieve thorough mixing of separatory funnel contents (available
      from Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4   Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
      (Associated Design and  Mfg. Co., Alexandria, VA. or other suppliers).
                                                                                         ' *rf
6.5   Boiling stones: Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033 or equiva-    '
      lent).  Heat at 400°C for 30 min prior to use. Cool and store in desiccator.
6.6   Water bath:  Heated, capable of temperature control (± 2°C).  The bath should be used in a
      hood.
6.7   Balance:  Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.8   Gas Chromatograph:  Analytical  system complete with temperature programmable GC suitable
      for use with capillary columns  and all required accessories including syringes, analytical
      columns, gases, detector and stripchart recorder. A data system is  recommended for measuring
      peak areas.  Table 1 lists retention times observed for method analytes using the columns and
      analytical conditions described  below.
      6.8.1   Column  1 (Primary column):  30 m long x 0.25 mm I.D. DB-5 bonded fused silica
               column,  0.25 jim film thickness (J&W Scientific)  or equivalent. Helium carrier gas
               flow is established at 30 cm/sec linear velocity and oven temperature is programmed
               from 60°C to 300°C at 4°C/min.  Data presented in this method were obtained using
               this column.  The injection volume was 2 uL in splitless mode with a 45 s delay.  The
               injector temperature was 250°C and  the detector temperature was 300°C.  Alternative
               columns may be used  in accordance  with the  provisions described in Sect. 10.4.
      6.8.2   Column  2 (Confirmation column): 30 m long x 0.25 mm I.D.DB-1701 bonded fused
               silica column, 0.25 /xm film  thickness (J&W Scientific) or equivalent. Helium carrier
               gas flow is established at 30  cm/sec  linear velocity and oven temperature is pro-
               grammed from 60C to 300°C at 4°C/min.
      6.8.3   Detector:  Nitrogen-phosphorus (NPD).  A NPD was used to generate the validation
               data presented  in this method. Alternative detectors, including a mass spectrometer,
               may be used in accordance with the  provisions described in Sect. 10.4.

 7.    REAGENTS AND CONSUMABLE MATERIALS

       WARNING:       When a solvent is purified,  stabilizers added by  the manufacturer
      are removed thus potentially making the solvent hazardous. Also,  when a solvent is
      purified, preservatives added by  the manufacturer are removed thus potentially reducing
       the shelf-life.

 7.1    Acetone, methylene chloride, methyl  tert.-butyl ether  (MTBE):  Distilled-in-glass quality or
       equivalent.
 7.2   Phosphate buffer, pH 7: Prepare by  mixing  29.6 mL 0.1  N Hcl and 50 mL 0.1  M dipotassi-
       um phosphate.
 90

-------
                                                                                    Method 507
7.3    Sodium chloride (NaCl), crystal, ACS grade:  Heat treat in a shallow tray at 450°C for a
       minimum of 4 hours to remove interfering organic substances.
7.4    Sodium sulfate, granular, anhydrous, ACS grade:  Heat treat in a shallow tray at 450°C for a
       minimum of 4 hours to remove interfering organic substances.
7.5    Sodium thiosulfate, granular, anhydrous, ACS grade.
7.6    Triphenylphosphate (TPP):  98% purity, for use as internal standard (available from Aldrich
       Chemical Co.).
7.7    l,3-Dimethyl-2-nitrobenzene:  98% purity, for use as surrogate standard (available  from
       Aldrich Chemical  Co.).
7.8    Mercuric Chloride:  ACS grade (Aldrich Chemical Co.) - for use as a bactericide.  If any
       other bactericide can be shown to work as well as mercuric chloride, it may be used instead.
7.9    Reagent water:  Reagent water is defined as a water that is reasonably free of contamination
       that would prevent the determination of any  analyte of interest.  Reagent water used to gener-
       ate the validation data  in this method was distilled water obtained from the Magnetic Springs
       Water Co., Columbus, Ohio.
7.10  Stock  Standard Solutions (1.00 /ig/^L):  Stock standard solutions may be purchased as certi-
       fied solutions or prepared from pure standard materials using the following procedure:
       7.10.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
               pure material.  Dissolve the material in MTBE and dilute to volume in a 10-mL
               volumetric flask. The stock solution for simazine should be prepared in methanol.
               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 indepen-
               dent source.
       7.10.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw cap amber
               vials.  Store at room temperature and protect from  light.
       7.10.3 Stock standard solutions should be replaced after two months or sooner  if comparison
               with laboratory fortified blanks, or QC samples indicate a problem.
7.11   Internal Standard Solution:  Prepare the internal standard solution by accurately weighing
       approximately 0.0500 g of pure TPP. Dissolve the TPP in  MTBE and dilute to  volume in a
       100-mL volumetric flask. Transfer the  internal standard solution to  a TFE-fluorocarbon-sealed
       screw  cap bottle and store at room temperature.  Addition of 50 ^L of the internal standard
       solution to 5 mL of sample extract results in a final TPP concentration of 5.0 /ng/mL.  Solu-
       tion should be replaced when ongoing QC (Sect. 10) indicates a problem. Note  that TPP has
       been shown to be  an effective internal standard  for the method analytes,' but other compounds
       may be used if the quality control requirements in Sect.  10 are met.
7.12  Surrogate Standard Solution: Prepare the surrogate standard solution by accurately weighing
       approximately 0.0250 g of pure l,3-dimethyl-2-nitrobenzene. Dissolve the 1,3-dimethyl-
       2-nitrobenzene in MTBE and dilute to volume in a 100-mL volumetric flask. Transfer the
       surrogate standard solution to a TFE-fluorocarbon-sealed screw cap bottle and store at room
       temperature.  Addition of 50 p,L of the surrogate standard solution to a 1-L sample  prior to

                                                                                            91

-------
Method 507
       extraction results in a 1,3-dimethyl-2-nitrobenzene concentration in the sample of 12.5
       Solution should be  replaced when ongoing QC (Sect. 10) indicates a problem. Note that
       1,3-dimethyl-2-nitrobenzene has been shown to be an effective surrogate standard for the
       method analytes,1 but other compounds may be used if the quality control requirements in
       Sect. 10 are met.
7.13  Laboratory Performance Check Solution:  Prepare the laboratory performance check solution
       by adding 5 /*L of the vernolate stock solution, 0.5 mL of the bromacil stock solution, 30 fiL
       of the prometon stock solution, 15 pL of the atrazine stock solution, 1.0 mL of the surrogate
       solution, and 500 pL of the internal standard solution to a 100-mL volumetric flask.  Dilute to
       volume with MTBE and thoroughly mix the solution.  Transfer to a TFE-fluorocarbon-sealed
       screw cap bottle and store at room temperature.  Solution should be replaced when ongoing
       QC (Section 10) indicates a problem.

8.     SAMPLE COLLECTION, PRESERVATION,  AND STORAGE

8.1    Grab samples must be collected in glass containers.  Conventional sampling practices6 should
       be followed; however, the bottle must not be prerinsed with sample before collection.
8.2    Sample Preservation and Storage
       8.2.1   Add mercuric chloride (See 7.8) to the sample bottle in amounts to produce a concen-
               tration of 10 mg/L.  Add 1 mL of a solution containing  10 mg/mL of mercuric
               chloride in reagent water to the sample bottle at the sampling site or in the laboratory
               before shipping to the sampling site.  A major disadvantage of mercuric chloride is
               that it is a highly toxic chemical; mercuric chloride must be handled with caution, and
               samples containing mercuric chloride  must  be disposed of properly.
       8.2.2   If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
               the sample bottle prior to collecting the  sample.
       8.2.3   After  the sample is collected in a bottle  containing preservative(s),  seal the bottle and
               shake vigorously for 1 min.
       8.2.4   The samples must be iced or refrigerated at 4°C away from light from the time of
               collection  until extraction. Preservation study results indicated that most method
               analytes present  in samples were stable  for 14 days when stored under these condi-
               tions.' The analytes disulfoton sulfoxide, diazinon,  pronamide, and terbufos exhibited
               significant aqueous instability, and samples to be analyzed for  these compounds must
               be extracted immediately.  The analytes carboxin, EPTC, fluridone, metolachlor,
               napropamide, tebuthiuron, and terbacil exhibited recoveries of less than 60% after 14
               days.  Analyte stability may be affected by the  matrix; therefore, the analyst should
               verify that the preservation technique  is applicable to the samples under study.
8.3   Extract Storage: Extracts should be stored at 4°C away  from light. Preservation study results
       indicate that most analytes are stable for  28 days; however, a 14-day maximum extract storage
       time is recommended.  The analyst should verify appropriate extract holding times applicable
       to the samples under study.
 92

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                                                                                     Method 507
9.     CALIBRATION

9.1    Establish GC operating parameters equivalent to those indicated in Sect. 6.8.  The GC system
       may be calibrated using either the internal standard technique (Sect. 9.2) or the external
       standard technique (Sect. 9.3).  Be aware that NPDs may exhibit instability (i.e., fail to hold
       calibration curves over time).  The analyst may, when analyzing samples for target analytes
       which are very rarely found, prefer to analyze on a daily basis a low level  (e.g.  5 to 10 times
       detection limit or 1/2 times the regulatory limit, whichever is less), sample (containing all
       analytes of interest) and require some minimum sensitivity (e.g. 1/2 full scale deflection) to
       show that if the analyte were present it would be detected.  The analyst may then quantitate
       using single point calibration (Sect.  9.2.5 or 9.3.4).

       NOTE: Calibration standard solutions must be prepared such that no unresolved
       analytes are mixed together.

9.2    Internal Standard Calibration Procedure:  To use this approach, the analyst must select one or
       more internal standards compatible 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.  TPP has been identified as a suitable internal standard.
       9.2.1   Prepare  calibration standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest by adding volumes of one or more stock standards
              to a volumetric flask.  If Merphos is to be determined,  calibrate with DBF (S,S,S-
              tributylphosphoro-trithioate). To each calibration standard, add a known constant
              amount of one or more of the internal standards, and dilute to volume with MTBE.
              The lowest standard should represent analyte concentrations near, but above, their
              respective EDLs. The remaining standards should bracket the analyte concentrations
              expected in the sample extracts, or should define the working range of the  detector.
       9.2.2  Analyze each calibration standard according to the procedure described  in Sect. 11.4.
              Tabulate response (peak height or area) against concentration for each compound and
              internal standard. Calculate the response factor (RF) for each analyte and surrogate
              using Equation 1.
                                          Equation 1
                                         RF
                     Where:
                     As = Response for the analyte.
                     Ais = Response for the internal standard.
                     Ca = Concentration of the internal standard \i.glL.
                     Cs = Concentration of the analyte to be measured
                                                                                             93

-------
Method 507
       9.2.3   If the RF value over the working range is constant (20%  RSD or less) the average RF
               can be used for calculations. Alternatively, the results can be used  to plot a calibration
               curve of response ratios (AS/A,5) vs. Cs.
       9.2.4   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 analyte
               varies from the predicted response by more than +20%,  the test must be repeated
               using a fresh calibration standard.  If the repetition also fails, a new calibration curve
               must be generated for that analyte using freshly prepared standards.
       9.2.5   Single point calibration is a viable alternative to a calibration curve.  Prepare single
               point standards from the secondary dilution standards in MTBE. The single point
               standard should be prepared at a concentration that produces a response that deviates
               from the sample extract response by no more than 20%.
       9.2.6   Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from  an independent source.
               Results from these analyses must be within the limits used to routinely check calibra-
               tion.
9.3    External Standard Calibration Procedure
       9.3.1   Prepare calibration  standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest and surrogate compound by adding volumes of one
               or more stock standards to a volumetric flask. If Merphos is to be determined,
               calibrate with DEF  (S,S,S-tributylphosphoro-trithioate).  Dilute to  volume with
               MTBE. The lowest standard should represent analyte concentrations near, but above,
               their respective EDLs. The remaining standards should bracket the analyte concentra-
               tions expected in the sample extracts,  or should define the working range of the
               detector.
       9.3.2   Starting with the standard of lowest concentration, analyze each calibration standard
               according to Sect.  11.4 and tabulate response (peak height or area) versus the concen-
               tration in the standard.  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 (20% RSD or less), linearity through the
               origin can be assumed and the average ratio or calibration factor can be used in place
               of a calibration curve.
       9.3.3   The working calibration curve or calibration factor must  be verified on each working
               day by the measurement of a minimum of two calibration check standards, one at the
               beginning and one at the end of the analysis day.  These check standards should be at
               two different concentration levels to verify the calibration curve. For extended periods
               of analysis (greater than 8 hrs.), it is strongly recommended that check standards be
               interspersed with samples at regular intervals during the course of  the analyses.  If the
               response for any analyte varies from the predicted response by more than ±20%, the
               test must be repeated using a fresh calibration standard.  If the results still do not
               agree, generate a new calibration curve.
       9.3.4   Single point calibration is a viable alternative to a calibration curve.  Prepare single
               point standards from the secondary dilution standards  in  MTBE.  The single point
94

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                                                                                    Method 507
               standard should be prepared at a concentration that produces a response that deviates
               from the sample extract response by no more than 20%.
       9.3.5   Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from an independent source.
               Results from these analyses must be within the limits used to routinely check calibra-
               tion.

10.   QUALITY CONTROL

10.1  Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
       determination of surrogate compound recoveries in each sample and blank, monitoring internal
       standard peak area or height in each sample and blank (when internal standard calibration
       procedures are being employed), analysis of laboratory reagent blanks,  laboratory fortified
       samples,  laboratory fortified blanks, and QC samples.
10.2  Laboratory Reagent Blanks.  Before processing any samples, the analyst must demonstrate that
       all glassware and reagent interferences are under control.  Each time a  set of samples is
       extracted or  reagents are changed, a LRB  must be analyzed.  If within the retention time
       window of any analyte of interest the LRB produces a peak that would  prevent the determina-
       tion of that analyte, determine the source of contamination and eliminate the interference
       before processing samples.
10.3  Initial Demonstration of Capability.
       10.3.1 Select a representative fortified concentration (about 10 times EDL or at the regulato-
              ry Maximum Contaminant  Level, whichever is lower) for each analyte.  Prepare  a
              sample concentrate (in methanol)  containing each analyte at 1000 times selected
              concentration.  With a syringe, add 1 mL of the  concentrate to each of at least four
               1-L  aliquots of reagent water, and analyze each aliquot according to procedures
              beginning  in Sect. 11.
       10.3.2 For  each analyte the recovery value for all  four of these samples must fall in the
              range of R ±  30% (or within R ± 3SR if broader) using the values for R and SR  for
              reagent water  in Table 2.  For those compounds  that meet the acceptance criteria,
              performance is considered acceptable and sample analysis may begin. For those
              compounds that fail these criteria, this procedure must be repeated using four fresh
              samples until satisfactory performance has been demonstrated.
       10.3.3 The initial demonstration of capability  is used primarily to preclude a laboratory from
              analyzing unknown samples via a new, unfamiliar method prior to obtaining some
              experience with it.  It is expected that as laboratory personnel gain experience with this
              method the quality of data will improve beyond those required  here.
10.4  The analyst is permitted to modify GC columns, GC detectors, GC conditions, continuous
       extraction techniques, concentration techniques  (i.e.  evaporation techniques),  internal standards
       or surrogate  compounds.  Each time such  method modifications are made, the analyst must
       repeat the procedures in Sect. 10.3.
                                                                                            95

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Method 507
10.5  Assessing Surrogate Recovery
       10.5.1  When surrogate recovery from a sample or method blank is <70% or > 130%, check
               (1) calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
               contamination, and (4) instrument performance.  If those steps do not reveal the cause
               of the problem, reanalyze the extract.
       10.5.2 If a blank extract reanalysis fails the  70-130% recovery criterion, the problem must
               be identified and corrected before continuing.
       10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
               for the reanalyzed extract.  If sample extract reanalysis continues to fail  the recovery
               criterion, report all data for that sample as suspect.
10.6  Assessing the Internal Standard
       10.6.1  When using the internal standard calibration procedure, the analyst is expected to
               monitor the IS response (peak area or peak height) of all samples during each analysis
               day. The IS response for any sample chromatogram should not deviate from the daily
               calibration check standard's IS response by more than 30%.
       10.6.2 If >30% deviation occurs with an individual extract, optimize instrument perfor-
               mance and inject a second aliquot of that extract.
               10.6.2.1 If the reinjected aliquot produces an acceptable internal standard response
                         report results for that aliquot.
               10.6.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
                         analysis of the sample should be repeated beginning with Sect. 11, provid-
                         ed the sample is still available. Otherwise, report results obtained from the
                         re-injected extract, but annotate as suspect.
       10.6.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
               calibration check standard.
               10.6.3.1 If the check standard provides a response factor (RF) within 20% of the
                         predicted value, then follow procedures itemized in Sect. 10.6.2 for each
                         sample failing the IS response criterion.
               10.6.3.2 If the check standard provides a response factor which deviates more than
                         20% of the predicted value, then the analyst must recalibrate, as  specified
                          in Sect. 9.
10.7  Assessing Laboratory Performance: Laboratory Fortified Blank
       10.7.1 The laboratory must analyze at  least one laboratory fortified blank (LFB) sample with
               every twenty samples or one per sample set (all samples extracted within a 24-h
               period) whichever is greater. The fortified concentration of each analyte in the LFB
               should be 10 times EDL or the MCL, whichever is less.  Calculate accuracy as
               percent recovery (X,). If the recovery  of any analyte falls outside the control limits
               (see Sect. 10.7.2), that analyte  is judged out of control, and the source of the problem
               should be identified and resolved before continuing analyses.
       10.7.2  Until sufficient data  become available from within their own laboratory, usually a
               minimum of  results from 20 to 30 analyses, the laboratory should assess laboratory

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                                                                                     Method 507
               performance against the control limits in Sect.  10.3.2 that are derived from the data in
               Table 2.  When sufficient internal performance data becomes available, develop
               control limits from the mean percent recovery (X) and standard deviation (S) of the
               percent recovery. These data are used to establish upper and  lower control limits as
               follows:

                         Upper Control Limit = X + 3S
                         Lower Control Limit =  X — 3S

               After each five to ten new recovery measurements, new control limits should be
               calculated using only the most recent 20-30 data points. These calculated control
               limits should never exceed those established in Sect. 10.3.2.
       10.7.3 It is recommended that the laboratory periodically determine and document its detec-
               tion limit capabilities for analytes of interest.
       10.7.4 At least quarterly, analyze a QC sample  from an outside source.
       10.7.5 Laboratories are encouraged to participate in external performance evaluation studies
               such as the laboratory certification programs offered by many states or the studies
               conducted by USEPA. Performance evaluation studies serve as independent checks
               on the analyst's performance.
10.8  Assessing Analyte Recovery:  Laboratory Fortified Sample Matrix
       10.8.1 The laboratory must add a known concentration to a minimum of 5% of the routine
               samples or one sample concentration per set, whichever is greater.  The fortified
               concentration should  not be less then the background concentration of the sample
               selected for fortification. Ideally, the concentration should be the same as that used
               for the laboratory fortified blank (Sect. 10.7).  Over time, samples from all routine
               sample sources should be fortified.
       10.8.2 Calculate the percent recovery, P of the concentration for each analyte, after correct-
               ing the analytical result, X, from the fortified sample for the background concentra-
               tion, b, measured in the unfortified sample,  i.e.,:
                                  p =      100  (X -  b)
                                       fortifying concentration
               and compare these values to control limits appropriate for reagent water data collected
               in the same fashion.  If the analyzed unfortified sample is found to contain NO back-
               ground concentrations, and the added concentrations are those specified in Sect. 10.7,
               then the appropriate control limits would be the acceptance limits in  Sect. 10.7. If,
               on the other hand, the analyzed unfortified sample is found to contain background
               concentration, b, estimate the standard deviation at the background concentration, sb,
               using regressions or comparable background data and, similarly, estimate the mean,
               Xa,  and standard deviation, sa, of analytical results at the total concentration after
               fortifying.  Then the appropriate percentage control limits would be  P  ± 3sP , where:
                                                                                             97

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Method 507
                               o               100 X
                               r -
                                    (b  + fortifying concentration)

                               and
                               sp  = 100
                                        fortifying concentration
               For example, if the background concentration for Analyte A was found to be 1 /ug/L
               and the added amount was also 1 jug/L, and upon analysis the laboratory fortified
               sample measured  1.6 /u./L, then the calculated P for this sample would be (1.6 /ig/L
               minus 1.0 /xg/L)/l /zg/L or 60%.  This calculated P is compared to control limits
               derived from prior reagent water data. Assume it is known that analysis of an  inter-
               ference free sample at 1 ^g/L yields an s of 0.12  /xg/L and  similar analysis at 2.0
               Hg/L yields X and s of 2.01 /xg/L and 0.20 //g/L, respectively.  The appropriate limits
               to judge the reasonableness of the percent recovery, 60%, obtained on the fortified
               matrix sample is computed  as follows:
                  100 (2.01

                      2.0 .  _

                                         + (0.20
                                      l.Opg/L
                100.5% ± 300 (0.233) =
                100.5% + 70% or 30% to 170% recovery of the added  analyte.
10.9  Assessing Instrument System—Laboratory Performance Check (LPC): Instrument performance
       should be monitored on a daily basis by analysis of the LPC sample. The LPC sample
       contains compounds designed to  indicate appropriate instrument sensitivity, column perfor-
       mance (primary column) and chromatographic performance.  LPC sample components and
       performance criteria are listed in Table 3.  Inability to demonstrate acceptable instrument
       performance indicates the need for reevaluation of the  instrument system.  The sensitivity
       requirements are set based on  the EDLs published in this method.  If laboratory EDLs differ
       from those listed in this method, concentrations of the  instrument QC standard compounds
       must be adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control 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.  For example, field or laboratory duplicates may be analyzed to assess
       the precision of the environmental measurements or field reagent blanks may be used to assess
       contamination of samples under site conditions, transportation and storage.
98

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                                                                                   Method 507
11.   PROCEDURE

11.1  Extraction (Manual Method)
      11.1.1 Mark the water meniscus on the side of the sample bottle for later determination of
              sample volume (Sect.  11.1.6).  Add preservative to blanks and QC check standards.
              Fortify the sample with 50 pL of the surrogate standard solution.  Pour the entire
              sample into a 2-L separatory funnel.
      11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
      11.1.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
      11.1.4 Add 60 mL methylene chloride to the sample bottle,  seal, and shake 30 s to rinse the
              inner walls. Transfer the solvent to the separatory funnel and extract the sample by
              vigorously shaking the funnel for 2 min with periodic venting to release excess pres-
              sure. Allow the organic layer to separate from the water phase for a minimum of 10
              min.  If the emulsion interface between layers  is more than one third the volume of
              the solvent layer, the analyst must employ mechanical techniques to complete the
              phase separation. The optimum technique depends upon the sample, but may include
              stirring, filtration of the emulsion through glass wool, centrifugation, or other physical
              methods.  Collect the methylene chloride extract in a 500-mL Erlenmeyer flask.
      11.1.5 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
              extraction procedure a second time,  combining the extracts in the Erlenmeyer flask.
              Perform  a third extraction in the same  manner.
      11.1.6 Determine the original sample volume  by refilling the sample bottle to the mark and
              transferring the water  to a 1000-mL graduated cylinder. Record the sample volume to
              the nearest 5 mL.
11.2  Extraction (Automated  Method): Data presented in this method were  generated  using the
      automated extraction procedure with the mechanical tumbler.
      11.2.1 Mark the water meniscus on the side of the sample bottle for later determination of
              sample volume (Sect.  11.2.6).  Add preservative to blanks and QC check standards.
              Fortify the sample with 50 /tL of the surrogate standard solution. If the mechanical
              separatory funnel shaker is used, pour  the entire sample into a 2-L separatory funnel.
              If the mechanical tumbler is used, pour the entire sample into a tumbler bottle.
      11.2.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.
      11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
      11.2.4 Add 300 mL methylene chloride to the sample bottle, seal, and  shake 30 s to rinse the
              inner walls. Transfer the solvent to the sample contained in the separatory funnel  or
              tumbler bottle, seal, and shake for 10 s, venting periodically. Repeat shaking and
              venting until pressure  release is not observed.  Reseal and place sample container in
              appropriate mechanical mixing device (separatory funnel shaker or tumbler).  Shake
              or tumble the sample for  1 hour. Complete mixing of the organic and aqueous phases
              should be observed within about 2 min after starting  the mixing device.
      11.2.5 Remove the sample container from the mixing device.  If the tumbler is used,  pour
              contents of tumbler bottle into a 2-L separatory funnel.  Allow the organic layer to

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Method 507
              separate from the water phase for a minimum of 10 min. If the emulsion interface
              between layers  is more than one third the volume of the solvent layer, the analyst
              must employ mechanical techniques to complete the phase separation.  The optimum
              technique depends upon the sample, but may include stirring, filtration through glass
              wool, centrifugation, or other physical methods. Collect the methylene chloride
              extract  in a 500-mL Erlenmeyer flask.
       11.2.6 Determine the original sample volume by refilling the sample bottle to the mark and
              transferring the water to a 1000-mL graduated cylinder.  Record the sample volume to
              the nearest 5 mL.
11.3  Extract Concentration
       11.3.1 Assemble a 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 Sect. 10.3 are met.
       11.3.2 Dry the extract by pouring it through a solvent-rinsed drying column containing about
              10 cm of  anhydrous sodium sulfate.  Collect the extract in the K-D concentrator, and
              rinse the column with 20-30 mL methylene chloride. Alternatively, add about 5 g
              anhydrous sodium sulfate to the extract in the Erlenmeyer flask; swirl flask to dry
              extract  and allow to sit for 15 min. Decant the methylene chloride extract into the
              K-D concentrator. Rinse the remaining sodium sulfate with two 25-mL portions  of
              methylene chloride and decant the rinses into the K-D concentrator.
       11.3.3 Add 1 to  2 clean boiling stones to the evaporative flask and attach a macro Snyder
              column.  Prewet the Snyder column by adding about 1 mL methylene chloride to the
              top. Place the  K-D apparatus on a hot water bath, 65 to 70°C, so  that the concentra-
              tor tube is partially immersed in the hot water,  and the entire lower rounded surface
              of the flask is bathed with hot vapor.   Adjust the vertical position of the apparatus and
              the water  temperature as required to complete the concentration in  15 to 20 min.   At
              the proper rate  of distillation the balls  of the column will actively chatter, but the
              chambers will not flood.  When the apparent volume of liquid reaches 2 mL, remove
              the K-D apparatus and allow it to drain and cool for at least 10 min.
       11.3.4 Remove the Snyder column and rinse the flask  and its lower joint into the concentra-
              tor tube with 1  to 2 mL of MTBE. Add 5-10 mL of MTBE and a fresh boiling stone.
              Attach  a micro-Snyder column to the concentrator tube and prewet the column by
              adding  about 0.5 mL of MTBE 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 com-
              plete concentration in 5 to 10 min. When the apparent volume of liquid reaches  2
              mL, remove the micro K-D from the bath and allow it to drain and cool. Add 5-10
              mL MTBE to the micro K-D and reconcentrate to 2 mL. Remove the micro K-D
              from the bath and allow it to drain and cool. Remove the micro Snyder column, and
              rinse the walls  of the concentrator tube while adjusting the volume to 5.0 mL with
              MTBE. NOTE:  If methylene chloride is not completely removed from the final
              extract, it may  cause detector problems.
       11.3.5 Transfer extract to an appropriate- sized TFE-fluorocarbon- sealed screw-cap vial and
              store, refrigerated at 4°C, until analysis by GC-NPD.

 100

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                                                                                    Method 507
11.4  Gas Chromatography
       11.4.1  Sect. 6.8 summarizes the recommended operating conditions for the gas chromato-
               graph. Included in Table 1 are retention times observed using this method. Other GC
               columns, chromatographic conditions, or detectors may be used if the requirements of
               Sect. 10.3 are met.
       11.4.2  Calibrate the system daily as described in Sect. 9. The standards and extracts must be
               in MTBE.
       11.4.3  If the internal standard calibration procedure is used, add 50 ftL of the internal stan-
               dard solution to the sample extract,  seal, and  shake to distribute the internal standard.
       11.4.4  Inject 2 ^L of the sample extract. Record the resulting peak size in area units.
       11.4.5  If the response for the peak exceeds the working range of the system, dilute the
               extract and reanalyze.
11.5  Identification of Analytes
       11.5.1  Identify a sample component by comparison of its retention time to the retention time
               of a reference chromatogram. If  the retention time of an unknown compound  corre-
               sponds, within limits, to the retention time of a standard compound, then identification
               is considered positive.
       11.5.2  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.
       11.5.3  Identification requires expert judgement when sample components are not resolved
               chromatographically.  When peaks obviously  represent more than one sample compo-
               nent (i.e., broadened peak with shoulder(s) or valley between two or more maxima),
               or any time doubt exists over the  identification of a peak on a chromatogram, appro-
               priate alternative techniques to help  confirm peak identification, need be employed.
               For example, more positive identification may be made by the use of an alternative
               detector which operates  on a chemical/physical principle different from that originally
               used, e.g., mass spectrometry, or the use of a second chromatography column. A
               suggested alternative column is described in Sect. 6.8.

12.   CALCULATIONS

12.1   Calculate analyte concentrations  in the sample from the response for the analyte using the
       calibration procedure described in Sect. 9.
12.2  If the internal standard calibration procedure is used, calculate the concentration (C) in  the
       sample using the response factor (RF) determined in Sect. 9.2 and Equation 2, or determine
       sample concentration from the calibration curve.
                                                                                           707

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Method 507
                                          Equation 2
                                                  (A
                                  C (pglL) =
                                               (X,,) (RF) (Vo)
                  Where:
                  A s =  Response for the parameter to be measured.
                  An =  Response for the internal standard.
                   / =  Amount of internal standard added to each extract
                  Vo =  Volume of water extracted (L).
12.3  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 determined in Sect.
       9.3.2.  The concentration (C) in the sample can be calculated from Equation 3.
                                          Equation 3

                                Concentration (us/L) =
                             Where:
                             A = Amount of material injected (ng).
                             V( = Volume of extract injected (fj.L) .
                             Vt = Volume of total extract (/iL).
                             Vt = Volume of water extracted (mL).
13.   PRECISION AND A CCURA c Y

13.1   In a single laboratory, analyte recoveries from reagent water were determined at five concen-
       tration levels.  Results were used to determine analyte EDLs and demonstrate method range.1
       Analytes were divided into five groups for recovery studies.  Analyte EDLs and analyte
       recoveries and standard deviation about the percent recoveries at one concentration are given
       in Table 2.
13.2  In a single laboratory, analyte recoveries from two standard synthetic ground waters were
       determined at one concentration level.  Results were used to demonstrate applicability of the
       method to different ground water matrices.'  Analyte recoveries from the two synthetic matri-
       ces are given in Table 2.
 102

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                                                                                 Method 5O7
                                     References

1.National Pesticide Survey Method No. 1: Determination of Nitrogen- and Phosphorus-Containing
Pesticides in Groundwater by Gas Chromatography with a Nitrogen-Phosphorus Detector.

2.ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
Preparation of Sample Containers and for Preservation", American Society for Testing and Materials,
Philadelphia, PA, 1986.

3. "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.

4. "OSHA Safety and Halth 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 11, Volume 11.01, D3370-82," Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA,  1986.
                                                                                       103

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Method 507
Table 1 . Retention Times for Method Analytes

Analyte
1 ,3-Dimethyl-2-nitrobenzene (surrogate)
Dichlorvos
Disulfoton sulfoxide
EPIC
Butylate
Mevinphos
Vernolate
Pebulate
Tebuthiuron
Molinate
Ethoprop
Cycloate
Chlorpropham
Atraton
Simazine
Prometon
Atrazine
Propazine
Terbufos
Pronamide
Diazinon
Disulfoton
Terbacil
Metribuzin
Methyl paraoxon
Simetryn
Alachlor
Ametryn
Prometryn
Terbutryn
Bromacil
Metolachlor
Triadimefon
MGK 264C
Diphenamid
Stirofos
Disulfoton sulfone
Butachlor
Fenamiphos
Napropamide
Tricyclazole
Merphos"
Carboxin
Norflurazon
Triphenyl phosphate (int. std.)
Hexazinone
Fenanmol
Flundone

Retention
Col. 1 |
14.48
16.54
19.08
20.07
22.47
22.51
22.94
23.41
25.15
25.66
28.58
28.58
29.09
31.26
31.49
31.58
31.77
32.01
32.57
32.76
33.23
33.42
33.79
35.20
35.58
35.72
35.96
36.00
36.14
36.80
37.22
37.74
38.12
38.73
38.87
41.27
41.31
41.45
41.78
41.83
42.25
42.35
42.77
45.92
47
46.58
51.32
56.68

Time'
Col. 2
b
15.35
_ b
16.57
18.47
21.92
19.25
19.73
42.77
22.47
26.42
29.67
_ b
29.97
31.32
30
31.23
31.13
b
32.63
b
30.9
b
34.73
34.1
34.55
34.1
34.52
34.23
34.8
40
35.7
37
36.73
37.97
39.65
42.42
39
41
b
44.33
39.28
42.05
47.58
45.4
47.8
50.02
59.07
704

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                                                                              Method 507
Table 1.  Retention Times for Method Analytes (com.)

3   Columns and analytical conditions are described in Sect, 6.8.1 and 6.8.2.
b   Data not available
0   MGK 264 gives two peaks; peak identified in this table used for quantification.
d   Merphos is converted to S,S,S-tributylphosphoro-trithioate (DEF) in the hot GC injection port;
    DEF is actually detected using these analyses conditions.

Table 2.  Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters3
Analyte
Alachlor
Ametryn
Ametraton
Atrazine
Bromacil
Butachlor
Butylate
Carboxin
Chlorpropham
Cycloate
Diazinon
Dichlorvos
Diphenamid
Disulfoton
Disulfoton
 sulfone
Disulfoton
  sulfoxide
EPIC
Ethoprop
Fenamiphos
Fenarimol
Fluridone
Hexazinone
Merphos
Methyl
  paraoxon
Metolachlor
Metribuzin
Mevinphos
MGK 264
Molinate
Napropamide
Norflurazon
Pebulate
Prometon
Prometryn
Pronamide
Propazine
 EDL"
 frg/L)
 0.38
 2
 0.6
 0.13
 2.5
 0.38
 0.15
 0.6
 0.5
 0.25
 0.25
 2.5
 0.6
 0.3
 3.8

 0.38

 0.25
 0.19
 1
 0.38
 3.8
 0.76
 0.25
 2.5

0.75
0.15
 5
 0.5
 0.15
0.25
0.5
0.13
0.3
0.19
0.76
0.13
                          Cone.
 3.8
20
 6
 1.3
25
 3.8
 1.5
 6
 5
 2.5
 2.5
25
 6
 3
 7.5

 3.8

 2.5
 1.9
10
 3.8
38
 7.6
 2.5
25

 7.5
 1.5
50
 5
 1.5
 2.5
 5
 1.3
 3
 1.9
 7.6
 1.3
Reagent
f¥ \
95
91
91
92
91
96
97
102
93
89
115
97
93
89
98
87
85
103
90
99
87
90
96
98
93
101
95
100
98
101
94
94
78
93
91
92
Water
s/
11
10
11
8
9
4
21
4
11
9
7
6
8
10
10
11
9
5
8
5
9
7
8
10
4
5
11
4
18
6
5
9
9
8
10
8
Synthetic
R
82
102
84
89
81
93
36
98
82
97
83
86
88
107
92
88
83
91
87
89
91
86
90
97
92
99
93
91
83
89
101
80
89
91
84
89
Water 1
SR
6
11
7
6
5
15
8
13
7
14
8
6
4
12
5
22
5
7
5
6
11
6
4
8
10
10
6
11
8
5
15
6
5
8
7
6
Synthetic
R
90
96
91
92
88
84
83
87
93
93
84
106
93
95
96
54
86
79
89
89
86
95
92
94
84
86
92
83
89
104
87
98
63
93
92
92
Water 2
SR
8
4
8
5
8
5
8
5
8
3
3
16
5
5
3
19
4
3
2
6
10
9

4
4
4
4
6
9
18
4
15
2
4
8
5
                                                                                    105

-------
Method 507
Table 2.   Single Laboratory Accuracy,  Precision and Estimated Detection Limits
(EDLs) for Analytes from  Reagent Water and Synthetic Groundwaters3 (cont.)
   Analyte

Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triadimefon
Tricyclazole
Vernolate
                 EDL"
0.075
0.25
0.76
1.3
4.5
0.5
0.25
0.65
1
0.13
Cone.
(fjg/U
0.75
2.5
7.6
13
45
5
2.5
6.5
10
1.3
Reagent
Ff |
100
99
98
84
97
97
94
93
86
93
Water
S"
•>R
7
5
6
9
6
4
9
8
7
6
Synthetic
*
86
88
84
85
86
80
91
94
90
79
Water 1
SR
5
4
6
10
5
6
8
5
6
9
Synthetic
"
103
103
95
98
102
77
92
95
90
81
Water 2
s,
14
14
10
13
12
7
4
5
11
2
    Data corrected for blank and represent the analysis of 7-8 samples using mechanical tumbling
    and internal standard calibration.
    EDL  =  estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136 —
    Definition and Procedure for the Determination of the Method Detection Limit—Revision 1.11)
    or a level of compound in a sample yielding a peak in the final extract with signal-to-noise ratio
    of approximately 5, whichever value is higher. The concentration used in determining the EDL
    is not the same as the concentration presented in this table.
    R =  average percent recovery.
    S =  standard deviation of the percent recovery.
    Corrected for amount found in blank; Absopure Nature Artesian Spring Water obtained from
    the Absopure Water Company in Plymouth, Michigan.
    Corrected for amount found in blank; reagent water fortified with fulvic acid  at the 1 mg/L
    concentration level.  A well-characterized  fulvic acid,  available from the International Humic
    Substances Society (associated with the United States Geological Survey in Denver, Colorado),
    was  used.
 106

-------
                                                                              Method 507
Table 3.  Laboratory Performance Check Solution

                                                Cone.
   Test                       Analyte          (fjg/mU           Requirements
   Sensitivity                   Vernolate          0.05     Detection of analyte; S/N> 3
   Chromatographic performance Bromacil           5.0      0.80 < PGF < 1.20a
   Column performance         Prometon          0.30     Resolution >  0.7b
                              Atrazine           0.15

a   PGF = peak Gaussian factor.  Calculated using the equation:
                                         1.83xW  -1
                                  PGF = 	     ' 2
             (•»                                       f    ~\
           .1   is the peak width at half height and W  Ur^r   is the peak  width at tenth height



b   Resolution between the two peaks as defined by the equation:
                                               w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
                                                                                     707

-------

-------
                                 Method 508
 Determination of Chlorinated Pesticides in
     Water by Gas Chromatography with an
                    Electron Capture Detector
                         Revision 3.0 — EPA EMSL-Ci
          J.J. Lichtenberg, J.E. Longbottom, T.A. Bellar, J.W. Eichelberger,
            and R.C. Dressman - EPA 600/4-81-053, Revision 1.0 (1981)
T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey Method 2,
                                       Revision 2.0 (1987)
                    R. L. Graves - Method 508, Revision 3.0 (1989)

-------

-------
                                Method  SOS
        Determination of Chlorinated Pesticides in Water by Gas
           Chromatography with an Electron  Capture Detector

1.    SCOPE AND APPLICA TION
1.1   This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
      nated pesticides in groundwater and finished drinking water.' The following compounds can
      be determined using this method:
               Compound
               Aldrin
               Chlordane-a
               Chlordane-7
               Chloroneb
               Chlorobenzilate*
               Chlorothalonil
               Chlorpyrifos
               DCPA
               4,4'-DDD
               4,4'-DDE
               4,4'-DDT
               Dieldrin
               Endosulfan I
               Endosulfan II
               Endosulfan sulfate
               Endrin
               Endrin aldehyde
               Etridiazole
               HCH-a
               HCH-/3
               HCH-5a
               HCH-7 (Lindane)
               Heptachlor
               Heptachlor epoxide
               Hexachlorobenzene
               Methoxychlor
               cis-Permethrin
               trans-Permethrin
               Propachlor
               Trifluralin
               Aroclor 1016*
               Aroclor 1221*
               Aroclor 1232*
               Aroclor 1242*
               Aroclor 1248*
               Aroclor 1254*
               Aroclor 1260*
               Toxaphene*
               Chlordane*
CAS No.
  309-00-2
 5103-71-9
 5103-74-2
 2675-77-6
  510-15-6
 1897-45-6
 2921-88-2
 1861-32-1
   72-54-8
   72-55-9
   50-29-3
   60-57-1
  959-98-8
33213-65-9
 1031-07-8
   72-20-8
 7421-93-4
 2593-15-9
  319-84-6
  319-85-7
  319-86-8
   58-89-9
   76-44-8
 1024-57-3
  118-74-1
   72-43-5
61949-76-6
61949-77-7
 1918-16-7
 1582-09-8
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11096-82-5
 8001-35-2
   57-74-9
          The extraction conditions of this method are comparable to USEPA Method
          608, which does measure the multicomponent constituents:  commercial
                                                                                777

-------
Method 508
            polychlorinated biphenyl (PCB) mixtures (Aroclors), toxaphene, and chlordane.
            The extract derived from this procedure may be analyzed for these
            constituents by using the GC conditions prescribed in either Method 608
            (packed column) or Method 505 (capillary column).  The columns  used in this
            method may well be adequate,  however, no data were collected for these
            constituents during methods development.

        d    These compounds are only qualitatively identified in the National Pesticides
            Survey (NFS) Program.  These compounds are not quantitated  because control
            over precision has not been accomplished.

1.2   This method  has been validated in a single laboratory and estimated detection limits (EDLs)
      have been determined for the analytes above (Sect. 13). Observed detection limits may vary
      between waters, depending upon  the nature of interferences in the sample matrix and the
      specific instrumentation used.
1.3   This method  is restricted to use by  or  under the supervision of analysts experienced in the use
      of GC 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 Sect.
       10.3.
1.4   Degradation of DDT and Endrin  caused by active sites in the injection port  and GC columns
      may occur.  This is not as much  a problem with new capillary columns as with packed
      columns. However, high boiling sample residue in capillary columns will create the same
      problem after injection of sample extracts.
1.5   Analytes that are not separated chromatographically,  i.e., analytes which have very similar
      retention times cannot be individually  identified and measured in the same calibration mixture
      or water sample unless an alternative technique for identification and quantitation exist (Sect.
       ll.5).
1.6   When this method  is used to analyze unfamiliar samples for any or all of  the analytes above,
      analyte identifications must be confirmed  by at least one additional qualitative technique.

2.    SUMMARY OF METHOD

2.1   A measured volume of sample of approximately 1  L  is solvent extracted with methylene
      chloride by shaking in a separatory funnel or mechanical tumbling in a bottle. The methylene
      chloride extract is isolated, dried and concentrated to a volume of 5 mL after solvent substitu-
      tion with methyl tert-butyl ether (MTBE).  Chromatographic conditions are  described which
      permit the separation and measurement of the  analytes in the extract by capillary column/GC
      with an electron capture detector (ECD).

3.    DEFINITIONS

3.1    Internal  standard: A pure analyte(s) added to a solution in known amount(s) and used to
      measure the relative responses of other method analytes and surrogates that are components of
      the same solution.  The internal standard  must be an analyte that is not a  sample component.
3.2   Surrogate analyte:  A pure analyte(s), which is extremely unlikely to be found in any sample,
      and which is added to a sample aliquot in known amount(s) before extraction and is measured
 772

-------
                                                                                     Method 508
       with the same procedures used to measure other sample components. The purpose of a
       surrogate analyte is to monitor method performance with each sample.
3.3   Laboratory duplicates (LD1 and LD2): Two sample aliquots taken in the analytical laboratory
       and analyzed separately with identical procedures. Analyses of LD1 and LD2 give a measure
       of the precision associated with laboratory procedures, but not with sample collection,
       preservation, or storage procedures.
3.4   Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the same throughout field and laboratory
       procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5   Laboratory reagent blank (LRB): An aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples.  The LRB is used to determine if method
       analytes or other interferences are present in the laboratory environment, the reagents,  or the
       apparatus.
3.6   Field reagent blank (FRB): Reagent  water placed in a sample container in the laboratory and
       treated as a sample in all respects, including exposure to sampling site  conditions, storage,
       preservation and all analytical procedures. The purpose of the FRB is to determine if method
       analytes or other interferences are present in the field environment.
3.7   Laboratory performance check solution (LPC): A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with respect to a defined set of method criteria.
3.8   Laboratory fortified blank (LFB): An aliquot of reagent water to which known quantities of the
       method analytes are added in the laboratory.  The LFB is analyzed exactly like a  sample, and
       its purpose is to determine whether the methodology is in control, and  whether  the laboratory
       is capable of making accurate and precise measurements at the required method detection limit.
3.9   Laboratory fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes  are added in the laboratory. The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results. The background concentrations of the analytes in the sample
       matrix must be determined in a separate  aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock standard solution: A concentrated solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
       an assayed  reference  compound.  Stock standard solutions are used to prepare primary dilution
       standards.
3.11  Primary dilution standard solution: A solution of several analytes prepared in the  laboratory
       from stock  standard solutions and diluted as needed to  prepare calibration solutions and other
       needed analyte solutions.
3.12  Calibration standard (CAL):  A solution prepared from the primary dilution standard solution
       and stock standard  solutions of the internal standards and surrogate analytes.  The CAL
       solutions are used to  calibrate the instrument response with respect to analyte concentration.
                                                                                            113

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Method 508
3.13  Quality control sample (QCS): A sample matrix containing method analytes or a solution of
       method analytes in a water miscible solvent which is used to fortify reagent water or
       environmental samples.  The QCS is obtained from a source external to the laboratory, and is
       used to check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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 Sect. 10.2.
       4.1.1   Glassware must be scrupulously cleaned.2  Clean all glassware as soon as possible
               after use  by thoroughly rinsing with the last solvent used in it. Follow by washing
               with hot water and detergent and thorough rinsing with tap and reagent water.  Drain
               dry, and  heat in an oven or muffle furnace at 400°C for 1 hour.  Do not heat
               volumetric ware.  Thermally stable materials such as PCBs might not be eliminated by
               this treatment.  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.
       4.1.2   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.

       WARNING:        When a solvent is purified, stabilizers added by the manufacturer
       are removed thus potentially making the solvent hazardous.  Also, when a solvent is
       purified, preservatives added by the manufacturer are removed thus potentially reducing
       the shelf-life.

4.2   Interferences by phthalate esters can pose a major problem in pesticide analysis when using the
       electron capture detector.  These compounds generally appear in the chromatogram as large
       peaks.  Common  flexible plastics contain varying amounts of phthalates that are easily
       extracted or leached during laboratory operations.   Cross contamination of clean glassware
       routinely occurs when plastics are handled during extraction steps, especially when
       solvent-wetted surfaces are handled.  Interferences from  phthalates can best 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
4.3   Interfering  contamination may occur when a sample containing low concentrations of analytes
       is analyzed immediately following a sample containing relatively high concentrations of
       analytes. Between-sample rinsing of the sample syringe  and associated equipment with MTBE
       can minimize sample cross contamination.   After analysis of a sample  containing high
       concentrations of analytes, one or more  injections of  MTBE should be made  to ensure that
       accurate values are obtained for the next sample.
 114

-------
                                                                                    Method 508
4.4    Matrix interferences may be caused by contaminants that are coextracted from the sample.
       Also,  note that all the analytes listed in the Scope and Application Section are not resolved
       from each other on any one column, i.e., one analyte of interest may be an interferant for
       another analyte of interest. The extent of matrix interferences will vary considerably from
       source to source, depending upon the water sampled.  Cleanup of sample extracts may be
       necessary.  Positive identifications should be confirmed (Sect. 11.5).
4.5    It is important that samples and standards be contained in the same solvent, i.e., the solvent
       for final working standards must be the same as the final solvent used in sample preparation. If
       this is not the case chromatographic comparability of standards to sample may be affected.
4.6    WARNING:  A dirty injector insert will cause the late eluting compounds to drop off.

5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations
       regarding the safe handling of the chemicals specified in this method. A reference file of
       material safety data 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.
5.2    WARNING:  When a solvent is purified stabilizers added by the  manufacturer are removed
       thus potentially making the solvent hazardous.

6.     APPARA TUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1    Sample Bottle: Borosilicate,  1-L volume with graduations (Wheaton Media/Lab bottle 219820
       or equivalent), fitted with screw caps  lined  with TFE-fluorocarbon. Protect samples from
       light.  The container must be washed  and dried as described in Sect. 4.1.1 before use to
       minimize contamination. Cap liners are cut to fit from sheets (Pierce Catalog No. 012736)
       and extracted with methanol overnight prior to use.
6.2    Glassware
       6.2.1   Separatory funnel:  2000-mL, with TFE-fluorocarbon stopcock,  ground glass or
               TFE-fluorocarbon stopper.
       6.2.2   Tumbler bottle 1.7-L (Wheaton Roller Culture Vessel or equivalent),  with
               TFE-fluorocarbon lined screw cap. Cap liners are cut to fit from sheets (Pierce
               Catalog No.  012736) and extracted with methanol overnight prior to use.
       6.2.3   Flask, Erlenmeyer:   500-mL.
       6.2.4   Concentrator tube, Kuderna-Danish (K-D) 10- or 25-mL, graduated (Kontes
               K-570050-1025 or K-570050-2525 or equivalent).  Calibration must be checked at the
               volumes employed in the test. Ground glass stoppers are used to prevent evaporation
               of extracts.
                                                                                           115

-------
Method 508
       6.2.5   Evaporative flask, K-D 500-mL (Kontes K-570001-0500 or equivalent). Attach to
               concentrator tube with springs.
       6.2.6   Snyder column, K-D three-ball macro (Kontes K-503000-0121 or equivalent).
       6.2.7   Snyder column, K-D two-ball micro (Kontes K-569001-0219 or equivalent).
       6.2.8   Vials:  Glass, 5- to  10-mL capacity with TFE-fluorocarbon lined screw cap.
6.3    Separatory Funnel Shaker:  Capable of holding 2-L separatory funnels and shaking them with
       rocking motion to achieve thorough mixing of separatory funnel contents (available from
       Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4    Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
       (Associated Design and Mfg. Co., Alexandria, VA or other suppliers.).
6.5    Boiling Stones Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033  or equivalent).
       Heat at 400°C for 30 min prior to use. Cool and store in a desiccator.
6.6    Water Bath:  Heated, capable of temperature control (± 2°C).  The bath should be used in a
       hood.
6.7    Balance:  Analytical, capable of accurately weighing to the nearest 0.0001  g.
6.8    Gas Chromatograph: Analytical system complete with temperature programmable GC suitable
       for use with capillary columns and all  required accessories including syringes, analytical
       columns, gases, detector and stripchart recorder. A data system is recommended for measuring
       peak areas.  Table 1 lists retention times observed  for method analytes using the columns and
       analytical conditions described below.
       6.8.1    Column  1 (Primary  column):  30 m long  x 0.25 mm I.D. DB-5 bonded fused silica
               column, 0.25 ^m film thickness (J&W Scientific).  Helium carrier gas  flow is
               established at 30 cm/sec linear velocity and oven temperature is programmed from
               60°C to 300°C at 4°C/min.  Data presented in this method were obtained using this
               column.  The injection volume was  2 /nL splitless mode with a 45 sec.  delay.  The
               injector temperature was 250°C  and the detector temperature was 320°C.  Column
               performance criteria are presented in Table 3 (See Section 10.9).  Alternative columns
               may be used in accordance with the provisions described in Sect.  10.4.
       6.8.2   Column 2 (Alternative column):   30 m long  X 0.25 mm I.D.DB-1701  bonded fused
               silica column, 0.25 /*m film thickness (J&W Scientific).  Helium carrier gas flow is
               established at 30 cm/sec linear velocity and oven temperature is programmed from
               60°C to 300°C at 4°C/min.
       6.8.3   Detector:  Electron capture.  This detector has proven effective in the analysis of
               spiked  reagent and artificial ground  waters.  An ECD was used to generate  the
               validation data presented in this  method.  Alternative detectors, including a  mass
               spectrometer, may be used in accordance with the provisions described in Sect.  10.4.

7.     REAGENTS AND CONSUMABLE MATERIALS

       WARNING:      When a solvent is purified, stabilizers added by the manufacturer
       are removed thus  potentially making the solvent hazardous.  Also, when a solvent is
116

-------
                                                                                    Method 508
       purified, preservatives added by the manufacturer are removed thus potentially reducing
       the shelf-life.

7.1    Acetone, methylene chloride, MTBE:  Distilled-in-glass quality or equivalent.
7.2    Phosphate Buffer, pH7:  Prepare by mixing 29.6 mL 0.1 N Hcl and 50 mL 0.1 M dipotassium
       phosphate.
7.3    Sodium chloride, crystal, ACS grade.  Heat treat in a shallow tray at 450°C for a minimum of
       4 hours to remove interfering organic substances.
7.4    Sodium sulfate, granular, anhydrous, ACS grade.  Heat treat in a shallow tray at 450°C for a
       minimum of 4 hours to remove interfering organic substances.
7.5    Sodium thiosulfate, granular, anhydrous, ACS grade.
7.6    Pentachloronitrobenzene (PCNB) 98%  purity, for use as internal standard.
7.7    4,4'-Dichlorobiphenyl (DCB) 96% purity, for use as surrogate standard (available from
       Chemicals Procurement Inc.).
7.8    Mercuric Chloride—ACS grade:  For use as a bactericide.  If any other bactericide can be
       shown to work as well as mercuric chloride,  it may be used instead.
7.9    Reagent Water: Reagent water is defined as water that is reasonably free of contamination that
       would prevent the determination of any analyte of interest.  Reagent water used to generate the
       validation data in this method was distilled water obtained from the Magnetic Springs Water
       Co.,  Columbus, Ohio.
7.10  Stock Standard Solutions (1.00 ^g//*L): Stock standard solutions may be purchased as
       certified solutions or prepared from pure standard materials using the following procedure:
       7.10.1  Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
               pure  material.  Dissolve the material in  MTBE and dilute to volume in a 10-mL volu-
               metric 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.
       7.10.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw cap amber
               vials.  Store at room temperature and protect from light.
       7.10.3 Stock standard solutions should be replaced after two months or sooner if comparison
               with laboratory fortified  blanks, or QC samples indicate a problem.
7.11   Internal Standard Solution:  Prepare an internal standard fortifying solution by accurately
       weighing approximately 0.0010 g of pure PCNB. Dissolve the PCNB in MTBE and dilute to
       volume in a 10-mL volumetric  flask.  Transfer the  internal standard solution to a TFE-fluoro-
       carbon-sealed  screw cap bottle  and store at room temperature.  Addition of 5 jiL of the
       internal standard fortifying  solution to 5 mL of sample extract results in a final internal
       standard concentration of 0.1 jug/mL.   Solution should be replaced when ongoing QC (Sect.
       10) indicates a problem.  Note  that PCNB has been shown to be an effective internal standard
       for the method analytes,' but other compounds may be used if the quality control requirements
       in Section 10  are met.

                                                                                           777

-------
Method 508
7.12  Surrogate Standard Solution: Prepare a surrogate standard fortifying solution by accurately
       weighing approximately 0.0050 g of pure DCB.  Dissolve the DCS in MTBE and dilute to
       volume in a 10-inL volumetric flask.  Transfer the surrogate standard fortifying solution to a
       TFE-fluorocarbon-sealed screw cap bottle and store at room temperature.  Addition of 50 /xL
       of the surrogate standard fortifying  solution to a 1-L sample prior to extraction results in a
       surrogate standard concentration in the sample of 25 ^ig/L and, assuming quantitative recovery
       of DCB, a surrogate standard concentration in the final extract of 5.0 jug/mL.  Solution should
       be replaced when ongoing QC (Sect. 10) indicates a problem.  Note DCB has been shown to
       be an effective surrogate standard for the method analytes,1 but other compounds may be used
       if the quality control requirements in Section 10 are  met.
7.13  Laboratory Performance  Check Solution:  Prepare by accurately weighing 0.0010 g each of
       chlorothalonil, chlorpyrifos, DCPA, and HCH-6.  Dissolve each analyte in MTBE and dilute
       to volume in individual 10-mL volumetric flasks.  Combine 2  fj.L of the chloropyrifos stock
       solution, 50 ^L of the DCPA stock solution, 50 /*L of the chlorothalonil stock solution, and
       40 /xL of the HCH-5 stock solution to a 100-mL volumetric flask and dilute to volume with
       MTBE.  Transfer to a TFE-fluorcarbon-sealed screw cap bottle and store at room temperature.
       Solution should be replaced  when ongoing QC (Section 10) indicates a problem.

8.     SAMPLE COLLECTION, PRESERVATION, AND  STORAGE

8.1    Grab samples must be collected in glass containers.  Conventional sampling practices (8)
       should be followed; however, the bottle must not be prerinsed with sample before collection.
8.2    Sample Preservation
       8.2.1   Add mercuric chloride (See 7.8) to the  sample bottle  in amounts to produce a
               concentration of 10 mg/L.  Add 1 mL of a  10 mg/mL solution of mercuric chloride in
               reagent water to the sample bottle at the sampling site or in the laboratory  before
               shipping to the sampling site.  A major disadvantage of mercuric  chloride is that it is
               a highly toxic chemical; mercuric chloride must be  handled with caution, and samples
               containing mercuric chloride must be disposed of properly.
       8.2.2   If residual chlorine  is present, add 80 mg of sodium thiosulfate per liter of sample to
               the sample bottle prior to collecting the sample.
       8.2.3   After adding the sample to the bottle containing preservative(s), seal the sample bottle
               and shake vigorously for 1  min.
       8.2.4   Samples must be iced or refrigerated at 4°C from the time of collection until
               extraction.  Preservation study results indicate that most of the target analytes present
               in the samples are stable for 7 days when stored under these conditions.' Preservation
               data for the analytes chlorthalonil, a-HCH,  6-HCH, 7-HCH,  cis-permethrin, trans-
               permethrin, and trifluralin are nondefinitive, and therefore if these are analytes of
               interest, it is recommended that the samples be analyzed immediately.  Analyte
               stability may be affected by the matrix; therefore, the analyst should verify that the
               preservation technique is applicable to the samples under study.
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                                                                                   Method 508
8.3    Extract Storage
       8.3.1  Sample extracts should be stored at 4°C away from light.  A 14-day maximum extract
              storage time is recommended.  However,  analyte stability may be affected by the
              matrix; therefore, the analyst should verify appropriate extract holding times
              applicable to the samples under study.

9.     CALIBRATION

9.1    Establish GC operating parameters equivalent to those indicated in Sect. 6.8.  The GC system
       must be calibrated using the internal standard technique (Sect. 9.2) or the external standard
       technique (Sect. 9.3).  WARNING: DDT and endrin are easily degraded in the injection port
       if the injection port or  front of the column is dirty. This is the result of buildup of high
       boiling residue from sample injection. Check for degradation problems by injecting  a mid-
       level standard containing only 4,4'-DDT and endrin.  Look for the degradation products of
       4,4'-DDT (4,4'-DDE and 4,4'-DDD) and endrin (endrin ketone and endrin aldehyde).   If
       degradation of either DDT or endrin exceeds 20%, take corrective action before proceeding
       with calibration.  Calculate percent breakdown as follows:
          % breakdown for 4,4' -DDT =
Total DDT degradation peak area (DDT + DDD)
  Total DDT peak area (DDT + DDE + DDD)
                                                                     x
          % breakdown for Endrin =

            Total endrin  degradation peak area  (endrin aldehyde + endrin ketone)    , ,,„
           -  X 1UU
             Total endrin peak area (endrin + endrin  aldehyde  + endrin ketone)
      NOTE:  Calibration standard solutions must be prepared such that no unresolved
      analytes are mixed together.

9.2   Internal  Standard Calibration Procedure:  To use this approach, the analyst must select one or
      more internal standards compatible 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.  PCNB has been identified as a suitable internal standard.
      Data presented in this method were generated using the internal standard calibration procedure.
      9.2.1    Prepare calibration standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest and surrogate compound by adding volumes of one
               or more stock standards to a volumetric flask. To each calibration standard, add a
               known constant amount  of one or more of the internal standards, and dilute to volume
               with MTBE.   The lowest standard should represent analyte concentrations near, but
               above, their respective EDLs.  The remaining standards should correspond to the
               range of concentrations expected in the sample concentrates, or should define the
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               working range of the detector. The calibration standards must bracket the analyte
               concentrations found in the sample extracts.
       9.2.2   Analyze each calibration standard according to the procedure (Sect. 11.4). Tabulate
               response ( peak height or area) against concentration for each compound and internal
               standard. Calculate the response factor (RF) for each analyte and surrogate using
               Equation 1.
                                          Equation 1
                    Where:
                    A s  =  Response for the analyte.
                    An  =  Response for the internal standard.
                    Cn  =  Concentration of the internal standard
                    Ct  =  Concentration of the analyte to be measured
       9.2.3   If the RF value over the working range is constant (20% RSD or less) the average RF
               can be used for calculations.  Alternatively, the results can be used to plot a
               calibration curve of response ratios (As/Ais) vs. Cs.
       9.2.4   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 analyte
               varies from the predicted response by more than  ±20%, the test must be repeated
               using a fresh calibration standard.  Alternatively, a new calibration curve must be
               prepared for that analyte.
       9.2.5   Single point calibration is a viable alternative to a calibration curve. Prepare single
               point standards from the secondary dilution standards in MTBE.  The single point
               standards should be prepared at a concentration that produces a response that deviates
               from the sample extract response by no more than 20%.
       9.2.6   Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from an independent source.
               Results from these analyses must be within the limits used to routinely check
               calibration.
9.3    External Standard Calibration Procedure
       9.3.1   Prepare  calibration standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest and surrogate compound by adding volumes of one
               or more stock  standards to a volumetric flask.  Dilute to volume with MTBE. The
               lowest standard should  represent analyte concentrations near, but above, their
               respective EDLs.  The  other concentrations should  correspond to the range of
               concentrations expected in the sample concentrates, or should define the working
               range of the detector. The calibration standards must bracket the analyte
               concentrations found in the sample extracts.
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       9.3.2   Starting with the standard of lowest concentration, analyze each calibration standard
               according to Sect. 11.4 and tabulate response (peak height or area) versus the
               concentration in the standard.  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 (20%  RSD or less), linearity
               through the origin can be assumed and the average ratio or calibration factor can be
               used in place of a calibration curve.
       9.3.3   The working calibration curve or calibration factor must be verified on each working
               day by the measurement of a minimum of two calibration check standards, one at the
               beginning and one at the end of the analysis day. These check standards should be at
               two different concentration levels to verify the calibration curve. For extended periods
               of analysis (greater than 8 hrs.), it  is strongly recommended that check standards be
               interspersed with samples at  regular intervals during the course of the analyses.  If the
               response for any analyte varies from the predicted response by more than ±20%,  the
               test must be repeated using a fresh  calibration standard.  If the results still do  not
               agree, generate a new calibration curve.
       9.3.4   Single point calibration is a viable alternative to a calibration curve.  Prepare single
               point standards from the secondary dilution standards in MTBE.  The single point
               standards should be prepared at a concentration that produces a response that deviates
               from the sample extract  response by no more than 20%.
       9.3.5   Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from an independent source.
               Results from these analyses must be within the  limits used to routinely check
               calibration.

10.   QUALITY CONTROL

10.1   Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
       determination of monitoring internal  standard peak area or height in each sample and blank
       (when internal standard calibration procedures are being employed), analysis of laboratory
       reagent blanks, laboratory fortified samples, laboratory fortified blanks, and QC samples.
10.2  Laboratory Reagent Blanks:  Before processing any samples,  the analyst must demonstrate  that
       all glassware and reagent interferences  are under control.  Each time a set of samples is
       extracted or  reagents are changed, a laboratory reagent blank (LRB) must  be analyzed.  If
       within the retention time window of any analyte of interest the LRB produces a peak that
       would prevent the determination of that analyte, determine the source of contamination and
       eliminate the interference before processing samples.
10.3  Initial Demonstration of Capability
       10.3.1  Select a representative fortified concentration (about  10 times EDL or at the
               regulatory Maximum Contaminant Level, whichever is lower) for each analyte.
               Prepare a sample concentrate (in methanol) containing each analyte at 1000 times
               selected concentration.  With a syringe, add  1 mL of the concentrate to each of at
               least four 1-L aliquots of reagent water, and analyze each aliquot according to
               procedures beginning in Section 11.

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       10.3.2 For each analyte the recovery value for all four of these samples must fall in the
               range of R  + 30% (or within R ± 3SR if broader) using the values for R and SR for
               reagent water in Table 2.  For those compounds that meet the acceptance criteria,
               performance is considered acceptable and sample analysis may begin. For those
               compounds that fail these criteria, this procedure must be repeated using four fresh
               samples until satisfactory performance has been demonstrated.
       10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it. It  is expected that as laboratory personnel gain experience with
               this method the quality of data will improve beyond those required here.
10.4  The analyst is permitted to modify GC columns, GC conditions, GC detectors, continuous
       extraction techniques, concentration techniques (i.e. evaporation techniques),  internal standards
       or surrogate compounds.  Each time such method modifications are made, the analyst must
       repeat the procedures  in Section 10.3.
10.5  Assessing Surrogate Recovery
       10.5.1 When surrogate recovery from a sample or method blank is <70% or > 130%, check
               (1) calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
               contamination or other obvious abnormalities, and (4) instrument performance.  If
               those steps do not reveal the cause of the problem, reanalyze the extract.
       10.5.2 If a blank extract reanalysis fails the 70-130% recovery criterion, the problem must
               be identified and corrected before continuing.
       10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
               for the reanalyzed extract. If sample extract reanalysis continues to fail the surrogate
               recovery criterion, report all data for that sample as suspect.
10.6  Assessing the Internal Standard
       10.6.1 When using the internal  standard calibration procedure, the analyst is expected to
               monitor the IS response  (peak area or peak height) of all samples during each analysis
               day. The IS response for any sample  chromatogram should not deviate from the daily
               calibration check standards IS response by more than 30%.
       10.6.2 If >30% deviation occurs with an  individual extract, optimize instrument
               performance and inject a second aliquot of that extract.
               10.6.2.1 If the reinjected aliquot produces an acceptable internal standard response
                         report results for that  aliquot.
               10.6.2.2 If a deviation of greater than 30%  is obtained for  the re-injected extract,
                         analysis of the sample should be repeated beginning with Section  11,
                         provided the  sample is still available.  Otherwise, report results obtained
                         from the re-injected extract, but annotate as suspect.
       10.6.3 If consecutive samples fail the IS response acceptance criterion, immediately  analyze a
               calibration check standard.
               10.6.3.1 If the check standard provides a response factor (RF) within 20%  of the
                         predicted value, then follow procedures itemized in Section 10.6.2 for each
                         sample failing the IS response criterion.

