PB-230  316

METHODS FOR  ORGANIC PESTICIDES IN WATER
AND WASTEWATER

National  Environmental Research  Center
Cincinnati,  Ohio

1971
                           DISTRIBUTED BY:
                           National Technical Information Service
                           U. S. DEPARTMENT  OF COMMERCE
                           5285  Port Royal Road, Springfield Va. 22151

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             METHODS

               FOR

       ORGANIC PESTICIDES

    IN WATER AND WASTEWATER



              1971
   ENVIRONMENTAL PROTECTION AGENCY

NATIONAL ENVIRONMENTAL RESEARCH CENTER
H
           CINCINNATI. OHIO

               45268

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                              PREFACE
The use of pesticides has become a routine practice in modern agri-
culture.  While these compounds have great advantages in the control
of predatory insects, they represent a possible danger to the aquatic
environment when present in even trace concentrations.

The National Technical Advisory Committee on Water Quality Criteria has
recommended "that environmental levels .   . . not be permitted to rise
above 50 nanograms/liter".  Many of the states have incorporated pesti-
cide criteria in their water quality standards.  Therefore, the moni-
toring of surface waters for pesticides is an essential part of our
measurement of water quality.

The Analytical Quality Control  Laboratory, assisted by Environmental
Protection Agency scientists experienced  in the determination of pesti-
cides, has prepared the  following method  for organochlorine pesticides.
In the opinion of the AQC Laboratory and  its advisors, this method is
the best available procedure at this time.

Because methods development and selection is a dynamic process, re-
quiring continual efforts toward  improvement, comments on  the appli-
cation of the method and suggestions for  improvements are  solicited
from the analysts in the field.   These comments should be  addressed to:

               Director,  Analytical  Quality Control  Laboratory
               Environmental  Protection Agency
               National Environmental Research Center
               Cincinnati, Ohio 45268

With  the  concurrence  of  the  Office  of  Pesticides,  the method has been
adopted as  the EPA  Method  for  Organochlorine  Pesticides  and is  recommen-
ded  for use  by all  laboratories in  acquiring  data  on the concentration
of these  materials  in  waters and  wastewaters  sampled by  EPA.
                                            Dwight G.  Ballinger,  Director
                                          Analytical Quality Control  Laboratory

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                     EPA COMMITTEE ON

        ANALYTICAL METHODS FOR PESTICIDES IN WATER
Chairman:   Lichtenberg, James J.
            National Environmental Research Center
            Analytical Quality Control Laboratory
            Cincinnati, Ohio  4S268

Members:    Boyle, Harvey
            Division of Field Investigations
            Denver Federal Center
            Building 22
            Denver, Colorado  80225

            Dressman, Ronald C.
            National Environmental Research Center
            Analytical Quality Control Laboratory
            Cincinnati, Ohio  45268

            Eichelberger, James W,
            National Environmental Research Center
            Analytical Quality Control Laboratory
            Cincinnati, Ohio  45268

            Garza, Mike
            Galveston  Bay Field Station
            Houston, Texas  77006

            Johnson, Dewitt
            Illinois Field Office
            Chicago,  Illinois  60605

            Kahn,  Lloyd
            Edison Water  Quality  Laboratory
            Edison,  New Jersey  08817

            Longbottom, James  E.
            National  Environmental Research Center
            Analytical Quality Control  Laboratory
            Cincinnati,  Ohio  45268

             Loy, William
             Southeast Water Laboratory
             Athens,  Georgia  30601

             Malueg, Nick
             Consolidated Laboratories
             Redman, Washington  98052
                             \l

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           Muth,  Gerald
           Surveillance and Analysis  Division
           Alameda,  California  94501

           Streck,  Larry
           Robert S.  Kerr Water Research Center
           Ada, Oklahoma  74820

           Tabri, Adib F.
           National  Field Investigations Center
           Cincinnati, Ohio  45213
The critical review of this manual by several members of the

Office of Pesticides, the Fish and Wildlife Service, and the

U.S. Geological Survey is gratefully acknowledged.
                            Ml

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                                                                            iv
                                 CONTENTS


