r/-o
                    METHODS FOR  BENZIDINE.  CHLORINATED  ORGANIC  COMPOUNDS.
                              PENTACHLORQPHENQL  AND  PESTICIDES
                                         K ANU'WASTEWATER
                                           INTERIM
                                     Pending Issuance of
                                 Methods for Organic Analysis
                                     of  Water and Wastes
                             U.S. ENVIRONMENTAL PROTECTION AGENCY
                       ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
                                   CINCINNATI, OHIO  42568
I
V
                                         September 1978

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                                                                             *•• v.
                                  FOREWORD
       This collection of methods for the  determination of benzidine,
chlorinated organic compounds, pentachlorophenol  and  pesticides  has  been
assembled by the staff of the Environmental Monitoring and Support
Laboratory - Cincinnati (EMSL-Cinti.) for  use by  the  NPOES Permits
Program.

       These methods are as referenced  in  the Federal Register of
December 1, 1976 and are being provided only for  the  interim period  until
the manual "Methods for Organic Analysis of Water and Wastes" becomes
available.
                      Dwight 6. Ballinger, Director
       Environmental Monitoring and.Support Laboratory - Cincinnati

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                                  DISCLAIMER

    The mention of trade names or commercial products  in ithis manual  is for
illustration purposes, and does not constitute endorsement or recommendatior
by the U. S. Environmental Protection Agency.
                                      111

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                                                                                't.
                                TABLE OF CCMTEi'ITS

 Method for Benzidine and Its Salts in Water and Wastewater
 Method for Chlorinated Hydrocarbons in Water and Wastewater
 Method for Organophosphorus Pesticides in Water and Waste-
      water
 Method for Polychlorinated Biphenyls (PC3s) in Water and Waste-
      water
 Method for Triazine Pesticides in Water and Wastewater
 Method for 0-aryl  Carbamate Pesticides in Water and Wastewater
 Method for N-aryl  Carbamate and Urea Pesticides in Water and'
      Wastewater
 Page
   1
   7

 25

 43
 83
 94

 T04
Method for  Chlorophenoxy Acid Pesticides in Water and Wastewater 115
Method for  Volatile  Chlorinated Organic Compounds in Water and
     Wastewater
                     O
Method for  Pentachlotfphenol  in Water and Wastewater
Appendix  I
Appendix  II
Appendix  III
Appendix  IV
Bibliography                                      '
130
140
141
146
149
151
154
                                     iv

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                                PAGE REFERENCES
F.R.#   Parameter
     EPA       14th ed.    ASTM    US6S*
This Manual  Std. Methods  (1975)
9
14



















94
95














Benzidine
Chlorinated orqanic
compounds:
Benzylchloride
Carbon tetrachloride
Chlorobenzene
Chloroform
Epichlorohydrin
Heptachloro epoxide
Methyl ene chloride
PCB-1016
PC8-1221
PCS- 1232
PCS- 1242
PCB-1248
PCS-1254
PCS-1260
1,1,2,2-Tetrachlorcethane
Tetr ach 1 oroethy 1 ene
1,2,4-Trichlorobenzene
1,1,2-Trichloroethane
Pentachlorophenol
Pesticides
Aldrin
Ametryn
Ami nocarb
Atraton
Atrazine
Azinphos methyl
Bar ban
BHC
Captan
Carbaryl
Carbophenothion
Chlordane
Chlorpropham
2,4-0
1

130
130
130
130
130'
_-
130
43
43
43
43
43
43
43
130
130
130
130
140

7
S3
94
S3
83
25
104
7
7
94
—
7
104
115
                                            555
                           529
30
                                            555
                            529     30
                                            555
                                            555
                                            555
                            529
                            529
                                                               30
                                                                35
        STORET
        NUMBER

        39120
32102
34301
32160

39420
34423
34671
39488
39492
39496
39500
39504.
39508

34475
        39032


        39330



        39033
         39640
         39750

         39350

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F.R.I   Parameter
     EPA       14th ed.    ASTM    USGS*    STORFT
This Manual  Std. Methods  (1975)           NUMBER
     DOD
     DDE
     DOT
     Demeton-0
     Diazinon
     Dicamba
     Dichlorofenthion
     Dichloran
     Dicofol
     Dieldrin
     Dioxathion
     Disulfoton
     Diuron
     Endosulfan
     Edrin
     Ethion
     Fenuron
     Fenuron - TCA
     Heptachlor
   "  tsodrin
     lindane
     Linuron
     Malathion
     Methiocarfa
     Methoxychlor
     Mexacarfaate
     Mirex
     Monuron
     Monuron-TCA
     Neburon
     Parathion methyl
    Parathion ethyl
    PCN8
    Perthane
    Prometon
    Prometryn
    Propazine
    Propham
    Proporur
    Secfaumeton
    Siduron
    Si 1vex,
    Simazine
    Strobane
    Swep
    2,4,5-T
    Terbuthylazine
      7
      7
      7
     25
     25
    115
     ;25
    1P4
      7
      7

    104
    104
      7

      7
    104
     25
     94
      7
     94
      7
    104
    104
    104
    25
    25
     7

    83
    83
    83
   104
    94
    83
   104
   115
    83
     7
   104
   ITS
    83
 555
 555
 555
                555
.529
 529
 529
 555
 555
 555

 555

 555

 555

 555
555
555
555
                           529
 529
 529
 529

 529



 529
           529
 30
 30
 30

 30

 30"
                                   30
                                   30
30
30
30
30
30

30

30
        30
555
529
                   35
                   35
 39360
 39365
 39370
 39560
 39570
                             39780
39010
39650
39388
39390
39398
39410
39430
39782

39530

39489

39755
         39600
         39540
         39029
         39034
         39056
         39057
         39024
         39052
                 39760
                 39055
                                    vi

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F.R.#   Parameter               EPA       14th ed.    ASTM    US6S*     STORET
                           This Manual  Std. Methods  (1975)            NUMBER


     Toxaphene          '         7         555        529     30        39400
     Trlfluraline                7           —          —       ~       39030

*Soerlitz, D. & Brown,  E. "Methods for Analysis of Organic  Substances  in
Water," U.S. Geological Survey Techniques of Water-Resources  Inv.  Book 5,  Ch.
A3 (1972).

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            METHQP FOR BENZIDINE AND ITS SALTS IN WASTEWATERS

1.   Scope and Application
    1.1  This method covers the determination for benzidine  and  its  salts
         in water and wastewaters.  The method can be modified to  apply
         also to the determination of closely related materials  as des-
         cribed under Interferences (4.2)..
    1.2  The salts of benzidine,  such as benzidine  sulfate,  are  measured
         and reported as benzidine,'STORET  NO. 39120.
    1.3  The method  detection  limit  is  0.2 Wl  when analyzing 1 liter of
         sample.
2.  Summary
    '2.1  The water  sample  is  made basic and the  benzidine is extracted
         with  ethyl  acetate.   Cleanup is accomplished by extracting the
         benzidine  from the ethyl acetate with hydrochloric acid.
         Chloramine-T is added to the acid solution to oxidize the benzi-
         dine.   The yellow oxidation product is extracted with ethyl
          acetate and measured with a scanning spectrophotometer.  The
          spectrum from 510 nm to 370 nm is used for qualitative  identi-
          fication.
 3.  Hazards
     3.1  Benzidine  is a known carcinogen.  All  manipulations  of -this
          method should be  carried out  in a hood with protection

                                        1

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          provided for the hands and arms of the analyst.  Consult'OSHA
          regulations (1)  before working with benzidine.
 4.   Interferences
     4.1   The  multiple extractions  effectively limit the  interferences to
          organic  bases.   The oxidation with Chloramine-T to form a  yellow
          product  is  very  selective and has been described in detail
          (2,3.).   The use  of the absorption spectrum for  the identi-
          fication of benzidine  results in a highly specific procedure.
     4.2   Some compounds having  a structure very similar  to benzidine  will
          interfere with the quantification,  if present.   Examples of
          these interfering compounds  are dichlorobenzidine,  o-tolidine,
          and dianisidine.
                  •                 i                  .    t>             _   .
     4.3   A general yellow background color in the  extract will  limit  the
          cell pathlength  that can  be employed and  thus limit the sensi-
          tivity of the method.
5.   Apparatus and Materials
     5.1   Spectrophotometer-visible, scanning (510-370 nm).
     5.2   Separatory  Funnels - 125  ml,  250 ml,  2000 ml.
     5.3   Cells -  1 to 5 cm pathlength,  20 ml  volume maximum.
6.   Reagents, Solvents and Standards
    6.1   Ethyl acetate
    6.2   Hydrochloric acid (1 N) - Add  83 ml  cone,  hydrochloric  acid  to
         water and-dilute  to one liter.
    6.3  Chloramine-T - 10%  solution.   Prepare  fresh daily  by dissolving
          l.Og Chloramine-T in 10 ml distilled water.

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    6.4  Stock standard (0.2 .ug/ul) - Dissolve  100.0 mg  purified  benzi-
         dine in about 30 ml 1 N HC1.  Dilute to 500 ml  with  water.
7.  Preparation of Calibration Curve
    7.1  To a series of 125-ml separatory  funnels,  add 45 ml  of hydro-
         chloric acid and 10 ml of ethyl acetate.   Shake for  one  minute
         to saturate the acid  layers.  Discard  the  solvent  layers.   Dose
         the series with volumes from  1.0  to 20.0 jul-of  stock standard,
         using syringes.
    7.2  Treat standards according to  the  Procedure beginning with  8.5.
8.  Quality Control
    8.1  Duplicate and spiked  sample analyses  are  recommended as  quality
         control checks.  Quality control  charts  should be developed
         and used as a check on the analytical  system.   Quality control
         check samples and  performance evaluation  samples  should  be
         analyzed on a regular basis.
    8.2  Each time a set of samples  is extracted,  a method blank  is
         determined on a volume of distilled  water equivalent to  that
         used to dilute the sample.
9.  Procedure
    9.1  Adjust  the sample  pH  to  8.5  to  9.0 with dilute NaOH or HC1.
    9.2  Transfer  1  liter of sample  to a 2000-ml  separatory funnel.  Add
          150 ml  ethyl  acetate  and shake  for two minutes.  Allow the
          layers  to  separate,•then drain  the water layer into a

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          second 2-liter separately funnel.   Drain  the  solvent  layer  into
          a 250-ml separatory funnel.
     9.3  Repeat the extraction of the water  layer  twice more with
          50-ml portions of ethyl acetate.  Combine all solvent  layers,
          then discard the water layer.
     9.4  Extract the solvent layer three times with 15-ml portions of
      . --hydrochloric acid fay shaking 2 minutes and allowing the phases
          to separate.   Combine the acid layers in a glass stoppered
          container for cold storage until time is available for analysis,
          or transfer the layers directly into a 125-ml separatory funnel.
     9.5  Prepare.the spectrophotometer so it is warmed and ready to use.
          The  remaining steps  of the procedure must be performed rapidly
          on one  sample at  a time.                   ' •
     9.6  To the  hydrochloric  acid solution in a V25 ml  separatory funnel,
          add  1.0 ml  chloramine-T solution and mix.   Add
          25.0-ml ethyl  acetate  with  a pipet  and snake for  two  rm'nuces.
          Allow the  layers  to  separate,  then  discard the. aqueous phase.
    9.7   Filter the  solvent  layer  through  coarse  filter paper  and fill  a
          5-cm cell with the filtrate.
    9.3   Scan the solvent  from  510 nm to  370  nm,   Ethyl  acetate is  used
         for a blank with  double beam instruments.  Shorter  pathlength
         cells should be used in cases where  absorbance  exceeds 0.8.
10.  Calculation of Results
    10.1 Benzidine is identified by its absorbance maximum at
         436 nm.  Dichlorobenzidine gives similar response but has: its
         absorfaance maximum at 445 nm..

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    10.2  Construct a hasaline from the absorbance minimum at about 470 nm
         to tha minimum at 350 nrn (or 420 nm minimum for samples with a
         high background).  Racord tha assorbanca ofthe peak maximum and
         the absorbanca of tha constructed baseline at the 436 nm.  Treat
         samples snd standards in ths sane fashion.
    10.3  Using the net absorbance values, prepare a calibration plot from
         the standards.  Determine the total micrograms in each sample
         from this plot.
    10.4  Divide the total micrograms by the sample volume, in liters, to
         determine pg/1.  Correct results for cell pathlength if
         necessary.
11.  Reporting Results
    11.1  Report results in micrograms per liter as benzidine without
         correction for recovery data.  When duplicate and spike samples
         are analyzed all data obtained should be reported.
12.  Accuracy and Precision
    12.1  When 1 liter samples of river water were dosed with 1.80 pg of
         benzidine, an average of 1.24jug was recovered.  The standard
         deviation was 0.092 ,ug/l (n=S).

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REFERENCES:
1-  Federal Register, Volume 39, Page 3779, Paragraph 1910.93; (January 29,
    19747.

2.  Classman, J. M., and Meigs, J. W., "Benzidine (4,4l-Diaminob-fphenyl) and
    Substituted Benzidines", Arch. Industr. Hyg., 4, 519, (1951),;

3.  Butt* L. T. and Strafford, N., "Papilloma of the Bladder in the Chemical
    Industry.  Anlaytical Methods for the Determination of Benzidine and
    B-Naphtylamine, Recommended by A.B.C.M. Sub-Committee", J. Appl. Chem.,
    £, 525 (1956).                                   .•••-.  	  	

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       METHOD FOR CHLORINATED HYDROCARBONS IN WATER AND WASTEWATER



1.   Scope and Application

    T.I   This method covers the determination of various organo-

          chlorine pesticides and heptachlor epoxide in water and

          wastewater.

    1.2   The following pesticides may be determined individually by  this

          method:

                 Parameter      .           Storet 'No.

                 Aldrin                      39330
                 8HC                         ——
               " Captan                      39640
                 Chlordane                   39350
                 ODD                         39360
                 DDE                      -  39365
                 DOT                         39370
                 Dichloran                   —-
                 Oieldrin                    39380
                 Endosulfan                  39388
                 Endrin                      39390
                 Heptachlor                  39.410
                 Lindane                     39782
                 Methoxychlor                39480
                 Mi rex                       39755
                 PCNB                        39029
                 Sfr-ohane                    ——
                 Toxaphene                   39400
                 Trifluralin                39030

     1.3    The following  chlorinated  organic  compound may be determined

           individually by  this  method:

                 Compound                  Storet  No.

                 Heptachlor epoxide         .  ——

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2.  Summary    .       .                   -.   .
    2.1   The method offers several analytical  alternatives, dependent on
          the analyst's assessment of the nature and extent of  interfer-
          ences and/or the complexity of the pesticide mixtures found,
          Specifically, -the procedure describes the use of an effective
          co-solvent for efficient sample extraction; provides, through
          use of column chromatography and  liquid-liquid partition,
          methods for elimination of non-pesticide  interferences  and  the
          pre-separation of pesticide mixtures.  Identification is made
          by selective gas chromatographic  separations and may  be corro-
          borated through the use of two or more unlike columns.
          Detection and measurement is accomplished by electron capture,
          microcoulometric or electrolytic  conductivity gas chromato-
          graphy.  Results are reported in  micrograms per  liter.
    2.2  . Confirmation of the identity of the compounds should  be made by
          GC-MS when a new or undefined sample  type is being analyzed and
          the concentration is adequate for such determination.
 '   2.3   This method is recommended for use only by experienced  pesti-
          cide analysts or under the close  supervision of  such  qualified
          persons.
3.  Interferences              .        .   .
    3.1   Solvents, reagents, glassware, and other  sample  processing
          hardware may yield discrete artifacts and/or elevated
          baselines, causing misinterpretation  of gas chromatograms.

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      All of these materials must be demonstrated to be free from
      interferences under the conditions of the analysis.  Specific
      selection of reagents and purification of solvents by distill-
      ation in all-glass systems may be required.  Refer to Appendix
      I.
3.2   The interferences in industrial effluents are high and varied
      and often pose great difficulty in obtaining accurate and
      precise measurement of organochlorine pesticides.  Sample
      clean-up procedures are generally required and may result  in
      the loss of certain organochlorine pesticides.  Therefore,
      great care should be exercised in the selection and use of
      methods for eliminating or minimfzing interferences.  It  is not
      possible to describe procedures for overcoming .all of the
      interferences that may be encountered in  industrial effluents.
3.3   Polychlorinated Biphenyls (PCSs)  - Special attention  is called
      to industrial plasticizers and hydraulic  fluids such  as the
      PCBs, which are a potential source of interference in pesticide
      analysis.  The presence of PCBs is indicated by a  large number
      of partially resolved or unresolved peaks which may occur
      throughout the entire chromatogram.  Particularly  severe  PCS
      interference will require special separation procedures (1, 2).
3.4   Phthalate Esters  - These compounds, widely used as
      plasticizers, respond to the  electron capture' detector  and ara
      a  source of  interference  in the determination  of  organochlorine
      pesticides using  this detector.   Water  leaches  these  materials
    • from  plastics, such  as polyethylene bottles  and  tygon tubing.

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          The presence of phthalate esters is implicated  in samples that
          respond to electron capture but not to the microcoulometric or
          electrolytic-conductivity halogen detectors'or  to the flame
          photometric detector.
    3.5   Organophosphorus Pesticides - A number of organophdsphorus
          pesticides, such as those containing a nitrp group, e.g.,, para-
          thion, also respond to the electron capture detector  and may
          interfere with the determination of the  organochlorine  pesti-
          cides.  Such compounds can. be  identified by their response  to.
          the flame photometric-detector (3).      .   • .
4.  Apparatus and Materials
    4.1   Gas Chromatograph  - Equipped with  glass  lined  injection port.
    4.2   Detector Options:           ,
          4.2.1  Electron  Capture  - Radioactive  (tritium or  m'ckel-63)
          4.2.2  Microcoulometric  Titration                      •
          4.2.3  Electrolytic Conductivity-
    4.3   Recorder -  Potentiometric strip  chart  (10  in.)  compatible with
          the detector.
    4.4   Gas Chromatographic  Column  Materials:
          4.4.1  Tubing  - Pyrex ()80  cm long X 4 mm ID)
          4.4.2  Glass Wool  -  Silanized              '        .
          4.4.3  Solid Support - Gas-Chrom-Q (100-120 mesh)
          4.4.4- Liquid Phases - Expressed as weight percent coated on
                     so.lid support*                               "
                 4.4.4.1  OV-1, 3%
                 .4.4.4.2  OV-2.10, '558:      -        '
                                       10

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                 4.4.4,3   OV-17,  1.5% plus QF-1 or OV-210, 1.95%
                 4.4.4.4   QF-1, 6% plus SE-30,  4%
   4.5    Kuderna-Danish  (K-D)  Glassware
          4.5.1   Snyder Column  -  three-ball (macro)  and two-ball
                    (micro)
          4.5.2   Evaporative Flasks - 500 ml
          4.5.3   Receiver Ampuls  - 10 ml, graduated
          4.5.4   AmpuT Stoppers
   4.6    Chromatographic Column  - Chromaflex (400 mm long x 19 mm ID)
          with coarse fritted plate on bottom and Teflon stopcock; 250-ml
          reservoir bulb  at top of column with flared out funnel shape at
          top of bulb - a special order (Kontes K-42054Q- 9011).
   4.7    Chromatographic Column  - pyrex (approximately 400 mm  long x 20
          mm ID) with coarse fritted plate on bottom.
   4.8    Micro  Syringes  - 10,  25, 50 and 100 jul.
   4.9-   Separatory funnels - 125 ml, 1000 ml and 2000 ml with teflon
          stopcock.
   4.10  Blender - High  speed, glass or stainless steel cup.
   4.11  Graduated cylinders - 100 and 250 ml.
   4.12  Florisil - PR Grade (60-100 mesh); purchase activated at
          1250°F and store in the dark in glass containers with glass
          stoppers or foil-lined screw caps.  Before use, activate each
          batch  overnight at 130°C in foil-covered glass container.
          Determine lauric-acid value (See Appendix  II).
5.  Reagents, Solvents, and Standards
    5.1    Sodium Chloride - (ACS) Saturated  solution in distilled water
                                       11    •

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          (pre-rinse Nad with hexane).
           *                       !                                   . •
    5.2   Sodium Hydroxide - (ACS) 10 N in distilled water.
    5.3   Sodium Sulfate - (ACS)  Granular, anhydrous (conditioned at 400
          C for 4 hrs.).
    5.4   SuIfuric Acid - (ACS) Mix equal volumes of cone. HgSO^ with
          distilled water.                                       :
    5.5   Diethyl Ether - Nanograde, redistilled  in glass, if necessary.
          5.5.1 Must be free of peroxides as indicated by EM Quant test
                strips.  (Test strips are available from EM Laboratories,
                Inc., 500 Executive Blvd., Elmsford, M.Y. 10523.)
          5.5.2 Procedures recommended for removal of peroxides  are
                provided with the test strips.
    5.6   A'cetonitrile, Hexane, Methanol, Methylene Chloride, Petroleum
          Ether (boiling range 30-60°C) - nanograde, redistill in glass
          if necessary.
    5.7   Pesticide Standards - Reference-grade.
6.  Calibration
    6.1   Gas chromatographic operating conditions are considered accept-
          able if the response to dicapthon  is  at least 50% of full  scale
          when < 0-06 ng is  injected for electron capture detection  and
          ^100 ng is injected for microcoulometric or electrolytic.
          conductivity detection.  For all quantitative measurements,  the
          detector must be operated within  its  linear response range and
          the detector noise level should be .less than 2% of  full  scale.
    '6.2   Standards are injected  frequently  as  a  check on the  stability
          of operating conditions.  Gas chromatograms of  several standard
                                      12