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               10.6.3.2 If the check standard provides a response factor which deviates more than
                         20% of the predicted value, then the analyst must recalibrate, as specified
                         in Section 9.
10.7 Assessing Laboratory Performance—Laboratory Fortified Blank
      10.7.1  The laboratory must analyze at least one  laboratory fortified blank (LFB) sample with
               every twenty samples or one per sample set (all samples extracted within a 24-h
               period) whichever is greater.   The fortified concentration of each analyte in the LFB
               should be 10 times EDL or the MCL, whichever is less.  Calculate accuracy as
               percent recovery (X,).   If the recovery of any analyte falls outside the control limits
               (see Sect. 10.7.2), that  analyte is judged  out of control, and the source  of the problem
               should be identified and resolved before continuing analyses.
      10.7.2  Until sufficient data become available from within  their own laboratory, usually a
               minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
               performance against the control limits in  Sect. 10.3.2 that are derived from the data in
               Table 2.  When sufficient internal  performance data becomes available, develop
               control limits from the mean percent recovery (x)  and  standard deviation (S) of the
               percent recovery. These data are used to establish upper and lower control limits as
               follows:

                            Upper Control Limit  = X + 3S
                            Lower Control Limit  = X — 3S

               After each five to ten new  recovery measurements, new control limits should be
               calculated using only the most recent 20-30 data points.  These calculated control
               limits should never exceed those established in Sect.  10.3.2.
      10.7.3  It is recommended that  the laboratory periodically document and determine its
               detection limit capabilities for the analytes of interest.
      10.7.4  At least quarterly, analyze  a QC sample from an outside source.
      10.7.5  Laboratories are  encouraged to participate in external performance evaluation studies
               such as the laboratory certification programs offered by many states or  the studies
               conducted by USEPA.  Performance evaluation studies serve as independent checks
               on the analyst's performance.
10.8 Assessing Method Performance—Laboratory Fortified Sample Matrix
      10.8.1  The laboratory must add a  known concentration to  a minimum of  10%  of the routine
               samples or one sample concentration per  set, whichever is greater. The added
               concentration should not be less then the  background concentration of the sample
               selected for fortification. Ideally, the fortified analyte concentrations should be the
               same as that used for the LFB (Section 10.7).  Over time, samples from all routine
               sample sources should be fortified.
      10.8.2  Calculate the percent recovery, P of the concentration for each analyte, after
               correcting the analytical result, X,  from the fortified sample for the background
               concentration, b, measured in the unfortified sample, i.e.,:
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Method 508
                                  p =      100 (X -  b)
                                       fortifying concentration
               and compare these values to control limits appropriate for reagent water data collected
               in the same fashion.  If the analyzed unfortified sample  is found to contain NO
               background concentrations, and the added concentrations are those specified in Sect.
               10.7, then the appropriate control limits would be the acceptance limits in Sect.  10.7.
               If,  on the other hand, the analyzed unfortified sample is found to contain background
               concentration, b, estimate the standard deviation at the background concentration, sb,
               using regressions or comparable background data and, similarly, estimate the mean,
               Xa and standard deviation, sa, of analytical results at the total concentration after
               fortifying. Then the appropriate percentage control limits would be P ±  3sp  , where:
                               p =            100 X
                                    (b + fortifying concentration)
                               and
                                        fortifying concentration
               For example, if the background concentration for Analyte A was found to be 1 jtg/L
               and the added amount was also 1 /ig/L, and upon analysis the laboratory fortified
               sample measured 1.6 /x/L, then the calculated P for this sample would be (1.6 fj,g/L
               minus 1.0 /xg/L)/l /xg/L or 60%.  This calculated P is compared to control limits
               derived from prior reagent water data. Assume it is known that analysis of an
               interference free sample at 1  /xg/L yields an s of 0.12 jig/L and similar analysis at 2.0
               jtg/L yields X and s of 2.01  /xg/L and 0.20 fig/L, respectively. The appropriate
               limits to judge the reasonableness of the percent recovery, 60%, obtained on the
               fortified matrix sample is computed as follows:
                  100  (2.01
                      2.0 fig/L
                + 3 no0)    -           +  (0.20
                100.5%  ± 300 (0.233)  =
                100.5%  ± 70%  or30% to  170%  recovery of the added analyte.
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                                                                                   Method 508
       10.8.3 If the recovery of any such analyte falls outside the designated range, and the
               laboratory performance for that analyte is shown to be in control (Sect. 10.7), the
               recovery problem encountered with the dosed sample is judged to be matrix related,
               not system related.  The result for that analyte in the unfortified sample is labeled
               suspect/matrix to inform the data user that the results are suspect due to matrix
               effects.
10.9  Assessing Instrument System—Laboratory Performance Check Sample: Instrument
       performance should be monitored on a daily basis by analysis of the LPC sample.  The LPC
       sample contains compounds designed to indicate appropriate instrument sensitivity, column
       performance (primary column) and chromatographic performance.  LPC sample components
       and performance criteria are listed in Table 3.  Inability to demonstrate acceptable instrument
       performance indicates the need for reevaluation of the instrument system.  The sensitivity
       requirements are set based on the EDLs published in this method.  If laboratory EDLs differ
       from those listed in this method, concentrations of the instrument QC standard compounds
       must be adjusted to be compatible with the laboratory EDLs.
10.10 The laboratory may adopt additional quality control 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. For example, field or laboratory duplicates may be analyzed to asses
       the precision of the environmental measurements or filed reagent blanks may be used to asses
       contamination of samples under site conditions, transportation and storage.

11.   PROCEDURE

11.1   Extraction (Manual Method)
       11.1.1  Mark the water meniscus on the side of the sample bottle for later determination of
               sample volume (Sect.  11.1.6). Add preservative to blanks and QC check standards.
               Fortify the sample with 50 jiL of the surrogate standard fortifying solution. Pour the
               entire sample into a 2-L separatory funnel.
       11.1.2 Adjust the sample to pH 7 by adding 50 mL of phosphate buffer.  Check pH: add
               H2SO4 or NaOH if necessary.
       11.1.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
       11.1.4 Add 60 mL methylene chloride to the sample bottle, seal, and shake 30 s to rinse the
               inner walls.  Transfer the solvent to the separatory funnel and extract the sample by
               vigorously shaking the funnel for 2 min with periodic venting to release excess
               pressure.  Allow the organic layer to separate from the water phase for a minimum of
               10 min.  If the emulsion interface between layers  is more than one third the volume of
               the solvent layer, the analyst must employ mechanical techniques to complete the
               phase separation. The optimum technique depends upon the sample, but may include
               stirring, filtration of the emulsion through glass wool, centrifugation, or other physical
               methods.  Collect the methylene chloride extract in a 500-mL Erlenmeyer flask.
       11.1.5  Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
               extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
               Perform a third extraction in the same manner.
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Method 508
       11.1.6 Determine the original sample volume by refilling the sample bottle to the mark and
               transferring the water to a 1000-mL graduated cylinder.  Record the sample volume to
               the nearest 5 mL.
11.2  Automated Extraction Method: Data presented in this method were generated  using the
       automated extraction procedure with the mechanical tumbler.
       11.2.1  Mark the water meniscus on the side of the sample bottle for later determination of
               sample volume (Sect. 11.2.6).  Add preservative to blanks and QC check standards.
               Fortify the sample with 50 /tL of the surrogate standard fortifying solution.  If the
               mechanical separately funnel shaker is used,  pour the entire sample into a 2-L
               separatory funnel.  If the mechanical tumbler is used, pour the entire sample into a
               tumbler bottle.
       11.2.2 Adjust the sample to pH 7 by adding 50 mL  of phosphate buffer.  Check pH:  add
               H2SO4 or NaOH if necessary.
       11.2.3 Add 100 g NaCl to the sample, seal, and shake to dissolve salt.
       11.2.4 Add 300 mL methylene chloride to the sample bottle, seal, and shake  30 s to rinse the
               inner walls. Transfer the solvent to the sample contained in the separately funnel or
               tumbler bottle, seal,  and shake for 10 s,  venting periodically.  Repeat  shaking and
               venting until pressure release is not observed during venting. Reseal and place sample
               container in appropriate mechanical mixing device (separately funnel shaker or
               tumbler). Shake or tumble the sample for 1 hour.  Complete mixing of the organic
               and aqueous phases should be observed within about 2 min after starting the mixing
               device.
       11.2.5 Remove  the sample container from the mixing device.  If the tumbler  is used, pour
               contents  of tumbler bottle into a 2-L separatory funnel.  Allow the organic layer to
               separate  from  the water phase for a minimum of 10 min. If the emulsion interface
               between  layers is more than one third the volume of the solvent layer, the analyst
               must employ mechanical techniques to complete the phase separation.  The optimum
               technique depends upon the sample,  but may  include stirring, filtration through glass
               wool, centrifugation, or other physical methods. Collect the methylene chloride
               extract in a 500-mL Erlenmeyer flask.
       11.2.6 Determine the original sample volume by refilling the sample bottle to the mark and
               transferring the water to a 1000-mL graduated cylinder.  Record the sample volume to
               the nearest 5 mL.
11.3  Extract Concentration
       11.3.1  Assemble a 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 Sect. 10.3 are met.
       11.3.2 Dry the extract by pouring it through a solvent-rinsed drying column containing about
               10 cm of anhydrous  sodium sulfate.   Collect  the extract in the K-D  concentrator, and
               rinse the column with 20-30 mL methylene chloride.  Alternatively, add about 5 g
               anhydrous sodium sulfate to  the extract in the Erlenmeyer flask; swirl flask to dry
               extract and allow to sit for 15 min.  Decant the methylene chloride extract into the
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                                                                                   Method 508
              K-D concentrator. Rinse the remaining sodium sulfate with two 25-mL portions of
              methylene chloride and decant  the rinses into the K-D concentrator.
       11.3.3 Add 1 to 2 clean boiling stones to the evaporative flask and attach a macro Snyder
              column.  Prewet the Snyder column by adding about 1 mL methylene chloride to the
              top. Place the K-D apparatus on a hot water bath, 65 to 70°C, so that the
              concentrator tube is partially immersed in the hot water, and the entire lower rounded
              surface of the flask is bathed with hot vapor. Adjust the vertical position of the
              apparatus and the water temperature as required to complete the concentration in  15 to
              20 min. At the  proper rate of  distillation the balls of the column will actively chatter,
              but the chambers will not flood.  When the apparent volume of liquid reaches 2 mL,
              remove the K-D apparatus and allow it to drain and cool for at least 10 min.
       11.3.4 Remove the Snyder column and rinse the flask and its lower joint into the
              concentrator tube with 1 to 2 mL of MTBE.  Add 5-10 mL of MTBE and a fresh
              boiling stone.  Attach a micro-Snyder column to the concentrator tube and prewet the
              column by adding about 0.5 mL of MTBE to the top.   Place the micro K-D apparatus
              on the water bath so that the concentrator tube is partially  immersed in the hot water.
              Adjust the vertical position of the apparatus  and the water  temperature as required to
              complete concentration in 5 to  10 min. When the apparent volume of liquid reaches 2
              mL, remove the micro K-D from the bath and allow it to drain and cool.  Add  5-10
              mL MTBE to the micro K-D and reconcentrate to 2 mL.   Remove the micro K-D
              from the bath and allow it to drain and cool. Remove the  micro Snyder column,  and
              rinse the walls of the concentrator tube while adjusting the volume to 5.0 mL with
              MTBE.
       11.3.5 Transfer extract  to an appropriate-sized TFE-fluorocarbon-sealed screw-cap vial and
              store, refrigerated at 4°C, until analysis by GC-NPD.
11.4  Gas Chromatography
       11.4.1 Sect. 6.8 summarizes the recommended operating conditions for the gas chromato-
              graph.  Included in Table 1 are retention times observed using this method.  Other
              GC columns, chromatographic  conditions, or detectors may be used if the
              requirements of  Sect. 10.3 are  met.
       11.4.2 Calibrate the system  daily as described in Sect. 9.  The standards and extracts must be
              in MTBE.
       11.4.3 If the internal standard calibration procedure is used, add 5 /xL of the internal standard
              fortifying solution to the sample extract, seal, and shake to distribute the internal
              standard.
       11.4.4 Inject 2 /xL of the sample extract.  Record the resulting peak size in area units.
       11.4.5 If the response for the peak exceeds the working range of the system, dilute the
              extract and reanalyze.
11.5  Identification of Analytes
       11.5.1 Identify a sample component by comparison of its retention time to the retention time
              of a reference chromatogram.  If the retention time of an unknown compound
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Method 508
               corresponds, within limits, to the retention time of a standard compound, then
               identification is considered positive.
       11.5.2 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.
       11.5.3 Identification requires expert judgment when sample components are not resolved
               chromatographically. When GC peaks obviously represent  more than one sample
               component (i.e.,  broadened peak with shoulder(s) or valley between two or more
               maxima), or any time doubt exists over  the identification of a peak on  a
               chromatogram, appropriate alternate techniques, to help confirm peak identification,
               need to be employed.  For example, more positive identification may be made by the
               use of an alternative detector which operates on a chemical/physical  principle  different
               from that originally used; e.g., mass spectrometry, or the use of a second
               chromatography column.  A suggested alternative column is described  in Sect. 6.8.

 12.   CALCULATIONS

 12.1  Calculate analyte concentrations in the sample from the response for the analyte using the
       calibration procedure described in Sect. 9.
 12.2  If the internal standard calibration procedure is used, calculate the concentration (C) in the
       sample using the calibration curve or response factor (RF) determined in Sect. 9.2 and
       Equation 2.
                                          Equation 2

                                   C (jiglL) =
                                               (A,)  (RF) (V)
                  Where:
                  As  = Response for the parameter to be measured.
                  Ais  = Response for the internal standard.
                   Is  = Amount of internal standard added to each extract
                  V  = Volume of water extracted  (L).
 12.3  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 determined in Section
       9.3. The concentration (C) in the sample can be calculated from Equation 3.
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                                                                                    Method 508
                                          Equation 3

                                Concentration (us/L)  =
                             Where:
                             A  = Amount of material injected (ng).
                             Vt  = Volume of extract injected
                             Vt  = Volume of total extract
                             V  = Volume of water extracted (mL).
13,  PRECISION AND A CCURA c Y

13.1  In a single laboratory, analyte  recoveries from reagent water were determined at five
      concentration levels. Results were used to determine analyte EDLs and demonstrate method
      range.1 Analytes were divided into two fortified groups for recovery studies. Analyte EDLs
      and analyte recoveries and standard deviation about the percent recoveries at one concentration
      are given in Table 2.
13.2  In a single laboratory, analyte  recoveries from two standard synthetic ground waters were
      determined at one concentration  level.  Results were used to demonstrate applicability of the
      method to different ground water matrices.'  Analyte recoveries from the two synthetic
      matrices are given in Table 2.
                                                                                           725

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Method 508
                                     References
I.     National Pesticide Survey Method No. 2: Determination of Chlorinated Pesticides in
       Groundwater by Gas Chromatography with a Electron Capture Detector.

2.     ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
       Preparation of Sample Containers and for Preservation", American Society for Testing and
       Materials, Philadelphia, PA,  1986.

3.     "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
       Public Health Service, Center for Disease Control, National Institute for Occupational Safety
       and Health, Publication No. 77-206, Aug.  1977.

4.     "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
       and Health Administration, OSHA 2206, (Revised, January 1976).

5.     "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
       Committee on Chemical  Safety, 3rd Edition, 1979.

6.     ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
       Sampling Water," American  Society for Testing and Materials, Philadelphia, PA,  1986.
 130

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                                                                               Method 508
Table 1 . Retention Times
Analyte
Etridiazole
Chloroneb
Propachlor
Trifluralin
HCH-a
Hexachlorobenzene
HCH-/3
HCH-i
PCNB (internal std.)
HCH-6
Chlorthalonil
Heptachlor
Aldrin
Chlorpyrifos
DCPA
Heptachlor epoxide
Chlordane-7
Endosulfan I
Chlordane-a
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
Chlorobenzilate
4,4'-DDD
Endrin aldehyde
Endosulfan sulfate
4,4'-DDT
Methoxychlor
cis-Permethrin
trans-Permethrin
DCB
for Method Analytes
Primary
23.46
25.50
28.90
31.62
31.62
31.96
33.32
33.66
34
35.02
35.36
37.74
40.12
40.6
41.14
42.16
43.52
44.20
44.54
45.90
45.90
46.92
47.60
47.94
48.28
48.62
49.98
50.32
53.38
58.48
58.82
64.1
Retention Time*
(minutes)
| Alternative
22.78
26.18
30.94
b
32.98
b
40.12
35.36
34
41.48
39.78
36.72
38.08
b
41.14
42.16
43.86
43.52
44.54
44.88
45.90
b
51.68
48.28
46.92
46.92
49.30
50.32
53.72
b
b
b
Columns and analytical conditions are described in Sect. 6.8.1 and 6.8.2.
Data not available.
                                                                                     131

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Method 508
Table 2. Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters3
EDL Cone. Reagent Water Synthetic Water T Synthetic Water 2'
Analyte
Aldrin
Chlordane-r*
Chlordane-y
Chloroneb
Chlorobenzilate
Chlorthalonil
DCPA
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrm
Endosulfan 1
Endosulfan
sulfate
Endrin
Endrin
aldehyde
Endosulfan II
Etridiazole
HCH-a
HCH-j3
HCH-5
HCH-y
Heptachlor
Heptachlor
epoxide
Hexachloro-
benzene
Methoxychlor
cis-Permethrin
trans-Permethrin
Propachlor
Trifluralin
(V9/U
0.075
0.0015
0.0015
0.5
5
0.025
0.025
0.0025
0.01
0.06
0.02
0.015
0.015

0.015
0.025

0.024
0.025
0.025
0.01
0.01
0.015
0.01
0.015

0.0077

0.05
0.5
0.5
0.5
0.025
(iig/U
0.15
0.15
0.15
5
10
0.25
0.25
0.25
0.1
0.6
0.2
0.15
0.15

0.15
0.25

0.15
0.25
0.05
0.1
0.1
0.15
0.1
0.15

0.05

0.5
5
5
5
0.25
RC
86
99
99
97
108
91
103
107
99
112
87
87
102

88
88

92
103
92
95
102
89
98
87

99

105
91
111
103
103
ss\
9.5
11.9
11.9
11.6
5.4
8.2
12.4
6.4
11.9
16.8
8.7
8.7
15.3

8.8
7.9

10.1
6.2
10.1
6.7
11.2
9.8
11.8
8.7

21.8

13.7
9.1
6.7
9.3
5.2
R
100
96
96
95
98
103
100
96
96
98
103
102
94

98
103

98
91
106
92
99
115
85
103

82

101
96
97
116
86
I *
11.0
12.5
12.5
6.7
10.8
10.3
13.0
8.6
12.5
11.8
9.3
8.2
1.3

9.8
11.3

10.8
6.4
7.4
5.5
11.9
6.9
11.1
7.2

9.8

10.1
11.5
9.7
4.6
10.3
/?
69
99
99
75
102
71
101
101
99
84
82
84
72

104
84

76
98
86
100
103
85
85
82

68

104
86
102
95
87
sa
9.0
7.9
6.9
8.3
9.2
9.2
6.1
7.1
6.9
8.4
7.4
8.4
12.2

9.4
9.2

6.8
3.9
7.7
6.0
6.2
7.7
7.7
9.8

4.8

6.2
9.5
7.1
7.6
9.6
    Data corrected for amount detected in blank and represent the mean of 7-8 samples.
    EDL=   estimated detection limit; defined as either MDL (Appendix  B to 40 CFR Part 136 —
    Definition and Procedure for the Determination of the Method Detection  Limit —Revision 1.11)
    or a level of compound in a sample yielding a peak in the final extract with signal-to-noise ratio
    of approximately 5,  whichever value is higher. The concentration level used in determining the
    EDL is not the same as the concentration level presented in this table.
    R   =   average percent recovery.
    SR   =   standard deviation of the percent recovery.
    Corrected for amount found in blank;  Absopure Nature Artesian Spring Water Obtained from
    the Absopure Water Company in Plymouth, Michigan.
    Corrected for amount found in blank;  reagent water fortified with fulvic acid at the 1 mg/L
    concentration level.  A  well-characterized fulvic acid, available from  the International Humic
    Substances Society  (associated with the United States Geological Survey in Denver, Colorado),
    was used.
732

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                                                                             Method 508
Table  3.  Laboratory Performance Check Solution

                                                 Cone.
Test                                Analyte      f/jg/U   Requirements
Sensitivity                       Chlorpyrifos      0.0020   Detection of analyte; S/N >  3
Chromatographic performance      DCPA           0.0500   PSF between 0.80 and 1.15a
Column performance              Chlorothalonil     0.0500   Resolution > 0.50b
                                HCH-6           0.0400

3    PGF - peak Gaussian factor.  Calculated using the equation:


                                        1.83xW fl
                                  PGF - 	     I 2

where W  I _   is the peak width at half height and W   _   is the peak width at tenth height



b    Resolution between the two peaks as defined by the equation:

                                          /?-JL

where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline,  of the two peaks.
                                                                                    133

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                               Method 515.1
Determination of Chlorinated Acids in Water
                 by Gas Chromatography with
                  an Electron Capture Detector
                           Revision 4.0 - EPA EMSL-Ci
   B.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
                    J.W. Hodgeson - Method 515, Revision 2.0 (1986)
        T. Engels (Battelle Columbus Laboratories) — National Pesticide Survey
                                 Method 3, Revision 3.0 (1987)
                     R.L. Graves - Method 515.1, Revision 4.0 (1989)

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                                 Method  515.1

           Determination of Chlorinated Acids in  Water by Gas

            Chromatography with an Electron Capture Detector



1.    SCOPE AND APPLICA TION

1.1   This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
      nated acids in ground water and finished drinking water.1 The following compounds can be
      determined by this method:

                  Analyte                                   CAS No.
                  Acifluorfen*                             50594-66-6
                  Bentazon                                25057-89-0
                  Chloramben*                               133-90-4
                  2,4-D                                      94-75-7
                  Dalapon*                                    75-99-0
                  2,4-DB                                     94-8.2-6
                  DCPA acid metabolites*
                  Dicamba                                 1918-00-9
                  3,5-Dichlorobenzoic acid                      51-36-5
                  Dichlorprop                                120-36-5
                  Dinoseb                                     88-85-7
                  5-Hydroxydicamba                        7600-50-2
                  4-Nitrophenol*                             100-02-7
                  Pentachlorophenol (PCP)                      87-86-5
                  Picloram                                 1918-02-1
                  2,4,5-T                                     93-76-5
                  2,4,5-TP                                    93-72-1

       a   DCPA monoacid and diacid metabolites included in method scope; DCPA
           diacid metabolite  used for validation studies.
       *   These compounds are only  qualitatively identified in the National Pesticides
           Survey (NPS) Program. These compounds are not quantitated because control
           over precision has not been accomplished.


1.2   This method may be applicable to the determination of salts and esters of analyte acids. The
      form of each acid is not distinguished by this method. Results are calculated and reported for
      each listed analyte as the total free acid.
1.3   This method has been validated in a single laboratory and estimated detection limits (EDLs)
      have been determined for the analytes above (Sect. 13). Observed detection limits may vary
      between ground waters, depending upon the nature of interferences in the sample matrix and
      the specific instrumentation used.

1.4   This method is restricted to use by or under the supervision of analysts experienced in the use
      of GC 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 Sect.
      10.3.
                                                                                     137

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Method 515.1
1.5    Analytes that are not separated chromatographically i.e., which have very similar retention
       times, cannot be individually identified and measured in the same calibration mixture or water
       sample unless an alternate technique for identification and quantitation exist (Sect.  11.8).
1.6    When this method is used to analyze unfamiliar samples for any or all of the analytes above,
       analyte identifications must be confirmed by at least one additional  qualitative technique.

2.     SUMMARY OF METHOD

2.1    A measured volume of sample of approximately  1 L is adjusted to pH 12 with 6 N sodium
       hydroxide and  shaken for 1  hr to hydrolyze derivatives. Extraneous organic material is
       removed by a solvent wash.  The sample is acidified, and the chlorinated acids are extracted
       with ethyl ether by shaking in a  separatory funnel or mechanical tumbling in a bottle.  The
       acids are  converted to their methyl esters using diazomethane as the derivatizing agent. Excess
       derivatizing reagent is removed, and the esters are determined by capillary column/GC using
       an electron capture detector (ECD).
2.2    The method provides a Florisil cleanup procedure to aid in the elimination of interferences that
       may be encountered.

3.     DEFINITIONS

3.1    Internal standard:  A pure analyte(s) added to a solution in known amount(s) and used to
       measure the relative responses of other method analytes and  surrogates that are components of
       the same  solution.  The internal standard must be an analyte  that is not a sample component.
3.2    Surrogate analyte:  A pure analyte(s), which is extremely unlikely to  be found in any sample,
       and which is added to a sample  aliquot in known amount(s) before  extraction and  is  measured
       with the same  procedures used to measure other sample components.   The purpose of a
       surrogate analyte is to monitor method performance with each sample.
3.3    Laboratory duplicates (LD1 and LD2):  Two sample aliquots taken in the analytical  laboratory
       and analyzed separately with identical procedures.   Analyses of LD1  and LD2 give a measure
       of the precision associated with  laboratory procedures, but not with sample collection, preser-
       vation, or storage procedures.
3.4    Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated  exactly the same throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory reagent blank (LRB):  An aliquot of reagent water that is treated exactly as a
       sample including exposure to  all glassware, equipment, solvents, reagents,  internal standards,
       and surrogates that are used with other samples. The LRB is used to determine if method
       analytes or other interferences are present in the laboratory environment, the reagents, or the
       apparatus.
3.6    Field  reagent blank (FRB):  Reagent water placed  in a sample container in  the laboratory and
       treated as a sample in all respects,  including exposure  to sampling  site conditions, storage,
       preservation and all analytical procedures. The purpose of the FRB is to determine if method
       analytes or other interferences are present in the field environment.


 138

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                                                                                   Method 515.1
3.7    Laboratory performance check solution (LPC):  A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with respect to a defined set of method criteria.
3.8    Laboratory fortified blank (LFB):  An aliquot of reagent water to which known quantities of
       the method analytes are added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to determine whether the methodology is in control, and whether the labora-
       tory is capable of making accurate and precise measurements at the required method detection
       limit.
3.9    Laboratory fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose  is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the sample
       matrix must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock standard solution: A concentrated solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
       an assayed  reference compound. Stock standard solutions are used to prepare primary dilution
       standards.
3.11  Primary dilution standard solution:  A solution of several analytes prepared in the  laboratory
       from stock  standard solutions and diluted  as needed to  prepare calibration solutions and other
       needed analyte solutions.
3.12  Calibration standard (CAL):  A solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards and surrogate analytes.  The  CAL
       solutions are used to calibrate the instrument response  with respect to analyte concentration.
3.13  Quality control sample (QCS):  A sample matrix containing method analytes or a solution of
       method analytes in a water miscible solvent which is used to fortify reagent water  or environ-
       mental samples.  The QCS is obtained from a source external to the laboratory, and is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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 Sect.  10.2.
       4.1.1   Glassware must be scrupulously cleaned.2  Clean all glassware as soon as  possible
               after use by thoroughly rinsing with the last solvent used in it.  Follow by washing
               with hot water and detergent and thorough rinsing with dilute acid, tap and reagent
               water.  Drain dry, and heat in an oven or muffle furnace at 400°C for 1 hr.  Do not
               heat volumetric ware.  Thermally stable materials such as PCBs might not be elimi-
               nated by this treatment.  Thorough rinsing with acetone may be substituted for the
               heating.  After drying and cooling, seal and store glassware in a clean environment to
                                                                                            753

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Method 515.1
               prevent any accumulation of dust or other contaminants.  Store inverted or capped
               with aluminum foil.
       4.1.2   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.
               WARNING:  When a solvent is purified, stabilizers added by the manufacturer are
               removed, thus potentially making the solvent hazardous.  Also, when a solvent is
               purified, preservatives added by the manufacturer are removed, thus potentially
               reducing the shelf-life.
4.2    The acid forms of the analytes are strong organic acids which react readily with alkaline
       substances and can be lost during sample preparation.  Glassware and glass wool must be
       acid-rinsed with IN hydrochloric acid and the sodium sulfate must be acidified with sulfuric
       acid prior to use to avoid analyte losses due to adsorption.
4.3    Organic acids and phenols, especially chlorinated compounds, cause the most direct interfer-
       ence with the determination.  Alkaline hydrolysis and subsequent extraction of the basic
       sample removes many chlorinated hydrocarbons and phthalate esters that might otherwise
       interfere with the electron capture analysis.
4.4    Interferences by phthalate esters  can pose a major problem in pesticide analysis when using the
       ECD.  These compounds generally appear in the chromatogram as large peaks.  Common
       flexible plastics contain varying amounts of phthalates, that are easily extracted or leached
       during  laboratory operations.  Cross contamination of clean glassware routinely occurs when
       plastics are handled during extraction steps, especially when solvent-wetted surfaces are
       handled. Interferences from phthalates can best be minimized by avoiding the use of plastics
       in the laboratory.  Exhaustive purification of reagents and glassware may be required to
       eliminate background phthalate contamination.3'4
4.5    Interfering contamination may occur when a sample  containing low concentrations of analytes
       is analyzed immediately following a sample containing relatively high concentrations of
       analytes. Between-sample rinsing of the sample syringe and associated equipment with methyl-
       t-butyl-ether (MTBE) can minimize sample cross contamination.  After analysis of a sample
       containing high concentrations of analytes, one or more injections of MTBE should be made to
       ensure  that accurate values are obtained for the next  sample.
4.6    Matrix  interferences may be caused by contaminants that are coextracted from the sample.
       Also, note that all analytes listed in the Scope and Application Section are not resolved from
       each other on any one column, i.e., one analyte of interest may be an interferant for another
       analyte of interest.   The extent of matrix interferences will vary considerably from source to
       source,  depending upon the water sampled.   The procedures in Sect.  11 can be used to over-
       come many of these interferences.  Positive identifications should be confirmed (Sect.  11.8).
4.7    It is important that samples and working standards be contained in the same solvent. The
       solvent for working standards must be the same as the final solvent used in sample prepara-
       tion. If this  is not the case, chromatographic comparability of standards to sample may be
       affected.
140

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                                                                                  Method 515.1
5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
       ing the safe handling of the chemicals specified in this method.   A reference file of material
       safety data 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.6"8
5.2    Diazomethane: A toxic carcinogen which can explode under certain conditions.  The follow-
       ing precautions must be followed:
       5.2.1   Use only a well ventilated hood:  do not breath vapors.
       5.2.2   Use a safety screen.
       5.2.3   Use mechanical pipetting aides.
       5.2.4   Do not heat above 90°C: EXPLOSION  may result.
       5.2.5   Avoid grinding surfaces, ground glass joints, sleeve bearings, glass  stirrers:  EX-
               PLOSION may result.
       5.2.6   Store away  from alkali metals:  EXPLOSION may result.
       5.2.7   Solutions of diazomethane decompose rapidly in the presence of solid materials such
               as copper powder, calcium chloride, and boiling chips.
       5.2.8   The diazomethane generation apparatus used in the esterification procedures (Sect.
               11.4 and 11.5) produces micromolar amounts of diazomethane to minimize safety
               hazards.
5.3    Ethyl Ether:  Nanograde, redistilled in glass, if necessary.
       5.3.1   Ethyl ether  is an extremely flammable solvent. If a mechanical device is used for
               sample extraction, the device should be equipped with an explosion-proof motor and
               placed in a hood to avoid possible damage and injury due to an explosion.
       5.3.2   Must be free of peroxides as  indicated by EM Quant test strips (available from Scien-
               tific Products Co., Cat. No.  P1126-8, and other suppliers).
5.4    WARNING: When  a solvent is purified, stabilizers added by the manufacturer are removed,
       thus potentially making the solvent hazardous.

6.     APPARA TUS AND EQUIPMENT
(All specifications are suggested. Catalog numbers are included for illustration only.)
6.1    Sample Bottle: Borosilicate, 1-L volume with graduations (Wheaton Media/Lab bottle 219820
       or equivalent), fitted with screw caps  lined with TFE-fluorocarbon.  Protect  samples from
       light.  The container must be washed  and dried as described in  Sect. 4.1.1 before use  to
       minimize contamination. Capliners are cut to fit from sheets (Pierce Catalog No. 012736) and
       extracted with methanol overnight prior to use.
                                                                                           141

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Method 515 1
6.2    Glassware
       6.2.1   Separatory funnel:  2000-mL, with TFE-fluorocarbon stopcocks, ground glass or
               TFE-fluorocarbon stoppers.
       6.2.2   Tumbler bottle:  1.7-L (Wheaton Roller Culture Vessel or equivalent), with
               TFE-fluorocarbon lined screw cap.  Cap liners are cut to fit from sheets (Pierce
               Catalog No. 012736) and extracted with methanol overnight prior to use.
       6.2.3   Concentrator tube, Kuderna-Danish (K-D):  10- or 25-mL, graduated (Kontes
               K-570050-2525 or Kontes K-570050-1025 or equivalent).  Calibration must be
               checked at the volumes employed in the test.  Ground glass stoppers are used to
               prevent evaporation of extracts.
       6.2.4   Evaporative flask, K-D:  500-mL (Kontes K-570001-0500 or equivalent).  Attach to
               concentrator tube with springs.
       6.2.5   Snyder column, K-D:  three-ball macro (Kontes K-503000-0121 or equivalent).
       6.2.6   Snyder column, K-D:  two-ball  micro (Kontes K-569001-0219 or equivalent).
       6.2.7   Flask, round-bottom:  500-mL with 24/40 ground glass joint.
       6.2.8   Vials: glass, 5- to 10-mL capacity with TFE-fluorocarbon lined screw cap.
       6.2.9   Disposable pipets: sterile plugged borosilicate glass, 5-mL capacity (Corning
               7078-5N or equivalent).
6.3    Separatory Funnel Shaker:  Capable of holding 2-L separatory funnels and shaking them with
       rocking motion to achieve thorough mixing of separatory funnel contents (available from
       Eberbach Co. in Ann Arbor, MI or other suppliers).
6.4    Tumbler: Capable of holding tumbler bottles and tumbling them end-over-end at 30 turns/min
       (Associated Design and Mfg. Co., Alexandria, VA and other suppliers).
6.5    Boiling Stones:  Teflon, Chemware (Norton Performance Plastics No. 015021 and other
       suppliers).
6.6    Water Bath:  Heated,  capable of temperature control (+  2°C).  The bath should be used in a
       hood.
6.7    Balance:  Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.8    Diazomethane Generator:  Assemble  from two 20 x 150 mm test tubes, two Neoprene rubber
       stoppers, and a source of nitrogen as shown in Figure  1 (available from Aldrich Chemical
       Co.).  When esterification is performed  using diazomethane solution, the diazomethane collec-
       tor is cooled in an approximately 2-L thermos for ice bath or a cryogenically cooled vessel
       (Thermoelectrics Unlimited Model SK-12 or equivalent).
6.9    Glass Wool:  Acid washed (Supelco 2-0383 or equivalent) and heated at 450°C for 4 hr.
6.10  Gas Chromatograph:  Analytical system complete with temperature programmable GC suitable
       for use with capillary columns and all required accessories including syringes, analytical
       columns, gases, detector and stripchart recorder.  A data system is  recommended for measur-
       ing peak areas.  Table I lists retention times observed for method analytes using the columns
       and analytical conditions described below.
 742

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                                                                                 Method 515.1
       6.10.1  Column 1 (Primary column):  30 m long x 0.25 mm I.D. DB-5 bonded fused silica
               column, 0.25 /^m film thickness (J&W Scientific).  Helium carrier gas flow is estab-
               lished at 30 cm/sec linear velocity and oven temperature is programmed from 60°C to
               300°C at 4°C/min. Data presented in this method were obtained using this column.
               The injection volume was 2 pL splitless mode with 45 second delay. The injector
               temperature was 250°C and the detector was  320°C.  Alternative columns may be
               used in accordance with the provisions described in Sect. 10.2.
       6.10.2  Column 2 (Confirmation column):  30 m long x 0.25 mm I.D. DB-1701 bonded
               fused silica column, 0.25 um film thickness (J&W Scientific). Helium carrier gas
               flow is established at 30 cm/sec linear velocity and oven temperature is programmed
               from 60°C to 300°C at 4°C/min.
       6.10.3  Detector: Electron capture.  This detector has proven effective in the analysis  of
               fortified reagent and artificial ground  waters.  An BCD was used  to generate the
               validation data presented in this method.  Alternative detectors, including a mass
               spectrometer, may be used in accordance with the provisions described in Sect. 10.3.

 7.     REAGENTS AND CONSUMABLE MATERIALS

       WARNING:       When a solvent is purified, stabilizers added by the manufacturer
       are removed, thus potentially making the solvent hazardous. Also, when a solvent is
       purified, preservatives added by the manufacturer are  removed, thus potentially reduc-
       ing the shelf-life.

7.1    Acetone, Methanol, Methylene Chloride, MTBE:  Pesticide quality or equivalent.
7.2    Ethyl Ether,  Unpreserved:  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. PI126-8, and other suppliers).  Procedures recommended for removal  of peroxides are
       provided with the test strips.
7.3    Sodium Sulfate, Granular, Anhydrous, ACS Grade:  Heat treat in a shallow tray at 450°C for
       a minimum of 4 hr to remove interfering organic substances. 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.  The pH must be below
       pH 4.  Store at 130°C.
7.4    Sodium Thiosulfate, Granular, Anhydrous:  ACS grade.
7.5    Sodium Hydroxide (NAOH), Pellets:  ACS grade.
       7.5.1    NaOH, 6 N:  Dissolve 216 g NaOH in 900 mL reagent water.
7.6    Sulfuric  Acid, Concentrated: ACS grade,sp. gr. 1.84.
       7.6.1    Sulfuric acid, 12 N: Slowly add 335 mL concentrated sulfuric acid to 665 mL  of
               reagent water.
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Method 515.1
7.7    Potassium Hydroxide (KOH), Pellets:  ACS grade.
       7.7.1  KOH, 37%  (w/v): Dissolve 37 g KOH pellets in reagent water and dilute to 100 mL.
7.8    Carbitol (Diethylene Glycol Monoethyl Ether): ACS grade. Available from Aldrich Chemical
       Co.
7.9    Diazald, ACS grade:  Available from Aldrich Chemical Co.
7.10  Diazald Solution:  Prepare a solution containing 10 g Diazald in  100 mL of a 50:50 by volume
       mixture of ethyl ether and carbitol.  This solution is stable for one month or longer when
       stored at 4°C in an amber bottle with a Teflon-lined screw cap.
7.11  Sodium Chloride (NACL), Crystal, ACS Grade:  Heat treat in a  shallow tray at 450°C for a
       minimum of 4 hr to remove interfering organic substances.
7.12  4,4-Dibromooctafluorobiphenyl (DBOB): 99% purity, for use as internal standard (available
       from Aldrich Chemical Co).
7.13  2,4-Dichlorophenylacetic ACID (DCAA):  99% purity, for use as surrogate standard (available
       from Aldrich Chemical Co).
7.14  Mercuric  Chloride:  ACS grade (Aldrich Chemical Co.) - for use as a bacteriocide.  If any
       other bactericide can be shown to work as well as mercuric chloride, it may be used instead.
7.15  Reagent Water:  Reagent water is defined as water that is reasonably free of contamination that
       would prevent the determination of any analyte of interest.  Reagent water used to generate the
       validation data in this method was distilled water obtained from the Magnetic Springs Water
       Co., Columbus, Ohio.
7.16  Silicic Acid, ACS Grade.
7.17  Florisil: 60-100/PR  mesh (Sigma No. F-9127). Activate by heating in a shallow container at
       150°C for at least 24 and not more than 48 hr.
7.18  Stock Standard Solutions (1.00 /*g//LtL):  Stock standard solutions may be purchased as certi-
       fied solutions or prepared from pure standard materials using the following procedure:
       7.18.1 Prepare stock standard solutions by accurately weighing  approximately 0.0100 g of
              pure material.  Dissolve the material in MTBE and dilute to volume in a 10-mL volu-
              metric 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 pre-
              pared stock  standards may be used at any concentration  if they are  certified by the
              manufacturer or by an independent source.
       7.18.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed  screw cap amber
              vials.  Store at  room temperature and protect from light.
       7.18.3 Stock standard  solutions should be replaced after two months or sooner if comparison
              with laboratory fortified blanks, or QC samples indicate a problem.
7.19  Internal Standard Solution: Prepare an internal standard solution by accurately weighing
       approximately 0.0010 g of pure DBOB.  Dissolve the DBOB in MTBE and  dilute to volume in
       a  10-mL volumetric flask.  Transfer the internal standard solution to a TFE-fluoro-
       carbon-sealed screw  cap bottle and store at room temperature.  Addition of  25 pL of the
       internal standard solution to 10 mL of sample extract results in a final internal standard

144

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                                                                                 Method 515.1
      concentration of 0.25 /xg/mL.  Solution should be replaced when ongoing QC (Sect. 10)
      indicates a problem.  Note that DBOB has been shown to be an effective internal standard for
      the method analytes, but other compounds may be used if the quality control requirements in
      Sect. 10 are met.1
7.20 Surrogate Standard Solution:  Prepare a surrogate standard solution by accurately weighing
      approximately 0.0010 g of pure DCAA.   Dissolve the DCAA in MTBE and dilute to volume
      in a 10-mL volumetric flask.  Transfer the surrogate standard solution to a TFE-fluoro-
      carbon-sealed screw cap bottle and store at room temperature.  Addition of 50 pL of the
      surrogate standard solution to a 1-L sample prior to extraction results in a surrogate standard
      concentration in the sample of 5 jug/L and, assuming quantitative recovery of  DCAA, a
      surrogate standard concentration in the final extract of 0.5 jtg/mL. Solution should be re-
      placed when ongoing QC (Sect. 10) indicates a problem.  Note DCAA has been shown to be
      an effective surrogate standard for the method analytes(l), but other compounds may be used
      if the quality control requirements in Sect. 10.4 are met.
7.21 Laboratory Performance Check Solutions: Prepare a diluted dinoseb solution by adding 10 pL
      of the 1.0 /ig/jttL dinoseb stock solution to the MTBE and diluting to volume  in a 10-mL
      volumetric flask. To prepare the  check solution, add 40 jtL of the diluted dinoseb solution, 16
      nL of the 4-nitrophenol stock solution, 6  /iL of the 3,5-dichlorobenzoic acid stock solution, 50
      /iL of the surrogate standard solution, 25 /xL of the internal standard solution, and 250 (j,L of
      methanol to a 5-mL volumetric flask and dilute to volume with MTBE.  Methylate sample as
      described in  Sects.  11.4 or 11.5.  Dilute  the sample to 10 mL in MTBE.  Transfer to a TFE-
      fluorocarbon-sealed screw cap bottle and  store at room temperature.  Solution should be
      replaced when ongoing QC (Sect. 10) indicates a problem.

8.    SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1   Grab samples must be collected in glass containers.  Conventional sampling practices should
      be followed; however, the bottle must not be prerinsed with sample before collection.8
8.2   Sample Preservation and Storage
      8.2.1   Add mercuric chloride (See 7.14) to the sample bottle in amounts to  produce a con-
               centration  of 10 mg/L. Add 1 mL of a 10 mg/mL solution of mercuric chloride in
               water to the  sample bottle at the sampling site or in the laboratory before shipping to
               the sampling site. A major disadvantage of mercuric chloride is that it is a highly
               toxic chemical; mercuric chloride must be handled with caution,  and samples contain-
               ing mercuric chloride must be disposed of properly.
      8.2.2   If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
               the sample bottle prior to collecting the sample.
      8.2.3   After the sample is collected in the bottle containing preservative(s),  seal the bottle
               and shake  vigorously for 1 min.
      8.2.4   The samples must be iced or refrigerated at 4°C away from light from the time of
               collection  until extraction.  Preservation study results indicate that the analytes (mea-
               sured as total acid) present in samples are stable for 14 days when stored under these
               conditions.1  However, analyte stability may be affected by the matrix; therefore, the
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               analyst should verify that the preservation technique is applicable to the samples under
               study.
8.3    Extract Storage
       8.3.1   Extracts should be stored at 4°C away from light.  Preservation study results indicate
               that most analytes are stable for 28 days; however, the analyst should verify appro-
               priate extract holding times applicable to the samples under study.'

9.     CALIBRA TION

9.1    Establish GC operating parameters equivalent to those indicated in Sect. 6.10. The GC system
       may be calibrated using either the internal standard technique (Sect. 9.2) or the external
       standard technique (Sect. 9.3).

       NOTE:  Calibration standard solutions must be prepared such that no unresolved
       analytes are mixed together.

9.2    Internal standard calibration procedure:   To use this approach, the analyst must select one or
       more internal standards compatible 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. DBOB has been identified as a  suitable internal standard.
       9.2.1   Prepare calibration standards at  a minimum of three (recommend five) concentration
               levels for each analyte of interest by adding volumes of one or more stock standards
               to a volumetric flask.  To each calibration  standard, add a known constant amount of
               one or more of the internal standards and 250 /*L methanol, and dilute to  volume with
               MTBE.  Esterify acids with diazomethane as described in Sect. 11.4 or 11.5.  The
               lowest standard should represent analyte concentrations near, but above, the respective
               EDLs. The remaining standards should bracket the analyte concentrations expected in
               the sample extracts, or should define the working range of  the detector.
       9.2.2   Analyze each calibration standard according to the procedure (Sect. 11.7). Tabulate
               response (peak height or area) against concentration for each compound and internal
               standard.  Calculate the response factor (RF) for each analyte and surrogate using
               Equation 1.
                                          Equation 1
                    where:
                    A^  =  Response for the analyte to be measured.
                    /4(1  =  Response for the internal standard.
                    CM  =  Concentration of the internal standard
                    C(  =  Contentration of the analyte to be measured
146

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                                                                                    Method 515.1
       9.2.3  If the RF value over the working range is constant (20% RSD or less) the average RF
               can be used for calculations.  Alternatively, the results can be used to plot a calibra-
               tion curve of response ratios (AS/A1S) vs. Cs.
       9.2.4  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 analyte
               varies from the predicted response by more than +20%, the test must be repeated
               using a fresh calibration standard.  If the repetition also fails, a new calibration curve
               must be generated for that analyte using freshly prepared standards.
       9.2.5  Single point calibration is a viable alternative to a calibration curve.  Prepare single
               point standards from the secondary dilution standards in MTBE.  The single point
               standards should be prepared at a concentration that produces a response that deviates
               from the sample extract response by no more than 20%.
       9.2.6  Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard  prepared from reference material obtained from an independent source.  Re-
               sults from these analyses must be within the limits used to routinely check calibration.
9.3    External Standard Calibration Procedure
       9.3.1   Prepare calibration standards at a minimum of three (recommend  five) concentration
               levels for each analyte of interest and surrogate compound by adding volumes of one
               or more  stock standards and 250 pL methanol to a volumetric flask.  Dilute to volume
               with MTBE. Esterify  acids with diazomethane as described in Sect.  11.4 or  11.5.
               The best  standard should represent analyte concentrations near, but above, the respec-
               tive EDL.  The remaining standards should bracket the analyte concentrations expect-
               ed in the sample extracts, or should define the working range of the detector.
       9.3.2  Starting with the standard of lowest concentration, analyze each calibration standard
               according to Sect.  11.7 and tabulate response (peak height or area) versus the concen-
               tration in the standard. 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 (20% RSD or less), linearity through the
               origin can be assumed  and the average ratio or calibration factor can be used  in place
               of a calibration curve.
       9.3.3   The working calibration curve or calibration factor must be verified on each working
               day by the measurement of a minimum of two calibration check standards, one at the
               beginning and one at the end  of the analysis day.  These check standards should be at
               two different concentration levels to verify the calibration curve.  For extended
               periods of analysis (greater than  8 hr), it is strongly recommended that check stan-
               dards  be  interspersed with samples at regular intervals during the  course of the analy-
               ses.  If the  response for any analyte varies from the predicted response by more than
               ±20%, the test must be repeated using a fresh calibration standard. If the results still
               do not agree, generate  a new  calibration curve or use a single point calibration
               standard  as described in Sect. 9.3.3.
       9.3.4  Single point calibration is a viable alternative to a calibration curve.  Prepare  single
               point standards  from the secondary dilution standards in MTBE.  The single point
                                                                                             147

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Method 515.1
               standards should be prepared at a concentration that produces a response that deviates
               from the sample extract response by no more than 20%.
       9.3.5   Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from an independent source.
               Results from these analyses must be within the limits used to routinely check
               calibration.

10.   QUALITY CONTROL

10.1   Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
       determination of surrogate compound recoveries in each sample and blank, monitoring internal
       standard peak area or height in each sample and blank (when internal standard calibration
       procedures are being employed), analysis of laboratory reagent blanks, laboratory fortified
       samples, laboratory fortified blanks, and QC samples.
10.2  Laboratory reagent blanks (LRB).  Before processing any samples, the analyst must
       demonstrate that all glassware and reagent interferences are under control. Each time a set of
       samples is extracted or reagents are changed, a LRB must be analyzed.  If within the retention
       time window of any analyte the LRB produces a peak that would prevent  the determination of
       that analyte, determine the source of contamination and eliminate the interference before
       processing samples.
10.3  Initial demonstration of capability
       10.3.1 Select a representative fortified concentration (about 10 times EDL)  for each analyte.
               Prepare a sample concentrate (in methanol) containing each analyte at  1000 times
               selected concentration.  With a syringe, add 1 mL of the concentrate to each of at
               least four 1-L aliquots of reagent water, and analyze each aliquot according to
               procedures beginning in Sect. 11.
       10.3.2 For each  analyte the recovery value for all four of these samples must fall in the
               range of R ± 30% (or within R ± 3SR if broader) using the values for R and SR for
               reagent water in Table 2.   For those compounds that meet the acceptable criteria,
               performance is considered  acceptable and sample analysis may begin.  For those
               compounds that fail these criteria, this procedure must be reported using five fresh
               samples until satisfactory performance has been demonstrated.
       10.3.3 The initial demonstration of capability is used primarily to preclude  a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it.  It is expected that as laboratory personnel gain experience with
               this method the quality of data will improve beyond those required here.
10.4  The analyst is permitted to modify GC columns, GC conditions, detectors, continuous
       extraction techniques, concentration techniques (i.e., evaporation techniques), internal standard
       or surrogate compounds. Each time such method modifications are made, the analyst must
       repeat the procedures in Sect. 10.3
10.5  Assessing Surrogate Recovery.
       10.5.1 When surrogate recovery from a sample or method blank is <70%  or  > 130%, check
               (1) calculations to locate possible errors, (2) spiking solutions for degradation, (3)
 748

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                                                                                    Method 515.1
               contamination, and (4) instrument performance.  If those steps do not reveal the cause
               of the problem, reanalyze the extract.
       10.5.2 If a blank extract reanalysis fails the 70-130% recovery criterion, the problem must
               be identified and corrected before continuing.
       10.5.3 If sample extract reanalysis meets the surrogate recovery criterion, report only data
               for the analyzed extract.  If sample extract continues to fail the recovery criterion,
               report all data for that sample as suspect.
10.6  Assessing the internal standard
       10.6.1 When using the internal standard calibration procedure, the analyst is expected to
               monitor the IS response (peak area or peak height) of all samples during each analysis
               day.  The IS response for any sample chromatogram should not deviate from the daily
               calibration check standard's IS response by more than 30%.
       10.6.2 If >30% deviation occurs with an individual extract, optimize instrument
               performance and inject a second aliquot of that extract.
               10.6.2.1  If the reinjected aliquot produces an acceptable internal standard response,
                          report results for that aliquot.
               10.6.2.2  If a deviation of greater than 30%  is obtained for the reinjected extract,
                          analysis of the samples should be repeated beginning with Sect. 11,
                          provided the sample is still available. Otherwise, report results obtained
                          from the reinjected extract, but annotate as suspect.
       10.6.3 If consecutive samples fail the IS response acceptance criterion,  immediately analyze a
               calibration check standard.
               10.6.3.1  If the check standard provides a response factor (RF) within 20% of the
                          predicted value, then follow procedures itemized in Sect. 10.6.2 for each
                          sample failing the IS response criterion.
               10.6.3.2  If the check standard provides a response factor which deviates more than
                          20% of the predicted value, then the analyst must recalibrate, as specified
                          in Sect. 9.
10.7  Assessing laboratory performance: Laboratory Fortified Blank
       10.7.1  The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
               every 20 samples or one per sample set (all samples extracted within a 24-hr period)
               whichever is greater.  The concentration of each analyte in the LFB should be  10
               times EDL or the MCL, whichever is less.  Calculate accuracy as percent recovery
               (X,).  If the recovery of any analyte falls outside the control limits (Sect. 10.7.2), that
               analyte is judged out of control, and the source of the problem should be identified
               and resolved before continuing analyses.
       10.7.2 Until sufficient data become available from within their own laboratory, usually a
               minimum of results from 20  to 30 analyses, the laboratory should assess laboratory
               performance against the control limits in Sect.  10.3.2 that are derived from the data in
               Table 2.  When sufficient internal performance data becomes available,  develop
               control limits from the mean percent  recovery (X) and standard deviation (S) of the
                                                                                            149

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Method 515.1
               percent recovery. These data are used to establish upper and lower control limits as
               follows:

                            Upper Control Limit = x  + 3S
                            Lower Control Limit = X  - 3S

               After each five to ten new recovery measurements, new control limits should be
               calculated using only the most recent 20-30 data points.  These calculated control
               limits should never exceed those established in Section 10.3.2.
       10.7.3 It is recommended that the laboratory periodically determine and document its
               detection limit capabilities for the analytes of interest.
       10.7.4 At least quarterly, analyze a QC sample from an outside source.
       10.7.5 Laboratories are encouraged to participate in external performance evaluation studies
               such as the laboratory certification programs offered by many states or the studies
               conducted by USEPA.  Performance  evaluation  studies serve as independent checks
               on the analyst's performance.
10.8  Assessing Analyte Recovery—Laboratory Fortified Sample Matrix
       10.8.1 The laboratory must add a known concentration to a minimum  of 10% of the routine
               samples or one sample concentration  per set, whichever is greater.  The concentration
               should not be less then the background concentration of the  sample selected for
               fortification.  Ideally, the concentration should be the same  as that used for the
               laboratory fortified blank (Sect.  10.7). Over time, samples  from all routine sample
               sources should be fortified.
10.8.2        Calculate the percent recovery,  P of the concentration for each analyte,  after
               correcting the analytical result, X, from the fortified sample for the background
               concentration, b, measured in the unfortified sample:
                                  p =       100 (X - V)
                                       fortifying concentration
               and compare these values to control limits appropriate for reagent water data collected
               in the same fashion.  If the analyzed unfortified sample is found to contain NO
               background concentrations, and the added concentrations are those specified in Sect.
               10.7,  then the appropriate control limits would be the acceptance limits in Sect. 10.7.
               If, on the other hand, the analyzed unfortified sample is found to contain background
               concentration, b, estimate the standard deviation at the background concentration, sb,
               using  regressions or comparable background data and, similarly, estimate the mean,
               xa  and standard deviation, sa, of analytical results at the total concentration after
               fortifying.  Then the appropriate percentage control limits would be  P + 3sP,  where:
150

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                                                                                 Method 515.1
                               p =            100X
                                    (b + fortifying concentration)

                              and
                              ,,  -  100 _        _
                                        fortifying concentration
              For example, if the background concentration for Analyte A was found to be 1 /xg/L
              and the added amount was also  1 /*g/L, and upon analysis the laboratory fortified
              sample measured  1.6 jig/L, then the calculated P for this sample would be (1.6 /*g/L
              minus 1.0 /^g/L)/l pig/L or 60%.  This calculated P is compared to control limits
              derived from prior reagent water data. Assume  it is known that analysis of an
              interference free sample at 1 ^g/L yields an s of 0.12 jtg/L and similar analysis at 2.0
              /ig/L yields X and s of 2.01 /xg/L and 0.20 /ig/L, respectively.  The appropriate limits
              to judge the reasonableness of the percent recovery, 60%, obtained on the fortified
              matrix sample is computed as follows:
                  100 (2.01 pg/L)
                     2.0 v-glL

                +  3 (100)  [(0-12 ng/L)2 + (0.20
                                      1.0  /ig/L
                100.5% ±  300 (0.233) =
                100.5% ±  70% or30% to 170% recovery of the added  analyte.
       10.8.3 If the recovery of any such analyte falls outside the designated range, and the
              laboratory performance for that analyte is shown to be in control (Sect. 10.7), the
              recovery problem encountered with the fortified sample is judged to be matrix related,
              not system related.  The result for that analyte in the unfortified sample is labeled
              suspect/matrix to inform the data user that the results are suspect due to matrix
              effects.
10.9   Assessing instrument system:  Laboratory Performance Check Sample.  Instrument
       performance should be monitored on a daily basis by analysis of the LPC sample.  The LPC
       sample contains compounds designed to indicate appropriate instrument sensitivity, column
       performance (primary column) and chromatographic performance.  LPC sample components
       and performance criteria are listed in Table 3.  Inability to  demonstrate acceptable instrument
       performance indicates the need for reevaluation of the instrument system.  The sensitivity
       requirements are set based on the EDLs published in this method.  If laboratory EDLs differ
       from those listed in this method, concentrations of the instrument QC standard compounds
       must be adjusted to be compatible with the laboratory EDLs.
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Method 515.1
10.10 The laboratory may adopt additional quality control 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.  For example, field or laboratory duplicates may be analyzed to assess
      the precision of the environmental measurements or field reagent blanks may be used to assess
      contamination of samples under site conditions, transportation and storage.

; 1.  PROCEDURE

11.1  Manual Hydrolysis, Preparation, and Extraction
      11.1.1  Add preservative to blanks and QC check standards. Mark the water meniscus  on the
               side of the sample bottle for later determination of sample volume (Sect.  11.1.9).
               Pour the entire sample into a 2-L separatory funnel. Fortify sample with 50 /*L of the
               surrogate standard solution.
      11.1.2  Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
      11.1.3  Add 17 mL of 6 N NaOH to the sample, seal, and  shake.  Check the pH of the
               sample with pH paper; if the sample does not have a pH greater than or equal to 12,
               adjust  the pH  by adding more 6 N NaOH.   Let the sample sit  at room temperature for
               1 hr, shaking  the separatory funnel and contents periodically.
      11.1.4  Add 60 mL methylene chloride to the sample  bottle to rinse the bottle, transfer  the
               methylene chloride to the separatory funnel and extract the sample by vigorously
               shaking the funnel for 2 min with periodic venting to release excess pressure.  Allow
               the organic layer to separate from the water phase for a minimum of 10 min. If the
               emulsion interface between layers is more than one-third the volume of the solvent
               layer,  the analyst must employ mechanical techniques to complete the phase
               separation.  The optimum technique depends upon the sample, but may include
               stirring, filtration through glass wool, centrifugation, or other  physical methods.
               Discard  the methylene chloride phase.
       11.1.5  Add a second 60-mL volume of methylene chloride to  the sample bottle and repeat the
               extraction procedure a second time, discarding the methylene chloride layer.  Perform
               a third extraction in the same manner.
       11.1.6  Add 17 mL of 12 N H2SO4 to the sample,  seal, and shake to mix.  Check the pH of
               the sample with pH paper; if the sample does not have a pH less than or equal to 2,
               adjust the pH  by adding more  12 N H2SO4.
       11.1.7  Add 120 mL  ethyl ether to the sample, seal, and extract the sample by vigorously
               shaking  the funnel for 2 min with periodic venting  to release excess pressure.  Allow
               the organic layer to separate from the water phase for  a minimum  of 10 min.  If the
               emulsion interface between layers is more than one third the volume of the solvent
               layer,  the analyst must employ mechanical techniques to complete  the phase
               separation.  The optimum technique depends upon the sample, but may include
               stirring, filtration through glass wool, centrifugation, or other physical methods.
               Remove the aqueous phase to a 2-L Erlenmeyer flask and collect the ethyl ether phase
               in a 500-mL round-bottom flask containing approximately  10 g of acidified anhydrous
               sodium sulfate.  Periodically, vigorously shake the sample and drying agent.  Allow
               the extract to remain in contact with the sodium sulfate for approximately 2 hours.

 152

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                                                                                  Method 515.1
       11.1.8 Return the aqueous phase to the separatory funnel, add a 60-mL volume of ethyl ether
              to the sample, and repeat the extraction procedure a second time, combining the
              extracts in the 500-mL erlenmeyer flask.  Perform a third extraction with 60 mL of
              ethyl ether in the same manner.
       11.1.9 Determine the original sample volume by refilling the sample bottle to the mark and
              transferring the water to a 1000-mL graduated cylinder.  Record the sample volume to
              the nearest 5 mL.
11.2  Automated Hydrolysis, Preparation, and Extraction:  Data presented in this method were
       generated using the automated extraction procedure with the mechanical separatory funnel
       shaker.
       11.2.1 Add preservative (Sect. 8.2) to any samples not previously preserved, e.g., blanks and
              QC check standards.  Mark the water meniscus on the side of the sample bottle for
              later determination of sample volume (Sect. 11.2.9).  Fortify sample with 50 /*L of
              the surrogate standard solution. If the mechanical separatory funnel shaker is used,
              pour the entire sample into a 2-L separatory funnel.  If the mechanical tumbler is
              used, pour the entire sample into a tumbler bottle.
       11.2.2 Add 250 g NaCl to the sample, seal, and shake to dissolve salt.
       11.2.3 Add 17 mL of 6 N NaOH to the sample, seal, and shake. Check the pH of the
              sample with pH paper; if the sample does not have a pH greater than or equal to 12,
              adjust the pH by adding more  6 N NaOH. Shake sample for 1 hr using the
              appropriate mechanical mixing device.
       11.2.4 Add 300 mL methylene chloride to the sample bottle to rinse the bottle, transfer the
              methylene chloride to the separatory funnel or tumbler bottle, seal, and shake for 10s,
              venting periodically.  Repeat shaking and venting until pressure release is not
              observed during venting. Reseal and place sample container  in appropriate
              mechanical mixing device.  Shake or tumble the sample for 1 hr. Complete and
              thorough mixing of the organic and aqueous phases should be observed at least 2 min
              after starting the mixing device.
       11.2.5 Remove the sample container from the mixing device.  If the tumbler is used, pour
              contents of tumbler bottle into a 2-L separatory funnel.  Allow the  organic layer to
              separate from the water phase  for a minimum of  10 min.  If the emulsion interface
              between layers is more than one third the volume of the solvent layer, the analyst
              must employ mechanical techniques to complete the phase separation.  The optimum
              technique depends upon the sample, but may include stirring, filtration through glass
              wool, centrifugation, or other  physical methods.  Drain and discard the organic phase.
              If the tumbler is used,  return the aqueous phase to the tumbler bottle.
       11.2.6 Add 17 mL of 12  N H2SO4 to the sample, seal, and shake to  mix.  Check the pH of
              the sample with pH paper; if the sample does not have a pH less than or equal to 2,
              adjust the pH by adding more  12 N H2SO4.
       11.2.7 Add 300 mL ethyl ether to the sample, seal,  and  shake for 10 s, venting periodically.
              Repeat shaking and venting until pressure release is not observed during venting.
              Reseal and place sample container in appropriate mechanical  mixing device. Shake or
                                                                                           153

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Method 515.1
               tumble sample for 1  hr.  Complete and thorough mixing of the organic and aqueous
               phases should be observed at least 2 min after starting the mixing device.
       11.2.8 Remove the sample container from the mixing device. If the tumbler is used, pour
               contents of tumbler bottle into a 2-L separatory  funnel.  Allow the organic layer to
               separate from the water phase for a minimum of 10 min.  If the emulsion interface
               between layers is more than one third the volume of the solvent layer, the analyst
               must employ mechanical techniques to complete the phase separation. The optimum
               technique depends upon the sample, but may include stirring, filtration through glass
               wool, centrifugation, or other physical methods.  Drain and discard the  aqueous
               phase.  Collect the extract in a 500-mL round-bottom flask containing about 10 g of
               acidified anhydrous sodium sulfate.  Periodically vigorously shake the sample and
               drying agent.  Allow the extract to remain in contact with the sodium sulfate for
               approximately 2 hr.
       11.2.9 Determine the original sample volume by refilling the sample bottle to the mark and
               transferring the water to a 1000-mL graduated cylinder.  Record the sample volume to
               the nearest 5 mL.
11.3  Extract Concentration
       11.3.1 Assemble a K-D  concentrator by attaching a concentrator tube to  a 500-mL
               evaporative flask.
       11.3.2 Pour the dried extract through a funnel plugged with acid washed glass  wool, and
               collect the extract in the K-D concentrator. Use a glass rod to crush  any caked
               sodium sulfate during the transfer. Rinse the round-bottom flask  and funnel with 20
               to 30 mL of ethyl ether to complete the quantitative transfer.
       11.3.3 Add  1 to 2 clean boiling stones to the evaporative flask and attach a macro 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. At the proper rate of distillation the balls of the
               column will actively chatter but the chambers will not flood.  When the apparent
               volume of liquid reaches 1  mL, remove the K-D apparatus and allow it  to drain and
               cool  for at least 10 min.
       11.3.4 Remove the Snyder  column and rinse the flask and its lower joint into the
               concentrator tube with 1 to 2 mL of ethyl  ether. Add 2 mL of MTBE and a fresh
               boiling stone.  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 concentration in 5 to 10 min.  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 250 /zL of methanol.  If the gaseous
               diazomethane procedure (Sect. 11.4) is used for esterification of pesticides,  rinse the
               walls of the concentrator tube while adjusting the volume to 5.0 mL with MTBE.  If
               the pesticides will be esterified using  the diazomethane solution (Sect. 11.5), rinse the
               walls of the concentrator tube while adjusting the volume to 4.5 mL with MTBE.