INTRODUCTION  	     1

PART I - RECOMMENDED PRACTICE FOR DETERMINATION OF ORGANIC
         PESTICIDES IN WATER AND WASTEWATER   	     3

     1.   General Information   	     3

     1.1  Introduction    	     3

     1.2  Sample Collection   	     3

     1.3  Sample Handling   	     4

     1.4  Glassware   	     4

     1.5  Standards, Reagents and Solvents  	     5

     1.6  Records   	     7

     2.   Common Analytical Operations   	     8

     2.1  Method Blank   	     8

     2.2  Sample Transfer    	     8

     2.3  Concentration  of Extracts    	     8

     3.   Gas-Liquid Chromatography    	     9

     3.1  Gas Chromatographic System   	     9

     3.2  Injection into the Gas Chromatograph	    16

     3.3  Qualitative Analysis   	    16

     3.4  Quantitative Analysis    	    17

     4.   Column Chromatography    	    19

     4.1  Adsorbents	    19

     4.2  Packing  the Column	    20

     4.3  Eluting  the Column	    20

     S.   Thin-Layer Chromatography  	    21

      5.1  Equipment    	    21

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      5.2  Layer Preparation  	   21

      5.3  Preparation of Developing Chamber  	   22

      5.4  Spotting the Layer   	   22

      5.S  Developing the Layer   	   22

      5.6  Visualizing and Sectioning the Layer   	   22

      5.7  Pesticide Removal from the TLC Plates	   23



PART II -  METHODS OF ANALYSIS    	24

      A.   METHODS FOR ORGANOCHLORINE PESTICIDES  	   24

      1.   Scope and Application	24

      2.   Summary	   24

      3.   Significance   	25

      4.   Interferences	25

      5,   Method for Analysis Using Electron Capture Gas
           Chromatography    	   27

      5.1  Extraction of Sample	27

      5.2  Clean-up and Separation Procedures 	   29

      5.3  Gas-Liquid Chromatography	33

      5.4  Confirmatory Evidence	34

      5.5  Calculation of Results 	   34

      5.6  Reporting  Results   	   35

      6.   Method  for Analysis Using Microcoulometric or
           Electrolytic Conductivity Gas Chromatography	35

      6.1  Extraction of Sample	35

      6.2  Clean-up and Separation  Procedures  	  37

      6.3  Gas-Liquid Chromatography	37

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                                                                              VI
     6.4  Confirmatory Evidence	38




     6.5  Calculation of Results	38




     6.6  Reporting Results 	   38




REFERENCES	48




APPENDIX	51

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               ORGANIC PESTICIDES  IN  WATER AND WASTEWATER
                               V


INTRODUCTION


    Advances in the science of analytical chemistry  in  recent years are

typified by constant new developments of methods which  yield greater

efficiency, selectivity, and sensitivity.  As a  result,  the analytical


chemist now has the means to measure  minute  quantities  of  pesticides,

either singly or in combination.   Laboratories  capable  of  this  degree of

measurement are now commonplace;  making possible pesticide pollution data

for every major stream in the nation.

    The increasing sophistication of  data  storage  and  retrieval systems

and the every expanding toxicological and  ecological information on the

impact of pesticides, permits data obtained  by  several  laboratories to be

used in the combined assessment of pollution in a  given river  system or

in the nation's major streams, no matter where  located.

    This growing use of data dictates development  of effective  means to


minimize procedural error within each laboratory and optimize  analytical

agreement between  laboratories through use of a standardized method.  The

Environmental Protection Agency's "Methods for Organic Pesticides in Water

and Wastewater", presented herein, arc designed to provide the means of

obtaining  such agreement and validity.

    Part I of this manual, entitled "Recommended Practice  for  the Deter-

mination of Organic Pesticides in Water'^presents a general  discussion,

helpful hints and  suggestions, and precautionary measures  required for

pesticide  analyses.   Succeeding chapters present stepwise  procedures ^or

various types of pesticides  and types of samples.  This format was chosen

to provide short,  concise,  and easy-to-follow methods, to facilitate the

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inclusion of additional methods and revisions of existing methods  as  they



are developed and found to be acceptable, and to emphasize the analytical



quality control aspects of pesticide analysis.



    The Environmental Protection Agency methods offer several analytical



alternatives, depending on the analyst's assessment of the nature and extent



of interferences and the complexity of the pesticide mixtures found.   They



are recommended for use only by experienced residue analysts or under the



close supervision of such qualified persons.