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          pesticides are shown in Figures  ls 2, 3  and 4  and  provide
          reference operating conditions for the four recommended  columns.
    6.3   The elution order and retention  ratios of various  organo-
          chlorine pesticides are provided  in Table 1, as  a  guide.
7.  Quality Control
    7.1   Duplicate and spiked sample  analyses  are recommended  as  quality
          control checks.  Quality control  charts  (4) should be developed
          and used as a-check on the analytical system.  Quality control
          check samples and performance evaluation samples should  be
          analyzed on a regular basis.
    7.2   Each time a set of samples is extracted, a method  blank  is
          determined on a volume of  distilled water  equivalent  to  that
          used to dilute the sample,
8.  Sample Preparation
    8.1   The sample size taken for  analysis  is dependent  on the type of
          sample and the sensitivity required for  the  purpose at hand.
          Background  information on  the pesticide  levels previously
          detected  at a given  sampling site will  assist  in determining
          the sample  size required,  as well as  the final volume to which
          the extract needs  to  be  concentrated.  A 1-liter sample is
          usually  taken for  drinking water and  ambient water analysis to
          provide  a detection  limit  of O.QSOto  O.lQO^g/1.  One-hundred
          milliliters  is  usually  adequate to provide a detection limit of
           1 jjg/1 for  industrial  effluents.
                                        13

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                                                &

                                               ~e
                                               S3
                                               aa

                                                re
                                               «-•  09
                                                OS  I—
                                                 .
                                        19 UJ   trt   «J
                                        "~ t   ra   oa
                                               •-   e.
                                                *ei "5
14

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                          10                 5
                      HETEXT10N TIME IH MINUTES
Figure  2. Column Picking:  5%  OV-210,  Carrier fias: Argon/Methane
          at 70 ml/rain, Column Temperature: 180 C, Detector:
          Electron Capture.
                             15

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        XHHIW
16

-------
               X3HIW
17

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                                     Table 1                  •
     RETENTION RATIOS OF VARIOUS ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Phase^ T.
Column Temp.
Argon/Methane
Carrier Flow
Pesticide
Trifluralin
«-BHC
PCNB
Lindane
Oichloran
Heptachlor .
Aldrin
Heptachlor Epoxide
Endosulfan I
p,p'-DDE
Dieldrin
Captan
Endrin
o,p'-DDT
p,p'-DOD
Endosulfan II
p,p'-ODT
Mirex
Methoxychlor
Aldrin
(Min. absolute)
1.5% OV-17
•f
95% OF-12
200 C
60 ml/min
RR
0.39
0.54
0.68
0.69
0.77
o;s2 .
1.00
1.54
1.95
2.23
2.40
2.59
2.93
3.16
3.48
3.59
4.18
6.T
7.6
3.5
5%
OV-210
180 C
70 ml/min
RR
1.11
0.64
0.85
0.81
1.29
0.87
1.00
1.93
2.48 ..
2. 10.
3.00
4.09
3.56
2.70
3.75
4.59
4.07
3.78
6.5.
2.6
3%
OV-1
180 C
70 ml/min
RR
0.33
0.35
0.49
0.44
0.49
0.78
1.00
1.28 '
1.62
2.00
1.93
1.22
2.18
2.69
2.61
2.25
3.50
6.6
5.7
4.0.
5% QF-1
»K
4% SE-30
200 C
60 ml/min
RR
0.57
0.49
0.63
0.60
0.70
• 0.83
,1.00
1.43
1.79
1.82
2.12
1.94
:2.42
2.39
2.55
2.72
3.12
4.79
4,60
5.6
     columns glass, 180 cm x 4 mm ID, solid support Gas-Chrom Q  (100/120
 mesh)
2OV-210 also may be used

                                      18

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    8.2   Quantitatively transfer the proper aliquot of sample from the
          sample container into a two-liter separatory funnel.   If less
          than 800 ml is analyzed, dilute to one  liter with  interference
          free distilled water.
9.  Extraction
    9.1   Add 60 ml of  15% methylene chloride  in  hexane (v:v)  to the
          sample in the separatory funnel and  shake vigorously for two
          minutes.                              .
    9.2   Allow the mixed  solvent  to separate  from the  sample, then  draw
          the water  into a one-liter Erlenmeyer flask.  Pour the organic
          layer into  a  100 ml  beaker and then  pass it  through a  column
          containing  3-4  inches of anhydrous  sodium sulfate, and collect
          it  in a  500 ml  K-0 flask equipped with'a 10  ml  ampul.   Return
          the water  phase  to the  separatory funnel.   Rinse the Erlenmeyer
          flask with a  second 60-ml  volume_of solvent;  add the solvent to
          the  separatory  funnel  and complete the extraction procedure a
          second  time.   Perform a third extraction in the same manner.
     9.3  Concentrate the extract in the K-D evaporator on a hot water
          bath.
     9.4  Analyze by gas chromatography unless a need for cleanup is
           indicated  (See Section 10).
 10. Clean-up and Separation Procedures
     10.1  Interferences in  the form of  distinct  peaks and/or  high back-
           ground  in  the initial gas chromatographic analysis, as well  as
           the physical characteristics  of  the  extract  (color, cloudiness,
           viscosity) and  background knowledge  of the sample will  indicate
                                      19

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      whether clean-up is required.  When these  interfere with
      measurement of the pesticides, or affect column life or
      detector sensitivity, proceed as directed below.
10,2  Acetonitrile Partition - This procedure is used to isolate fats
      and oils from thel sample extracts.  It should be noted that not
         •
      all pesticides are quantitatively recovered by this procedure.
      The analyst must be aware of this and demonstrate the effi-
      ciency of the partitioning for specific pesticides.  All of the
      pesticides listed in Scope (-1.2)  with the exception of mi rex
      are efficiently recovered.
      10.2.1  Quantitatively:transfer the previously concentrated
              extract to  a 125-ml separatory funnel with enough
              hexane to bring  the final volume to 15 ml.  Extract the
              sample four times by shaking vigorously for one minute
              with  30-ml  portions of hexane-saturated acetonitrile.
      10.2.2  Combine and transfer the  acetonitrile phases  to a
              one-liter separatory funnel  and add 650 ml of distilled
              water  and 40 ml  of  saturated sodium chloride  solution.
              Mix thoroughly for  30-45  seconds.   Extract with two
              100-ml  portions  of  hexane by vigorously shaking about
              15 seconds.
      10.2.3   Combine  the hexane  extracts  in  a one-liter separatory
              funnel  and wash  with  two  100-ml portions of distilled
              water.   Discard  the water layer and  pour the  hexane
              layer through  a  3-4  inch  anhydrous  sodium  sulfate
              column  into a  500-ml  K-0  flask  equipped with  a  10-ml
                                 20

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              ampul.   Rinse the separatory funnel and column with
              three 10-ml portions of hexane.
      10.2.4  Concentrate the extracts to 6-10 ml in the K-0 evapor-
              ator in a hot water bath.
      10.2.5  Analyze by gas chromatography unless a need for further
              cleanup is indicated.
10.3  Florisil Column Adsorption Chromatography
      10.3.'1  Adjust the sample extract volume to 10 ml.
      T0o3.2  Place a charge of activated Florisil (weight  determined
              by lauric-acid value, see Appendix  II) in a   Chromaflex
             . column.  After settling  the Florisil by  tapping the
              column, add about one-half inch  layer of anhydrous
              granular-sodium  sulfate  to the top.
      10.3.3  Pre-elute  the column, after cooling, with 50-60 ml of
              petroleum  ether.  Discard the  eluate and just prior  to
              exposure of the  sulfate  layer  to air, quantitatively
              transfer the  sample  extract  into the column  by
              decantation and  subsequent petroleum ether wash-  ings.
              Adjust  the elution  rate  to about 5  ml  per minute  and,
              separately, collect  up  to three  eluates  in 500-ml K-0
              flasks  equipped  with 10-ml ampuls  (see  Eluate
              Composition  10.4.).   Perform the first  elution with
              200  ml  of  6%  ethyl  ether in  petroleum  ether,  and  the
              second  elution with 200 ml of 15%  ethyl  ether in
                                   21

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                   petroleum ether.  Perform the third elution with 200 ml
                   of 50% ethyl ether - petroleum ether and the fourth
                   elution with 200 ml of 100% ethyl ether.
           10.3.4  Concentrate the eluates to 5-10 ml in the IC-D eva-
                   porator in a hot water bath.
           10.3.5  Analyze by gas chromatography.
     70.4  Eluate Composition  - By using an equivalent quantity of any
           batch of Florisil, as determined by its lauric acid value, the
           pesticides will be separated into the eluates indicated below:
                               6% Eluate
              Aldrin
              BHC
              Chlordane  •
              ODD
              ODE
             15% Eluate
              Endosulfan I
              Endrin
              Dieldrin
              Dichloran
DDT                 Mi rex
Heptachlor          PCNB
Heptachlor Epoxide  Strobane
Lindane             Toxaphene
Methoxychlor        Trifluralin
    :         50% Eluate
              Endosulfan II
              Captan
           Certain thiophosphate pesticides will occur in each of the
           above fractions as well  as the 100% fraction.   For additional
           information  regarding eluate composition, refer to the FDA
           Pesticide Analytical  Manual (5)».
'11. Calculation of Results
    11.1   Determine the pesticide  concentration by using the absolute
           calibration  procedure described below or the relative cali-
           bration procedure  described in-Appendix III.
                                      22

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             (1)    Miprograms/liter = (A)  (B) • (vt)
                                        (V-\) l»S'
                   A - nq standard
                       Standard area

                   B 3 Sample aliquot area
                   V-fs Volume of extract  injected
                   V-t= Volume of total extract  (jul)
                   Vss Volume of water extracted (ml)

12.   Reporting Results

     12.1  Report results in micrograms per  liter without  correction  for

           recovery data.  When duplicate and spiked  samples  are ana-

           lyzed, all data obtained should be reported.
                                      23

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REFERENCES:
1.  Monsanto Methodology for Aroclors - Analysis of Environmental  Materials
    for Biphenyls, Analytical Chemistry Method 71-35, Monsanto Company,
    St. Louis, Missouri, 63166, 1970.

2.  "Method for Polychlorinated Biphenyls in Water and Wastewater", this
    manual, p. 43.

3.  "Method for Organophosphorus Pesticides in Water and Wastewater", this
    manual, p. 25.

4.  "Handbook for Analytical Quality Control in Water and Wastewater
    Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
    Agency, National Environmental Research Center, Analytical Quality Con-
    trol Laboratory, Cincinnati, Ohio, 45268, 1973.

5.  "Pesticide Analytical Manual", U. S. Dept. of Health, Education and
    Welfare, Food and Drug Administration, Washington, 0. C.
                                     24

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       METHOD FOR ORSANOPHOSPHORUS PESTICIDES IN WATER AND WASTEWATER
1.  Scope and Application
    1.1  This method covers the determination of various organophosphorus
         pesticides in water and wastewater.
    1.2  The following pesticides may be determined  individually by this
         method:
                     Parameter                    Storet No.
                     Azinphos methyl
                     Demeton-0                       33560 .
                     Demeton-S                        —
                     Diazinon    "                   - 39570
                     Disulfoton                      39010
                     Malathion                       39530
                     Parathion methyl                39600
                     Parathion ethyl                 39540
2,  Summary
    2.1  The method offers several analytical alternatives,  dependent  on
         the analyst's assessment of the nature and  extent of  interferences
         and the complexity of the pesticide mixtures found.   Specifically,
         the procedure describes the use of an effective co-solvent for
         efficient sample extraction; provides, through use  of the column
         chromatography and liquid-liquid partition, methods for the
         elimination of non-pesticide interferences  and the  preseparation
         of pesticide mixtures.  Identification is made by selective gas
         chromatographic separation and may be corroborated  through the use
         of two or more unlike columns.  Detection and measurement are best
         accomplished by flame photometric gas chromatography  using a
                                      25

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         phosphorus specific filter.  The electron  capture  detector, though
         non-specific, may also be used for those compounds to which it
         responds.  Results are reported  in micrograms  per  liter,
    2.2  Confirmation of the identity of the compounds  should be made by
         GC-MS when a new or undefined sample  type  is being analyzed and
         the concentration is adequate for such determination.
    2.3  This method is recommended for use only by experienced pesticide
         analysts or under the close supervision of such qualified persons.
3.  Interferences
    3.1  Solvents, reagents, glassware, and other sample processing hard-
         ware may yield discrete artifacts and/or elevated baselines, caus-
         ing misinterpretation of gas chromatograms.  All of these  .
         materials must be demonstrated to be  free  from interferences under
         the conditions of the analysis.  Specific  selection of reagents'
         and purification of solvents by distillation in all-glass systems'"
         may be required.  Refer to Appendix I.
    3.2  The- interferences in industrial effluents  are  high and varied and
         often pose great difficulty in obtaining accurate  and precise
         measurement} of organophosphorus pesticides.  Sample clean-up
         procedures are generally;required and may  result in the loss of
         certain organophosphorus pesticides.  Therefore, great care should
         be exercised in the selection and use of methods for elfminating
         or minimizing interferences.  It is not possible to describe
         procedures for overcoming all of the  interferences that may be
         encountered in industrial effluents.
                                      26

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   3.3  Compounds  such  as  organochlorine  pesticides,  polychTor-mated
        biphenyls  and phthalate  esters  Interfere  with the analysis  of
        organophosphorus pesticides  by  electron capture gas  chro-
        matography.  When  encountered,  these interferences are overcome by
        the  use of the  phosphorus specific flame  photometric detector.   If
        such a detector is not available, these  interferences may be
        removed from the  sample by using the clean-up procedures described
         in the EPA methods for those compounds (1, 2).
    3.4  Elemental  sulfur  will interfere with the determination of organo-
         phosphorus 'pesticides by flame photometric and electron capture
         gas chromatography.  The elimination of elemental sulfur as  an
         interference is described in Section 10.5, Clean-up and Separation
         Procedures.
4.  Apparatus and Materials
    4.1  Gas Crhomatograph  -  Equipped with  glass  lined  injection port.
    4.2  Detector options:
         4.2.1     Flame Photometric  -  526  mu phosphorus  filter.
         4.2.2     Electron Capture  - Radioactive (tritium or  nickel-63).
    4.3  Recorder  -  Potentiometric strip  chart (10 in.)  compatible  with the
         detector.
    4.4  Gas Chromatographic  Column  Materials:
         4.4.1      Tubing  - Pyrex (180  cm long x  4 mm ID)
         4.4.2      Glass Wool - Silanized
         4.4.3     Solid Support - Gas  Chrom Q (100-120 mesh)
         4.4.4     Liquid  Phases - Expressed as  weight percent coated on
                    solid  support.
                                       27

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          4.4.4.1   OV-1, 3St  • •     .
          4.4.4.2   OV-210, 5%
          4.4.4.3.  OV-17,  1.5% plus QF-1  or OV-210,  1
          4.4.4.4   QF-1 or OV-210, 6% plus SE-30, 4%
4.5  Kuderna-Danish (K-0) Glassware
     4.5.1     Snyder Column  - three ball  (macro)  and two  ball  (micro)
     4.5.2     Evaporative Flasks  - 500 ml-.
     4.5.3     Receiver Ampuls - 10 ml, graduated
     4.5.4     Ampul Stoppers.
4.6  Chromatographic Column -. Chromaflex  (400  mm x 19 mm ID)  with
     coarse fritted plate and Teflon stopcock  on bottom;  250  ml
     reservoir bulb at top of column with  flared out  funnel shape  at
     top of bulb - a special  order (Kontes K-420540-9011).
4.7  Chromatographic Column - pyrex (approximately 400 mm long  x 20 mm
     ID) with coarse fritted  plate on bottom.
4.8  Micro Syringes - 10, 25, 50 and TOOjjl.
4.9  Separatory funnels - 125 ml,  1000 ml  and  2000 ml with Teflon
     stopcock.
4.10 Micro-pipets - disposable (140 mm  long x  5 mm. ID).
4.11 Blender  - High speed,  glass or stainless  steel cup. ,
4.12 Graduated cylinders -  100 and 250 ml.
4.13 Florisil - PR Grade (50-100 mesh); purchase  activated at 1250°F
     and store in the dark  in glass containers with glass stopper? or
     foil-lined screw caps..   Before use,  activate  each batch overnight
     at  130°C in foil-covered glass container.  Determine lauric-acid
     value (See Appendix II).
                                   28

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    4.14 Alumina - Woelm, neutral; deactivate by pipeting 1 ml of distilled
         water into 125 ml ground glass-stoppered Erlenmeyer flask.  Rotate
         flask to distribute water over surface of glass.   Immediately  add
         19.0 g fresh alumina through small powder funnel.  Shake flask
         containing mixture for two hours on a mechanical shaker (3).
5.  Reagents, Solvents, and Standards
    5.1  Sodium Chloride  - (ACS) Saturated  solution  in  distilled water
         (pre-rinse NaCl with hexane).
    5.2  Sodium Hydroxide - (ACS)  10.N in distilled  water.
    5.3 . Sodium Sulfate - (ACS) Granular, anhydrous  (conditioned at  400*C
         for 4 hrs.).                     .
 ..  5.4  Sulfuric Acid -'(ACS) Mix equal  volumes  of  cone. H2S04-with
       -  distilled water.
    5.5  Oiethyl Ether -Nanograde, redistilled  in glass,  if  necessary.
         5.5.1     Must be free of peroxides as  indicated by  EM Quant  test
                   strips.  (Test  strips  are available  from EM
                   Laboratories,  Inc., 500  Executive Blvd., Emslford,  N.Y-.
                   10523.)
         5.5.2     Procedures  recommended for  removal of peroxides  are
                   provided with  the  test strips.
    5.6  Acetonitrile, Hexane, Methanol,  Methylene  Chloride,  Petroleum
         Ether  (boiling  range  30-60toC)  - nanograde,  redistill  in  glass if
         necessary.
    5.7  Pesticide Standards  - Reference grade.
                                        29

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 6.  Calibration
     6.1  Gas chromatographic operating conditions are considered acceptable
          If the response to dicapthon is at least 50% of full scale when <1.5
          ng 1s Injected for flame photometric detection and <"0.06 ng is
          Injected for electron capture detection.  For all quantitative
          measurements, the detector must be operated within its linear
          response range and the-detector noise level should be less than 2%
          of full  scale.
     6.2-  Standards are injected frequently as a check on the stability of
          operating conditions.  Gas chromatograms of several standard
          pesticides are shown in Figures 1, 2, 3 and 4 and provide reference
          operating conditions for the four recommended columns.
     6.3   The elution order and retention ratios of various organophosphorus
          pesticides are provided in Table 1,  as a guide.
7.   Quality Control
     7.1   Duplicate and spiked sample analyses are recommended as quality
          control  checks.   Quality control charts (4)  should be developed and
          used  as  a check  on the analytical  system.   Quality control check
          samples  and performance evaluation samples  should be analyzed on a
          regular  basis.
     7.2   Each  time a set  of samples is  extracted,  a  method blank is
          determined on a  volume of distilled  water equivalent to that used
          to  dilute the sample.
8.   Sample  Preparation
    8.1   The sample size  taken  for analysis is dependent  on the type of
          sample and  the sensitivity required  for the  purpose at hand.
                                      30                         :

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0         2         4        8         8        10
                RETENTION TIME IN MINUTES
Figure 1. Column Packing:  1.5% OY-17 + 1.95 %  QM?
Carrier Gas: Nitrogen at 70 ml/min,  Column Temperature: 213 C,
Detector: Flame Photometric [Phospliorus).
                            31

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-CJJ
 0
10
           24         8        8
             RETENTION TIME IN MINUTES
Figure 2. Column Packing: 5% OY-210, Carrier Gas: litrogen
at 60 ml/min, Column Temperature: 200 C, Detector:
Flame Photometric  [Phosphorus].
                         32

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  fl        2         4         8'8        10
                 RETENTION TIME IN MINUTES
Figure 3. Column Packing: 6% QF-1 +4% SE-30, Carrier Gas: Nitrogen
at 70 mi/min, Column Temperature: 215 C, Detector: FSame
Photometric (Phosphorus).
                               33

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            2        4         6         8        10
               RETENTION TIME IN  MINUTES
Figure 4. Column Packing:  3% OY-1, Carrier Gas: Nitrogen at
60 ml/min, Column Temperature:  200 C, Detector: Flame
Photometric (Phosphorus).
                          34

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                                   TABLE  1

             RETENTION TIMES OF  SOME ORGANOPHOSPHOROUS  PESTICIDES
                            RELATIVE TO PARATHION
Liquid Phase1

Column Temp. 	 ^
Nitrogen
Carrier Flow
Pesticide 	 ,
Demeton^

Diazinon
Disulfoton
Ma lath ion
Parathion methyl •
Parathion ethyl
Azinpnos methyl
Parathion
(min absolute) 	
1.5% OV-17
1.95£ QF-12
215 C
70 ml/min
RR
0.46

0.40
0.46
0.86
0.82
1.00
6.65
4.5

6% QF-1^
4% SE-30
215 C
70 ml/min
RR
0.25
0.43
0.38
0.45
0.78
0.80
1.00
4.15
6.6

5%
OV-210
200 C
60 ml/min
RR
0.20
.38
0.25
0.31
0.73
0.81
1.00
4.44
5.7

7%
OV-1
200 C
60 ml/min
RR
0.74

0.59
0.62
0.92-
0.79
1.00
4.68
wo I
n /ic>n
_  mesh.
2May substitute OV-210 for QF-1.
^Anomalous, multipeak response often encountered.
                                      35