 154

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                                                                                  Method 515.1
11.4  Esterification of Acids Using Gaseous Diazomethane:  Results presented in this method were
       generated using the gaseous diazomethane derivatization procedure.  See Section 11.5 for an
       alternative procedure.
       11.4.1  Assemble the diazomethane generator (Figure 1) in a hood.
       11.4.2 Add 5 mL of ethyl ether to Tube 1.  Add  1 mL of ethyl ether, 1 mL of carbitol, 1.5
               mL of 37% aqueous KOH, and 0.2 grams Diazald to Tube 2.  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 1 min.  Remove
               first sample.  Rinse the tip of the diazomethane generator with ethyl ether after
               methylation of each sample. Bubble diazomethane through the second sample extract
               for  1 min.  Diazomethane reaction mixture should be used to esterify only two
               samples; prepare new reaction mixture in Tube 2 to  esterify each two additional
               samples.  Samples should turn yellow after addition  of diazomethane and remain
               yellow for at least 2 min. Repeat methylation procedure if necessary.
       11.4.3 Seal concentrator tubes with stoppers.  Store at room temperature in a hood for 30
               min.
       11.4.4 Destroy any unreacted diazomethane by adding 0.1 to 0.2 grams silicic acid to the
               concentrator tubes. Allow to stand until the evolution of nitrogen gas has stopped (ap-
               proximately 20 min).  Adjust the sample volume to 5.0 mL with MTBE.
11.5  Esterification of Acids Using Diazomethane Solution:  Alternative procedure.
       11.5.1  Assemble the diazomethane generator (Figure 2) in a hood.  The collection vessel is a
               10-  or 15-mL vial, equipped with a Teflon-lined screw cap and maintained at 0-5C.
       11.5.2  Add a sufficient amount of ethyl ether to tube 1 to cover the first impinger.  Add  5
               mL  of MTBE to the collection vial.  Set the nitrogen flow at 5-10 mL/min.  Add  2
               mL  Diazald solution (Sect. 7.10) and 1.5 mL of 37% KOH solution to the second
               impinger.  Connect the  tubing  as shown and allow the nitrogen flow to purge the
               diazomethane from the reaction vessel into the collection vial for 30 min.  Cap the
               vial  when collection is complete  and maintain at 0-5°C.  When stored at 0-5°C this
               diazomethane solution may be  used over a period of 48  hr.
       11.5.3  To each concentrator tube containing sample or standard, add 0.5 mL diazomethane
               solution.  Samples should turn yellow after addition of the diazomethane solution and
               remain yellow for at least 2 min.  Repeat methylation procedure if necessary.
       11.5.4  Seal concentrator tubes  with stoppers.  Store at room temperature in a hood for 30
               min.
       11.5.5  Destroy any unreacted diazomethane by adding 0.1 to 0.2 grams silicic acid to the
               concentrator tubes. Allow  to stand until the evolution of nitrogen gas has stopped (ap-
               proximately 20 min).  Adjust the sample volume to 5.0  mL with MTBE.
11.6  Florisil Separation
       11.6.1  Place a small plug of glass  wool into a 5-mL disposable glass pipet. Tare the pipet,
               and  measure 1 g of activated Florisil into the pipet.
       11.6.2  Apply 5 mL of 5 percent methanol in MTBE to the Florisil.  Allow the liquid to just
               reach the top of the Florisil. In this and subsequent  steps, allow the liquid level to

                                                                                          155

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Method 515.1
              just reach the top of the Florisil before applying the next rinse, however, do not allow
              the Florisil to go dry.  Discard eluate.
       11.6.3 Apply 5 mL methylated sample to the Florisil leaving silicic acid in the tube.  Collect
              eluate in K-D tube.
       11.6.4 Add  I mL of 5 percent methanol  in MTBE to the sample container, rinsing walls.
              Transfer the rinse to the Florisil column leaving silicic acid in the tube.  Collect eluate
              in a K-D tube. Repeat with 1-mL and  3-mL aliquots of 5 percent methanol in MTBE,
              collecting eluates in K-D tube.
       11.6.5 If necessary, dilute eluate to 10 mL with 5 percent methanol in MTBE.
       11.6.6 Seal the vial and store in a refrigerator if further processing will not be performed
              immediately.  Analyze by GC-ECD.
11.7  Gas Chromatography
       11.7,1 Sect. 6.10 summarizes the recommended operating conditions for the  GC.  Included
              in Table 1 are retention times observed using this method. Other GC columns,
              chromatographic conditions, or detectors may be used if the requirements of Sect.
               10.4 are met.
       11.7.2 Calibrate the system daily as described in Sect. 9.  The standards and extracts must be
              in MTBE.
       11.7.3 If the internal standard calibration procedure is used, fortify the extract with 25 /iL of
              internal standard solution.  Thoroughly mix sample and place aliquot in a GC  vial for
              subsequent analysis.
       11.7.4 Inject 2 ;iL  of the sample extract.  Record the resulting peak size in area units.
       11.7,5 If the response for the peak exceeds the working range of the system, dilute the
              extract and  reanalyze.
11.8  Identification of Analytes
       11.8.1 Identify a sample component by comparison of its retention time to the retention time
              of a reference chromatogram.  If the retention time of an unknown compound
              corresponds, within limits, to the retention time of a standard compound, then
              identification is considered positive.
       11.8.2 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.
       11.8.3 Identification requires expert judgement when sample components are not resolved
              chromatographically.  When GC peaks obviously represent more than one sample
              component (i.e.,  broadened peak  with  shoulder(s) or valley between two or more
               maxima, or any time doubt exists  over the identification of a peak on a
               chromatogram, appropriate alternative  techniques, to help confirm peak identification,
               need to be employed.  For example, more positive identification may be made by the
               use of an alternative detector which operates on a chemical/physical principle different
 756

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                                                                                  Method 515.1
               from that originally used, e.g., mass spectrometry, or the use of a second
               chromatography column.  A suggested alternative column in described in Sect. 6.10.

 12.   CALCULATIONS

 12.1  Calculate analyte concentrations in the sample from the response for the analyte using the
       calibration procedure described in Sect. 9.
 12.2  If the internal standard calibration procedure is used,  calculate the concentration (C) in the
       sample using the response factor (RF) determined in Sect.  9.2 and Equation 2, or determine
       sample concentration from the calibration curve.
                                          Equation 2

                                    C (»glL) = -
                  where:
                  As = Response for the parameter to be measured.
                  Ais = Response for the internal standard.
                   Is = Amount of internal standard added to each extract
                  Vo =  Volume of water extracted (L).
12.3  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 determined in Sect.
       9.3. The concentration (C) in the sample can be calculated from Equation 3.
                                         Equation 3
                             where:
                             A  = Amount of material injected (ng).
                             V,  = Volume of extract injected (/xL).
                             V;  = Volume of total extract (pL).
                             Vs  = Volume of water extracted (mL).
13.  PRECISION AND ACCURACY

13.1  In a single laboratory, analyte recoveries from reagent water were determined at five
      concentration levels.  Results were used to determine analyte EDLs and demonstrate method
      range.' Analyte EDLs and analyte recoveries and standard deviation about the percent
      recoveries at one concentration are given in Table 2.
13.2  In a single laboratory, analyte recoveries from one standard synthetic ground waters were
      determined at one concentration level.  Results were used to demonstrate  applicability of the
                                                                                          757

-------
Method 515.1
       method to different ground water matrices.'  Analyte recoveries from the one synthetic matrix
       are given in Table 2.
 758

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                                                                                Method 515.1
                                     References

1.     National Pesticide Survey Method No. 3, "Determination of Chlorinated Acids in Water by
       Gas Chromatography with an Electron Capture Detector."

2.     "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No. 68-03-2697.
       Available from U.S. Environmental Protection Agency, Environmental Monitoring and
       Support Laboratory, Cincinnati, Ohio 45268.

3.     ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
       Preparation of Sample Containers and for Preservation," American Society for Testing and
       Materials, Philadelphia, PA, p. 86, 1986.

4.     Giam, C. 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).

5.     Giam, C. S., and Chan, H. S. "Control of Blanks in the Analysis of Phthalates in Air and
       Ocean Biota Samples," U.S. National Bureau of Standards, Special Publication 442, pp.
       701-708, 1976.

6.     "Carcinogens - Working with Carcinogens," Department of Health,  Education, and Welfare,
       Public Health  Service, Center for Disease Control, National Institute for Occupational Safety
       and Health, Publication No. 77-206, Aug. 1977.

7.     "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
       and Health Administration, OSHA 2206, (Revised, January 1976).

8.     "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
       Committee on Chemical Safety,  3rd Edition, 1979.

9.     ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
       Sampling Water," American Society for Testing and Materials, Philadelphia, PA, p. 130,
       1986.
                                                                                         759

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Method 515.1
Table 1.  Retention Times for Method Analytes

                                              Retention Time* (minutes)
Analyte
Dalapon
3,5-Dichlorobenzoic acid
4-Nitropheno(
DCAA (surrogate)
Dicamba
Dichlorprop
2,4-D
DBOB (int. std.)
Pentachlorophenol (PCP)
Chloramben
2,4,5-TP
5-Hydroxydicamba
2,4,5-T
2,4-DB
Dinoseb
Bentazon
Picloram
DCPA acid metabolites
Acifluorfen

3    Columns and analytical conditions are described in Sect. 6.10.1 and 6.10.2.
Primary
3.4
18.6
18.6
22.0
22.1
25.0
25.5
27.5
28.3
29.7
29.7
30.0
30.5
32.2
32.4
33.3
34.4
35.8
41.5
Confirmation
4.7
17.7
20.5
14.9
22.6
25.6
27.0
27.6
27.0
32.8
29.5
30.7
30.9
32.2
34.1
34.6
37.5
37.8
42.8
 160

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                                                                            Method 515.1
Table 2.  Single Laboratory Accuracy, Precision and Estimated Detection Limits
(EDLs) for Analytes from Reagent Water and Synthetic Groundwaters8
                           EDL   Concentrate
    Analyte               fag/if      fag/L)
    Acifluorfen              0.096        0.2
    Bentazon               0.2          1
    Chloramben             0.093        0.4
    2,4-D                  0.2          1
    Dalapon                1.3          10
    2,4-DB                 0.8          4
    DCPA acid metabolites   0.02         0.2
    Dicamba                0.081        0.4
    3,5-Dichlorobenzoic      0.061        0.6
    acid
    Dichlorprop             0.26         2
    Dinoseb                0.19         0.4
    5-Hydroxydicamba       0.04         0.2
    4-Nitrophenol            0.13         1
    Pentachlorophenol       0.076        0.04
    (PCP)
    Picloram                0.14         0.6
    2,4,5-T                 0.08         0.4
    2,4,5-TP                0.075        0.2
Reagent Water
If
121
120
111
131
100
87
74
135
102
107
42
103
131
130
91
117
134

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Method 515.1
Table 3.  Laboratory Performance Check Solution


                                                    Cone
Test                       Analyte                 (pg/mL)  Requirements
Sensitivity                  Dinoseb                   0.004  Detection of analyte; S/N > 3
Chromatographic performance 4-Nitrophenol              1.6    0.70 0.40b
                           4-Nitrophenol              1.6


a    PGF =  peak Gaussian factor.  Calculated using the equation:


                                        1.83xW  fl
                                  PGF  = 	LI
where W  | _
                            WIJS
is the peak width at half height and W I _L I is the peak width at tenth height
           2         '                   "| TO
b    Resolution between the two peaks as defined by the equation:


                                          R-l.
                                               w

where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
762

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                                                                   .1
Nitrogen
    Tubel
                       Tube 2
                              r
                                         Sample
                                          Tube
                                                                52-015-27
        Figure 1. Gaseous Diazorhethane Generator
                                                                   163

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  Method 515.1
                                                               Glass Tubing
    Nitrogen
Rubber Stopper
                    Tubel
                                          Tube 2
Collection
                                                                                52-015-28
                                                               Thermos or
                                                            Cryogenic Cooler
                      Figure 2.  Diazomethane Solution Generator
764

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                               Method 515.2
Determination of Chlorinated Acids in Water
                   Using Liquid-Solid Extraction
                      and Gas Chromatography
            with an Electron Capture Detector
                           Revision 1.0 - EPA EMSL-Ci
                                         August 1992
    B.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
                     J.W. Hodgeson - Method 515, Revision 2.0 (1986)
         T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey
                                 Method 3, Revision 3.0 (1987)
                      R.L. Graves - Method 515.1, Revision 4.0 (1989)
                    J.W. Hodgeson - Method 515.2, Revision 1.0 (1992)

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                                 Method 515.2

    Determination of Chlorinated Acids in Water Using Liquid-Solid
                Extraction and Gas Chromatography with an
                            Electron Capture Detector



1.    SCOPE AND APPLICA TION

1.1   This is a gas chromatographic (GC) method applicable to the determination of certain chlori-
      nated acids in ground water and finished drinking water. The following compounds can be
      determined by this method:

                Analyte                                   CAS No.
                Acifluorfen                               50594-66-6
                Bentazon                                25057-89-0
                2,4-D                                      94-75-7
                2,4-DB                                     94-82-6
                Dacthal3                                   1861-32-1
                Dicamba                                   1918-00-9
                3,5-Dichlorobenzoic acid                      51-36-5
                Dichlorprop                                 120-36-5
                Dinoseb                                    88-85-7
                5-Hydroxydicamba                          7600-50-2
                Pentachlorophenol (PCP)                      87-86-5
                Picloram                                   1918-02-1
                2,4,5-T                                     93-76-5
                2,4,5-TP  (Silvex)                            93-72-1

       3   Dacthal monoacid and diacid metabolites included in method  scope; Dacthal
           diacid metabolite used for validation studies.


1.2   This method  is applicable to the determination of salts and esters of analyte acids.  The form
      of each acid is not distinguished by this method.  Results are calculated and reported for each
      listed analyte as  the total free acid.
1.3   Single laboratory accuracy and precision data and method detection limits (MDLs) have been
      determined for the analytes above (Sect.  13). Observed detection limits may vary  among
      water matrices, depending upon the nature of interferences  in the sample matrix and the
      specific instrumentation used.

1.4   This method  is restricted to use by or under the supervision of analysts experienced in the use
      of GC 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 Sect.
      9.3.

1.5   Analytes that are not separated chromatographically, (i.e., have very similar retention times)
      cannot be individually identified and measured in the same  calibration mixture or water sample
      unless an alternative technique for identification and  quantitation exists (Sect. 11.6).
                                                                                     167

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Method 515.2
1.6    When this method is used to analyze unfamiliar samples for any or all of the analytes above,
       analyte identifications should be confirmed by analysis on a second gas chromatographic
       column or by gas chromatography/mass spectrometry (GC/MS).

2.     SUMMARY OF METHOD

2.1    A 250-mL measured volume of sample is adjusted to pH 12 with 6 N sodium hydroxide for 1
       hr to hydrolyze derivatives. Extraneous organic material is removed by a solvent wash.  The
       sample is acidified, and the chlorinated acids are extracted  with a 47 mm resin based extrac-
       tion disk.  The acids are converted to their methyl esters using diazomethane.  Excess
       derivatizing reagent is removed, and the esters are determined by capillary column GC using
       an electron capture detector (ECD).

3.     DEFINITIONS

3.1    Internal Standard (IS):   A pure analyte(s) added to a sample, extract, or standard solution in
       known amount(s), and used to measure the relative responses of other method analytes and
       surrogates that are components of the same sample or solution.  The IS must be an analyte that
       is not a sample component.
3.2    Surrogate Analyte (SA): A pure analyte(s), which  is extremely unlikely to be found in any
       sample, and which is added to a sample aliquot in known amount(s) before extraction or other
       processing, and is measured with the same procedures used to measure other sample compo-
       nents. The purpose of the SA is to monitor method performance with each sample.
3.3    Laboratory Duplicates (LD1 and LD2):  Two aliquots  of the same sample taken in the analyti-
       cal laboratory and analyzed separately with identical procedures. Analyses of LD1 and LD2
       indicate the precision associated with laboratory procedures, but not with sample collection,
       preservation, or storage procedures.
3.4    Field Duplicates (FD1 and FD2):   Two  separate samples collected at the same time and place
       under identical circumstances and treated exactly the same  throughout field and laboratory
       procedures.  Analyses of FD1  and FD2  give a measure of  the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory Reagent Blank (LRB):  An aliquot of reagent water or other blank matrix that is
       treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents,
       internal standards, and surrogates that are used with other samples.  The LRB  is used to
       determine if method analytes or other interferences are present in the laboratory environment,
       the reagents, or the apparatus.
3.6    Field Reagent Blank (FRB): An aliquot of reagent water or other blank matrix that is placed
       in a sample container in the laboratory and treated  as a sample in all respects,  including
       shipment to the sampling site,  exposure to sampling site conditions, storage, preservation and
       all analytical procedures. The purpose of the FRB is to determine if method analytes or other
       interferences are present in the field environment.
3.7    Instrument Performance Check Solution (IPC): A solution of one or more method analytes,
       surrogates, internal standards, or other test substances used to evaluate the performance of the
       instrument system with  respect to a defined set of criteria.
 168

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                                                                                   Method 515.2
3.8   Laboratory Fortified Blank (LFB): An aliquot of reagent water or other blank matrix to which
       known quantities of the method analytes are added in the laboratory.  The LFB is analyzed
       exactly like a sample, and its purpose is to determine whether the methodology is in control,
       and whether the laboratory is capable of making accurate and precise measurements.
3.9   Laboratory Fortified Sample Matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the sample
       matrix must be determined in a separate aliquot, and the measured values in the LFM correct-
       ed for background concentrations.
3.10  Stock Standard Solution (SSS):  A concentrated solution containing one or more method
       analytes prepared in the laboratory using assayed reference materials or purchased from a
       reputable commercial source.
3.11  Primary Dilution Standard Solution (PDS):  A solution of several analytes prepared in the
       laboratory from stock standard solutions, and diluted as needed to prepare calibration solutions
       and other needed analyte solutions.
3.12  Calibration Standard (CAL):  A solution prepared from the primary dilution standard solution
       or stock standard solutions and the internal standards and surrogate analytes.  The CAL
       solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13  Quality Control Sample (QCS): A solution of method analytes of known concentrations which
       is used to fortify an aliquot of LRB or sample matrix.  The QCS is obtained from a source
       external to the laboratory and different from the source of calibration standards. It is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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 analytical conditions by analyzing laboratory reagent blanks as described in
       Sect. 9.2.
       4.1.1   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 water and detergent and thorough rinsing with dilute acid, tap and reagent
               water.  Drain dry,  and heat in an oven or muffle furnace at  400°C for 1 hr. Do not
               heat volumetric ware. Thermally stable materials  such as PCBs might not be elimi-
               nated by this treatment.  Thorough rinsing with acetone may be substituted for the
               heating. After glassware is dry and cool, store it in a clean environment to prevent
               any accumulation of dust or other contaminants. Store inverted or capped with
               aluminum  foil.
       4.1.2   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.
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Method 515.2
       WARNING:        When a solvent is purified, stabilizers and preservatives added by
       the manufacturer are removed, thus potentially making the solvent hazardous and
       reducing the shelf life.

4.2   The acid forms of the analytes are  strong organic acids which react readily with alkaline
       substances and can be lost during sample preparation.  Glassware and glass wool must be
       acid-rinsed with I N hydrochloric acid and the sodium sulfate must be acidified with sulfuric
       acid prior to use to  avoid analyte losses due to adsorption.
4.3   Organic acids and phenols, especially chlorinated compounds, cause the most direct  interfer-
       ence with the determination.  Alkaline hydrolysis and subsequent extraction of  the basic
       sample removes many chlorinated hydrocarbons and phthalate esters that might otherwise
       interfere with the electron capture analysis.
4.4   Interferences by phthalate esters  can pose a major problem in pesticide analysis when using the
       ECD.  Phthalates generally appear in the chromatogram as large peaks.  Common flexible
       plastics  contain varying amounts  of phthalates, that are easily extracted or leached during
       laboratory operations. Cross-contamination of clean glassware routinely occurs when plastics
       are handled during extraction steps, especially when solvent-wetted surfaces are handled.
       Interferences from phthalates can best be minimized by avoiding the use of plastics in the
       laboratory. Exhaustive purification of reagents and glassware may be required to eliminate
       background phthalate contamination.2-3
4.5   Interfering contamination may occur when a sample containing low concentrations of analytes
       is analyzed immediately  following a sample containing relatively high concentrations of
       analytes. Between-sample rinsing of the sample syringe and associated equipment with methyl-
       tert-butyl-ether  (MTBE) can minimize sample cross- contamination.  After analysis of a sample
       containing high concentrations of analytes, one or more injections of MTBE should be made to
       ensure that accurate values are obtained for the next sample.
4.6   Matrix interferences may be caused by contaminants that are coextracted from the sample.
       Also, note that all analytes listed in the Scope and Application Section are not resolved from
       each other on any one column, i.e., one analyte of interest may interfere with another analyte
       of interest. The extent of matrix interferences will vary considerably from source to source,
       depending upon the water sampled. The procedures in Sect. 11 can be used to overcome
       many of these interferences.  Tentative  identifications should be confirmed (Sect. 11.6).
4.7   It is important that samples and working standards  be contained in the same solvent. The
       solvent for working standards must be the same as  the final solvent used in sample prepara-
       tion.  If this is not the case,  chromatographic  comparability of standards to sample extracts
       may  be  affected.

5.     SAFETY

5.1   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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining  a current awareness file of OSHA regulations regard-
 770

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                                                                                  Method 515.2
       ing the safe handling of the chemicals specified in this method.  A reference file of material
       safety data 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.
5.2    Diazomethane:  A toxic carcinogen which can explode under certain conditions. The follow-
       ing precautions must be followed:
       5.2.1   Use the diazomethane generator behind a safety shield in a well ventilated fume hood.
               Under no circumstances  can the generator be heated above 90°C, and all grinding
               surfaces such as ground glass joints, sleeve bearings, and glass stirrers must be
               avoided.  Diazomethane  solutions must not be stored.  Only generate enough for the
               immediate needs.  The diazomethane generator apparatus used in the esterification
               procedure  (Sect.  11.4) produces micromolar amounts of diazomethane in solution to
               minimize safety hazards.  If the procedure is followed exactly, no possibility for
               explosion exists.
5.3    Methyltertbutyl Ether: Nanograde, redistilled in glass,  if necessary.  Must be free of perox-
       ides as indicated by EM  Quant test strips (available from Scientific Products Co., Cat. No.
       PI 126-8, and other  suppliers).
5.4    WARNING:  When a solvent is purified, stabilizers added by the manufacturer are removed,
       thus potentially making the solvent hazardous.

6.     EQUIPMENT AND SUPPLIES
(All specifications are suggested.  Catalog numbers are included for illustration only.)
6.1    Kontes Filter Funnels: Fisher Cat. No. 953755-0000 or equivalent.
6.2    Vacuum Flasks:  1000 mL with glass side arm
6.3    Vacuum Manifold:   The  manifold should be capable of holding 6-8 filter flasks in series with
       house vacuum.  Commercial manifolds are available from a number of suppliers, e.g., Baker,
       Fisher, and Varian.
6.4    Culture Tubes (25 x 200 mm) With Teflon-lined Screw Caps:  Fisher Cat. No. 14-933-1C, or
       equivalent.
6.5    Pasteur Pipets: Glass disposable  (5 mL)
6.6    Large Volume Pipets: Disposable, Fisher Cat.  No. 13-678-8 or equivalent.
6.7    Balance:  Analytical, capable of weighing to .0001 g.
6.8    pH Meter:  Wide range capable of accurate measurements in the pH = 1-12  range.
6.9    Diazomethane Generator: See Figure 1 for a diagram of an all glass system custom made for
       these validation studies.  A micromolar generator is also available from Aldrich Chemical.
6.10  Analytical Concentrator:   Six or twelve positions, Organomation N-EVAP Model No.  111-
       6917 or equivalent.
6.11   Gas Chromatography: Analytical system complete with gas chromatograph equipped with
       ECD, split/splitless capillary injector, temperature programming, differential  flow control and
       all required accessories.  A data system is recommended for measuring peak  areas. An
       autoinjector is recommended to improve precision of analysis.

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Method 515.2
6.12  GC Columns and Recommended Operating Conditions
       6.12.1  Primary:  DB-5 or equivalent, 30 m x .32 mm ID, 0.25 /xm film thickness.  Injector
               Temp. = 200°C, Detector Temp.  = 280°C, Helium linear velocity is 30 cm/sec at
               200°C and 10 psi, 2 piL splitless injection with purge on 3 min.  Program:  Hold at
               60°C 1 min., increase to 260°C at 5°C/min. and hold 5 min.
       6.12.2  Confirmation: DB-1701 or equivalent, 30 m x .32 mm ID, 0.25 itm film thickness.
               Injector Temp.  = 200 °C, Detector Temp. = 280°C, Helium linear velocity is 30
               cm/sec at 200°C and 10 psi, 2 /xL splitless injection with purge on 3 min. Program:
               Hold at 60°C 1 min., increase to 260°C at 5°C/min. and hold 5 min.
6.13  Glass Wool:  Acid washed with IN HCl  and heated at 450°C for 4 hr.
6.14  Short Range pH Paper (pH=0-3).
6.15  Volumetric Flasks:  50 mL,  100 mL, and 250 mL
6.16  Microsyringes:  25 /xL, 50 /xL, 100 pL, 250 /xL, 500 /xL
6.17  Amber Bottles:  15 mL, with Teflon-lined screw caps
6.18  Graduated Cylinder:  250 mL
6.19  Separatory Funnel:  500 mL
6.20  Graduated Centrifuge Tubes:  15 mL or  10 mL Kuderna Danish Concentrator tubes

7.     REAGENTS AND STANDARDS

7.1    Extraction Disks, 47 mm:  Resin based polystyrenedivinylbenzene
7.2    Reagent  Water:  Reagent water is defined as a water in which an interference is not observed
       at the MDL of each analyte of interest.
       7.2.1    A Millipore Super-Q water system or its equivalent may be used to  generate deionized
               reagent water. Distilled water that has  been passed through granular charcoal may
               also be suitable.
       7.2.2   Test reagent water each day it is used by analyzing according to Sect. 11.
7.3    Methanol: Pesticide quality or equivalent.
7.4    Methyltertbutyl Ether (MTBE):  Nanograde, redistilled in glass if necessary.  Ether must be
       demonstrated to be free of peroxides. One test kit (EM Quant Test Strips), is available from
       EM Science,  Gibbstown, NJ. Procedures for removing peroxides from the ether are provided
       with the  test strips.  Ethers must be periodically tested (at least monthly) for peroxide forma-
       tion during use.  Any reliable test kit may be used.
7.5    Sodium Sulfate:  (ACS) Granular, Anhydrous:  Heat in a shallow tray at 400°C for a mini-
       mum of  4 hr  to remove phthalates and other interfering organic substances.  Alternatively,
       extract with methylene chloride in a Soxhlet apparatus for 48 hr.
       7.5.1    Sodium sulfate drying tubes: Plug the bottom of a large volume disposable pipet with
               a minimum amount of acidified glass wool (Supelco Cat. No. 20383 or equivalent).
               Fill the pipet halfway (3 g) with acidified sodium sulfate (See Sect. 7.9).
772

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                                                                                  Method 515.2
7.6    Sulfuric Acid:  Reagent grade.
       7.6.1  Sulfuric acid, 12 N: Slowly add 335 mL concentrated sulfuric acid to 665 mL of
              reagent water.
7.7    Sodium Hydroxide:  ACS reagent grade or equivalent.
       7.7.1  Sodium hydroxide IN:  Dissolve 4.0 g reagent grade sodium hydroxide in reagent
              water and dilute to  100 mL in volumetric flasks.
       7.7.2  Sodium hydroxide 6N
7.8    Ethyl Ether, Unpreserved: 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. PI126-8, and other suppliers). Procedures recommended for removal of peroxides are
       provided with the test strips.
7.9    Acidified Sodium Sulfate: Cover 500 g sodium sulfate (Sect.  7.5) with ethyl ether (Sect. 7.8).
       While agitating vigorously, add dropwise approximately 0.7 mL concentrated sulfuric acid.
       Remove the ethyl ether overnight under vacuum and store the sodium sulfate in a 100°C oven.
7.10  Carbitol, ACS Grade: Available from  Aldrich Chemical.
7.11  Diazald, ACS Grade:  Available from Aldrich Chemical.
7.12  Diazald Solution:  Prepare a solution containing 10 g Diazald in 100 mL of a 50:50 by volume
       mixture of ethyl ether and carbitol.  This solution is stable for 1-month or longer when stored
       at 4°C in an amber bottle with a Teflon-lined screw cap.
7.13  4,4'-Dibromooctafluorobiphenyl (DBOB):  99% purity, for use as internal standard.
7.14  2,4-Dichlorophenylacetic Acid (DCAA):  99% purity, for use as surrogate standard.
7.15  Potassium Hydroxide: ACS reagent grade or equivalent.
       7.15.1 Potassium hydroxide solution,  37%:  Using extreme caution, dissolve 37 g reagent
              grade potassium hydroxide in reagent water and dilute to 100 mL.
7.16  Stock Standard Solutions (1.00-2.00 /xg//xL):  Stock standard solutions may be purchased as
       certified solutions or prepared from pure standard materials using the following procedure:
       7.16.1 Prepare stock standard solutions by accurately weighing approximately  0.0100-0.0200
              g of pure material.  Dissolve the material in 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.
       7.16.2 Transfer the  stock standard solutions into 15-mL TFE-fluorocarbon-sealed screw cap
              amber vials.  Store  at 4°C or less when not in use.
       7.16.3 Stock standard solutions should be replaced after 2 months or sooner if comparison
              with laboratory fortified blanks, or QC samples indicate a problem.
       7.16.4 Primary Dilution Standards:  Prepare two sets of  standards according to the sets
              labeled A and B in Table 1.  For each set, add approximately 25 mL of methanol to a
              50 mL volumetric flask.  Add  aliquots of each stock standard in the range of approxi-
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Method 515.2
              mately 20 to 400 /zL and dilute to volume with methanol.  Individual analyte concen-
              trations will then be in the range of 0.4 to 8 /xg/mL (for a 1.0 mg/mL stock). The
              minimum concentration would be appropriate for an analyte with strong electron
              capture detector (ECD) response, e.g. pentachlorophenol.  The maximum concentra-
              tion is for an analyte with weak response, e.g., 2,4-DB.  The concentrations given in
              Table 2 reflect the relative volumes of stock standards used for the primary  dilution
              standards used in generating the method validation data.  Use these relative  values to
              determine the  aliquot volumes of individual stock standards above.
7.17  Internal Standard Solution:  Prepare a stock internal standard solution by accurately weighing
       approximately 0.050 g of pure DBOB.  Dissolve the DBOB in methanol  and dilute to volume
       in a 10-mL volumetric flask.  Transfer the DBOB solution to a TFE-fluorocarbonsealed screw
       cap bottle and store at  room temperature. Prepare a primary dilution standard at approximate-
       ly 1.00 /xg/mL by the addition of 20 /xL  of the stock standard to 100 mL of methanol. Addi-
       tion of 100 jxL of the primary dilution standard solution to the final 5 mL of sample  extract
       (Sect.  11) results in a final  internal standard concentration of 0.020 jig/mL. Solution should
       be replaced when ongoing QC (Sect. 9) indicates a problem. Note that DBOB has been shown
       to be an effective internal standard for the method analytes, but other compounds may be used
       if the QC requirements in Sect. 9 are met.
7.18  Surrogate Analyte Solution: Prepare a surrogate analyte stock standard solution by accurately
       weighing approximately 0.050 g of pure DCAA.  Dissolve  the DCAA in methanol and dilute
       to volume  in a 10-mL  volumetric flask.   Transfer the surrogate analyte solution to a
       TFE-fluorocarbon-sealed screw cap bottle and store at room temperature. Prepare a primary
       dilution standard at approximately 2.0 /xg/mL by addition of 40 /xL at the stock standard to
       100 mL of methanol.  Addition of 250 /xL of the surrogate  analyte solution to a 250-mL
       sample prior to extraction results in a surrogate concentration in the sample of 2 uglL and,
       assuming quantitative recovery of DCAA, a surrogate analyte concentration in the final  5 mL
       extract of 0.1 /xg/mL.  The surrogate standard solution should be replaced when ongoing QC
       (Sect.  9) indicates a problem.  DCAA has been shown to be an effective surrogate standard for
       the method analytes, but other compounds may be used if the QC requirements in Sect. 10 are
       met.
7.19  Instrument Performance Check Solution:  Prepare a diluted dinoseb solution by adding 10 itL
       of the 1.0  /xg//zL dinoseb stock solution to the MTBE and diluting to volume in a 10-mL
       volumetric flask.  To prepare the check solution, add 40 /xL of the diluted dinoseb solution,  16
       ixL of the 4-nitrophenol stock solution, 6 /xL of the 3,5-dichlorobenzoic acid stock solution, 50
       /xL of the surrogate standard solution, 25 iiL of the internal standard solution, and 250 tiL of
       methanol to a 5-mL volumetric flask and dilute to volume with MTBE.   Methylate sample as
       described in Sect. 11.4.  Dilute the sample to 10 mL in MTBE. Transfer to a TFE-fluorocar-
       bon-sealed screw cap bottle and store at  room temperature.  Solution should be replaced when
       ongoing QC (Sect. 9) indicates a problem.

8.     SAMPLE  COLLECTION,  PRESERVATION, AND  STORAGE

8.1    Grab samples should be collected in 1-L amber glass containers.  Conventional sampling
       practices should be followed; however,  the bottle must not  be prerinsed with sample before
       collection.7
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                                                                                   Method 515.2
8.2    Sample Preservation and Storage
       8.2.1   Add hydrochloric acid (diluted 1:1  in water) to the sample at the sampling site in
               amounts to produce a sample pH < 2.  Short range (0-3) pH paper (Sect. 6.14) may
               be used to monitor the pH.
       8.2.2   If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
               the sample bottle prior to collecting the sample.
       8.2.3   After the sample is collected in the bottle containing preservative(s), seal the bottle
               and shake vigorously for 1 min.
       8.2.4   The samples must be iced or refrigerated at 4°C away from light from the time of
               collection until extraction. Preservation study results  indicate that the sample analytes
               (measured as  total acid), except 5-hydroxy-dicamba, are stable in water for 14 days
               when stored under these conditions (Tables 8 and 9).  The concentration of 5-
               hydroxydicamba is seriously degraded over 14 days in a biologically active matrix.
               However, analyte stability will very likely be affected by the matrix; therefore, the
               analyst should verify that the preservation technique is applicable to the samples under
               study.
8.3    Extract Storage
       8.3.1   Extracts should be stored at 4°C or less away from light.  Preservation study results
               indicate that most analytes are stable for 14 days (Tables 8 and 9); however, the
               analyst should verify appropriate extract holding times applicable to the samples under
               study.

9.     QUALITY  CONTROL

9.1    Minimum QC requirements are initial demonstration of laboratory capability, determination of
       surrogate compound recoveries in each sample and blank, monitoring internal standard peak
       area or height in each  sample and blank (when internal standard calibration procedures are
       being employed),  analysis of laboratory reagent blanks, laboratory fortified samples,  laborato-
       ry fortified blanks, and QC samples.
9.2    Laboratory Reagent Blanks (LRB): Before processing any  samples, the analyst must demon-
       strate that all glassware and reagent interferences are under control.  Each time a set of
       samples is extracted or reagents are changed, a LRB must be analyzed. If within the retention
       time window of any analyte the LRB produces a peak that would prevent the determination of
       that analyte, determine the source of contamination and eliminate  the interference before
       processing  samples.
9.3    Initial Demonstration of Capability
       9.3.1   Select a representative fortified concentration (about 10 to 20 times MDL) for each
               analyte.  Prepare a sample concentrate (in methanol) containing each analyte at 1000
               times selected concentration. With a syringe, add  250 pL of the concentrate to each
               of at least four 250 mL aliquots of reagent water,  and analyze each aliquot according
               to  procedures beginning in Sect.  11.
       9.3.2   For each analyte the recovery value for all four of these samples must  fall in the
               range of  + 40%  of the fortified concentration.  For those compounds that meet the

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Method 515.2
               acceptance criteria, performance is considered acceptable and sample analysis may
               begin.  For compounds failing this criteria, this procedure must be repeated using five
               fresh samples until satisfactory performance has been demonstrated for all analytes.
       9.3.3   The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it.  As laboratory personnel gain experience with this method the
               quality of data should improve beyond those required here.
9.4    The analyst is permitted to modify GC columns, GC conditions, detectors, concentration
       techniques (i.e., evaporation techniques), internal standard or surrogate compounds.  Each
       time such method modifications are made, the analyst must repeat the procedures in Sect. 9.3.
9.5    Assessing Surrogate Recovery
       9.5.1   When surrogate recovery from a sample or a blank is  <60% or > 140%, check (1)
               calculations to locate possible errors, (2) fortifying solutions for degradation, (3)
               contamination, and (4) instrument performance.  If those steps do not reveal the cause
               of the problem, reanalyze the extract.
       9.5.2   If a blank extract reanalysis fails the 60-140% recovery criteria, the problem must be
               identified and corrected before continuing.
       9.5.3   If sample extract  reanalysis meets the surrogate recovery criteria, report only data for
               the reanalyzed extract. If sample extract continues to  fail the recovery criteria, report
               all data for that sample as suspect.
9.6    Assessing the Internal Standard
       9.6.1   When using the internal standard (IS) calibration procedure, the analyst is expected to
               monitor the IS response (peak area or peak height) of  all samples during each analysis
               day.  The IS response for any sample chromatogram should not deviate from the daily
               calibration check standard's IS response by more than 30%.
       9.6.2   If >30% deviation occurs with an individual extract,  optimize instrument perfor-
               mance and inject a second aliquot of that extract.
               9.6.2.1    If the  reinjected aliquot produces an acceptable internal standard response,
                          report results for that aliquot.
               9.6.2.2   If a deviation of greater than 30% is obtained for the reinjected extract,
                          analysis of the samples should be repeated beginning with Sect. 11,  pro-
                          vided the sample is still available.  Otherwise, report results obtained from
                          the reinjected extract, but annotate as suspect.
       9.6.3   If consecutive samples fail the IS response acceptance criteria, immediately analyze a
               medium  calibration standard.
               9.6.3.1    If the standard provides a response factor (RF) (Sect.  10.2.2) within 20%
                          of the predicted value,  then follow procedures itemized in Sect. 9.6.2 for
                          each sample failing the IS response criterion.
               9.6.3.2   If the check standard provides a response factor which deviates more than
                          20% of the predicted value, then the analyst must recalibrate as specified
                          in Sect. 10.
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                                                                                   Method 515.2
9.7    Assessing Laboratory Performance:  Laboratory Fortified Blank
       9.7.1   The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
               every 20 samples or one per sample set (all samples extracted within a 24-hr period)
               whichever is greater.  The concentration of each analyte in the LFB should be 10
               times the MDL.  Calculate percent recovery (X,).  If the recovery of any analyte falls
               outside the control limits (See Sect. 9.7.2),  that analyte is judged out of control, and
               the source of the problem should be identified and resolved before continuing analy-
               ses.
       9.7.2   Until sufficient data become available, usually a minimum of results from 20 to 30
               analyses, each laboratory should assess laboratory performance against the control
               limits in Sect. 9.3.2 that are derived from the data in Table 2.  When sufficient
               internal performance data become available, develop control limits from the mean
               percent recovery (X) and standard deviation (S) of the percent recovery. These data
               are used to establish upper and lower control limits as follows:

                            Upper Control Limit = X + 3S
                            Lower Control Limit = X - 3S

               After each five to ten new recovery measurements, new control limits should be
               calculated using only the most recent 20-30 data points. These calculated control
               limits should never exceed those established in Sect. 9.3.2.
       9.7.3   Method detection limits (MDL) must be determined using the procedure given in
               reference 8. The MDLs must be sufficient to detect analytes at the required levels
               according to SDWA regulations.
       9.7.4   At least quarterly, analyze a QCS (Sect. 3.13) from an outside source.
       9.7.5   Laboratories are encouraged to participate  in external performance evaluation studies
               such as the laboratory certification programs offered by many states or the studies
               conducted by USEPA.
9.8   Assessing Analyte Recovery -  Laboratory Fortified Sample Matrix
       9.8.1   Each laboratory must analyze a LFM for 10% of the samples or one sample con-
               centration per set, whichever is greater. The concentration should not be less then the
               background concentration of the sample selected for fortification.  Ideally, the concen-
               tration should be the same as that used for the laboratory fortified blank (Sect. 9.7).
               Over time, samples from all routine sample sources should be fortified.
       9.8.2   Calculate the percent recovery,  P of the concentration for each analyte, after correct-
               ing the measured concentration, X, from the fortified sample for the background
               concentration, b,  measured in the unfortified sample.
                                   p =       100 (X - b)
                                       fortifying concentration
                                                                                             177

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Method 515.2
                 and compare these values to control limits appropriate for reagent water data
                 collected in the same fashion. If the analyzed unfortified sample is found to con-
                 tain NO background concentrations and the added concentrations are those speci-
                 fied in Sect. 9.7, then the appropriate control limits would be the acceptance limits
                 in Sect. 9.7. If, on the other hand, the analyzed  unfortified sample is found to
                 contain background concentration, b, estimate the standard deviation at the back-
                 ground concentration, sh, using regressions or comparable background data and,
                 similarly, estimate the mean, Xa  and standard deviation, sa,  of analytical results at
                 the total concentration after fortifying.   Then the  appropriate percentage control
                 limits would be P  ±  3sP , where:
                                                100 X
                                     (b + fortifying concentration)
                               and
                               ,,.1.0 _        _
                                         fortifying concentration
                  For example, if the background concentration for Analyte A was found to be 1
                  pg/L and the added amount was also 1 Mg/L, and upon analysis the laboratory
                  fortified sample measured 1.6 /xg/L, then the calculated P for this sample would be
                  (1.6 /ig/L minus 1.0 ^g/L) /I pig/L or 60%.  This calculated P is compared to
                  control limits derived from prior reagent water data.  Assume that analysis of an
                  interference free sample at 1 fig/L yields an s of 0.12 jtg/L and similar analysis at
                  2.0 /xg/L yields X and  s of 2.01 jig/L and 0.20 /xg/L, respectively.  The appropri-
                  ate limits to judge the reasonableness of the percent recovery, 60%, obtained on
                  the fortified matrix sample is computed as follows:
                   100 (2.01
                      2.0
                    3
                                            (0.20
                                       1.0 pg/L
                 100.5% ±  300 (0.233) =
                 100.5% ±  70% or 30% to 170% recovery of the added analyte.
       9.8.3  If the recovery of any such analyte falls outside the designated range, and the labora-
               tory performance for that analyte is shown to be in control (Sect. 9.7), the recovery
               problem encountered with the fortified sample is judged to be matrix related, not
 775

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                                                                                   Method 515.2
               system related.  The result for that analyte in the unfortified sample is labeled sus-
               pect/matrix to inform the data user that the results are suspect due to matrix effects.
9.9    Assessing Instrument System/Instrument Performance Check (IPC) Sample:  Instrument
       performance should be monitored on a daily basis by analysis of the IPC sample.  The IPC
       sample contains compounds designed to indicate appropriate instrument sensitivity, column
       performance (primary column) and chromatographic performance.  IPC sample components
       and performance criteria are listed in Table 11. Inability to demonstrate acceptable instrument
       performance indicates the need for reevaluation of the instrument system. The sensitivity
       requirements are set based on the MDLs published in this method. MDLs will vary from
       laboratory to laboratory.
9.10  The laboratory may adopt additional QC 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. For example, field  or laboratory duplicates may be analyzed to assess the preci-
       sion of the environmental measurements or field reagent blanks may be used to assess contami-
       nation of samples under site conditions, transportation, and storage.

1 0.   CALIBRA TION AND STANDARDIZA TION

10.1  Establish GC operating parameters equivalent to those indicated in Sect. 6.12.  This calibration
       procedure employs procedural standards, i.e., fortified aqueous standards which are processed
       through most of the method (Sect. 11).  The GC system is calibrated by means of the internal
       standard technique (Sect. 10.2).  NOTE: Calibration standard solutions must be prepared such
       that no unresolved analytes  are mixed together (See Table 1).
10.2  Internal Standard Calibration Procedure:  To  use this approach, the analyst must select one or
       more internal standards compatible 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.  DBOB (Sect.  7.13) has been identified as a suitable
       internal standard.
       10.2.1 Prepare aqueous calibration standards at a minimum of three (five are recommended)
               concentration levels for each method analyte as follows: for each concentration, fill a
               250-mL volumetric flask with 240 mL of reagent water at pH 1 and containing 20%
               by  weight of dissolved sodium sulfate. Add an appropriate aliquot  of the  primary
               dilution standard (Sect. 7.16.4) and dilute to 250 mL with the same reagent water.
               Process each aqueous calibration sample through the analytical procedure  beginning
               with Sect.  11.2, i.e.,  omit the hydrolysis and cleanup step (Sect.  11.1). The lowest
               calibration standard should represent analyte concentrations near, but above, the
               respective MDLs.  The remaining standards should bracket the analyte concentrations
               expected in the sample extracts, or should define the working range of the detector.
               The internal standard is added to the final 5 mL extract as specified in Sect. 11.
       10.2.2 Analyze each calibration standard according to the procedure beginning in Sect. 11.2.
               Tabulate response (peak  height or area) against concentration for each compound and
               internal standard.  Calculate the response factor (RF) for  each analyte and surrogate
               using Equation 1.
                                                                                           179

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Method 515.2
                                          Equation  1
                    where:
                    As = Response for the analyte to be measured.
                    Ais = Response for the internal standard.
                    Ca = Concentration of the internal standard
                    Cs = Concentration of the analyte to be measured (/j.g/L).
       10.2.3 If the RF value over the working range is constant (30% RSD or less) the average RF
              can be used for calculations.  Alternatively, the results can be used to plot a calibra-
              tion curve of response ratios (As/Ais) vs. Cs.  A data station may be used to collect the
              chromatographic data, calculate response factors and generate linear or second order
              regression curves.
       10.2.4 The working calibration curve or RF must be verified on each working shift by the
              measurement of one or more calibration standards. A new calibration standard need
              not be derivatized each day. The same standard extract can be used up to 14 days. If
              the response for any analyte varies from the predicted response by more than ±30%,
              the test must be repeated using a fresh calibration  standard.  If the repetition also
              fails,  a new calibration curve must be generated for that analyte using freshly pre-
              pared standards.
       10.2.5 Verify calibration standards periodically, at least quarterly is recommended, by ana-
              lyzing a standard  prepared from reference material obtained from an independent
              source.  Results from these analyses must be within the limits used to routinely check
              calibration.

1 1.   PROCEDURE

11.1   Manual Hydrolysis and Clean-up
       11.1.1 Remove the sample bottles  from cold storage and  allow them to equilibrate to room
              temperature.  Acidify and add sodium thiosulfate to blanks and QC check standards as
              specified in Sect.  8.
       1 1 .1 .2 Measure a 250-mL aliquot of each  sample with a 250-mL graduated cylinder and pour
              into a 500-mL separatory funnel.  Add 250 pL of the surrogate primary dilution stan-
              dard (Sect. 7.18)  to each 250-mL sample.  The surrogate will be  at a  concentration of
              2 ng/L.  Dissolve  50 g sodium sulfate in the sample.
       1 1 .1 .3 Add 4 mL of 6 N NaOH to each sample, seal, and shake.  Check the pH of the
              sample with pH paper or a  pH meter; if the sample does not have a pH greater than
              or equal to 12, adjust the pH by adding more 6 N NaOH.  Let the sample sit at room
              temperature for 1 hr, shaking the separatory funnel and contents periodically.
       1 1 .1 .4 Add 15 mL methylene chloride to the graduated cylinder to rinse the walls, transfer
              the methylene chloride to the separatory funnel and extract the sample by vigorously
              shaking the  funnel for 2 min with periodic venting to release excess pressure.  Allow

180

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                                                                                 Method 515.2
              the organic layer to separate from the water phase for a minimum of 10 min.  If the
              emulsion interface between layers is more than one-third the volume of the solvent
              layer, the analyst must employ mechanical techniques to complete the phase separa-
              tion.  The optimum technique depends upon the sample, but may include stirring,
              filtration through glass wool, centrifugation, or other physical methods.  Discard the
              methylene chloride phase.
       11.1.5 Add a second  15-mL volume of methylene chloride to the separatory funnel and
              repeat the extraction procedure a second time, discarding the methylene chloride
              layer.  Perform a third extraction in the same manner.
       11.1.6 Drain the contents of the separatory funnel into a 500-mL beaker.  Adjust the pH to
              1.0 + 0.1 by the dropwise addition of concentrated sulfuric acid with constant stir-
              ring. Monitor the pH with a pH meter (Sect. 6.8) or short range (0-3) pH paper
              (Sect. 6.14).
11.2  Sample Extraction
       11.2.1 Vacuum Manifold:  Assemble a manifold (Sect. 6.3) consisting of 6-8 vacuum flasks
              with filter funnels (Sect. 6.1,6.2).  Individual vacuum control, on-off and vacuum
              release valves  and vacuum gauges are desirable.   Place the  47 mm extraction disks
              (Sect. 7.1) on  the filter frits.
       11.2.2 Add 20 mL of 10% by volume of methanol  in MTBE to the top of each disk without
              vacuum and allow the solvent to remain for  2 min.  Turn on full vacuum and pull the
              solvent through the disks, followed by room air for 5 min.
       11.2.3 Adjust the vacuum to  approximately 5 in. (mercury) and add the following in  series to
              the filter funnel (a) 20 mL methanol (b) 20 mL reagent water (c) sample.  Do not
              allow the disk to dry between steps and maintain the vacuum at 5 in.
       11.2.4 After the sample is extracted completely, apply maximum vacuum and draw room air
              through the disks for 20 min.
       11.2.5 Place the culture tubes (Sect. 6.4) in the vacuum tubes to collect the eluates.   Elute
              the disks with two each 2-mL aliquots of 10% methanol in  MTBE.  Allow each
              aliquot to remain on the disk for one min before applying vacuum.
       11.2.6 Rinse each 500-mL beaker  (Sect. 11.1.6) with 4 mL of pure MTBE and elute the disk
              with this solvent as in Sect. 11.2.5.
       11.2.7 Remove the culture tubes and cap.
11.3  Extract Preparation
       11.3.1 Pre-rinse the drying tubes (Sect. 7.5.1) with 2 mL of MTBE.
       11.3.2 Remove the entire extract with a 5-mL pipet and drain the lower aqueous layer back
              into the culture tube.  Add  the organic layer to the sodium  sulfate drying tube (Sect.
              7.5.1).  Maintain liquid in the drying tube between this and subsequent steps.   Collect
              the dried extract in a  15-mL graduated centrifuge tube or a 10-mL Kuderna-Danish
              tube.
       11.3.3 Rinse the culture tube with an additional 1 mL of MTBE and repeat Sect. 11.3.2.
                                                                                           787

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Method 515.2
       11.3.4 Repeat step Sect. 11.3.3 and finally add a 1-mL aliquot of MTBE to the drying tube
               before it empties.  The final volume should be 6-9 mL.  In this form the extract is
               esterified as described below.
11.4  Extract Esterification
       11.4.1  Assemble the diazomethane generator (Figure 1) in a hood.
       11.4.2 Add 5 mL of ethyl ether to Tube  1.  Add 4 mL of Diazald solution (Sect. 7.12) and
               3 mL of 37% KOH solution (Sect. 7.15.1) to the reaction tube 2.  Immediately place
               the exit tube into the collection tube containing the sample extract.  Apply nitrogen
               flow (10 mL/min) to bubble diazo-methane through the extract.  Each charge of the
               generator should be sufficient to esterify four samples.  The appearance of a persistent
               yellow color is an indication that esterification is complete. The first sample should
               require 30 sec to 1 min and each subsequent sample somewhat longer.  The final
               sample may require 2-3 min.
       11.4.3 Cap each collection tube and allow to remain stored at room temperature in a hood
               for 30 min.  No significant fading of the yellow color should occur during this period.
               Fortify each  sample with 100 /*L of the internal standard primary dilution solution
               (Sect. 7.17) and reduce the volume to 5.0 mL with the analytical concentrator (Sect.
               6.10), a  stream of dry nitrogen, or an equivalent concentration technique.  NOTE:
               The excess diazomethane is volatilized from the extract during the concentration
               procedure.
       11.4.4 Cap the tubes and store in a refrigerator if further processing will not be performed
               immediately. Analyze by  GC-ECD.
11.5  Gas Chromatography
       11.5.1  Sect. 6.12 summarizes the recommended GC operating conditions.  Included in Table
               1 are retention times observed using this method.  Figures 2A and 2B illustrate the
               chromatographic performance of the primary column (Sect. 6.12.1) for groups A  and
               B of the method analytes.  Other GC columns, chromatographic conditions, or detec-
               tors may be used if the requirements of Sect.  9.3 are met.
       11.5.2 Calibrate the system daily as described in Sect. 10.
       11.5.3 Inject 2 pL of the sample extract.  Record the resulting peak size in area units.
       11.5.4 If the response for any sample peak exceeds the working range of the detector, dilute
               the extract and reanalyze.
11.6  Identification of Analytes
       11.6.1  Identify a sample component by comparison of its retention time to  the retention time
               of a reference chromatogram.  If the retention time of an unknown compound
               corresponds, within limits, to the  retention time of a standard  compound, then an
               analyte is considered to be identified.
       11.6.2 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 interpretation of chromatograms.

752

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                                                                                  Method S15.2
       11.6.3 Identification requires expert judgment when sample components are not resolved
               chromatographically.  When GC peaks obviously represent more than one sample
               component (i.e., broadened peak with shoulder(s) or valley between two or more
               maxima, or any time doubt exists over the identification of a peak in a chromatogram,
               appropriate alternative techniques to help confirm peak identification need to be
               employed. For example, more positive identification may be made by the use of an
               alternative detector which operates on a chemical/physical principle different from that
               originally used, e.g., mass spectrometry, or the use of a second chromatography
               column. A suggested alternative column is described in Sect. 6.12.2.

72.   DA TA ANAL vs/s AND CALCULA TIONS

12.1   Calculate analyte concentrations in the sample from the response for the analyte using the
       calibration procedure described in Sect. 10.
12.2   Calculate the concentration (C) in the sample using the response factor (RF) determined in
       Sect. 10.2.2 and Equation 2, or determine sample concentration from the calibration curve
       (Sect.  10.2.3).
                                         Equation 2
                                               (Ais)(RF)(Vo)
                  where:
                  As = Response for the parameter to be measured.
                  Au = Response for the internal standard.
                   Is ~ Amount of internal standard added to each extract (/j.g).
                  V  = Volume of water extracted  (L).
13.  METHOD PERFORMANCE

13.1  In a single laboratory, analyte recoveries from reagent water were determined at three
      concentration levels, Tables 2-4.  Results were used to determine the analyte MDLs8 listed in
      Table 2.
13.2  In a single laboratory, analyte recoveries from dechlorinated tap water were determined at two
      concentrations, Tables 5 and 6.  In addition, analyte recoveries were determined at two
      concentrations from an ozonated surface (river) water, Tables 7 and 8, and at one level from a
      high humectant surface (reservoir) water,  Table  10. Finally, a holding study was  conducted on
      the preserved, ozonated surface water and recovery data are presented for day 1 and day 14 of
      this study, Tables 8 and 9.   The ozonated surface water was chosen as the matrix in which to
      study analyte stability during a 14-day holding time because it was very biologically active.
                                                                                          183

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Method 515.2
14.  POLLUTION PREVENTION

14.1  This method utilizes the new liquid-solid extraction technology which requires the use of very
      small quantities of organic solvents. This feature eliminates the hazards involved with the use
      of large volumes of potentially harmful organic solvents needed for conventional liquid-liquid
      extractions. Also, mercuric chloride, a highly toxic and environmentally hazardous chemical,
      has been replaced with hydrochloric acid as the sample preservative.  These features make this
      method much safer and a great deal less harmful to the environment.  Some of the phenolic
      herbicides on the analyte list are very difficult to methylate and diazomethane is still required
      to derivatize these compounds.
14.2 For information about pollution prevention that may be applicable to laboratory operations,
      consult "Less is Better:  Laboratory Chemical Management for Waste Reduction"  available
      from the American Chemical Society's Department of Government Relations and Science
      Policy, H55 I6th Street N.W., Washington, D.C. 20036.

15.   WASTE MANAGEMENT

15.1  Due to the nature of this method, there is little need for waste management.  No large volumes
      of solvents or hazardous chemicals are used.  The matrices of concern are finished drinking
      water or source water. However, the  Agency requires that laboratory waste management
      practices be conducted consistent with all applicable rules and regulations, and  that laboratories
      protect the air,  water, and land by minimizing and controlling all releases from fume hoods
      and bench operations.  Also, compliance is required with any sewage discharge permits and
      regulations, particularly the hazardous waste identification rules and land disposal restrictions.
      For further information on waste management, consult "The Waste Management Manual for
      Laboratory Personnel," also available  from the American Chemical Society at the address in
      Sect.  14.2.
 184

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                                                                               Method 515.2
                                     References

1.    ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard Practice for
      Preparation of Sample Containers and for Preservation," American Society for Testing and
      Materials, Philadelphia, PA, p. 86, 1986.

2.    Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for Determination of Phthalate
      Ester Plasticizers in Open-Ocean Biota Samples," Analytical Chemistry. 47,  2225 (1975).

3.    Giam, C.S., and H.S. Chan. "Control of Blanks in the Analysis of Phthalates in Air and
      Ocean Biota Samples," U.S. National Bureau of Standards, Special Publication 442, pp.
      701-708, 1976.

4.    "Carcinogens - Working with Carcinogens," Department of Health, Education, and Welfare,
      Public Health Service, Center for Disease Control, National Institute for Occupational Safety
      and Health, Publication No. 77-206, Aug.  1977.

5.    "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational Safety
      and Health Administration, OSHA 2206, (Revised, January 1976).

6.    "Safety  in Academic Chemistry Laboratories," American Chemical Society Publication,
      Committee on Chemical Safety, 3rd Edition, 1979.

7.    ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
      Sampling Water," American Society for Testing and Materials, Philadelphia, PA, p. 130,
      1986.

8.    Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L.,  "Trace Analyses
      for Wastewaters," Environ. Sci. Technol. 1981,15, 1426-1435.