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                                                                           3
PART I - RECOMMENDED PRACTICE FOR DETERMINATION OF ORGANIC PESTICIDES
         IN WATER AMD WASTEWATER


1.  General Information

    1.1  Introduction - Part I of this manual is intended to provide

general information, helpful hints and suggestions, and precautionary

measures which experience has shown to be important in producing con-

sistently reliable results.  Part I is by no means complete and other  ref-

erences, such as the Food and Drug Administration's "Pesticide Analytical

Manual" (1), The Canadian Department of Agriculture's "Guide to the

Chemicals Used in Crop Protection" (2), and "Official Methods of Analysis

of the Association of Official Analytical Chemists" (3), should also be

consulted by pesticide residue analysts.  Other helpful references for

general practice and analytical  quality control are ASTM Part 30 "Tentative

Recommended Practice for General  Gas Chromatography Procedures"  (4), and the

Environmental Protection Agency  manual  "Control of Chemical Analyses in

Water  Pollution  Laboratories"  (S).  Additional  recommended  references giving

fundamentals of  Chromatography  in general,  and gas chromatography  in parti-

cular,  are  listed  in  the bibliography.


     1.2  Sample  Collection - Wide mouth glass  bottles equipped  with Teflon-

 lined screw caps should  be used for  sample  collection.   Plastic bottles  must

 not be used since  they are known to  introduce  interference and  absorb  pesti-

 cides.  The size of the sample is dictated  by  the sensitivity required

 for a particular purpose and the detection  system to  be employed.   The

 normal sample volume requirements are given in the individual methods  of

 Part II.   If analysis by more than one method  is to be performed,  sufficient

 sample must be collected to supply the need of each analysis.  In addition,

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4




     sufficient sample should be collected to permit  running  of .duplicate and




     spiked analyses.  Breakage of glass sample bottles  is  overcome  by  shipping



     them in expanded  polystyrene containers molded to  fit the bottles.  Refer




     to ASTM Standards, Part 23, D510 for further sampling recommendations  (6).






         1.3  Sample Handling - The sample collector should provide the following



     information in writing:  date, time, location (coordinates or river mile,



     city, etc.)i depth, suspected contaminants, type of sample (surface water,



     waste discharge,  etc. ), name of sample collector, as well as any other



     information that may be helpful in selecting the analytical approach as



     well as in interpreting results.  Upon receipt in the laboratory, samples



     should be  logged in immediately.  Due to the instability of many of the



     pesticides in water  (7)  (8)  (9), samples should be extracted and analyzed




     as  soon as possible after  collection.   If samples must be stored, they



     should be  placed in a cool  dark place, preferably in a refrigerator.



     Holding time and conditions of  storage  should be reported along with



     results.






          1.4   Glassware



          1.4.1  Cleaning  Procedure - It  is  particularly important  that glass-



     ware  used in pesticide  residue  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



      soap  water, rinsing  with tap water,  distilled water,  redistilled  acetone



      and finally with pesticide quality hexane.   Heavily  contaminated  glassware



      may require muffling at 400C for 15 to 30 minutes.   High boiling materials,

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such as some of the polychlorinated biphenyls (PCB's)  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.



    1.4.2  Calibration - Individual Kuderna-Danish concentrator tubes  and/



or centrifuge tubes used for final concentration of extracts must be



accurately calibrated at the working volume.  This is  especially important



at volumes below 1 ml.  Calibration should be made using a precision micro-



syringe, recording the volume required to bring the liquid level to the



individual graduation marks.  Class A volumetric ware should be used for



preparing all standard solutions.






    1.5  Standards, Reagents and Solvents




    I.S.I  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  sen-




sitive 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.



    1.5.1.1  Stock Standards - Pesticide stock standards solutions should



be prepared in 1 ug/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  volumet-




 ric flask.  The  stock solution is  transferred  to  ground  glass  stoppered




 reagent bottles.   These standards  should  be checked frequently for  signs

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of degradation and concentration,  especially just prior to preparing working




standards from them.




    1.5.1.2  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/vl) may be




prepared by diluting 1 ul of the 1 ug/ul stock solution to .volume in a



10 ml ground glass  stoppered volumetric flask.  The standard solutions




should be transferred to ground glass stoppered reagent bottles.  Prepa-




ration 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.



    1.5.1.3   Identification of  Reagents -  All  stock and working  standards




should be  labeled as  follows:   name  of compound, concentration,  date prepa-



red, solvent  used,  and name of  person who  prepared it.




    1.5.1.4   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.



    1.5.1.5   Cotton used at the top  of the sodium  sulfate column must be




pre-extracted for  about 40 hours  in  soxhlet using  the  appropriate sol-



vent.   A cheap grade  of cotton  is  recommended.   Red Cross cotton is not



 recommended.



     1.5.2  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 concen-

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trated to the minimum final volume.   Interferences are noted in terras of




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.