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         Background  information  on  the  pesticide levels  previously detected
         at a given  sampling  site will  assist  in determining  the sample
         size required,  as well  as  the  final volume to which  the extract
         needs to be concen-  trated.  A 1-liter sample is  usually taken for
         drinking water  and ambient water  analysis  to provide a-detection
         limit of 0.050  to 0.100;jg/l.   One-hundred milliliters is usually
         adequate to provide  a detection limit of 1 ^ig/1 for  industrial
         effluents.
    8.2  Quantitatively  transfer the  proper  aliquot of sample from the
         sample container into a two-liter separatory funnel.  If less than
         a 800 ml is analyzed, dilute to one  liter  with  interference free
         distilled water.
9.  Extraction
    9.1  Add 60 ml of  15% methylene chloride  in hexane (v:v)  to the sample
         in the separatory funnel and shake  vigorously for two minutes.
    9.2  Allow the mixed solvent to separate from the sample, then draw the
         water into  a  one-liter  Erlenmeyer flask.   Pour  the organic layer
         into a 100  ml beaker and then  pass  it through a column containing
         3-4 inches  of anhydrous sodium sulfate,  and collect  it in a 500 ml
         K-D flask equipped with a  10 ml ampul.   Return  the water phase to
         the separatory  funnel.  Rinse  the Erlenmeyer flask with a second
         60 ml volume  of solvent; add the  solvent to the separatory funnel
         and complete  the extraction  procedure a second  time.  Perform a
         third extraction in  the same manner.
    9.3  Concentrate the extract in the K-D  evaporator on  a hot water bath.
                                       36

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    9.4  Analyze by gas chromatography unless a need for cleanup is indi-
         cated.  (See Section 10).
10.  Clean-up and Separation Procedures
    10.1 Interferences in the form of distinct peaks and/or high background
         in the iaitial gas chromatographic analysis, as well as the
         physical characteristics of the extract (color, cloudiness,
         viscosity) and background knowledge of the sample source will
                          •
         indicate whether clean-up is required.  When these interfere with
         measurement of the pesticides, or affect column life or detector
         sensitivity, proceed as directed below.  The use of these
         procedures is not required for samples free of  interferences.
         They  are provided as options to the analyst to be used when'needed.
    10.2' Acetonitrile Partition - This procedure  is used to separate  fats
         and oils from the sample extracts.  It should be noted that  not
         all pesticides are quantitatively recovered by  this procedure.
         The analyst must be  aware of this and demonstrate the efficiency
         of the partitioning  for  specific pesticides.
         10.2.1    Quantitatively transfer the previously concentrated
                   extract to a  125-ml separatory funnel with  enough  hexane
                   to bring the final volume to  15 ml.   Extract the sample
                   four times by  shaking  vigorously for  one minute  with 30
                   ml portions of hexane-saturated  ace-tonitrile.
         10.2.2    Combine and transfer  the  acetonitrile phases  to  a
                   one-liter  separatory  funnel  and  add  650 ml  of  distilled
                   water  and  40 ml  of saturated  sodium chloride  solution.
                   Mix  thoroughly for 30-45  seconds.   Extract  with  two
                                       37

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               100  ml  portions of hexane by vigorously shaking about 15
               seconds.
     10.2.3     Combine the hexane extracts in a one-liter separatory
               funnel  and wash with two 100 ml portions of distilled
               water.   Discard the water layer and pour the hexane
               layer through a 3-4 inch anhydrous sodium sulfate column
               into a 500-ml K-D flask equipped with a 10-ml ampul.
               Rinse the separatory funnel and column with three 10 ml
               portions of hexane.
     10.2.4     Concentrate the extracts to 6-10 ml in the K-D
               evaporator in a hot water bath.
     10.2.5     Analyze by gas chromatography unless a need for further
               clean-up is indicated.
10.3 Florisil Column Adsorption Chromatography
     10.3.1     Adjust the sample extract volume to 10 ml.
     10.3.2     Place a charge of activated Florisil (weight  determined
               fay lauric-acid value, see Appendix II) in  a Chromaflex .
              •column.  After settling  the Florisil by tapping the
               column, add about one-half  inch  layer of  anhydrous
               granular sodium sulfate  to  the  top.
     10.3.3     Pre-elute the column, after cooling, with 50-SO ml  of
               petroleum ether.   Discard the eluate and just prior to
               exposure of the sulfate  layer  to  air, quantitatively
               transfer the sample extract into  the column  by
               decantation  and subsequent  petro-  leum  ether  washings.
               Adjust the elution  rate  to  about 5 ml  per; minute  and,
                                  38                          -

-------
               separately,  collect  up  to  four eluates  In 500-ml  K-0
               flasks equipped with  10-inl  ampuls.   (See Eluate Compos-
               ition, 10.4.)  Perform  the  first  elution with 200 ml of
               6% ethyl ether in petroleum ether,  and.the second
               elution with 200 ml of  15%  ethyl  ether  in petroleum
               ether.  Perform the third elution with  200 ml of  50%
               ethyl ether  - petroleum ether  and the fourth  elution
               with 200 ml of 100% ethyl ether.
     10.3.4    Concentrate the eluates  to  6-10 ml  in the K-0 evaporator
               in a hot water bath.
     10.3.5    Analyze by gas chromatography.
10.4 Eluate Composition --By iTsing an  equivalent quantity of any batch
     of Florisil as determined by its  lauric-acid  value,  the pesticides
     will be separated into the eluates indicated  below:
             6% Eluate                  15% Eluate
             Demeton                    Diazinon
             Oisulfoton                 Malathion  (trace)
                                        Parathion  Methyl
             50% Eluate       '          100%  Eluate
             Malathion                  Azinphos methyl  (30%)
             Azinphos methyl (20%)
     For additional information regarding  eluate composition,  refer
     to the FDA Pesticide Analytical Manual (5).
10.5 Removal of Sulfur - If elemental  sulfur  interferes  with  the  gas
     chromatographic analysis, it can be removed by the  use  of an
     alumina microcolumn.
     10.5.1 Adjust the sample extract volume  to 0.5 ml  in  a  K-0
                                  39

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                apparatus, using  a two-ball  Snyder microcolumn.
         10.5.2 Plug a disposable pipet with a  small quantity of  glass
                wool.  Add enough alumina to produce a 3-cm column  after
                settling.  Top the alumina with a 0.5-cm  layer of
                anhydrous sodium  sulfate.
         10.5.3 Quantitatively transfer the concentrated  extract  to the
                alumina microcolumn using a  100 jjl syringe.  Rinse  the
                ampul with 200 pi of hexane and add to the microcolumn.
         10.5.4 Elute the microcolumn with 3 ml of hexane and discard the
                first eluate which contains the elemental sulfur*
         10.5.5 Next elute the column with 5 ml of 10% hexane in
                methylene chloride.  Collect the eluate in a 10 ml
                graduated ampul.
         10.5.6 Analyze by gas chromatography.               -
         NOTE:  If the electron capture detector is to be used methylene
              .  chloride must be removed... To do this, attach the ampul
                to a K-D apparatus (500-ml flask and 3-ball Snyder
                column) and concentrate to about 0.5 ml.   Adjust volume
                as required prior to analysis.     -
11.  Calculation of Results
    11.1 Determine the pesticide concentration by using the absolute
         calibration procedure described below or the relative cali-
         bration procedure described in Appendix III.                   •
                                      40

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        (1)   Micrograms/liter = iA)Jll_(ltl
        V                           (ViJ  ivs)   '
        A  = no standard
            Standard  area
        B  = Sample  aliquot  area
        V-j =  Volume of extract Injected (.ul)                      a
        Vt =  Volume of total extract Ul)
         Vs s  Volume of water extracted (ml)
12.  Reporting  Results
    12.1 Report results in micrograms per liter without correction for
         recovery data.  When 'duplicate and spiked samples are analyzed
         all  data obtained  should be reported.
                                        41

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REFERENCES:
1.  "Method for Chlorinated Hydrocarbons in Water and Wastewater", this
   • manual, p. 7.

2.  "Method for Polychlorinated EJiphenyls (PCBs) in Water and Wastewater",
    this manual, p. 43.

3.  Law, L. M. and Georlitz, D. F., "Microcolumn Chromatographic Clean-up
    for the Analysis of Pesticides in Water", Journal of the Association
    for Analytical Chemists, 53_, 1276 (1970).                        ~~

4.  "Handbook for Analytical Quality Control in Water and .Wastewater
    Laboratories", Chapter 6, Section 6.4, U. S! Environmental Protection
    Agency, National Environmental Research Center, Analytical Quality Con-
    trol Laboratory, Cincinnati, Ohio, 45268, 1973.

5.  "Pesticide Analytical Manual", U. S. Dept. of Health, Education and
    Welfare, Food and Drug Administration, Washington, D. C.
                                     42

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     METHOD  FOR  POLYCHLORINATEO BIPHENYLS (PCBs)  IN WATER AND WASTEWATER

1.  Scope and Application
    1.1  This method  covers  the determination  of  various polych-lorinated
         biphenyl  (PCS) mixtures in  water and  wastewater.
    1.2  The following mixtures of chlorinated biphenyls (Aroclors)  may be
         determined by this  method:
                       Parameter                 Storet  No.
                       PCS-1016                    34671
                       PCS-1221                    39488
                       PC8-1232                    39492
                       PCS-1242                    39496
                       PCB-1248                    39500
                       PCS-1254                    39504
                       PCB-1260                    39508
    1.3   The method  is an extension of  the Method  for Chlorinated
          Hydrocarbons in Water and Wastewater (1).   It  is designed  so
          that determination of both the  PCBs  and the organochlorine
          pesticides may be made on the  same sample.
2.  Summary
    2.1   The PCBs and the organochlorine pesticides are co-extracted by
          liquid-liquid extraction and,  insofar as  possible,  the  two
          classes of compounds  separated  from  one another prior to gas
          chromatographic determination.  A combination  of the standard
          Florisil column cleanup procedure and a silica gel  microcolumn
          separation procedure  (2)(3) are employed.  Identification  is
                                      43

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          made from gas chromatographic patterns obtained through the use
          of two or more unlike columns.  Detection and measurement  is
          accomplished using an electron capture, microcoulometric,  or
          electrolytic conductivity detector.  Techniques for confirming
          qualitative identification are suggested.
3.  Interferences
    3.1   Solvents, reagents, glassware, and other sample processing
                                                             *          A
          hardware may yield discrete artifacts and/or elevated baselines
          causing misinterpretation of gas chromatograms.  All of these
          materials must be demonstrated to be free from interferences
          under the conditions of the analysis.  Specific selection  of
          reagents and the purification of solvents by distillation  in
          all-glass systems may be required.'  Refer to Appendix I.
    3.2   The interferences in industrial effluents are high and varied
          and pose great difficulty in obtaining accurate and precise
          measurement of PCBs and organochlorine pesticides.  Separation
          and clean-up procedures are generally required and may result
          in the loss of certain organochlorine compounds.  Therefore,
         'great care should be exercised in the selection and use of
          methods for eliminating or minimizing interferences.  It is not
          possible to describe procedures for overcoming all of the
          interferences that may be encountered in industrial effluents.
    3.3   Phthalate esters, certain organophosphorus pesticides, and
          elemental sulfur will interfere when using electron capture for
          detection.  These materials do not interfere when the
                                      44

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          nricrocoulometric or electrolytic conductivity detectors are
          used in the halogen mode.
    3.4   Organochlorine pesticides and other halogenated compounds
          constitute interferences in the determination of PCBs.  Most of
          these are separated by the method described below.  However,
          certain compounds, if present in the sample, will occur with
          the PCSs.  Included are:  Sulfur, Heptachlor, aldrin, DDE,
          technical chlordane, mirex, and to some extent, o,p'-DDT and
          p,p'-ODT.
4.  Apparatus and Materials
    4.1   Gas Chromatograph  - Equipped with glass lined  injection port.
    4.2   Detector Options:
          4.2.1  Electron Capture  - Radioactive  (tritium  or nickel-63)
          4.2.2  Microcoulometric  Titration
          4.2.3  Electrolytic Conductivity
    4.3   Recorder  - Potentiometric  strip chart  (10  in.)  compatible  with
          the detector.
    4.4   Gas Chromatographic Column  Materials:
          4.4.1  Tubing  - Pyrex  (180  cm  long  X 4 mm  ID)
          4.4.2  Glass  Wool - Silanized
          4.4.3  Solid  Support  - Gas-Chrom  Q  (100-120 mesh)
          4.4.4  Liquid Phases  - Expressed  as weight percent  coated on
                  solid  support.
                 4.4.4.1      SE-30 or.OV-1,  3%
                  4.4.4.2      OV-17,  1.5% + QF-1 or OV-210, 1.95%
                                      45

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4.5   Kuderna-Danish (K-D) Glassware
      4.5.1.  Snyder Column - three-ball  (macro)  and  two-ball  (micro)
      4.5.2  Evaporative Flasks - 500 ml
      4.5.3  Receiver Ampuls - 10 ml, graduated
      4.5.4  Ampul Stoppers
4.6   Chromatographic Column - Chromaflex  (400 mm  long x  19 mm  ID)
  •  '"with coarse fritted plate on bottom  and Teflon  stopcock;  250-ml
      reservoir bulb at top of column with flared  out funnel  shape  at
      top of bulb - a special order (Kontes K-420540-9011)..
4.7   Chromatographic Co-lumn - pyrex  (approximately  400 mm  'long x 20
      mm ID) with coarse fritted plate on  bottom.
4.8   Micro Co.lumn Pyrex - constructed according to  Figure  T.
4.9   Capillary pipets disposable (5-3/4 in.) with rubber bulb
      (Scientific Products P5205-1).                  ,
4.10  Low pressure regulator - 0 to 5 PSIG - with  low-flow needle
      valve (see Figure 1, Matheson Model  70).
4.11  Beaker - 100 ml
4.12  Micro Syringes - 10, 25, 50 and 100  ul.
4.13  Separatory funnels - 125 ml, 1000 ml and 2000  ml with Teflon
      stopcock.
4.14  Blender - High speed, glass or  stainless steel  cup.
4.15  Graduated cylinders - 100 and 250 ml.
4.16  Florisil - PR Grade (60-100 mesh); purchase  activated1 at
      1250°F and store in the dark in glass containers with glass
      stoppers or foil-lined screw caps.   Before use, activate  each
                                  46

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                                             PRESSURE
COMPRESSED
AIR	
SUPPLY
I
               SHUT-OFF
                 VALVE
0-5
PSIG
           REGULATOR
                                             NEEDLE
                                              VALVE
                                        FLEXIBLE
                                         TUBING
               SILICA  GEL
                   5 cm
                     i cm
                                g
                                    $  10/30
                                 „  !5mi
                                  RESERVOIR
                                    §  10/30
                                    23cm  x 4.2mm i,D.
                                    2 cm   x  2 mm l.D.
            FIGURE  I.   MICROCOLUMN  SYSTEM
                            47

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          batch overnight  at  130°C  in foil-covered glass container.
          Determine lauric-acid value (See Appendix  II).
    4.17  Silica gel - Davison code 950-08008-226 (60/200 mesh).
    4.18  Glass Wool - Hexane extracted.
    4.19  Centrifuge Tubes - Pyrex calibrated  (15 ml).
5.  Reagents, Solvents, and Standards
    5.1   Sodium Chloride  - (ACS) Saturated solution  in distilled water
          (pre-rinse NaCl with hexane).
    5.2   Sodium Hydroxide - (ACS)  10 N  in distilled  water.
    5.3   Sodium Sulfate - (ACS) Granular, anhydrous .(conditioned at 400°
          C for 4 hrs.K
    5.4   Sulfuric Acid -  (ACS) Mix equal volumes of  cone. H2S04 w1t}l
          distilled water.                    .                 -
  .  5.5   Diethyl Ether - Nanograde, redistilled in glass, if  necessary.
          5.5.1  Must be free of peroxides as  indicated by EM  Quant test
                 strips.   (Test strips are-available  from EM Labora-
                 tories, Inc., 500 Executive Blvd.,   Elmsford, N.Y.
                 10523).
          5.5.2  Procedures recommended  for removal of peroxides are
                 provided with the test  strips.
    5.6   n-Hexane - Pesticide quality (NOT MIXED HEXANES).
    5.7   Acetonitrile, Hexane, Methanol, Methylene Chloride,  Petroleum
          Ether (boiling range 30-60°C)  - pesticide quality, redistill in
          glass if necessary.
   '5.8   Standards - Aroclors 1221, 1232, 1242, 1248, 1254, 1260,, and
          .1016.
                                      48

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    5,9   Anti-static Solution - STATNUL,  Daystrom,  Inc.,  Weston Instru-
          ment Division, Newark, N.J.,  95212.
6.  Calibration
    ^•^•^•••^••"^^••••••••^••^                      •
    6.1   Gas chromatographic operating conditions are considered accept-
         i         -                                         •             '
        '' able if the response to  dicapthon is at least 50% of full scale
  :        when < 0.06 ng  is  injected for electron capture detection and <
          100 ng is  injected for microcoulometric or electrolytic con-
          ductivity  detection.   For all quantitative measurements, the
          detector must be operated within its linear response range and
          the detector  noise level should be less than 2% of full  scale.
    6.2   Standards  are injected frequently as a check on the stability
          of operating  conditions, detector and column.   Example  chro-
          matograms  are shown  In Figures 3 through 8  and  provide
           reference  operating conditions.
 7.   Quality Control
     7.1    Duplicate  and spiked sample  analyses are recommended  as quality
           control checks.  Quality  control charts  (4)  should  be developed
           and used as  a check on  the analytical  system.   Quality control
           check samples and performance evaluation  samples  should be
           analyzed on  a regular basis.
     7.2   Each  time a  set of samples  is extracted,  a method blank is
           determined on a volume  of distilled water equivalent to that
           used  to dilute  the sample.
 8.  Sample  Preparation
     8.1   Blend the  sample  if  suspended matter is present and adjust pH
                                       49

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           to  near  neutral  (pH 6.5-7.5)  with 50% sulfuric acid or 10 N
           sodium hydroxide.
    8.2    For sensitivity  requirement of 1  ug/1,  when using micro-
           coulometric  or electrolytic conductivity methods for detection
           take 1000 ml  of  sample for analysis.   If interferences pose no
           problem, the  sensitivity of the electron capture detector
           should permit as little as 100 ml of  sample to be used.  Back-
           ground information  on  the extent  and  nature of interferences
           will  assist  the  analyst in choosing the required sample size
           and  preferred detector.
    8.3    Quantitatively transfer the proper aliquot  into a two-liter
           separatory funnel and  dilute  to. one liter.
9.  Extraction                 •  ...
    9.1    Add  60 ml of  15^ methylene chloride in  hexane  (v:v)  to the
           sample in the  separatory  funnel and shake vigorously for  two
          minutes.
    9.2   Allow the mixed  solvent  to separate from the sample,  then draw
          the water into a one-liter Erlenmeyer flask.   Pour the organic
           layer into a  100-ml  beaker and  then pass  it through  a column
          containing 3-4 inches  of  anhydrous  sodium sulfate, and collect
           it in a 500-ml K-0 flask  equipped  with  a 10 mi-ampul.   Return
          the water phase to the separatory  funnel.   Rinse  the  Erlenmeyer
          flask with a  second  60-ml  volume  of solvent; add  the  solvent to
          the separatory funnel  and  complete  the  extraction procedure  a
          second time.   Perform  a third extraction  in the  same  manner.
                                      50

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9.3   Concentrate the extract  in the K-D evaporator  on  a hot  water
      bath.
9.4   Qualitatively analyze the sample by  gas  chromatography  with an
      electron capture detector.  From the response  obtained  decide:
      a.  If there are any organochlorine  pesticides present.
      b.  If there are any PCBs present.
      d  If there is a combination of a and b.
      d.  If elemental sulfur  is present.
      e.  If the response  is too complex to  determine a, b or c.
      f.  If no response, concentrate to 1.0 ml  or less, as required,
          and repeat  the analysis  looking  for  a,  b,  c,  d, and e.
          Samples containing Aroclors with a low percentage of
          chlorine, e.g.,  1221 and  1232, may require this concentra-
          tion  in order to  achieve  the detection limit of 1 ug/1.
          Trace quantities  of  PCBs  are often masked by background
          which usually occur  in  samples.
9.5    If  condition _a  exists, quantitatively  determine the organo-
       chlorine  pesticides  according to  (1).
                       N
9.6    If  condition b_ exists, PCBs  only  are present; no further
       separation  or  cleanup is necessary.   Quantitatively determine
       the PCBs  according  to step  11.
9.7    If  condition £ exists, compare peaks obtained from the sample
       to  those  of standard Aroclors and  make a judgment  as to wh'ich
       Aroclors  may be present.  To separate the PCBs from the organo-
       chlorine  pesticides,  continue as outlined in  10.4.
                                   51

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    9.8   If condition d_ exists,  separate the sulfur from the sample
          using the method outlined in 10.3 followed by the method in
          10.5.
    9.9   If condition e, exists,  the following macro cleanup and separa-
  i        tion procedures (10.2 and 10.3) should be employed and, if
          necessary, followed by the micro separation procedures (10.4
          and 10.5).
10.  Cleanup and Separation Procedures
    10.1  Interferences in the form of distinct peaks and/or high back-
          ground in the initial gas chromatographic analysis, as well as
          the physical characteristics of the extract (color, cloudiness,
          viscosity) and background knowledge of the sample will Indicate
          whether clean-up is required.  When these interfere with
          measurement of the PCBs, or affect column life or detector
          sensitivity, proceed as directed below.
    10.2  Acetonitrile Partition - This  procedure  is used  to'remove fats
          and oils from the sample extracts.  It should be noted that not
          all pesticides are quantitatively recovered by this procedure.
          The analyst must be aware of this and demonstrate the  effi-
          ciency of the partitioning for the compounds of  interest.
          10.2.1 Quantitatively transfer the previously concentrated
                 extract to a  125-ml  separatory funnel with  enough  hexane
                 to bring the final volume to 15 ml.  Extract the  sample
                 four times fay shaking vigorously  for one  minute with
                 30-ml portions of  hexane-saturate'd  acetonitrile.
                                       52