9.    40 CFR, Part 136, Appendix B.
                                                                                        185

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Method 515.2
                                                     Retention Time (min.f
Table 1.   Retention Data
  Analyte
  3,5-Dichlorobenzoic acid
  2,4-Dichlorophenylacetic acid (SA)
  Dicamba
  Dichlorprop
  2,4-D
  4,4'-Dibromooctafluorobiphenyl (IS)
  Pentachlorophenol
  Silvex
  5-Hydroxydicamba
  2,4,5-T
  2,4-DB
  Dinoseb
  Bentazon
  Picloram
  Dacthal
  Acifluorfen
*   Analytes were divided into two groups during method development to avoid chromatographic
    overlap.
b   Columns and chromatographic conditions are described in Sect. 6.12.
Table 2.   Single Laboratory Recovery, Precision Data and Method Detection Limit
           With Fortified Reagent Water —Level 1
Group'
A
A,B
B
A
B
A,B
A
B
B
A
B
A
B
B
A
B
Primary
16.72
19.78
20.18
22.53
23.13
24.26
25.03
25.82
26.28
26.57
27.95
28.03
28.70
29.93
31.02
35.62
I Confirmation
18.98
22.83
23.42
25.90
27.01
26.57
27.23
29.08
30.18
30.33
31.47
33.02
33.58
35.90
34.32
40.58
                       Fortified Cone.  Mean* Recovery   Relative Std.
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
(itg/U
  0.50
  2.50
  0.25
  2.50
  0.25
  0.75
  1.25
  0.25
  0.50
  0.75
  0.25
  0.75
  0.25
  0.25
 70
 70
 96
 79
 96
109
126
106
 87
 90
103
 95
116
 98
Dev. (%)
     21
     11
     38
     12
     16
     11
     24
     15
     22
     12
     18
     15
     18
      9
 MDL
fag/U
 0.25
 0.63
 0.28
 0.72
 0.13
 0.28
 1.23
 0.13
 0.28
 0.25
 0.16
 0.35
 0.16
 0.06
    Based on the analyses of seven replicates.
    Measurement includes the mono- and diacid metabolites.
 756

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                                                                           Method 515.2
Table 3.   Single Laboratory Recovery and Precision Data For Fortified Reagent
           Water-Level 2
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified Cone.
    frg/U
     0.80
     4.0
     0.40
     4.0
     0.40
     1.20
     2.00
     0.40
     0.80
     1.20
     0.40
     1.20
     0.40
     0.40
                                                 Mean* Recovery    Relative Std. Dev.
 61
 81
 96
 90
 96
109
126
 76
 87
 90
 66
 68
116
105
27
 8
38
13
16
11
24
21
22
12
26
21
18
 7
    Based on the analyses of six-seven replicates.
    Measurement includes the mono- and diacid metabolites.
Table 4.   Single Laboratory Recovery and Precision Data For Fortified Reagent
           Water—Level 3
Analyte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified Cone.
    (V9/U
       2.0
     10.0
       1.0
     10.0
       1.0
       3.0
       5.0
       1.0
       2.0
       3.0
       1.0
       3.0
       1.0
       1.0
                                                 Mean* Recovery   Relative Std. Dev.
 59
 68
 90
 74
 60
 75
 62
 97
 63
 77
 69
 66
 64
 68
13
 8
20
 6
10
 9
18
17
10
 8
11
 9
15
 8
    Based on the analyses of six-seven replicates.
    Measurement includes the mono- and diacid metabolites.
                                                                                   757

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 Method 515.2
Table 5.  Single Laboratory Recovery and Precision Data For Fortified,
Dechlorinated Tap Water —Level  1

                              Fortified Cone.     Mean' Recovery    Relative Std. Dev.
Analyte                          (f/g/LJ                (%)                 (o/o}
Acifluorfen                         0.50                 117                21
Bentazon                           2.50                  96                12
2,4-D                              0.25                  59C                55
2,4-DB                             2.50                 112                15
Dacthal"                            0.25                 101                 10
Dicamba                           0.75                  91                 14
3,5-Dichlorobenzoic acid              1.25                 103                15
Dichlorprop                         0.25                 218d                37
Dinoseb                            0.50                 134                10
5-Hydroxydicamba                   0.75                  90                14
Pentachlorophenol                   0.25                  91                  8
Picloram                            0.75                  76                28
2,4,5-T                             0.25                 118                16
2,4,5-TP                           0.25                  99                10

a    Based on the analyses of six-seven replicates.
b    Measurement includes the mono- and diacid metabolites.
'    2,4-D background value was 0.29 /vg/L.
d    Probable interference.
Table 6.  Single Laboratory Recovery and Precision  Data For Fortified,
Dechlorinated Tap Water—Level 2

                              Fortified Cone.      Mean* Recovery    Relative Std. Dev.
Analyte                           fag/L)                (%)                 (%)
Acifluorfen                           2.0                150                  7
Bentazon                           10.0                112                  9
2,4-D                               1.0                 90                 16
2,4-DB                             10.0                111                 10
Dacthal"                             1.0                118                  8
Dicamba                             3.0                 86                 10
3,5-Dichlorobenzoic acid               5.0                111                  5
Dichlorprop                          1.0                 88                 30
Dinoseb                             2.0                121                  6
5-Hydroxydicamba                    3.0                 96                  6
Pentachlorophenol                    1.0                 96                  6
Picloram                             3.0                132                 12
2,4,5-T                             1.0                108                 10
2,4,5-TP                             1.0                115                  7
2,4-Dichlorophenylacetic acidc          1.0                120                 19

a    Based on the analyses of six-seven replicates.
b    Measurement includes the mono- and diacid metabolites.
c    Surrogate analyte.
188

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                                                                          Method 515.2
Table 7.  Single Laboratory Recovery and Precision Data For Fortified, Ozonated
Surface Water—Level 1

                             Fortified Cone.     Mean' Recovery    Relative Std. Dev.
Analyte                           frg/U               (%)                 (%)
Acifluorfen                           .50               172                 14
Bentazon                           2.50                 92                 22
2,4-D                              0.25               127                 13
2,4-DB                             2.50               154                 19
Dacthal"                            0.25               113                 17
Dicamba                            0.75               107                 13
3,5-Dichlorobenzoic acid              1.25               100                 17
Dichlorprop                         0.25               115                 20
Dinoseb                            0.50               134                 28
5-Hydroxydicamba                   0.75                 89                 13
Pentachlorophenol                   0.25               110                 22
Picloram                            0.75               109                 27
2,4,5-T                             0.25               102                 19
2,4,5-TP                           0.25               127                  8
2,4-Dichlorophenylacetic acidc         0.25                 72                 31

a   Based on the analyses of six-seven replicates.
b   Measurement includes the mono- and diacid metabolites.
c   Surrogate analyte.
Table 8.  Single Laboratory Recovery and Precision Data For Fortified, Ozonated
Surface Water—Level 2, Stability Study Day  1°

                             Fortified Cone.     Mean* Recovery   Relative Std. Dev.
Analyte                           (f/g/U               (%)                 (%)
Acifluorfen                           2.0               173                 11
Bentazon                           10.0               122                  7
2,4-D                               1.0               126                 10
2,4-DB                             10.0               130                  7
Dacthal"                             1.0               116                 11
Dicamba                             3.0               109                  9
3,5-Dichlorobenzoic acid               5.0               115                 11
Dichlorprop                          1.0               116                 11
Dinoseb                             2.0               116                  9
5-Hydroxydicamba                    3.0               121                  9
Pentachlorophenol                    1.0               118                 10
Picloram                             3.0               182                 14
2,4,5-T                              1.0               112                  9
2,4,5-TP                            1.0               122                 10
2,4-Dichlorophenylacetic acid"          1.0               110                 26

a   Based on the analyses of six-seven replicates.
b   Measurement includes the mono- and diacid metabolites.
c   Samples preserved at pH = 2.0.
d   Surrogate analyte.
                                                                                  755

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Method 515.2
Table 9.  Single Laboratory Recovery and Precision Data  For Fortified, Ozonated
Surface Water-Level 2, Stability Study Day  14°

                              Fortified Cone.     Mean* Recovery   Relative Std. Dev.
Analyte                           (jjg/U               (%)                 (%)
Acifluorfen                           2.0               151                 18
Bentazon                           10.0                 97                   9
2,4-D                               1.0                 84                 11
2,4-DB                             10.0               128                 10
Dacthal6                             1.0               116                   7
Dicamba                             3.0               103                   9
3,5-Dichlorobenzoic acid               5.0                 81                 12
Dichlorprop                          1.0               107                 11
Dinoseb                             2.0               118                   7
5-Hydroxydicamba                    3.0                 20                 14
Pentachlorophenol                    1.0                 94                   7
Picloram                             3.0               110                 32
2,4,5-T                              1.0               113                   8
2,4,5-TP                            1.0               113                 11
2,4-Dichlorophenylacetic acid"          1.0                 87                   6

3    Based on the analyses of six-seven replicates.
b    Measurement includes the mono- and diacid metabolites.
0    Samples preserved at pH =  2.0.
d    Surrogate analyte.
Table 10.  Single Laboratory Recovery and Precision Data For Fortified, High Humic
Content Surface Water

                             Fortified Cone.      Mean" Recovery   Relative Std. Dev.
Analyte                           (tig/U                (%)                (%)
Acifluorfen                           2.0                120                13
Bentazon                           10.0                 87                11
2,4-D                               1.0                 59                  7
2,4-DB                             10.0                 80                14
Dacthal"                             1.0                100                  6
Dicamba                             3.0                 76                  9
3,5-Dichlorobenzoic acid               5.0                 87                  4
Dichlorprop                          1.0                110                22
Dinoseb                             2.0                 97                  6
5-Hydroxydicamba                    3.0                 82                  9
Pentachlorophenol                    1.0                 70                  5
Picloram                             3.0                124                  9
2,4,5-T                             1.0                101                  4
2,4,5-TP                            1.0                 80                  6

a   Based on the analyses of six-seven replicates.
b   Measurement includes the mono- and diacid metabolites.
 190

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                                                                             Method 515.2
Table 11.  Laboratory Performance Check Solution
Test
Sensitivity
Analyte
Dinoseb
Chromatographic performance 4-Nitrophenol
Column performance         3,5-Dichlorobenzoic acid
                           4-Nitrophenol
 Cone,
(ug/mL)   Requirements
   0.004  Detection of analyte; S/N >
          3
   1.6     0.70 0.40b
   1.6
    PGF = peak Gaussian factor. Calculated using the equation:
                                         1.83xW
                                  PGF =
where W | _   is the peak width at half height and W   — |  is the peak width at tenth height
"   Resolution between the two peaks as defined by the equation:
                                           R-J-
                                               w
where t is the difference  in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
                                                                                     191

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Method 515.2
                   N2 Flow
                                        Flat Joint with O Ring and Clamp
       Diethyl Ether Level
                                                   Flat Joint with O Ring and Clamp
                        Diazald Level
                          KOH Level
                                                                                      52-015-1
                            Figure 1.  Diazomethane Generator
192

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                                                                            Method 515.2
    4.0 -
   3.0 -
v>

I
o

X
             35DCBA
    2.0 -
    1.0 -
            1.5
                                       PCP
                                                       Dacthal
2.0
    2.5

x101 Minutes
3.0
3.5
               Figure 2A. Chromatogram of Group A Analytes Extracted from
                          Ozonated Surface Water (bottom Chromatogram is
                          the laboratory reagent blank)
                                                                                 52-015-2
                                                                                    193

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 Method 515.2
   3.0 —
 ID
 *-
 o
 >  2.0-

'o
   1.0 —
              Dicamba
                  I
              SURR
                 2.0
                                         Bentazon
                                    Silvex
                            IS
                           24D
                                            24DB
                                       5HD
                                   2.5
3.0
                                        x101 Minutes
                                                                     ACIP
3.5
                                                                                 52-015-3
             Figure 2B. Chromatogram of Group B Analytes Extracted from
                        Ozonated Surface Water (bottom Chromatogram is
                        the laboratory reagent blank)
194

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                      Method  525.1
   Determination of Organic Compounds
                       in Drinking Water
               by Liquid-Solid Extraction
                   and Capillary Column
Gas Chromatography/Mass Spectrometry
                   Revision 2.2 - EPA EMSL-Ci
                                 May 1991
        J.W. Eichelberger, T.D. Behymer, W.L. Budde — Method 525,
                         Revision 1.0, 2.0, 2.1 (1988)

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                                Method  525.1
        Determination of Organic Compounds in Drinking Water
              by Liquid-Solid Extraction and Capillary Column
                 Gas Chromatography/Mass  Spectrometry
1.    SCOPE AND APPLICA TION

1.1   This is a general purpose method that provides procedures for determination of organic
      compounds in finished drinking water, raw source water, or drinking water in any treatment
      stage.  The method is applicable to a wide range of organic compounds that are efficiently
      partitioned from the water sample onto a C,g organic phase chemically bonded to a solid silica
      matrix in a cartridge or disk, and sufficiently volatile and thermally stable for gas chromatog-
      raphy.  Single-laboratory accuracy and precision data have been determined at two concentra-
      tions with two instrument systems for the following compounds:
         Compound
         Acenaphthylene
         Alachlor
         Aldrin
         Anthracene
         Atrazine
         Benz[a]anthracene
         Benzo[6]fluoranthene
         Benzo[Ar]fluoranthene
         Benzo[a]pyrene
         Benzo[<7,/?,/]perylene
         Butylbenzyl phthalate
         Chlordane Components
         a-Chlordane
         7-Chlordane
         trans-Nonachlor
         2-Chlorobiphenyl
         Chrysene
         Dibenz[a,/7]anthracene
         Di-n-butyl phthalate
         2,3-dichlorobiphenyl
         Diethyl phthatate
         Bis(2-ethylhexyl) adipate
         Bis(2-ethylhexy) phthalate
         Dimethyl phthalate
         Endrin
         Fluorene
         Heptachlor
         Heptachlor epoxide
         2,2'3,3',4,4',6-Heptachlorobiphenyl
         Hexachlorobenzene
         2,2',4,4',5,6'-Hexachlorobiphenyl
         Hexachlorocyclopentadiene
         lndeno[1,2,3,c,£/]pyrene
MW
CAS No.
152
269
362
178
215
228
252
252
252
276
312
406
406
440
188
228
278
278
222
222
222
390
194
378
166
370
386
392
282
358
270
276
208-96-8
15972-60-8
309-00-2
120-12-7
1912-24-9
56-55-3
205-99-2
207-08-9
50-32-8
191-24-2
85-68-7
5103-71-9
5103-74-2
39765-80-5
2051-60-7
218-01-9
53-70-3
84-72-2
16605-91-7
84-66-2
103-23-1
117-81-7
131-11-3
72-20-8
86-73-7
76-44-8
1024-57-3
52663-71-5
118-74-1
6-145-22-4
77-47-4
193-39-5
                                                                                   757

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Method 525.1
Compound
Lindane
Methoxychlor
2,2',3,3',4,5',6,6'-Octachlorobiphenyl
2,2,3',4,6-Pentachlorobiphenyl
Pentachlorophenol
Phenanthrene
Pyrene
Simazine
2,2',4,4'-Tetrachlorobipheneyl
Toxaphene mixture
2,4,5-Trichlorobiphenyl
MW
288
344
426
324
264
178
202
201
290

256
CAS No.
58-89-9
72-43-5
40186-71-8
60233-25-2
87-86-5
85-01-8
1 29-00-0
122-34-9
2437-79-8
8001-35-2
15862-07-4
        a    Monoisotopic molecular weight calculated from the atomic masses of the iso-
            topes with the smallest masses.

      A laboratory may use this method to identify and measure additional analytes after the labora-
      tory obtains acceptable (defined in Sect.  10) accuracy and precision data for each added
      analyte.
1.2   Method detection limit (MDL) is defined as the statistically calculated minimum amount that
      can be measured with 99% confidence that the reported value is greater than zero.1  The MDL
      is compound dependent and is particularly dependent on extraction efficiency and sample
      matrix.  For the listed  analytes, MDLs vary from 0.01 to 15 ng/L.  The concentration calibra-
      tion range of this method is 0.1 jttg/L to  10 ng/L.

2.    SUMMARY OF METHOD

2.1   Organic  compound analytes, internal standards, and surrogates are extracted from a water
      sample by passing 1 liter of sample water through a cartridge or disk containing a solid inor-
      ganic matrix coated with a chemically bonded CI8 organic phase (liquid-solid extraction, LSE).
      The organic compounds are eluted from  the LSE cartridge or disk with a small quantity of
      methylene chloride, and concentrated further by evaporation of some of the solvent.  The
      sample components are separated, identified, and measured by injecting an aliquot of the
      concentrated methylene chloride extract into a high resolution fused silica capillary column of
      a gas chromatography/mass spectrometry (GC/MS) system.  Compounds eluting from the GC
      column are identified by comparing  their measured mass  spectra and retention times to refer-
      ence spectra  and retention times in a data base.  Reference spectra and retention times for
      analytes  are obtained by the measurement of calibration standards under the same conditions
      used for samples.  The concentration of each identified component is measured by relating the
      MS response of the quantitation ion  produced by that compound to the MS response of the
      quantitation ion produced by a compound that is used as an internal standard.  Surrogate
      analytes, whose concentrations are known in every sample, are measured with the same
      internal standard calibration procedure.
198

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                                                                                   Method 525.1
3.     DEFINITIONS

3.1    Internal standard:  A pure analyte(s) added to a solution in known amount(s) and used to
       measure the relative responses of other method analytes and surrogates that are components of
       the same solution.  The internal standard must be an analyte that is not a sample component.
3.2    Surrogate analyte:  A pure analyte(s), which is extremely unlikely to be found in any sample,
       and which is added to a sample aliquot  in known amount(s) before extraction and is measured
       with the same procedures used to measure other sample components.  The purpose of a surro-
       gate  analyte is to monitor method performance with each sample.
3.3    Laboratory duplicates (LD1 and LD2):  Two sample aliquots taken in the analytical laboratory
       and analyzed separately with identical procedures.  Analyses of LD1 and LD2 give a measure
       of the precision associated with laboratory procedures, but  not with sample collection, preser-
       vation, or storage procedures.
3.4    Field duplicates (FD1 and FD2):  Two  separate samples collected at the same time and place
       under identical circumstances and treated exactly the same  throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation, and storage, as well as with laboratory procedures.
3.5    Laboratory reagent blank (LRB):  An aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware,  equipment, solvents, reagents, internal standards,
       and surrogates that are  used with other  samples. The LRB is used to determine if method
       analytes or  other interferences are present in the laboratory environment, the reagents, or the
       apparatus.
3.6    Field reagent blank (FRB):  Reagent water placed in a sample container in the laboratory and
       treated as a sample in all respects, including exposure to sampling site conditions, storage,
       preservation, and all analytical procedures.  The purpose of the FRB is to determine if method
       analytes or  other interferences are present in the field environment.
3.7    Laboratory  performance check solution (LPC): A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with  respect to a defined set of method  criteria.
3.8    Laboratory  fortified blank (LFB):  An aliquot of reagent water to which known quantities of
       the method  analytes are added in  the laboratory. The LFB is analyzed  exactly like a sample,
       and its purpose is to determine whether the methodology is in control, and whether the labora-
       tory is  capable of making accurate and precise measurements at the required method detection
       limit.
3.9    Laboratory  fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory. The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the sample
       matrix  must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock standard solution: A concentrated solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared  in the laboratory with
                                                                                           199

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Method 525. 1
       an assayed reference compound.  Stock standard solutions are used to prepare primary dilution
       standards.
3.11   Primary dilution standard solution:  A solution of several analytes prepared in the laboratory
       from stock standard solutions and diluted as needed to prepare calibration solutions and other
       needed analyte solutions.
3.12  Calibration standard (CAL):  A solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
       tions are used to calibrate the instrument response with respect to analyte concentration.
3.13  Quality control sample (QCS):  A sample matrix containing method analytes or a solution of
       method analytes  in a water miscible solvent which is used to fortify reagent water or environ-
       mental samples.  The QCS is obtained from a source external to the laboratory, and is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    During analysis, major contaminant sources are reagents and liquid-solid extraction columns.
       Analyses of field and laboratory reagent blanks provide information about the presence of
       contaminants.
4.2   Interfering contamination may occur when a sample containing low concentrations of com-
       pounds is analyzed immediately after  a sample containing relatively high concentrations of
       compounds. Syringes and splitless injection port liners must be cleaned carefully or replaced
       as needed.  After analysis of a  sample containing high concentrations of compounds,  a labora-
       tory reagent blank should be analyzed to ensure that accurate values are obtained for the next
       sample.

5.     SAFETY

5.1    The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
       fined; each chemical should be treated as a potential health hazard, and exposure to these
       chemicals should be minimized.  Each laboratory is responsible for maintaining awareness of
       OSHA regulations regarding safe handling of chemicals used in this method.  Additional
       references to laboratory  safety  are cited.2~*
5.2   Some method analytes have been tentatively classified as known or suspected human or mam-
       malian carcinogens. Pure standard materials and stock standard solutions of these compounds
       should be handled with suitable protection to skin, eyes, etc.

6.     APPARATUS AND EQUIPMENT

6.1    All glassware must be meticulously cleaned. This may  be  accomplished by washing with
       detergent and water, rinsing with water, distilled water, or solvents, air-drying, and heating
       (where appropriate) in an oven.  Volumetric glassware is never heated.
6.2   Sample  containers.  1-liter or 1-quart amber glass bottles fitted with a Teflon-lined screw cap.
       (Bottles in which high purity solvents were received can be used as sample containers without
       additional cleaning if they have been  handled carefully to avoid contamination during use and
       after use of original contents.)

200

-------
                                                                                  Method 525.1
6.3   Separatory funnels.  2-liter and 100-mL with a Teflon stopcock.
6.4   Liquid chromatography column reservoirs.  Pear-shaped 100- or 125-mL vessels without a
       stopcock but with a ground glass outlet joint sized to fit the liquid-solid extraction column.
       (Lab Glass, Inc. part no. ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom inner
       joint, or equivalent).  A 14/35 outlet joint fits some commercial cartridges.
6.5   Syringe  needles. No. 18 or 20 stainless steel.
6.6   Vacuum flasks. 1- or 2-liter with solid rubber stoppers.
6.7   Volumetric flasks, various sizes.
6.8   Laboratory or aspirator vacuum system.  Sufficient capacity to maintain a slight vacuum of
       13 cm (5 in.) of mercury in the vacuum flask.
6.9   Micro syringes, various sizes.
6.10  Vials. Various sizes of amber vials with Teflon-lined screw caps.
6.11  Drying column. Approximately 1.2 cm x 40 cm with 10 mL graduated collection vial.
6.12  Analytical balance.  Capable of weighing 0.0001 g accurately.
6.13  Fused silica capillary gas chromatography column.  Any capillary column that provides ade-
       quate resolution, capacity, accuracy,  and precision (Sect. 10) can be used.  A 30 m  x
       0.25 mm id fused silica capillary column coated with a 0.25 /mi bonded film of polyphenyl-
       methylsilicone  is recommended (J&W DB-5 or equivalent).
6.14  Gas chromatograph/mass spectrometer/data system (GC/MS/DS)
       6.14.1  The GC must be capable of temperature programming and  be equipped for
               splitless/split or on-column capillary injection. The injection tube liner should be
               quartz and about 3 mm in diameter. The injection system must not allow the analytes
               to contact hot stainless steel  or other metal surfaces that promote decomposition.
       6.14.2  The GC/MS interface should allow the capillary column or transfer line exit to be
               placed within a few mm of the ion source.  Other interfaces, for example the open
               split interface,  are acceptable as long as the system has adequate sensitivity (see
               Sect. 9 for calibration requirements).
       6.14.3  The mass spectrometer must be capable of electron ionization at a nominal electron
               energy of 70 eV.  The spectrometer must be capable of scanning from 45 to 450 amu
               with a complete scan cycle time (including scan overhead) of 1.5 sec or less.  (Scan
               cycle time  = Total  MS data acquisition time in sec divided by number of scans in the
               chromatogram). The spectrometer must produce a mass spectrum that meets all
               criteria in Table 1 when 5 ng or less of DFTPP is introduced into the GC.  An aver-
               age spectrum across the DFTPP GC peak may be used to test instrument
               performance.
       6.14.4  An interfaced data system is required to  acquire, store, reduce, and output mass spec-
               tral data.  The  computer software must have the capability of processing stored
               GC/MS data by recognizing  a GC peak within any given retention time window,
               comparing the mass spectra from the GC peak with spectral data in a user-created data
              base, and generating a list of tentatively  identified compounds with their retention
              times and scan  numbers.  The software must also allow integration of the ion abun-

                                                                                          207

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Method 525.1
               dance of any specific ion between specified time or scan number limits, calculation of
               response factors as defined in Sect. 9.2.6 (or construction of a second or third order
               regression calibration curve), calculation of response factor statistics (mean and
               standard deviation), and calculation of concentrations of analytes using either the
               calibration curve or the equation in Sect. 12.
6.15  Millipore Standard Filter Apparatus, All Glass. This will be used if the disks are to be used to
       carry out the extraction instead of the cartridges.

7.     REAGENTS AND CONSUMABLE MATERIALS

7.1    Helium carrier gas, as contaminant free as possible.
7.2    Liquid-solid extraction (LSE) cartridges.  Cartridges are inert non- leaching  plastic, for exam-
       ple polypropylene, or glass,  and must not contain plasticizers, such as phthalate esters or
       adipates, that leach into methylene chloride.  The cartridges are packed with about 1 gram of
       silica, or other inert inorganic support, whose surface is modified by chemically bonded
       octadecyl (C,8) groups.  The packing must have a narrow size distribution and must not leach
       organic compounds into methylene chloride.  One liter of water should pass  through the
       cartridge in about 2 hrs with the assistance of a slight vacuum of about 13 cm  (5 in.) of
       mercury.  Sect. 10 provides criteria for acceptable LSE cartridges which are available from
       several commercial suppliers.The extraction disks contain approximately 0.5 grams of 8 um
       octadecyl bonded silica uniformly enmeshed in a matrix of  inert PFTE fibrils.  The size of the
       disks is 47mm x 0.5mm.  As with cartridges, the disks should not contain any organic com-
       pounds, either from the PFTE or the bonded silica, which will leach into the methylene chlo-
       ride eluant.  One liter of reagent water should pass through the disks in 5-20 minutes using a
       vacuum of about 66cm (26 in.) of mercury.  Section 10 provides criteria for acceptable LSE
       disks which are available commercially.
7.3    Solvents
       7.3.1   Methylene chloride, acetone, toluene and methanol.  High purity pesticide quality or
               equivalent.
       7.3.2   Reagent water.  Water  in which an interferent is not observed at the method detection
               limit of the compound of interest. Prepare reagent water  by passing tap water through
               a filter bed containing about 0.5 kg of activated carbon or by using  a  water purifica-
               tion system.  Store in clean, narrow-mouth bottles with Teflon-lined septa and screw
               caps.
7.4    Hydrochloric acid. 6N.
7.5    Sodium sulfate, anhydrous.  (Soxhlet extracted with methylene chloride for a minimum of 4
       hrs.)
7.6    Stock standard solutions. Individual solutions of analytes,  surrogates, and internal standards
       may be  purchased as certified solutions or prepared from pure materials.  To prepare,  add 10
       mg (weighed on an analytical balance to 0.1  mg) of the pure material to  1.9 mL of methanol
       or acetone in a 2-mL volumetric flask, dilute to the mark,  and transfer the solution to an
       amber glass vial.  If the analytical standard is available only in quantities smaller than  10 mg,
       reduce the volume of solvent accordingly. Some polycyclic aromatic hydrocarbons are not
       soluble  in methanol or acetone, and their stock standard solutions are prepared in toluene.

202

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                                                                                    Method 525.1
       Methylene chloride should be avoided as a solvent for standards because its high vapor pres-
       sure leads to rapid evaporation and concentration changes.  Methanol and acetone are not as
       volatile as methylene chloride,  but their solutions must also be handled with care to avoid
       evaporation.  Compounds 10, 11, and 35 in Table 2 are soluble in acetone.  Compounds 12,
       13, and 20 in Table 2 are soluble in toluene. If compound purity is certified by the supplier at
       >96%, the weighed amount can be used without correction to calculate the concentration of
       the solution (5 ftg//*L).  Store the amber vials in a dark cool place.
7.7   Primary dilution standard solution.  The stock standard solutions are used to prepare a primary
       dilution standard solution that contains multiple analytes.  The recommended solvent for this
       dilution is acetone.   Aliquots of each of the stock standard solutions are combined to produce
       the primary dilution in which the concentration of the analytes is at least equal to the concen-
       tration of the most concentrated calibration solution, that is, 10 ng//*L.  Store the primary
       dilution standard solution in an amber vial in a dark cool place, and check frequently for signs
       of deterioration or evaporation, especially just before preparing calibration solutions.
7.8   Fortification solution of internal standards and surrogates. Prepare  a solution of
       acenaphthene-D10, phenanthrene-D,0, chrysene-D,2,  and perylene-D,2 in methanol or acetone at
       a concentration of 500 /*g/mL of each.  This solution is used in the preparation of the calibra-
       tion solutions.  Dilute a portion of this solution by 10 to 50 /xg/mL  and use this solution to
       fortify the actual water samples (see Sect.  11.2).  Other surrogates, for example, caffeine-'5N2
       and pyrene-D10 may be included in this solution as needed (a 100-/*L aliquot of this 50 /ig/mL
       solution added to 1  liter of water gives a concentration of 5 /xg/L of each internal standard or
       surrogate).  Store this solution in an amber vial in a dark cool  place.
7.9   MS performance check solution.  Prepare a 5 ng//xL solution of DFTPP  in methylene chlo-
       ride.  Store this solution in an amber vial in a dark  cool place.
7.10  Calibration solutions (CAL1 through CAL6). Prepare a series of six concentration calibration
       solutions in acetone which contain all analytes except pentachlorophenol and toxaphene at
       concentrations of 10, 5, 2, 1, 0.5, and 0.1  ng//*L, with a constant concentration of 5 ng//iL of
       each internal standard and surrogate in each CAL solution. CAL1 through CAL6 are prepared
       by combining appropriate aliquots of the primary dilution standard solution (7.7) and the
       fortification solution (500 /^g/mL) of internal standards and surrogates (7.8). Pentachlorophe-
       nol is included in this solution at a concentration four times the other analytes.  Toxaphene
       CAL solutions should be prepared as separate solutions at concentrations of 250, 200,  100, 50,
       25, and 10 ng//xL.  Store these  solutions in amber vials in a dark cool place.  Check these
       solutions regularly for signs of deterioration,  for example, the appearance of anthraquinone
       from the oxidation of anthracene.
7.11  Reducing agents.  Sodium sulfite or sodium arsenite.  Sodium thiosulfate is not recommended
       as it may produce a residue of elemental sulfur that can interfere with some analytes.
7.12  Fortification solution for optional recovery  standard. Prepare a solution of terphenyl-D,4 in
       methylene chloride at a concentration of 500 /xg/mL.  An aliquot of this solution may  be  added
       (optional) to the extract of the LSE  cartridge  to check on the recovery of the internal standards
       in the extraction process.
                                                                                             203

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Method 525.1
8.     SAMPLE COLLECTION,  PRESERVATION, AND HANDLING

8.1    Sample collection.  When sampling from a water tap, open the tap and allow the system to
       flush until the water temperature has stabilized (usually about 2-5 min).  Adjust the flow to
       about 500 mL/min and collect samples from the flowing stream.  Keep samples sealed from
       collection time until analysis.  When sampling from an open body of water,  fill the sample
       container with water from a representative area.  Sampling equipment, including automatic
       samplers, must be free of plastic tubing, gaskets, and other parts that  may leach analytes into
       water.  Automatic samplers that composite samples over time must use refrigerated glass
       sample containers.
8.2    Sample dechlorination and preservation.  All samples should be iced or refrigerated at 4°C
       from the time of collection until extraction.  Residual chlorine should  be  reduced at the sam-
       pling site by addition of a reducing agent. Add 40-50 mg of sodium sulfite  or sodium arsenite
       (these may be added as solids with stirring until dissolved) to each liter of water.  Hydrochlo-
       ric acid should be used at the sampling site to retard the microbiological degradation of some
       analytes in unchlorinated water.  The sample pH is adjusted to <2 with 6 N hydrochloric
       acid. This is the same pH used in the extraction, and is required to support  the recovery of
       pentachlorophenol.
8.3    Holding time.  Samples must be extracted within 7 days and the extracts  analyzed within 30
       days of sample collection.
8.4    Field blanks.
       8.4.1   Processing of a field reagent blank (FRB) is recommended along with each sample
               set,  which  is composed of the samples collected from the same general sample site at
               approximately the same time.  At the laboratory, fill a sample container with reagent
               water, seal, and ship to the sampling site along with the empty sample containers.
               Return the FRB  to the laboratory with filled sample bottles.
       8.4.2   When hydrochloric acid is added to samples, use the same procedures to add the same
               amount to the FRB.

9.     CALIBRATION

9.1    Demonstration and documentation of acceptable initial calibration is required before any
       samples are analyzed and is required intermittently throughout sample analysis as dictated by
       results of continuing calibration checks.  After initial calibration  is successful, a continuing
       calibration check is required at the beginning of each 8 hr. period during which analyses are
       performed. Additional periodic calibration checks are good laboratory practice.
9.2    Initial Calibration
       9.2.1   Calibrate the mass and abundance scales of the MS with calibration  compounds and
               procedures prescribed by the manufacturer with any modifications necessary to meet
               the requirements in Sect. 9.2.2.
       9.2.2   Inject into the GC a l-/xL  aliquot of the 5 ng//iL DFTPP solution and acquire a mass
               spectrum that includes data for m/z 45-450.  Use GC conditions that produce a nar-
               row (at least five scans per peak) symmetrical peak.  If the spectrum does not meet all
               criteria (Table 1),  the MS must be retuned and adjusted to meet all  criteria before

204

-------
                                                                           Method 525.1
        proceeding with calibration.  An average spectrum across the GC peak may be used to
        evaluate the performance of the system.
9.2.3  Inject a 1-pL aliquot of a medium concentration calibration solution, for example
        0.5-2 /ig/L, and acquire and store data from m/z 45-450 with a total cycle time
        (including scan overhead time) of  1.5 sec or less.  Cycle time should be adjusted to
        measure at least five or more spectra during the elution of each GC peak.
        9.2.3.1   Multi-ramp temperature program GC conditions. Adjust the helium carrier
                   gas flow rate to about 33 cm/sec.  Inject at 45°C and hold in splitless
                   mode for 1  min.  Heat rapidly to 130°C.  At 3 min start the temperature
                   program: 130-180°C at 12°/min; 180-240°C at 7°/min; 240-320°C at
                   12°/min. Start data acquisition at 5 min.
        9.2.3.2   Single ramp linear temperature program.  Adjust the helium carrier gas
                   flow rate to about 33 cm/sec. Inject at 40°C and hold in splitless mode
                   for 1 min.  Heat rapidly to  160°C.  At 3 min start the temperature pro-
                   gram:  160-320°C at 6°/min; hold at 320° for 2 min.  Start data acqui-
                   sition at  3 min.
9.2.4  Performance criteria for the medium calibration. Examine the.stored GC/MS data
        with the data system software.  Figure 1  shows an acceptable total ion chromatogram.
        9.2.4.1   GC performance.   Anthracene and phenanthrene should be separated by
                   baseline.  Benz[a]anthracene and chrysene should be separated by a valley
                   whose height is less than 25% of the average peak height of these two
                   compounds.  If the valley between benz[a]anthracene and chrysene exceeds
                   25%, the GC column requires maintenance.  See Sect. 9.3.6.
        9.2.4.2   MS sensitivity. The GC/MS/DS peak identification software should be
                   able to recognize  a GC peak in the appropriate retention time window for
                   each of the compounds in calibration solution, and make correct tentative
                   identifications. If fewer than 99% of the compounds are recognized,
                   system maintenance is required.  See Sect.  9.3.6.
        9.2.4.3   Lack of degradation of endrin.  Examine a plot of the abundance of m/z  67
                   in the region of 1.05-1.3 of the retention time of endrin.  This is the re-
                   gion of elution of endrin aldehyde,  a product of the thermal isomerization
                   of endrin.  Confirm that the abundance of m/z 67 at the retention time of
                   endrin aldehyde is < 10% of the abundance of m/z 67 produced by endrin.
                   If more than 10% endrin aldehyde is observed, system maintenance is
                   required  to correct the problem.  See Sect.  9.3.6.
9.2.5  If all performance criteria are met,  inject a  1-/*L aliquot of each of the other CAL
        solutions using the same GC/MS conditions.
9.2.6  Calculate a response factor (RF) for each analyte and surrogate for each CAL solution
        using the internal standard whose retention  time is nearest the retention time of the
        analyte or surrogate. Table 2 contains suggested internal standards for each analyte
        and surrogate, and quantitation ions for all compounds.  This calculation is supported
        in acceptable GC/MS data system software  (Sect. 6.14.4), and many other software
                                                                                   205

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Method 525.1
               programs.  RF is a unitless number, but units used to express quantities of analyte and
               internal standard must be equivalent.
               where:
                A^ = intergrated abundance of the quantitation ion of the analyte.
                An = integrated abundance of the quantitation ion internal standard.
               (2t =  quantity  of analyte injected in  ng or concentration units.
                Qn = quantity  of internal injected in ng or concentration units.


               9.2.6.1   For  each analyte and surrogate, calculate the mean RF from the analyses
                         of the six CAL solutions.  Calculate the standard deviation (SD) and the
                         relative standard deviation  (RSD) from each mean: RSD = 100 (SD/M).
                         If the RSD of any analyte or surrogate mean RF exceeds 30%, either ana-
                         lyze additional aliquots of appropriate CAL  solutions to obtain an accep-
                         table RSD of RFs over the entire concentration range, or take action to
                         improve GC/MS performance.  See Sect. 9.2.7.
       9.2.7   As an alternative to calculating mean response factors and applying the RSD test, use
               the GC/MS data system software or other available software to generate a linear,
               second, or third order regression calibration curve.
9.3   Continuing calibration check. Verify the MS tune and initial calibration at the beginning of
       each 8 hr. work shift during which analyses are performed using the  following procedure.
       9.3.1   Inject a l-/nL aliquot of the 5ng//xL DFTPP solution and acquire a mass spectrum that
               includes data for m/z 45-450. If the spectrum does not meet all criteria (Table 1), the
               MS must be retuned and adjusted to meet all criteria before proceeding with the
               continuing calibration check.
       9.3.2   Inject a 1-/*L aliquot of a medium concentration calibration solution and analyze  with
               the same conditions used during the initial calibration.
       9.3.3   Demonstrate acceptable performance for the criteria shown in Sect. 9.2.4.
       9.3.4   Determine that  the absolute areas of the quantitation ions of the internal standards and
               surrogate(s) have not decreased by more than 30% from the areas measured  in the
               most recent continuing calibration check, or by more than 50% from the areas mea-
               sured during initial calibration.  If these areas have decreased by more than these
               amounts, adjustments must be made to restore system sensitivity.  These adjustments
               may require cleaning of the MS ion source, or other maintenance as indicated in Sect.
               9.3.6, and recalibration. Control charts are useful aids in documenting system sen-
               sitivity changes.
       9.3.5   Calculate the RF for each analyte and surrogate from the data measured in the con-
               tinuing calibration check.  The RF for each analyte and surrogate must be within 30%
               of the mean value  measured in the initial calibration. Alternatively, if a second or
               third order regression is used, the point from the continuing calibration check for each

206

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                                                                                   Method 525.1
               analyte and surrogate must fall, within the analyst's judgement, on the curve from the
               initial calibration. If these conditions do not exist, remedial action must be taken
               which may require reinitial calibration.
       9.3.6   Some possible remedial actions.  Major maintenance such as cleaning an ion source,
               cleaning quadrupole rods, etc. require returning to the initial calibration step.
               9.3.6.1   Check and adjust GC and/or MS operating conditions; check the MS
                         resolution, and calibrate the mass scale.
               9.3.6.2   Clean or replace the splitless injection liner; silanize a new injection liner.
               9.3.6.3   Flush the GC column with solvent according to manufacturer's instruc-
                         tions.
               9.3.6.4   Break off a short portion (about 1 meter) of the column from the end near
                         the injector; or replace GC column.  This action will cause a change in
                         retention times.
               9.3.6.5   Prepare fresh CAL solutions, and repeat the initial  calibration step.
               9.3.6.6   Clean the MS  ion source and rods (if a quadrupole).
               9.3.6.7   Replace any components that allow analytes to come into contact with hot
                         metal surfaces.
               9.3.6.8   Replace the MS electron multiplier,  or any other faulty components.

10.   QUALITY CONTROL

10.1   Quality control (QC) requirements are the initial demonstration of laboratory capability fol-
       lowed by regular analyses of laboratory reagent blanks,  laboratory fortified blanks, and
       laboratory fortified matrix samples.  The laboratory must maintain records to document the
       quality of the data generated.  Additional quality control practices are recommended.
10.2   Initial demonstration of low system background and acceptable particle size and packing.
       Before any samples are analyzed, or any time a new supply of cartridges or disks is received
       from a supplier, it must be demonstrated that a laboratory  reagent blank (LRB) is reasonably
       free of contamination that would prevent the determination of any analyte of concern.  In this
       same experiment, it must be demonstrated that the particle size and packing of the LSE
       cartridge or disk are acceptable. Consistent flow rate is an indication of acceptable particle
       size distribution and packing.
       10.2.1  A major source of potential  contamination is the liquid-solid extraction (LSE) car-
               tridge which could contain phthalate esters, silicon compounds, and other contami-
               nants that could prevent the  determination of method analytes.5 Although disks are
               made of a  teflon matrix, they may still contain phthalate materials. Generally, phtha-
               late  esters  will be leached from the cartridges into methylene chloride and produce a
               variable background that is equivalent to < 2 /xg/L in the water sample.  If the back-
               ground contamination is sufficient to prevent accurate and precise analyses, the con-
               dition must be corrected before proceeding with the initial demonstration.  Figure 2
               shows unacceptable background contamination from a poor quality commercial LSE
                                                                                           207

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Method 525.1
               cartridge.  The background contamination is the large broad peak, and the small peaks
               are method analytes present at a concentration equivalent to 2 /*g/L.  Several sources
               of LSE cartridges may be evaluated before an acceptable supply is identified.
       10.2.2 Other sources of background  contamination are solvents, reagents, and glassware.
               Background contamination must be reduced to an acceptable level before proceeding
               with the next section.  In general, background from method analytes should be below
               the method detection limit.
       10.2.3 One  liter of water should pass through a cartridge in about 2 hrs with a partial vacu-
               um of about 13 cm (5 in.) of mercury. The flow rate through a disk should be about
               5-20 minutes for a liter of drinking water, using full aspirator or pump vacuum. The
               extraction time should not vary unreasonably among a set of LSE cartridges or disks.
10.3  Initial demonstration of laboratory accuracy and precision.  Analyze four to seven replicates of
       a laboratory fortified blank containing each analyte of concern at a concentration in the range
       of 2-5 fig/L (see regulations and maximum contaminant levels for guidance on appropriate
       concentrations).
       10.3.1 Prepare each replicate by adding an appropriate aliquot of the primary dilution stan-
               dard solution, or another certified quality control sample, to reagent water.  Analyze
               each replicate according to the procedures described in Sect. 11 and on a schedule that
               results in the analyses of all replicates over a period of several days.
       10.3.2 Calculate the measured concentration of each analyte in each replicate, the mean
               concentration of each analyte in all replicates, and mean accuracy (as mean percentage
               of true value)  for each analyte, and the precision (as  relative standard deviation, RSD)
               of the measurements for each analyte.  Calculate the MDL of each analyte using the
               procedures described in Sect. 13.1.2.1
       10.3.3 For each analyte and surrogate, the mean accuracy, expressed as a percentage of the
               true  value, should be 70-130% and the RSD should be  <30%.  Some analytes, par-
               ticularly the polycyclic aromatic hydrocarbons with molecular weights > 250, are
               measured  at concentrations below 2 ftg/L, with a mean accuracy of 35-130% of true
               value.  The MDLs should be sufficient to detect analytes at the regulatory levels.  If
               these criteria are not met for  an analyte, take remedial action  and repeat the measure-
               ments for that analyte to demonstrate acceptable performance  before samples are
               analyzed.
       10.3.4 Develop and maintain a system of control charts to plot the precision and accuracy of
               analyte and surrogate measurements as a function of time.  Charting of surrogate
               recoveries is an especially valuable activity since these are present in every sample
               and the analytical results will form a significant record of data quality.
10.4  Monitor the integrated areas of the quantitation ions of the internal standards and surrogates in
       continuing calibration checks (see Sect.  9.3.4). In laboratory fortified blanks or  samples, the
       integrated areas of internal standards and surrogates will not be constant because the volume of
       the extract will vary (and is difficult to keep constant). But the ratios  of the areas should be
       reasonably constant in laboratory fortified blanks and samples.  The addition of 10 /*L of the
208

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                                                                                   Method 525.1
       recovery standard, terphenyl-D,4 (500 jig/mL), to the extract is optional, and may be used to
       monitor the recovery of internal standards and surrogates in laboratory fortified blanks and
       samples. Internal standard recovery should be in excess of 70%.
10.5  Laboratory reagent blanks. With each batch of samples processed as a group within a work
       shift, analyze a laboratory reagent blank to determine the background system contamination.
       Any time a new batch of LSE cartridges or disks is received, or new supplies of other reagents
       are used, repeat the demonstration of low background described in 10.2.
10.6  With each batch of samples processed as a group within a work shift, analyze a single labora-
       tory fortified blank (LFB) containing each analyte of concern at a concentration as determined
       in 10.3.  If more than 20 samples are included in a batch, analyze a  LFB for every  20 sam-
       ples.  Use the procedures described in 10.3.3 to evaluate the accuracy of the measurements,
       and to estimate whether the method detection limits can be obtained. If acceptable accuracy
       and method detection limits cannot be achieved, the problem must be located and corrected
       before further samples are analyzed.  Add these results to the on-going control charts to
       document data quality.
10.7  Determine that the sample matrix does not contain materials that adversely affect method
       performance.  This is accomplished by analyzing replicates of laboratory fortified matrix
       samples and ascertaining that the precision, accuracy, and method detection limits of analytes
       are in the same range as obtained with laboratory fortified blanks.  If a variety of different
       sample matrices are analyzed regularly, for example, drinking water from groundwater and
       surface water sources, matrix independence should be established for each.   A laboratory
       fortified sample matrix should be analyzed for every 20 samples processed in the same batch.
10.8  With each set of field samples a field reagent blank (FRB) should be analyzed.  The results of
       these analyses will help define contamination resulting from field sampling and transportation
       activities.
10.9  At least quarterly, replicates of laboratory fortified blanks should be  analyzed to determine the
       precision of the laboratory measurements.  Add these results to the on-going control charts  to
       document data quality (as in Sect. 10.3).
10.10 At least quarterly, analyze a quality control sample from an  external  source.  If measured
       analyte concentrations are not of acceptable accuracy (Sect.  10.3.3),  check the entire analytical
       procedure to locate and correct the problem source.
10.11 Numerous other quality control measures are incorporated into other parts of this procedure,
       and serve to alert the analyst to  potential problems.

7 1.   PROCEDURE

11.1  Cartridge Extraction
       11.1.1  Setup the extraction apparatus shown in Figure 3A.  The reservoir is not required, but
               recommended for convenient operation.  Water drains from  the reservoir through the
               LSE cartridge and into a syringe needle which is inserted through a rubber  stopper
               into the suction flask.  A slight vacuum of 13 cm (5 in.) of mercury is used during all
                                                                                            209

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Method 525.1
               operations with the apparatus. The pressure used is critical as a vacuum > than 13
               cm may result in poor precision.  About 2 hrs is required to draw a liter of water
               through the system.
       11.1.2 Pour the water sample into the 2-L separatory funnel with the stopcock closed, add 5
               mL methanol, and mix well.  Residual chlorine should not be present as a reducing
               agent should have been added at the time of sampling.  Also the pH of the sample
               should be about 2.  If residual chlorine is present and/or the pH is >2, the sample
               may be invalid.   Add a 100-/iL aliquot of the fortification solution (50 /ig/mL) for
               internal  standards and surrogates, and mix immediately until homogeneous.  The
               concentration of these compounds in  the water should be 5 /zg/L.
       11.1.3 Flush each cartridge with two 10 mL aliquots of methylene chloride, followed by two
               10 mL  aliquots of methanol,  letting the cartridge drain dry after each flush. These
               solvent flushes may be accomplished by adding the solvents directly to the solvent
               reservoir in Figure 3A.   Add 10 mL of reagent water to the solvent reservoir, but
               before the reagent water  level drops below the top edge of the packing in the LSE
               cartridge, open the stopcock of the separatory funnel and begin adding sample water
               to the solvent reservoir.  Close the stopcock when an adequate amount of sample is in
               the reservoir.
       11.1.4 Periodically open the stopcock and drain a portion of the sample water into the solvent
               reservoir.  The water sample will drain into the cartridge, and from the exit into the
               suction flask. Maintain the packing material in the cartridge immersed in water at all
               times.  After all of the sample has passed through the LSE cartridge,  wash the separa-
               tory funnel and cartridge with 10 mL of reagent water, and draw air through the
               cartridge for 10 min.
       11.1.5 Transfer the 125-mL solvent reservoir and LSE cartridge (from Figure 3A) to the
               elution apparatus (Figure 3B). The same 125-mL solvent reservoir is used for both
               apparatus. Wash the 2-liter separatory funnel with 5 mL of methylene chloride and
               collect  the washings. Close the stopcock on the 100-mL separatory funnel of the
               elution apparatus, add the washings to the reservoir and enough additional methylene
               chloride to bring the volume back up to 5 mL and elute the LSE cartridge.  Elute the
               LSE cartridge with an additional 5 mL of methylene chloride (10-mL total).  A small
               amount of nitrogen positive pressure may be used to elute the cartridge.  Small
               amounts of residual water from the LSE cartridge will form an immiscible layer with
               the methylene chloride in the 100-mL separatory funnel.  Open the stopcock and
               allow the methylene chloride to pass through the drying column packed with an-
               hydrous sodium sulfate (1-in) and into the collection vial.  Do not allow the water
               layer to enter the drying column.  Remove the 100 mL separatory funnel and  wash
               the drying column with 2 mL of methylene chloride. Add this to the extract.   Con-
               centrate the extract to 1  mL under a gentle stream of nitrogen. If desired, gently
               warm the extract in a water bath to evaporate to between 0.5-1.0 mL (without gas
               flow).  Do not concentrate the extract to less than 0.5 mL (or dryness) as this will
2/0

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                                                                                  Method 525.1
               result in losses of analytes.  If desired, add an aliquot of the recovery standard to the
               concentrated extract to check the recovery of the internal standards (see Sect. 10.4).
11.2  Disk Extraction (This may be manual or automatic)
       11.2.1  Preparation of Disks
               11.2.1.1  Insert the disk into the 47mm filter apparatus as shown in Figure 4.  Wash
                         the disk with 5mL methylene chloride (MeC12) by adding the MeCl2 to the
                         disk, drawing about half through the disk, allowing it to soak the disk for
                         about a minute, then drawing the remaining MeC12 through the disk.
               11.2.1.2  Pre-wet  the disk with 5 mL methanol (MeOH) by adding the MeOH to the
                         disk and allowing it to soak for about a minute,  then drawing most of the
                         remaining MeOH through.  A layer of MeOH must be left on the surface
                         of the disk, which should not be allowed to go dry from this point until the
                         end of the sample extraction. THIS IS A CRITICAL STEP FOR A UNI-
                         FORM FLOW AND GOOD RECOVERY.
               11.2.1.3  Rinse the disk with 5 mL reagent water by adding the water to the disk
                         and drawing most through, again leaving a layer on the surface of the disk.
       11.2.2  Add 5 mL MeOH per liter of water sample.  Mix well.
       11.2.3  Add the water sample to the reservoir and  turn on the vacuum to begin the extraction.
               Full aspirator vacuum may be used.  Particulate-free water  may pass  through the disk
               in as little as ten minutes or less.  Extract  the entire  sample, draining as much water
               from the sample container as possible.
       11.2.4  Remove the filtration top from the vacuum flask, but do not disassemble the  reservoir
               and fritted base.  Empty the water from the flask, and insert a suitable sample tube to
               contain the eluant.  The only constraint on the sample tube  is that it fit around the
               drip tip of the fritted base.  Reassemble the apparatus.
       11.2.5  Add 5 mL methylene chloride to the sample bottle, and rinse the inside walls thor-
               oughly.  Allow the methylene chloride to settle to the bottom of the bottle, and trans-
               fer to the disk with  a pipet or syringe, rinsing the sides of the glass filtration reservoir
               in the process. Draw about half of the methylene chloride through the disk,  release
               the vacuum, and allow the disk to soak for a minute.  Draw the remaining methylene
               chloride through the disk.
       11.2.6  Repeat the above step twice.  Pour the combined eluates through a small funnel with
               filter paper containing three grams of anhydrous sulfate.  Rinse the test tube  and
               sodium sulfate with two 5 mL portions of methylene chloride.  Collect all the extract
               and washings in a concentrator tube.
       11.2.7  Concentrate the extract to 1 mL under a gentle  stream of nitrogen. If desired, gently
               warm the extract in a water bath or  heating block to  concentrate to between 0.5 and 1
               mL. Do not concentrate the extract to less than 0.5 mL, since this will result in
               losses of analytes.
11.3  Analyze  a 1-2 juL aliquot with the GC/MS system under the same conditions used for the
       initial and continuing calibrations (Sect. 9.2.3).
                                                                                          211

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Method 525.1
11.4  At the conclusion of data acquisition, use the same software that was used in the calibration
       procedure to tentatively identify peaks in retention time windows of interest.  Use the data
       system software to examine the ion abundances of components of the chromatogram.  If any
       ion abundance exceeds the  system working range, dilute the sample aliquot and analyze the
       diluted aliquot.
11.5  Identification of analytes.   Identify a sample  component by comparison of its mass spectrum
       (after background subtraction) to a reference spectrum in the user-created data base.  The GC
       retention time of the sample component should be within 10 sec of the time observed for that
       same compound when a calibration solution was analyzed.
       11.5.1 In general, all ions that are present above 10% relative abundance in the mass spec-
              trum of the standard should be present in the mass spectrum of the sample component
              and should agree within absolute 20%. For example, if an ion has a relative abun-
              dance of 30% in the standard spectrum, its  abundance in the sample spectrum should
              be  in the range of  10 to 50%.  Some ions, particularly the  molecular ion,  are of
              special importance, and should be evaluated even if they are below 10% relative abun-
              dance.
       11.5.2 Identification is hampered when sample components are not resolved chromatograph-
              ically and produce mass spectra containing  ions contributed by more than  one analyte.
              When GC peaks obviously represent more than one sample component (i.e., broad-
              ened peak with shoulder(s) or valley between two or more  maxima), appropriate
              analyte spectra and background spectra can be  selected by examining plots of charac-
              teristic ions for tentatively identified components.  When analytes coelute  (i.e., only
              one GC  peak is apparent), the identification criteria can be met but each analyte
              spectrum will contain extraneous ions  contributed by the coeluting compound.
       11.5.3 Structural isomers that produce very similar mass spectra can be explicitly identified
              only if they have sufficiently different GC retention times.  See Sect. 9.2.4.1.  Accep-
              table resolution is  achieved if the height of  the  valley between two isomer peaks is
              less than 25% of the average height of the two peak heights.  Otherwise, structural
              isomers  are identified as isomeric pairs.  Benzofb] and benzo[k]fluoranthene are
              measured as  an isomeric pair.
       11.5.4 Phthalate esters and other background components appear in variable quantities in
              laboratory and field reagent blanks,  and generally cannot be accurately measured at
              levels below about 2 /xg/L. Subtraction of the  concentration in the blank from the
              concentration in the sample at or below the 2 ^ig/L level is not recommended because
              the concentration of the background in the blank is highly variable.

12.   CALCULATIONS

12.1  Complete chromatographic resolution is not necessary for accurate and precise measurements
       of analyte concentrations if unique ions with adequate intensities are available for quantitation.
       For  example, although two listed analytes, dibenz[a,h]anthracene and indeno[l,2,3,c,d]pyrene,
       were not  resolved with the  GC conditions used, and produced mass spectra containing common
       ions, concentrations (Tables 3-6) were calculated  by measuring appropriate characteristic ions.
       12.1.1 Calculate analyte and surrogate concentrations.

272

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                                                                                   Method 525.1
                                        c  =
                                              (AJRF V
       where:
        Cx = concentration  of analyte or surrogate in ng/L in the water sample.
        Ax = integrated abundance of the quantitation ion of the analyte in the  sample.
       Ais = integrated abundance of the quantitation ion of the analyte in the  sample.
       Qis = total quantity  (in micrograms) of internal standard added to the water sample.
         V = original water sample volume in liters.
       RF = mean response factor of analyte from the initial calibration.
       12.1 .2 Alternatively, use the GC/MS system software or other available proven software to
               compute the concentrations of the analytes and surrogates from the second or third
               order regression curves.
       1 2.1 .3 Calculations should utilize all available digits of precision, but final reported concen-
               trations should be rounded to an appropriate number of significant  figures (one digit
               of uncertainity).  Experience indicates that three significant figures may be used for
               concentrations above 99 /tg/L, two significant figures for concentrations between 1-99
                    and one significant figure for lower concentrations.
13.  METHOD PERFORMANCE
13.1  Single laboratory accuracy and precision data (Tables 3-7) for each listed analyte was obtained
      at two concentrations with the same extracts analyzed on more than two different instrument
      systems.  Seven 1-liter aliquots of reagent water containing 2 /xg/L of each analyte, and five to
      seven 1-liter aliquots of reagent water containing 0.2 /xg/L of each analyte were analyzed with
      this procedure.  Tables 8-10 list data gathered using C-18 disks.  These data were results from
      different extracts generated by a volunteer laboratory, Environmental Health Laboratories.
      13.1.2 With these data, method detection limits (MDL) were calculated using the formula:
                                    MDL =  $Vi, .-0 = 0.99,
    where:
    l
-------
Method 525.1
       13.2.2 Some polycyclic aromatic hydrocarbons are rapidly oxidized and/or chlorinated in
               water containing residual chlorine.  Therefore residual chlorine must be reduced
               before analysis.
       13.2.3 In water free of residual chlorine, some polycyclic aromatic hydrocarbons (for exam-
               ple, compounds 9, 12, 13, 20, and 35) are not accurately measured because of low
               recoveries in the extraction process.
       13.2.4 Pentachlorophenol No. 40 and hexachlorocyclopentadiene No. 34 may not be accu-
               rately measured.  Pentachlorophenol is a strong acid and elutes as a broad weak peak.
               Hexachlorocyclopentadiene is susceptible to photochemical and thermal decom-
               position.
214

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                                                                               Method 525.1
                                    References
1.     Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Quave, and W. L. Budde,  "Trace Analyses
      for Wastewaters," Environ. Sci. Technol. 198115, 1426-1435.

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, Aug. 1977.

3.     "OSHA Safety and Health Standards, General Industry," (29CFK1910), 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.     Junk, G.A.,  M. J. Avery, J. J. Richard, "Interferences in Solid-Phase Extraction Using C-18
      Bonded Porous Silica Cartridges," Anal. Chem. 1988, 60, 1347.
                                                                                       215

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Method 525.1
Table  1.  Ion Abundance Criteria for Bis(perfluorophenyl)phenyl Phosphine (Deca-
fluorotriphenylphosphine, DFTPP)
Mass
(M/z)   Relative Abundance Criteria
   51    10-80% of the base peak
   68    <2% of mass 69
   70    < 2% of mass 69
 127    10-80% of the base peak
 197    <2% of mass 198
 198    base peak or  >50% of 442
 199    5-9% of mass 198
 275    10-60% of the base peak
 365    > 1 % of the base peak
 441    Present and < mass 443
 442    base peak or  >50% of 198
 443    15-24% of mass 442
Purpose of Checkpoint1
Low mass sensitivity
Low mass resolution
Low mass resolution
Low-mid mass sensitivity
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mass resolution and isotope ratio
Mid-high mass sensitivity
Baseline threshold
High mass resolution
High mass resolution and sensitivity
High mass resolution and isotope ratio
    All ions are used primarily to check the mass measuring accuracy of the mass spectrometer
    and data system, and this is the most important part of the performance test. The three
    resolution checks, which include natural abundance isotope ratios, constitute the next most
    important part of the performance test. The correct setting of the baseline threshold, as
    indicated by the presence of low intensity ions, is the next most important part of the perfor-
    mance test. Finally, the ion abundance ranges are designed to encourage some standardization
    to fragmentation patterns.
276

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                                                                               Method 525.1
Table 2.  Retention Time Data, Quantitation Ions, and Internal Standard References
for Method Analytes
                                               Retention
Compound

Internal standard
Acenapththene-D10
Phenanthrene-D10
Chrysene-D12

Surrogate
Perylene-D12

Target analytes
Acenphthylene
Aldrin
Anthracene
Atrazine
Benz[a]anthracene
Benzo[6]fluoranthene
Benzo[/r]fluoranthene
Benzofajpyrene
Benzo[5r,/7,/]perylene
Buthylbenzyl phthalate

Chlordane Components
a-Chlordane
•y-Chlordane
trans-Nonachlor
2-Chlorobiphenyl
Chrysene
Dibenz[a,/;]anthracene
Di-/?-butyl phthalate
2,3-Dichlorobiphenyl
Diethyl phthalate
Di(2-ethylhexyl) phthalate
Di(2-ethylhexyl) adipate
Dimethyl phthalate
Endrin
Fluorene
Heptaclor
Heptachlor epoxide
2,2',3,3',4,4',6-Hepta-
   chlorobiphenyl
Hexachlorobenzene
2,2',4,4',5,6'-Hexa-
   chlorobiphenyl
Hexachloro-
   cyclopentadiene
lndeno[1,2,3,c,cflpyrene
Compound
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
lime (i
A'
4:49
8:26
18:14
23:37
4:37
11:21
8:44
7:56
18:06
22:23
22:28
23:28
27:56
16:40
13:44
13:16
13:54
4:56
18:24
27:15
10:58
7:20
5:52
19:19
17:17
4:26
15:52
6:00
10:20
12:33
18:25
7:37
14:34
3:36
27:09
mn:sec/
I *
7:45
11:08
19:20
22:55
7:25
13:36
11:20
10:42
19:14
22:07
22:07
22:47
26:44
18:09
15:42
15:18
15:50
7:55
19:23
25:57
13:20
10:12
8:50
20:01
18:33
7:21
16:53
8:53
12:45
14:40
19:25
10:20
16:30
6:15
25:50
Quantitatk
Ion (m/z)
164
188
240
264
152
66
178
200/215
228
252
252
252
276
149
375
375
409
188
228
278
149
222
149
149
129
163
81
166
100/160
81/353
394/396
284/286
360
237
276
 Internal
 Standard
Reference
    1
    2
    2
    1/2
    3
    3
    3
    3
    3
    2/3
   2/3
   2/3
   2/3
    1
    3
    3
    2
    1
    1
   2/3
   2/3
    1
   2/3
    1
    2
    2

    3
   1/2
    1
    3
                                                                                       277

-------
Method 525.1
Table 2.  Retention Time Data, Quantitation Ions, and Internal Standard References
for Method Analytes (cont.)
Compound
Lindane
Methoxychlor
2,2',3,3',4,5',6,6'-
  Octachlorobiphenyl
2,2',3',4,6-Penta-
  chlorobiphenyl
Pentachlorophenol
Phenanthrene
Pyrene
Simazine
2,2',4,4'-Tetrachloro-
  biphenyl
Toxaphene
2,4,5-Trichlorobiphenyl
Alachlor
Compound_
 Number
  36
  37

  38

  39
  40
  41
  42
  43
                                            Retention
                                          Time (min:sec)
   A'
 8:17
18:34

18:38

12:50
 8:11
 8:35
13:30
 7:47
  44        11:01
  45  11:30-23:30
  46         9:23
  47
      10:57
      19:30

      19:33

      15:00
      10:51
      11:13
      15:29
      10:35

      13:25
13:00-21:30
      11:59
      13:19
              Internal
.Quantitation  Standard
  Ion (m/zj   Reference
   181/183       1/2
     227         3
     430

     326
     266
     178
     202
     201

     292
     159
     256
     160
 2
 2
 2
2/3
1/2

 2
 2
 2
 2
    Single ramp linear temperature program conditions (Sect. 9.2.3.2).
    Multi-ramp linear temperature program conditions (Sect. 9.2.3.1).
218

-------
                                                                     Method 525.1
 Table 3.  Accuracy and Precision Data from Seven Determinations of the Method
 Analytes at 2 //g/L With Liquid-Solid Extraction and the Ion Trap Mass
 Spectrometer
                                                           Mean

Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean"


True Cone.
(ug/L)
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Mean
Observed
Cone.
(U9/U
5.0
1.9
1.6
1.7
2.2
1.8
not separated
4.2
0.8
0.7
2.0
2.0
2.2
2.7
1.9
2.2
0.3
2.2
2.3
2.0
1.9
1.6
1.9
1.8
2.2
2.2
2.3
1.4
1.7
1.6
1.1
0.4
2.1
1.8
1.8
1.9
8.2
2.4
1.9
2.1
1.5
28.
1.7
1.8


Method
Pel. Std. Accuracy
Std. Dev.
(ug/L)
0.3
0.2
0.2
0.1
0.3
0.2
from No. 1 1;
0.3
0.2
0.1
0.3
0.2
0.3
1.0
0.1
0.1
0.3
0.3
0.1
0.3
0.2
0.3
0.2
0.1
0.2
0.3
0.2
0.2
0.2
0.4
0.1
0.2
0.2
0.2
0.2
0.1
1.2
0.1
0.1
0.2
0.1
4.7
0.1
0.2
Dev.
(%)
6.0
11.
13.
5.9
14.
11.
measured
7.1
25.
14.
15.
10.
14.
37.
5.2
4.5
100.
14.
4.3
15.
11.
19.
11.
5.5
9.1
14.
8.7
14.
12.
25.
9.1
50.
9.5
11.
11.
5.3
15.
4.2
5.3
9.5
6.7
17.
5.9
15.
(% of True
Cone.)
100
95
80
85
110
90
with No. 1 1
105
40
35
100
100
110
135
95
110
15
110
115
100
95
80
95
90
110
110
115
70
85
80
55
20
105
90
90
95
102
120
95
105
75
112
85
91
Method
Detection
Limit (MDL)
(ug/L)
_ a
_ a
_ a
_ a
_ a
a

_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
__ a
_ a
_ a
_ a
_ "
_ a
_ a
_ a
__ a
_ a
_ a
__ a
_ a
__ a
_ a
_ a
_^ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
_ a
15.
_ a
6.6
8   See Table 4.
"   Compounds 4, 40, and 45 excluded from the means.
                                                                           219

-------
Method 525.1
Table 4.  Accuracy and Precision Data from Five to Seven Determinations of the
Method Anaytes at 0.2 //g/L with Liquid-Solid Extraction and the Ion Trap Mass
Spectrometer
Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean3

True Cone.
(ug/L)
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Mean
Observed
Cone. (u/L)
0.45
0.13
0.13
0.13
0.24
0.14

0.25
0.03
0.03
0.32
0.17
0.19
0.17
0.19
0.21
0.03
0.48
0.20
0.45
0.39
0.31
0.21
0.12
0.21
0.22
0.19
0.19
0.16
0.19
0.04
0.04
0.22
0.11
0.19
0.13
0.78
0.20
0.18
0.25
0.14

Std. Dev
(ug/L)
0.6
0.03
0.03
0.01
0.03
0.01
not separated
0.04
0.01
0.02
0.07
0.04
0.03
0.08
0.03
0.01
0.02
0.09
0.03
0.21
0.16
0.16
0.01
0.12
0.05
0.01
0.04
0.03
0.04
0.03
0.01
0.03
0.02
0.01
0.05
0.02
0.08
0.004
0.005
0.04
0.04
not measured at this
0.2
0.2
0.13
0.18
0.02
0.04
RelStd.
Dev.
(%)
13.
23.
23.
7.7
13.
7.1
from No. 1
16.
33.
67.
22.
24.
16.
47.
16.
4.8
67.
19.
15.
47.
41.
52.
4.8
100.
24.
4.5
21.
16.
25.
16.
25.
75.
9.1
9.1
26.
15.
10.
2.0
2.8
16.
29.
level
15.
25.
Mean Method
Accuracy
(% of True Cone.)
90
65
65
65
120
70
Method
Detection Limit
(MDU (ug/L)
0.1
0.1
0.1
0.04
0.1
0.04
1; measured with No. 1 1
62
15
15
160
85
95
85
95
105
150
240
100
225
195
155
105
60
105
110
95
95
80
95
20
20
110
55
95
65
97
100
90
125
70

65
95
0.2
0.04
0.1
0.3
0.2
0.1
0.3
0.1
0.04
0.1
0.3
0.1
0.8
0.6
0.6
0.04
0.5
0.2
0.04
0.2
0.1
0.1
0.1
0.03
0.1
0.1
0.04
0.2
0.1
0.3
0.01
0.02
0.02
0.1

0.06
0.16
   "Compound 4, 40, and 45 excluded from the means.
220

-------
                                                                   Method 525.1
Table 5.  Accuracy and Precision data From Five to Seven Determinations of the
Method Anaytes at 0.2 /yg/L with Liquid-Solid Extraction and a Magnetic Sector
Mass Spectrometer
Compound
Number
(Table 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean3

True Cone.
(fjg/U
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
8
2
2
2
2
25
2
2
Afea/i
Observed
Cone. (u/L)
5.7
1.9
1.6
2.2
2.4
2.2
Pel. Std. Mean Method
Std. Dev
(ug/L)
0.34
0.22
0.18
0.67
0.46
0.87
not separated from No.
4.0
0.85
0.69
2.0
2.2
2.1
1.9
2.0
2.1
0.75
2.5
2.0
3.5
2.0
1.4
2.9
1.7
2.6
1.2
2.6
1.5
1.5
1.9
0.89
0.83
2.2
2.0
1.5
1.6
12.
2.3
2.0
2.5
1.6
28.
1.9
1.8
0.37
0.15
0.12
0.20
0.41
0.38
0.10
0.29
0.32
0.18
0.32
0.23
1.8
0.28
0.16
0.70
0.45
1.0
0.10
0.42
0.19
0.35
0.17
0.11
0.072
0.10
0.88
0.11
0.14
2.6
0.18
0.26
0.34
0.17
2.7
0.073
0.32
Dev.
<%) (%
6.0
12.
11.
30.
19.
40
1 1 ; measured
9.3
18.
17.
10.
19.
18.
5.2
14.
15.
24.
13.
12.
51.
14.
11.
24.
26.
38.
8.3
16.
13.
23.
8.9
12.
8.7
4.5
44.
7.3
8.8
22.
7.8
13.
14.
11.
10.
3.8
16.
Accuracy
of True Cone.)
114
95
80
110
120
110
with No. 1 1
100
43
35
100
110
105
95
100
105
38
125
100
175
100
70
145
85
130
60
130
75
75
95
45
42
110
100
75
80
150
115
100
125
80
112
95
88
Method
Detection Limit
(MOD (ug/U
a
_ a
_ a
_ a
_ a
a

_ a
_ a
_ a
_ a
a
a
_ a
a
a
a
_ a
_ a
_ a
_ a
_ a
_ a
a
a
a
a
_ a
_ a
a
_ a
a
a
a
_ a
_ a
_ a
_ a
_ a
a
a
9.
a
1.
3 See Table 4.
    Compound 4, 40, and 45 exluded from the means.
                                                                           221

-------
Method 525.7
Table 6. Accuracy and Precision Data from Six or Seven Determinations of the
Method Anaytes at 0.2 jjg/L with Liquid-Solid Extraction and a Magnetic Sector
Spectrometer
Compound
Number
(Tab/e 2)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Mean8
True
Cone.
(ug/L)
0.5
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.8
0.2
0.2
0.2
0.2
Mean
Observed
Conc.(p/L)
0.67
0.11
0.11
0.14
0.26
0.24
Pel. Std. Mean Method Method Detection
Std.Dev
(ug/L)
0.07
0.03
0.02
0.02
0.08
0.06
not separated from
0.40
0.08
0.07
0.33
0.19
0.17
0.19
0.17
0.27
0.09
1.1
0.18
0.29
0.42
0.32
0.20
0.53
0.18
0.11
0.33
0.17
0.11
0.17
0.05
0.08
0.27
0.24
0.15
0.13
1.8
0.21
0.19
0.27
0.13
0.10
0.02
0.01
0.16
0.02
0.08
0.04
0.02
0.08
0.01
1.2
0.05
0.17
0.23
0.16
0.09
0.30
0.03
0.05
0.08
0.01
0.04
0.03
0.02
0.06
0.03
0.09
0.02
0.02
0.82
0.07
0.04
0.07
0.03
Dev.
(%) (%
9.4
24.
21.
17.
31.
26.
No. 1 1 ; measured
25.
27.
22.
48.
13.
45.
18.
13.
28.
15.
109.
30.
59.
55.
50.
47.
57.
15.
42.
26.
7.1
40.
15.
35.
8.1
11.
39.
12.
13.
46.
33.
23.
27.
22.
Accuracy
of True Cone.)
134
55
56
70
130
120
with No. 1 1
100
38
33
160
95
85
95
85
135
46
550
90
145
210
160
100
265
90
55
165
85
55
85
24
40
135
120
75
65
225
105
95
135
65
Limit (MDL)
(ug/L)
0.2
0.1
0.1
0.1
0.3
0.2

0.3
0.1
0.1
0.5
0.1
0.3
0.1
0.1
0.3
0.1
4.
0.2
0.6
0.8
0.5
0.3
1.
0.1
0.2
0.3
0.04
0.2
0.1
0.1
0.02
0.1
0.3
0.1
0.1
3.
0.2
0.1
0.2
0.1
not measured at this point
0.2
0.2
0.16
0.21
0.04
0.09
23.
28.
80
102
0.12
0.3
       Compound 4, 40, and 45 excluded from the means.
222

-------
                                                                    Method 525.1
Table 7.  Accuracy and Precision Data from Seven Determinations at 2 jug/L with
Liquid-Solid Extraction and a Quadrupole Mass Spectrometer

 Compound   True     Mean            Rel. Std.  Mean Method  Method Detection
  Number    Cone.   Observed Std. Dev  Dev.      Accuracy       Limit (MDL)
  (Table 2)   (ug/U  Cone. (u/LJ  (ug/L)    (%)   (% of True Cone.)      (ug/L)
     47        2        2.4      0.4     16.         122             1.0
                                                                           223

-------
 Method 525.1
Table 8. Accuracy and Precision Data from Seven Replicates at 0.2 /vg/L with
Liquid-solid C-1
Compound
Number
(Table 2)
1
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
8 Disk Extraction and an ion Trap Mass Spectrometer
Target
Concentration
(ug/L)
0.2
5.0
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
2.0
0.2
0.2
0.2
0.2
20.0
0.2
0.2
Standard
Deviation
(ug/L)
0.01
0.37
0.03
0.03
0.04
0.07
0.16
0.03
0.04
0.03
0.07
0.12
0.06
0.18
0.01
0.02
0.05
0.08
0.02
0.02
0.50
0.04
0.02
0.05
0.01
0.05
0.08
0.08
0.04
0.06
0.01
0.05
0.01
0.03
0.04
0.03

0.02
0.02
0.04
0.06
2.47
0.04
0.03
Relative
Deviation
<%)
5.3
7.4
13.2
13.7
22.4
33.2
77.6
13.7
21.7
14.9
32.5
61.1
31.9
91.3
7.2
10.9
22.9
40.3
9.7
11.9
252.0
20.8
7.6
25.4
7.3
22.9
38.9
38.0
17.7
31.9
5.2
27.0
6.5
13.4
21.1
15.1

12.2
10.2
18.8
28.1
12.3
21.4
14.7

Mean
(ng/U
0.22
5.55
0.26
0.22
0.21
0.29
0.40
0.21
0.26
0.23
0.37
0.19
0.19
0.55
0.16
0.27
0.18
0.47
0.17
0.27
1.54
0.36
0.23
0.23
0.20
0.28
0.36
0.28
0.22
0.19
0.34
0.29
0.22
0.20
0.20
0.17

0.20
0.24
0.19
0.21
24.80
0.19
0.11

Accuracy
(% of target)
110
111
130
108
105
147
199
107
128
115
183
95
93
276
78
136
90
233
87
133
771
180
117
117
101
139
181
141
109
96
170
143
110
100
99
84

102
121
94
107
123
95
55
224

-------
Method 525.1
Table 9. Accuracy and Precision Data from
Seven Replicates at 2.
0 //g/L with
Liquid-Solid C-18 Disk Extraction and an ion Trap Mass Spectrometer
Compound
Number
(Table 2)
1
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Target
Concentration
(ug/L)
2
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20
2
2
2
2
100
2
2
Standard
Deviation
(ug/U
0.18
0.45
0.30
0.17
0.47
0.21
0.62
0.57
0.31
0.28
0.33
0.62
1.02
1.39
0.22
0.23
0.27
0.23
0.38
0.22
0.38
0.26
0.69
0.12
0.19
0.30
0.15
0.64
0.85
0.52
0.22
0.37
0.42
0.34
0.77
0.29
15.16
0.20
0.17
0.27
0.15
3.36
0.58
0.07
Relative
Deviation
(%)
9.2
9.1
14.8
8.6
23.5
10.4
30.9
28.7
15.6
13.9
16.7
31.1
51.2
69.3
11.2
11.6
13.4
11.3
18.9
11.1
19.1
12.8
34.6
6.1
9.7
15.0
7.4
32.2
42.3
25.9
11.0
18.3
21.2
16.8
38.5
14.7
75.8
9.9
8.3
13.3
7.4
3.4
28.8
3.5

Mean
(ug/L)
2.00
5.22
2.14
2.25
2.78
2.21
2.84
2.30
2.61
2.28
2.92
1.21
1.92
3.29
2.52
1.99
2.25
2.45
2.35
2.23
3.25
2.49
1.80
1.97
2.15
2.10
2.41
2.46
1.96
2.05
1.42
2.31
2.69
2.34
0.97
2.11
19.51
2.20
2.34
2.37
2.11
98.33
1.65
1.55

Accuracy
(% of target)
100
104
107
112
139
111
142
115
130
114
146
61
96
164
126
100
113
123
117
111
163
124
90
98
108
105
121
123
98
102
71
115
134
117
49
106
98
110
117
119
106
98
82
77
        225

-------
Method 525.1
Table  10.  Minimum Detection Limits From Seven Replicates Using Liquid-Solid
Extraction  C-18 Disks and an  Ion Trap Mass Spectrometer

                 Chemical Name             Minimum Detection Limits
                 Acenaphytiene                                 0.033
                 Alachlor                                       0.092
                 Aldrin                                         0.083
                 Anthracene                                    0.086
                 Atrazine                                       0.140
                 Benz[a]anthracene                              0.224
                 Benzo|6]fluoranthene                            0.488
                 Benzo[*]fluoranthene                            0.086
                 Benzo[a]pyrene                                 0.137
                 BenzolgA/lperylene                             0.094
                 Butylbenzyl phthalate                            0.204
                 Chlordane-a                                    0.384
                 Chlordane-7                                    0.200
                 Chlordane (trans-Nonachlor)                      0.574
                 Chrysene                                      0.068
                 Dibenz[a,/?]anthracene                           0.144
                 Di-n-butyl phthalate                             0.253
                 Diethyl phthalate                               0.075
                 Di(2-ethylhexyl)  phthalate                         1.584
                 Di(2-ethlyhexyl)  adipate                          0.131
                 Dimethyl phthalate                              0.048
                 Endrin                                         0.160
                 Fluorene                                      0.046
                 Heptachlor                                     0.144
                 Heptachlorepoxide                              0.244
                 Hexachlorobenzene                             0.111
                 Hexachlorocyclopentadiene                      0.039
                 lndeno[1,2,3,c,rf]pyrene                         0.170
                 Lindane                                       0.041
                 Methoxychlor                                  0.084
                 PCB-mono-CI-isomer                            0.045
                 PCB-di-CI-isomer                                0.061
                 PCB-tri-CI-isomer                                0.135
                 PCB-tetra-CI-isomer                              0.177
                 PCB-penta-CI-isomer                             0.200
                 PCB-hepta-CI-isomer                             0.239
                 PCB-octa-CI-isomer                              0.133
                 Pentachlorophenol                              47.648
                 Phenanthrene                                   0.076
                 Pyrene                                         0.064
                 Simazine                                       0.118
                 Toxaphene                                     7.763
 226

-------
                                                                            Method 525.1
     TIC
 100-

 80-

 60-


 40-

 20-
34    26 J
i     U

                                                 44
                                                                A°
                                                                         8925696
                                                                         33
                                  27

                                  A
    Scan
    R.T.
          100
          4:55
300
9:46
400
12:12
500
14:38
    TIC
100-

 80-

 60-


 40-

 20-
          25
                   38
                       24
              600
              17:04
                          700
                          19:29
                                         10 11
                900
                24:21
                                                                         2513216
                                                                            13
                                                                      35 20  A
                                                                  -uv^A-J^
              1000
              26:47
                                                                                52-015-11
              Figure 1. Total Ion Chromatogram of Two Nanograms of Analytes
                       And Five Nanograms of Surrogates and Internal Standard
                                                                                  227

-------
  Method 525.1
ioo%H
 TOT-
                               600
                               10:01
900
15:01
1200
20:01
                                                                              52-015-13
               Figure 2, Total Ion Chromatogram from a Laboratory Blank
                        With an Unacceptably High Background
228

-------
                                                                               Method 525.1
HD
                            2 Liter
                            Separatory Funnel
                                                HO
                                                HD
                            125 ml.
                            Solvent
                            Reservoir
                            Ground Glass
                            Stopper 14/35

                            LSE Cartridge

                            Rubber Stopper


                            No. 18-20 Luer-lok
                            Syringe Needle
HD
                           125ml_
                           Solvent
                           Reservoir

                           Ground Glass
                           Stopper 14/35

                           LSE Cartridge
                           100mL
                           Separatory
                           Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
                                                                           10 mL
                                                                           Graduated
                                                                           Vial
                                                                                   52-015-14
       A. Extraction Apparatus
            B.  Elution Apparatus
                                      Figure 3.
                                                                                       229

-------
Method §25.1
  Source
  Vacuum
                                                                             1 Liter
                                                                             Suction Flask
                                                    Pinch Clamp
                                                                                    52-015-15
                           Figure 4. Disk Extraction Apparatus
230

-------
                                                                  Method 525.1
                               Appendix
 Detection Limits for Precision and Accuracy for the Analysis
         of Pesticide  Compounds by EPA Method 525.1


Table 7.  Method 525.1  Detection Limits —507 & 508 Compounds
Compound
Alachlor
Aldrin
Ametryn
Atraton
Atrazine
BHC, a
BHC, 0
BHC, 6
BHC, y
Bromacil
Butachlor
Butylate
Carboxin
Chlordane, a
Chlordane, 7
Chloroneb
Chlorobenzilate
Chlorothalonil
Chlorpropham
Cycloate
DCPA
ODD, 4,4'-
DDE, 4,4'-
DDT, 4,4-
Diazinon
Dichlorvos
Dieldrin
Diphenamid
Disulfoton
Disulfoton sulfone
Disulfoton sulfoxide
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin  aldehyde
EPIC
Ethoprop
Etridiazole
Fenamiphos
Fenarimol
Fluridone
Target
(pg/U
0.2
0.2
0.1
2.0
0.2
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.2
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.1
0.1
0.1
2.0
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
2.0
#
Reps
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
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
%
Rec
73
33
68
51
78
143
76
78
73
117
98
78
65
61
61
62
315
77
84
67
78
73
143
74
98
75
86
87
75
86
131
72
101
79
85
92
56
152
61
140
136
105

SD
7
10
14
8
7
31
9
9
12
13
8
23
9
11
11
8
25
9
9
14
23
14
16
17
14
28
4
7
16
8
11
10
30
13
16
11
20
2
24
12
9
19
MS
MDL
0.044
0.060
0.043
0.533
0.041
0.970
0.027
0.029
0.072
0.041
0.025
0.071
0.027
0.069
0.066
0.026
0.077
0.028
0.028
0.043
0.071
0.045
0.051
0.052
0.043
0.089
0.265
0.022
0.050
0.025
0.723
0.031
0.096
0.042
0.102
0.036
0.061
0.007
0.076
0.037
0.028
1.166
EC-NPD
MDL
0.380
0.075
2.000
0.600
0.130
0.025
0.010
0.010
0.015
2.500
0.380
0.150
0.600
0.002
0.002
0.500
5.000
0.025
0.500
0.025
0.025
0.003
0.010
0.060
0.250
2.500
0.020
0.600
0.300
3.800
0.380
0.015
0.024
0.015
0.015
0.025
0.250
0.250
0.190
1.000
0.380
3.800
                                                                         231

-------
Method 525.1
Table 7. Method 525.1 Detection Limits— 507 & 508 Compounds (cont.)

Compound
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexazinone
Merphos
Methoxychlor
Methyl paraoxon
Metolachlor
Metribuzin
Mevinphos
MKG-264
Molinate
Napropamide
Norflurazon
Pebulate
Permethrin, cis-
Permethrin, trans-
Prometon
Prometryn
Pronamide
Propachlor
Propazine
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triademefon
Tricyclazole
Trifluralin
Vernolate

Target
(V9/U
0.2
0.2
0.2
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.05
0.15
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
2.0
0.2
0.1

#
Reps
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
7
7
7
7
7
7
7
Avg
%
Rec
54
71
80
124
119
15
122
81
49
98
64
64
89
108
72
76
80
78
98
73
92
77
81
82
101
114
104
162
83
208
150
68
54
91

SD
12
10
16
12
15
3
11
5
14
14
4
13
7
7
19
20
20
58
16
7
11
5
8
34
8
14
6
19
12
84
77
13
14
15
MS
MDL
0.074
0.061
0.102
0.038
0.049
0.020
0.036
0.015
0.045
0.044
0.013
0.041
0.021
0.023
0.061
0.031
0.095
0.181
0.050
0.021
0.071
0.015
0.026
0.108
0.025
0.044
0.019
0.060
0.038
0.264
4.861
0.082
0.045
0.16
EC-NPD
MDL
0.010
0.015
0.008
0.760
0.250
0.050
2.500
0.750
0.150
5.000
0.500
0.150
0.250
0.500
0.130
0.500
0.500
0.300
0.190
0.760
0.500
0.130
0.075
0.250
0.760
1.300
4.500
0.500
0.250
0.650
1.000
0.025
0.130
0.67
232

-------
Method 525.1
Table 9. Method 525.1 -Laboratory Fortified Blank

Compound
Alachlor
Aldrin
Ametryn
Atraton
Atrazine
BHC, a
BHC, 0
BHC, 6
BHC, 7
Bromacil
Butachlor
Butyl ate
Carboxin
Chlordane, a
Chiordane, 7
Chlorneb
Chlorobenzilate
Chlorothalonil
Chlorpropham
Cycloate
DCPA
ODD, 4,4'-
DDE, 4,4'-
DDT, 4,4'-
Diaznion
Dichlorvos
Dieldrin
Diphenamid
Disulfoton
Disulfoton sulfone
Disulfoton sulfoxide
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
EPTC
Ethoprop
Etridiazole
Fenamiphos
Fenarimol
Fluridone
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexazinone
Merphos
Methoxychlor
Methyl paraoxon

Target (pg/L)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

# Reps
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
9
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
Mean
Obser.
1.039
1.114
0.850
0.313
0.932
1.070
1.075
1.136
1.105
0.879
1.126
0.843
0.757
1.017
1.144
1.018
1.211
1.211
1.121
0.639
0.822
1.084
1.221
1.128
1.335
0.749
0.819
1.383
0.978
0.880
0.490
1.229
1.358
1.194
1.223
1.331
1.159
1.134
0.911
1.088
1.074
1.108
1.102
1.218
1.024
0.988
0.955
1.182
2.334
Data, 1 Liter

SD
0.15
0.58
0.09
0.10
0.13
0.64
0.61
0.66
0.67
0.21
0.27
0.23
0.07
0.69
0.61
0.60
0.65
0.65
0.67
0.24
0.16
0.63
0.60
0.66
0.84
0.09
0.12
0.79
0.12
0.11
0.47
1.47
0.67
0.56
0.66
0.77
0.70
0.19
0.11
0.58
0.47
0.20
0.22
0.63
0.65
0.60
0.28
0.31
0.83

%RSD
13
15
13
13
14
12
13
12
12
13
9
18
16
9
9
10
11
13
15
10
11
9
13
13
13
28
11
12
16
17
19
24
12
15
11
10
14
29
12
17
11
14
14
12
9
24
10
20
34

Accuracy
104
111
85
31
93
107
108
114
110
88
113
84
76
102
114
102
121
121
112
64
82
108
122
113
134
75
82
138
98
88
49
123
136
119
122
133
116
113
91
109
107
111
110
122
102
99
95
118
233
        233

-------
Method 525.1
Table 9. Method 525.1 —Laboratory Fortified Blank Data, 1 Liter (cont.)

Compound
Metolachlor
Metribuzin
Mevinphos
MKG-264
Molinate
Napropamide
Norflurazon
Pebulate
Permethrin, cis-
Permethrin, trans-
Prometon
Prometryn
Pronamide
Propachlor
Propazine
Simazine
Simetryn
Stirofos
Tebuthiuron
Terbacil
Terbufos
Terbutryn
Triademefon
Tricyctazole
Trifluralin
Vernolate


Target ffjg/U
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0


# Reps
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
13
Avg
Mean
Obser.
0.888
1.043
0.727
0.822
0.751
0.827
0.972
1.043
1.123
0.860
1.124
0.562
1.118
0.932
2.179
0.853
0.708
1.043
1.036
1.119
0.913
0.908
1.172
0.931
2.215
0.901
1.05

SO
0.18
0.15
0.13
0.13
0.19
0.18
0.12
0.15
0.24
0.96
0.22
0.11
0.13
0.13
0.90
0.20
0.06
0.21
0.23
0.18
0.13
0.10
0.19
0.42
0.78
0.18
0.40

%RSD
14
13
18
21
12
12
12
11
16
18
11
8
18
14
14
14
9
13
30
17
13
13
15
54
15
16
15

Accuracy
89
104
73
82
75
83
97
104
112
86
112
56
112
93
218
85
71
104
104
112
91
91
117
93
221
90
105
234

-------
                          Method 531.1
Measurement of N-Methylcarbamoyloximes
          and N-Methylcarbamates in Water
          by Direct Aqueous Injection HPLC
            with Post-Column Derivatization
                       Revision 3.0 - EPA EMSL-Ci
                    D. L. Foerst - Method 531, Revision 1.(1985)
           T. Engels (Battelle Columbus Laboratories) — National Pesticide
                        Survey Method 5, Revision 2.0 (1987)
                 R. L. Graves - Method 531.1, Revision 3.0 (1989)

-------

-------
                                Method 531.1
                 Measurement of N-Methylcarbamoyloximes
 and N-Methylcarbamates in Water by Direct Aqueous Injection HPLC
                        with Post Column Derivatization


 1.    SCOPE AND APPLICA TION

 1.1   This is a high performance liquid chromatographic (HPLC) method applicable to the determi-
      nations of certain N-methylcarbamoyloximes and N-methylcarbamates in ground water and
      finished drinking water.' The following compounds can be determined using this method:

                 Analyte                            CAS No.
                 Aldicarb                              116-06-3
                 Aldicarb sulfone                      1646-88-4
                 Aldicarb sulfoxide                    1646-87-3
                 Baygon                               114-26-1
                 Carbaryl                               63-25-2
                 Carbofuran                          1563-66-2
                 3-Hydroxycarbofuran                16655-82-6
                 Methiocarb                          2032-65-7
                 Methomyl                          16752-77-5
                 Oxamyl                            23135-22-0

 1.2   This method has been validated in a single laboratory and estimated detection limits (EDLs)
      have been determined for the analytes above (Sect. 12).  Observed detection limits may vary
      between ground waters, depending upon the nature of interferences in the sample matrix and
      the specific  instrumentation used.
 1.3   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 Sect. 10.3.
 1.4   When this method is used to analyze unfamiliar samples  for any or all of the analytes above,
      analyte identifications should be confirmed by at least one additional qualitative technique.

2.    SUMMARY OF METHOD

2.1   The water sample is filtered and a 400-/iL aliquot is injected into a reverse phase HPLC
      column. Separation of the analytes is achieved using gradient elution chromatography.  After
      elution from the HPLC column, the analytes are hydrolyzed with 0.05 N sodium hydroxide
      (NaOH) at 95°C.  The methyl amine formed during hydrolysis is reacted with o-phtalaldehyde
      (OPA) and 2-mercaptoethanol  to form a highly fluorescent derivative which is detected by a
      fluorescence detector.2
                                                                                  237

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Method 531.1
3.     DEFINITIONS

3.1    Internal standard: A pure analyte(s) added to a solution in known amount(s) and used to
       measure the relative responses of other method analytes and surrogates that are components of
       the same solution.  The internal standard must be an analyte that is not a sample component.
3.2    Surrogate analyte:  A pure analyte(s), which is extremely unlikely to be found in any sample,
       and which is added to a sample aliquot in known amount(s) before extraction and is measured
       with the same procedures used to measure other sample components.  The purpose of a
       surrogate analyte is to monitor method performance with each sample.
3.3    Laboratory  duplicates: (LDl and LD2):   Two sample aliquots taken in the analytical laborato-
       ry and  analyzed separately with  identical procedures.  Analyses of LDl and LD2 give a
       measure of the precision associated with laboratory procedures, but not with sample collection,
       preservation, or storage procedures.
3.4    Field duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the same throughout field and laboratory
       procedures.  Analyses of  FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory  reagent blank  (LRB): An aliquot of reagent water that is treated exactly as a
       sample  including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples. The LRB is used to determine if method
       analytes or  other interferences are present in the laboratory environment, the reagents, or the
       apparatus.
3.6    Field reagent blank (FRB):  Reagent water placed in a sample container in the laboratory  and
       treated  as a sample in all  respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures.  The purpose of the FRB is to determine if method
       analytes or  other interferences are present in the field environment.
3.7    Laboratory  performance check solution (LPC):  A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with respect to a defined set of method criteria.
3.8    Laboratory  fortified blank (LFB):  An aliquot of reagent water to which known quantities of
       the method  analytes are added in the laboratory. The LFB is analyzed exactly like a sample,
       and its  purpose is to determine whether the methodology is in control, and whether the labora-
       tory is  capable of making accurate and precise measurements at the required method detection
       limit.
3.9    Laboratory  fortified sample matrix (LFM): An aliquot of an environmental sample to which
       known  quantities of the method analytes are added in the laboratory.  The  LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to  the analytical results. The background concentrations of the analytes in the sample
       matrix  must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock standard solution:   A  concentrated  solution containing a single certified standard that is a
       method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
238

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                                                                                    Method 531.1
       an assayed reference compound.  Stock standard solutions are used to prepared primary
       dilution standards.
3.11  Primary dilution standard solution:  A solution of several analytes prepared in the laboratory
       from stock standard solutions and diluted as needed to prepare calibration solutions and other
       needed analyte solutions.
3.12  Calibration standard (CAL):  A solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards and surrogate analytes. The CAL solu-
       tions are used to calibrate the instrument response with respect to analyte concentration.
3.13  Quality control sample (QCS):  A sample matrix containing method analytes or a solution of
       method analytes in a water miscible solvent which is used to fortify reagent water or environ-
       mental samples.  The QCS is obtained from a source external to the laboratory, and is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    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.  Specific sources of contamination have not been identified. 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 Sect. 10.2.
       4.1.1   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 through rinsing with tap and reagent water.  Drain
               dry, and heat in  an oven or muffle furnace at 450°C for 1 hour. Do not heat volu-
               metric ware.   Thermally stable materials might not be eliminated by this treatment.
               Through 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.
       4.1.2   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.  WARN-
               ING: when a solvent is purified,  stabilizers added by the manufacturer are removed,
               thus potentially making the solvent hazardous.  Also, when a solvent is purified,
               preservatives added by the manufacturer are removed, thus potentially reducing the
               shelf-life.
4.2    Interfering contamination may occur when a sample containing low concentrations of analytes
       is analyzed immediately following a sample containing relatively high concentrations of
       analytes.  A preventive technique is between-sample rinsing of the sample syringe and filter
       holder with two portions of reagent water.  After analysis of a sample containing high concen-
       trations of analytes, one or more laboratory method blanks should be analyzed.
4.3    Matrix interference may be caused by contaminants that are present in the sample.  The extent
       of matrix  interference will vary considerably from source to  source,  depending upon the  water
       sampled.  Positive identifications must be confirmed.
                                                                                            239

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Method 531.1
5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
       ing the safe handling of the chemicals specified in this method.   A reference file of material
       safety data 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.
5.2    WARNING: When a solvent is purified, stabilizers added by the manufacturer are removed,
       thus potentially making the solvent hazardous.

6.     APPARATUS AND EQUIPMENT
(All specifications are suggested.  Catalog numbers are included for illustration only.)
6.1    Sampling Equipment
       6.1.1   Grab sample bottle:  60-mL screw cap vials (Pierce No. 13075 or equivalent) and
               caps equipped with a PTFE-faced silicone septa (Pierce No. 12722 or equivalent).
               Prior to use, wash vials and septa as described in Sect.  3.1.1.
6.2    Balance:  Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.3    Filtration Apparatus
       6.3.1   Microfiltration: To filter derivatization solutions and mobile phases used in HPLC.
               Recommend using 47 mm filters (Millipore Type HA,  0.45 ftm for water and Millip-
               ore Type FH, 0.5 pm  for organics or equivalent).
       6.3.2  Microfiltration: To filter samples prior to HPLC analysis. Use 13 mm filter holder
               (Millipore stainless steel XX300/200 or equivalent), and 13 mm diameter 0.2 /xm
               polyester filters (Nuclepore  180406 or equivalent).
6.4    Syringes and Syringe Values
       6.4.1   Hypodermic syringe:   10-mL glass, with Luer-Lok tip.
       6.4.2  Syringe value: 3-way (Hamilton HV3-3 or equivalent).
       6.4.3  Syringe needle: 7- to  10-cm long, 17-gauge, blunt tip.
       6.4.4  Micro syringes: various sizes.
6.5    Miscellaneous
       6.5.1   Solution storage bottles:  Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-
               Hned screw cap.
       6.5.2  Helium, for degassing solutions and solvents.
6.6    High Performance Liquid Chromatograph (HPLC)
       6.6.1  HPLC  system capable of injecting 200- to 400-/uL aliquots, and performing binary
               linear gradients at a constant flow rate.  A data system is recommended for measuring
               peak areas. Table  1 lists retention times observed for  method analytes using  the
               columns and analytical conditions described below.

240

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                                                                                Method 531.1
       6.6.2  Column 1 (Primary column):  150 mm long  x 3.9 mm I.D. stainless steel packed
              with 4 pm NovaPak CIS.  Mobil Phase is established at 10:90 methanol:water, hold 2
              min., then program as a linear gradient to 80:20 methanol:water in 25 min.  Alterna-
              tive columns may be used in accordance with the provisions described in Sect.  10.4.
       6.6.3  Column 2 (Alternative column):*  250 mm long x 4.6 mm I.D. stainless steel
              packed with 5 jtm Beckman Ultrasphere ODS. Mobile phase is established at 1.0
              mL/min as a linear gradient from 15:85 methanol:water to methanol in 32  min.  Data
              presented in this method were obtained using this column.
       6.6.4  Column 3 (Alternative column):  250 mm long x  4.6 mm I.D. stainless steel packed
              with 5 /*m Supelco LC-1.  Mobile phase is established at 1.0 mL/min as a linear
              gradient from 15:85 methanol:water to methanol in 32 min.
       6.6.5  Post—column reactor:  Capable of mixing reagents into the mobile phase.  Reactor
              should be constructed using PTFE tubing and equipped with pumps to deliver 0.1 to
              1.0 mL/min of each reagent; mixing tees;  and two 1.0-mL delay coils, one thermosta-
              ted at 95°C  (ABI URS  051 and URA  100 or equivalent).
       6.6.6  Fluorescence detector:  Capable of excitation at 230 run and detection of emission
              energies greater than 418 nm. A Schoffel Model 970 fluorescence detector was used
              to generate the validation data presented in this method.

7.     REAGENTS AND CONSUMABLE MATERIALS

       WARNING:       When a solvent is purified, stabilizers added by the manufacturer
       are removed, thus potentially making the solvent hazardous. Also, when a solvent is
       purified, preservatives added by the manufacturer are removed, thus potentially redu-
       cing the shelf-life.

7.1    Reagent Water:  Reagent water is defined as water that is reasonably free of contamination that
       would prevent the determination of any analyte of interest.  Reagent water used to generate the
       validation data in this method was distilled water obtained from the Magnetic Springs Water
       Co., 1801 Lone Eagle St., Columbus, Ohio 43228.
7.2    Methanol:  Distilled-in-glass quality or equivalent.
7.3    HPLC Mobile Phase
       7.3.1   Water:  HPLC grade (available from Burdick and  Jackson).
       7.3.2  Methanol: HPLC grade. Filter and degas with helium before use.
7.4    Post-Column Derivatization Solutions
       7.4.1   Sodium hydroxide, 0.05 N: Dissolve 2.0  g of sodium hydroxide (NaOH) in  reagent
              water.  Dilute to 1.0 L  with reagent water. Filter and degas with helium just before
              use.
    *Newer manufactured columns have not been able to resolve aldicarb sulfone from oxamyl.

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Method 531  1
       7.4.2   2-Mercaptoethanol (1 + 1):  Mix 10.0 mL of 2-mercapto-ethanol and 10.0 mL of
               acetonitrile.  Cap.  Store in hood (CAUTION—stench).
       7.4.3   Sodium borate (0.05 N): Dissolve 19.1 g of sodium borate (Na2B4O7 10H2O) in
               reagent water. Dilute  to 1.0  L with reagent water.  The sodium borate will complete-
               ly dissolve at room temperature if prepared a day before use.
       7.4.4   OPA reaction solution: Dissolve 100 + 10 mg of o-phthalaldehyde (mp 55-58°C) in
               10 mL of methanol. Add to  1.0 L of 0.05 N sodium borate.  Mix, filter, and degas
               with helium.  Add 100 ^L  of 2-mercaptoethanol  (1 +1) and mix.  Make up fresh
               solution daily.
7.5    Monochloroacetic Acid Buffer (pH3):  Prepare by mixing 156 mL of 2.5 M monochloroacetic
       acid and 100 Ml 2.5 M potassium acetate.
7.6    4-Bromo-3,5-Dimethylphenyl N-Methycarbamate (BDMC):  98% purity, for use as internal
       standard (available from Aldrich Chemical Co.).
7.7    Stock Standard Solutions (1.00 /*g//xL):  Stock standard solutions may be purchased as cer-
       tified solutions or prepared from pure standard materials using the following procedure:
       7.7.1   Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
               pure material. Dissolve the material in HPLC 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.
       7.7.2   Transfer the stock standard solutions into TFE-fluoro-carbon-sealed screw cap vials.
               Store at room temperature  and protect from light.
       7.7.3   Stock  standard solutions should be replaced after two months or sooner if comparison
               with laboratory fortified blanks, or QC samples indicate a problem.
7.8    Internal Standard Solution:  Prepare an internal standard fortification solution  by accurately
       weighing approximately 0.0010 g of pure BDMC.  Dissolve  the BDMC in pesticide-quality
       methanol and dilute  to volume  in a  10-mL volumetric flask.  Transfer the internal standard
       fortification solution to a TFE-fluorocarbon-sealed screw  cap bottle and store at room tempera-
       ture. Addition of 5  ^L of the internal standard fortification solution to  50 mL of sample
       results  in a final internal standard concentration of 10 /xg/L.  Solution should be replaced when
       ongoing QC (Sect. 10) indicates a problem.  Note:  BDMC has  been shown to be  an effective
       internal standard for the method analytes,1 but other compounds may be used, if the quality
       control requirements in Sect. 9 are  met.
7.9    Laboratory Performance Check Solution:  Prepare  concentrate by adding 20 /^L of the 3-
       hydroxycarbofuran stock standard solution, 1.0 mL of the aldicarb sulfoxide stock standard
       solution, 200 ^L of the methiocarb stock standard solution, and\mL of the internal standard
       fortification solution to a  10-mL volumetric flask.  Dilute to  volume with methanol.  Thor-
       oughly mix concentrate.  Prepare check solution by placing 100 pL  of the concentrate solution
       into a 100-mL volumetric flask.  Dilute to volume with buffered reagent water.  Transfer to a
       TFE-fluorocarbon-sealed  screw cap bottle and store at room  temperature.   Solution should be
       replaced when ongoing QC (Sect.  10) indicates a problem.

242

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                                                                                   Method 531.1
8.     SAMPLE COLLECTION, PRESERVATION AND HANDLING

8.1    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.
8.2    Sample Preservation/PH Adjustment: Oxamyl, 3-hydroxycarbofuran, aldicarb sulfoxide, and
       carbaryl can all degrade quickly in neutral and basic waters held at room temperature.6'7 This
       short term degradation is of concern during the time samples are being shipped and the  time
       processed samples are held at room temperature in autosampler trays.  Samples targeted for
       the analysis of these three analytes must be preserved at pH 3. The pH adjustment also
       minimizes analyte biodegradation.
       8.2.1   Add 1.8 mL of monochloroacetic acid buffer to the 60-mL sample bottle.  Add buffer
               to the sample bottle at the sampling site or in the laboratory before shipping to the
               sampling site.
       8.2.2   If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
               the sample bottle prior to collecting the sample.
       8.2.3   After sample is collected in bottle containing buffer, seal the sample bottle and shake
               vigorously for 1 min.
       8.2.4   Samples must be iced or refrigerated at 4°C from the time of collection until storage.
               Samples must be stored at -10°C until analyzed. Preservation study results indicate
               that method analytes are stable in water samples for at least 28 days when adjusted to
               pH 3 and stored at -10°C.  However, analyte stability may be effected by the matrix;
               therefore, the analyst should verify that the preservation technique is applicable to the
               samples under study.

9.     CALIBRATION

9.1    Establish HPLC operating parameters equivalent to those indicated  in Sect. 6.6.  The  HPLC
       system may be calibrated using either the internal standard technique (Sect. 9.2) or the external
       standard technique (Sect. 9.3).
9.2    Internal Standard Calibration Procedure.  The analyst must select one or more internal stan-
       dards similar in analytical behavior to the analytes of interest.  The analyst must further
       demonstrate that the measurement of the internal standard is not affected by method or matrix
       interferences. BDMC has been identified as a suitable internal standard.
       9.2.1   Prepare calibration standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest by adding volumes of one or of the more stock
               standards to a volumetric flask.  To each calibration standard, add a known constant
               amount of one or more  internal standards, and dilute to volume with buffered reagent
               water.  To prepare buffered reagent water, add  10 mL of 1.0 M monochloroacetic
               acid buffer to 1  L of reagent water. The lowest standard should represent analyte
               concentrations near,  but above, their respective  EDLs.  The remaining standards
               should bracket the analyte concentrations expected in the sample extracts, or should
               define the working range of the detector.
       9.2.2   Analyze each calibration standard according to the procedure (Sect. 11.2).  Tabulate
              peak height or area responses against concentration for each compound and internal

                                                                                           243

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Method 531.1
               standard.  Calculate response factors (RF) for each analyte, surrogate and internal
               standard usino Rniiatinn 1
                      r
standard using Equation 1.
                                          Equation  1
                                               CU (Q
                      Where:
                      As = Response for the analyte to be measured.
                      AK = Response for the internal standard.
                      Ca = Concentration of the internal standard
                      Cs = Concentration of the analyte to be measured /x/L.
       9.2.3   If the RF value over the working range is constant (20% RSD or less) the average RF
               can be used for calculations.  Alternatively, the results can be used to plot a calibra-
               tion curve of response ratios (A,/A1S) vs. Cs.
       9.2.4   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 analyte
               varies from the predicted response by  more than ±20%, the test must be repeated
               using a fresh  calibration standard.  If the repetition also fails, a new calibration cure
               must be generated for that analyte using freshly prepared standards.
       9.2.5   Single point calibration is a viable alternative to a calibration curve.  Prepare single
               point standards from the secondary dilution standards. The single point  standards
               should be prepared at a concentration that deviates from the sample extract response
               by no more than 20%.
9.3    External Standard Calibration Procedure
       9.3.1   Prepare calibration standards at a minimum of three (recommend five) concentration
               levels for each analyte of interest by adding volumes  of one or more  stock standards
               to a volumetric flask. Dilute to volume with buffered reagent water.  The lowest
               standard should represent analyte concentrations near, but above, the respective
               EDLs.  The remaining standards should bracket the analyte concentrations expected in
               the sample  extracts, or should  define the working range of the detector.
       9.3.2   Starting with  the standard of lowest concentration, analyze each calibration standard
               according to Sect. 11.2 and tabulate responses (peak height or area) versus the con-
               centration in the standard. 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  <20% 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.
       9.3.3   The working  calibration curve or calibration factor must be verified on each working
               day by the  measurement of a minimum of two calibration check standards,  one at the
               beginning and one at the end of the analysis day.  These check standards should be at
244

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                                                                                   Method 531.1
               two different concentration levels to verify the concentration curve. For extended
               periods of analysis (greater than 8 hr), it is strongly recommended that check stan-
               dards be interspersed with samples at regular intervals during the course of the analy-
               ses. If the response for any analyte varies from the predicted response by more than
               ±20%, the test must be repeated using a fresh calibration standard. If the results still
               do not agree, generate a new calibration curve or cue a single point calibration stan-
               dard as described in Sect. 9.3.3.
       9.3.4   Single point calibration is a viable alternative to a calibration curve. Prepare single
               point standards from the secondary dilution standards.  The single point standards
               should be prepared at a concentration that deviates from the sample extract response
               by no more than 20%.
       9.3.5   Verify calibration standards periodically, recommended at least quarterly,  by analyz-
               ing a standard prepared from reference material  obtained from an independent source.
               Results from these analyses must be within the limits used to routinely check calibra-
               tion.

10.   QUALITY  CONTROL

10.1   Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
       monitoring  internal standard peak area or height in each sample and blank (when internal
       standard calibration procedures are being employed),  analysis of laboratory reagent blanks,
       laboratory fortified samples, laboratory fortified blanks and QC samples.
10.2   Laboratory  Reagent Blanks:  Before processing any samples, the analyst must demonstrate that
       all glassware and reagent interferences are under control.  Each time a set of samples is
       extracted or reagents are changed, a laboratory reagent blank (LRB) must be analyzed. If
       within the retention time window of any analyte of interest the LRB produces a peak that
       would prevent the determination of that analyte, determine the source of contamination and
       eliminate the interference before processing samples.
10.3   Initial Demonstration of Capability
       10.3.1  Select a representative concentration (about 10 times EDL) for each analyte.  Prepare
               a sample concentrate (in methanol) containing each analyte at 1000 times selected
               concentration.  With a syringe, add 50 /xL of the concentrate to each of at least four
               50-mL aliquots of reagent water,  and analyze each aliquot according to procedures
               beginning in Sect. 11.
       10.3.2  For each analyte the recovery value of all four of these samples must fall in the range
               of R + 30% (or within R ±  3SR if broader) using the values for R and  SR for reagent
               water in Table 2.  For those compounds that meet the acceptance criteria,  perfor-
               mance is judged acceptable and sample analysis may begin.  For those compounds
               that meet the acceptance criteria,  performance is judged acceptable and sample analy-
               sis may begin.  For those compounds that fail these criteria, this procedure must be
               repeated using four fresh samples until satisfactory performance has been demonstrat-
               ed.
       10.3.3  The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some

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Method 531.1
               experience with it. It is expected that as laboratory personnel gain experience with
               this method the quality of data will improve beyond those required here.
10.4  The analyst is permitted to modify HPLC columns, HPLC conditions, internal standards or
       detectors to improve separations or lower analytical costs.  Each time such method modifica-
       tions are made, the analyst must repeat the procedures in Sect.  10.3.
10.5  Assessing the Internal Standard
       10.5.1  When using the internal standard calibration procedure, the analyst is expected to
               monitor the IS response (peak area or peak height) of all samples during each analysis
               day.  The IS response for any sample chromatogram should not deviate from the daily
               calibration check standard's IS response by more than 30%.
       10.5.2 If >30% deviation occurs with an individual extract, optimize instrument perfor-
               mance and inject a second aliquot.
               10.5.2.1 If the reinjected aliquot produces an acceptable internal standard response,
                         report results for that aliquot.
               10.5.2.2 If a deviation of greater than 30% is obtained for the reinjected extract,
                         analysis of the sample should be repeated beginning with Sect. 11, provid-
                         ed the sample is still available.  Otherwise, report results obtained from the
                         reinjected extract, but annotate as suspect.
       10.5.3 If consecutive samples fail the IS response acceptance criterion, immediately analyze a
               calibration check standard.
               10.5.3.1 If the check standard provides a response factor (RF) within 20% of the
                         predicted  value, then follow procedures itemized in Sect. 10.5.2 for each
                         sample failing the IS response criterion.
               10.5.3.2 If the check standard provides a response factor which deviates more than
                         20% of the predicted value,  then the analyst must recalibrate, as specified
                         in Sect. 9.
10.6  Assessing Laboratory Performance—Laboratory  Fortified Blanks
       10.6.1  The laboratory must  analyze at least one laboratory fortified blank (LFB) sample with
               every 20 samples or  one per sample set (all sample analyzed within a 24-h period)
               whichever is greater.  The fortification concentration of each analyte in the LFB
               should be 10 times EDL or the  MCL, whichever is less.  Calculate accuracy as
               percent recovery (Xj).  If the recovery of any analyte falls outside the control limits
               (see Sect. 10.7.2), that analyte is judged out of control, and the source of the problem
               must be identified and resolved before continuing analyses.
       10.6.2 Until sufficient data become available from within their own laboratory, usually a
               minimum of results from 20 to 30 analyses, the laboratory should assess laboratory
               performance against the control limits in Sect. 10.3.2 that are derived from the data in
               Table 2.  When sufficient internal performance data becomes available, develop
               control limits from the mean percent recovery (X) and standard deviation (S) of the
246

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                                                                                   Method 531.1
              percent recovery.  These data are used to establish upper and lower control limits as
              follows:
                              Upper Control Limit = X + 3S
                              Lower Control Limit =  X - 3S

              After each five to ten new recovery measurements,  new control limits should be
              calculated using only the most recent 20-30 data points.  These calculated control
              limits should never exceed those established in Sect. 10.3.2.
       10.6.3 It is recommended that the laboratory periodically determine and document its detec-
              tion limit capabilities for analytes of interest.
       10.6.4 At least quarterly, analyze a QC sample from an outside source.
       10.6.5 Laboratories are encouraged to participate in external performance evaluation studies
              such as the laboratory certification programs offered by many states or the studies
              conducted by USEPA.  Performance evaluation  studies serve as independent checks
              on the analyst's performance.
10.7  Assessing Analyte Recovery—Laboratory Fortified Sample Matrix
       10.7.1 The laboratory must add a known concentration  to a minimum of 5% of the routine
              samples or one sample concentration per set, whichever is greater.  The concentration
              should not be less than the background concentration of the sample selected for
              fortification. Ideally, the concentration should be the same as that used for the labora-
              tory fortified blank (Sect. 10.6).  Over time, samples from all routine sample sources
              should be fortified.
       10.7.2 Calculate the percent recovery, P, of the concentration for each analyte, after correct-
              ing the analytical result, X, from the fortified sample for the background concentra-
              tion, b, measured  in the unfortified sample, i.e.,:
                                  p =      100 (X - b)
                                       fortifying concentration
               and compare these values to control limits appropriate for reagent water data collected
               in the same fashion.  If the analyzed unfortified sample  is found to contain NO back-
               ground concentrations, and the added concentrations are those specified in Sect. 10.7,
               then the appropriate control limits would be the acceptance limits in Sect. 10.7. If,
               on the other hand, the analyzed unfortified sample is found to contain background
               concentration, b, estimate the standard deviation at the background concentration, sb,
               using regressions  or comparable background data and, similarly, estimate the mean,
               xa, and standard deviation, sa, of analytical results at the total concentration after for-
               tifying.  Then the appropriate percentage control limits would be P ± 3SP, where:
                                                                                            247

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Method 531.1
                               P  =
                                               100 X
                                    (b + fortifying concentration)
                               ,,-100 _
                                        fortifying concentration
               For example, if the background concentration for Analyte A was found to be 1 pig/L
               and the added amount was also 1 /*g/L, and upon analysis the laboratory fortified
               sample measured 1.6 p/L, then the calculated P for this sample would be (1.6 /xg/L
               minus 1.0 Mg/L)/l ftg/L or 60%.  This calculated P is compared to control limits
               derived from prior reagent water data.  Assume it is known that analysis of an inter-
               ference free  sample at  1 /xg/1 yields an s of 0.12 jig/L and similar analysis at 2.0 jig/L
               yields X and s of 2.01  /tg/L and 0.20 /ig/L, respectively. The appropriate limits to
               judge the reasonableness of the percent recovery, 60%, obtained on the fortified
               matrix sample is computed as follows:
                   100 (2.01 pg/L)
                      2.0
                            K°-12Pg/L)2 +  (O-20
                                      1.0 ftgIL
                 100.5%  +  300 (0.233)  =
                 100.5%  ±  70%  or 30% to  170%  recovery of the added analyte.
       10.7.3    If the recovery of any such analyte falls outside the designated range, and the
               laboratory performance for that analyte is shown to be in control (Sect. 10.6), the
               recovery problem encountered with the dosed sample is judged to be matrix related,
               not system related.  The result for that analyte in the unfortified sample is labeled
               suspect/matrix to inform the data user that results are suspect due to matrix  effects.
 10.8  Assessing Instrument System—Laboratory Performance Check Sample:  Instrument perfor-
       mance should be monitored on a daily basis by analysis of the LPC sample. The LPC sample
       contains compounds designed to indicate appropriate instrument sensitivity, column perfor-
       mance (primary column) and chromatographic performance. LPC sample components and
       performance criteria are listed in Table 3.  Inability to demonstrate acceptable instrument
       performance indicates the need for reevaluation of the instrument system. The sensitivity
       requirements are set based on the EDLs published in this method, concentrations of  the
       instrument QC standard components must be adjusted to be compatible with the laboratory
       EDLs.
 10.9  The laboratory may adopt additional quality control 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.  For example, field or laboratory duplicates may be analyzed  to assess
 245

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                                                                                 Method 531.1
      the precision of the environmental measurements or field reagent blanks may be used to assess
      contamination of samples under site conditions, transportation and storage.

7 7.  PROCEDURE

11.1  pH Adjustment and Filtration
      11.1.1 Add preservative to any samples not previously preserved (Sect. 8). Adjust the pH of
              the sample or standard to pH 3  ±0.2 by adding  1.5 mL of 2.5 M monochloroacetic
              acid buffer to each 50 mL of sample.  This step  should not be necessary if sample pH
              was adjusted during sample collection as a preservation precaution.  Fill a 50-mL
              volumetric flask to the mark with the sample.  Add 5 /*L of the internal standard
              fortification solution (if the internal standard calibration procedure is being employed)
              and mix by inverting the flask several times.
      11.1.2 Affix the three-way valve to a 10-mL syringe. Place clean filter in the filter holder
              and affix the filter holder and the 7- to  10-cm syringe needle to the syringe valve.
              Rinse the needle and syringe with reagent water.  Prewet the filter by passing 5 mL of
              reagent water through the filter.  Empty the syringe and check for leaks.  Draw 10
              mL of sample into the syringe and expel through the filter.  Draw another 10 mL of
              sample into the syringe, expel through the filter, and collect the last 5 mL for analy- •
              sis.  Rinse the syringe with reagent water. Discard the filter.
11.2  Liquid Chromatography
      11.2.1 Sect. 6.6 summarizes the recommended operating conditions for the liquid chromato-
              graph.  Table 1 lists retention times observed using this method. Other HPLC col-
              umns,  chromatographic conditions, or detectors may be used if the requirements of
              Sect. 10.4 are met.
      11.2.2 Calibrate the system daily as descried in Sect. 10.  The standards and samples must be
              in pH3 buffered water.
      11.2.3 Inject 400 /xL of the sample.  Record the volume injected and the resulting peak size
              in area units.
      11.2.4 If the response for the peak exceeds the working range of the system, dilute the
              sample with pH 3 buffered reagent water and reanalyze.
11.3  Identification of Analytes
      11.3.1 Identify a sample component by comparison of its retention time to the retention time
              of a reference chromatogram. If the  retention tune of an unknown compound corre-
              sponds, within limits, to the retention time of a standard compound, then identification
              is considered positive.
      11.3.2 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 tune 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.
      11.3.3 Identification requires expert judgement when sample components are not resolved
              chromatographically.  When peaks obviously represent more than one sample compo-

                                                                                          249

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Method 531.1
               nent (i.e., broadened peak with shoulder(s) or valley between two or more maxima),
               or any time doubt exists over the identification of a peak on a chromatogram, appro-
               priate alternate techniques, to help confirm peak identification, need to be employed.
               For example, more positive  identification may be made by the use of an alterative
               detector which operates on a chemical/physical principle different from that originally
               used; e.g., mass spectrometry, or the use of a second chromatography  column. A
               suggested alternative column is described in Sect. 6.6.3.

12.   CALCULATIONS
Determine the concentration of individual compounds in the sample using the following equation:
                                         c  -
                                              As .  RF

  where:
  Cx  = analyte concentration  in micrograms per liter,
  Ax  = response of the sample analyte;
  A  = response of the standard (either internal or external), in units consistent with those used
        for the analyte response;
  RF  = response factor (with  an  external standard, RF = 1 , because the standard is the same
        compound as the measured analyte);
  Qs  = concentration of internal standard present or concentration of external standard
        that produced As, in micrograms per liter.
 13.   PRECISION AND A CCURA c Y

 13.1  In a single laboratory, analyte recoveries from reagent water were determined at five concen-
       tration levels.  Results were used to determine analyte EDLs and demonstrate method range.'
       Analyte recoveries and standard deviation about the percent recoveries at one concentration are
       given in Table 2.
 13.2  In a single laboratory, analyte recoveries from two standard synthetic ground waters were
       determined at one concentration level.  Results were used to demonstrate applicability of the
       method to different ground water matrices.'  Analyte recoveries from the two synthetic matri-
       ces are given in Table 2.
250

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                                                                               Method S31.1
                                     References

 1.     National Pesticide Survey Method No. 5., "Measurement of N-Methylcarbamoyloximes and
       N-Methylcarbamates in Groundwater by HPL with Post Column Derivatization."

2.     Moye, H.A., S. J. Sherrer, and P.A. St. John, "Dynamic Labeling of Pesticides for High
       Performance Liquid Chromatography: Detection of N-Methylcarbamates and o-Phthaladehy-
       de," Anal. Lett. 10. 1049, 1977.

3.     ASTM Annual Book of Standards, Part 11, Volume 11.02,  D3694-82,  "Standard Practice for
       Preparation of Sample Containers and for Preservation,"  American Society for Testing and
       Materials, Philadelphia, PA, p. 86,  1986.

4.     "Carcinogens — Working with Carcinogens," Department of Health, Education, and Welfare,
       Public Health Service, Center For Disease Control, National Institute for Occupational Safety
       and Health, Publication No. 77-206, Aug.  1977.

5.     "OSHA Safety and Health Standards, General Industry,"  (29 CFR 1910), Occupational Safety
       and Health Administration, OSHA 2206, (Revised, January  1976).

6.     "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
       Committee on Chemical Safety, 3rd Edition,  1979.

7.     Foerst, D.L. and H.A. Moye, "Aldicarb in Drinking Water via Direct Aqueous Injection
       HPLC with Post column Derivatization," Proceedings of the 12th annual AWWA Water
       Quality Technology conference, 1984.

8.     Hill, K.M., R.H. Hollowell, and  L.A. DalCortevo,  "Determination of N-Methylcarbamate
       Pesticides in Well Water by Liquid Chromatography and Post Column Fluorescence Derivatiz-
       ation,"  Anal. Chem. 56, 2465 (1984).

9.     ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard Practice for
       Sampling Water,"  American Society for Testing and Materials, Philadelphia, PA, p. 130,
       1986.
                                                                                       257

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Method 531.1
Table 1.  Retention Times for Method Analytes
                                              Retention Time '
                                                  (minutes)
 Analyte

 Aldicarb sulfoxide
 Aldicarb sulfone
 Oxamyl
 Methomyl
 3-Hydroxcarbofuran
 Aldicarb
 Baygon
 Carbofuran
 Carbaryl
 Methiocarb
 BDMC
 Primary1

 6.80
 7.77
 8.20
 8.94
13.65
16.35
18.86
19.17
20.29
24.74
25.28
Alternative2

  15.0
  15.2
  17.4
  18.4
  23.3
  27.0
  29.3
  29.6
  30.8
  34.9
  35.5
Alternative3

 17.5
 12.2
 14.6
 14.8
 19
 21.4
 24.4
 23.4
 25.4
 28.6
     Columns and analytical conditions are described in Sect. 6.6.2 and 6.6.3.
     Waters NovaPack C18
     Beckman Ultrasphere ODS
     Superlco LC-1
252

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     Table 2.  Single Laboratory Accuracy, Precision and Estimated Detection Limits (EDLS)
     For Analytes From Reagent Water and Synthetic Ground Waters3
Water 2f                 EDL
Analyte                 (pg/Lf

Aldicarb                  1.0
Aldicarb sulfone            2.0
Aldicarb sulfoxide          2.0
Baygon                   1.0
Carbaryl                  2.0
Carbofuran                1.5
3-Hydroxycarbofuran       2.0
Methiocarb                4.0
Methomyl                 0.5
Oxamyl                   2.0
Concentration Level
V9/L \ ff
5 115
10 101
10 97
5 106
10 97
7.5 102
10 102
20 94
2.5 105
10 100
Reagent Water
SR" ^
3.5 106
4.0 98
4.9 105
3.2 96
5.8 94
5.1 102
4.1 98
1.9 102
4.2 98
4.0 97
Synthetic Water T
SR fl
3.2 102
3.9 95
4.2 94
4.8 97
4.7 104
3.1 100
4.9 101
4.1 112
3.9 105
2.9 102
                                                                                                                   Synthetic
                                                                                                                       8.2
                                                                                                                       9.5
                                                                                                                      10.3
                                                                                                                       5.8
                                                                                                                      10.4
                                                                                                                       7.0
                                                                                                                      10.1
                                                                                                                       3.4
                                                                                                                       9.5
                                                                                                                      10.2
          Data corrected for amount detected in blank and represent the mean of 7-8 samples.
          EDL = Estimated detection limit; defined as either MDL (Appendix B to 40 CFR Part 136 - Definition and Procedure for the
          Determination of the Method Detection Limit - Revision 1.11) or a level of compound in a sample yielding a peak in the final extract
          with the signal-to-noise ratio of approximately 5, whichever value is higher. The concentration level used in determining the EDL is
          not the same as the concentration  level presented  in this table.
          R  = Average percent recovery.
          SR =  Standard deviation of the percent recovery.
          Corrected for amount found in blank; Absopure Nature Artesian Spring Water Obtained from the Absopure Water Company in
          Plymouth, Michigan.
          Corrected for amount found in blank; reagent water fortified with fulvic acid at the 1  mg/L concentration level.  A well-characterized
          fulvic acid, available from the International Humic Substances Society  (associated with the United States Geological Survey in
          Denver, Colorado) was used.
                                                                                                                                     I
01
Co
                                                                                                                                     wi
                                                                                                                                     Co

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Method 531.1
Table 3. Laboratory Performance Check Solution

Test Analyte
Sensitivity 3-Hydroxycarbofuran
Chromatographic Aldicarb sulfoxide
performance
Column performance Methiocarb
4-Bromo-3,5-dimethylphenyl
N-methylcarbamate (IS)
Cone.
f/jg/mL)
2
100

20

10

Requirements
Detection of analyte; S/N>3
0.90,1.0b


*   PGF = peak Gaussian factor.  Calculated using the equation:
                                   PGF =
 where W 11
                                             W   J-
                                                 10
is the peak width at  half height and W | — |  is the peak width at tenth height
                                       1
b   Resolution between the two peaks as defined by the equation:
                                            R = -L
                                                w
where t is the difference in elution times between the two peaks and W is the average peak width,
at the baseline, of the two peaks.
254

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                          Method  547
 Determination of Glyphosate in Drinking
Water by Direct-Aqueous-Injection HPLC,
         Post-Column Derivatization, and
                  Fluorescence Detection
                                EPA EMSL-Ci
                                  July 1990
           T.W. Winfield, W.J. Bashe (Technology Application, Inc.),
                   T.V. Baker (Technology Applications, Inc.)

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                                   Method 547
  Determination of Glyphosate in Drinking Water by Direct-Aqueous-
     Injection HPLC, Post-Column Derivatization, and Fluorescence
                                       Detection


 1.     SCOPE AND A PPLICA TION

 1.1    This method describes a procedure for the identification and measurement of Glyphosate (N-
       phosphonomethyl glycine) in drinking water matrices. Single laboratory accuracy and preci-
       sion data have been determined for this method.
 1.2    Glyphosate was found to rapidly decompose in chlorinated waters.1  It is therefore unlikely
       that the analyte will be evidenced in tap water except as separate glycine and N-phosphonome-
       thyl moieties, neither of which is applicable to this method.

                              Analyte         CAS No.
                              Glyphosphate    1071-83-6

 1.3    The method detection limits (MDL, defined in Section 13) for glyposate are listed in Table I.2
       The MDLs for a specific sample may differ from those listed.

 2.     SUMMARY OF METHOD

 2.1    A water sample is filtered and a 200 /*L aliquot injected into a cation exchange HPLC column.
       Separation is achieved by using an isocratic elution. After elution from the analytical column
       at 65°C,  the analyze is oxidized with calcium hypochlorite and the product (glycine) coupled
       with o-phtaladehyde-2-mercaptoethanol complex at 38° C to give a fluorophor detected by a
       fluorometer with excitation at 340 ran and detection of emission measured > 455 nm.1'3

3.     DEFINITIONS

3.1    Laboratory Duplicates (LD1 and LD2):  Two sample aliquots taken in the analytical laboratory
       and analyzed separately with identical procedures.  Analyses of LD1 and LD2 give a measure
       of the precision associated with laboratory procedures, but not with sample collection,  preser-
       vation, or storage procedures.
3.2    Field Duplicates (FD1 and FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the same throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.3    Laboratory Reagent Blank (LRB):  An aliquot of reagent water that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples. The LRB is used to determine if method
       analytes or other interferences are present in the laboratory environment, the reagents, or the
       apparatus.
                                                                                     257

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Method 547
3.4    Field Reagent Blank (FRB):  Reagent water placed in a sample container in the laboratory and
       treated as a sample in all  respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures.  The purpose of the FRB is to determine if method
       analytes or other interferences are present in the field environment.
3.5    Laboratory Performance Check Solution (LPC): A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the  performance of the instrument system
       with respect to a defined set of method criteria.
3.6    Laboratory Fortified  Blank (LFB):  An aliquot of reagent water to which known quantities of
       the method analytes are added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to determine whether the method is in control, and  whether the laboratory  is
       capable of making accurate and precise measurements at the required  method detection limit.
3.7    Laboratory Fortified  Sample Matrix (LFM): An aliquot on an environmental sample to which
       known quantities of the method analytes  are added in the laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose is  to determine whether the sample matrix contributes
       bias to the analytical  results. The background  concentrations of the analytes in the sample
       matrix must be determined in a separate  aliquot and  the measured values in the LFM corrected
       for background concentrations.
3.8    Stock  Standard Solution:  A concentrated solution containing a single  certified standard that is
       a method analyte, or  a concentrated solution of a single analyte prepared in the laboratory with
       an assayed reference  compound.  Stock standard solutions are used to prepare primary dilution
       standards.
3.9    Calibration Standard  (CAL): A solution prepared from the primary dilution standard solution
       and stock standard solutions of the internal standards and surrogate analytes.  The CAL solu-
       tions are used  to calibrate the instrument response with respect to analyte concentration.
3.10  Quality Control Sample (QCS):   A sample matrix  containing method analytes or a solution of
       method analytes in a water miscible solvent which is used to fortify reagent water or environ-
       mental samples. The QCS is obtained from a source external to the laboratory, and is used  to
       check laboratory performance with externally  prepared  test materials.

4.     INTERFERENCES
4.1    Method interferences may be caused by  contaminants in solvents, reagents, glassware, and
       other  sample processing hardware that lead to discrete artifacts and/or elevated baselines in the
       chromatograms.  All of these materials must be routinely demonstrated to be free from inter-
       ferences under the conditions of the analysis by analyzing laboratory  reagent blanks as re-
       quired by  Section 10.2.
       4.1.1     Glassware must be scrupulously cleaned."  Clean all glassware as soon as possible
                  after use  by rinsing with the last solvent used in it. This  should be followed by
                  detergent washing with hot water, and rinses with tap water and distilled water.
                  Glassware should then be drained  dry, and  heated in a laboratory oven at 400°C
                  for several hours before use.  After drying and cooling, glassware should be stored
                  in  a clean environment to prevent  any  accumulation of dust or other contaminants.
 258

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                                                                                    Method 547
       4.1.2     The use of high purity reagents and solvents helps to minimize interference prob-
                 lems.  Purification of solvents by distillation in all-glass systems may be required
                 to achieve necessary purity.
4.2    Samples may become contaminated during shipment or storage.  Field blanks must be analyzed
       to determine that sampling and storage procedures have prevented contamination.
4.3    The extent of matrix interferences may vary considerably from source to source, depending
       upon the nature and diversity of the matrix being sampled. No interferences have been
       observed in the matrices studied.
4.4    The extent of interferences that may be encountered using liquid chromatographic techniques
       has not been fully assessed. Although the HPLC conditions described allow for a unique
       resolution of the compound covered in this method,  other matrix components may interfere.

5.     SAFETY

5.1    The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
       fined. Each  chemical should be treated as a potential health hazard, and exposure to these
       chemicals should be minimized.  Each laboratory is  responsible for  maintaining awareness of
       OSHA regulations regarding safe handling of chemicals used in this method.5 A reference file
       of material data handling sheets should be made available to all personnel involved in the
       chemical analysis.

6.     APPARA TUS AND EQUIPMENT
(All specifications are suggested.  Catalog numbers are included for illustration only).
6.1    Sampling Equipment (for discrete or composite sampling)
       6.1.1     Grab sample bottle:  60mL screw cap bottles (Pierce No. 13075 or equivalent) and
                 caps equipped with a PTFE-faced silicone septa (Pierce No.  12722 or equivalent).
                 Prior to use, wash vials  and septa as described in Section 4.1.1.
6.2    Glassware
       6.2.1     Autosampler vials:  Glass, 3.7 ml,  with PTFE-lined septa  and screw caps. (Supel-
                 co, #2-3219, or equivalent)
       6.2.2     Volumetric flask:   1000  mL and 100 mL
6.3    Balance: Analytical, capable of accurately weighing 0.0001 g.
6.4    pH Meter:  Capable of measuring pH  to 0.01 units.
6.5    Filtration Apparatus
       6.5.1     Macrofiltration:  To filter mobile phase derivatization  solutions used in HPLC
                 system. Membrane filter,  0.2 /x mesh, 47 mm diameter, Nylon 66 (Alltech, #2034
                 or equivalent)
       6.5.2     Microfiltration: To filter samples prior to HPLC analysis.  Use 0.45 \t. filters
                 (Gelman Acrodisc—CR or equivalent)
       6.5.3     Helium, for degassing solutions and solvents.
                                                                                          259

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Method 547
6.6   Syringes
      6,6.1     One 250 yiL glass syringe, with blunt tip needle for manual injection.
      6.6.2     3-5 mL disposable hypodermic syringes with Luer-Lok tip.
      6.6.3     Micro syringes, various sizes.
6.7   Instrumentation: A schematic diagram of the analytical system is shown in Figure 1.
      6.7.1     A high performance liquid chromatograph (HPLC) capable of injecting 200 /xL
                 aliquots and utilizing and  isocratic pumping system with constant flow rate of 0.5
                 mL/min.
      6.7.2     Column:  250 x 4 mm, Bio-Rad, Aminex A-9. Column specifications:  K+ form,
                 packed at 65 °C, pH = 1.9.  This column  was used to generate the method perfor-
                 mance statements in Section 13. Different HPLC columns may be used if require-
                 ments described in Section 10.3 are met.  Use of guard columns is recommended.
      6.7.3     Guard Column:  C18 packing:  (Dupont, Zorbax Guard Column or equivalent).
      6.7.4     Column Oven, (Fiatron, Model CH-30 and controller, Model TC-50, or equiva-
                 lent).
      6.7.5     Post Column Reactor (PCR):  Capable  of mixing reagents into the mobile phase.
                 Reactor to be equipped with pumps to deliver 0.5 mL/min of each reagent; mixing
                 tees; two  1.0 mL delay coils, both thermostated at 38°C; and constructed using
                 PTFE tubing. (Kratos Model URS 051 and URA 200 or equivalent).
      6.7.6     Fluorescence Detector: Capable of excitation at 340 nm and detecting of emission
                 >  455 nm.  A Schoeffel  Model 970 fluorescence detector was used to generate the
                 validation data presented in this method.
      6.7.7     Data System:  A strip chart recording of the detector response  must be provided as
                 a minimum requirement.  The use of a data system to calculate retention times and
                 peak areas is recommended but not required. The system used to generate the data
                 in Table 1 is as follows.
              6.7.7.1  IBM AT computer with 640 KB of RAM, a 20 MB  hard disk, and en-
                        hanced graphics monitor or equivalent.
              6.7.7.2  Nelson Analytical Interface and Software Model 2600, Version 4.1 or
                        equivalent to perform data computations.
              6.7.7.3  Printer, Epson FX-286 or equivalent for report generation.