-------
     10.2.2 Combine and transfer the acetonitrile phases to a
            one-liter separatory funnel and add 650 ml of distilled
            water and 40 ml of saturated sodium chloride solution.
            Mix thoroughly for 30-45 seconds.  Extract with two
            100-ml portions of hexane by vigorously shaking about  15
            seconds.
     10.2.3 Combine the hexane extracts  in  a  one-liter  separatory
            funnel and wash with two 100-ml portions  of  distilled
            water.  Discard the water  layer and  pour  the hexane
            layer  through  a 3-4 inch anhydrous sodium sulfate column
            into  a 500-ml  K-0 flask equipped  with a 10-ml  ampul.
            Rinse  the separatory funnel  and column with three 10-ml
            portions  of  hexane.
      10.2.4 Concentrate  the extracts to 6-10 ml  in the K-0 eva-
            porator  in a hot  water bath.,
      10.2.5 Analyze  by gas chromatography unless a need for further
             cleanup  is indicated.
10.3  Florisil Column Adsorption Chromatography
      10;3.1 Adjust the sample extract volume  to  10 ml.
      10..-3.2 Place a charge of activated Florisil  (weight determined
             by lauric-acid value,  see Appendix  II) in a Chromaflex
             column.  After settling the Florisil  by  tapping  the
             column,  add about one-half  inch  layer  of anhydrous
             granular sodium  sulfate to  the top.
                                   53

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10.3.3 Pre-elute the column, after cooling, with 50-60 ml of
       petroleum ether.  Discard the eluate and just prior to
       exposure of the sulfate layer to air, quantitatively
       transfer the sample extract into the column by
       decantation and-subsequent petroleum ether washings.
       Adjust the elution rate to about 5 ml per minute and,
       separately, collect up to three eluates  in 500-ml K-D
                              •                         ,
       flasks equipped with 10-ml ampuls (see Eluate Composi-
       tion 10.4.).  Perform the first elution,with 200 ml of
       6% ethyl ether  in petroleum ether, and the second
       elution with 200 ml of 15% ethyl ether in petroleum
       ether.  Perform the third elution with 200 ml of 50%
       ethyl"ether - petroleum ether  and the fourth elution
       with 200 ml of  100% ethyl ether.                        •
       10.3.3.1     Eluate Composition - By using an equivalent
                    quantity of  any batch of Florisil  as  deter-
                    mined by  its  lauric acid value, the  pesti-
                    cides will be separated  into the eluates
                    indicated as follows.
                                 6%  Eluate
Aldrin
8HC
Chlordane
ODD
ODE
DDT
Heptach 1 or
Heptachlor Epoxide
Lindane *
Methoxych 1 or
Mi rex
Pentachloro-
nitrobenzene
Strobane
Toxaphene
TrifTuralin
PCBs
                    15% Eluate
                    Endosulfan I
                    Endrin
                    Dieldrin
                    Dichloran
                    Phthalate esters
                             54
50% Eluate
Endosulfan I!
Captan

-------
                      Certain thiophosphate pesticides will occur in
                      each of the above fractions as well as the 100%
                      fraction.  For additional information regarding
                      eluate composition, refer to the FDA Pesticide
                      Analytical Manual (5).
      10.3.4 Concentrate the eluates to 6-10 ml in the K-0 evaporator
             in a hot water bath.
      10.3.5 Analyze by gas chromatography.
10.4  Silica Gel Micro-Column Separation Procedure (6)
      10.4.1 Activation for Silica Gel
             10.4.1.1 Place about 20 gm of silica gel in a 100-ml
                      beaker.  Activate at 180°C for. approximately
                      16 hours.  Transfer the silica gel to a 100-ml
               .    .   glass-stoppered bottle.  When cool, cover with
                      about 35 ml of 0.50% diethyl ether in benzene
                      (volume:volume). -Keep bottle well sealed.  If
                      silica gel collects on the ground glass
                      surfaces, wash off with the above solvent
                      before resealing.  Always maintain an excess of
                      the mixed solvent in bottle (aproximately 1/2
                      in. above silica gel).  Silica gel can be
                      effectively stored in this manner for several
                      days.
      10.4.2 Preparation of the Chromatographic Column
             10.4.2.1 Pack the lower 2 mm ID section of the micro-  •
                      column with glass wool.  Permanently mark
                                  55

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         the column 120 mm above the glass wool.  Using
         a clean rubber bulb from a disposable pipet
         seal the lower end of the microcolumn.  Fill
         the microcolumn with 0.50% ether in benzene
         (v:v) to the bottom of the- 10/30 joint (Figure
         1).  Using a disposable capillary pipet,
         transfer several aliquots of the silica gel
         slurry into the microcolumn.  After approxi-
         mately 1 cm of silica gel collects in the
         bottom of the microcolumn, remove the rubber
         bulb seal, tap the column to insure that the
         silica gel reaches the 120 ± 2 mm mark.  Be
         sure that there are no air bubbles in the
         column.  Add about 10 mm of sodium sulfate to
         the top of the silica gel.  Under low humidity
         conditions, the s'ilica gel may coat the sides
         of the column and not settle properly.  This
         can be minimized by wiping the outside of the
         column with an anti-static solution.
10.4.2.2 Deactivation of the Silica Gel
         a.  Fill the microcolumn to the base of the
             10/30 joint with the 0.50% ether-benzene
             mixture, assemble reservoir (using spring
             clamps) and fill with approximately 15 ml
             of the 0.50% ether-benzene mixture.:  Attach
             the air pressure device (using spring
                     56

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   clamps) and adjust the  elution  rate  to
   approximately  1 ml/min. with  the  air
   pressure control.  Release  the  air pressure
   and  detach reservoir  just as  the  last of
   the  solvent enters the  sodium sulfate.
   Fill  the column with  n-hexane (not mixed
   hexanes) to the base  of the 10/30 fitting.
   Evaporate  all  residual  benzene from the
   reservoir,  assemble  the reservoir section
    and  fill with  5 ml  of n-hexane.  Apply air
    pressure  and  remove the reservoir just as
    the  n-hexane  enters the sodium sulfate.
    The  column is now ready for' use.
b.  Pipet a 1.0 ml aliquot of the concentrated
    sample extract (previously reduced  to  a
    total volume of 2.0 ml) on to the column.
    As the last of the sample  passes  into  the
    sodium sulfate layer,  rinse  down  the
    internal wall of the column  twice with 0.25
    ml of n-hexane.  Then  assemble the  upper
    section of the column.  As the last of the
    n-hexane rinse reaches the surface of the
    sodium sulfate,  add  enough n-hexane (volume
    predetermined, see  10.4.3) to just elute
    all  of the PCBs  present in the sample.
    Apply air pressure  and adjust until the
             57

-------
                    flow is 1 ml/min.  Collect the desired
                    volume of eluate (predetermined, see
                    10.4.3) in an accurately calibrated ampul.
                    As the last of the n-hexane reaches the
                    surface of the sodium sulfate, release the
                    air pressure and change the collection
                    ampul.   "                     '     .
                c..  Fill the column with 0.50% diethyl ether  in
                    benzene, again apply air pressure  and
                    adjust flow to 1 ml/min.  Collect  the
                    eluate until all of the organochlorine
                    pesticides of interest have been eluted
                    (volume predetermined, see 10.4.3).
                d.  Analyze the eluates by gas chromatography.
10.4.3 Determination of Elution Volumes
       10.4.3.1 The elution volumes for the PCBs and the
                pesticides depend upon a number of factors
                which are difficult to control.  These include
                variation in:
                a.  Mesh size of the silica gel
                b.  Adsorption properties of the silica gel
                c.  Polar contaminants present in the eluting
                    solvent
                d.  Polar materials present in the sample and
                    sample solvent
                            58

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        e.  The dimensions  of  the  microcolumns
            Therefore,  the  optimum elution  volume must
            be experimentally  determined  each  time a
            factor  is changed.   To determine the
            elution  volumes,  add standard mixtures of
            Aroclors and  pesticides to the  column and
            serially collect  1-ml  elution volumes.
            Analyze  the individual eluates  by  gas
            chromatography  and determine  the cut-off
            volume  for  n-hexane and for ether-benzene.
            Figure  2 shows  the retention  order of the
            various PCS components and of the  pesti-
            cides.   Using this information, prepare the
            mixtures required for calibraton of the
            microcolumn.
10.4.3.2 In determining  the*volume of hexane required to
         elute the PCBs  the sample volume (1 ml) and the
         volume of n-hexane used to rinse the column
         wall  must be considered.  Thus,  if  it is
         determined that a  10.0-ml elution volume  is
         required to elute the PCBs, the  volume of
         hexane to be added in addition to the sample
         volume but  including the  rinse volume should be
         9.5 ml.
                    59

-------
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                                       60

-------
             10.4.3.3  Figure 2  shows  that  as  the  average chlorine
                      content of  a  PCB  mixture  decreases the solvent
                     .volume for  complete  elution increases.  Quali-
                      tative determination (9.4)  indicates which
                      Aroclors  are  present and  provides the basis for
                      selection of  the  ideal  elution volume.  This
                      helps to  minimize the quantity of organo-
                      chlorine  pesticides  which will elute along with
                      the  low percent chlorine  PCBs and insures the
                      most efficient  separations  possible for
                      accurate  analysis.
             10.4.3.4  For  critical  analysis where the PCBs'and pesti-
                      cides  are not separated completely, the column
                      should be accurately calibrated according to
                      (10.4.3.1)  to determine the percent of material
                      of interest that eTutes in  each fraction.  Then
                      flush  the column with an  additional 15 ml of
                      0.50%  ether in benzene  followed by 5 ml of
                      n-hexane  and use this reconditioned column for
                      the  sample separation.   Using this technique
                      one  can  accurately predict  the amount (%) of
                      materials in each micro column fraction.
10.5  Micro Column Separation  of Sulfur, PCBs,  and Pesticides
      10.5.1 See procedure for preparation and packing micro column
             in PCB analysis section (10.4.1  and 10.4.2).
                                  61

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10.5.2 Microcolumn Calibration
       10.5.2.1 Calibrate the microcolumn for  sulfur  and  PCB
                separation by collecting  1.0-ml fractions and
                analyzing them by gas chromatography  to
                determine the following:
                1}  The fraction with the first eluting PCBs
                    (those present  in 1260),
                2)  The fraction with the last eluting PCBs
                    (those present  in 1221),
                3)  The elution volume for sulfur,
                4)  The elution volume for the pesticides of
                    interest in the 0.50% ether-benzene
                    fraction.
                From these data determine the following:
                1)  The eluting volume containing only sulfur
                    (Fraction I)r   ,
                2)  The eluting volume containing the last of
                    the sulfur and the early eluting  PCBs
                    (Fraction II),
                3)  The eluting volume containing the remaining
                    PCBs (Fraction  III),
                4)  The ether-benzene eluting volume  containing
                    the pesticides of interest (Fraction  IV).
10.5.3  Separation Procedure
       10.5.3.1 Carefully concentrate the 6% eluate from  the
                            62

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         florisil column to 2.0 ml in the graduated
         ampul on a warm water bath.
10.5.3.2-Place 1.0 ml (50%) of the concentrate into the
         microcolumn with a 1-ml pipet.  Be careful not
         to get any sulfur crystals into the pipet.
10.5.3.3 Collect Fractions I and II in calibrated
         centrifuge tube's.  Collect Fractions  III  and  IV
         in calibrated ground glass stoppered  ampuls.
10.5.3.4 Sulfur Removal (7) - Add  1 to 2 drops of
         mercury to Fraction II stopper and place  on a
         wrist-action shaker.  A black precipitate
         indicates the presence of sulfur.  After
         approximately 20 minutes  the-mercury  may  become
         entirely reacted or deactivated by the
         precipitate.  The sample  should be quanti-
         tatively transferred to  a clean centrifuge .tube
         and  additional mercury  added.  When  crystals
         are  present  in the  sample,  three  treatments may
         be necessary to  remove  all  the  sulfur.   After
         all  the sulfur has  been  removedfrom  Fraction  II
         (check  using gas chromatography)  combine
         Fractions  II and III.   Adjust the volume to 10
         ml  and  analyze by gas  chromatography.  Be sure
         no mercury is transferred to the  combined
         Fractions  II and III,  since it can react with
         certain pesticides.
                      63

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                           By  combining  Fractions  II  and  III,  if PCBs are
                           present,  it is possible  to identify the
                           Aroclor(s) present  and  a quantitative analysis
                           can be performed  accordingly.   Fraction  I  can
                           be  discarded  since  it only contains the  bulk of
                           the sulfur.   Analyze Fractions  III  and IV  for
                           the PCBs  and  pesticides.   If DDT and its
                           homologs, aldrin, heptachlor, or technical
                           chlordane are present along with the PCBs,  an
                           additional microcolumn separation can be
                           performed which may help to further separate
                           the PCBs from the pesticides (See 10.4),,.
11.  Quantitative Determination
    U.I  Measure the volume of n-hexane eluate containing the PCBs  and
          inject 1 to 5/
-------
                Microgram/ liter =
                A = ng of Standard Injected
                                                 mm
                                                2
                B =   of Sample Peak Areas - (mm )
                V.  = Volume of sample injected
       •         vt  = Vo1ume of Extract (vl)  from which sample
                       is injected into gas  chromatograph
                V  = Volume of water sample  extracted (ml)
                N = 2 when micro column used
                    1 when micro column not  used
                Peak Area = Peak height (mm  x Peak Width at 1/2
                   height
11.2.2 For complex  situatons, use the calibration method
       described below (8).  Small variations in components
       between different Aroclor batches make it necessary to
       obtain samples of several specific Aroclors.  These
       reference Aroclors can be obtained from the Southeast
       Environmental Research Laboratory, EPA, Athens, Georgia,
       30601.  The procedure is as follows:
       11.2.2.1 Using the OV-1 column, chromatograph a known
                quantity of each Aroclor reference standard.
                Also chromatograph a sample of p,p'-DOE.
                Suggested concentration of each standard  is 0.1
                ng/ul for the Aroclors and 0.02 ng/yl for the
                p9p'-ODE.
                            65

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 11.2.2.2 Determine the relative retention  time  (RRT)  of
          each PCB peak in the resulting chromatograms
          using p,p'-DDE as 100.                i
                   x 10°
          RRT =
                  DDE
          RRT = Relative Retention Time
          RT  = Retention time of peak of interest
          RTODE = Retention time of p,p'-DDE
          Retention time is measured as that distance in
          mm between the first appearance of the solvent
          peak  and the maximum for the compound.
 11.2.2.3  To calibrate the instrument for each PCB
          measure the area of each peak.
          Area  =  Peak height -(mm)  x Peak  width at 1/2
          height.   Using Tables  1  through 6  obtain the
          proper  mean weight factor,  then determine the
response factor .ng/mm .
                 V
         ng/rnn^
                                 (mean weight percent)
                                          100   '
                                     (Area)
         rig.,- = ng of Aroclor Standard  Injected
         Mean weight percent - obtained from Tables  1
         through 6.
11.2.2.4 Calculate the RRT value and the area for each
         PCB peak in the sample chromatogram.  Compare
       •  the sample chromatogram to those obtained for
         each reference Aroclor standard.   If it; is
                     66

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                                 Table 1

                     Composition of Aroclor 1221  (8)
RRTa
n
14
16
19
21
28

32

37"
40
Mean
Weight
Percervt
31.8
19.3
10.1
2.8
20.8
5.4

1.4

1.7

Relative
Std. Dev.b
15.8
9.1
9.7
9.7
9.3
13.9

30.1

48.8

Number of
Chlorines0
1
1
2
2
2
2 85%
3 15%
2 10%
3 90%
3

aRetention time relative to p,p'-DDE=100.  Measured from first appearance
 of solvent.  Overlapping peaks that are quantitated as one peak are
 bracketed.

bStandard deviation of seventeen results as a percentage of the mean of
 the results.

cFrom GC-MS data.  Peaks containing mixtures of  isomers of different
 chlorine numbers are bracketed.
                                      67

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                                  Table 2

                      Composition  of Aroclor  1232  (8)
RRTa
n
14
16
20
21
28

32
37
•40
47
54

58
70

78
Mean
Weight
Percent '
16.2
• 9.9
7.1
17.8

9.6

3.9
6.8
6.4
4.2
3.4

2.6
4.6

1.7
Relative .
Std. Dev.
3.4
2.5
6.8
.2.4

3.4

4.7
2.5
2.7
4. 1
3.4

3.7
3.1

7.5
Number of
Chlorines
1
1
2
2

2
3
3
3
3
4
3
4
4
4
• 5
4





40%
60%




33%
67%

90%
10%,

Total
94.2
Detention time relative to p,p'-QDE=100.  Measured from first appearance
 of solvent.  Overlapping peaks that are quantitated as one peak are
 bracketed.

bStandard deviation of four results as a mean of the results.

cFrom SC-MS data.  Peaks containing mixtures of isomers of different
 chlorine numbers are bracketed.
                                      68

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                                 Table 3

                     Composition of Aroclor 1242 (8)
RRTa
n
16
21
28

32
37
40
47
54

58
70

78
84
98
104
125

146

Mean
Weight
Percent
1.1
2.9
11.3
n.o

6.1
11.5
n.i
8.8
6.8

5.6
10.3

3.6
2o7
1.5
• 2.3
1.6

1.0

Relative ,
Std. Dev.
35.7
4.2
3.0
5.0

4.7
5.7
6.2
4.3
2.9

3.3
2.8

4.2
9.7
9:4
16.4
20.4

19.9

Number of
Chlorines
1
2
2
2
3
3
3
3
4
3.
4
4
4
5
4
5
5
5
5
6
5
6



25%
75%




33%
67%

90%
10%




85%
15%
75%
25%
Detention time relative to p,p'-ODE=100.  Measured from first appearance
 of solvent.

bStandard deviation of six results as a percentage of the mean of the
 results.

cFrom GC-MS data.  Peaks containing mixtures of isomers of different
 chlorine numbers are bracketed.
                                     69

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                                 Table 4

                     Composition of Aroclor 1248 (8)
RRTa
21
28
32
47
40

47 '
54

58
70

78
84
98
104

.112
125

146

Mean
Wei ght
Percent
1.2
5.2
3.2
8.3
8.3

15.6
9.7

. 9.3 -
19.0

6.6
4.9
3.2
3.3

1.2
2.6
•
1.5

Relative .
Std. Dev.
23.9
3.3
3.8
3.6
3.9

1.1
6.0

5.8
1.4

2.7
2.6
3.2
3.6

"6.6
5.9

10.0

Number of
Chlorines
2
3
3
3
3
4
4
3
4
4
4
. 5 •••
4
5
5
4
5
5
5
6
5
6




85%
15%

10%
90%

80%
20%



10%
90%

90%
10%
85%
15%
 Total
103.1
Detention time relative to p,p'-DDE-100.  Measured from first appearance
 of solvent.

^Standard deviation of six results as a percentage of the mean of the.
 results.

cFrom GC-MS data.  Peaks containing mixtures of isomers of different
 chlorine numbers are bracketed.
                                     ,70

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                                 Table 5

                     Composition of Aroclor  1254  (8)
RRTa
47
54
58
70

84
98
104
125

146 •

160
174
203
232
Mean
Weight
Percent
6.2
2.9
1.4
13.2

17.3
7.5
13.6
15.0
-
10.4

1.3
8.4
1.8
1.0
Relative .
Std. Dev.
3.7
2.6
2.8
2.7

1.9
5.3
3.8
2.4

2.7

' 8 ..4
5.5
18.6
26.1
Number of
Chlorines0
4
4
4
4
5
5
5
5
5
6
5
6
6
6
6
7



25%
75%



70%
80%
30%
70%




 Total              100.0
Detention time relative to p,p'-DDE=100.  Measured from first appearance
 of solvent.


bStandard deviation of six results as a percentage of the mean of the
 results.


cFrom GC-MS data.  Peaks containing mixtures of isomers are bracketed.
                                      71

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                                 Table 6

                     Composition of Aroclor 1260 (8)
RRTa
70
84
98
104

117
125

146
160

174
203

232
244

280
332
372
448
528
Mean
Wei ght
Percent
2.7
4.7
3.8


3.3
12.3

14.1
4.9

12.4
9.3


9.8

11.0
4.2
4.0
.6
1.5
Relative , Number of
Std. Dev. Chlorines
6.3
1.6
3.5

• •
6.7
3.3

3.6
, 2.2 .
-
2.7
4.0 ,-


: " 3.4

-2.4
5.0
8.6
25.3
10.2
5
5
ci
5
6
6
5
6
6
- 6
7
6
6
7
a
6
7
7;
7
8
8
8



60%
40%

15%
85%

50%
50%

10%
90%

10%
90%





 Total
98.6
Detention time relative to p,p'-DDE=100.  Measured from first appearance
 of solvent.  Overlapping peaks that are quantitated as one peak are
 bracketed.
^Standard deviation of six results as a mean of the results.
cFrom GC-MS data.  Peaks containing mixtures of isomers of different
 chlorine numbers are bracketed.
dComposition determined at the center of peak 104.
eComposition determined at the center of peak 232.
                                      72

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 apparent  that the PCB peaks present are due to
 only one  Aroclor, then calculate the concen-
 tration of each PCB using the following formula:
               ?
 ng PCB =  ng/mm  x Area
                      2
 Where Area = Area (mm ) of sample peak
 ng/mm  =  Response factor for that peak       :
      measured.
 Then add  the nanograms of PCBs present in the
 injection to get the total number of nanograms
 of PCBs present.  Use the following formula to
 calculate the concentration of PCBs in the
 sample:
 Micrograms/Liter =
- V  = volume  of water  extracted  (ml)
 Vt = volume  of extract  (yl)
 V. = volume  of samp-le injected  (uD
 ng = sum of  all  the  PCBs  in  nanograms  for  that
      Aroclor identified
 N = 2 when microcolumn  used
 N =  1 when microcolumn not used
 The value  can then be reported  as  micro-
 grams/liter  PCBs or  as the Aroclor.   For
 samples  containing more than one Aroclor,  use
 Figure 9 chromatogram divisional flow chart to
 assign a proper response factor to each peak
 and also identify the "most likely" Aroclors
              73

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                           present.   Calculate the ng of each PCJ3 isomer
                           present  and  sum them according to the
                           divisional flow chart.   Using the formula
                           above, calculate the concentration of the
                           various Ar.oclors  present  in  the sample.
12.  Reporting Results
    12.1  Report results in micrograms per  liter  without correction for
          recovery data.  When duplicate  and  spiked  samples  are  analyzed,
          all data obtained should be reported.
                                     74

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   \
               37
                  AROCLOR 1242
Figure  3.  Column: 3% OV-1, Carrier Gas: Nitrogen at 60 ml/min,
          Column Temperature: 170 C, Detector:  Electron Capture
                              75

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I
                 70
                    84
                              AROCLOR 1254
                         104
                             125
                                  146
Figure  4. Column:  3%  OY-1, Carrier Gas: Nitrogen at 61) mi/min,
         Column Temperature: 170 C, Detector: Electron Capture.
                              76

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           146
        125 (I  174
 AROCLOR 1260
280
5.  Column:  3% OV-1, Carrier Gas: Nitrogen at 60 ml/mm,
   Column Temperature:  170  G,  Detector: Electron Capture.
                        77

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                       AROCLOR 1242
I
I
I
                                     I
I
I
1
3
                                    21
                             24
               6       9      12      15      18
                       RETENTION TIME  IN MINUTES
Figure  6. Column: 1.5%  OV-17  +  1.95%  QF-1, Carrier fias: Mitrogen
at 60  ml/min,  Column  Temperature: 200 C, Detector: Electron Capture.
                        78

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                RRT of  first peak < 47?
             YES
                         NO
     Is there  a distinct
     peak with RRT 78?
      YES
/      V
                       RRT 47-58?
YES
  Use 1242  for
 peaks 1 RRT 84
          Use  1242 for
         peaks -  RRT  70
 Use 1254
 for peaks
1  RRT 104
                                              NO
                RRT- 70?
           Is there a distinct
           peak with  RRT 117?
         YES
               NO
                    Use 1254 for  all
                    peaks- RRT 174
     Use 1260 for
     all other peaks
                               Use 1260 for
                                 all peaks
Figure  9. Chromatogram Division Flowchart  (8).
                          81

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REFERENCES:
1.  "Method for Chlorinated Hydrocarbons in Water and Wastewater", this
    manual, p. 7.

2.  Leoni, V., "The Separation of Fifty Pesticides and Related Compounds and
    Polychlorinated Biphenyls into Four Groups by Silica Gel Microcolumn
    Chromatography", Journal of Chromatographv, 62, 63 (1971).

3.  McClure, V. E., "Precisely Deactivated Adsorbents Applied to the Separa-
    tion of Chlorinated Hydrocarbons", Journal of Chromatographv, 70, 168
    (1972).                            	a__*_j.  _

4.  "Handbook for Analytical Quality Control in Water and Wastewater
    Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
    Agency, National  Environmental Research Center, Analytical Quality
    Control Laboratory, Cincinnati, Ohio, 45268, 1972.

5.  "Pesticide Analytical Manual", U. S.'Dept. of Health, Education and
    Welfare, Food and Drug Administration, Washington, D. C.

6.  Bellar, T. A. and Lichtenberg, J.. J., "Method for the Determination of
  .  Polychlorinated Biphenyls 'in Water and Sediment", U. S. Environmental
    Protection Agency', National Environmental Research Center, Analytical
    Quality Control Laboratory, Cincinnati, Ohio, 45268, 1973.

7.  Goerlitz, D. F. and Law, L. M., "Note on Removal of Sulfur Interferences
    from Sediment Extracts for Pesticide Analysis", Bulletin of Environmental
    Contamination and Toxicology, 6_, 9 (1971).                .  !

8.  Webb, R. G. and McCall, A.  C,, "Quantitative PCB Standards for Electron
    Capture Gas Chromatography", Journal  of Chromatographic Science, 11, 366
    (1973).	
                                     82

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             METHOD FOR TRIAZINE PESTICIDES IN WATER AND WASTEWATER
V.   Scope and Application
    1.1  This method covers the determination of various symmetrical triazine
         pesticides in water and wastewaters.
    1.2  The following pesticides may be determined  individually by this
         method:
                    Parameter                 Storet No.
                    Ametryn
                    Altraton
                    Atrazine                    39033
                    Prometon              .      39056
                    Prometryn                   39057
                    Propazine                   39024
                    Secbumeton                    —
                    Simazine                    39055
                    Terbuthylazine                —
 2.   Summary
     2.1   The method describes  an  efficient sample extraction procedure
          and provides, through  use  of column chromatography, a method
          for the elimination  of non-pesticide interferences and the
          pre-separation of pesticide mixtures.   Identification is made
          by selective gas chromatographic separation, and measurement
          is accomplished by the use of an electroytic conductivity
          detector (CCD) in the nitrogen mode or a nitrogen  specific
          thermionic detector.  Results are reported in micrograms per
          liter.
                                       83

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    2.2  This method is recommended for use only by experienced
         pesticide analysts or under the close supervision of such
         qualified persons.
3.  Interferences
    3.1  Solvents, reagents, glassware, and other sample processing
         hardware may yield discrete artifacts and/or elevated
         baselines causing misinterpretation of gas chromatograms.
         All of these materials must be demonstrated to be free from
         interferences under the conditions of the analysis.  Specific
         selection of reagents and purification of solvents by
         distillation in all-glass systems may be required.  Refer to
         Appendix I.                                •
    3.2  The interferences  in  industrial effluents" are high and varied
         and often pose great difficulty in obtaining accurate and
         precise measurement of triazine pesticides.  The use of  a
         specific detector  supported by an .optional column cleanup
         procedure will eliminate many of these interferences.'
    3.3  Nitrogen containing compounds other than the triazines may
         interfere.
4.  Apparatus and Materials
   - 4.1  Gas Chromatograph  - Equipped with glass  lined injection  port.
    4.2  Detector Options
         4.2.1  Electrolytic Conductivity.
         4.2.2  Nitrogen specific thermionic
    4.3  Recorder - Potentiometric strip chart  (10  in.)  compatible
         with the detector.
                                       84

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4.4  Gas Chromatographic Column Materials:
     4.4,1  Tubing - Pyrex (180 cm long x 4 mm  ID)
     4.4.2  Glass Wool •- Silam'zed
     4.4.3  Solid Support - Gas Chrom Q (100-120 mesh)
     4.4.4  Liquid Phases - Expressed as weight percent  coated  on
            solid support.
            4.4.4.1 - Garbowax 20M, 1%
4.5  Kuderna-Danish (K-0) Glassware
     4.5.1  Snyder Column - three ball  (macro)  and  two  ball  (micro)
     4.5.2  Evaporative Flasks  - 500 ml
     4.5.3  Receiver Ampuls -  10 ml, graduated
     4.5.4  Ampul Stoppers
4.6  Chromatographic Column -  Chromaflex  (400 mm  x  19-mm ID)  with
     coarse fritted plate and  Teflon stopcock on  bottom; 250  ml
     reservoir  bulb at top of  column with  flared  out funnel  shape
     at  top of  bulb - a special order  (Kontes K-420540-9011).
4.7  Chromatographic Column -  Pyrex  (approximately  400  mm long x
     20  mm  ID)  with coarse fritted plate  on  bottom,
4.8  Micro  Syringes -  10, 25,  50  and  100 jul.
4.9  Separatory funnels - 2000 ml with  Teflon  stopcock.
4.10 Blender  -  High speed,  glass  or  stainless  steel cup.
4.11 Graduated  Cylinders  -  1000 ml.
4.12 Florisil  - PR  Grade  (60-100  mesh); purchase activated at
     1250°F and store  in  the  dark in  glass containers with glass
     stoppers  or foil-lined  screw caps.  Before use, activate each
                                   85

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         batch  overnight at 130°C in foil-covered glass container.
         Determine  1 auric acid  value (See Appendix II).
5.  Reagents, Solvents,  and Standards
    5.1  Sodium Hydroxide - (ACS)  10 N in distilled water.
    5.2  Sodium Sulfate  •• (ACS)  Granu-lar, anhydrous (conditioned at
         400 C  for  4  hrs.).
    5.3  Sulfuric Acid - (ACS)  Mix  equal  volumes  of cone.  H2S04
         with distilled  water. '
    5.4' Diethyl Ether - Pesticide  Quality,  redistilled in  glass,  if
         necessary
         5.4.1   Must  be  free  of  peroxides as indicated  fay  EM Quant:
                 Test  strips.  (Test strips are  available from EM
                 Laboratories, Inc.,  500 Executive Blvd., Elmsford.,
                 N.Y.  10523.)
         5.4.2   Procedures  recommended for removal  of peroxides are
                 provided  with the test strips.
    5.5  Hexane, Methanol,  Methylene Chloride,  Petroleum Ether
         (boiling range  30-60°C)  -  pesticide quality, redistill in
         glass  if necessary.                    .
    5.6  Pesticide  Standards  - Reference  grade.
6.  Calibration           .
    6.1  Gas chromatographic  operating conditions are considered
         optimum when an  injection  of^ 20 ng of  each triazine will "
         yield  a peak at  least 50%  of full scale  deflection  with the
         modified Coulson detector  (1).   For all  quantitative
                                       86

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         measurements, the detector must be operated within  its  linear
         response range and the detector noise level should  be less
         than 2% of ful.l scale.
    6.2  Inject standards frequently as a check on the stability of
         operating conditions.  A chromatogram of a mixture  of several
         pesticides is shown  in Figure 1 and provides reference
         operating conditions for the-recommended column.
    6.3  The elution order and retention ratios of various
         organophosphorus pesticides are provided in Table 1, as a
         guide.
7.  Quality Control
    7.1  Duplicate and spiked sample analyses are recommended as
         quality control checks.  Quality control charts  (2) should be
         developed and used as a check on the analytical  system.
         Quality control check samples and performance evaluation
         samples should be analyzed on a regular basis.
    7.2  Each time a set of samples is extracted, a method blank is
         determined on a volume of distilled water equivalent to that
         used to dilute the sample.
8.  Sample Preparation
    8.1  Blend the sample if  suspended matter is present  and adjust pH
         to near neutral (pH  6.5-7.5) with 50% sulfuric acid or  ION
         sodium hydroxide.
    8.2  Quantitatively transfer a 1000 ml aliquot into a two-liter
         separatory funnel.
                                      87

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                            ce
                  4         6         8         10

                         RETENTION  TIME  IN MINUTES
14
Figure 1. Column Packing: 1% Carbowax 20M on Gas-Ghrom Q (100/120 mesh],
        Column Temperature: 155 C, Carrier Gas: Helium at 80 ml/min,
        Detector:  Electrolytic Conductivity.
                                    88

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                                TABLE 1
                  RETENTION RATIOS OF VARIOUS TRIAZINE
                    PESTICIDES RELATIVE TO ATRAZINE
             Pesticide                  Retention  Ratio
             Prometon                       0»52
             Atraton                        0-67
             Propazine                      0«71
             Terbuthylazine                0.78
             Secbumeton                    0-88
             Atrazine                       1.00
             Prometryne                    1-^0
             Simazine                       T-35
             Ametryne '                     1-48
Absolute retention time of atrazine = 10.1 minutes
                                       89

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9.  Extraction
    9.1  Add 60 ml methylene chloride to the  sample  in  the  separatory
         funnel and shake vigorously for two  minutes.
    9.2  Allow the solvent to separate from the sample,  draw the ;
         organic layer into a 100-ml beaker,  then pass  the  organic
         layer through a chromatographic column containing  3-4  inches
         anhydrous sodium sulfate, and collect it in a  500-ml K-D
         flask equipped with a 10 ml ampul.   Add a second 60-ml volume
         of solvent to the separatory funnel  and complete the
         extraction procedure a second time.  Perform a third
         extraction in the same manner.
    9.3 ' Concentrate the extract to 10 ml.  in  a K-D evaporator on a  hot
         water bath.  Disconnect the Snyder column just long enough  to
         add 10 ml hexane to the K-D flask and then  continue the
         concentration to about 5-6 ml.  This operation is  to displace
         methylene chloride and give a final  hexane  solution.   If the
         need for cleanup is indicated, continue to  Florisil Column
         Cleanup (10 below).
    9.4  If further cleanup is not required,  replace the Snyder column
         and flask with a micro-Snyder column and continue  the
         concentration to 0.5-1.0 ml.  Analyze this  final concentrate
         by gas chromatography.
10. Florisil Column Adsorption Chromatography
    10.1 Adjust the sample extract volume  to  10 ml.
                                       90

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10.2  Place a charge of activated Florisil (weight determined by
      lauric acid value, see Appendix II) in a Chromaflex column*
      After settling the Florisil by tapping the column, add about
      oner-half inch layer of anhydrous granular sodium sulfate to
      the top.
10.3  Pre-elute the column, after cooling, with 50-60 ml of
      petroleum ether.  Discard the eluate and just prior to
      exposure of the sulfate layer to air, quantitatively
      transfer the sample extract into the column by decantation
      and subsequent petroleum ether washings.  Adjust the elution
      rate to about 5 ml per minute and, separately, collect up to
      four eluates in 500-ml K-D flasks equipped with 10-ml
      ampuls.  (See Eluate Composition, 10.4.)  Perform the first
      elution with 200 ml of 6% ethyl ether in petroleum ether,
      and the second elution with 200 ml of 15% ethyl ether in
      petroleum ether.  Perform the third elution with 200 ml of
      50% ethyl ether - petroleum ether and the fourth elution
      with 200 ml of 100% ethyl ether.
10.4  Eluate Composition - By using an equivalent quantity of any
      batch of Florisil as determined by its lauric acid value,
      the pesticides will be separated into the eluates indicated
      as follows:
                                  91

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                 Eluate
                                   50% Eluate
100% Eluate
           Propazine (90%)       Propazine (10%)      Atraton
           Terbuthylazine (30%)  Terbuthylazine(70%)  Secbumeton
           Atrazine (20%)        Atrazine (80%)       Prometon
                                 Ametryne
                                 Prometryne
                                 Simazine
    10.5 Concentrate the eluates to 6-10 ml in the K-D evaporator in a
         hot water bath.  Change to the micro-Snyder column and continue
         concentration to 0.5-1.0 ml.
    10.6 Analyze by gas chromatography.
11.  Calculation of Results
    11.1 Determine the pesticide concentration by using the absolute
         calibration procedure .described below or the relative
         calibration procedure described in Appendix- III.
         (1)   Micrograms/liter = (A)   (B)   (Vtl
         A = ng standard
             Standard area
         B = Sample aliquot area
         Vj = Volume of extract injected (yl)
         Vt = Volume of total extract G-il)
         v<
12.  Reporting Results
    12.1 Report results in micrograms per  liter without correction for
         recovery data.  When duplicate and spiked samples  are  analyzed
         all data obtained should be reported.            "
          s   Volume of water extracted (ml)
                                       92

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REFERENCES:
    Patchett, G. 6., "Evaluation of the Electrolytic Conductivity Detector
    for Residue Analyses of Nitrogen-Containing Pesticides", Journal of
    Chroma tographic Science, 8., 155 (1970).

    "Handbook for Analytical Quality Control in Water and Wastewater
    Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
    Agency, National Environmental Research Center, Analytical Quality
    Control Laboratory, Cincinnati, Ohio, 45268, 1972.  (Revised edition
    to be available soon.)
                                      93

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   METHOD  FOR  0-ARYL CARBAMATE PESTICIDES IN WATER AND WASTEWATER
Scope and  Application
1.1   This method  covers  the  determination  of various 0-aryl. carbamate
      pesticides in  water and wastewater.                  •<
1.2   The  following  pesticides may  be  determined  individually by this
      method:
                   Parameter                 Storet  No.
                  Aminocarfa
                  Carbaryl
                  Methiocarb
                  Mexacarbate
                  Propoxur
39750
Summary
2.1   A measured volume of water  is extracted with methylene
      chloride.  The concentrated extract is cleaned up with  a
      Florisil column.  Appropriate fractions from the column are
      concentrated and portions are separated by'thin-layer
      chromatography.  The carbamates are hydrolyzed on the layer and
      the hydrolysis products are reacted with 2,6-dibromoquinone
      chlorimide to yield specific colored products.  Quantitative
      measurement is achieved by visually comparing the responses of
      sample extracts to the responses of standards on the same
      thin-layer.  Identifications are confirmed by changing the pH
      of the layer and observing color changes of the reaction
      products.   Results are reported in micrograms per liter.

                                 94

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    2.2  • This method is recommended for use only by experienced
          pesticide analysts or under the close supervision of such
          qualified persons.
3.  Interferences
    3.1   Direct interferences may be encountered from phenols that may
          be present in the sample.  These materials react with the
          chromogenic reagent and yield reaction products .sim-ilar to
          those of the carbamates.  In cases where phenols are suspected
          of interfering with a determination, a different solvent system
          should be used to attempt to isolate the carbamates.
    3.2   Indirect interferences may be encountered from naturally
          colored materials whose presence masks the chromogenic reaction.
4.  Apparatus and Materials
 .4.1   Thin-layer plates - Glass plates ('200 x 200 mm) coated with
          0.25 mm layer of Silica Gel G (gypsum binder),
    4.2   Spotting Template
    4.3   Developing Chamber
    4.4   Sprayer - 20 ml capacity
    4.5   Kuderna-Danish (K-D) Glassware (Kontes)
          4.5.1  Snyder Column - three ball  (K-503000)
       .   4.5.2  Micro-Snyder Column -two ball (K-569001)
          4.5.3  Evaporative Flasks - 500 ml  (K-570001)
          4.5.4  Receiver Ampuls - 10 ml graduated  (K-570050)
          4.5.5  Ampul Stoppers
                                      95

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    4.6    Chromatographic  Column - Chromaflex (400 mm long x 19 mm ID)  with
           coarse fritted plate  on bottom and Teflon stopcock;  250 ml
           reservoir  bulb at  top of column with flared out funnel shape  at
           top of bulb  - a  special order (Kontes K-420540-9011).
    4.7    Chromatographic  Column - Pyrex (approximately 400 mm long x 20 mm
           ID) with coarse  fritted plate on bottom.
    4.8    Micro Syringes - 10,  25,  50  and 100 jul.
                                 *
    4.9    Separator/ Funnel  - 2000  ml,  with Teflon stopcock.
    4.10   Blender -  High speed,  glass  or stainless steel cup.
    4.11   Florisil - PR Grade (60-80 mesh); purchase activated at 1250°F
           and store  in the dark  in  glass containers with glass stoppers  or
           foil-lined screw caps.   Before use activate each batch overnight
           at 130°C in foil-covered  glass container.   Determine lauric
           acid value (see  Appendix  II).                          !
5.  Reagents, Solvents, and  Standards
    5.1    Sodium Hydroxide - (ACS)  10  N in distilled water.
    5.2    Sodium Sulfate - (ACS)  Granular,  anhydrous.
    5.3    Sulfuric Acid -  (ACS)  Mix equal  volumes of cone.  H2S04 with
           distilled water.                                         .
    5.4    Diethyl Ether -  Nanograde, redistilled  in glass,  if  necessary.
          5.4.1  Must be free of  peroxides  as  indicated  by EM  Quant test
                 strips.   (Test  strips  are  available from EM Laboratories,
                 Inc.,  500 Executive Blvd.,  Elmsford,  N.Y.  10523.)
          5.4.2  Procedures recommended  for removal  of peroxides are
                 provided with the  test  strips.
                                      96

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    5.5   Hexane, Methanol, Methylene Chloride, Petroleum  Ether  -
          nanograde, redistill in glass  if necessary.
    5.6  .Pesticide Standards - Reference grade.
          5.8.1  TLC Standards - 0.100 ^ig/ul in chloroform.
    5.7   Chromogenic agent - Dissolve 0.2 g 2,6-dibromoquinone  chlorimide
          in 20 ml chloroform.
    5.8   Buffer solution - 0.1 N sodium borate in water.
6.  Calibration
    6.1   To insure even solvent travel  up the  layer,  the  tank used  for-
          layer development must be thoroughly  saturated with developing
          solvent before it is used.  This may  be achieved by lining the
          inner walls of the tank with chromatography  paper and  introducing
          the solvent 1-2 hours before use.
    6.2   Samples and standards should be introduced to the layer  using a
          syringe, micropipet or other suitable device that permits  all the
          spots to be about the same  size and as small as  possible..  An air
          stream directed on the layer during spotting will speed  solvent
          evaporation and help to maintain small spots.
    6.3   For qualitative and quantitative work, spot  a series representing
          0.1-1.0 jjg of a pesticide.  Tables  1  and 2 present color
          responses and Rf values for several solvent  systems.
7.  Quality Control
    7.1   Duplicate and spiked sample analyses  are recommended as  quality
          control checks.  Quality control charts  should be developed
          and used  as a check on the  analytical system.   Quality control
                                      97

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                                    Table 1

         Values of 0-Aryl Carbamate Pesticides in Several Solvent Systems

Carbaryl
Aminocarb
Mexacarbate
Methiocarb
Propoxur
A
0.26
0.26
0.34
0.31
0.27
B
0.22
0.02
0.22
0.31
0.10
.C
0.48
0.46
0.54
0.55
0.53
D
0.41
0.52
0.53
0.55
0.59
E
0.58
0.54
0.60
0.59
0.60
F
0.24
0.04
0.24
0.28
0.13
Solvent Systems:

A.  Hexane/acetone (3:1)
B.  Methylene chloride
C.  Benzene/acetone (4:1)
D.  Benzene/cyclohexane/diethylamine (5:2:2)
E.  Ethyl acetate
F.  Chloroform
                                     98

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                                Table 2

      Color Responses  and  Detection Limit for 0-Aryl  Carbamates


                    ~                'Colors;               ""•  Detection
                            Before            After         '    Limit
	Buffer  	Buffer	(ug)

 Carbaryl                    Brown            Red-Purple          0.1
 Aminocarb                   Gray             Green               0.1
 Mexacarbate                 Gray             Sreen         •      0.1
 Methiocarb                 Brown            Tan                 O.Z
 Propoxur                    Blue             Blue                0,1
                                  99

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          check samples  and performance  evaluation  samples  should  be
          analyzed on a  regular basis.
    7.2   Each time a set of  samples  is  extracted,  a method blank  is
          determined on  a volume of distilled water equivalent  to  that  used
          to dilute the  sample.                          ,        i
8.  Sample Preparation                                     .   '
    8.1   Blend the sample if  suspended  matter  is present and adjust  pH to
          near neutral (pH 6.5-7.5) with 50% sulfuric acid  or 10 N sodium
          hydroxide.
    8.2   quantitatively transfer  a one-liter aliquot into,  a two-liter
          separatory funnel.
9.  Extraction
    9.1   Add 60 ml of methylene chloride  to the  sample  in  the  separatory
          funnel and shake vigorously for  two minutes.
    9.2   Allow the solvent to separate  from the  sample, draw the  organic  •
          layer .into a 100-ml  beaker, then pass the organic layer  through  a
          chromatographic column containing 3-4 inches anhydrous sodium
          sulfate, and collect it  in  a 500-ml K-0 flask  equipped with a
          10-ml ampul.   Add a  second  60-ml volume of solvent to the
          separatory funnel and complete the extraction  procedure  a second
          time.  Perform a third extraction in  the  same  manner.
             ?
    9.3   Concentrate the extract  to  10  ml in a K-D evaporator  on  a hot
          water bath.  Disconnect  the Snyder column just long enough  to add
          10 ml of hexane to the K-D  flask and  then continue the
          concentration  to about 5-6  ml.   If the  need for cleanup  is
          indicated, continue  to Florisil  Column  Cleanup (10 below).