7.    REAGENTS AND CONSUMABLE MATERIALS

7.1   HPLC Mobile Phase
      7.1.1     Reagent Water:  Reagent  water is defined  as water of very high purity, equivalent
                 to distilled-in-glass solvents.
      7.1.2     Mobile Phase:  0.005 M KH2PO4 (0.68 gm)  in 960 mL reagent water, add 40 mL
                 HPLC grade methanol, adjust pH of solution to 1.9 with concentrated phosphoric
                 acid then  filter with 0.22  /* filter and degas with helium before use.
260

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                                                                                   Method 547
7.2   Post Column Derivatization Solutions
      7.2.1     Calcium hypochlorite solution:  Dissolve 1.36 g KH2PO4, 11.6 g NaCl and 0.4 g
                 NaOH in 500 mL deionized water.  Add 15 mg Ca(C10)2 dissolved in 50 mL
                 deionized water and dilute solution to 1000 mL with deionized water.  Filter
                 solution through 0.22 /x membrane filter and degas with helium before use.
      7.2.2  O-phthalaldehyde (OPA) reaction solution
              7.2.2.1   2-Mercaptoethanol (1 + 1):  Mix 10.0 mL of 2-mercaptoethanol and 10.0
                         mL of acetonitrile.  Cap store in hood.  (Caution - stench).
              7.2.2.2   Sodium borate (0.05N): Dissolve 19.1  g of sodium borate (Na^O, 10
                         H20) in 1.0 L of reagent water.  The sodium borate will completely dis-
                         solve at room temperature if prepared a day before use.
              7.2.2.3   OPA Reaction Solution: Dissolve 100 + 10 mg of o-phthalaldehyde (mp
                         55-58°C) in 10 mL of methanol.  Add to 1.0 L of 0.05 N sodium borate.
                         Mix, filter through 0.45 /x membrane filter,  and degas.  Add 100 n of 2-
                         mercaptoethanol (1 + 1) and mix.  Make up  fresh solution daily unless the
                         reagent solution is protected from atmospheric oxygen.  The solution can
                         be stored in glass bottles under atmospheric conditions at 4°C for up to
                         two weeks without appreciable increases in background fluorescence or
                         stored under nitrogen for indefinite periods.

      NOTE: Fluoraldehyde (Pierce Chemical), a commercially formulated OPA reaction
      solution, may be substituted for Steps 7.2.2.1 through 7.2.2.3.

7.3   Sample Preservation Reagents
      7.3.1     Sodium thiosulfate:  Granular, ACS grade or better (Fisher, S-446).
7.4   Stock Standard Solution (1.00 /* g/mL)
      7.4.1     Accurately weigh and dissolve 0.1000 g of pure glyphosate in 1000 mL of deion-
                 ized water. Larger or smaller volumes may  be used at the convenience of the
                 analyst. If compound purity  is certified  at 96%  of greater, the weight may be used
                 without correction to calculate the concentration of the stock standard.

8.    SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1   Collect samples in glass containers  (6.1.1).  Conventional sampling practices6  are to be
      followed.
8.2   Sample Preservation:  Treatment of samples to remove residual  chlorine will eliminate the
      possibility of glyphosate losses due to chlorine during storage. Chlorine is destroyed by
      adding  100 mg/L of sodium thiosulfate to the sample.
8.3   Sample Storage:  Samples should be stored at 4°C away from light and  analyzed within 2
      weeks.  A preservation study7 has demonstrated the stability of glyphosate in frozen samples
      for up to 18 months.  The analyst should verify appropriate sample holding times applicable to
      the sample under study.
                                                                                         261

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Method 547
9.     CALIBRATION

9.1    Establish liquid chromatographic operating conditions indicated in the Table 1.
9.2    Prepare a minimum of three calibration standards of glyphosate by serial dilution of the stock
       standard solution in deionized water.  One of the calibration standards should correspond to a
       glyphosate concentration near to, but above the MDL. The other concentrations should
       comprise the range of concentrations expected for the samples, or, otherwise, define the
       working range of the detector.
9.3    Analyze each calibration standard and tabulate peak area against concentration (in /xg/L) inject-
       ed.  The results may be used to prepare a calibration curve for glyphosate.
       Alternatively, if the ratio of response to concentration (response  factor) is constant over the
       working range (<  10% relative standard deviation), linearity through the origin can be
       assumed and the average ratio or response factor can be used in  place of a calibration curve.
9.4    The working calibration curve must be verified on each working day by the measurement of a
       minimum of two calibration check standards, one at the beginning and one at the end of the
       analysis day.  These check standards should be at two different concentration levels to verify
       the calibration curve.  For extended periods of analysis (greater  than 8 hr), it is strongly
       recommended that check standards be interspersed with samples  at regular intervals during the
       course  of the analyses. If the response for the analyte varies from the predicted response by
       more than ±20%, the test must be repeated using a fresh calibration standard.  If the results
       still do not agree, generate a new calibration curve.

10.   QUALITY CONTROL

10.1  Minimum quality control (QC) requirements are initial demonstration of laboratory capability,
       analysis of laboratory reagent blanks, laboratory fortified matrix samples, laboratory fortified
       blanks  and QC samples.
10.2  Laboratory Reagent Blanks: Before processing any samples, the analyst must demonstrate that
       all glassware and reagent interference are under control.  Each time a set of samples is extract-
       ed or reagents are changed, a laboratory reagent blank (LRB) must be analyzed. If within the
       retention time window of the analyte of interest the LRB produces a peak that would prevent
       the determination of that analyte, determine the source of contamination and eliminate the
       interference before processing samples.
10.3  Initial Demonstration of Capability
       10.3.1 Prepare laboratory fortified blanks (LFBs) at an analyte concentration of 250 /xg/L.
               With a syringe, add .250 mL of the stock standard (Section 7.4) to at least four - 100
               mL aliquots of reagent water and analyze each aliquot according to procedures begin-
               ning in Section 11.
       10.3.2 The glyphosate recovery (R) values determined  in 10.3.1 should be within ± 30% of
               the R values listed in Table 2 for at least three of four consecutive samples. The
               relative standard deviation (Sr) of the mean recovery (R) should be less than 30%.  If
               the analyte of interest meets the acceptance  criterion, performance is judged accept-
262

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                                                                                    Method 547
               able and sample analysis may begin.  For analytes that fail this criterion, initial
               demonstration procedures should be repeated.
       10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples via a new, unfamiliar method prior to obtaining some
               experience with it.  It is expected that as laboratory personnel gain experience with
               this method the quality of the data will improve beyond the requirements stated in
               Section 10.3.2.
10.4  The analyst is permitted to modify HPLC column, HPLC  conditions, or detectors to improve
       separations or lower analytical costs.  Each time such method modifications are made, the
       analyst must repeat the procedures in Section 10.3.
10.5  Laboratory Fortified Blanks
       10.5.1  The laboratory must analyze at least one laboratory fortified blank (LFB) sample per
               sample set (all samples analyzed within  a 24-h period). The fortified concentration of
               glyphosate in the LFB should be 10 times the MDL. Calculate accuracy as percent
               recovery (R). If R falls  outside the control limits (See  Section 10.5.2.),  the analysis
               is judged out of control, and the source  of the problem must be identified and re-
               solved before continuing analyses.
       10.5.2 Until sufficient data become available from within their own laboratory,  usually a
               minimum of results from 20 to 30  analyses, the laboratory should assess laboratory
               performance against the control limits in Section 10.3.2.  When sufficient internal
               performance data become available, develop control limits from the mean percent
               recovery (R) and SR of the percent recovery.  These data are used to establish upper
               and lower control limits  as follows:

                            Upper Control Limit = R + 3SR
                            Lower  Control Limit = R - 3SR

               After each five to ten new  recovery measurements,  new control limits should be
               calculated using only  the most recent 20-30 data points.
10.6  Laboratory Fortified Sample Matrix
       10.6.1  The laboratory must add a known fortified concentration to a minimum of  10% of the
               routine samples or one fortified sample per set, whichever is greater.  The fortified
               concentration should not be less than the background concentration of the original
               sample.  Ideally, the fortified concentration should be the same as that used for the
               laboratory fortified blank (Section  10.5).  Over time, samples from all routine samples
               sources should be fortified.
       10.6.2 Calculate the accuracy as R for the analyte, corrected for background concentrations
               measured in the original sample, and compare these values to the control limits
               established in Section 10.5.2 from  the analyses of LFBs.
       10.6.3 If recovery of any sample falls  outside the designated range, and the laboratory
               performance of the analyte is shown to be in control (Section 10.5), the recovery
               problem encountered  with the dosed sample is judged to be matrix related, not system
                                                                                           263

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Method 547
               related.  The result for the analyte in the original sample is labeled suspect/matrix to
               inform the data user that the results are suspect due to matrix effects.
 10.7  Quality Control Samples (QCS): Each quarter the laboratory should analyze at least one QCS
       (if available).  If criteria provided with the QCS are not met, corrective action should be taken
       and documented.
 10.8  The laboratory may adopt additional quality control 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.  For example, field or laboratory duplicates may be analyzed to assess
       the precision of the environmental measurements of field reagent blanks may be used to assess
       contamination of samples under site  conditions, transportation and storage.

 11.   PROCEDURE

 11.1  Sample Cleanup:  The cleanup procedure for this direct aqueous  injection HPLC method is
       limited to the filtration procedure described in Section 11.2.3. Applying only filtration, no
       interferences were evidenced in the analysis of tap  water, ground water and municipal effluent.
       If particular circumstances  demand the use of an alternative cleanup procedure, the analyst
       must demonstrate that the recovery of the analyte is within limits specified by the method.
 11.2  Analysis
       11.2.1 Table 1 details the recommended HPLC-PCR operating conditions.  An example of
              the chromathography achieved under these conditions is  shown in Figure 2.
       11.2.2 Calibrate the system daily as described in  Section 9.
       11.2.3 Filter samples using 0.45  fi  Acrodisc filters (6.5.2) and inject 200 jtL of sample into
              the HPLC-PCR system for analysis.
       11.2.4 Record resulting peak sizes  in area units.
       11.2.5 If the response for a glyphosate peak in a sample chromatogram exceeds the working
              calibration range, dilute the  sample with reagent water and reanalyze.
 11.3  Identification of Analytes
       11.3.1 Identify a sample component by comparison of its retention  time to the retention time
              of  a reference chromatogram. If the retention time of an unknown compound corre-
              sponds, within limits  (11.3.2), to the retention time of the standard,  then identification
              is considered positive.
       11.3.2 The width of the retention time window used to make identification should be based
              upon measurements of actual retention time variations of standards over the course of
              a day. Three times the standard deviation in 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.
       11.3.3 Identification requires expert judgement when sample components are not  resolved
              chromatographically.  When peaks obviously represent more then one sample compo-
              nent (i.e., broadened  peak with shoulder(s) or valley between two or more maxima),
              or  any time doubt exists over the identification of a peak in a chromatogram, appro-
              priate confirmatory techniques such as use of an alternative detector which operates on
              a physical/chemical principle different from that originally used, e.g., mass spec-

264

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                                                                                  Method 547
              trometry, or the use of an alternative separation technology, e.g., anion exchange
              chromatography, must be employed.

12.   CALCULATIONS

12.1  Determine the concentration (C) of glyphosate in the sample by direct comparison with the
      calibration curve described in Section 9, or alternatively, by means of the equation below
      derived from the calibration data.
12.2  For samples processed as part of a set where laboratory fortified blank and/or laboratory
      fortified matrix recoveries fall outside control limits in Section 10.5 &  10.6, data for the
      affected samples must be  labeled  as suspect.
                                                  -

                      where:
                       A = Area of glyphosate peak in sample
                      RF = Response factor derived from calibration data
13.  METHOD PERFORMANCE

13.1  Method Detection Limits:  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 the background level.2 The concentrations listed in Table  1 were obtained using
      reagent water, ground water and dechlorinated tap water.
13.2  Single-laboratory precision and accuracy  results at several concentrations  in drinking water
      matrices are presented in Table 2.
                                                                                         255

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Method 54 7
                                    References

 1.     Bashe, W.J., T.V. Baker, "Analysis of Glyphosate in Drinking Water by Direct Aqueous
       Injection HPLC with Post Column Derivatization," in preparation, Technology Applications,
       Inc.,  1988.

2.     Glasser, J.A., D.L. Foerst, G.M. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
       Wastewaters," Environ. Sci. Technol., 15, 1426, 1981.

3.     Cowell, J.E., "Analytical Residue Method for N-phosphonomethyl Glycine and Aminomethyl
       Phosphonic Acid in Environmental Water," Monsanto Company, Method Number 86-63-1,
       1987.

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, PA, p. 679, 1980.

5.     "OSHA Safety and Health Standards, General Industry," (29CF/?1910), Occupational Safety
       and Health Administration, OSHA 2206, (Revised, January 1976).

6.     ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for
       Sampling Water,"  American Society for Testing and Materials, Philadelphia, PA, 1986.

7.     Cowell, J.E., "Storage Stability of Glyphosate in Environmental Water," Monsanto Company,
       1988.
266

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                                                                              Method 547
Table  1.  Analytical Conditions and Method Detection Limits for Glyphosate
Matrix1
RW
GW
TW-T
Retention Time (min)
13.5
13.7
11.8
MDL,3 fjg/L
6.00
8.99
5.99
Conditions:
           Column:    250 x 4 mm, Bio-Rad, Aminex A-9 (Specifications as per Subsection 6.7)
                      thermostated at 65°C.
       Mobile Phase:    0.005 M KH2P04 - watermethanol (24:1) buffered at pH = 1.9 (Sec-
                      tion 7).

       Elution Mode:    Isocratic

         Flow Rate:    0.5 mL/min.

   Injection Volume:    200 /yL

              PCR:    Calcium Hypochlorite Flow rate  = 0.5 mL/min., OPA solution flow rate =
                      0.5 mL/min., reactor temperature =  38°C.

           Detector:    Excitation wavelength at 340 nm and detection emission at 455 nm.

1  RW = Reagent water, GW = ground water, TW-T = tap water spiked after dechlorination
  treatment.
2  All MDL data were generated from spiked samples at 25 fjg/L.
                                                                                    267

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Method 547
Table 2.  Recovery of Glyphosate In Representative Drinking Water Matrices
 Fortified
 Concentration
 (ug/U
 2500
 700
 250
 25
Matrix1

RW
GW
TW-T

RW
GW
TW-T

RW
GW
TW-T

RW
GW
TW-T
 Number
    of
Replicates

    8
    8
    8

    8
    8
    8

    8
    8
    8

    8
    8
    8
                                  Mean
                                Recovery
102
103
 99.2

101
 98.7
 96.4

 95.6
101
 98.0

 96.0
 96.0
108
                Relative
                Standard
               Deviation
 1.96
 1.25
 1.74

 2.65
 2.01
 1.80

 3.91
 1.77
 1.75

 9.07
12.3
 6.57
'RW = Reagent water, GW = Ground Water, TW-T = Tap water spiked after dechlorination
treatment.
268

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                                                                    Method 547
 Buffer
Reservoir
     HPLC
     Pump
             Hypochlorite
              Reservoir
           /  OPA   \
           \ Reservoir
   \
   /
Autoinjector
7
                                                  Fluorescence
                                                    Detector



Nelson
Analytical
Data
System

Computer

                                                   Quantitation
                                                                       52-015-30
            Figure 1. HPLC, Post-Column Reactor System
                                                                        269

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    Method 547
       uV
145.367—
130.671 —
115.975 —
101.280—
 86.584 —
 71.888 —
 57.192
          1.20  2.08
           i     i
7.25
 i
                                              13.69
                  21.72   24.35 25.60 26.78 28.10
                     i       i    i   i    i
                      5.0
         10.0
15.0
20.0
25.0
                                        Retention Time (Minutes)
                                                                                          52-015-29
                    Figure 2.  Liquid Chromatogram of Glyphosate at 250
                               (conditions are as stated in Table 1).
    270

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                     Method 548
Determination of Endothall in Drinking
    Water by Aqueous Derivatization,
              Liquid-Solid Extractionf
           and Gas Chromatography
      with Electron-Capture Detection
                 July 1990 - EPA EMSL-Ci
                           J.W. Hodgeson

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                                  Method 548
        Determination of Endothall in Drinking Water by Aqueous
   Derivatization, Liquid-Solid Extraction, and Gas Chromatography
                        with Electron-Capture Detection
1.    SCOPE AND APPLICA TION

1.1   This method covers the determination of endothall in drinking water sources and finished
      drinking water. The following analyte can be determine by this method:


                      Analyte                    CAS No.
                       Endothall                    145-73-3

1.2   This is a gas chromatographic (GC) method applicable to the determination of the compound
      listed above. When this method is used to analyze unfamiliar samples, compound identifica-
      tion should be supported by at least one additional qualitative technique.  A gas chromato-
      graph/mass  spectrometer (GC/MS) may be used for the qualitative confirmation of results for
      Endothall using the extract produced by this method.
1.3   The method detection limit' (MDL, defined in Section 12) for endothall is listed in Table 1.
      The MDL for a specific sample may differ from the listed value, depending upon the nature of
      interferences in the sample matrix and the amount of sample used in the procedure.
1.4   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.
1.5   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 11.

2.    SUMMARY OF METHOD

2.1   A 5.0 mL volume of liquid sample is placed in a Kuderna-Danish tube and the volume is
      reduced to less than 0.75 mL using a heating block.  The tube is charged with glacial acetic
      acid and sodium acetate, followed by a solution of the derivatization reagent, pentafluoro-
      phenylhydrazine (PFPH), in glacial acetic acid.  After heating at 150°C for 90 minutes the
      derivative is extracted by a solid sorbent from the reaction solution, followed by elution with
      5.0 mL of methyl-tert-butyl ether (MTBE).  The MTBE extract is analyzed by gas chromatog-
      raphy with electron capture detection (GC/ECD).
                                                                                    273

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Method 548
3.     DEFINITIONS

3.1    Internal  Standard:  A pure analyte(s) added to a solution in known amount(s) and used to
       measure the relative responses of other method analytes and surrogates that are components of
       the same solution.  The internal standard must be analyte that is not a sample component.
3.2    Surrogate Analyte:  A pure analyte(s), which is extremely unlikely  to be found in any sample,
       and which is added to a sample aliquot in known amount(s) before extraction and is measured
       with the same procedures used  to measure other sample components. The purpose of a
       surrogate analyte is to monitor method performance with  each sample.
3.3    Laboratory  Duplicates (LDl and LD2):  Two sample aliquots taken in the analytical laboratory
       and analyzed separately with identical procedures.  Analyses of LDl and  LD2  give a measure
       of the precision associated with laboratory procedures,  but not with sample collection, preser-
       vation, or storage procedures.
3.4    Field Duplicates (FD1 and FD2):  Two separate samples  collected at the same time and place
       under identical circumstances and treated exactly the same throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory  Reagent Blank (LRB):  An aliquot of reagent  water that is treated exactly as a
       sample including exposure to all glassware, equipment, solvents, reagents, internal standards,
       and surrogates that are used with other samples.  The LRB is used to determine if method
       analytes  or  other interferences are  present in the laboratory environment,  the reagents, or the
       apparatus.
3.6    Field Reagent Blank (FRB):  Reagent water placed in a sample container  in the laboratory and
       treated as a sample in all respects, including exposure to sampling site conditions, storage,
       preservation and all analytical procedures.  The purpose of the FRB is to  determine if method
       analytes  or  other interferences are  present in the field environment.
3.7    Laboratory  Performance Check Solution (LPC):  A solution of method analytes, surrogate
       compounds, and internal standards used to evaluate the performance of the instrument system
       with respect to a defined set of method criteria.
3.8    Laboratory  Fortified Blank (LFB):  An aliquot of reagent water to which  known quantities of
       the method  analytes are added in the laboratory.  The LFB is analyzed exactly like a sample,
       and its purpose is to determine whether the methodology is in control, and whether the labora-
       tory is capable of making  accurate and precise measurements at the required method detection
       limit.
3.9    Laboratory  Fortified Sample Matrix (LFM):  An aliquot of an environmental sample to  which
       known quantities of the method analytes are added in the  laboratory.  The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample  matrix contributes
       bias to the analytical results.  The  background concentrations of the analytes in the sample
       matrix must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock Standard Solution:  A concentrated solution containing a single certified standard  that is
       a  method analyte, or a concentrated solution of a single analyte prepared in the laboratory with
274

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                                                                                     Method 548
       an assayed reference compound. Stock standard solutions are used to prepare primary dilution
       standards.
3.11  Primary Dilution Standard Solution:   A solution of several  analytes prepared  in the laboratory
       from stock standard solutions and diluted as needed to prepare calibration solutions and other
       needed analyte solutions.
3.12  Calibration Standard (CAL):  A solution prepared from the  primary dilution standard solution
       and stock standard solutions of the  internal standards and surrogate analytes.   The CAL solu-
       tions are used to calibrate the instrument response with respect to analyte concentration.
3.13  Quality Control Sample (QCS): A sample matrix  containing method analytes or a solution of
       method analytes  in a water  miscible solvent which  is used to fortify reagent water or environ-
       mental samples.  The QCS  is obtained from a source external to the laboratory, and is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    Method interference may be caused by contaminants in solvents, reagents, glassware, and
       other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the
       chromatograms.  All of these materials must be routinely demonstrated to be free from inter-
       ferences under the conditions of the analysis  by running laboratory reagent blanks as described
       in Section 10.2.
       4.1.1   Glassware must be scrupulously clean.2  Clean all glassware as soon as possible after
               used by rinsing with the last solvent used in it.  This should be followed by detergent
               washing with hot water, and rinses with tap water and distilled water.  It should then
               be drained dry, and heated in a laboratory oven at 40°C for several hours before use.
               Solvent  rinses with methanol  may be substituted for the oven heating.  After drying
               and cooling, glassware should be stored in a clean environment to prevent any accu-
               mulation of dust or other contaminants.
       4.1.2   The use of high purity regents and solvents helps to minimize interference problems.
               Purification of solvents by distillation in all-glass systems may be required.
4.2    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 matrix being sampled.  If significant interferences occur in
       subsequent samples, some additional cleanup may be necessary to achieve the  MDL listed in
       Table 1.
4.3    The extent of interferences  that may be encountered using gas chromatographic techniques has
       not been  fully assessed.  Although the GC conditions described allow for a unique resolution
       of the specific compound covered by this method, other  matrix components may interfere.

5.     SAFETY

5.1    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-
                                                                                            275

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Method 548
      ness file of OSHA regulations regarding the safe handling of the chemical 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. Additionally references to laboratory safety are
      available.

6.    APPARA TUS AND MA TERIALS
6.1   Sampling Equipment (for discrete or composite sampling).
      6.1.1   Grab sample bottle: Amber glass fitted with screw caps lined with Teflon.  If amber
               bottles are not available, protect samples from light. The container must be washed,
               rinsed with methanol, and dried before use to minimize contamination
6.2   Glassware
      6.2.1   Volumetric flasks:  5 mL, 25 mL
      6.2.2   Vials:  Glass,  1 mL, with Teflon-lined caps
      6.2.3   Glass syringes, 250 jtL, 500 /*L
      6.2.4   Pipets:  1 mL, 4 mL
6.3   Balance:  Analytical, capable of accurately  weighing 0.0001 g.
6.4   Solid Sorbent Cartridges: CIS, Baker 7020-6 or equivalent
6.5   Vacuum manifold for extraction using solid sorbent cartridges:  Supelco 5-7030 or equivalent
6.6   Kuderna-Danish (K-D) concentrator tubes:  10 or 25 mL graduated (Kontes K-570050-1025 or
      K-570050-2525)
      6.6.1   Snyder column, Kuderna-Danish:  2-ball micro (Kontes, K-569001-0219)
6.7   Tube heater for 25 mL K-D tubes:  Kontes
6.8   Boiling chips: Carborundum, #12 granules (Arthur H. Thomas Co. #1590-033 or equivalent).
      Heat at 400°C for 30 minutes prior to use.  Cool and stored in dessicator.
6.9   Gas chromatographic system capable of temperature programming
      6.9.1   Autosampler
      6.9.2   Electron capture detector
      6.9.3   Column 1: Supelco SPB-5, 0.25 mm x 30 m or equivalent
               Column 2: J&W DB-1, 0.32 mm  x 30 mm or equivalent
      6.9.4   Strip-chart recorder compatible with detector. Use of a data system with printer for
               measuring and recording peak areas and retention times is recommended.

7.    REAGENTS AND SOLUTIONS
7.1   Reagent Water:  reagent water is defined as a water of very high purity, equivalent to distilled
      in glass solvents
7.2   Pentafluorophenylhydrazine(PFPH):  Aldrich
7.3   Sodium Acetate:  Anhydrous, Aldrich
7.4   Sodium Thiosulfate: Baker
276

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                                                                                   Method 548
7.5    Acetic Acid:  Glacial, Baker
7.6    Methyl-Tert-Butyl Ether (MTBE):  Distilled in glass (Burdick & Jackson)
7.7    Endothall-PFPH Derivative:  See Appendix for synthesis procedure
7.8    EndosulfanI: USEPA Repository
7.9    Endothall, monohydrate:  USEPA Repository
7.10  Stock Standard Solutions
       7.10.1    Endothall:  10 ^g/mL in reagent water
       7.10.2    Endothall:  50 ^g/mL in reagent water
       7.10.3    Stock standard solutions must be replaced after six months, or sooner, if com-
                 parison with check standards indicates a problem.
7.11  Reaction Solutions
       7.11.1 PFPH solution:  4 mg/mL in glacial acetic acid
       7.11.2 Internal standard stock solution:  10  /tg/mL endosulfan I in MTBE

8.     SAMPLE COLLECTION, PRESERVATION, AND HANDLING
8.1    Grab samples must be collected in glass containers.  Conventional sampling practices should
       be followed, except that the bottle must not be prewashed with sample before collection.
       Composite samples should be collected  in refrigerated glass containers in accordance with the
       requirements of the program.  Automatic sampling equipment must be as free as possible of
       Tygon tubing and  other potential sources of contamination.
8.2    The samples must be iced or refrigerated at 4°C from the time of collection until
       derivatization.  The analyte measured here is not known to be light sensitive, but excessive
       exposure to light and heat should be avoided.
8.3    Some samples are likely to be biologically  active and the stability of samples upon storage will
       be different for each matrix.  All samples should be derivatized within 7 days of collection,
       and analysis completed within 1 day of derivatization.  If these criteria are not met, the analyst
       must demonstrate the stability of the stored sample by performing suitable holding time
       studies.

9.     CALIBRATION
9.1    Establish gas chromatographic operating parameters to produce  a retention time equivalent to
       that indicated in Table  1.  The chromatographic system can be calibrated using the internal
       standard technique (Section 9.2)
       9.1.1  Due to the complex nature of the sample chromatogram, the analyst should periodical-
              ly inject a solution containing only pure endothall-PFPH (See Appendix) to verify the
              retention time  of the derivative.
9.2    Internal Standard Calibration Procedure:
       9.2.1  Use 250 and 500 yiL syringes to add sufficient quantities of 7.10.1 or 7.10.2 stock
              solutions to reagent water in 25 mL volumetric flasks to produce endothall standard
                                                                                          277

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Method 548
               solutions at the following concentrations in /*g/L:  500 (250 ^L of 7.10.2 stock), 200
               (100 jiiL of 7.10.2 stock),  100 (50 /xL of 7.10.2 stock) and 50(125 jiL of 7.10.1
               stock).
       9.2.2   Process each  standard as per Section 11.2.  It is recommended that triplicate samples
               of each standard be processed.
       9.2.3   Before analyzing matrix samples, the analyst must process a series of calibration
               standards to validate elution patterns and the absence of interferences from reagents.
       9.2.4   Analyze each calibration standard and tabulate the ratio of the peak area of the en-
               dothall-PFPH derivative versus that  of the internal standard against endothall con-
               centration.  The results may be used to prepare a calibration curve for endothall.
       9.2.5   The working  calibration curve must  be verified on each working day by processing
               and analyzing one or more calibration standards.  If the response varies from the
               previous response by more than ±20%, the test must be repeated using a fresh
               calibration standard. Should the retest fail, a new calibration curve must be  gene-
               rated.

10.   QUALITY CONTROL

10.1   Each laboratory that uses this method is required to operate a formal quality control (QC)
       program.  The minimum QC requirements are initial demonstration of laboratory capability,
       analysis of laboratory  reagent blanks, laboratory fortified blanks, laboratory fortified matrix
       samples and QC check standards.
10.2  Laboratory Reagent Blanks.  Before processing any samples, the analyst must demonstrate that
       all glassware and reagents interferences are under control.  Each time a set of samples is
       analyzed or reagent are changed, a method blank must be analyzed.  For this method, the
       method blank is filtered  reagent water.  If within the retention time window of an analyte of
       interest, the method blank produces a peak which prevents the measurement of that analyte,
       determine the source of  contamination and eliminate the interference before processing sam-
       ples.
10.3  Initial Demonstration of Capability
       10.3.1  Select a representative fortified concentration (about 10 times MDL) for endothall.
               Prepare a concentrate (in reagent water) containing the analyte at 10 times the selected
               concentration.  Using a pipet, add 1.00 mL of the concentrate to each of at least  four
               10 mL aliquots  of reagent water and analyze each aliquot according to procedures
               beginning in Section 11.
       10.3.2 The recovery value should for at least three out of four consecutively analyzed sam-
               ples fall in the range of R ± 30% (or within R ± 3SR, if broader) using the  values
               for R and SR for reagent water. If the recovery value meets the acceptance criteria,
               performance is  acceptable  and  sample analysis may begin. If the recovery value  fails
               these criteria, initial demonstration of capability should be repeated.
       10.3.3 The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples by a new, unfamiliar method prior to evidencing a basal
               level of skill at  performing the technique.  It is expected that as laboratory personnel
278

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                                                                                     Method 543
               gain experience with this method the quality of the data will improve beyond the
               requirements stated in Section 10.3.2.
10.4  The analyst is permitted to modify GC columns, GC conditions, or detectors to improve
       separations or lower analytical costs. Each time such method modifications are made, the
       analyst must repeat the procedures in Section 10.3.
10.5  Assessing the Internal Standard:  In  using the IS calibration procedure, the analyst is expected
       to monitor the IS response (peak area or peak height) of all samples during each analysis day.
       The IS response for any sample chromatogram  should not deviate from the calibration standard
       IS response by more than 30%.
       10.5.1  If a deviation of greater than 30% is encountered for a sample, re-inject the extract.
               10.5.1.1  If acceptable IS  response is achieved for the re-injected extract, then report
                         the results for that sample.
               10.5.1.2 If a deviation of greater than 30% is obtained for the re-injected extract,
                         analysis of the sample should be repeated beginning with Section 11,
                         provided the sample  is still  available.  Otherwise, report results obtained
                         from the re-injected extract, but annotate  as suspect.
       10.5.2 If consecutive  samples fail the IS response acceptance criterion, immediately analyze a
               calibration check standard.
               10.5.2.1  If the check standard provides a response factor (RF) within 20% of the
                         predicated value, then follow  procedures  itemized in Section 10.5.1 for
                         each sample failing the IS response criterion.
               10.5.2.2 If the check standard provides a response factor (RF) with deviates more
                         than 20% of the predicted value, then the analyst must recalibrate, as
                         specified in Section 9.2.
10.6  Assessing Laboratory Performance
       10.6.1  The laboratory must analyze at least one  LFB per sample set (all  samples analyzed
               within a 24 hour period). The fortifying concentration in the LFB should be 10 times
               the MDL.  Calculate accuracy as percent recovery (X,). If the recovery falls outside
               the control limits (See Section 10.6.2), the system is judged out of control, and the
               source of the problem must be identified  and resolved before continuing analyses.
       10.6.2 Until sufficient LFB data become available, usually a minimum of results from 20 to
               30 analyses,  the laboratory should assess its performance against the control limits
               described in Section 10.3.2. When sufficient laboratory performance data becomes
               available, develop control limits from the mean percent recovery (X) and standard
               deviation (S) of the percent  recovery.  These data are used to establish upper and
               lower control limits as follows:

                            Upper  Control Limit =  X  + 3S
                            Lower Control Limit  =  X - 3S

               After each group of five to  ten new  recovery measurements, control limits  should be
               recalculated using only the most recent 20 to 30 data points.
                                                                                            279

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Method 548
       10.6.3 It is recommended that the laboratory periodically determine and document its detec-
               tion limit capabilities for endothall.
       10.6.4 Each quarter the laboratory should analyze QCS (if available).  If criteria provided
               with the QCS are not met, corrective action should be taken and  documented.
10.7  Assessing Analyte Recovery
       10.7.1 The laboratory must add a known fortified concentration to a minimum of 10% of the
               routine samples or one fortified sample per set, whichever is greater.  The fortified
               concentration should not be less than the background concentration of the sample
               selected for spiking. The fortified concentration should be the same as that used for
               the LFB (Section 10.6).  Over time, samples from all routine sample sources should
               be fortified.
       10.7.2 Calculate the percent recovery (R,) for each analyte, corrected for background con-
               centrations measured in the unfortified sample, and compare these values to the
               control limits established in Section 10.6.2 for the analyses of LFBs.
       10.7.3 If the recovery of any analyte falls outside the designated range,  and the laboratory
               performance for that analyte is shown to be in control (Section 10.6), the recovery
               problem encountered with that dosed sample is judged to be matrix related, not system
               related.  The result for that analyte in the  unfortified sample must be labelled sus-
               pect/matrix to inform the data user that the results are suspect due to matrix effects.

7 7.   PROCEDURE

11.1  Cleanup and Separation: Cleanup procedures may not be necessary for a relatively clean
       samples matrix.  If particular circumstances demand the use of an alternative cleanup proce-
       dure, the analyst must demonstrate that the recovery of endothall is within the limits specified
       by the method.
       11.1.1 If the sample is not clean, or the complexity is unknown, the entire sample should be
               centrifuged at 2500 rpm for 10 minutes.  The supernatant is decanted from the centri-
               fuge bottle and passed through glass fiber filter paper into a container which can be
               tightly sealed.
       11.1.2 Store all samples at 4°C.
11.2  Sample Extraction and Analysis
       11.2.1 Measure out a 5.0 mL aliquot of the sample and place it in a 25 mL K-D tube.  Add
               boiling chips.
       11.2.2 Place on tube heater at maximum setting and concentrate sample to less than 0.5 mL.
       11.2.3 Add 4 mL glacial acetic acid, 200 mg sodium acetate and  1 mL of glacial acetic acid
               containing 4 mg PFPH.  Use glass stirring rod to break-up the sodium acetate solid.
               Place a Micro Snyder column on each K-D tube.
       11.2.4 Heat at  !50°C for 90 minutes.
       11.2.5 Dilute the reaction mixture with 50 mL reagent water.  Wash the residue in the tube
               into the aqueous solution.
280

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                                                                                   Method 548
      11.2.6 Assemble the vacuum manifold.  Rinse the solid sorbent cartridge by passing 5 mL of
              reagent water though the cartridge. Discard the water. Extract the aqueous sample
              from 12.5 by passing the sample through the solid sorbent cartridge at a rate of
              5-6 mL per minute.
      11.2.7 Wash the cartridge with 5 mL reagent water.  Elute the cartridge with two 2 mL
              aliquots of MTBE.  Combine the eluates with  .05 mL of the internal standard stock
              solution (7.11.2) and dilute to 5 ml in a volumetric flask with MTBE.
      11.2.8 Analyze the eluates by GC/ECD using conditions described in Table  1. This table
              includes the retention time and MDL  that were obtained under these conditions.
              Sample chromatograms of an endothall standard and a LRB both with internal stan-
              dard are  represented in Figures  1 and 2.  Other columns, chromatographic conditions,
              or detectors may be used if the requirements of Section 10.3 are met.
11.3 Identification of Analytes
      11.3.1 Identify a sample component by comparison of its retention time to the retention time
              of a reference chromatogram.  If the retention time of an unknown compound corre-
              sponds, within limits, to the retention time of a standard compound, then identification
              is considered positive.  However, positive identifications should be confirmed by
              retention time comparisons  on the second GC column, or by using GC/MS.
      11.3.2 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.
      11.3.3 Identification requires expert judgement when sample components are not resolved
              chromatographically, that is, when GC peaks obviously represent more than one
              sample component (i.e., broadened peak with shoulder(s) or valley between two or
              more maxima, or any time  doubt exists over the identification of a peak as a chroma-
              togram, appropriate techniques such as use of an alternative detector which operates
              on a chemical/physical principle different from that originally used, e.g., mass  spec-
              trometry, or the use of a second chromatography column must be  used.
11.4 If the peak area exceeds the linear range of the calibration curve, a smaller sample volume
      should be used. Alternatively, the final solution may be diluted with MTBE and reanalyzed.
11.5 If the peak area measurement is prevented by  the presence of interferences, further cleanup is
      required.

12.  CALCULATIONS

12.1 Determine the peak area ratio for endothall  in the injected sample.
      12.1.1 Calculate the concentration of endothall injected using the calibration curve in Section
              9.2. The concentration in a liquid sample can be calculated from  Equation 1:
                                                                                          281

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Method 548
                                          Equation 1

                                Concentration
                                                         (VS)
                       where:
                        A  = Concentration of endothall in extract, in fig/L
                       VF  = Final volume of MTBE, in mL
                       VS  = Sample volume, in mL
12.2  Report results as micrograms per liter. When duplicate and fortified samples are analyzed,
       report all data obtained with the sample results.
12.3  For samples processed as part of a set where the laboratory fortified sample recovery falls
       outside of the control limits established in Section 10.3, data must be labeled as suspect.

13.   METHOD PERFORMANCE

13.1  Method Detection Limits:  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 the background
       level.  The estimated MDL concentration listed in Table 1 was obtained using reagent water.
       Similar results were achieved using representative matrices.
13.2  This method has not been tested for linearity of recovery from fortified reagent water.
13.3  In a single laboratory using dechlorinated tap and reagent water fortified matrices, the average
       recoveries presented in Table 2 were obtained.  The standard deviation of the percent recovery
       is also included  in Table 2.
282

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                                                                       Method 548
                             References
40 CFR Part 136, Appendix B.

ASTM Annual Book of Standards, Part 31, D3694-78.  "Standard Practices for Preparation of
Sample Containers and for Preservation of Organic Constituents", American Society for
Testing and Materials, Philadelphia, PA.
                                                                             283

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Method 548
Table 1.  Gas Chromatography Conditions and Method Detection Limits


 Analyte                     Ret. Time (mm.)             MDL (pg/L)

 Endothall                     42.3                        11.5

GC conditions: 0.25 mm x 30 m SPB-5 column; 2 //L injection; (Split, 40:1) hold one minute at
60°C, program to 300°C at 4°C/minutes, hold at 300°C for 15 minutes.
284

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                                                                             Method 548
Table 2.  Single Operator Accuracy and Precision
Analyte
Endothall

Matrix
Type
Reagent Water
Dechlorinated Tap
Water
Average
Percent
Recovery
120
108
84.0
94.0
Standard
Deviation
{percent}
25.3
15.3
13.8
13.3
Fortified
Cone.
(fjg/L)
15
150
15
150
Number of
Analyses
8
8
8
8
100 mg/L sodium thiosulfate (Na2S203) added to prior to fortifying with endothall
                                                                                  285

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Method 548
                                     Appendix
        Preparation of Endothall-Pentafluorophenylhydrazine

1.    Prepare solution A of endothall by dissolving 0.204 g of endothall monohydrate (1.0 mmol) in
      14 mL of methylene chloride and  3.6 mL of dry tetrahydrofuran (THF).

2.    Prepare solutions B of dicyclohexylcarbodiimide (DCC) by dissolving 0.206 g (1.0 mmol) in
      3.4 mL of dry THF.

3.    Mix solution A and B and cover with a watchglass.  (Note:  a white precipitate will form in 3
      to 5 minutes).

4.    Gently stir the mixture from Step  3 with  a magnetic stirrer for 4.5 hours at ambient tempera-
      ture.

5.    Prepare solution C by dissolving 0.206 g of DCC and 0.198 g of pentafluorophenylhydrazine
      (PFPH) in 18 mL of dry THF.

6.    Mix solution C with the mixture from step 4, cover with a watchglass and stir the mixture
      overnight (16 hours) at ambient temperature.

7.    Filter the mixture and dry the filtrate under reduced pressure to yield a  beige powder.

8.    Recrystallize the beige powder with  20 mL of warm (40°C) methanol: H2O (8:2 v/v).

9.    Filter the solution from Step 8 to remove the insoluble material.

10.   Allow the filtrate from Step 9 to cool to room temperature.  A precipitate will form immedi-
      ately upon cooling.

11.   Filter and wash the precipitate formed in Step 10 with two 1 mL portions of cold methanol:
      H2O (8:2).  Save the filtrate.

12.   Allow the filtrate from Step 11  to stand overnight covered with a watchglass at ambient
      temperature. A precipitate will form on  standing.

13.   Filter and wash the precipitate from Step 12 with two 1 mL portions of cold methanol: H20
      (8:2).

14.   Recrystallize the off white precipitate from Step 13 with 20 mL of warm methanol:  H20
      (8:2).  Filter the warm solution and  allow the filtrate to cool, producing a white, crystalline
      precipitate.

15.   Filter the white precipitate from Step 14, wash  with two 1 mL portions of cold methanol: H20
      (8:2) and dry under vacuum.

286

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                                                                                       Method 548
16.    Determine the melting point of the precipitate of Step 15.  The melting point of the endothall-
       pentafluorophenylhydrazine derivative is 201.OC.  If the melting point of the precipitate is not
       within 1.0 C of this melting point, recrystallize again as per Step 14-15.
                                                                                             287

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-------
                                                                                Method 548
   Eo>
                         Endosulfan I (40.42)
   600 —
0)
c
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Q.
(/)
0>
cc
2
o
s
0
Q


500 —



400 —
300 —


200 —
100 —












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                                       Elution Time (Minutes)
                                                                                   52-015-34
          Figure 2. Representative Chromatogram of a Laboratory Reagent Blank
                                                                                       289

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                    Method  548.1
Determination of Endothall in Drinking
    Water by Ion-Exchange Extraction,
         Acidic Methanol Methylation
            and Gas Chromatography/
                   Mass Spectrometry
                Revision 1.0 - EPA EMSL-Ci
                            August 1992
                           Jimmie W. Hodgeson
              Jeffrey Collins (Technology Applications, Inc.)
               W. J. Bashe (Technology Applications, Inc.)

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                                 Method 548.1
              Determination of Endothall in Drinking Water by
          Ion Exchange Extraction, Acidic Methanol Methylation
                and Gas Chromatography/Mass Spectrometry


 1.    SCOPE AND APPLICA TION

 1.1   This method is for the identification and simultaneous measurement of endothall in drinking
      water sources and finished drinking water.  The following analyte can be determined by this
      method:


                   Analyte                                    CAS No.
                   Endothall                                    145-73-3

 1.2   This is a gas chromatographic/mass spectrometric (GC/MS) method.  However, a flame
      ionization detector (FID) may be utilized for the determination, but must be supported by an
      additional analysis using a confirmatory gas chromatographic column.
 1.3   The method detection limit1 (MDL, defined in Sect.  13) for endothall is listed in Table 1 for
      both GC/MS and FID.  The MDL may differ from the listed value depending upon the nature
      of interferences in the sample matrix.  In particular, water sources containing high levels of
      dissolved calcium, magnesium and sulfate may require sample dilution before extraction to
      obtain adequate endothall recovery. Guidelines (Sect. 4.2 and Sect.  11.2.1) are provided on
      levels of these ions above which dilution is recommended, as well as appropriate dilution
      factors.
 1.4   In this ion exchange liquid-solid extraction  procedure, endothall may be esterified directly in
      the elution solvent, acidic methanol.
 1.5   The method performance data provided in this method were obtained using both a GC/MS
      system and a gas chromatograph with a flame ionization detector (FID). Modern GC/MS
      instruments have sensitivities at least equivalent to the FID. If the analyst has access to a
      GC/MS system meeting the specifications described in Sect. 6.10, it should be as the primary
      means of identification and measurement.

2.    SUMMARY OF METHOD

2.1   Liquid-solid extraction (LSE) cartridges containing an intermediate strength, primarily tertiary
      amine anion exchanger are mounted on a vacuum manifold and conditioned with appropriate
      solvents. LSE disks may be used instead of cartridges of all quality control criteria specified
      in Sect.  9 are met. A 100-mL sample is extracted and the analyte is eluted with 8-mL of
      acidic methanol.  After addition of a small  volume of methylene chloride as a co-solvent, the
      dimethyl ester of endothall is formed within 30 min with modest heating (50°C).  After
      addition of salted reagent water, the ester is partitioned into 8-10 mL of methylene chloride.
      The extract volume is reduced to 1 mL with nitrogen purge for a concentration  factor of 100.
      The extract is analyzed by GC/MS or GC/FID with a megabore capillary column.

                                                                                     293

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 Method 548. 1
 3.    DEFINITIONS

 3.1   Internal Standard (IS):  A pure analyte(s) added to a sample, extract, or standard solution in
       known amount(s) and used to measure the relative responses of other method analytes and
       surrogates that are components of the same sample or solution.  The internal standard must be
       an analyte that is not a sample component.
 3.2   Surrogate Analyte (SA):  A pure analyte(s), which is extremely unlikely to be found in any
       sample, and which is added to a sample aliquot in known amount(s) before extraction or other
       processing and is measured with the same procedures used to measure other sample com-
       ponents.  The purpose of the SA  is to monitor method performance  with each sample.
 3.3   Laboratory Duplicates (LDl  and  LD2): Two aliquots of the same sample taken in the labora-
       tory and analyzed separately with  identical procedures.  Analyses of LDl and LD2 indicate the
       precision associated with laboratory procedures, but not with sample collection, preservation,
       or storage procedures.
 3.4   Field Duplicates  (FD1 and FD2):  Two separate samples collected at the same time and place
       under identical circumstances and  treated exactly the same throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
 3.5   Laboratory Reagent Blank (LRB):  An aliquot of reagent water or other blank matrix that is
       treated exactly as a sample including exposure to all glassware,  equipment, solvents, reagents,
       internal standards, and surrogates  that are used with other samples.  The LRB is used to
       determine if method analytes or other interferences are present in the laboratory environment,
       the reagents, or the apparatus.
 3.6   Field Reagent Blank (FRB):  An aliquot of reagent water or other blank matrix that is placed
       in a sample container in  the labora-tory and treated as a sample  in all respects, including
       shipment to the sampling site, exposure to sampling site conditions,  storage, preservation, and
       all analytical procedures. The purpose of the FRB is to determine if method analytes or other
       interferences are  present in the field environment.
 3.7   Instrument Performance  Check Solution (IPC):  A solution of one or more method analytes,
       surrogates,  internal standards, or other test substances used to evaluate the performance of the
       instrument system with respect to a defined set of method criteria.
 3.8   Laboratory  Fortified Blank (LFB):  An aliquot of reagent water  or other blank matrix to which
       known quantities of the method analytes are added in the laboratory.  The LFB is analyzed
       exactly like a sample,  and its purpose is to determine whether the methodology is in control,
       and whether the laboratory is capable of making accurate and precise measurements.
 3.9   Laboratory  Fortified Sample Matrix (LFM): An aliquot of an environmental sample to which
       known quantities of the method analytes are added in the laboratory.  The LFM is analyzed
       exactly like a sample,  and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the  analytes  in the  sample
       matrix  must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
254

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                                                                                    Method 548.1
3.10  Stock Standard Solution (SSS):  A concentrated solution containing one or more method
       analytes prepared in the laboratory using assayed reference materials or purchased from a
       reputable commercial source.
3.11  Primary Dilution Standard Solution (PDS):  A solution of several analytes prepared in the
       laboratory from stock standard solutions and diluted as needed to prepare calibration solutions
       and other needed analyte solutions.
3.12  Calibration Standard (CAL): A solution prepared from the primary dilution standard solution
       or stock standard solutions and the internal standards and surrogate analytes.  The CAL
       solutions are used to calibrate the instrument response with respect to analyte concentration.
3.13  Quality Control Sample (QCS):  A solution of method analytes in known concentrations which
       is used to fortify an aliquot of LRB or sample matrix.  The QCS is obtained from a source
       external to  the laboratory and different from the source  of calibration standards. It is used to
       check laboratory performance with externally prepared test materials.

4.     INTERFERENCES

4.1    Method interference may be caused by contaminants in solvents, reagents, glassware, and
       other sample processing hardware  that lead to discrete artifacts and/or elevated  baselines  in the
       chromatograms. All of these materials must be routinely demonstrated to be free from inter-
       ferences under the analytical conditions by analyzing laboratory reagent blanks  as described in
       Sect. 9.2.
       4.1.1   Glassware must be scrupulously cleaned2 as soon as possible after use by rinsing with
               the last solvent used in it.  This should be followed by detergent washing with hot
               water,  and rinses with tap water and distilled water.  It should then be  drained dry,
               and heated in a laboratory oven at 400°C for several hours before use.  Solvent  rinses
               with methanol may be substituted for the oven heating. After drying and cooling,
               glassware should be stored in a clean environment to prevent any accumulation of dust
               or other contaminants.
       4.1.2   The use of high purity reagents and solvents is  absolutely necessary to  minimize
               interference problems. Purification of solvents by distillation in all-glass systems
               immediately prior to use may be necessary.
4.2    The major potential interferences in this ion-exchange procedure are other naturally occurring
       ions in water sources, namely, dissolved calcium, magnesium and sulfate.  These are the only
       ions thus far demonstrated to be interferences when present at  concentrations possibly occur-
       ring in drinking water sources.  For example, the sources identified in Tables 3 and 4 con-
       tained elevated concentrations of these ions and reduced recoveries were observed.  Sulfate is
       an effective counter ion, and displaces endothall from the column when present at high concen-
       trations.  On the other hand, both calcium and magnesium complex the endothall anion, which
       then is no longer available in ionic form for ion-exchange extraction.  Table 4 illustrates  that
       sample dilution or the addition of ethylenediamine tetraacetic acid for complexing the cations,
       or a combination of the two, may be used.  Figure 1 illustrates quantitatively the separate
       effects  of these  ions on recovery.
4.3    The extent of interferences that may be encountered using this  method has not been fully
       assessed. Although the GC conditions described allow  for a unique resolution of endothall,

                                                                                            295

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Method 548.1
       other matrix components may interfere. Matrix interferences may be caused by contaminants
       that are coextracted from the sample.  Matrix interferences will vary considerably from source
       to source, depending on the nature of the matrix being sampled.  A distinct advantage of this
       method is that the anion exchange cartridge provides an effective clean-up mechanism for
       many potential organic matrix interferences. Many  neutral and basic organics retained by the
       column are removed by the methanol  wash step of Sect.  11.2.3.  The most probable matrix
       interferences are other organic acids or phenols retained  by the column.  For the cartridge to
       effectively serve for both sample clean-up and analyte extraction, it is critical that the condi-
       tioning steps described in Sect. 11.2.1 be followed exactly.

5.     SAFETY

5.1    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 minimized.  The laboratory  is
       responsible for maintaining a current awareness file  of OSHA regulations regarding the safe
       handling of the chemical 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.
       Additionally references to laboratory safety are available.3"5

6.     EQUIPMENT AND SUPPLIES

6.1    Sampling Equipment (for discrete or composite sampling). Amber glass bottles (250 mL or
       larger) fitted with screw caps lined with Teflon.  If amber bottles are not available,  protect
       samples from light.  The container must be washed, rinsed with methanol,  and dried before
       use to minimize contamination.
6.2    Separator Funnels: 125 mL, with Teflon stopcocks, ground  glass or Teflon stoppers.
6.3    Screw Cap:  125 x  13 mm, culture tubes. Screw caps should have Teflon liners.
6.4    Graduated 15 mL centrifuge tubes with #13 ground  glass stoppers
6.5    Pasteur Pipets:  Glass, disposable 5-3/4" length
6.6    Balance:  Analytical, capable of weighing to .0001 g.
6.7    Six or twelve position analytical concentrator (Organomation, N-EVAP model # 111/6917 or
       equivalent).
6.8    pH Meter
6.9    Gas Chromatograph:  Analytical system complete with GC suitable for flame ionization
       detection, split/splitless capillary injection temperature programming, and all required
       accessories including syringes, analytical columns, gases and strip chart recorder.  A data
       system is recommended for measuring peak areas. An auto  injector is recommended for
       improved precision of analysis.
6.10  Gas Chromatograph/mass Spectrometer/data System (GC/MS/DS):
       6.10.1 The GC must be capable of temperature programming and be equipped for
               split/splitless or on-column capillary injection.   The injection tube liner should be
296

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                                                                                 Method 548.1
              quartz and about 3 mm in diameter.  The injection system must not allow the analytes
              to contact hot stainless steel or other metal surfaces that promote decomposition.
       6.10.2 The GC/MS interface should allow the capillary column or transfer line exit to be
              placed within a few mm of the ion source. Other interfaces, for example, the open
              split interface, are acceptable as long as the system has adequate sensitivity (See Sect.
              10 for calibration requirements).
       6.10.3 The mass spectrometer must be capable of electron ionization at a nominal electron
              energy of 70 eV and of scanning from 45 to 450 amu with a complete scan cycle time
              (including scan overhead) of 1.5 sec or less.  (Scan cycle time = Total MS data
              acquisition time in sec divided by  total number of scans in the chromatogram).  The
              spectrometer must produce a mass spectrum that meets all criteria in Table 5 when 5
              to 10 ng of DFTPP is introduced into the GC.  An average spectrum across the
              DFTPP GC peak may be used to test instrument performance.
       6.10.4 An interfaced data system is required to acquire, store, reduce, and output mass
              spectral data.  The computer software must have the capability of processing stored
              data by recognizing a GC peak within any given retention time window, comparing
              the mass spectra from the GC peak with spectral data in a user-created data base, and
              generating a list of tentatively identified compounds with their retention times and
              scan numbers. The software must also allow integration of the ion abundance of any
              specific ion between specified time or scan number limits, calculation of response
              factors as defined in Sect. 10.3.6 (or construction of a second or third order
              regression calibration curve), calculation of response factor statistics (mean and
              standard deviation), and calculation of concentrations of analytes as described in
              Sect. 12.
6.11   GC Columns
       6.11.1 GC/MS:  DBS, 30 m x 0.25 mm, 0.25 /mi film thickness
       6.11.2 FID Primary: RTX Volatiles, 30 m  x 0.53 mm. ID, 2.0 urn film thickness, Restek
              Catalog No. 10902.
       6.11.3 FID Confirmation:  DBS, 30 m x 0.32 mm ID, 0.25 pm film thickness
6.12  Liquid-solid Extraction Vacuum System:  May be used.
6.13  8 mL Liquid-solid Extraction Cartridges With Frits: Also available from a  number of
       commercial suppliers.  Appropriate liquid-solid extraction disks may also be used in this
       method if equivalent or better quality assurance data can be demonstrated (See  Sect. 9).
6.14  Liquid-solid Extraction 70 mL Reservoirs And Adapters: Baxter Catalog # 9442 (adapter
       catalog # 9430) or equivalent.

7.     REAGENTS AND CONSUMABLE MATERIALS

7.1    Reagent Water:  Reagent water is defined as water in which an interference is not observed at
       the endothall method detection.
       7.1.1  A Millipore Super-Q Water System or its equivalent may be used to generate
              deionized reagent water.  Distilled water that has been charcoal filtered may also be
              suitable.

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Method 548.1
7.2   Methanol:  Pesticide quality.
7.3   Methylene Chloride: Pesticide quality or equivalent.
7.4   Sodium Sulfate—ACS Granular:  Heat in a shallow tray for 4 hrs at 400°C to remove
      phthlates and other interfering organic substances or extract with methylene chloride in a
      Soxhlet apparatus for 48 hrs.
7.5   10% Sulfuric Acid In Methanol:  Using extreme caution, slowly dissolve reagent grade
      sulfuric (10% v/v) acid in methanol.
7.6   Sodium Hydroxide (NAOH) 1 N:  Dissolve 4 g ACS grade in reagent water and dilute up to
      100 mL in a 100 mL volumetric flask.
7.7   10% Sodium Sulfate In Reagent Water:  Dissolve  100 g sodium sulfate in reagent water and
      dilute to volume in a 1-L volumetric flask.
7.8   Biorex 5 Anion Exchange Resin:  BioRad Laboratories Catalog No. 140-7841.
7.9   Disodium Ethylenediamine Tetraacetate  (EDTA):  Certified ACS Fisher or equivalent.
7.10 Endothall, Monohydrate: Available as neat material from Ultra Scientific, North Kingston, RI
      or as a concentrated solution from NSI Environmental Solutions, Research Triangle Park, NC.
7.11 Acenapthene-dlO:  Available from MSD Isotopes or Cambridge Chemicals.
7.12 Stock Standard Solutions
      7.12.1 Endothall:  50/xg/mL in methanol
      7.12.2 Acenaphthene-dlO:  500 pg/mL in methanol.  Dissolve 25 mg (approximately 32.2
              /xL) Acenapthnene-dlO in 50 mL methanol. Prepare a working standard at 10 ^g/mL
              by a 1:50 dilution of the stock standard.
      7.12.3 Decafluorotriphenylphosphine(DFTPP):  5/xg/mL.
      7.12.4 Stock standard solutions  must be replaced after 6 months, or sooner if comparison
              with check standards indicates a problem.

8.    SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1   Grab samples must be collected in glass containers.  Conventional sampling practices should
      be  followed, except that the bottle must not be prewashed with sample before collection.
      Composite samples should be collected in refrigerated glass containers.  Automatic sampling
      equipment must  be as free as possible of plastic tubing and other potential sources of
      contamination.
8.2   Sample Preservation
      8.2.1   If residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample to
              the sample bottle prior to collecting the sample.
      8.2.2  After adding the sample  to the bottle containing the sodium thiosulfate, seal the bottle
              and shake vigorously for 1 min.
      8.2.3  The samples must be iced or refrigerated at 4°C from the time of collection until
              extraction and analysis.  Endothall is not known to be light sensitive, but excessive
              exposure to light and heat should be avoided.
298

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                                                                                    Method 548.1
       8.2.4  A graphical representation of the results of a 14-day holding stability study on
               endothall in three different water matrices is presented in Figure 2.  These matrices
               were a dechlorinated tap water sample, a filtered river water sample containing
               considerable biological activity and the same river water biologically preserved at
               pH 2. These data indicate that the samples may be held for 7 days before extraction
               under the conditions of Sect. 8.2.3.  Endothall appears to be biologically stable over
               7 days.  However, the chemical  and  biological stability of endothall may be matrix
               dependent.   The analyst may verify analyte stability in the matrix of interest by
               conducting appropriate holding studies.  Samples with unusually high biological
               activity  should be acidified to pH 1.5 to 2.0 with  1:1 HC1:H20.
8.3   Extract Storage:  Sample extracts should  be stored in the dark at 4°C or less. A maximum
       extract holding time of 14 days is recommended.

9.     QUALITY CONTROL

9.1    Each laboratory that uses this method is required to  operate a formal quality control (QC)
       program.  The minimum QC requirements are initial demonstration of laboratory capability,
       analysis of laboratory reagent blanks, laboratory fortified blanks, laboratory fortified matrix
       samples and QC  check standards.
9.2   Laboratory Reagent Blanks:  Before processing any  samples, the analyst must demonstrate that
       all glassware and reagent interferences are under control.  Each time a set of samples is
       analyzed or reagents are changed, a laboratory reagent blank must be analyzed.  For this
       method, the blank matrix is filtered reagent water.  If within the retention time window of
       endothall,  the reagent blank produces a peak which prevents the measurement of endothall,
       determine  the source of contamination and eliminate the  interference before processing
       samples.
9.3   Initial Demonstration Of Capability
       9.3.1   Select a  representative fortified concentration for endothall. Prepare a methanol
               solution containing endothall at 1000  times the selected concentration.  The concen-
               trate must be prepared independently  from the standards used to prepare the calibra-
               tion curve (Sect. 10.2).  With a syringe, add 100 /iL of the concentrate to each of
               four  to seven 100-mL aliquots of reagent water and analyze each aliquot according to
               procedures  in Sect.  11.
       9.3.2   Calculate the mean percent recovery (R), the relative standard deviation of the  recov-
               ery (RSD in Table 2), and the MDL (1).  The mean recovery must fall in the range of
               R ±  20% using the values for R (Recovery) for reagent water (Table 2).  The  stan-
               dard  deviation should be less than 30%.  If  these  acceptance criteria are met, perfor-
               mance is acceptable and sample analysis  may begin. If either of these criteria fails,
               initial demonstration of capability should be repeated until satisfactory performance
               has been demonstrated.
       9.3.3   The initial demonstration of capability is used primarily to preclude a laboratory from
               analyzing unknown samples by a new, unfamiliar method prior to demonstrating a
              basic level of skill at performing  the technique.  As laboratory personnel gain experi-
                                                                                            299

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Method 548.1
               ence with this method the quality of the data should improve beyond the requirements
               stated in  Sect. 9.3.2.
9.4    The analyst is permitted to modify GC columns or GC conditions to improve separations or
       lower analytical costs.  Each time such method modifications are made, the analyst must repeat
       the procedures in  Sect. 9.3.
9.5    Assessing the Internal Standard:  In using the IS calibration procedure, the analyst is expected
       to monitor the IS  response (peak area) of all samples during each analysis day.  The IS
       response for any sample chromatogram should not deviate from the most recent calibration
       check standard IS response by more than 30%.
       9.5.1   If a deviation of greater than 30% is encountered for a sample, reinject the extract.
               9.5.1.1   If acceptable IS  response is achieved for the reinjected extract, then report
                         the results for that sample.
               9.5.1.2   If a deviation of greater than 30% is obtained for the reinjected extract,
                         analysis of the sample should be repeated beginning with Sect. 11, pro-
                         vided the sample is still available. Otherwise, report results obtained from
                         the reinjected extract, but annotate as suspect.
       9.5.2   If consecutive samples fail the IS response acceptance  criterion, immediately analyze a
               medium calibration check standard.
               9.5.2.1   If the check standard provides a response factor (RF) within 20% of the
                         predicted value, then follow procedures itemized in Sect. 9.5.1  for each
                         sample failing the IS response criterion.
               9.5.2.2   If the check standard provides a response factor (RF) which deviates more
                         than 20% from the predicted value, then the analyst must recalibrate, as
                         specified in Sect. 10.2.
9.6    Assessing Laboratory Performance
       9.6.1   The laboratory must analyze at least one laboratory fortified blank (LFB) per sample
               set (all samples extracted within a 24-hr period). The fortifying  concentration in the
               LFB should  be  10 to 20 times the MDL.  Calculate accuracy as percent recovery (Rj).
               If the recovery falls outside the  control limits (See Sect. 9.6.2), the system is judged
               out of control, and the source of the problem must be  identified and resolved before
               continuing analyses.
       9.6.2   Until sufficient  LFB data become available, usually a minimum of results from 20 to
               30 analyses, the laboratory should assess  its performance against  the control limits
               described in Sect. 9.3.2. When sufficient laboratory performance data become avail-
               able, develop control limits from the mean percent recovery (R) and standard devia-
               tion (S) of the percent recovery. These data are used to establish upper and lower
               control limits as follows:

                         Upper Control  Limit = R + 3S
                         Lower Control  Limit = R -  3S

               After each group of five to ten new recovery measurements, control limits should be
               recalculated  using only the  most recent 20 to 30 data points.

300

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                                                                                    Method 548.1
       9.6.3   Each laboratory should periodically determine and document its detection limit capa-
               bilities for endothall.
       9.6.4   Each quarter the laboratory should analyze quality control samples (if available).  If
               criteria provided with the QCS are not met, corrective action should be taken and
               documented.
9.7    Assessing Analyte Recovery
       9.7.1   The laboratory  must add a known fortified concentration to a minimum of 10% of
               samples or one  fortified matrix sample per set, whichever is greater.  The fortified
               concentration should not be less than the background concentration of the sample
               selected for fortification. The fortified concentration should be the same as that used
               for the LFB (Sect. 9.6).  Over time, samples from all routine sample sources should
               be fortified.
       9.7.2   Calculate the percent recovery for endothall, corrected for background concentrations
               measured in the unfortified sample, and compare these values to the control limits
               established in Sect. 9.6.2 for the analyses of LFBs.
       9.7.3   If the recovery  falls outside the designated range and the laboratory performance for
               that sample set  is shown to be in control (Sect. 9.6), the recovery problem encoun-
               tered with the fortified sample is judged to be matrix related, not system related.  The
               result in the unfortified sample must be labelled suspect/matrix to inform  the data user
               that the results  are suspect due to matrix effects.