                                      TOO

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    9.4   If further cleanup Is not required, replace the Snyder column  and
          r
          flask with a micro-Snyder column and continue the concentration
          to 0.5-1.0 ml.  Analyze this final concentrate by thin-layer
          chromatography (Section 11).
10.  Florisil Column Cleanup
    10.1  Adjust the sample extract to 10. ml with hexane.
    10.2  Place a charge of activated Florisil (weight determined by
          lauric-acid value, see Appendix II) in a Chromaflex  column.
          After settling the Florisil by'tapping the column, add about
          one-half inch layer of anhydrous granular sodium sulfate  to the
          top.                          .
    10.3  Pre-elute the column, after cooling, with 50-60 ml of petroleum
          ether.  Discard the eluate  and  just prior to exposure of  the
          sulfate layer to  air, quantitatively transfer the sample  extract
          into the column by decantation.  and .subsequent petroleum ether
          washings.  Adjust the elution rate to  about 5 ml per minute  and,
          separately collect the fo'ur eluates in 500-ml K-D flasks  equipped
          with  10-ml ampuls-.  Perform the first  elution with 200 ml of  6%
          ethyl ether in petroleum ether, and the  second elution with 200
          ml of 15% ethyl ether in petroleum ether.  Perform the third
          elution with  200  ml of 50%  ethyl ether - petroleum ether  and  the
          fourth elution with 200 ml  of  100% ethyl ether.
          10.3.1 Eluate Composition  - By  using an  equivalent quantity  of
                 any batch  of Florisil  as determined by  its  1 auric  acid
                 value, the pesticides will  be separated  into  the  eluates
                 indicated  as follows:
                                      101

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                         50% E1uate            100% Eluate
                         Carbaryl (70%)        Carbaryl (30%)
                         Mexacarbate           Aminocarb
                                               Propoxur
    10.4 Concentrate the eluates to 6 - 10 ml in the K-D evaporator in a
         hot water bath.  Change to the micro-Snyder column and continue
         concentration to 0.5-1.0 ml.
    10.5 Analyze according to 11. below.
11.  Separation and Detection
    11.1 Carefully spot 10% of the extract on a thin layer.  On the same
         plate spot several pesticides or mixtures for screening
         purposes, or a series of 1, 2, 4, 6, 8 and 10 jjl of specific
         standards for quantitative analysis.
    11.2 Develop the'layers 10 cm in a tank  saturated with  solvent
         vapors.  Remove the plate and allow it to dry.
    11.3 Spray the layer rapidly and evenly  with about  10-15 ml
         chromogenic reagent.  Heat the layer-in an oven  at 110° C for  15
         minutes.  The pesticides will appear with colors as  indicated in
         Table 2.  Make quantitative estimates by visually  comparing  the
         intensity and  size of the spots with those of  the  series of
         standards.
    11.4 Spray the layer with sodium borate  reagent and  observe the color
         shift of the reaction products.  The color shift'must be the
         same for sample and  standard  for  identification  to be confirmed.
                                      102

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12. Calculation of Results
    12.1. Determine the concentration of pesticide  in  a  sample  by
         comparing the response in a sample to that of  a quantity of
         standard treated on the same  layer.  Divide  the result, in
         micrograms, by the fraction of extract spotted to convert to
         micrograms per liter.
13. Reporting Results
    '13.1 Report results in micrograms per liter without correction for
         recovery data.  When duplicate and spiked samples are analyzed
         all  data obtained should be reported.
                                    103

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  METHOD FOR N-ARYL CARBAMATE AND UREA PESTICIDES IN WATER AND WASTEWATER

1.  Scope and Application

    1.1   This method covers the determination of various N-aryl carbamate

          and urea pesticides in water and wastewater.

          The following pesticides may be determined individually by this

          method:                •
1.2
                     Parameter

                     Barban
                     Chlorpropham
                     Diuron
                     Fenuron
                     Fenuron-TCA
                     Linuron
                     Monuron
                     Monuron-TCA
                     Neburon
                     Propham
                     Siduron
                     Swep
                                            Storet No.
                                              39650
                                               39052
2.  Summary

    2.1  A measured  volume  of water  is  extracted with methylene chloride

         and the  concentrated extract is cleaned up with a Florisil

         column.   Appropriate fractions from the column are concentrated

         and portions  are separated  by thin-layer chromatography.  The

         pesticides  are hydrolyzed to primary amines, which in turn are

         chemically  converted to diazonium salts.  The layer is sprayed

         with  1-naphthol and the products appear as colored spots.

         Quantitative  measurement is achieved by visually comparing the
                                       104

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         responses of sample extracts to the responses of standards on the
         same thin layer.  Results are reported in micrograms per liter.
    2.2  This method is recommended for use, only by experienced pesticide
         analysts or under the close supervision of such qualified persons.
3.  Interferences           .
    3.1  Direct interferences may be encountered from aromatic amines that
         may be present in the sample.  These materials react with the
         chromogenic reagent and yield reaction products similar to those
         of the pesticides.  In cases where amines are suspected of
       '  interfering with a determination,  a different solvent system
         should be used to attempt to isolate the pesticides on the layer.
    3,2  Indirect interferences may be encountered from naturally colored
         materials whose presence masks the chromogenic reaction.
4.  Apparatus and Materials
    4.1  Thin-layer plates - Glass plates  (200 x 200 mm) coated with 0.25
         mm  layer of Silica Gel 6 (gypsum  binder).
    4.2  Spotting Template
    4.3  Developing Chamber
    4.4  Sprayer - 20 ml capacity
    4.5  Kuderna-Danish  (K-D) Glassware  (Kontes)
         4.5.1  Snyder Column - three ball (K-503000)
         4,5.2  Micro-Snyder Column  - two  ball  (K-569001)
         4.5.3  Evaporative Flasks  T 500 ml  (K-570001)
         4.5.4  Receiver Ampuls -  10 ml  graduated  (K-S70050)
         4.5.5  Ampul  Stoppers
                                      105

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5.
4.6  Chromatographic Column - Chromaflex  (400 mm  long x  19 mm  ID) with
     coarse fritted plate on bottom and Teflon  stopcock; 250 ml
     reservoir bulb at top of column with flared  out funnel shape at
     top of bulb - a special order (Kontes K-420540-9011).
                                            4
4.7  Chromatographic Column - Pyrex (approximately 400 mm long x 20 mm
     ID) with coarse fritted plate on bottom.
4.8  Micro Syringes - 10, 25, 50 and 100 jjl.
4.9  Separatory Funnel - 2000 ml, with Teflon stopcock.
4.10 Blender - High speed, glass or stainless steel cup.
4.11 Florisil - PR Grade (60-80 mesh); purchase activated at 1250°F
     and store in the dark in glass containers  with glass stoppers or
     foil-lined screw caps.  Before use activate  each batch overnight
     at 130°C in foil-covered glass container.  Determine lauric acid
     value (see Appendix II).
Reagents, Solvents, and Standards
5.1  Sodium Chloride - (ACS) Saturated solution in distilled water
     (pre-rinse Nad with hexane).
5.2  Sodium Hydroxide - (ACS) 10 N in distilled water.
5.3  Sodium Sulfate - (ACS) Granular, anhydrous (conditioned at 400* C
     for 4 hrs.).
5.4  Sulfuric Acid - (ACS) Mix equal volumes of cone. HgSO^ with
     distilled water.
5.5  Diethyl Ether - Nanograde, redistilled in  glass, if necessary.
     5.5.1  Must be free of peroxides as  indicated by EM Quant test
            strips.  (Test strips are available from.EM  Laboratories,
            Inc., 500 Executive Blvd., Elmsford,  N.Y. 10523,,)

                                  106

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         5,5:2  Procedures recommended for  removal  of  peroxides are
                provided with the test strips.
    5.6  Hexane, Methanol, Methylene Chloride,  Petroleum Ether - nanograde,
         redistill in glass  if necessary.
    5.7  -Pesticide Standards - Reference  grade.
         5.9.1  TLC Standards - 0.100 ^ug/jul  in  chloroform.
    5.8  Nitrous acid - prepare just before  use fay  mixing 1  g  NaN02 with
         20 ml 0.2 N HC1.                                                  '
    5.9  Chromogenic agent - dissolve  1.0 g  1-Naphthol  in 20 ml ethanol.
         Prepare fresh daily.
6.  Calibration
    6.1  To insure even solvent travel up the  layer, the tank  used  for
         layer development must be thoroughly  saturated  with developing
         solvent before it is used.  This may  be achieved by lining the
         inner walls of the  tank with chromatography paper and introducing
         the solvent 1-2 hours before use.
    6.2  Samples and standards should be  introduced to the layer using a
         syringe, micropipet or other  suitable  device  that permits  all the
         spots to be about the same size  and as small  as possible.   An air
         stream directed on  the layer  during spotting  will  speed solvent
         evaporation and help to maintain small  spots.
    6.3  For qualitative and quantitative work,  spot a series  representing
         0.1-1.0 jug of a pesticide.  Tables  1  and 2 present  color responses
         and R-. values for several solvent  systems. •
                                      107

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                                    TABLE 1

               Rf VALUES OF N-ARYL CARBAMATE AND UREA PESTICIDES
                           IN SEVERAL SOLVENT SYSTEMS
Carbamates
Propham
Chloropropham
Barban
Swep
Urea
Fenuron
Fenuron-TCA
Monuron
Monuron-TCA
Diuron
Linuron
Neburon
Siduron
A
0.49
0.57
0.61
0.48

0.03
0.03
0.04
0.04
0.05
0.40
0.21
0.02
B
0.54
0.60
0.59
0.44
-
0.04
0.04
0.05
0.06
0.09
0.43
0.28
0.07
C
0.73
0.73
0.72
0.70

0.38
0.36
0.37
0.34
0.38
0.62
0.64
0.68
D
0.48
0.49
0.41
0.41

- 0.22
0.22
0.24
0.24
0.28
0.39
0.41
0.39
E
0.36
0.37
0.28
0.28

0.10
0.10
0.10
0.10
.a. 13
0.24
0.26
0.25
F
0.68
0.70
0.70
0.67

0.41
0.41
0.47
0.46
0.54
0.66 ,
0.68
0.62
G
0.69
0.73
0.74
0.66

0.30
0.30
0.34
0.34
0.44
0.64
0.65
0.55
Solvent Systems:

A.  Methylene chloride
B.  Chloroform
C.  Ethyl Acetate
D.  Hexane/acetone (2:1)
E.  Hexane/acetone (4:1)
F.  Chloroform/acetonitrile (2:1)
G.  Chloroform/acetom'trile (5:1)
                                      108

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                           TABLE 2

COLOR RESPONSES AND DETECTION LIMIT FOR THE N-ARYL CARBAMATES
                          AND UREAS
• • G-arbamates
Prop ham
Chlorpropham
Barb an
Swap
Ureas
Fenuron
Fenuron-TCA
Monuron
Monuron-TCA
Diuron
Linuron
Neburon
Siduron
Color Detection Limit (ug)
Red-purple
Purple
Purple
Blue-.purple

Red -purple
Red-purple
Pink -orange
Pink -orange
Blue-purple
Blue-purple
Blue-purple
Red-purple
0,2
0.1
0.05
0.2

0.05
0.1
0.05
0.1
0.1
0.1
0.1
0.05
                               109

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7.  Quality Control
    7.1  Duplicate and spiked sample analyses are recommended as quality
         control checks.  Quality control charts (1) should be developed
         and used as a check on the analytical system.  Quality control
         check samples and performance evaluation samples should be
         analyzed on a regular basis.
    7.2  Each time a set of samples is extracted, a method blank is
         determined on a volume of distilled water equivalent to that used
         to dilute the sample.
8.  Sample Preparation
    8.1  Blend the sample if suspended matter is present and adjust pH to
         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 H sodium
         hydroxide.
    8.2  Quantitatively transfer a one-liter aliquot into a two-liter
         separatory funnel.
9.  Extraction                            ..
    9.1  Add 60 ml of methylene chloride to the sample  in the separatory
         funnel,and shake vigorously for two minutes.
    9.2  Allow the solvent to separate from the sample, draw the organic
         layer into a 100-ml beaker, then pass the organic layer through a
         chromatographic column containing 3-4 inches anhydrous sodium
         sulfate, and collect it in a 500-ml K-D flask  equipped with a
         10-ml ampul.  Add a second 60-ml volume of solvent to the
         separatory funnel and complete the extraction  procedure a second
         time.  Perform a third extraction in the same  manner.
                                      110

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    9.3  Concentrate the extract to  10 ml  in a K-D evaporator  on  a  hot
         water bath.  Disconnect the Snyder column just  long enough to  add
         10-ml hexane to the K-D flask and then continue the concentration
         to about 5-6 ml.  If the need for cleanup is indicated,  continue
         to Florisil Column Cleanup  (10 below).
    9.4  If further cleanup is not required, replace the Snyder column  and
         flask with a micro-Snyder column and continue the concentration to
         0.5-1.0 ml.  Analyze this final concentrate by thin-layer
         chromatography (Section 11).
10.  Florisil Column Cleanup
    10.1 Adjust the sample extract to 10 ml with hexane.
    10.2 Place a charge of activated Florisil (weight determined  by lauric
         acid value, see Appendix II) in a Chromaflex column.  After
         settling the Florisil by tapping the column, add about one-half
         inch layer of anhydrous granular sodium sulfate to the top.
    10.3 Pre-elute the column, after cooling,'with 50-60 ml of petroleum
         ether.  Discard the eluate and just prior to exposure of the
         sulfate layer to air, quantitatively transfer the sample'extract
         into the column by decantation and subsequent petroleum  ether
         washings.  Adjust the elution rate to about 5 ml per minute and,
         separately, collect up to four eluates in 500-ml K-D flasks
         equipped with 10-ml  ampuls.   (See Eluate Composition, 10.3.1.)
         Perform the first elution with 200 ml of 6% ethyl ether  in
         petroleum ether, and the second elution with 200 ml of 15% ethyl
         ether in petroleum ether.   Perform the third elution with 200 ml
                                      111

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         of 50%  ethyl  ether - petroleum ether and the fourth elution with
         200 ml  of  100%  ethyl ether.
         10.3.1  Eluate Composition  -  By using an  equivalent quantity of any
                 batch  of Florisil as  determined by its lauric acid value,
                 the pesticides will be separated  into the eluates indicated
                 below:     '                                  .     •.
                         15%  Eluate
                         Chlorpropham
                         Propham
                         Barban (95%)
50% Eluate
Barban (5%)'
Linuron
Neburon (8%)
100% Eluate
Neburon (92%)
Diuron
Moniuron
Siduron
                CAUTION:  Fenuron and Fenuron-TCA  are  not  recovered  from
                the Florisil column.
    10.4 Concentrate the eluates to 6-10 ml  in the K-0 evaporator  in a  hot
         water bath.  Change to the micro-Snyder column  and continue
         concentration to 0.5-1.0 ml.
    10.5 Analyze according to 11. below.
11.  Separation and Detection
    11.1 Carefully spot 10% of the extract on a thin layer.  On the  same
         plate spot several pesticides or mixtures for screening purposes,
         or a series of 1, 2, 4, 6,  8 and 10 ^1 of specific standards for
         quantitative analysis.
    11.2 Develop the layers 10 cm in a tank saturated with solvent vapors.
         Remove the plate and allow it to dry.
    11.3 Spray the layer rapidly and evenly with about 10-15 ml sulfuric
         acid solution.   Heat the layer in an oven at  110°C for 15
         minutes.          •

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    11.4 When the layer is cool, spray it with nitrous acid reagent and
         allow 1t to dry.  Spray the layer with 1-naphthol reagent and
         allow it to dry again.  The pesticides will appear as purple spots
         (see Table 2).  Identifications are made by comparison of colors
         and R~ values.  Quantitative estimates are made by visually
         comparing the intensity and size of the spots with those of the
         series of standard.
12.  Calculation of Results
    12.1 Determine the concentration of pesticide in a sample by comparing
         the response in a sample to that of a quantity of standard treated
         on the same layer.  Divide the result, in micrograms, by the
         fraction of extract spotted to convert to micrograms per liter.
13.  Reporting Results
    -13.1 Report results in micrograms per liter-without correction for
         recovery data.  When duplicate and spiked samples are analyzed all
         data obtained should be reported.
                                      113

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REFERENCES:
1.  "Handbook for Analytical Quality Control  in Water and Wastewater
    Laboratories", Chapter 6, Section 6.4,  U.  S.  Environmental  Protection
    Agency, National Environmental  Research Center, Analytical  Quality
    Control Laboratory, Cincinnati, Ohio, 45268, 1972.
                                    114

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     METHOD FOR CHLOROPHENOXY ACID PESTICIDES IN WATER AND WASTEWATERS
1.   Scope and Application
    1.1   This method covers the determination of various chlorinated
          phenoxy acid pesticides in water and wastewater.
    1.2   The following pesticides may be determined individually by this
          method:
                 Parameter             .          Storet No.
                254-D
                Dicamba                             —
                Si 1vex                             39760
           . .    2,4,5-T                             —
    1.3   Since these compounds may occur in water in various  forms
          (i.e., acid, salt, ester, etc.). a hydrolysis step  is included
          to permit the determination of  the .active part of  the herbicide.
2.  Summary
    2.1   Chlorinated, phenoxy  acids and their esters are extracted  from
          the  acidified water  sample with ethyl  ether.  The  esters  are
          hydrolyzed  to acids  and extraneous organic material  is removed
          by a  solvent wash.   The acids are converted  to methyl esters
          which  are extracted  from  the aqueous  phase.  The  extract  is
          cleaned by  passing  it  through a micro-adsorption  column.
           Identification  of the  esters  is made  by  selective gas
           chromatographic separations  and may be corroborated  through  the
           use  of two  or more  unlike columns.   Detection  and'measurement
           is  accomplished by  electron  capture,  microcoulometric or
                                      115

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          electrolytic conductivity gas chromatography (1).  Results are
          reported in micrograms per liter.
    2.2   This method is recommended for use only by experienced
          pesticide analysts or under the close supervision of such
          qualified persons.
3.  Interferences
    3.1   Solvents, reagents, glassware, and other sample  processing
          hardware may yield discrete artifacts and/or elevated baselines
          causing misinterpretation of gas chromatograms.  All of these
          materials must be demonstrated to'be free from interference
          under the conditions of the analysis.  Specific  selection of
          reagents and purification of solvents by distillation in
          all-glass systems may be required.  Refer to Appendix I-.
    3.2   The  interferences in industrial  effluents are high  and varied
          and  often pose great difficulty  in obtaining accurate arid
          precise measurement of chlorinated phenoxy  acid  herbicides.
          Sample clean-up  procedures  are generally required  and may   •
          result in loss of certain of these herbicides.   It  is not
          possible to describe procedures  for  overcoming  all  of the
          interferences that may be encountered  in  industrial affluents.
    3.3   .Organic  acids, especially chlorinated  acids, cause the  most
          direct interference with the  determination.  Phenols including
          chlorophenols will  also  interfere  with  this procedure.
    3.4   Alkaline hydrolysis  and  subsequent extraction  eliminates  many
          of the predominant  chlorinated  insecticides which  might
          otherwise  interfere  with  the  test.
                                      116

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    3.5   The herbicides, being strong organic  acids,  react  readily  with
          alkaline substances and may be  lost during analysis.   Glassware
          and glass wool should be acid-rinsed  and  sodium  sulfate  should
          be acidified with sulfuric acid to avoid  this possibility.
4.  Apparatus and Materials
    4.1   Gas Chromatpgraph - Equipped with glass lined injection  port.
    4.2   Detector Options:
          4.2.1  Electron Capture - Radioactive  (tritium or  nickel-63)
          4.2.2  Microcoulometric Titration
          4.2.3  Electrolytic Conductivity
    4.3'  Recorder - Potentiometric strip chart  (10 in.) compatible with
          the detector.        .
    4.4   Gas Chromatographic Column Materials:
          4.4.1  Tubing - Pyrex (180 on long X 4 mm ID)
          4.4..2  Glass Wool - Silanized
          4.4.3  Solid Support - Gas-Chrom-Q (100-120 mesh)
          4.4.4  Liquid Phases - Expressed as weight percent coated on
                    solid support.
                  4.4.4.1  OV-210, 5%
                  4.4.4 2  OV-17,  1.556 plus QF-1 or OV-210,  1.95%
    4.5   Kuderna-Oanish (K-D) Glassware
          4.5.1   Snyder Column - three ball (macro) and two ball
                  (micro)                  •
          4.5.2  Evaporative Flasks - 250 ml
                                      117

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          4.5.3  Receiver Ampuls -  10 ml, graduated
          4.5.4  Ampul Stoppers
    4.6   Blender - High speed, glass or stainless steel cup.
 •   4.7   Graduated cylinders - 100 and 250 ml.
    4.8  . Erlenmeyer flasks - 125 ml, 250 ml ground glass 3 24/40
    •4.9   Microsyringes - 10, 25, 50 and 100   1.
   " 4.10  Pipets - Pasteur, glass disposable (140 mm  long X 5 mm  I'D).
    4.11  Separatory Funnels - 60 ml and 2000  ml with Teflon stopcock.
    4.12  Glass wool - Filtering grade, acid washed.
    4.13  Diazald Kit - Recommended for the generation of diazomethane
        '  (available from Aldrich Chemical Co., Cat. #210,025-2).
5.   Reagents, Solvents and Standards
    5.1   Boron Trifluoride-Methanol-esterification-reagent, 14 percent
          boron trifluoride by weight.
    5.2   N-methyl-N-nitroso-p-toluenesulfonamide (Diazald) - High
          purity, melting point range 60-62^C.  Precursor for the
          generation of diazomethane (see Appendix IV).
    5.3   Potassium Hydroxide Solution - A 37  percent aqueous solution
          prepared from reagent grade potassium hydroxide pellets and
          reagent water.
    5.4   Sodium Chloride - (ACS) Saturated solution  (pre-rinse NaCl with
          hexane) in distilled water.
    5.5   Sodium Hydroxide - (ACS)  10 N in distilled water.
                                      118.