1 0.   CALIBRA TION AND STANDARDIZA TION

10.1   Preparation Of Calibration Standards
       10.1.1  Calibration standards as dimethyl esters are prepared by addition of aliquots of the
               endothall stock  standard (Sect. 7.12.1) to the esterification reaction mixture,
               consisting of 8  mL of 10% H2SO4/methanol and 6 mL of methylene  chloride in the
               screw cap culture tubes (Sect. 6.3). The standards are then esterified and partitioned
               into the organic phase according to Sect.  11.4.  Prepare endothall acid standards
               equivalent to aqueous  standards at  100, 50, 25 and 5 jig/L by addition of the
               following aliquots of the stock standard solution (Sect. 7.12) to the esterification
               reaction mixture—200 /*L, 100 /*L, 50 /xL and 10 /*L.  By way of illustration, 200 /xL
               of the 50 fj.g/mL stock contains  10 /ig of endothall.  When dissolved in 100 mL of
               water, the aqueous concentration is 100 /ig/L.
       10.1.2 Process each standard as described in Sect. 11.4.1 and Sect. 11.4.2.  The internal
               standard is added as described in Sect. 11.4.3. Triplicate samples should be prepared
               at each concentration level.
10.2  Demonstration and documentation of acceptable initial calibration are required before any
       samples are analyzed and intermittently throughout sample analyses as dictated by  results of
       continuing calibration checks.  After  initial  calibration is successful, a continuing calibration
       check is required at the  beginning of each 8-hr period during which analyses are performed.
       Additional periodic calibration checks are good laboratory practice.
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Method 548.1
10.3  Initial Calibration
       10.3.1  Calibrate the mass spectrometer with calibration compounds and procedures
               prescribed by the manufacturer with any modifications necessary to meet the
               requirements in Sect. 10.3.2.
       10.3.2 Inject into the GC a 1-or 2-fj.L aliquot of the 5 ng/juL DFTPP solution and acquire a
               mass spectrum that includes data for m/z = 45-450.  Use GC conditions that produce
               a narrow (at least five scans per peak) symmetrical peak.  If the spectrum does not
               meet all criteria (Table 5), the MS must be retuned  to meet all criteria before
               proceeding with calibration.  An average spectrum across the GC peak may be used to
               evaluate the performance of the  system.
       10.3.3 Inject a 1-/*L aliquot of a medium concentration calibration solution, for example 50
               Mg/L, and acquire and store data from m/z 45-450 with a total cycle time (including
               scan overhead time) of 1.5 sec or less.  Cycle time should be adjusted to measure at
               least five or more spectra during the elution of the GC peak.  Figure 3 illustrates a
               total ion chromatogram and mass spectrum of endothall and the internal  standard,
               acenaphthene-dlO, using the prescribed conditions.
       10.3.4 If all performance criteria are met,  inject a l-/xL aliquot of each of the other
               calibration solutions using the same GC/MS conditions.
       10.3.5 Calculate a response factor (RF) for endothall for each calibration solution by use of
               the  internal standard response as expressed below. This calculation is supported in
               acceptable GC/MS data system software (Sect. 6.10.4),  and many other  software
               programs.  The RF  is a unitless number, but units used to express quantities of
               analyte and internal standard must be equivalent.
          Where:
          Ax  = integrated abundance of the quantitation ion of the analyte (m/z 183).
          Ais  = integrated abundance of the quantitation ion internal standard (m/z 164).
          Qx  = quantity of analyte injected in ng or concentration units.
          Qis  = quantity of internal standard injected in ng or concentration units.
               10.3.5.1 Calculate the mean RF from the analyses of the calibration solutions.
                         Calculate the standard deviation (SD) and the relative standard deviation
                         (RSD) from each mean:  RSD =  100 (SD/M).  If the RSD of any analyte
                         or surrogate mean RF exceeds 30%, either analyze additional aliquots of
                         appropriate calibration solutions to obtain an acceptable RSD of RFs over
                         the entire concentration range or take action to improve GC/MS perfor-
                         mance.  See Sect. 10.4.5 for possible remedial actions.
 302

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                                                                                   Method 548.1
       10.3.6 As an alternative to calculating mean response factors and applying the RSD test, use
               the GC/MS data system software or other available software to generate a linear or
               second order regression calibration curve.
10.4  Continuing calibration check.  Verify the MS tune and initial calibration at the beginning of
       each 8-hr work shift during which analyses are performed using the following procedure.
       10.4.1  Inject a 1-/*L aliquot of the 5 ng//xL DFTPP solution and acquire a mass spectrum that
               includes data for m/z 45-450.  If the spectrum does not meet all criteria (Table 5), the
               MS must  be retuned to  meet all criteria before proceeding with the continuing
               calibration check.
       10.4.2 Inject a 1-^L aliquot of a medium concentration calibration solution and analyze with
               the same  conditions used during the initial calibration.
       10.4.3 Determine that the  absolute area of the quantitation ion of the internal standard has not
               decreased by more  than 30% from the area measured in the most recent continuing
               calibration check, or by more than 50% from the area measured during initial calibra-
               tion.  If the area has decreased by more than these amounts, adjustments must be
               made to restore system  sensitivity.  These adjustments may require cleaning of the MS
               ion source, or other maintenance as indicated in Sect. 10.4.5, and recalibration.  Con-
               trol charts are useful aids in documenting system sensitivity changes.
       10.4.4 Calculate  the RF for endothall from the data measured  in the continuing calibration
               check.  The RF must be within 30% of the mean value measured in the initial
               calibration.  Alternatively, if a linear or second order regression is used, the
               concentration measured  using the calibration curve must be within 30% of the true
               value of the concentration in the medium calibration  solution.  If these conditions do
               not exist,  remedial action must be taken which may require repeating the initial
               calibration.
       10.4.5  Some possible remedial  actions: major maintenance such as cleaning an ion source,
               cleaning quadrupole rods, etc. require returning to the initial calibration step.
               10.4.5.1  Check and adjust GC and/or MS operating conditions; check the MS
                         resolution, and calibrate the mass scale.
               10.4.5.2 Clean or replace the splitless injection liner; silanize a new injection  liner.
               10.4.5.3 Flush the GC column with solvent according to manufacturer's
                         instructions.
               10.4.5.4 Break off a short portion (about 1 meter)  of the column from the end near
                        the injector;  or replace GC column.  This action will cause a slight change
                         in retention times.
               10.4.5.5 Prepare  fresh CAL solutions, and repeat the initial  calibration step.
               10.4.5.6 Clean the MS ion source and rods (if a quadrupole).
               10.4.5.7 Replace  any  components that allow analytes to come into contact with hot
                        metal surfaces.
               10.4.5.8 Replace  the MS electron multiplier or any other faulty components.
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Method 548.1
 11.   PROCEDURE

 11.1  Preparation Of Anion Exchange Cartridges
       11.1.1 Prepare a 50% (v/v) slurry of Bio-Rex 5 resin and reagent water.
       11.1.2 Attach the required number of 8-mL extraction cartridges (Sect. 6.13) to the vacuum
               manifold (Sect. 6.12), and insert bottom fritted disks into each cartridge.
       11.1.3 Fill the cartridges completely with Bio-Rex 5 slurry. Draw off excess water with
               vacuum.  The final wet resin bed height should be 3.5 + 0.1  cm.  Adjust the height
               by adding more slurry and repeating procedure, or add more reagent water to
               reservoir and remove excess resin slurry.
       11.1.4 After the bed heights are adjusted to 3.5 cm and  with excess water removed under
               vacuum,  insert a fritted disk on top of the resin bed. The fritted disk should press
               firmly into the resin and be horizontal to the reservoir to prevent sample channeling
               around the disk.  Fill the cartridges with reagent  water and draw half of the water into
               the resin.  Maintain the resin cartridges in this condition until ready  for use.
               NOTE:    The use of liquid-solid extraction disks instead of cartridges is permissible
                         as long as all the quality control criteria specified in Sect. 9 of this method
                         are met.
11.2  Sample Preparation
       11.2.1  As discussed above (Sect. 1.3  and Sect. 4.2), reduced recoveries will be observed if
               the sample contains elevated levels of Call, Mgll or sulfate.  If facilities are available,
               measure the concentrations of these ions.  Figure 1 graphically presents analyte
               recovery  versus individual  ion concentration.  Reduced recoveries  may be anticipated
               when the combined Call -I- Mgll exceeds approximately 100 mg/L or sulfate exceeds
               approximately 250 mg/L.  If measurement of ion concentration is not feasible,
               determine the actual  recovery for a laboratory fortified sample matrix as described in
               Sect. 9.7. In the event of anticipated or measured low recoveries, treat the sample  as
               described in Sect. 11.2.2.
       11.2.2 For samples containing moderately high levels of these ions, add 186 mg of EDTA
               (Sect. 7.9) per 100-mL sample (0.005 M). The treated ground water characterized in
               Table 3 is an example  of a matrix successfully treated this way. For samples
               containing very high levels of sulfate, sample dilution may be  required in addition to
               the EDTA.  The  western surface water characterized in Table 3 (ca.  2000 mg/L
               sulfate) was successfully analyzed after dilution by  a factor of 10 and the addition of
               75 mg EDTA per 100  mL of the diluted sample (0.002 M).  Samples containing
               intermediate levels of sulfate can be analyzed with smaller dilution factors.
               Guidelines on dilution  factors and EDTA addition are given below.

                                                                Added EDTA
                Sulfate fmg/U         Dilution Factor         (mg/100 mL)
                     < 250                    1:1                     186
                   250-500                  1:2                    125
                   500-1250                  1:5                    75
                    > 1250                  1:10                    75
304

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                                                                                 Method 548.1
       NOTE: Dilution should not be employed if adequate recovery is attained by the
       addition ofEDTA alone.

       11.2.3 The addition of EDTA results in a large reagent peak near the end of the temperature
              program.  Therefore, complete the entire program described in Table 1.
       11.2.4 If the ionic nature of the samples being processed is completely unknown, the analyst
              as an option may routinely dilute all samples by a factor of 10 and add EDTA as
              above. However, the analyst should  be able to demonstrate reagent water MDLs of 2
              /ig/L or lower.  In this event the MDL will be 20 /tg/L or less for the diluted sample,
              still a factor of 5 below the regulated maximum contaminant level.
11.3  Sample Extraction
       11.3.1 Attach the 70-mL reservoir to the  resin cartridge with the adapter (Sect. 6.14).
       11.3.2 Condition the resin  cartridge by drawing the following reagents through the cartridge
              in the following order:
                         1.  10 mL methanol
                        2.  10 mL reagent water
                        3.  10 mL 10% H2S04  in methanol
                        4.  10 mL reagent water
                        5.  20 mL 1 N NaOH
                        6.  20 mL reagent water
              Do not allow the cartridge to become dry between steps.  Draw each reagent through
              the cartridge at a rate of 10 mL/min. Leave a 1-cm layer of reagent water over  the
              resin bed.
       11.3.3 Fill the 70-mL reservoir with 60 mL of the sample.  Adjust sample flow rate to 3
              mL/min. Add the balance of sample when needed to prevent the reservoir from  going
              dry.
       11.3.4 After the sample passes through the  cartridge, remove the 70-mL reservoir and the
              adapter.  Draw 10 mL of methanol through the resin cartridge.  Make sure that any
              visible water  inside  the cartridge dissolves in methanol.  Next draw room air through
              the cartridge for 5 min under a vacuum of 10-20 in. Hg.  Position the culture tube
              (Sect. 6.3) inside the manifold to collect the eluent.
       11.3.5 Elute the cartridge with 8 mL of 10% H2S04 in methanol, followed by 6 mL of
              methylene chloride under vacuum  over a 1  min period.
11.4  Sample Derivatization, Partition And Analysis
       11.4.1 Cap the culture tube and hold at 50°C for 1 hr in a heating block or water bath.
              Remove from heat and allow the tube to cool for 10 min.
       11.4.2 Pour the contents of the culture tube into a 125-mL separatory funnel.  Rinse the tube
              with two x 0.5 mL aliquots  of methylene chloride and add the rinsings to the separa-
              tory funnel.  Add 20 mL of 10% sodium sulfate in reagent water to the separatory

                                                                                         305

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Method 548.1
               funnel. Shake the funnel three times vigorously, venting with the stopcock, and then
               shake  vigorously for an additional 15 sec.  After the phases have separated, drain the
               lower  organic layer into a 15-raL graduated centrifuge tube (Sect. 6.4).  Repeat the
               extraction procedure above with two additional 2-mL aliquots of methylene chloride,
               adding the organic phase to the centrifuge tube each time.
       11.4.3 Fortify the extract with 250 jiL of the internal standard working solution (Sect.
               7.12.2) and concentrate to a final volume of 1.0 mL, using the N-EVAP (Sect. 6.7)
               and dry nitrogen.
       11.4.4 Inject  2 p.L of the concentrated extract (Sect. 11.4.3) and analyze by GC/MS using
               the conditions described in Table 1.  This table includes the retention time and MDL
               that were obtained under these conditions.  A sample total ion chromatogram of
               endothall and d-10 acenaphthene illustrating retention times, and the mass spectrum of
               the dimethylated endothall are shown in Figure 3.  Other columns, chromatographic
               conditions, or detectors may be used if the requirements of Sect. 9.3 are met.
       11.4.5 If the  peak area exceeds the linear range of the calibration curve, a smaller sample
               volume should be used.
11.5  Identification Of The Analyte
       11.5.1 Identify endothall by comparison of its mass spectrum (after background subtraction)
               to a reference spectrum in a user created spectral library.  The GC retention time of
               the sample component should be within 10 sec of the retention time of endothall in the
               latest calibration standard.  If a FID is used, identifications should be confirmed by
               retention time comparisons on the second GC column (Table  1).
       11.5.2 In general, all ions present above 10% relative abundance in the mass spectrum of the
               standard should be present in the mass spectrum of the sample component and should
               agree  within absolute 20%.  For example,  if an ion has a relative abundance of 30%
               in the  standard spectrum, its abundance in  the sample spectrum should be in the range
               of 10-50%. However, the experience of the analyst should weigh heavily in the
               interpretation of spectra and chromatograms.
       11.5.3 Identification requires expert judgement when sample components are not resolved
               chromatographically, that is, when GC peaks from interferences are present.  When
               endothall coelutes with an interference, indicated by a broad peak or a shoulder on the
               peak,  the identification criteria can usually be  met, but the endothall spectrum will
               contain extraneous ions contributed by the  coeluting interfering compound.

 12.   DATA ANALYSIS AND CALCULATIONS

12.1  When using GC/MS, complete chromatographic resolution is not necessary for accurate and
       precise measurements of analyte concentrations if unique ions with adequate intensities are
       available for quantitation. However, when using FID, complete resolution is essential.
       12.1.1 Calculate endothall concentration.
306

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                                                                                  Method 548.1
                                         c  =
                                          *    (A,)RF V
where:
  C = concentration of endothall in \nglL in the water sample.
 Ax = integrated abundance of thequantitation ion of endothall (mlz 183) in the sample.
Ais = integrated abundance of the quantitation ion of the internal standard (mlz 164) in the sample.
Qa = total quantity (in micrograms) of internal standard added to the water sample.
  V = original water sample volume in liters.
RV = mean response factor endothall the initial calibration.
       12.1.2 Alternatively, use the GC/MS data system software or other  available proven
              software to compute the concentration of the endothall from the linear calibration or
              the second order regression curves.
       12.1.3 Calculations should utilize all available digits of precision, but final reported
              concentrations should be rounded to an appropriate number of significant figures (one
              digit of uncertainty).  Experience indicates that three significant figures may be used
              for concentrations above 99 j*g/L, two significant figures for concentrations between
              1-99 (j.g/L, and  one significant figure for lower concentrations.

13.   METHOD PERFORMANCE

13.1   Method Detection Limits: 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 the background
       level.' The MDLs listed in  Table 1 were obtained using reagent water for detection by
       GC/MS and FID.
13.2  In a single laboratory study on fortified reagent water and ground water matrices, the mean
       recoveries and relative standard deviations presented in Table 2 were obtained.  Table 3
       provides the concentrations of Call, Mgll and sulfate for two high ionic strength drinking
       water sources studied.  Table 4 presents mean recovery data for these fortified sources with
       and without the addition  of EDTA and/or sample dilution.

14.   POLLUTION PREVENTION

14.1   This method utilizes the  new liquid-solid  extraction technology which requires the use of very
       little organic solvent thereby eliminating the hazards involved with the use of large volumes of
       organic solvents in conventional liquid-liquid  extractions. It also uses acidic methanol as the
       derivatizing reagent in place of the highly toxic and explosive diazomethane.  These features
       make this method  much safer for the analyst to employ and a great deal less harmful to the
       environment.
                                                                                          307

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Method 548.1
15.   WASTE MANAGEMENT

15.1  Due to the nature of this method, there is very little need for waste management. No large
      volumes of solvents or hazardous chemicals are used.  The matrices are drinking water or
      source water, and can be discarded down the sink.
 308

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                                                                               Method 548.1
                                    References
1.    40 CFR Part 136, Appendix B.
2.    ASTM Annual Book of Standards, Part 31, D3694-78.  "Standard Practices for Preparation of
      Sample Containers and for Preservation of Organic Constituents," American Society for
      Testing and Materials, Philadelphia, PA.

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," (29CF/J1910), 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.
                                                                                       309

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Method 548.1
Table 1.  Retention Times And Method Detection Limits
                               Retention Time (min.)
Method Detection Limit
        frg/U'
Compound            \  Column A\  Column B  \  Column C      GC/MS   |     FID
Endothall                  16.02        19.85        18.32         1.79         0.7
d10-Acenaphthene          14.69
 'Based on 7 replicate analyses of a reagent water fortified at 2 //g/L
Column A:DB-5 fused silica capillary for GC/MS, 30 m x 0.25 mm, 0.25 micron film

MS inlet temperature = 200°C
Injector temperature = 200°C

Temperature Program: Hold 5 min at 80°C, increase to 260°C at 10°/min, hold 10 min.
Column B:FID primary column, RTX Volatiles, 30 m  x 0.53 mm I.D., 2 micron film thickness.

Detector temperature = 280°C
Injector Temperature  =  200°C

Carrier gas velocity = 50 cm/sec.

Temperature program: Same as Column A


Column C:FID confirmation column, DB-5, 30 m x 0.32 mm ID, 0.25 micron film

Carrier Gas velocity = 27 cm/sec

Same injector, detector  and temperature program as Column A.
370

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                                                                        Method 548.1
Table 2.  Endothall Method Development Data

                            Cone.              Recovery1              RSD2
Matrix                      frg/L)                 f%)                  (%)
Reagent Water                   2                   101                  10
Reagent Water                  10                    86                  10
Reagent Water                 100                    95                   3
Ground Water3                   2                    91                  25
Ground Water                   10                    82                  14
Ground Water                  100                    88                   6

1    Based on analysis of 7 replicates.
2    Relative Standard Deviation.
3    High Humic Content Florida Ground Water.
                                                                                111

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Method 548.1
Table 3.  Matrix Analyses1
                                Western Surface,           Eastern Ground
            Major Ion                (mg/L)                    (mg/L)

                Ca                       330                        122
                Mg                       132                         33
                Na                       400                         23
              Sulfate                    1850                        102

    Determination by  inductively coupled plasma - mass spectrometry for cations and ion
    chromatography for sulfate.
372

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                                                                         Method 548.1
Table 4.  Endothall Method Validation Data
                             Cone.      EOT A1        Recovery1       RSD
      Matrix                 (pg/L)      (Mole/L)          (%)          (%)
      WS3                    25           0                9        19
      WS-1/10*              50           0               66        13
      WS-1/10               50         0.002             88         5
      EG5                     25           0               43        17
      EG                      25         0.005             97         6
      EG-1/5                 25           0               97         5
    Ethylenediamine Tetraacetic Acid
    Based on 7 Replicates
    WS - Treated Western Surface Water
    Dilution Factor in Reagent Water
    WG - Eastern Ground Water
                                                                                313

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Method 548.1
Table  5.  Ion Abundance Criteria For Bis(Perfluorophenyl)Phenyl Phosphine
(Decafluorotriphenylphosphine, DFTPP)
Mass
(M/z)
 51
 51
 68
 70
127
197
198
199
275
365
441
442
443
Relative Abundance Criteria

10-80% of the base peak
<2% of mass 69
<2% of mass 69
2% of mass 69
10-80% of the base peak
<2% of mass 198
base peak or >50% of 442
5-9% of mass 198
10-60% of the base peak
> 1 % of the base peak
Present and < mass 443
base peak or >50% of 198
15-24% of mass 442
Purpose of Checkpoint1

Low mass sensitvity
Low mass sensitvity
Low mass resolution
Low mass resolution
Low-mid mass sensitivity
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mss resolution and isotope ration
Mid-high mass sensitivity
Baseline threshold
High mass resolution
High mass resolution and sensitivity
High mass resolution and isotope ration
    All ions are used primarily to check the mass measuring accuracy of the mass spectrometer
    and data system, and this is the most important part of the performance test.  The three
    resolution checks, which include natural abundance isotope ratios, constitute the next most
    important part of the performance test.  The correct setting of the baseline threshold, as
    indicated by the presence of low intensity ions, is the next most important part of the
    performance test. Finally, the ion abundance ranges are designed to encourage some
    standardization to fragmentation patterns.
314

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                                                                  Method 548.1
25
50
  S04
  NA2SO4
100     150     200     250     300

              Concentration mg/L
350
400
600
            A  CAII
                CACL.2
                          O  MGII
                              MGCL2
                                                                      52-015-4A
       *  SOd
          CASO4
                 Endothall Recovery from Reagent Water
                   with 804, MGII and CAII Ions Present
         Figure 1. Endothall Recovery versus Ion Concentrations
                                                                         375

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  Method 548.1
    100
     84
 r
 o
 8
oc
                                  1                         7



                                       Holding Time Days
                                                                                  52-015-5A
    +  Tap

       Dechlorinated
A  River

    Preserved
O  River
                       Figure 2. Endothall Sample Holding Study
376

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                                                                            Method 548.1
200000—
150000—
100000—
 50000-
            TIC: 716ML10.D


         D-10Acenaphthene
                                   Endothall
     Time   13.5    14.0     14.5    15.0    15.5    16.0    16.5    17.0    17.5


                          Scan 1737 (16.024 Min):  716ML10.D (•)

3000-
2500-
2000-
0)
c
m
ID
c
^ 1500-
1000-



500-






































67
59


•
111



ti






























123








113
95

85
81

Illl

I,


hi




nl lid LI





,


1{


27




145




I ll




i:
55







183




I TNI lib
     M/Z
               60
80
100
120
140
160
180
       Figure 3. Endothall GC/MS
                 Upper:  Total Ion Chromatography Endothall:  16.02 Min., 10 ng
                 Lower:  Relative Ion Abundance
                                                                                52-015-66
                                                                                    317

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                          Method 553
Determination of Benzidines and Nitrogen-
           Containing Pesticides in Water
               by Liquid-Liquid Extraction
                 or Liquid-Solid Extraction
     and Reverse Phase High Performance
    Liquid Chromatography/Particle Beam/
                      Mass Spectrometry
                    Revision 1.1 - EPA EMSL-Ci
                               August 1992
     Thomas D. Behymer, Thomas A. Bellar, James S. Ho, William L. Budde

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                                  Method  553
  Determination of Benzidines and Nitrogen-Containing Pesticides in
       Water by Liquid-Liquid Extraction or Liquid-Solid Extraction
and Reverse Phase High Performance Liquid Chromatography/Particle
                           Beam/Mass Spectrometry


1.    SCOPE AND APPLICA TION

1.1   This is a general purpose method that provides procedures for determination of benzidines and
      nitrogen-containing pesticides in water and wastewater.  The method is applicable to wide
      range of compounds that are efficiently partitioned from a water sample into methylene
      chloride or onto a liquid-solid extraction device.  The compounds must also be amenable to
      separation on a reverse phase liquid chromatography column and transferable to the mass
      spectrometer with a particle beam interface.  Paniculate bound organic matter will not be
      partitioned onto the liquid-solid extraction system, and more than trace levels or particulates in
      the water may disrupt the partitioning process.  The compounds listed below are potential
      method analytes and single-laboratory accuracy and precision data have been determined for
      the compounds as described in Sect.  13. The specific analytical conditions given in the
      method are applicable to those compounds for  which accuracy and precision data are given.
      Other analytes (Sect. 1.2) may require slight adjustments of analytical conditions.  A laborato-
      ry may use this method to identify and measure additional analytes after the laboratory obtains
      acceptable (defined in Sect.9) accuracy and precision data for each added analyte.

                                        Abbre-
           Compound                  viation      MW      CAS No.
           Benzidine                       BZ         184         92-87-5
           Benzoylprop ethyl               BP         365     33878-50-1
           Caffeine                        CF         194         58-08-2
           Carbaryl                        CL         201         63-25-2
           o-Chlorophenyl thiourea          PT         186       5344-82-1
           3,3'-Dichlorobenzidine           DB         252         91-94-1
           3,3'-Dimethoxybenzidine          MB         244        119-90-4
           3,3'-Dimethylbenzidin            LB         212        119-93-7
           Diuron                         Dl         232        330-54-1
           Ethylene thiourea                ET         102         96-45-7
           Linuron (Lorox)                  LI         248        330-55-2
           Monuron                      MO         198        150-68-5
           Rotenone                       RO         394         83-79-4
           Siduron                        SI         232       1982-49-6

        a   Monoisotopic molecular weight calculated from the atomic masses of the
           isotopes with the smallest masses.

1.2   Preliminary investigation indicates that the following compounds may be amendable to this
      method: Aldicarb sulfone, Carbofuran, Methiocarb, Methomyl (Lannate), Mexacarbate
      (Zectran), and N-(l-Naphthyl) thiourea.  Caffeine, Ethylene thiourea and o-Chlorophenyl

                                                                                   527

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Method 553
       thiourea have been successfully analyzed by HPLC/PB/MS,  but have not been successfully
       extracted from  a water matrix.
1.3    Method detection limit (MDL) is defined as the statistically calculated minimum amount that
       can be measured with 99% confidence that the reported value is greater than zero.'  The MDL
       is compound dependent and is particularly dependent on extraction efficiency and sample
       matrix.  For analytes listed in Tables 3-5,  the estimated MDLs range from 2 to 30 fj.g/L.

2.     SUMMARY OF METHOD

2.1    Organic compound analytes and surrogates are extracted from  1 L of water sample by liquid-
       liquid extraction (LLE) with methylene chloride or by passing  1 L of sample water through a
       cartridge or disk containing a solid inorganic matrix coated with a chemically bonded C
       organic phase or a neutral polystyrene/divinylbenzene polymer (liquid-solid extraction, LSE).
       If LLE is used, the analytes are concentrated in methanol by evaporation of the methylene
       chloride and addition of methanl (solvent exchange).  If LSE is used, the analytes are eluted
       from the  LSE cartridge or disk with a small quantity of methanol  and concentrated  further by
       evaporation of  some  of the solvent.  The sample components are separated, identified and
       measured by injecting an aliquot of the concentrated methanol  solution into a high performance
       liquid chromatograph (HPLC) containing a reverse phase HPLC column and interfaced to a
       mass spectrometer (MS) with a particle beam (PB) interface. Compounds  eluting from the
       HPLC column  are identified by comparing their measured mass spectra and retention times  to
       reference spectra and retention times in a data base.  Reference spectra and retention times for
       analytes are obtained by measurement of calibration standards under the same conditions used
       for samples. The concentration of each identified component is measured  by relating the MS
       response of the quantitation ion produced by that compound  to the MS response of  the quan-
       titation ion produced by the same compound in a calibration standard (external standard).
       Surrogate analytes, whose concentration are known in every sample, are measured  with the
       same external standard calibration procedure.  An optional isotope dilution procedure is
       included for samples which contain interfering matrix or coeluting compounds.

3.     DEFINITIONS

3.1    External Standard (ES):  A pure analyte(s) that is measured  in an  experiment separate from the
       experiment used to measure the analyte(s) in the sample. The signal observed for a known
       quantity of the  pure external standard(s) is  used to calibrate the instrument response for the
       corresponding analyte(s).  The instrument response is used to calculate the concentrations of
       the anayte(s) in the sample.
3.2    Surrogate Analyte (SA): A pure analyte(s), which is extremely unlikely to be found in any
       sample, and which is added to a sample aliquot in known amount(s) before extraction and is
       measured with  the same procedures  used to measure other sample components.  The purpose
       of a surrogate analyte is to monitor method performance with each sample.
3.3    Laboratory  Duplicates  (LD1 and LD2):  Two  aliquots of the same sample  taken in  the labora-
       tory and analyzed separately with identical procedures.  Analyses  of LD1 and LD2 indicate the
       precision associated with laboratory  procedures, but not with samples  collection, preservation,
       or storage procedures.
322

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                                                                                     Method 553
3.4    Field Duplicates (FD1 and FD2):  Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the same throughout field and laboratory
       procedures.  Analyses of FD1 and FD2 give a measure of the precision associate with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.5    Laboratory Reagent Blank (LRB):  An aliquot of reagent water or other blank matrix that is
       treated exactly as a sample including exposure to all glassware, equipment, solvents,  reagents,
       and surrogates that are used with other samples. The LRB is used to determine  if method
       analytes or other interferences are present in the laboratory environment, the  reagents,  or the
       apparatus.
3.6    Field Reagent Blank (RFB):  An aliquot of reagent water or other blank matrix that is placed
       in a sample container in the laboratory and treated as a sample in all respects, including
       shipment to the sampling site, exposure to sampling site conditions, storage, preservation and
       all analytical procedures. The purpose of the FRB is to determine if method  analytes or other
       interferences are present in the field environment.
3.7    Instrument Performance Check Solution (IPC):  A solution of one or more method analytes,
       surrogates, internal standards, or other test substances used to evaluate the performance of the
       instrument system with respect to a defined  set of method criteria.
3.8    Laboratory Fortified Blank (LFB):  An aliquot of reagent water or other blank matrix to which
       known quantities of the method  analytes are added in the laboratory. The LFB is analyzed
       exactly like a sample,  and its purpose is to determine whether the methodology  is in control,
       and whether the laboratory is capable of making accurate and precise measurements.
3.9    Laboratory Fortified Sample Matrix (LFM):  An aliquot of an environmental  sample to which
       known quantities of the method  analytes are added in the laboratory. The LFM is analyzed
       exactly like a sample,  and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the sample
       matrix must be determined in a  separate aliquot and the measured values in the LFM corrected
       for background concentrations.
3.10  Stock Standard Solution (SSS):  A concentration solution containing one or more method
       analytes prepared in the laboratory using assayed reference materials or purchased from a
       reputable commercial source.
3.11  Primary Dilution Standard Solution (PDS):  A solution of several analytes prepared in the
       laboratory from the standard solutions and diluted  as needed  to prepare calibration solutions
       and other needed analyte solutions.
3.12  Calibration Standard (CAL):  A solution prepared  from the primary dilution standard solution
       or stock standard solutions and the internal standards and surrogate analytes.  The CAL
       solutions are used to calibrate the instrument response with respect to analyte  concentration.
3.13  Quality Control Samples (QCS): A solution of method analytes of known concentrations
       which is used to fortify an aliquot of LRB or sample matrix.  The QCS is obtained from a
       source external to the  laboratory and different from the source of calibration standards. It is
       used to check laboratory performance with externally prepared test materials.
                                                                                            323

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Method 553
3.14  Instrument Detection Limit (IDL):  The minimum quantity of analyte or the concentration
       equivalent which gives an analyte signal equal to three times the standard deviation of the
       background signal at the selected wavelength, mass, retention time, absorbance line, etc.

4.     INTERFERENCES

4.1    When two compounds coelute, the transport efficiency of both compounds thought the particle
       beam interface generally improves and enhanced ion abundances are observed in the mass
       spectrometer.2 The degree of signal enhancement by coelution is compound dependent.  This
       coelution effect invalidates the external calibration curve and, if not recognized, will result in
       incorrect concentration measurements. Procedures given in this method to check for coeluting
       compounds must be followed to preclude inaccurate measurements (Sect.  10.2.6.5 and Sect.
       12.1).  An optional isotope dilution calibration procedure has been included for use when
       interfering matrix or coeluting compounds are present.
4.2    During analysis,  major contaminant sources are reagents, chromatography columns, and liquid-
       solid extraction columns or disks.  Analyses of field and laboratory reagent blanks provide
       information about the presence of contaminants.
4.3    Interfering contamination may occur when a sample containing low concentrations of com-
       pounds is analyzed immediately after a sample containing relatively high concentrations of
       compounds.   Syringes, injectors, and other equipment must be cleaned carefully or replaced as
       needed.  After analysis of a sample containing high concentrations of compounds, a laboratory
       reagent blank should be analyzed to ensure that accurate values are obtained for the next
       sample.

5.     SAFETY

5.1    The toxicity or carcinogenicity of chemicals used in this method has not been precisely de-
       fined; each chemical should be treated as a potential health hazard, and exposure to these
       chemicals should be minimized.  Each laboratory is responsible for maintaining awareness of
       procedures and regulations for safe handling of chemicals uses in this method.3"5
5.2    Some method analytes have been tentatively classified as known or suspected human or
       mammalian carcinogens.  Pure standard materials and stock standard solutions of all analytes
       should be handled with suitable protection to skin, eyes, etc.

6.     EQUIPMENT AND SUPPLIES

6.1    All glassware must be meticulously cleaned.  This may be accomplished by washing with
       detergent and water, rinsing with water, distilled water, or solvents, air-drying, and heating
       (where appropriated) in an oven. Volumetric glassware is never heated.
6.2   Sample Containers: 1-L or 1-qt amber glass bottles fitted with a Teflon-lined screw cap.
       (Bottles in which high purity solvents were received can be used as sample containers without
       additional cleaning if they have been handled carefully to avoid contamination during use and
       after use of original contents.)
6.3   Separatory Funnels: 2-L and 100-mL with a Teflon stopcock.
 324

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                                                                                   Method 553
6.4    Liquid Chromatography Column Reservoirs:  Pear-shaped 100- or 125-mL vessels without a
       stopcock but with a ground glass outlet sized to fit the liquid-solid extraction column.  (Lab
       Glass, Inc., Part No. ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom inner
       joint, or equivalent.)  A 14/35 outlet joint fits some commercial cartridges.
6.5    Syringe  Needles: No. 18 or 20 stainless steel.
6.6    Vacuum Flasks:  1 or 2 L with solid rubber stoppers.
6.7    Volumetric Flasks:  Various sizes.
6.8    Laboratory or Aspirator Vacuum System:  Sufficient capacity to maintain a slight vacuum of
       13 cm (5 in) of mercury in the vacuum flask.
6.9    Micro Syringes:  Various  sizes.
6.10  Vials: Various sizes  of amber vials with Teflon-lined screw caps.
6.11  Drying Column:  0.6 cm x 40 cm with 10 mL graduated collection vial.
6.12  Concentrator Tube:  Kuderna-Danish (K-D) 10 mL graduated with ground glass stoppers.
6.13  Analytical Balance:  Capable of weighing 0.0001 g accurately.
6.14  Liquid Chromatograph Column:  A 15-25 cm x  2 mm (i.d.) stainless steel tube (e.g., Waters
       C—18 Novapak or equivalent) packed with silica particles (4-10 jim) with
       octadecyldimethylsilyl (C-18) groups chemically bonded to the silica surface.  Residual acidic
       sites should be blocked (endcapped) with methyl  or other non-polar groups and the stationary
       phase must be bonded to the solid support to minimize column bleed.  Column selection for
       minimum bleeding is  strongly recommended.  The column must be conditioned over night
       before each use by pumping a  75-100% v/v acetonitrile: water solution through it at a rate of
       about 0.05 mL/min.   Other packings and column sizes may be used if equivalent or better
       performance can be achieved.
6.15  Guard column of similar packing used in the analytical column is  recommended.
6.16  Liquid Chromatograph/Mass Spectrometer/Data System (LC/MS/DS)
       6.16.1  The LC must accurately maintain flow rates between 0.20-0.40 mL/min while perfor-
               ming a gradient elution from 100% solvent A to 100% solvent B. Pulse dampening is
               recommended but not required. An autoinjector is highly desirable and should be
               capable of accurately delivering 1-10 /xL injections without affecting the chromatog-
               raphy.
       6.16.2  The system should include a post-column mixing tee and  an additional LC pump for
               post-column  addition of acetonitrile at a constant rate of 0.1-0.7 mL/min.
       6.16.3  The particle beam LC/MS interface must reduce the system pressure to a level fully
               compatible with the generation of classical electron ionization (El) mass spectra, i.e.,
               about 1 x 106 to 1  x  104 Torr, while delivering sufficient quantities of analytes to
               the conventional El source to meet sensitivity, accuracy, and precision requirements.
               All significant background components with mass greater than 62  Daltons should be
               removed to a level that does not produce ions greater than a relative abundance of
               10% in the mass spectra of the analytes.
       6.16.4  The mass spectrometer must be capable of electron ionization at a nominal electron
               energy of 70 eV.  The spectrometer must be  capable of scanning from 45 to 500 amu

                                                                                          325

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Method 553
               with a complete scan cycle time (including scan overhead) of 1.5 sec or less.  (Scan
               cycle time = Total MS data acquisition time in seconds divided by number of scans in
               the chromatogram).  The spectrometer must produce a mass spectrum that meets all
               criteria in Table 1 when 500  ng or less of DFTPPO (Sect. 7.11) is introduced into the
               LC.  An average spectrum across the DFTPPO LC peak may be used to test instru-
               ment performance.
       6.16.5 An interfaced data system is required to acquire, store, reduce, and output mass
               spectral data.  The computer  software should have the capability of processing stored
               LC/MS data by integration of the ion abundance of any specific ion between specified
               time or scan number limits, construction of a first or second order regression calibra-
               tion curves, calculation of response factors as defined in Sect. 10.2.9, calculation of
               response factor statistics (mean and standard deviation),  and calculation of concentra-
               tions of analytes from the calibration curve or the equation in Sect.  12.
6.17  Millipore Standard Filter Apparatus, All Glass:  This will be used if the disks are to be used to
       carry out the extraction instead of cartridges.

7.     REAGENTS AND STANDARDS

7.1    Helium nebulizer/carrier gas as contaminant free as possible.
7.2   Liquid-Solid Extraction (LSE) Materials
       7.2.1   Cartridges are inert  non-leaching plastic, for example polypropylene, or glass and
               must not contain contaminants that leach into methanol.  The cartridges are packed
               with various amounts of sorbents such as Clg or a neutral polystrene/divinylbenzene
               polymer.  The packing must have a narrow size distribution and must not leach
               organic compounds  into  methanol.  One liter of water should pass through the car-
               tridge in about 2 hr  with the assistance of  a slight vacuum of about 13 cm  (5 in) of
               mercury.   Faster flow rates are acceptable if equivalent accuracy and precision are
               obtained.   Robotic systems topically pump the sample through a cartridge in less than
               2 hr.  These systems are also acceptable if equivalent accuracy and precision are
               obtained.   Sect. 9 and Tables 4 and 5 provide criteria for acceptable LSE cartridges
               which are available  from several commercial  suppliers.
       7.2.2   Extraction disks (Empore) are thin filter-shaped materials with C18 modified silica,  or
               neutral polystyrene/divinylbenzene polymer, impregnated in Teflon or other inert
               matrix. As with cartridges, the disks should not contain any organic compounds,
               either from the Teflon or the bonded silica, which  will leach into the methanol eluant.
               One liter  of reagent  water should pass through the  disks in 5-20 min using a vacuum
               of about 66 cm (26  in) of mercury. Sect.  9 provides criteria for acceptable LSE disks
               which are available  commercially.
7.3   Solvents
       7.3.1   Acetonitrile, methylene chloride, and methanol:  HPLC  grade and pesticide quality or
               equivalent.
       7.3.2   Reagent water:  Water in which an interferant is not observed at the MDL of the
               compound of interest. Prepare reagent water by passing tap water through a filter bed
326

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                                                                                     Method 553
               containing about 0.5 kg of activated carbon or by using a water purification system.
               Store in clean, narrow-mouth bottles with Teflon-lined septa and screw caps.
7.4   Hydrochloric acid, concentrated.
7.5   Sodium sulfate, anhydrous.
7.6   Reducing Agents for Chlorinated Water:  Sodium sulfite, sodium thiosulfate or sodium arse-
       nite.
7.7   Ammonium Acetate, Sodium Chloride, and Sodium Hydroxide (IN): ACS reagent grade.
7.8   Stock Standard Solutions (SSS):  Individual solution of analytes, surrogates, and isotopically
       labelled analogues  of the anlytes may be purchased as certified solutions or prepared from pure
       materials.  To prepare, add 10 mg (weighed on an analytical balance to 0.1 mg) of the pure
       material to 1.9 mL of methanol or acetonitrile in a 2-mL volumetric flask, dilute to the mark,
       and transfer the solution to an amber glass vial.  Certain analytes, such as 3,3' -dimethoxy-
       benzidine, may require dilution in 50% v/v acetonitrile or methanol:  water solution.   If the
       analytical standard is available only in quantities smaller than 10 mg, reduce the volume of
       solvent accordingly.  If compound purity is certified by the supplier at >96%, the weighed
       amount can be used without correction to calculate the concentration of the solution (5 /xg//xL).
       Store the amber vials in a freezer at < 0°C.
       7.8.1   Benzidines as the free base or as acid chlorides may be used for calibration purposes.
               However,  the concentration of the standard must be  calculated as the  tree base.
7.9   Primary Dilution Standard Solution (PDS):  The stock standard  solutions are used to prepare a
       primary dilution standard solution that contains multiple analytes.  The recommended solvent
       for this dilution is a 50% v/v acetonitrile:water mixture. Aliquot of each of the stock standard
       solutions are combined to produce the primary dilution in which the concentration of the
       analytes is a least equal to the concentration of the most concentrated calibration solution.
       Store the primary dilution standard solution in an amber vial  in  a freezer at < 0°C, and check
       frequently for signs of deterioration or evaporation, especially just before preparing calibration
       solutions.
7.10  Fortification Solution of Surrogates:  The analyst should monitor the performance of the
       extraction,  cleanup (when used), and  analytical system and the effectiveness of the method in
       dealing with each sample matrix by spiking each sample, standard,  and blank with 1 or 2
       surrogates recommended to encompass the range of the gradient elution program used  in this
       method.  The compounds recommended as surrogates for the analysis of benzidines and
       nitrogen-containing pesticides are benzidine-D8 (DBZ),  caffeine-15N2 (NCF), 3,3'-dichloro-
       benzidene-D6 (DCB), and bis-(perfluorophenyl)-phenylphosphine oxide (OD) unless their
       unlabelled counterpart is being analyzed or they will be used  for isotope dilution calibration
       (Abbreviations in parentheses are used in Figure 4).  Prepare a solution of the surrogates in
       methanol or acetonitrile at a concentration of 5 mg/mL of each.  Other surrogates may be
       included in this solution as needed. (A 10-/*L  aliquot of this  solution added to 1L of water
       gives a concentration of 50 /ug/L of each surrogate).  Store the surrogate fortifying solution in
       an amber vial in a freezer at <0°C.
7.11   MS  Performance Check Solution: Prepare a 100 ng/p.L solution of bis-(perfluorophenyl)-
       phenylphosphine oxide (DFTPPO) in  acetonitrile.  Store this  solution in an amber vial in a
       freezer at < 0° C° DFTPPO is not currently commercially available. For this method

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       development work, DFTPPO was synthesized from bis-(perfluorophenyl) phenyl phosphine
       (DFTPP) in solution by adding a slight excess ui hydrogen peroxide (DFTPP + H2O2 -»
       DFTPPO +  H2O). The solvent was removed and the resulting crystals were thoroughly
       washed with water to remove any residual hydrogen peroxide.  It is critical to remove all
       residual hydrogen peroxide before adding the DFTPPO to the CAL solution.  Any residual
       hydrogen peroxide will  degrade some analytes.
7.12  Calibration Solutions (CAL1-CAL6): Prepare a series of six concentration calibration solu-
       tions in acetonitrile which contain all analytes at concentrations of 2, 5,  10, 25, 50 and  100
       times the instrument detection limit of each compound with a constant concentration of each
       surrogate in each CAL solution.  This calibration range may be optimized by the operator, but
       each analyte must be bracketed by at least 2 calibration points.  CAL1 through CAL6 are
       prepared by combining  appropriate aliquots of the primary dilution standard solution (Section.
       7.9) and the  fortification solution of surrogates (Sect. 7.10).  DFTPPO may be added to one  or
       more CAL solutions to  verify MS tune (See Sect. 10.3.1.).  Store these solutions in amber
       vials in a freezer at < 0°C.  Check these solutions quarterly for signs of deterioration.
       7.12.1  For isotope dilution calibration, prepare the calibration solutions as described above
               with the addition  of one coeluting isotopically labelled analog for each analyte of
               interest.  The concentration for each coeluting labelled standard should be approxi-
               mately  25 to 50 times the instrument detection limit of the analyte of interest and must
               be constant in all calibration solutions (CAL1 through CAL6).  These solutions permit
               the relative response (unlabelled to labelled) to be measured as a function of the
               amount of analyte injected.  If more than one labelled compound is used, one spiking
               solution containing all labelled compounds should be prepared.
7.13  Mobile Phase:   Solvent A is a 75:25 v/v watenacetonitrile solution containing ammonium
       acetate at a concentration of 0.01 M.  This composition is used to eliminate biological activity
       in the A Phase.   Solvent B is acetonitrile.  Both solvents are degassed in an ultrasonic bath
       under reduced pressure and maintained by purging with a low flow of helium.

8.     SAMPLE COLLECTION,  PRESERVATION AND STORAGE

8.1    Sample Collection:  When sampling from a water tap, open the tap and allow the system to
       flush until the water temperature has stabilized (usually about 2-5 min). Adjust the  flow to
       about 500 mL/min and  collect samples from the flowing stream.  Keep samples sealed from
       collection time until analysis.  When sampling from an open body of water, fill the sample
       container with water from a representative area.  Sampling equipment,  including automatic
       sampler, must be free of plastic tubing, gaskets, and other parts that may leach analytes into
       water.  Automatic samplers that composite samples over time must use refrigerated  glass
       sample containers.
8.2   Sample Dechlorination and Preservation:  All samples should be iced or refrigerated at  4°C
       from the time of collection until extraction. Residual chlorine should be reduced at  the
       sampling site by addition of a reducing agent.  Add  40-50 mg of sodium sulfite or  sodium
       thiosulfate (these may be added as solids with stirring until dissolved) to each liter of water.
8.3   Holding Time:  Samples must be extracted within 7 days and the extracts analyzed within 30
       days of sample  collection.  Extracts should be stored in an amber vial in a freezer at <  0°C.
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8.4   Field Blanks
       8.4.1   Processing of a field reagent blank (FRB) is recommended along with each sample
               set, which is composed of the samples collected from the same general sample site at
               approximately the same time. At the laboratory, fill a sample container with reagent
               water, seal, and ship to the sampling site along with the empty sample containers.
               Return the FRB to the laboratory with filled sample bottles.

9.     QUALITY  CONTROL

9.1    Quality control (QC) requirements are the initial demonstration of laboratory capability fol-
       lowed by regular analyses  of LRBs, LFBs, and laboratory fortified matrix samples.  The
       laboratory must maintain records to document the quality of the data generated. Additional
       QC practices are recommended.
9.2   Initial demonstration of low system background and acceptable particle size and packing.
       Before any  samples are analyzed, or any time a new supply of LSE cartridges or disks is
       received from a supplier, or  a new column is installed, it must be demonstrated that a LRB is
       reasonably free of contamination that  would prevent the determination of any analyte of
       concern.  In this same experiment it must be demonstrated that the particle size and packing of
       the LSE cartridge are acceptable.  Consistent flow rate is an indication of acceptable particle
       size distribution and packing.
       9.2.1   A source of potential contamination may be the liquid-solid extraction (LSD) car-
               tridges and  disks and columns which may contain silicon compounds  and  other con-
               taminants that could prevent the determination of method analytes. Generally, con-
               taminants will  be leached from the cartridges,  disks, or columns into  the solvent and
               produce a variable background.  If the background contamination  is sufficient to
               prevent accurate and precise  analyses, this condition must be corrected before pro-
               ceeding with the initial demonstration.  Figure 1 show unacceptable background
               contamination  from  a column with stationary phase bleed.
       9.2.2   Other sources of background contamination are solvents, reagents, and glassware.
               Background contamination must be reduced to an acceptable level before proceeding
               with the next section.  In general, background for method analytes should be below
               the MDL.
       9.2.3   One liter of water should pass through the cartridge in about 2 hr (faster flow rates
               are acceptable  if precision and accuracy are acceptable) with a partial vacuum of about
               13  cm (5 in) of mercury.  The extraction time should not vary unreasonably among
               LSE cartridges. Robotic systems typically pump the sample through  a cartridge in
               less than 2 hr.  These systems are also acceptable if equivalent accuracy and precision
               are obtained.  Extraction disks may be used at a faster flow rate (See Sect. 7.2.2).
9.3   Initial Demonstration of Laboratory Accuracy and Precision:  Analyze 5-7 replicates of a LFB
       containing each analyte of  concern at  a concentration in the range of 10-50 times the instru-
       ment detection limits (see regulation and maximum contaminant levels for  guidance on appro-
       priate concentrations).
       9.3.1   Prepare each replicate by adding an appropriated aliquot of the PDS,  or another certi-
               fied quality control sample, to reagent water.  Analyze each replicate according to the

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               procedures described in Sect. 11 and on a schedule that results in the analyses of all
               replicates with 48 hr.
       9.3.2   Calculate the measured concentration of each analyte in each replicate, the mean
               concentration of each analyte in all replicates, and mean accuracy (as mean percentage
               of true value) for each  analyte, and the precision (as relative standard deviation, RSD)
               of the measurements for each analyte.  Calculate the MDL of each  analyte using the
               referenced procedures.1
       9.3.3   For each analyte and surrogate, the mean accuracy, expressed as a  percentage of the
               true value, should be 70-130% and the RSD should be <  30%. The MDLs  should
               be sufficient to  detect analytes at the  regulatory levels.  If these criteria are not met
               for an analyte, take remedial action and repeat the measurements for the analyte to
               demonstrate acceptable performance before samples are analyzed.
       9.3.4   Develop and maintain a system of control charts to plot the precision and accuracy of
               analyte and surrogate measurements as a function of time.  Charting of surrogate
               recoveries is an especially valuable activity since these are present in every sample
               and the  analytical results will form a significant record of data quality.
9.4   Laboratory Reagent Blanks (LRBs):  With each batch of samples processed  as a group within a
       work shift, analyze a laboratory reagent blank to determine the background  system contami-
       nation.  Any time a new batch of LSE cartridges or disks is used, or new supplies of other
       reagents are used,  repeat the demonstration of low background described in Sect. 9.2.
9.5   With each batch of samples processed as a group within a work shift, analyze a single LFB
       containing each analyte of concern at a concentration as determined in Sect. 9.3.  Evaluate the
       accuracy of the measurements (Sect. 9.3.3), and estimate whether acceptable MDLs can be
       obtained.  If acceptable accuracy and MDLs cannot be achieved, the problem must be located
       and corrected before further samples are analyzed.  Add these results to  the ongoing control
       charts to document data quality.
9.6   Determine that the sample matrix does not contain materials that adversely affect method
       performance. This is accomplished by analyzing replicates of laboratory fortified matrix
       samples and ascertaining that the precision, accuracy, and MDLs of analytes are  in the same
       range as obtained with LFBs.  If a variety of different sample matrices are analyzed regularly,
       for example, drinking water from groundwater and surface water sources, matrix independence
       should be established for each.
9.7   With each set of field samples  a FRB should be analyzed. The results of these analyses will
       help define contamination resulting from field sampling and transportation activities.
9.8   At least quarterly, replicates of LFBs should be analyzed to determine the precision of the
       laboratory measurements. Add these results to the ongoing control charts to document data
       quality.
9.9   At least quarterly, analyze a QCS from an external source.  If measured analyte concentrations
       are  not of acceptable accuracy  (Sect. 9.3.3), check the entire analytical procedure to locate  and
       correct the problem source.
9.10  Numerous other specific QC measures are incorporated into other parts of this procedure, and
       serve to alert the analyst to potential problems.
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10.   CALIBRA TION AND STANDARDIZA TION

10.1   Demonstration and documentation of acceptable initial calibration and system optimization are
       required before any samples are analyzed and is required intermittently during sample analysis
       as indicated by results of continuing calibration checks. After initial calibration is successful,
       a continuing calibration check is required at the beginning of each 8-hr period during which
       analyses are performed.  Additional periodic calibration checks are good laboratory practice.
10.2   Initial Calibration
       10.2.1    Optimize the interface according to the manufacturer's instructions. This usually is
                 accomplished on initial installation by flow injection with caffeine or benzidine and
                 should utilize a mobile phase of 50% v/v acetonitrile:water.  Major maintenance
                 may require reoptimization.
       10.2.2    Calibrate the MS mass and abundance scales using the calibration compound and
                 manual (not automated) ion source tuning procedures specified by the manufactur-
                 er.  Calibration must be accomplished while a 50%  v/v acetonitrile:water mixture
                 is pumped through the LC column and the optimized particle beam interface.  For
                 optimum long-term stability and precision, interface and ion  source parameters
                 should be set near the  center of a broad signal plateau rather than at the peak of a
                 sharp maximum (sharp maxima vary short term with particle beam interfaces and
                 gradient elution conditions).
       10.2.3    Fine tune the interface by making a series of injections into the LC column of a
                 medium level CAL standard and adjusting the operating parameters until optimum
                 sensitivity and precision are obtained for the maximum number of target com-
                 pounds.6  Suggested additional operating conditions are:
                 mobile phase purge: helium at 30 mL/min continuous,
                 mobile phase flow  rate:  0.3 mL/min through the column,
                 gradient elution: hold for 1 min at  25 % acetonitrile, then linearly program to
                         = 70% acetonitrile in 29 min, start data acquisition immediately,
                 post-column addition:  acetonitrile at 0.1-0.7 mL/min, depending on the interface
                        requirements.  Maintain a minimum of 30% acetonitrile  in the interface to
                        improve system precision and possibly sensitivity,
                 desolvation chamber temperature:  45°-80°C,
                 ion source temperature:  250°-290°C,
                 electron energy: 70 eV, and
                 scan range:  62-465 amu at 1-2 sec/scan.
       10.2.4 The medium level standard (CAL) used in  Sect. 10.2.3 should contain DFTPPO, or
              separately inject into the LC a 5-^.L aliquot of the  100 ng/jtl DFTPPO solution and
              acquire a mass  spectrum that includes data form M/Z 62-465.  Use  LC conditions that
              produce a narrow (at least ten scans per peak) symmetrical peak.  If the spectrum
              does not meet all  criteria  (Table 1), the MS ion source must be returned and adjusted
              to meet all criteria before proceeding with calibration.  An average  spectrum across
              the LC peak may be used to evaluate the performance of the system.  Figure 2 repre-
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Method 553
               sents the average composite spectrum obtained for DFTPPO from a multilaboratory
               study involving 5 different particle beam interfaces from 13 laboratories.
       10.2.5 Inject a 5 /xL aliquot of a medium concentration calibration solution, for example 50
               ng/fiL,  and acquire and store data from m/z 62-465 with a total cycle time (including
               scan overhead time) of  1.5 sees or less.  Cycle time should be adjusted to measure at
               least ten spectra during the elution of each LC peak.
               10.2.6.1  LC Performance: 3,3'-dimethyl- and 3,3'-dimethoxybenzidine should be
                         separated by a valley whose height is less than 25% of the average peak
                         height of these two compounds. If the valley between them exceeds 25%,
                         modify the gradient.  If this fails, the LC column requires maintenance.
                         (See Sect. 10.3.6)
               10.2.6.2 Peak tailing: Examine a total ion chromatogram and determine the degree
                         of peak tailing.  Severe tailing indicates a major problem and system main-
                         tenance is required to correct the problem. (See Sect. 10.3.6)
               10.2.6.3 MS sensitivity:  Signal/noise in any analyte mass spectrum should be at
                         least 3:1.
               10.2.6.4 Column bleed:  Figure  1 shows an unacceptable chromatogram with
                         column bleed.  Figure 3 is the mass spectrum of dimethyloctadecylsilanol,
                         a common stationary phase bleed product.  If unacceptable column bleed is
                         present, the  column must be  changed or conditioned to produce an accept-
                         able background (Figure 4).
               10.2.6.5 Coeluting  compounds:  Compounds which coelute cannot be measured
                         accurately because of carrier effects in the particle beam interface. Peaks
                         must be examined carefully for coeluting substances and if coeluting com-
                         pounds are present at greater than  10% the concentration of the target
                         compound, condition must be adjusted to resolve the components, the
                         target compound must be flagged as positively biased, or isotope dilution
                         calibration should be used.
       10.2.7 If all performance criteria are met, inject a 5 /xL aliquot of each of the other CAL
               solutions using the same LC/MS conditions.
       10.2.8 The general method of calibration (external) is a second order regression of integrated
               ion abundances of the quantitiation ions  (Table 2) as a function of amount injected.
               For second order regression, a sufficient number of calibration points must be ob-
               tained to accurately determine the equation of the curve. For some individual analytes
               over a short concentration range, reasonable linearity may be observed and response
               factors  may be used.  Calculate response factors  using the equation below.  Second
               order regressions and response factor calculations are supported in acceptable LC/MS
               data system software (Sect.  6.16.5), and may  other software programs.
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                                                                                    Method 553
                where:
                Ax = integrated abundance of the quantitation ion of the analyte.
                Qx = quantity of analyte injected in ng or concentration units.
       10.2.9 If response factors are used (i.e., linear calibration with the line going through the
               origin), calculate the mean RF from the analyses of the six CAL solutions for each
               analyte and surrogate. Calculate the standard deviation (SD) and the relative standard
               deviation (RSD) from each mean (M): RSD = 100  (SD/M). If the RSD of any
               analyte or surrogate mean RF exceeds 20%, either analyze additional aliquots of
               appropriate CAL solutions to obtain an acceptable RSD of RFs over the entire concen-
               tration range, take action to improve LC/MS performance, or use the second order
               regression calibration. (See Sect. 10.2.8)
10.3  Continuing Calibration Check:  Verify the MS tune and initial calibration at the beginning of
       each 8-hr work shift during which analyses are performed using the following procedure:
       10.3.1  Inject a 5-/iL aliquot of the 100 ng//xL DFTPPO solution (this may be contained in the
               medium level CAL solution used in Sect.  10.3.2) and acquire a mass spectrum that
               includes data for m/z 62-465. If the spectrum does not meet all criteria (Table 1), the
               MS must be  returned and adjusted to meet all criteria before proceeding with the
               continuing calibration check.
       10.3.2 Inject a 5-/xL aliquot of a medium level CAL solution and analyze with the same
               conditions used during the initial  calibration.  One or more additional CAL solutions
               should be analyzed.
       10.3.3 Demonstrate acceptable performance for the criteria shown in Sect. 10.2.6.
       10.3.4 Determine that the absolute areas of the quantitation ions of the external standards and
               surrogate(s) have not changed by more than 20% from the areas measured during
               initial calibration.  If these areas have changed by more than 20%, recalibration and
               other adjustment are necessary.  These adjustments may  require cleaning of the MS
               ion source, or other maintenance  as indicated in Sect. 10.3.6, and recalibration.
               Control charts are useful aids in documenting system sensitivity changes.
       10.3.5 Using the previously generated second order regression curve, calculate the concentra-
               tions  in the medium level CAL solution and compare the results to the known values
               in the CAL solution. If calculated concentrations deviate by more than 20% from
               known values, recalibration of the system with the 6 CAL solutions is required. If
               response factors were used, calculate the RF for each  analyte and surrogate from the
               data measured in the continuing calibration check.  The RF for each analyte and
               surrogate must be within 20% of the mean value measured in the initial calibration.
       10.3.6  Some possible remedial actions of Major maintenance such as cleaning  an ion source,
               cleaning quadrupole rods, etc. require returning to the initial calibration step.
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Method 553
               10.3.6.1 Check and adjust LC and /or MS operating conditions: check the MS
                         resolution, and calibrate the mass scale.
               10.3.6.2 Replace the mobile phases with fresh solvents.  Verify that the combined
                         flow rate from the LC  and post-column addition pumps is constant.
               10.3.6.3 Flush the  LC column with acetonitrile.
               10.3.6.4 Replace LC column; this action will cause a change in retention  times.
               10.3.6.5 Prepare fresh CAL solutions and  repeat the initial calibration step.
               10.3.6.6 Clean the MS ion source, entrance lens, and rods (if a quadrupole).
               10.3.6.7 Replace any component that leak.
               10.3.6.8 Replace the MS electron multiplier or any other faulty components.
               10.3.6.9 Clean the interface  to eliminate plugged components  and/or replace skim-
                         mers according to the manufacturer's instructions.
               10.3.6.10  If automated peak areas are being used, verify values by manual integra-
                         tion.
               10.3.6.11  Increasing ion source temperature can reduce peak tailing, but excessive
                         ion source temperature can affect the quality of the spectra for some com-
                         pounds.
               10.3.6.12  Air leaks into the interface may affect the quality of the spectra (e.g.
                         DFTPPO), especially when ion source  temperatures are operated in excess
                         of 280°.
10.4  Calibration with Isotope Dilution (Optional): For samples  with interfering matrix or coeluting
       peaks, the most reliable method for quantitiation is the use of coeluting isotope labelled
       internal standards.7  Isotope dilution calibration will be limited by the availability and cost of
       the labelled species and the requirement that each analyte must coelute with the labelled
       internal standard.  Because the labelled internal standard must coelute with the analyte, the
       quantitation ion for the internal standard must be larger than that of the analyte and not present
       in the anlayte's mass spectrum. In addition, it must be verified that the labelled internal
       standard is not contaminated by its unlabelled counterpart.
       10.4.1  A calibration curve encompassing  the concentration range is prepared for each com-
               pound to  be  determined.  The relative response (analyte integrated ion abundances to
               labelled integrated ion abundance) vs. amount of analyte injected is plotted using
               linear regression.  A minimum of five data points  are employed for this type of
               calibration.
       10.4.2 To calibrate, inject a 5.0 p.L aliquot of each of the calibration standards (Sect. 7.12.1)
               and compute the relative response (analyte integrated ion abundances  to labelled
               compound integrated ion abundance).  Plot this versus the amount of analyte injected
               by linear regression.   This plotted line or the equation of this line should be used for
               quantitative calculations.  Unless this line goes through the origin, the response  factors
               at each point will not constant  and therefore, average response factors cannot be used.
               These calculations are supported in acceptable LC/MS data system software (Sect.
               6.15.5), and in many other software programs.
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                                                                                     Method 553
       10.4.3 Following Sect. 10.3 to verify calibration at the beginning of each 8-hr work shift by
               injecting a 5.0 /xL aliquot of a medium CAL solution and analyze it with the same
               conditions used during the initial calibration. Using the previously generated first
               order regression line (relative response versus amount of analyte injected), calculate
               the concentrations in the medium level CAL solution and compare the results to the
               known values in the CAL solution.  If calculated concentrations deviate by more than
               20% from know values,  recalibration of the system with the CAL solutions, contain-
               ing the isotopically labelled analogues, is required.

11.   PROCEDURE

11.1   The extraction procedure depends on the anlaytes selected and the nature of the sample. LSE
       (cartridge or disk) is limited to particulate-free water, e.g., drinking water. Consult Table 3-5
       to determine which analytes are amenable to  liquid-solid and liquid-liquid extraction.  Sect.
       11.2 provides the LSE procedure using cartridges and Sect.  11.3 provides the LSE procedure
       using disks.  Sect.  11.4 provides  the procedure for LLE. After the extraction is complete,
       proceed to Sect. 11.5 to continue with the method.
11.2   Liquid-Solid Extraction (LSE) Procedure Using Cartridges (This procedure may be manual or
       automated).
       11.2.1  Set up the extraction apparatus shown in Figure 5.  The reservoir is not required but
               recommended for convenient operation.  Water drains from the reservoir through  the
               LSE cartridge and into a syringe needle which is inserted through a rubber stopper
               into the suction flask. A slight vacuum of 13 cm (5 in) of mercury is used during all
               operations with the apparatus. The pressure used is critical as a vacuum  greater than
               13 cm may result in poor precision.  About 2 hr is required to draw a liter of water
               through the cartridge, but faster flow rates are acceptable if precision and accuracy
               are acceptable.  The use  of robotic extraction systems is acceptable if equivalent
               MDLs, precision and accuracy are obtained.
       11.2.2 Mark the water meniscus on the side of the sample bottle for later determination of
               the sample volume.  A 1-L sample is recommended. Pour the water sample into the
               2-L  separator funnel with the stopcock closed.  Adjust the pH to 7.0 by the dropwise
               addition of hydrochloric  acid or  1 N sodium hydroxide. Residual chlorine must not
               be present, as a reducing agent should have been added at the time of sampling.  For
               extractions using C,8  cartridges,  add 0.01 M ammonium acetate (0.77  g in 1 L) to the
               water sample and mix until homogeneous.  Add a 10-/xL aliquot of the fortification
               solution for surrogates, and mix until homogeneous. The concentration of surrogates
               in the water should be 10-50 times the instrument detection limit.
       11.2.3 Flush each cartridge with two 10-mL aliquots of methanol, letting the  cartridge drain
               dry after each flush.  This  solvent flush may be accomplished by adding methanol
               directly to the solvent reservoir in Figure 5. Add 10 mL of reagent water to the
               solvent reservoir, but before the reagent water level drops below the top edge of the
               packing in the LSE cartridge, open the stopcock of the separatory funnel  and begin
               adding sample water to the solvent reservoir. Close the stopcock when an adequate
               amount of sample is in the reservoir.
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Method 553
       11.2.4 Periodically open the stopcock and drain a portion of the sample water into the solvent
               reservoir.  The water sample will drain into the cartridge, and from the exit into the
               suction flask.  Maintain the packing material in the cartridge immersed in water at all
               times.  Wash the separatory funnel and cartridge with 10 mL of reagent water, and
               draw air through the cartridge  for 10  min.
       11.2.5 Transfer the LSE cartridge to the elution apparatus shown in Figure 5B.  Wash the
               2-L separatory funnel with 15 mL of  methanol, close the stopcock of the 100-mL
               separatory funnel of the elution apparatus, and elute the cartridge with two 7.5-mL
               aliquots of the methanol washings. Concentrate the extract to the desired volume
               under a gentle stream of nitrogen. Record the exact volume  of the extract.
               11.2.5.1  If isotope dilution calibration is used, spike the extract with the isotopically
                         labelled standards prior to solvent evaporation.  The  concentration of these
                         isotopically  labelled compounds after the desired extract volume is reached
                         should be the same  as the  concentration in each CAL solution.
       11.2.6 Determine the original  sample  volume by  refilling the sample bottle to the mark and
               transferring the liquid to a 1000-mL graduated cylinder. Record the sample volume
               to the nearest 5 mL.
11.3  Liquid-Solid Extraction (LSE) Procedure Using Disks (This procedure may be manual or
       automated).
       11.3.1  Mark the water meniscus on the side  of the sample bottle for later determination of
               the sample volume. A  1-L sample is  recommended.  Pour the water sample into the
               2-L separatory funnel with the  stopcock closed. Adjust the pH to 7.0 by the dropwise
               addition of hydrochloric acid or 1 N sodium hydroxide.  Residual chlorine must not
               be present because a reducing agent should have been added at the time of sampling.
               For extractions using C18 disks, add 0.01 M ammonium  acetate (0.77 g in 1 L) to the
               water sample and mix until homogeneous.  Add a 10-/*L aliquot of the fortification
               solution for surrogates, and mix until  homogeneous.  The concentration of surrogates
               in the water should be  10-50 times the instrument detection limit.
       11.3.2 Preparation of Disks
               11.3.2.1  Inset the disk into the 47 mm filter apparatus (See Figure 6).  Wash and
                         pre-wet the  disk with  10 mL methanol (MeOH) by adding the MeOH to
                         the disk and allowing  it to soak for about a min, then pulling most of the
                         reaming MeOH through.   A layer of MeOH must be left on the surface of
                         the disk, which should not be allowed to go dry from this point until the
                         end of the sample extraction. THIS IS A CRITICAL STEP FOR A UNI-
                         FORM FLOW AND GOOD RECOVERY.
               11.3.2.2 Rinse the disk with  10 mL reagent water by adding the water to the disk
                         and pulling  most through,  again leaving a layer of water on the surface of
                         the disk.
       11.3.3 Add the water sample to the reservoir and turn on the vacuum to begin the extraction.
               Full aspirator vacuum may be used.   Particulate-free water may  pass through the disk
               in as little as 10 minutes or less.   Extract  the entire sample, draining as much water
               from the sample container as possible.