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5.6   So'dium Sulfate, Acidified  -  (ACS)  granular  sodium sulfate,
      treated as follows:  Add 0.1 ml  of cone,  suIfuric acid  to  lOOg
      of sodium sulfate slurried with  enough  ethyl ether to just
      cover the solid.  Remove the ether with the vacuum.  Mix  1  g  of
      the resulting solid with 5 ml of reagent  water  and ensure the
      mixture to have a pH below 4.  Store  at 130*C.
5.7   Sulfuric acid - (ACS) concentrated, Sp. Gr. 1.84.
5.8   Florisil - PR grade (60-100 mesh)  purchased activated at  1250°F
      and stored at 130°C.
5.9   Carbitol (diethylene glycol monoethyl ether).
5.10  Diethyl Ether - Nanograde, redistilled  in glass,  if necessary.
      5.1.0.1 Must be free of peroxides as indicated by  EM Quant test
             strips.  (Test strips are available from EM
             Laboratories, Inc., 500 Executive  Blvd., Elmsford, N.Y.
             10523.)
      5.10.2 Procedures recommended for 'removal of peroxides  are
             provided with the test strips.
5.11  Benzene Hexane - Nanograde, redistilled in glass,  if necessary.
5.12  Pesticide Standards - Acids and  Methyl  Esters,  reference grade.
      5.12.1 Stock standard solutions  -  Dissolve 100  mg  of each
             herbicjde in 60 ml ethyl  ether;  then make  to 100 ml  with
             redistilled hexane.  Solution  contains 1 mg/ml.
      5.12.2 Working standard - Pipet  1.0 ml  of each  stock solution .
             into a single 100 ml volumetric  flask.   Make to  volume
             with a mixture of ethyl ether  and  hexane (1:1).
             Solution contains 10 jug/ml  of  each standard.

                                  119

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          5.12.3 Standard for Chromatography (Diazomethane Procedure) -
                 Pipet 1.0 ml of the working standard into a glass
                 stoppered test tube and evaporate the solvent using a
                 steam b.ath.  Add 2 ml diazomethane to the residue.  Let
                 stand 10 minutes with occasional shaking, then allow the
                 solvent to evaporate spontaneously.  Dissolve the
                 residue in 200 ^1 of hexane for gas chromatography.
          5.12.4 Standard for Chromatgraphy (Boron Trifluoride Proce-
                 dure) - Pipet 1.0 ml of the working standard into  a
                 glass stoppered test tube.  Add 0.5 ml of benzene  and
                 evaporate to 0.4 ml using a two-ball Snyder microcolumn
                 and a steam bath.  Proceed as  in  11.3.1.  Esters  are
              •   then ready for gas chromatography.
6.  Calibration                                      -
    6.1   Gas chromatographic operating  conditions  are  considered
          acceptable  if the response  to  dicapthon  is  at least 50%  of full
          scale when < 0.06  ng  is  injected for  electron capture  detection
          and < 100 ng is  injected  for microcoulometric or electrolytic
          conductivity detection.   For  all quantitative measurements,  the
          detector must be operated within  its  linear response  range and
          the detector noise level  should  be  less  than  .2% of full  scale.
    6.2   Standards,  prepared from methyl  esters of phenoxy  acid
          herbicides  calculated as the  acid  equivalent, are  injected
          frequently  as  a check on the  stability of operating
          conditions.  Gas chromatograms of several chlorophenoxys are
          shown in  Figure 1.

                                      120

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   _L
    6        4         2-0
     RETENTION  TIME IN  MINUTES
Fig. I Column: 1.5% 0V -17 * 1.95 % QF- I,
Carrier Gas : Argon (5%) /Methane: 70mt/min.s
Column Temp. 185 C, Detector: Electron Capture .
                            121

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8.
6.3   The elution order and retention  ratios  of methyl  esters  of
      chlorinated phenoxy acid herbicides  are provided  in Table  1,  as
      a guide.
Quality Control
7.1   Duplicate and spiked sample  analyses  are recommended  as  quality
      control checks.  Quality control charts (2)  should be developed
      and used as a check on the analytical system.   Quality control
      check samples and performance  evaluation samples  should  be
      analyzed on a regular basis.
7.2   Each time a set of samples is  extracted, a method blank  is
      determined on a volume of distilled  water equivalent  to  that
      used to dilute the sample.
Sample Preparation
8.1   The sample size taken for analysis  is dependent on the type of
      sample and the sensitivity required  for the  purpose at hand.
      Background information on the  pesticide levels  previously
      detected at a given sampling site will  assist  in  determining
      the sample size required, as well as  the final  volume to which
      the extract needs to be concentrated.   A 1-liter  sample  is
      usually taken for drinking water and  ambient water analysis to
      provide a detection limit of 0.050  to 0.100^jg/l.  One-hundred
      milliliters is usually adequate  to  provide  a detection limit  of
      1 ;jg/l for industrial effluents.
                                      122

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                         Table  1

 RETENTION RATIOS FOR METHYL ESTERS OF  SOME  CHLORINATED
       PHENOXY ACID HERBICIDES RELATIVE TO 2,4-D
Liquid Phase1

Column Temp.
Argon /Methane
Carrier Flow
Herbicide
dicamba
2,4-D
si 1 vex
2,4, 5-T
2,4-D
(minutes absolute)
1.5% OV-17
1.95% QF-12
185°C

70 ml/min
RR
0.60
1 = 00
1.34
1.72

2.00

5% OV-210
185°C

70 ml/min
RR
0.61
1.00
1.22
1.51

1.62
     columns glass, 180 cm x 4- mm ID, solid support
 Gas Chrom Q (100/120 mesh)

2OV-210 may be substituted
                             123

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    8.2   Quantitatively transfer the proper  aliquot  of  sample  from  the
          sample container into a two-liter separatory funnel.   If less
          than 800 ml is analyzed, dilute to  one  liter with  interference
          free distilled water.
9.  Extraction
    9.1   Add 150 ml of ether to the sample in the  separatory funnel  and
          shake vigorously for one minute.
    9.2   Allow the contents to separate for  at  least ten minutes.,   After
          the layers have separated, drain the water  phase  into a 1-liter
          Erlenmeyer flask.  Then collect the extract in a  250-ml
          ground-glass Erlenmeyer flask containing  2  ml of  37 percent
         .aqueous potassium hydroxide.
    9.3   Extract the sample two more times"using 50  ml of  ether each-
          time, and combine the extracts in the  Erlenmeyer  flask.  (Rinse
          the 1-liter flask with each additional  aliquot of  extracting
          solvent.)                       -
10. Hydrolysis
    10.1  Add 15 ml of distilled water and a  small  boiling  stone to  the
          flask containing the ether extract, and fit the flask with  a
          3-ball Snyder column.  Evaporate the ether  on  a steam bath  and
          continue heating for a total of 60  minutes.
    10.2  Transfer the concentrate to a 60-ml separatory funnel.  Extract
          the basic solution two times with 20 ml of  ether  and  discard
          the ether layers.  The herbicides remain  in the aqueous phase.
                                      124

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    10.3  Acidify the contents of the separatory funnel by adding 2 ml  of
          cold (4°C) 25 percent sulfuric acid (5.9).  Extract  the
          herbicides once with 20 ml of ether and twice with 10 ml of
          ether.  Collect the extracts in a 125-ml Erlenmeyer  flask
          containing about 0.5 g of acidified anhydrous sodium sulfate
          (5.8).  Allow the extract to remain in contact the the sodium
          sulfate for approximately two hours.
11-  Esterification (3,4)
    11.1  Transfer the ether extract, through a funnel plugged with glass
          wool, into a Kuderna-Danish flask equipped with a 10-ml
          graduated ampul.  Use liberal washings of ether.  Using a glass
          rod, crush any caked sodium sulfate during the transfer.
          11.1.1 If esterification is to be done with diazomethane,
                 evaporate to approximately 4 ml on a steam bath (do not
                 immerse the ampul in water) and proceed as directed in
                 Section 11.2.  Prepare diazomethane as directed in
                 Appendix IV.
          11.1.2 If esterification is to be done with boron trifluoride,
                 add 0.5 ml benzene and evaporate to about 5 ml on a
                 steam bath.  Remove the ampul from the flask  and further
                 concentrate the extract to 0.4 ml using a two-ball
                 Snyder microcolumn and proceed as in 11.3.
    11.2 Diazomethane Esterification
          11.2.1 Disconnect the ampul from the K-D flask and place in a
                 hood away from steam bath.  Adjust volume to  4 ml with
                                      125

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             ether, add 2 ml diazomethane, and let stand 10 minutes
             with occasional swirling.
      11.2.2 Rinse inside wall of ampul with several hundred
             microliters of ethyl ether.  Take sample to
             approximately 2 ml to remove excess diazomethane by
             allowing solvent to evaporate spontaneously (room
             temperature.
      11.2.3 Dissolve residue in 5 ml of hexane.  Analyze by gas
             chromatography.
      fl.2.4 If further clean-up of the sample is required, proceed
             as in 11.3.4 substituting hexane for benzene.
11.3  Boron Trifluoride Esterification
      11.3.1 After the benzene solution in the ampul has cooled, add
             0.5 ml of borontrifluoride-methanol reagent.  Use the
             two-ball Snyder microcolumn as an air-cooled condenser
             and hold the contents of .the ampul at 50°C for 30
             minutes on the steam bath.
      11.3.2 Cool and add about 4.5 ml of a neutral 5 percent aqueous
             sodium sulfate solution so that the benzene-water
             interface is in the neck of the Kuderna-Oanish ampul.
             Seal the flask with a ground glass stopper and shake
             vigorously for about one minute.  Allow to stand for
             three minutes for phase separation.            '
      11.3.4 Pipet the solvent layer from the ampul to the top of a
             small column prepared by plugging a disposable Pasteur
                                  126

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                 pipet with glass wool and packing with 2.0 cm of sodium
                 sulfate over 1.5 cm of Florisil adsorbent.  Collect the
                 eluate in a graduated ampul.  Complete the transfer by
                 repeatedly rinsing the ampul with small quantities of
                 benzene and passing the rinses through the column until
                 a final volume of 5.0 ml of eluate is obtained.  Analyze
                 by gas chromatography.
12.  Calculation of Results
    12.1  Determine the methyl ester concentration by using the absolute
          calibration procedure described below or the relative calibra-
          tion procedure described in Appendix  III.
         (1)    .  Mi crograms/ liter = (A)  (B^
                                       (V-f)  (s
                  A = nq standard
                      standard area
                  B = Sample aliquot area
                .  V-| = Volume of extract  injected
                  V-t= Volume of total extract  Cul)
                  Vs= Volume of water extracted (ml)
    12.2  Molecular weights for the calculation of methyl  esters  as the
          acid equivalents.
         2,4-D                 222.0      Dicamba                  221.0
         2,4-D methyl ester    236.0      Dicamba methyl ester     236.1
         Silvex                269.5      2S4,5-T                  255.5
         SiIvex methyl  ester   283.5      2,4,5-T methyl ester     269.5
                                       127

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13.  Reporting Results
    13.1  Report results in micrograms per liter as the acid equivalent
          without correction for recovery data.  When duplicate and
          spiked samples are analyzed all data obtained should be
          reported.
                                      128

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REFERENCES:


1.  Goerlitz, D. G., and Lamar, W. L., "Determination of Phenoxy and
    Herbicides in Water by Electron-Capture and Microcoulometric Gas
    Chromatography", U. S.- Geol. Survey Water-Supply Paper 1817-C (1967).

2   "Handbook for Analytical Quality Control in Water and Wastewater
    Laboratories" (1972), U. S. Environmental Protection Agency, National
    Environmental Research Center, Analytical Quality Control Laboratory,
    Cincinnati, Ohio, 45268.

3.  Metcalf, L. D. and Schmitz, A. A., "The Rapid Preparation of Fatty Acid
    Esters for Gas Chromatographic Analysis", Analytical Chemistry, 33,
    363 (1961).                        .

4.  Schlenk, H. and Gellerman, J. L., "Esterifi cation of Fatty Acids with
    Diazomethane on a Small Scale", Analytical Chemistry, 32, 1412  (1960).
                                      129

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  METHOD FOR VOLATILE CHLORINATED ORGANIC -COMPOUNDS IN WATER AND WASTEWATERS
1.  Scope and Application
    1.1  This method covers the determination of various chlorinated organic
         compounds in water and wastewater.
    1.2  The following chlorinated organic compounds may be determined
         individually by this method:
                   Parameter
                   Benzylchloride
                   Carbon tetrachloride
                   Chlorobenzene
                   Chloroform
                   Epichlorohydrin
                   Methylene Chloride
                   1,1,2,2-Tetrachloroethane
                   Tetrach1oroethy1ene
                   1,2,4-Trichlorobenzene
                   1,1,2-Trich1oroethane
Storet No.
  32102
  34301
  32106
  34423
  34475
2.  Summary
    2.1  If the sample is turbid, it  is  initially centrifuged or filtered
         through a fiber glass filter in order to remove suspended matter.
         A three to ten micro liter aliquot of the sample is  injected  into
         the gas chromatograph equipped with a halogen specific detector.
         The resulting chromatogram is used to identify and  quantitate
         specific components in the sample.  Results are reported in
         micrograms per liter.  Confirmation of qualitative  identifications
         are made using two or more dissimilar columns.
                                      130

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3.  Interferences
    3.1  The use of a halogen specific detector minimizes the possibility of
         interference from compounds not containing chlorine, bromine, or
         iodine.  Compounds containing bromine or iodine will interfere with
         the determination of organochlorine compounds.  The use of two
         dissimilar chromatographic columns helps to eliminate this
         interference and, in addition, this procedure helps to verify all
         qualitative identifications.  When concentrations are sufficiently
         high, unequivocal identifications can be made using infrared or
         mass spectroscopy.  Though non-specific, the flame  ionization
         detector may be used for known systems where interferences are not
         a problem.                             •
    3.2  Ghosting is usually attributed to the history of the
         chromatographic system.  Each time a sample is  injected,  small
         amounts of various compounds are adsorbed on active sites  in the
         inlet and at the head of the column.  Subsequent injections of
         water tend to steam clean these sites resulting  in
         non-representative peaks or displacement of the baseline.  This
         phenomenon normally occurs when an analysis of  a series of highly
         concentrated samples is followed by a low level  analysis.  The
         system  should be checked for ghost peaks prior  to each quantitative
         analysis by injecting distilled water in a manner identical to the
         sample  analysis  (1).  If excessive ghosting occurs, the following
         corrective measures should be applied,  as required, in the order
         listed:
         1)  Multiple flushes with distilled water
                                      131

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        2)  Clean or replace the glass Injector liner
        3)  Replace the chromatographic column.
4.  Apparatus and Materials
   4.1  Gas Chromatograph - Equipped with programmed oven temperature
        controls and glass-lined injection port.  The oven should be
        equipped with a column exit port and heated transfer line for
        convenient attachment to the halogen specific detector.
   4.2  Detector Options:
        4.2.1  Microcoulometric Titration
        4.2.2  Electrolytic Conductivity
        4.2.3  Flame lonization
   4.3  Recorder - Potentiometric strip chart recorder  (10 in) compatible
        with the detector.
   4.4  Syringes - 1 ^il, lOjjl, and SOjul.
   4.5  BOO type bottle or 40 ml screw cap vials sealed.with Teflon faced
        silicone septa.
   4.6  Volumetric Flasks - 500 ml, 1000 ml.                      ;
   4.7  Syringe - Hypodermic Lur-lock type (30  ml).
   4.8  Filter glass fiber filter - Type A (13  mm).
   4.9  Filter holder - Swinny-type hypodermic  adapter  (13.mm).
   4.10 Glass stoppered ampuls  - 10 ml
   4.11 Chromatographic columns
        4.11.1    Moderately-Polar Column -  23  ft x 0.1 in  ID  x  0.125  in OD
                  stainless steel column #304 packed with 5% Carbowax  20 M
                  on Chromosorb-W (60-80 mesh).
                                      132

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         4.11.2    Highly-Polar Column - 23 ft x 0,1 in ID x 0.125 in OD
                   stainless steel #304 packed with 5% l,2s3-Tris-(2-cyano-
                   ethoxy) propane on Chromosorb-W (60-80 mesh).
         4.11.3    Porous Polymer Column - 6 ft- x 0.1 in ID x 0.125 in 00
                   stainless steel #304 packed with Chromosorb-101 (60-80
                   mesh).
         4.11.4    Carbopack Column - 8 ft x 0.1 in ID x 0.125  in OD
                   stainless steel #304 packed with Carbopack-C (80-100
                   mesh) + 0.2% Carbowax 1500.
5.  Reagents
    5.1  Chlorinated hydrocarbon reference standards
         5.1.1          Prepare standard. mixtures in volumetric flasks using
                        contaminant-free distilled water as solvent.  Add a
                        known amount of the chlorinated compounds with a
                        micro liter syringe.  Calculate the concentration of
                        each component as follows:
        mg/1 = (Density of Compound) (pi -injected) v - ^-^ - /
                                                  (•Dilution Volume  (ml))
6.  Quality Control
    6.1  Duplicate quantitative analysis on dissimilar columns should  be
         performed.  The duplicate quantitative data should agree within
         experimental error (+6 percent).   If not, analysis on a third
         dissimilar column should be performed.   Spiked sample analyses
         should be routinely performed to  insure  the integrity of the  method.
                                       133

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7.  Selection Gas Chromatographic Column
    7.1  No single column can efficiently resolve all chlorinated
         hydrocarbons.  Therefore, a specific column must be selected to
         perform a given analysis.  Columns providing only partially or
         non-resolved peaks are useful only for confirmatory
         identifications.  If the qualitative nature of the sample  is known,
         an efficient column selection can be made by reviewing the
         literature (2).  In doing this, one must remember that injection of
         large volumes of water can cause two serious problems not  normally
         noted using common gas Chromatographic techniques:
         1)  Water can cause early column failure due to  liquid phase
             displacement.                                   •
         2)  Water passing through the column causes retention times  and
             orders to change when compared to common sample solvent media,
             i.e., hexane or air.
         For these reasons, column life  and the separations obtained  by
         direct aqueous  injection may  not be  identical  to those suggested  in
         literature.
8.  Sample  Collection and Handling
    8.1  The sample containers  should  have  a  total  volume in excess of 25  to
         40 ml ,  although larqer narrow-mouth bottles may be used.