336

-------
                                                                                    Method 553
       11.3.4 Remove the filtration top from the flask, but do not disassemble the reservoir and
               fritted base.  Empty the water from the flask, and insert a suitable sample tube to
               contain the eluant.  The only constraint on the sample tube is that it must fit around
               the drip tip of the fritted base.  Reassemble the apparatus.
       11.3.5 Add 5 mL MeOH to the sample bottle, and rinse the inside walls thoroughly.  Allow
               the MeOH to settle to the bottom of the bottle, and transfer to the disk with a dis-
               posable pipet, rinsing the sides of the glass filtration reservoir in the process.  Pull
               about half of the MeOH through the disk, release the vacuum, and  allow the disk to
               soak for a minute.  Pull the remaining  MeOH through the disk.
       11.3.6 Repeat the above step twice. Concentrate the combined extracts to the desired volume
               under  a gentle stream of nitrogen. Record the extract volume of the extract. (Prelim-
               inary investigation indicates that acetonitrile is a better extraction solvent for rotenone
               when extracting water, containing high levels of particulate matter,  with LSE disks.)
               11.3.6.1  If isotope dilution calibration is used,  spike the extract with the isotopically
                         labelled standards prior to solvent evaporation. The  concentration of these
                         isotopically labelled compounds after the desired extract volume is reached
                         should be the same as the concentration in each GAL solution.
       11.3.7 Determine the original sample volume  by refilling the sample bottle to the mark and
               transferring the  liquid to a 1000-mL graduated cylinder.  Record the sample volume
               to the  nearest 5  mL.
11.4  Liquid-Liquid Extraction (LLE) Procedure
       11.4.1  Mark the water  meniscus on the side of the sample bottle for later determination of
               the sample volume.  A 1-L sample is recommended.  Pour the water sample into a
               2-L separatory funnel with the stopcock closed. Residual chlorine should not be
               present as a reducing agent should have been added at the time of sampling.  Add a
               10-jtL aliquot of the fortification solution for surrogates, and mix until homogeneous.
               The concentration of surrogates in the water should be 10-50 times the instrument
               detection limit.
       11.4.2 Adjust the pH of the sample to 7.0 by  dropwise addition of hydrochloric acid or  1 N
               sodium hydroxide.  Add 100 g of sodium chloride to the sample and shake to dissolve
               the salt.
       11.4.3 Add 60 mL of methylene chloride to the sample bottle, shake, and transfer the  solvent
               to the  separatory funnel and extract the sample by vigorously shaking the funnel for  2
               min with periodic venting to release excess pressure. Allow  the organic  layer to
               separate from the water phase for a minimum of  10 min.  If the emulsion interface
               between layers is more than one-third the volume of the solvent layer, mechanical
               techniques must be employed to complete the phase separation.  The optimum tech-
               nique depends on the sample, but may  include stirring, filtration of the emulsion
               through glass wool,  centrifuging, etc.   Collect  the methylene chloride extract in a 500-
               mL Erlenmeyer  flask.
       11.4.4 Add a second 60-mL volume of methylene chloride and repeat the extraction a  second
               time, combining the extracts in the Erlenmeyer flask.  Perform a third extraction in
               the same manner.

                                                                                           337

-------
Method 553
       11.4.5 Assemble a K-D concentrator by attaching a 10-mL concentrator tube to a 500-mL
              evaporative flask.  Dry the extract by pouring it through a solvent-rinsed drying
              column containing about 10 cm of anhydrous sodium sulfate.  Collect the extract in
              the K-D concentrator, and rinse the column with 20-30 mL of methylene chloride.
       11.4.6 Add 1 or 2 clean boiling stones to the evaporative flask and attach a macro Snyder
              column.  Pre-wet the Snyder column by adding about  1 mL of methylene chloride to
              the top.  Place the K-D apparatus on a hot water bath, 65-70°C, so that the con-
              centrator tube is partially immersed in the hot water, and the entire lower rounded
              surface of the flask is bathed with hot vapor.   Adjust the vertical position of the
              apparatus and the water temperature as required to complete the concentration in
               15-20 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 the liquid
              reaches 2 mL, add 20 mL of methanol through the Snyder column using a syringe and
              needle.  Raise the temperature of the hot water bath to 90°C,  and concentrate the
              sample to about 2 mL.  Concentrate the extract to the desired volume under a gentle
              stream of nitrogen.  Record the exact volume of the concentrated extract.
               11.4.6.1 If isotope dilution calibration is used, spike the extract with the isotopically
                         labelled  standards prior to solvent evaporation. The concentration of these
                         isotopically labelled compounds after the desired extract volume is reached
                         should be the same as the concentration in each CAL solution.
       11.4.7 Determine the original sample volume by  refilling the sample  bottle to the mark and
              transferring the liquid to a 1000-mL gradulated cylinder.  Record the sample volume
              to the nearest 5 mL.
11.5  Liquid Chromatography/Mass Spectrometry (LC/MS)
       11.5.1 Analyze a 5-/tL aliquot with the LC/MS system under the same conditions used for
              the initial and continuing calibrations (Sect. 10.2).
11.6  Identification of Analytes
       11.6.1 At the conclusion of data acquisition, use the system software to display the chro-
              matogram, mass spectra and retention times of the peaks  in the chromatogram.
       11.6.2 Identify a sample component by comparison of its mass spectrum (after background
              subtraction) to a reference spectrum in the user-created data base. The LC retention
              time of the sample component should be within 10 sec of the time observed for that
              same compound when a calibration solution was analyzed.  In  general, all ions that
              are present above 10% relative abundance in the mass spectrum of the standard should
              be present in the mass spectrum of the sample component and  should agree within
              absolute 20%.  For example,  if an ion has a relative abundance of 30% in the stan-
              dard spectrum, its abundance  in the sample spectrum should be in the range of 10 to
              50%.  Some ions, particularly the molecular ion, are of special importance, and
              should be evaluated even if they are below 10% relative abundance.
       11.6.3  Use the data system software  to examine the  ion abundances of components of the
              chromatogram.  If any ion abundance exceeds the system working range, dilute the
               sample aliquot and  analyze the diluted aliquot.
338

-------
                                                                                    Method 553
       11.6.4 Identification is hampered when sample components are not resolved chromatographi-
               cally and produce mass spectra containing ions contributed by more than one analyte.
               When LC peaks obviously represent more than one sample component (i.e., broad-
               ened peak with shoulder(s) or valleys between two or more maxima), appropriate
               analyte spectra and background spectra can be selected  by examining plots of charac-
               teristic ions for tentatively identified components.  When analytes coelute (i.e., only
               one LC peak is apparent), the identification criteria can be met but each analyte
               spectrum will contain extraneous ions contributed  by the coeluting compound.
       11.6.5 Structural isomers that produce very similar mass  spectra can be explicitly identified
               only if they have sufficiently different LC retention times.  (See Sect. 10.2.6.1.)
               Acceptable resolution is achieved  if the height of the valley between two isomer peaks
               is less than 25% of the average height of the two peak heights. Otherwise, structural
               isomers are identified as isomeric pairs.
       11.6.6 Background components appear in variable quantities in laboratory and field reagent
               blanks, and generally subtraction of the concentration in the blank from the concentra-
               tion in the sample is not recommended  because the concentration of the background in
               the blank is highly variable.  If method analytes appear in the blank, then resample.

12.  DATA ANALYSIS AND CALCULATIONS

12.1  Complete chromatographic resolution is necessary for accurate and precise measurements of
      analyte concentrations.  Compounds which coelute cannot be measured accurately because of
      carrier effects in the particle beam interface.2 Peaks must be examined carefully for coeluting
      substances and if coeluting compounds are present at greater than 10% the concentration of the
      target compound, either conditions must be adjusted to resolve the components, or the target
      compound must be removed from the list of quantitative analytes.
12.2  Use the LC/MS system software or other available proven software to compute the concentra-
      tions of the analytes  and surrogates from the second order  regression  curves.  Manual verifica-
      tion of automated integration is recommended.
       12.2.1 For isotope dilution calculations, use the first  order plot of relative response (analyte
               integrated ion abundances to labelled integrated ion abundance) vs. amount of analyte
               injected or the equation of the line to compute concentrations. If the plotted line does
               not go through the origin, response factors will not be constant at each calibration
               point; therefore, average response factors cannot be used.
12.3  If appropriate, calculate analyte and surrogate concentrations from response factors and the
      following equation.
                                                                                           339

-------
Method 553
                                        c  .
                                             RF V V
         where:
          CA = Concentration of analyte  or surrogate in pglL in the water sample.
          Ax = integrated abundance of the quantitation ion of the analyte in the sample.
           V = original water sample volume in liters.
         RF = mean response factor of analyte the initial the initial calibration.
          Ve = volume of final extract in pL
          V = injection  volume in \iL
 13.   METHOD PERFORMANCE
 13.1   Single laboratory accuracy and precision data (Tables 3-5) for each listed analyte were ob-
       tained.  Five to seven 1-L aliquots of reagent water containing approximately 5 times the MDL
       of each analyte were analyzed with this procedure.  (For these experiments, the final extract
       volume was 0.5 mL.)
       13.1.2 With these data, MDLs were calculated using the formula:
                                                 .,.-0=099,
    where'.
    l(n-\ i-a = 099)  = sfudent's t value for the 99% confidence level with n-\ degrees of freedom
             n  = number of replicates
             S  = standard deviation of replicate analyses.

13.2  A multilaboratory (12 laboratories) validation of the determinative step was done for four of
       the analytes: benzidine (BZ), 3,3'-dimethoxybenzidine (MB), 3,3'-dimethylbenzidine (LB),
       and 3,3'-dichlorobenzidine (DB).  Table 6 gives the results from this study for single labora-
       tory precision,  overall  laboratory precision, and overall laboratory accuracy.  The two con-
       centration levels shown represent the two extremes of the concentration range  studied.

14.   POLLUTION PREVENTION

14.1  Although this method allows the  use of either  LLE or LSE, LSE is highly recommended
       whenever possible.  Only small amounts of methanol are used with this procedure as compared
       to much larger amounts of methylene chloride used for LLE. All other compounds used are
       neat materials used to prepare standards and sample preservatives.  All compounds are used in
       small amounts  and pose minimal threat to the  environment if properly disposed.
14.2  For information about  pollution prevention that may be applicable to laboratory operations,
       consult "Less Is Better:  Laboratory Chemical Management for Waste Reduction" available
       from the American Chemical Society's Department of Government Relations and  Science
       Policy, 1155 16th Street, N.W.,  Washington,  D.C., 20036.

340

-------
                                                                                   Method 553
15.    WASTE MANAGEMENT

15.1  There are generally no waste management problems involved with discarding spent or left over
      samples in this method since most often the sample matrix is drinking water.  If a sample is
      analyzed which appears to be highly contaminated with chemicals, analyses should be carried
      out to assess the type and degree of contamination so that the samples may be discarded
      properly. All other expired standards should be discarded properly.  It is the laboratory's
      responsibility to comply with all applicable regulations for waste disposal.  The Agency
      requires that laboratory waste management practices be conducted consistent with all applicable
      rules and regulations, and that laboratories protect the air, water, and land by minimizing and
      controlling all releases  from fume hoods and bench operations.  Also, compliance is required
      with any sewage discharge permits and regulations, particularly the hazardous waste identi-
      fication rules and land disposal restrictions. For further information on waste management,
      consult "The Waste Management Manual for Laboratory Personnel" also available from the
      American Chemical Society at the address in Sect.  14.2.
                                                                                          341

-------
Method 553
                                    References
1.     Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
      Wastewaters," Environ. Sci. Techno!. 1981, 15, 1426-1435.

2.     Bellar, T.A., T.D. Behymer, and W.L.  Budde, "Investigation of Enhanced Ion Abundances
      from a Carrier process in High-Performance Liquid Chromatography Particle Beam Mass
      Spectrometry," J. Am. Soc.  Mass Spectrom.. 1990, 1, 92—98.

3.     "Carcinogens—Working With Carcinogens," Department of Health, Education, and Welfare,
      Public Health Service, Center for Disease Control, National Institute for Occupational Safety
      and Health, Publication No.  77-206, Aug. 1977.

4.     "OSHA Safety and Health Standards, General Industry," (29CFK1910), 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.     Behymer,  T.D., T.A. Bellar, and W.L.  Budde, "Liquid Chromatography/Particle Beam/Mass
      Spectrometry of Polar Compounds of Environmental Interest," Anal. Chem.. 1990, 62,
      1686-1690.

7.     Ho, J.S., T.D. Behymer, W.L. Budde, and T.A. Bellar, "Mass Transport and Calibration in
      Liquid Chromatography/Particle Beam/Mass Spectrometry," J. Am. Soc. Mass Spectrom..
      1992, 3, 662-671.
342

-------
                                                                              Method 553
Table  1.  Ion Abundance Criteria for BlS(Perfluorophenyl) Phenylphosphine Oxide
(Decafluorotriphenylphosphine Oxide, DFTPPO)

Mass         Relative Abundance
(M/z)         Criteria                   Purpose of Checkpoint
77            Present, major ion          Low mass sensitivity
168           Present, major ion          Mid-mass sensitivity
169           4-10% of 168             Mid-mass resolution and isotope ratio
271           Present, major ion          Base peak
365           5-10% of base  peak       Baseline threshold check
438           Present                   Important high mass fragment
458           Present                   Molecular ion
459           15-24% of mass 458       High mass resolution and isotope ratio

All ions are used primarily to check the mass measuring accuracy of the mass spectrometer and a
data  system, and is the most important part of the performance test. There resolution checks,
which include natural abundance isotope rations, constitute the next most important part of the
performance test.  The correct setting of the baseline threshold, as indicated  by the presence of
low intensity ions, is the next most important part of the performance test.  Finally, the ion abun-
dance ranges are designed to encourage some standardization of fragmentation patterns.
                                                                                    343

-------
Method 553
Table 2.  Retention Time Data and Quantitation Ions for Method Analytes
                                                  Retention
                                               Time (min: sec)
                                                                          Quantitation
Compound                                   A'               Bf           Ion (m/z)
Benzidine                                      4.3             4.9             184
Benzoylprop ethyl                              24.8            31.3             105
Caffeine                                       1.4             1.6             194
Carbaryl                                      10.1             14.7             144
o-Chlorophenyl thiourea                         2.7             3.0             151
3,3'-Dichlorobenzidine                          16.6            22.7             252
3,3'-Dimethoxybenzidine                        8.1             11.5             244
3,3'-Dimethylbenzidine                          8.5            12.4             212
Diuron                                       11.0            16.1               72
Ethylene thiourea                               1.2             1.4             102
Linuron                                       16.0            21.9             161
Rotenone                                     21.1             27.4             192
Siduron                                       14.8            20.6              93
Surrogates:0
    Benzidine-D8                               4.2             4.8             192
    Caffeine-15N2                               1.3             1.6             196
    3,3'-Dichlorobenzidine-D6                   16.5            22.6             258
    Bis(perfluorophenyl)-phenylphosphine         22.0            28.9             271
    oxide

a    These retention times were obtained on a Hewlett-Packard 1090 liquid chromatograph with a
    Waters C18 Novapak 15 cm  x 2 mm  column using gradient conditions given in Sect. 10.2.3.
h    These retention times were obtained o a Waters 600 MS liquid chromatograph with a Waters
    C18 Novapak 15 cm x 2 mm column  using gradient conditions given in Sect. 10.2.3.
c    These compounds cannot be used if unlabelled compounds are presents (See Sect. 4.1)
344

-------
        Table 3.  Accuracy and Precis
        Using Liquid-Liquid Extraction
        Compound
        Benzidine
        Benzoylprop ethyl
        Caffeine
        Carbaryl
        o-Chlorophenyl thiourea
        3,3'-Dichlorobenzidine
        3,3'-Dimethoxybenzindine
        3,3'-Dimethylbenzindine
        Diuron
        Ethylene thiourea
        Linuron
        Monuron
        Rotenone
        Siduron
Ol
ata from Six


True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
24.8
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
nge (See Sect.


Determinations
Mean
Observed
Cone.
(ug/U
20.5
33.0
10.5
52.2
15.3
21.7
29.2
31.8
26.2
0.0
89.5
31.8
44.9
29.6
9.3.3)


of the
Method Analytes
in Reagent
Pel. Mean Method
Std.
Dev.
(ug/U
0.8
1.1
0.9
2.9
2.2
0.7
2.3
1.0
1.3
0.0
3.9
1.2
9.4
1.4



Std.
Dev. 1
(%)
3.3
3.3
6.3
5.1
6.8
2.9
7.3
3.1
5.1
0.0
4.1
3.8
18.8
5.2



Accuracy
% of True
Cone.)
89.6
101.6
72.6
92.3
47.0
89.6
92.3
100.4
104.8
0.0
94.2
101.9
89.3
106.3



Water
Method
Detection
Limit (MDU
(ug/U
2.5
3.7
3.1
9.8
7.4*
2.4
7.7
3.3
4.4
*
13.1
4.0
31.6
4.7























1
Ul
01
Co

-------
Co
Table 4.  Accuracy and Precision Data from Seven Determinations of the Method Analytes In Reagent Water
Using Liquid-Solid Extraction (C18 LSE Cartridge)
        Compound
        Benzidine
        Benzoylprop ethyl
        Caffeine
        Carbaryl
        o-Chlorophenyl thiourea
        3,3'-Dichlorobenzidine
        3,3'-Dimethoxybenzindine
        3,3'-Dimethylbenzindine
        Diuron
        Ethylene thiourea
        Linuron
        Monuron
        Rotenone
        Siduron


True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
5.0
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
Mean
Observed
Cone.
(ug/L)
12.2
29.3
6.4
53.9
0.0
4.4
25.5
31.4
24.4
0.0
88.9
30.5
45.0
24.8

Std.
Dev.
(ug/U
1.7
2.0
1.4
1.8
0.0
0.4
1.8
1.0
1.4
0.0
4.8
2.9
2.4
2.0
Pel.
Std.
Dev.
(%)
13.7
6.9
21.4
3.3
0.0
10.0
7.1
3.1
5.6
0.0
5.4
9.6
5.4
7.9
Mean Method Method
Accuracy
(% of True
Cone.)
53.2
90.2
44.2
95.2
0.0
89.6
80.8
99.0
97.6
0.0
93.6
97.8
89.6
88.9
Detection
Limit (MOD
(ug/U
5.3*
6.3
4.4*
5.7
*
1.4
5.7
3.0
4.4
*
15.1
9.1
7.5
6.3
I
o
Q.
Wl
01
Co
         'Recovery was not in the 70-130% range (See Sect. 9.3.3)

-------
Table 5. Accuracy and
Precision Data from
Six Determinations of the Method Analytes in Reagent Water
Using Liquid-Solid Extraction (Neutral Polystrene/Divinylbenzene Polymer



Compound
Benzidine
Benzoylprop ethyl
Caffeine
Carbaryl
o-Chlorophenyl thiourea
3,3'-Dichlorobenzidine
3,3'-Dimethoxybenzindine
3,3'-dimethylbenzindine
Diuron
Ethylene thiourea
Linuron
Monuron
Rotenone
Siduron


True Cone.
(ug/U
22.9
32.5
14.4
56.6
32.6
5.0
31.6
31.7
25.0
32.0
95.0
31.2
50.3
27.9
•Recovery was not in the 70-130% range (See Sect










Mean
Observed
Cone.
(ug/U
24.7
31.1
0.7
59.5
0.0
5.0
32.8
31.5
26.1
0.0
97.9
34.4
40.5
26.8
. 9.3.3)






Std.
Dev.
(ug/U
2.4
3.0
0.5
4.7
0.0
0.5
2.2
2.1
1.8
0.0
8.7
2.5
6.0
1.0






LSE Disk)
Pel.
Std.
Dev.
(%)
9.8
9.6
72.5
7.9
0.0
9.4
6.7
6.7
7.0
0.0
9.0
7.3
14.8
3.6







Mean Method
Accuracy
(% of True
Cone.)
108.0
95.8
5.2
105.1
0.0
101.7
103.8
99.4
104.5
0.0
103.0
110.4
80.5
96.1







Method
Detection
Limit (MDL)
(ug/U
8.1
10.1
1.8*
15.8
*
1.6
7.4
7.1
6.1
*
29.3
8.4
20.2
3.4



























(6
O
Q.
Wl
Co

-------
Method 553
Table 6.  Mean Recoveries, Multilaboratory Precision and Estimates of Single
Analyst Precision for the Measurements of Four Benzidines by LC/PB/MS



Compound
10 ug/mL

Recovery
(%)

RSD
Multi-Lab
RSD
Single
Analyst
100 ug/mL

Recovery
<%)

RSD
Multi-Lab
RSD
Single
Analyst
BZ 96 10 5.6 97 10 9.1
MB 104 20 18 95 10 7.0
LB 98 14 10 97 8.6 4.9
DB 96 18 9.4 97 9.1 4.6
 348

-------
                                                                      Method 553
50000 -
45000 -
40000 -
35000 -
30000 -
25000 -
20000 -
15000-
10000-
 5000-
    0-
 Dimethyloctadecylsilanol
 (Major Ions — M/Z, 75,313)
                            r
                           12
 T
16
 l
20
 I
24
 I
28
4       8
C18 Column Following Exposure to Ammonium Acetate
 l
32
                           100
                          -90
                          -80
                          -70
                          -60
                          -50
                          -40
                          -30
                          -20
                          - 10
                          -0
24000

20000 -
16000-

12000-
 8000-
 4000-
    0-
                     i
                     8
 l
16
 i
20
 I
24
 4      8      12
 C18 Column Maintained with Acetonitrile Flushing
n
 28
 i
32
                                 - 100
                                 -90
                                 -80
                                 -70
                                 -60
                                 -50
                                 -40
                                 -30
                                 -20
                                 - 10
                                 -0
                                                                        52-015-35B
      Figure 1. Unacceptable Chromatogram with Column Bleed and
               Acceptable Chromatogram Following Column Flushing
                                                                            349

-------
Method 553
100-
90-
80
70-
60-
50-

40-
30-

20-
10-
o
271









77






69































168

99








117


I








20


I ' I









6 22







255




>4









F 	 F o ^ 	 F
/ \ II / \
F\_fP\_/f
F F Jv F F
Hl MH
Hi^jH
H
438

291
45
365
|
I ' I ' I ' I ' I
          100       150       200       250       300       350       400       450
                                                                            52-015-37A
                                      Mass
         Figure 2. Average Spectrum of DFTPPO from Multilaboratory Study
350

-------
                                                                                 Method 553
100%
             75
  INT_
         61
      50
          CH3


          I
     HO- Si-



          CHo
                                          Dimethyloctadecylsilanol
                                                                         M-Methyl
                                                                           313
                                                                281
                                                    295   I

                                                   —lU.	ttJk_
100
150
200

M/Z
250
300
                                                                                  52-015-38A
                              Mass Spectrum of C18 Column Bleed
                   Figure 3. Mass Spectrum of Dimethyloctadecylsilanol,
                             A Common Stationary Phase Bleed Product
                                                                                      351

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Method 553
1200000
                                            CB + DCB
     100   200  300   400  500   600   700   800  900  1000  1100  1200  1300 1400
                         0.3 mL/min 0.01 M Ammonium Acetate
                          Post Column 0.1 mL/min Acetonitrile
            Figure 4.  Total Ion Chromatogram of Analytes and Surrogates
                      (140-950 ng injected)
352

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                                                                            Method 553
HD
                           2 Liter
                           Separatory Funnel
                                             HD
              \7
                                             HD
                           125mL
                           Solvent
                           Reservoir
                           Ground Glass
                           Stopper 14/35

                           LSE Cartridge

                           Rubber Stopper


                           No. 18-20 Luer-lok
                           Syringe Needle
HD
125ml_
Solvent
Reservoir

Ground Glass
Stopper 14/35

LSE Cartridge
                          100mL
                          Separatory
                          Funnel
Drying Column
(Na2SO4)
1.2 cm x 40 cm
                                                                       10 mL
                                                                       Graduated
                                                                       Vial
       A. Extraction Apparatus
           B. Elution Apparatus
     Figure 5. Schematic Diagram of a Liquid-Solid Extraction (LSE) Apparatus
                                                                                  353

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 Method 553
   Source
   Vacuum
                                                                           1 Liter
                                                                           Suction Flask
                                                  Pinch Clamp
                                                                                 52-015-41
         Figure 6.  Schematic Diagram of Liquid-Solid Disk Extraction Apparatus
354

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                             Method 555
Determination of Chlorinated Acids in Water
                 by High Performance Liquid
   Chromatography with a Photodiode Array
                         Ultraviolet Detector
                       Revision 1.0 - EPA EMSL-Ci
                                  August 1992
      James W. Eichelberger, Winslow J. Bashe (Technology Applications, Inc.)

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                                  Method  555
                Determination of Chlorinated Acids in  Water
                by High Performance Liquid Chromatography
                 with a Photodiode Array Ultraviolet Detector
 1.    SCOPE AND APPLICA TION

 1.1   This is a high performance liquid chromatographic (HPLC) method for the determination of
      certain chlorinated acids in ground water and finished drinking water.  The following com-
      pounds can be determined by this method:

                   Analyte                          CAS No.
                   Acifluorfen                        50594-66-6
                   Bentazon                          25057-89-0
                   Chloramben3                         133-90-4
                   2,4-D                                94-75-7
                   2,4-DB                              94-82-6
                   Dicamba                           1918-00-9
                   3,5-Dichlorobenzoic acid               51-36-5
                   Dichlorprop                         120-36-5
                   Dinoseb                             88-85-7
                   5-Hydroxydicambaa                  7600-50-2
                   MCPA                               94-74-6
                   MCPP                              7085-19-0
                   4-Nitrophenola                       100-02-7
                   Pentachlorophenol" (PCP)               87-86-5
                   Picloram"                           1918-02-1
                   2,4,5-T                              93-76-5
                   2,4,5-TP                             93-72-1

       8   Analytes measurable from 20 ml sample volume only.
       b   Use a  100 mL sample  for pentachlorophenol in order to attain a MDL of
           0.3 /yg/L. The MLC for this compound is 1.0 jt/g/L


1.2   This method is applicable to  the determination of salts and esters of analyte acids.  The form
      of each analyte is not  distinguished by this method.  Results are calculated and reported for
      each listed analyte as the total free acid.
1.3   This method has been validated in a single laboratory and method detection limits (MDLs)
      have been determined from a 20-mL sample for the analytes above.1 Observed MDLs may
      vary among ground waters, depending on the nature of interferences in the sample matrix and
      the specific instrumentation used.

1.4   This method is restricted to use by or under the supervision of analysts experienced in the use
      of HPLC and in the interpretation of chromatograms.  Each analyst must demonstrate the abi-
      lity to generate acceptable  results with this method using the procedure described in Sect. 9.3.
                                                                                   357

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Method 555
1.5    Analytes that are not separated chromatographically cannot be individually identified and
       measured in the same calibration mixture or water sample unless an alternative technique for
       identification and quantitation exists (Sect. 11.3).
1.6    When this method is used to analyze unfamiliar samples, analyte identifications must be con-
       firmed by at least one additional qualitative technique.

2.     SUMMARY OF METHOD

2.1    A measured sample volume of approximately 100 mL is adjusted to pH 12 with 6 N sodium
       hydroxide,  shaken, and allowed  to set for  1 hr to  hydrolyze chlorinated esters.  The sample is
       acidified with H3PO4, filtered, and the chlorinated acids are extracted from a 20-mL aliquot.
       The 20-mL aliquot is pumped through an HPLC cartridge (containing Clg-silica), trapping the
       chlorinated acids. The  concentrator cartridge is valved in-line with the C,8 analytical column
       following extraction.  The analytes are separated and measured by photodiode array-ultraviolet
       detection (PDA-UV).

       NOTE: A liquid-solid extraction disk is perfectly acceptable for use in the in-line
       extraction of the analytes providing all quality control (QC) criteria in Sect.  9 are met
       or exceeded.

2.2    The method measures the analytes from 20-mL volumes.  Volumes of up to 100 mL may be
       analyzed by this procedure for certain analytes. The analytes which may not be determined in
       a  larger volume are indicated in  Sect. 1.1.

3.     DEFINITIONS

3.1    Laboratory duplicates (LD1 AND LD2):  Two aliquots of the same sample taken in the
       laboratory and analyzed separately with identical procedures.  Analyses of LD1 and LD2
       indicate the precision associated  with laboratory procedures, but not with sample collection,
       preservation, or storage procedures.
3.2    Field duplicates (FD1 AND FD2): Two separate samples collected at the same time and place
       under identical circumstances and treated exactly the  same throughout field and laboratory
       procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample
       collection, preservation and storage, as well as with laboratory procedures.
3.3    Laboratory reagent blank (LRB): An aliquot of reagent water or other blank matrix that is
       treated exactly as a sample including exposure to all glassware, equipment, solvents, and
       reagents that are used with other samples. The LRB is used to determine if method analytes
       or other interferences are present in the laboratory environment,  the reagents, or the apparatus.
3.4    Field reagent blank (FRB):  An  aliquot of reagent water or  other blank matrix that is placed in
       a  sample container in the laboratory and treated as a  sample in all respects,  including shipment
       to the sampling site, exposure to sampling site conditions, storage, preservation, and all
       analytical procedures.   The purpose of the FRB is to determine if method analytes or other
       interferences are present in the field environment.
358

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                                                                                     Method 555
 3.5   Laboratory fortified blank (LFB):  An aliquot of reagent water or other blank matrix to which
       known quantities of the method analytes are added in the laboratory.  The LFB is analyzed
       exactly like a sample, and its purpose is to determine whether the methodology is in control,
       and whether the laboratory is capable of making accurate and precise measurements.
 3.6   Laboratory fortified sample matrix (LFM):  An aliquot of an environmental sample to which
       know quantities of the method analytes are added in the laboratory.   The LFM is analyzed
       exactly like a sample, and its purpose is to determine whether the sample matrix contributes
       bias to the analytical results.  The background concentrations of the analytes in the  sample
       matrix must be determined in a separate aliquot and the measured values in the LFM corrected
       for background concentrations.
 3.7   Stock standard solution (SSS):  A concentrated solution containing one or more method
       analytes prepared in the laboratory using assayed reference materials  or purchased from a
       reputable commercial supplier.
 3.8   Primary dilution standard solution (PDS):  A solution of several analytes prepared in the
       laboratory from stock standard solutions and diluted as  needed to prepare calibration solutions
       and other needed analyte solutions.
 3.9   Calibration standard (CAL):  A solution prepared from the primary dilution standard solution
       or stock standard solutions and the internal standards and surrogate analytes. The CAL
       solutions are used to calibrate the instrument response with respect to analyte concentration.
 3.10  Quality control sample (QCS):  A solution of method analytes of known concentrations which
       is used to fortify an aliquot of LRB or sample matrix.   The QCS  is obtained from a source
       external to the laboratory and different from the source of calibration standards, It is used to
       check laboratory performance with externally prepared  test materials.
 3.11  Method detection limit (MDL): The minimum concentration of an analyte that can be identi-
       fied, measured and reported with 99% confidence that the analyte concentration is greater than
       zero.
 3.12  External standard (ES):  A pure analyte(s)  that is measured in an  experiment separate from the
       experiment used to measure the analyte(s) in the sample. The signal observed for a known
       quantity of the external standard(s) is used to calibrate the instrument  response for the corre-
       sponding analytes(s). The instrument response is used to calculate the concentrations of the
       analyte(s) in the sample.

4.     INTERFERENCES

4.1    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 analyzing laboratory reagent  blanks as
       described in Sect. 9.2.
       4.1.1  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 dilute acid,  tap and reagent
              water.  Drain dry, and heat in an oven or muffle furnace  at 400°C  for 1  hr. Do  not
                                                                                            359

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Method 555
               heat volumetric ware.  Thermally stable materials such as PCBs might not be elimi-
               nated by this treatment.  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.
       4.1.2   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.

       WARNING:       When a solvent is purified, stabilizers added by the manufacturer
       are removed, thus potentially making the solvent hazardous.  Removal of preservatives
       by distillation may  also reduce the shelf-life of the solvent.

4.2    The acid forms  of the analytes are strong organic acids which react readily with alkaline
       substances and can be lost during sample preparation. Glassware must be  acid-rinsed with 1 N
       hydrochloric acid prior to use to avoid analyte losses due to adsorption.
4.3    Matrix interferences may be caused by contaminants that are coextracted from the sample.
       Also, note that all method analytes are not resolved from each other on a single column, i.e.,
       one analyte of interest may interfere with another analyte of interest.  The  extent of matrix
       interferences will vary considerably from source to source, depending upon the water sampled.
       The procedures in  Sect.  11 can be used to overcome many of these interferences.   Tentative
       identifications should always be confirmed (Sect.  11.3).

5.     SAFETY

5.1    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.
       Accordingly, exposure to these chemicals must be reduced to the lowest possible level. The
       laboratory is responsible for maintaining a current awareness file of OSHA regulations regard-
       ing the safe handling of the chemicals specified in this method. A reference file of material
       safety data sheets should also be made available to all personnel involved in the chemical
       analysis.
5.2   WARNING:  When a solvent is purified, stabilizers added by the manufacturer  are removed,
       thus  potentially making the solvent hazardous.  Therefore, storage of large volumes of purified
       solvents may be hazardous.  Therefore, only small volumes of solvents should be purified just
       before use.

6.      EQUIPMENT  AND SUPPLIES

6.1    Ssmple bottle:  Borosilicate, 125-mL volume, graduated, fitted with Teflon-lined screw cap.
       Protect samples from light. The container must be washed and dried as described in Sect.
       4.1.1 before use to minimize  contamination.  Cap liners may be cut to fit  from Teflon sheets
       and extracted with methanol overnight prior to use.
6.2   Glassware
       6.2.1    Volumetric flask, Class A:   100 mL, with ground glass stoppers.
 360

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                                                                                   Method 555
       6.2.2  Graduated cylinder:  100 mL
       6.2.3  Disposable pipets, Transfer: borosilicate glass
       6.2.4  Glass syringe:  50 mL, with Luer-Lok fitting
       6.2.5  Volumetric pipette, Class A:  20 mL
6.3    Balance:  Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.4    Liquid chromatograph:  Analytical system complete with gradient programmable HPLC
       suitable for use with analytical HPLC columns and all required accessories including an
       injector, analytical column, semi-prep guard column, and photodiode array UV detector. A
       data system is necessary for measuring the peak areas and for assessing the confirmation of the
       peak identification. A personal computer (PC) of at least the AT-class is generally needed  to
       control and collect data from the photodiode array UV detector.  Table 1 lists the retention
       times observed for the method analytes using the column and analytical conditions described
       below.  Figure 1  is a schematic drawing of the analytical system including the sample concen-
       trator column (semi-prep guard column).
       6.4.1   Primary  Column:  250 mm x 4.6 mm I.D. ODS-AQ, 5 jon spherical (YMC Ltd.).
              Any column may be used if equivalent or better performance (better peak shape,
              better analyte efficiency, or more complete separation of analytes) can be demonstrat-
              ed.  Mobile phase flow rate is established at 1.0 mL/min (linear velocity of 6.0
              cm/min). Two mobile phase components are used: A—0.025 M H3PO4; B—Aceto-
              nitrile.  A gradient solvent program is used to separate the analytes:  90:10 A:B to
              10:90 A:B in 30 min, linear ramp / hold at 10:90 for  10 min.  Reverse the gradient
              and establish initial conditions:  10:90 A:B to 90:10 A:B in 10 min,  linear ramp.
              Allow column back pressure to restablize for 5 to 10 min before beginning the next
              analysis.  Total restabilization time will be determined by each analyst.
       6.4.2  Confirmation Column: 300 mm  x 3.9  mm I.D. Nova-Pak C18, 4 /mi spherical (Wa-
              ters Chromatography Division, Millipore).  Any column may be used if equivalent or
              better performance (better peak shape, better analyte efficiency, or more complete
              separation of analytes) can be demonstrated.   Mobile phase and  conditions same as
              primary column.
       6.4.3  Sample Concentrator Column:  30 mm  x 10 mm I.D. ODS-AQ, 5 pm  spherical
              (YMC Ltd).  An alternative concentrator column may  be used if all QC criteria in
              Sect. 9 can be equalled or improved.  Also, a liquid-solid extraction  disk may be used
              if all QC criteria in Sect. 9 can be equalled or improved.
       6.4.4  6-port Switching Valve:  Rheodyne Model 7000 (Rheodyne Corp).
       6.4.5   Sample Delivery Pump:  A piston-driven pump capable of delivering aqueous sample
              at a flow rate of 5.0 mL/min. An analytical HPLC pump may serve as the sample
              delivery pump. A Waters Model 6000A was used to generate the data presented in
              this method.
       6.4.6   Detector: Photodiode Array-Ultraviolet (PDA-UV),  LKB-Bromma Model 2140
              Rapid Spectral Detector or equivalent. Detector parameters: Scan Range - 210 to
              310 nm at 1  scan/sec, detector integration—1 sec.
                                                                                         361

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Method 555
       6.4.7   Data Handling System: DOS-based Personal Computer, AT-class machine or machine
               of greater capability with 640 K RAM or more, an 80 Mb hard disk or larger, VGA
               monitor or equivalent.

7.     REAGENTS AND STANDARDS
7.1    Acetonitrile: HPLC Grade or equivalent.
7.2    Sodium sulfite, granular, anhydrous:  ACS Grade.
7.3    Sodium hydroxide (NaOH), pellets:  ACS Grade.
       7.3.1   NaOH, 6 N:  Dissolve 216 g NaOH in 900 mL reagent water.
7.4    Phosphoric acid, 85% AR,:  ACS grade.
       7.4.1   0.025 M: Mix 2.0 mL of H3PO4 in 998 mL of reagent water.
7.5    Stock standard solutions (1.00 /ig/jtL):  Stock standard solutions may be purchased as certified
       solutions or prepared from pure standard materials using the following procedure:
       7.5.1   Prepare stock standard solutions by accurately weighing approximately 0.0100 g of
               pure material.  Dissolve the material in acetonitrile and dilute to volume in a 10-mL
               volumetric flask.  Larger volumes may be used at the convenience of the analyst.  If
               compound purity is certified at  96% or greater, the weight may be used without
               correction to calculate the concentration of the stock standard. Commercially pre-
               pared stock standards may be used at any concentration if they are certified by the
               manufacturer or by an independent source.
       7.5.2   Transfer the stock standard solutions into Teflon-lined sealed screw cap amber vials.
               Store at room temperature and protect from light.
       7.5.3   Stock standard solutions should be replaced after two months or sooner if comparison
               with laboratory fortified blanks, or QC samples indicate a problem.
7.6    Hydrochloric acid:  ACS grade.
       7.6.1   HCl, 1 N :  Dilute 50 mL in 600 mL of reagent water.
7.7    Filters, 0.45 /mi,  Nylon, 25 mm i.d. (Gelman Sciences)

8.     SAMPLE COLLECTION, PRESERVATION,  AND  STORAGE
8.1    Grab samples must be collected in glass containers.  Conventional sampling practices should
       be followed; however, the bottle must not be prerinsed with sample before collection.2
8.2    Sample Preservation and Storage
       8.2.1   Add hydrochloric acid (1:1) to the sample to produce a pH of 2. The pH may be
               measured in the field using pH  indicator strips.
       8.2.2   Residual  chlorine should be reduced at the sampling site by the addition of a reducing
               agent.  Add 4-5 mg of sodium  sulfite (this may be added as a solid with shaking until
               dissolved) to each 100 mL of water.
       8.2.3   The samples must be iced or refrigerated at 4°C away from light from the time of
               collection until extraction.  The samples must be analyzed within 14 days  of collec-
               tion.  However, analyte stability may be affected by the matrix. Therefore, the analyst
               should verify that the preservation technique is  applicable to the samples under study.

362

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                                                                                     Method 555
               If the 14-day holding time is exceeded, the data should be flagged so that the data
               user is aware of possible analyte degradation.
       8.2.4  Field reagent blanks (FRB):  Processing a (FRB)  is recommended along with each set,
               which is composed of the samples collected from the same general sample site at
               approximately the same time. At the laboratory,  fill a sample container with reagent
               water, seal, and ship to the sampling site along with the empty sample containers.
               During sample collection, open the FRB and add  HC1 (Sect. 8.2.1) and sodium sulfite
               (Sect. 8.2.2)  Return the FRB to the laboratory with filled sample bottles.

9.     QUALITY  CONTROL

9.1    Minimum QC requirements are initial demonstration of laboratory capability, analysis of
       laboratory reagent blanks, laboratory  fortified samples, laboratory fortified blanks,  and QC
       samples.
9.2    Laboratory reagent blanks (LRB): Before processing any  samples, the analyst must demon-
       strate that all glassware and reagent interferences are under control.  Each time a set of
       samples is extracted or reagents are changed, a LRB must be analyzed.  If within the retention
       time window of any analyte the LRB  produces  a peak that would  prevent the determination of
       that analyte, determine the source of contamination and eliminate  the interference before
       processing samples.
9.3    Initial demonstration of capability
       9.3.1   Select a representative fortified concentration for each analyte.  Prepare  a sample
               concentrate (in acetonitrile) containing each  analyte at 1000 times the selected concen-
               tration. With a syringe, add 100 fiL of the concentrate to each of at least four 100-
               mL aliquots of reagent water, and analyze each aliquot according to procedures
               beginning in Sect. 11.
       9.3.2  Calculate the recoveries, the  relative standard deviation, and the MDLs.5 For each
               analyte the recovery value for all four  of these samples must fall in the range of R  ±
               30%, using the  value for R for reagent water in Table 2.  As the calibration proce-
               dure employs a  fortified reagent water blank for the determination of the calibration
               curves or factors, the recovery values for the analytes should, by definition, be within
               this range.  If the mean recovery of any analyte fails this demonstration, repeat the
               measurement of that analyte to demonstrate acceptable performance.
       9.3.3  The initial demonstration of capability  is used primarily to preclude a laboratory from
               analyzing unknown samples using a new, unfamiliar method prior to obtaining some
               experience with it.  As laboratory personnel gain  experience with this method the
               quality of data should improve beyond what is required here.
9.4    The analyst is permitted  to modify LC columns, LC  conditions, and detectors.  Each time such
       method modifications are made, the analyst  must repeat the procedures in Sect. 9.3.

       NOTE:  The LC column  and guard cartridge used to generate the data in this method
       were found to be unique C/g-silica columns.  Before substituting other CI8 columns,  a
       careful review of the literature  is recommended.
                                                                                           363

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Method 555
9.5    Assessing laboratory performance:  Laboratory fortified blank
       9.5.1   The laboratory must analyze at least one laboratory fortified blank (LFB) sample with
               every 20 samples or one per sample set (all samples analyzed within a 24-hr period)
               whichever is greater.  The concentration of each analyte in the LFB should be 10
               times the MDL or the MCL, whichever is less.  Calculate accuracy as percent recov-
               ery (X,).  If the recovery of any analyte falls outside the control limits (See Sect.
               9.5.2), that analyte is judged out of control, and the source of the problem should be
               identified and resolved before continuing analyses.
       9.5.2   Until  sufficient data become available from within the laboratory, usually a minimum
               of results from 20 to 30 analyses, the laboratory should assess laboratory performance
               against the control limits in Sect. 9.3.2 that are derived from the data in Table 2.
               When sufficient internal performance data become available, develop control limits
               from the mean percent recovery (X) and standard deviation (S) of the percent recov-
               ery.  These data are used to establish upper and lower control limits as follows:

                         Upper Control Limit  = X +  3S
                         Lower Control Limit  = X - 3S

               After each five to ten  new recovery measurements, new control limits  should be
               calculated using only the most recent 20-30 data points.  These calculated control
               limits should never exceed those established in Sect. 9.3.2.
       9.5.3   It is recommended that the laboratory periodically determine and document its detec-
               tion limit capabilities for the analytes of interest.
       9.5.4   At least quarterly, analyze a QC sample from an outside source.
       9.5.5   Laboratories are encouraged to participate in external performance evaluation studies
               such as the laboratory certification programs offered by many states or the studies
               conducted by USEPA. Performance evaluation studies serve as independent checks
               on the analyst's performance.
9.6    Assessing analyte recovery:  Laboratory fortified sample matrix
       9.6.1   The laboratory should add a known concentration to a minimum of 10% of the routine
               samples or one sample per set, whichever is greater.  The concentration should not be
               less than the background concentration of the sample selected for fortification.  Ideal-
               ly, the concentration should be the same as that  used for the laboratory fortified blank
               (Sect. 9.5).  Over time, samples from all routine sample sources should be fortified.
       9.6.2   Calculate the percent recovery,  P of the concentration for each analyte, after correct-
               ing the analytical result, X, from the fortified sample for the background concentra-
               tion, b, measured in the unfortified sample,
                                   p =       100 (X - b}
                                       fortifying concentration
 364

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                                                                               Method 555
        and compare these values to control limits appropriate for reagent water data collected
        in the same fashion.  If the analyzed unfortified sample is found to contain NO back-
        ground concentrations, and the added concentrations are those specified in Sect. 9.5,
        the appropriate control limits would be the acceptance limits in Sect. 9.5.  If, on the
        other hand, the analyzed unfortified sample is found to contain background concentra-
        tion,  b, estimate the standard deviation at the background data, sb, using regressions
        or comparable background data and similarly, estimate the mean, Xa, and standard
        deviation, sa, of analytical  results or the total concentration after fortifying.  Then the
        appropriate percentage control limits would be P  + 3sp, where:
                         P =             100 x
                              (b + fortifying concentration)
                         and

                         sp  =  100 _	^L^	
                                  fortifying concentration
           For example, if the background concentration for Analyte A was found to be 1
           /xg/L and the added amount was also 1 /xg/L, and upon analysis the laboratory
           fortified sample measured 1.6 /*g/L, then the calculated P for this sample would
           (1.6 /xg/L minus 1.0 /tg/L)/ 1.0 /xg/L or 60%.  This calculated P is compared to
           control limits derived from prior reagent water data.  Assume it is  known that
           analysis of an interference free sample at 1.0 /ig/L yields an s of 0.12 /xg/L and
           similar analysis at 2.0 /xg/L yields X and S of 2.01 /xg/L and 0.20  /xg/L, respec-
           tively.  The appropriate limits to judge the reasonableness of the percent recovery,
           60%, obtained on the fortified matrix sample is computed as follows:
            100 (2.01 /xg/L)
               2.0 Mg/L

          +  3 (100) [(Q-12 WW +  (0.20  /xg/L)2]*  _
                                1.0/xg/L
          100.5% ± 300  (0.233)  =
          100.5% ± 70% or 30% to  170%  recovery of the added analyte.
9.6.3  If the recovery of any such analyte falls outside the designated range, and the labora-
        tory performance for that analyte is shown to be in control (Sect. 9.5), the recovery
        problem encountered with the fortified sample is judged to be matrix related, not
        system related.  The result for that analyte in the unfortified sample is labeled sus-
        pect/matrix to inform the data user that the results are suspect due to matrix effects.
                                                                                      365

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Method 555
9.7    The laboratory may adopt additional QC 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. For example, field or laboratory duplicates may be analyzed to assess the
       precision of the environmental measurements.  The field reagent blanks may be used to assess
       contamination of samples under site conditions, transportation and storage.

10.   CALIBRA TION AND  STANDARDIZA TION

10.1   Establish HPLC operating parameters equivalent to those indicated in Sect. 6.4.1.  The HPLC
       system should be calibrated using the external  standard technique (Sect. 10.2).

       NO TE:  Calibration standard solutions must be prepared such that no unresolved
       analytes are mixed together.  The method analytes have been separated into two cali-
       bration solutions (See Table 1 for Groups A and B).  The analytes in these solutions
       have been found to be  resolved under the LC conditions listed.   Mixtures of these
       analytes at  concentration levels of 100 pg/mL  (in acetonitrile) are suggested as a
       possible secondary dilution standard.  Figures 2 and 3  are typical chromatograms of
       Groups A and B as separated on the primary HPLC column.

10.2  External standard calibration procedure
       10.2.1  Prepare calibration standards (CAL) at a minimum of three (five are recommended)
               concentration  levels  for each analyte of  interest by  adding volumes of one or more
               stock standards to volumetric flasks.   Alternatively, add various volumes of a primary
               dilution standard solution of Group A or B (Sect. 10.1) to a volumetric flask. Dilute
               to volume with the aqueous mobile phase (0.025 M H3P04). The lowest standard
               should contain analyte concentrations  near, but above, the  respective MDL.  The
               remaining  standards should bracket the analyte concentrations expected in the sample
               extracts, or should define the working range of the detector.
       10.2.2 Starting with the standard of the lowest  concentration,  process each calibration stan-
               dard according to Sect.  11.1  and tabulate response  (peak area) versus injected quantity
               in the standard.  The results can be used to prepare a calibration curve for each
               compound. Alternatively, if the ratio of response to concentration (response factor) is
               a constant over the working range  (20% RSD  or less),  linearity through the origin can
               be  assumed and the average ratio or response factor can be used in place of a calibra-
               tion curve.
       10.2.3 The working calibration curve or response factor must be verified on each working
               day by the measurement of a CAL, analyzed at the beginning of the analysis day. It
               is highly recommended that an additional check standard be analyzed at the end of the
               analysis day.  For extended periods of analysis (greater than 8 hr), it is strongly
               recommended that check standards be interspersed  with samples at regular intervals
               during analyses.  If the response for any analyte varies from the predicted response by
               more than ±25%, the test must be repeated using a fresh calibration standard.  If  the
               results still do not agree, generate  a new calibration curve.
366

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                                                                                   Method 555
       10.2.4 Verify calibration standards periodically, recommend at least quarterly, by analyzing a
               standard prepared from reference material obtained from an independent source.
               Results from these analyses must be within the limits used to routinely check calibra-
               tion.

 7 7.   PROCEDURE

 11.1  Hydrolysis, preparation, and extraction
       11.1.1  Add preservative to blanks and QC check standards.  Mark the water meniscus on the
               side of the sample bottle for later determination of sample volume (Sect. 11.1.5).
       11.1.2 Add 1.7 mL of 6 N NaOH to the sample, seal, and shake. Check the pH of the
               sample with pH paper; if the sample does not have a pH greater than or equal to 12,
               adjust the pH by adding more 6 N NaOH.  Let the sample sit at room temperature for
               1 hr, shaking the sample bottle and contents periodically.
       11.1.3  Add 2 mL of concentrated H3PO4 to the sample, seal, and shake to mix. Check the
               pH of the sample with pH paper; if the sample does not have a pH less  than or equal
               to two, adjust the pH by adding more H3PO4.
       11.1.4  From the homogeneous sample, remove a 20-mL aliquot for  analysis. Filter the
               aliquot through a 0.45 /*m filter into a graduated cylinder or  other convenient graduat-
               ed container. Using an HPLC pump (or HPLC reagent delivery pump), pump the 20-
               mL aliquot through the on-line concentrator column at a flowrate of 5.0 mL/min (See
               Figure 1).  The use of a liquid-solid extraction disk is perfectly acceptable providing
               all QC criteria in Sect. 9 are met or exceeded.  After passing the sample through the
               concentrator column, follow with an additional  10-mL of the aqueous mobile phase
               (0.025 M H3PO4).
       11.1.5  After analysis is completed, determine the original sample volume by refilling the
               sample bottle to the mark and transferring the water to a 100-mL graduated cylinder.
               Record the sample volume to the nearest 1 mL.
11.2 High performance liquid chromatography
       11.2.1  Sect. 6.4.1 summarizes the recommended operating conditions for the HPLC.  Includ-
               ed in Table 1 are retention times observed using this method.  Other HPLC columns,
               chromatographic conditions, or detectors may be used if the requirements of Sect. 9.3
               are met.
       11.2.2  Calibrate the system daily as described in Sect.  10.
       11.2.3 After loading the sample (or calibration standard) onto the concentrator column, valve
              the sample into the analytical stream, backflushing the concentrator column.  The
              photodiode array detector (PDA-UV) is set to scan and record from 210 to 310 nm, 1
              scan per second during the entire chromatographic run (40 min).  Extract the 230 ran
              trace from the stored data and record the resulting peak size in area units for all
              analytically significant peaks.
      11.2.4 If the responses for the peaks exceed the working range of the system, dilute an
              additional 20-mL aliquot of the sample with reagent water, adjust the pH to 12 with
              NaOH, and reanalyze according to Sect. 11.1.2.

                                                                                         367

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Method 555
11.3  Identification of analytes
       11.3.1  Identify a sample component by comparison of its retention time to the retention time
               of a reference chromatogram.  If the retention time of an unknown compound corre-
               sponds, within limits, to the retention time of a standard compound, then identification
               is considered positive.
       11.3.2  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.
       11.3.3  Identification requires expert judgment when  sample components are not resolved
               chromatographically.  When peaks obviously represent more that one sample compo-
               nent i.e.,  broadened peak with shoulder(s) or vollies between two or more maxima, or
               any time doubt exists over the  identification or a peak on a chromatogram, appropriate
               alternative techniques, to help confirm peak identification, should be used.   For this
               method, the use of the PDA-UV detector  affords the  analyst the option of using a
               secondary wavelength for the analysis of the questionable identification.  The response
               ratio for a compound of interest at two wavelengths may  be determined from stan-
               dards of known purity. If the wavelength response ratio and the retention time match-
               es a given unknown to a method analyte, more certainty may  be assigned to the
               identification of the unknown.  If this method of compound confirmation is employed,
               each analyst will need to determine the wavelength response ratio for each analyte.
               Table 3 lists suggested alternative wavelengths for each analyte in the scope of the
               method.   An alternative LC column may be used to separate and confirm the identifi-
               cation of unknown peaks.  A suggested alternative column is described in Sect. 6.4.2.

12.   DA TA ANAL YSIS AND CALCULA TIONS

12.1  Calculate analyte concentrations in  the sample from the response for the analyte using the
       calibration procedure described in Sect. 10.
12.2  Calculate the amount of sample analyte injected from the peak response using the calibration
       curve or calibration response factor determined in  Sect.  10.2.  The concentration (C) in the
       sample can be calculated from Equation 1.
                                         Equation 1

                                Concentration (ng/L) =
                                                       ^ /'^ S?
                             Where:
                             A = Amount of material injected (ng).
                             V = Volume of standard injected (mL).
                             V( = Volume of sample injected (mL).
                             V = Volume of water sample (mL).
368

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                                                                                   Method 555
13.  METHOD PERFORMANCE

13.1  In a single laboratory, analyte recoveries from reagent water were determined at two concen-
      tration levels. Results were used to determine analyte MDLs and demonstrated method range.5
      Analyte MDLs and analyte recoveries and  standard deviations about the percent recoveries at
      one concentration are given in Table 2.
13.2  In a single laboratory, analyte recoveries from dechlorinated tap water and ground waters were
      determined at one concentration level, 10 /*g/L.  Results were used to demonstrate appli-
      cability of the method to different tap and ground water matrices.  Analyte recoveries from tap
      water and ground water are given in Table 4. MDLs calculated from results of analyses of six
      100 mL reagent water samples at 0.5 /tg/L concentrations for each analyte are listed in
      Table 5.

14.  POLLUTION PREVENTION

14.1  This method utilizes the new in-line liquid-solid extraction technology which requires the use
      of very small quantities of organic solvents.  This feature eliminates the hazards involved with
      the use of large volumes of potentially harmful organic solvents needed for conventional
      liquid-liquid extractions.  Also, this method uses no  derivatizing reagents, which are toxic or
      explosive, to form gas chromatographable derivatives. These features make this method much
      safer for use by the analyst in the laboratory and a great deal less harmful to the environment.
14.2  For information about pollution prevention that may be applicable to  laboratory operations,
      consult "Less is Better:  Laboratory Chemical Management for Waste Reduction," available
      from the American Chemical Society's Department  of Government Relations and  Science
      Policy, 1155 16th Street N.W., Washington, D.C. 20036.

15.  WASTE MANAGEMENT

15.1  Due to the nature of this method, there is little need for waste management.  No large volumes
      of solvents or hazardous chemicals are used. The matrices of concern are finished drinking
      water or source water.  However, the Agency requires that laboratory waste management
      practices be consistent with all applicable rules and  regulations, and that laboratories protect
      the air, water, and land by  minimizing and controlling all releases from fume hoods and bench
      operations.  Also, compliance is required with any sewage discharge permits and  regulations,
      particularly the hazardous waste identification rules  and land disposal restrictions.  For further
      information on waste management,  consult "The Waste Management Manual for Laboratory
      Personnel," also available from the American Chemical Society at the address in Sect.  14.2.
                                                                                          369

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Method 55o
                                    References
1.    Giazer, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., Environ. Sci.
      Technol. 15, 1981, pp.  1426-1435.

2.    "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract No. 68-03-2697.
      Available from U.S. Environmental Protection Agency, Environmental Monitoring and
      Support Laboratory, Cincinnati, Ohio 45268.

3.    ASTM Annual Book of Standards,  Part 11, Volume  11.02, D3694-82,  "Standard Practice for
      Preparation of Sample Containers and for Preservation," American Society for Testing and
      Materials, Philadelphia, PA, p.  86,1986.

4.    Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for Determination of Phthalate
      Ester Plasticizers in Open-Ocean Biota Samples," Analytical Chemistry. 47, 2225 (1975).

5.    40 CFR,  Part 136, Appendix B.
370

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                                                                             Method 555
Table  1.  Retention Times for Method Analytes
                                                 Retention Times'
                                                     (minutes)

Group
(A)
(A)
(A)
(B)
(A)
(A)
(B)
(A)
(B)
(B)
(A)
(B)
(B)
(A)
(A)
(B)
(B)

Primary Column
19.0
19.7
21.1
21.6
24.0
25.2
25.5
25.6
26.7
27.2
27.3
27.5
28.0
29.2
30.7
32.8
33.4
Confirmation
Column
12.8
13.5
14.8
5.0
18.2
19.5
20.1
20.1
21.3
21.8
21.8
22.4
22.8
23.9
25.5
27.7
28.3
Analyte
Picloram
5-Hydroxydicamba
Chloramben
4-Nitrophenol
Dicamba
Bentazon
MCPA
2,4-D
3,5-Dichlorobenzoic acid
MCPP
Dichloroprop
2,4,5-T
2,4-DB
2,4,5-TP
Acifluorfen
Dinoseb
Pentachlorophenol
   Columns and analytical conditions are described in Sect. 6.4.1 and Sect. 6.4.2.
                                                                                   377

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Method 555
Table 2. Single Laboratory
(MDLs) for Analytes From
Accuracy,
Precision and Method
Detection Limits
Reagent Water a
MDL
Analyte d
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic
acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol
(PCP)
Picloram
2,4,5-T
2,4,5-TP
ig/Lf
1.7
4.6
3.1
1.3
1.9
2.1
2.1

1.7
1.5
2.2
0.8
1.7
1.2
1.6

0.5
1.3
1.8
Concentra tion
(fjg/U
10.0
10.0
10.0
10.0
10.0
10.0
10.0

10.0
10.0
10.0
10.0
10.0
10.0
10.0

10.0
10.0
10.0
Reagent Water
ff
104
126
83
112
92
104
94

108
97
132
93
95
95
99

104
93
90
S d
1.7
14.6
10.0
4.2
5.9
6.6
6.7

5.4
4.8
7.0
2.5
5.5
4.0
5.2

1.7
4.1
5.8
    Data represent the average of 6-7 samples.  Sample volume =  20 mL.
    MDL = method detection limit; defined in Appendix B to 40 CFR Part 136 —Definition and
    Procedure for the Determination of the Method Detection Limit—Revision 1.11.
    R =  average percent recovery.
    S, = standard deviation of the percent recovery
372

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                                                                          Method 555
Table 3.  Confirmation Wavelengths and Area Response Ratios for Method

                                           Confirmation            Area Response
                                         Wavelength (nm)              Ratio'
Acifluorfen                                      293                      1 .72
Bentazon                                        240                      1 .08
Chloramben                                     214                      0.61
2,4-D                                          285                      4.02
2,4-DB                                         285                      5.93
Dicamba                                        220                      0.66
3,5-Dichlorobenzoic acid                           285                      5.15
Dichlorprop                                     285                      4.07
Dinoseb                                         268                      0.48
5-Hydroxydicamba                                293                      1 .89
MCPA                                          285                      6.66
MCPP                                          285                      6.49
4-Nitrophenol                                    310                      0.56
Pentachlorophenol (PCP)                           290                      5.65
Picloram                                        223                      0.82
2,4,5-T                                         290           .           4.00
2,4,5-TP                                        293                      3.84
    Area Response Ratio = Peak Area for 230 nm / Peak Area for Conf . Wavelength
                                                                                373

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Method 555
Table 4.  Single Laboratory Precision and Accuracy Data From Tap Water And
Ground Water3
                                             Dechlorina ted
    Analyte

    Acifluorfen
    Bentazon
    Chloramben
    2,4-D
    2,4-DB
    Dicamba
    3,5-Dichlorobenzoic acid
    Dichlorprop
    Dinoseb
    5-Hydroxydicamba
    MCPA
    MCPP
    4-Nitrophenol
    Pentachlorophenol 
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                                                                            Method 555
Table 5.  Single Laboratory Recovery and Precision Data and Method Detection
Limits (MDLs) for Analytes from Reagent Water3
     Analyte
     Acifluorfen
     Bentazon
     Chloramben
     2,4-D
     2,4-DB
     Dicamba
     3,5-Dichlorobenzoic acid
     Dichlorprop
     Dinoseb
     5-Hydroxdicamba
     MCPA
     MCPP
     4-Nitrophenol
     Pentachlorophenol (PCP)
     Picloram
     2,4,5-T
     2,4,5-TP
MDL
dig/if
0.40
0.12
NR
0.34
0.31
0.24
0.38
0.33
0.26
NR
0.35
0.19
NR
0.15
NR
0.21
0.37
Concentration
(tig/U
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Reagent Water
ff
114
91
NR
121
99
80
105
110
99
NR
124
125
NR
93
NR
80
77
S,<
23.7
7.3
NR
20.2
18.5
14.1
22.5
19.4
15.5
NR
21.0
11.1
NR
8.6
NR
12.7
21.7
a   Data represent the average of six samples.  Sample Volume = 100 ml
b   MDL=  Method detection limit; defined in Appendix B to 40 CFR 136—Definition and
           Procedure for the Determination of  the Method Detection Limit—Revision 1.11.
c   R  =  Average percent recovery.
d   S,  =  Standard deviation of the percent recovery.
    NR =  Not Recovered.
                                                                                   375

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Method 555
                         Precolumn Extraction Hardware
             Analysis Mode


                      Sample Pump
          Extraction Mode
 Precolumr
                                  Waste
   Column
                                  Rheodyne 7000 Valve
                                        (6-Port)
                                           Precolumn
   Analytical     Analytical Pump(s)
                               Waste
Analytical
Column
Analytical Pump(s)
              Detector
                                                       L
             Detector
                                                                              52-015-8
             Figure 1.  Schematic Diagram of Sample Concentration and
                       Analytical HPLC Hardware
 376

-------
                                                                               Method 555
 0200-
 0 150-
 0.100-
0.050 -
                                                 c
                                                 0)
                                                 .O
                                                 E
                                                 6
                                         S
                                         a
                                         o
                                         'S.
                                         g
                                         |
                                                              S
                                                              Q
                                                                       I
-0050
	1	1	1	1	1	1	1	1	1	1	
0.0       5.0       10.0       15.0        20.0        25.0

                                      C1 (Minutes)
                                                                    300
              Figure 2. Typical HPLC Chromatogram of Group AAnalytes
35!o    4OO
                                                                                   52-015-9
                                                                                    377

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  Method 555
                                                                                 Method 555
0.20 -
0.15-
0.10-
0.05-
  0-!
 -0.05
                           o
                           c
                           
-------