         8.1.1  Narrow mouth  screw cap bottles  with the TFE fluorocarbon
                face  silicone  septa  cap-liners  are  strongly recommended.
                Crimp-seal  serum  vials with TFE  fluorocarbon  faced septa or
                                        134

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           ground glass stoppered bottles  are  acceptable  if  the  seal  is
           properly made and maintained  during shipment.
8.2  Sample Bottle Preparation
     8.2.1  Wash all sample bottles  and  TFE  seals  in  detergent.   Rinse
            with tap water and finally with  distilled water.
     8.2.2 'Allow the bottles and seals  to air dry at room temperature.
     :8.2.3  Place the bottle in  a 200°C  oven for one  hour, then  allow
            to cool in  an area known  to  be free of organics.
     8.2.4  When cool,  seal the  bottles  using  the  TFE seals  that will be
            used for sealing the samples.
8.3  The sample is best preserved by protecting it from phase
     separation.  Since the majority of  the  chlorinated solvents are
     volatile and relatively insoluble  in  water,  it is  important that
     the sample bottle  be filled completely  to minimize air  spaca over
     the sample.  Acidification  will  minimize  the  formation  of
     nonvolatile salts  formed from  chloro'brganic  acids  and certain
     chlorophenols.  However,  it may interfere with the detection of
     acid  degradable compounds  such  as  chloroesters.  Therefore, the
     sample history must be known before any chemical or physical
     preservation steps can be  applied.   To  insure sample integrity,  it
     is best  to analyze the sample  within  1  hour  of collection.
8.4  Collect  all samples  in duplicate.
8.5  Fill  the sample bottles  in  such a  manner  that no air bubbles pass
     through  the sample as the  bottle is filled.
             /
8.6  Seal  the bottles  so  that  no air bubbles are  entrapped in it.
8.7  Maintain the hermetic  seal  on  the  sample  bottle until analysis.
                                  135

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    8.8  Sampling from a water tap.
         8.8.1 Turn on water and allow the system to flush.  When the
               temperature of the water has stabilized, adjust the flow to
               about 500-ml/minute and collect duplicate samples from the
               flowing stream.
    8.9  Sampling from an open body of water.
         8.9.1 Fill a 1-quart wide-mouth bottle with sample from a repre-
               sentative area.  Carefully fill duplicate 25 to 40 mi-sample
               bottles from the 1-quart bottle.
9.  Sample Preparation
    9.1  If the sample is turbid,  it  should be filtered or centrifuged to
         prevent syringe plugging  or  excessive ghosting problems.  Filtering
         the  sample is accomplished by filling a 30-mT hypodermic  syringe
         with sample and attaching the Swinny-type  hypodermic filter  adaptor
         with a glass fiber filter "Type A"  installed.  Discard  the first  5..
         ml of sample then collect the filtered sample in  a glass  stoppered
         sample filled to the  top.   (One should occasionally .analyze  the
         non-filtered sample  to insure that  the filtering  technique does  not
         adversely  affect the  sample).
10.  Method of Analysis
     10.1 Daily, analyze  a standard containing 10.0  mg/1 of each  compound  to
         be analyzed  as  a quality check  sample before  any  samples  are
         analyzed.   Instrument status checks and  lower limit  of  detection
         estimations  based  upon response factor calculations  at  two  times
         the  signal  to  noise  ratio are obtained  from these dflta.   In
                                       136

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         addition, response factor data obtained from this standard can be
         used to estimate the concentration of the unknowns.
    10.2 Analyze the filtered sample of unknown composition by injecting 3 to
         10 ^il into the gas chromatograph.  Record the injection volume and
         detector sensitivity.
    10.3 Prepare a.standard mixture consisting of the same compounds in
         concentrations approximately equal to those detected in the sample.
         Chromatograph the standard mixture under condition's identical to the
         unknown.
11.  Calculation or Results
    11.1 Measure the area of each unknown peak and each reference standard
         peak as follows:                                      •
         Area = (Peak Height)(Width of Peak at 1/2 Height)
    11.2 Calculate the concentration of each unknown as follows:
          (Area of Sample peak)Cul of Standard Injectedj(Conc'n of Standard)
  mg/1 =   (jul of Sample injected)(Area of Standard Peak)
12.  Reporting Results
    12.1 Report results in mg/1.  If a result is negative, report the minimum
         detectable limit (see 10.1).  When duplicate and spiked samples are
         analyzed, all data obtained should be reported.
                                      137

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      CS
                     as e»
        t I I I ! I!  Illltllf t)
              RETENTION TIME IN MINUTES
Figure 1. Column: Cliromosorls-101, Temperature Program: 125 C
for 4 min  then 4C/min up to 280 C., Carrier Gas: Nitrogen at
36ml/min,  Detector:  Microcoulometric.
                            138

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REFERENCES:                     .


1.   Dressman, R.  C., "Elimination of Memory Peaks Encountered in Aqueous-
    Injection Gas Chromatography", Journal of Chroma to graphic Science s 8.,
    265 (1970).

2.   "Gas Chromatography Abstracts", Knapman, C, E. H.s Editor, Institute of
    Petroleum,. 61 New Cavendish Street, London W1M8AR, Annually 1958 to date,
    since 1970,  also includes Liquid Chromatography Abstracts.
                                    139

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             METHOD FOR PENTACHLOROPHENOL  IN  WATER  AND  WASTEWATER
1.  Scope and Application
    1.1  This method covers the determination of  pentachlorophenol  (PCP)  in
         water and wastewater.
2.  Summary                                        •        .
    2.1  Pentachlorophenol is extracted from  the  acidified water sample  (pH
         3) with toluene, methylated with  diazomethane,  and  analyzed  by
         electron-capture gas chromatography,  using the columns  listed in
         the organochlorine pesticide method.  (Page 7,  this manual)
    2.2  Further identification of pentachlorophenol  is made  with  a mass
         spectrometer.
3.  Interferences
    3.1  Chlorinated pesticides and ot.her  high boiling  chlorinated  organic
         compounds may interfere with the  analysis  of PCP.
    3.2  Injections of samples not treated with diazonmethane indicate,  to a
         certain degree, whether interfering  substances are present.
4.  Precision and Accuracy
    4.1  Single laboratory accuracy and precision reported for this method
         when analyzing five replicates of tap water  spiked with 0.05 to
         0.07 ;ig/l of PCP is as follows:
              Recovery - mean 95.9%, range 88.1 to  100.2%
              'Standard Deviation - 6.0%
                                      140

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REFERENCE:
    "Analysis of Pentachlorophenol Residues in Soil, Water and Fish," Stark,
    Ac, Agricultural and Food Chemistry, 17, 871  (July/August 1969).
                                       141

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                                  APPENDIX I
     CONSIDERATIONS FOR GLASSWARE AND REAGENTS USED IN ORGANIC ANALYSIS*
1.  Glassware
    1.1  Cleaning Procedure - It is particularly important that glassware
         used in trace organic analyses be scrupulously cleaned before
         initial use as well as after each analysis.  The glassware should
         be cleaned as soon as possible after use, first rinsing with water
         or the solvent that was last used in it.  This should be followed
         by washing with hot soap water, rinsing with tap water, distilled
         water, redistilled acetone and finally with pesticide quality
         hexane.  Heavily contaminated glassware may require muffling at
            e
         400C for 15-to 30-minutes.  High boiling materials, such as some of
         the polychlorinated biphenyls (PCBs) may not be eliminated by such
         heat treatment.  NOTE:  Volumetric ware should not be muffled.  The
         glassware should be stored immediately after drying to prevent
         accumulation of dust or other contaminants.  Store inverted or
         cover mouth with foil.
    1.2  Calibration - Individual Kuderna-Danish concentrator tubes and/or
         centrifuge tubes used for final concentration of extracts must be
*Methods for Organic Pesticides  in Water and Wastewater,"  1971,
Environmental Protection Agency, National Environmental Research  Center,
Cincinnati, Ohio, 45268

                                       142

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         accurately calibrated at the working volume-   This  is  especially
         important at volumes below 1 ml*  Calibration should be made  using
         a precision microsyringe, recording the volume required to  bring
         the liquid level to the individual graduation marks.  Glass A
         volumetric ware should be used for preparing all  standard solutions,
2.  Standards, Reagents and Solvents
    2.1  Analytical Standards and Other Chemicals  - Analytical reference
         grade standards should be used whenever available.  They should be
         stored according to the manufacturer's instructions.  Standards and
         reagents sensitive to light should be stored in dark bottles  and/or
         in a cool dark place.  Those requiring refrigeration should be
        'allowed to come to room temperature before opening.  Storing  of
         such standards under nitrogen is advisable.
         2.1.1     Stock Standards - Pesticide stock standards solutions
                   should be prepared in 1 jjg'/^ul concentrations  by
                   dissolving 0.100-grams of the standard  in pesticide-
                   quality hexane or other appropriate solvent (Acetone
                   should not be used since some pesticides degrade  on
                   standing in this solvent) and diluting  to volume  in a 100
                   ml ground glass stoppered volumetric flask.   The  stock
                   solution is transferred to ground glass stoppered reagent
                   bottles.  These standards should be checked frequently
                   for signs of degradation and concentration, especially
                   just prior to preparing working standards from them.
                                       143

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 2.1.2      Working Standards - Pesticide working standards are
           prepared from  the stock  solutions  using a micro syringe
           preferably  equipped with a Chaney  adapter.   The
           concentration  of  the working  standards  will  vary
           depending on the  detection system  employed and the level
           of pesticide in the samples to be  analyzed.   A typical
           concentration  (0.1  ng/jul)  may be prepared by diluting  1
          jul of the 1 jug/ul  stock  to volume  in  a  10-ml ground glass
           stoppered volumetric flask.   The standard solutions
           should  be transferred  to ground glass stoppered reagent
           bottles.  Preparation  of a fresh working standard  each
           day will  minimize  concentration through evaporation of
           solvent.  These standards  should be stored  in the  same
          manner  as the  stock  solutions.
2.1.3      Identification of  Reagents -  All stock  and working
           standards should be  labeled as  follows:   name of
          compound, concentration, date prepared,  solvent used, and
          name of person who  prepared it.
2.1.4     Anhydrous sodium sulfate used as a drying agent for
          solvent extracts should  be prewashed with the solvent or
          solvents  that  it comes in  contact  with  in  order to remove
          any interferences that may be present.
2.1.5     Glass wool used at  the top of the  sodium sulfate column
          must be pre-extracted for  about 40-hours  in  soxhlet using
          the appropriate solvent.
                              144

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2.2  Solvents - Organic solvents must be of pesticide quality  and-
     demonstrated to be free of interferences,in a manner compatible
     with whatever analytical operation is to  be performed.  Solvents
     can be checked by'analyzing a volume equivalent to that used  in the
                                                                  •
     analysis and concentrated to the minimum  final volume.
     Interferences are note.d in terms or gas chromatographic response  -
     relative retention time, peak geometry, peak intensity and  width  of
     solvent response.  Interferences noted under these conditions  can
     be considered maximum.  If necessary, a solvent must be redistilled
     in glass using a  high efficiency distillation  system.  A  60-cm
     column packed with 1/8  inch glass  helices is effective.
     2.2.1     Ethyl Ether - Hexane  - It  is particularly  important that
               these two  solvents, use'd for extraction of organochlorine
               pesticides from water, be  checked for  interferences just
               prior to use.  Ethyl  ether, in  particular, can  produce
               troublesome  interferences.' (NOTE:   The formation of
               peroxides  in  ethyl  ether creates a potential  explosion
               hazard.  Therefore  it must  be checked  for  peroxides
               before  use.)   It  is recommended that the  solvents be
               mixed just prior  to use  and only in  the  amount  required
               for  immediate use  since  build-up of  interferences often
               occurs  on  standing.
     2.2.2     The  great  sens-itivity of the  electron  capture detector
               requires  that al-1  solvents  used for  the  analysis be of
               pesticide  quality.   Even these  solvents  sometimes require
                                  145

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redistillation in an all glass system prior to use.  The
quality of the solvents may vary from lot to  lot and even
within the same lot., so that each bottle of solvent must
be checked before use.
                   146

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                             •APPENDIX II
   STANDARDIZATION OF FLORISIL COLUMN BY WEIGHT ADJUSTMENT BASED ON
                       ADSORPTION OF LAURIC ACID
1.   Scope
    T.I  A rapid method for determining adsorptive capacity of
         Florisil is based on adsorption of lauric acid from hexane
         solution.  An excess of lauric acid is used and amount not
         adsorbed is measured by alkali titration.  Weight of lauric
         acid adsorbed is used to calculate, by simple proportion,
         equivalent quantities of Florisil for batches having
         different adsorptive capacities.
2<,   Apparatus
    2.1  Buret — 25 ml with 1/10 ml graduations.
    2.2  Erlenmeyer flasks — 125 ml narrow mouth and 25 ml,-glass
         stoppered.
    2.3  Pipet ~ 10 and 20 ml transfer.
    2.4  Volumetric flasks — 500 ml.
3.   Reagents and Solvents
    3.1  Alcohol, ethyl.  ~ USP or  absolute, neutralized  to
         phenolphthalein.
    3.2  Hexane   — Distilled from all glass apparatus.
    3.3  Lauric  acid — Purified, CP.
                                      147

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    3.4  Laurie  acid  solution  - Transfer 10.000 g lauric acid to 500
         ml volumetric  flask,  dissolve in hexane, and dilute to 500 ml
         (1 ml = 20 mg).
    3.5  Phenolphthalein  Indicator - Dissolve 1 g in alcohol and
         dilute  to 100  ml.      I
    3.6  Sodium  hydroxide. -  Dissolve 20 g NaOH (pellets, reagent
         grade)  in water  and dilute to 500 ml (IN.).   Dilute 25 ml IN.
         NaOH to  500  ml with water (O.OSJ^).   Standardize as follows:
         Weigh 100-200  mg lauric  acid into 1250 ml  Erlenmeyer flask.
         Add 50  ml neutralized ethyl alcohol  and 3  drops         ;
         phenolphthalein  indicator; titrate to permanent end point.
         Calculate mg lauric acid/ml 0.05, jN NaOH (about 10 rag/ml).
4.  Procedure              .      .
    4.1  Transfer 2.000 g Florisil  to 25 ml  glass stoppered Erlenmeyer
         flasks.  Cover loosely with aluminum foil  and heat overnight
         at 130°C.  Stopper, cool  to room temperature, add 20.0 ml
         lauric  acid  solution  (400 mg),  stopper,  and shake
         occasionally for 15 min.   Let adsorbent settle and pipet 10.0
         ml of supernatant into 125 ml Erlenmeyer flask.  Avoid
         inclusion of any Florisil.
    4.2  Add 50-ml neutral alcohol  and 3 drops indicator solution;
         titrate with O.OSjN  to a  permanent end point.
5.  Calculation  of Lauric Acid Value and Adjustment  of Column Weight
    5.1  Calculate amount of lauric acid adsorbed on Florisil as
         follows:
                                       148

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         Laurie Acid value = mg lauric acid/g Florisil = 200  -  (ml
         required for titration X mg lauric acid/ml O.OBN^ NaOH).
    5.2  To obtain an equivalent quantity of any batch of Florisi-1,
       .  divide 110 by lauric acid value for that batch and multiply
         fay 20 g.  Verify proper elution of pesticides by 6.
6.  Test for Proper Elution Pattern and Recovery of Pesticides
    6.T  Prepare a test mixture containing aldrin, heptachlor epoxide,
         p,p'-DDE, dieldrin, Parathion and malathion.  Dieldrin and
         Parathion should elute in the 15% eluate; all but  a  trace of
         malathion in the 50% eluate and others  in the 6% eluate.
7.  References
    7.1  "Pesticide Analytical Manual," U«S. Department of  Health,
         Education and Welfare, Food and Drug Administration,
         Washington, D.C.
    7.2  Mills,  P.A.,  "Variation  of Florisil Activity:   Simple  Method
         for Measuring Adsorbent  Capacity  and- Its  Use in  Standardizing
         Florisil Columns,"  Journal of the Association  of Official
         Analytical  Chemists,  5J,  29  (1968).
                                       149

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                                  APPENDIX III                       '
                     CHROMATOGRAPHIC CALIBRATION TECHNIQUE
 Relative" Calibration (Internal Standardization):
 A relative calibration curve is prepared by simultaneously chromatographing
 mixtures of the previously identified sample constituent and a reference
 standard in known weight ratios and plotting the weight ratios against- area
 ratios.   An accurately known amount of the reference material is then  added
 to the  sample and the mixture chromatographed.   The area ratios are
 calculated and the weight ratio is  read from the curve.  Since the amount of
 reference material added is known,  the amount of the sample constituent can
 be calculated as  follows:
                                            Rw x Ws
                         micrograms/liter =   Vs
                         Rw = Weight ratio of component to standard
                              obtained from calibration curve
                         Ws = Weight of internal standard added to •'
                              sample in nanograms
                         Vs = Volume of sample in milliliters
Using this  method,  injection  volumes need not be accurately measured the
detector  response  need  not remain constant since changes in response will
not alter  the  ratio.  This method is preferred  when  the internal  standard
meets the  following conditions:
          a^  well-resolved from other  peaks
         b)  elutes close  to  peaks  of  interest

                                      150

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         c)  approximates concentration of unknown

         d)  structurally similar to unknown.


"Methods for Organic Pesticides in Water and Wastewater," U.S.
Environmental Protection Agency, National Environmental Research
Center, Cincinnati, Ohio 45268
                                       151

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                                  APPENDIX IV
                      PREPARATION  OF  DIAZOMETHANE IN ETHER
1.  Scope
    1.1  Diazomethane is  prepared by reaction  of Carhitol and Diazald in the
         presence of  KOH.   Solutions of diazomethane decompose rapidly in
         the presence of  solid  material such  as copper powder, calcium
         chloride, boiling  stones, etc.'  These solid materials cause solid
         .polymethylene and  nitrogen  gas to  form.
2.  Apparatus  .
    2.1  .Distilling flask with  condenser,  125  ml,  long neck with dropping
         funnel.
    2.2  Erlenmeyer flasks  - 500  ml  and 125 ml.
    2.3  Water bath.
3.  Reagents .and Solvents
    3.1  Ether
    3.2  Potassium hydroxide pellets.
    3.3  Carbitol (diethylene glycol  monoethyl  ether).
    3.4  Diazald in ether.  Dissolve 21.5 g of Diazald in 140 ml ether.
4,  Procedure
                                                           /
    4.1  Use a we 11-ventilated  hood  and cork stoppers for all connections.
         Fit a 125-ml  long-neck distilling flask with a dropping funnel  and
         an efficient  condenser set  downward for  distillation.  Connect  the
         condenser to  two receiving  flasks in  a  series  - a 500-ml  Erlenmeyer

                                        152

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         followed by a 125-ml Erlenmeyer containing jO ml ether.  The  inlet
         to the 125-ml Erlenmeyer should,dip below the ether.   Cool  both
         receivers to 0°C.  As a water bath for the distilling  flask,  set
         up a 2-liter beaker on a stirplate (hot plate and  stirreH,
         maintaining temperature at 70°C.
    4.2  Dissolve 6-g KOH in 10 ml water in the distilling  flask  (no heat).
         Ad 35 ml Carbitol (diethylene glycol monoethyl ether),  stirring
         bar, and another 10 ml ether.  Connect the distilling  flask to the
         condenser and immerse distilling flask in water bath.,   By means of
         the dropping funnel, add a .solution of 21.5 g Diazald  in 140  ml
         ether over a period of 20 minutes.  After distillation  is
         apparently complete, add another 20 ml ether and continue
         distilling until distillate  is colorless.  Combine the  contents of
         the two receivers in a glass bottle (WITHOUT ground glass neck),
         stopper with cork, and freeze overnight.  Decant the diazomethane
         from the ice crystals into a glass bpttle, .stopper with cork, and
         store in freezer until ready for use.  The final solution may be
         stored up to six months without marked deterioration.   The  21.5 g
         of .Diazald reacted in this manner produce about 3  g of Diazomethane,
5.  Cautions
    5.1  Diazomethane is very toxic.  It can explode under  certain
         conditions.  The following precautions should be observed.
         5.1.1     Use only in we 11-ventilated hood.
         5.1.2     Use safety screen.
         5.1.3     Do not pipette solution of diazomethane  by mouth.
                                       153

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         5.1.4'     For pouring solutions of diazomethane, use of gloves  is
                   optional.
         5.1.5     Do not heat solutions at 100°C (EXPLOSIONS).
         5.1.6     Store solutions of gas at low temperatures (freezer
                   compartment of explosion-proof refrigerators),,
         5.1.7     Avoid ground glass apparatus, glass stirrers and sleeve
                   bearings where grinding may occur (EXPLOSIONS).
         5.1.8     Keep solutions away from alkali metals (EXPLOSIONS).
6.  Reference
    6.1  "Pesticide Analytical Manual," U.S. Department of Health, Education
         and Welfare, Food and Drug Administration, Washington, D;C.
                                      154

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BIBLIOGRAPHY

1.   "Analysis  of Pesticide Residues in Human and Environmental
     Samples,"  U.S.  Environmental Protection Agency, Perrine Primate
     Research Laboratories, Perrine, Florida, 33157, 1971.

2.   Mills,  P;A.,  "Variation of Florisil Activity:  Simple Method for
     Measuring  Adsorbent Capacity and Its Use in Standardizing Florisil
     Columns,"  Journal  of the Association of Official Analytical
  '   Chemists,  51., 29  (1968).

3.   Goerlitz,,  O.F.  and Brown,  E.,  "Methods for Analysis of Organic
     Substances in Water," Techniques of Water Resources Investigations
     of the  United States Geological Survey, Book 5, Chapter A3, U.S.
     Department of the  Interior,  Geological Survey,  Washington,. D.C.
     20242,  1972, pp. 24-40.

4.   Steere, N»V., editors "Handbook of Laboratory Safety," Chemical
     Rubber  Company,  18901 Cranwood Parkway, Cleveland, Ohio, 44128,
     1971, pp.  250-254.

3.   Cochrane,  W. P.  and Wilson,  B. P.,  "Electrolytic conductivity
     detection  of some  nitrogen-containing herbicides," Journal of
     Chromatography, 63,  364 (1971).,

4.   "Standard  Practice for Measuring Volatile Organic Matter in Water
     by Aqueous - Injection Gas Chromatography," 02908 Annual Book of
     ASTM Standards, Part 31, Water; American Society for Testing and
     Materials,  1916 Race Street,  Philadelphia,  PA,  19103.

5.   Gas-liquid Chromatographic Techniques for Petro Chemical
     Wastewater Analysis,  Sugar,  J.W.  and Conway, R.A., Journal of
     WPCF, 40,  (Annual  Conference Issue)  1622 (1968).

6.   "Handbook  of Chemistry and Physics," 48th Edition, the Chemical
     Rubber  Company, 18901 Cranwood Parkway, Cleveland, Ohio, 44128.
     (1967-1968).
                        U. S. GOVERNMENT PRINTING OFFICE: 1979 — 657-060/1624
                                      155

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