    1.5.2.1  Ethyl Ether - Hexane - It  is particularly important that



these two solvents, used 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.




    The great  sensitivity  of  the electron  capture detector requires  that




all solvents used for the  analysis be of pesticide quality.  Even  these



solvents sometimes require 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.








     1.6 Records



     1.6.1  The progress of the  sample  through the analysis  should be




recorded in  permanently bound notebooks or on  laboratory  data  cards.



Dates of extraction,  clean-up and  separation  and G.C.  analysis  and




date of reporting results  should be  recorded.




     1.6.2   All evidence accumulated during the analysis:   gas  chroma-



 tograms,  photographs of thin-layers, infrared spectra, etc.  should be

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retained for as long as may be required to fulfill the purpose for the



analysis.




    1.6.3  All results should be recorded on laboratory data cards or



bound notebook so as to provide a permanent laboratory record.  Where



appropriate, the data should be entered into STORET.








2.  Common Analytical Operations




    2.1  Method Blank - A method blank must be determined whenever a



sample or group of  samples  is analyzed.  This is done by following



the procedure  step  by step  including all reagents,  solvents,  and




other materials in  the  quantity required by the method concurrently



and under conditions  identical to  those for the samples.  Additional



blanks  are  required whenever  a new supply  of any  of the  reagents,




solvents, etc. is  introduced.



     2.2  Sample Transfer  -  The utmost  in  care and technique must  be




exercised in order  to assure  quantitative  transfer of extract solutions



 from  one vessel to  another throughout  the  analysis.  Careless technique



will  introduce determinate errors  and  produce inaccurate results.  The



 internal wall of  the  vessel must be carefully rinsed several  times



 (usually three) with  a volume of  the particular solvent  appropriate  for



 the analysis involved.   Final flushing while pouring into the receiving



 vessel is  recommended.








     2.3  Concentration of Extracts



     2.3.1  Kudema-Danish  (K-D)  Evaporation -  A Snyder column, evaporative



 flask and calibrated receiver ampul are employed.  The evaporative flask



 should be filled to no more than 60% capacity.   Set the K-D assembly over



 a vigorously boiling water bath or a live steam bath.  The evaporation

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                                                                            9
must be carefully attended to avoid loss of pesticides.   The water level
should be maintained just below the lower joint, and the apparatus mounted
so that the lower rounded surface of the flask i;  bathed in steam.  Carry
out the evaporation in a hood so that solvent vapors are exhausted.  When
the solvent no longer actively distills, the K-D apparatus is removed
from the bath and allowed to cool.  The condensed solvent is allowed to
drain into the ampul before dismantling.
    2.3.2  h'inal Concentration - Concentration below 5 ml is usually
required when analyzing surface water samples.  Final evaporation to a
minimum of 0.2 ml may be  accomplished in the ampul with the aid of a
gentle stream of clean dry nitrogen or  air  in a warm water bath,  adjusted
to  the temperature prescribed by the method.  Final evaporation may also
be  accomplished  in the ampul using a micro  Snyder column  to give  a
final  volume of  0.2-0.4 ml.  In the latter  case, a small  sand-size
boiling  chip is  added to  the ampul prior to evaporation.  The extract
volume is  reduced  to  0.1  ml  below  the volume sought so  that  the internal
wall  of  the  ampul  may be  rinsed.   This  step is  carried  out  at  least
 three times.   Great  care  must  be  exercised  to prevent the extract from
going to dryness.


 3.   Gas-Liquid Chromatography
     3.1   Gas Chromatographic System  - The  gas chromatographic  system em-
 ployed must  be demonstrated to be suitable for the  determination  of pes-
 ticides  with a minimum  of decomposition and loss  of compounds  of  interest.
 The analyst  must evalup'... the  individual  system to  demonstrate  such capa-
 bility.   Detectors,  col-uP Backings,  column conditioning as well  as sug-
 gested operating conditions are given  below.

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10




          3.1.1   Injection  Systems  - Many  organic  compounds, pesticides  in par-



      ticular,  decompose  at elevated temperatures  when they  come  in  contact with




      stainless  steel.  To  prevent  this  decomposition, the inlet  port  of the gas



      chromatograph must  be capable of accepting a quartz  or pyrex glass insert.



      To avoid bleed off  that may cause high background or discrete  interferences,




      the septa should be preconditioned prior to use.  The  septa can be treated




      by heating in a vacuum oven at  250 C for two hours;  changing ...he septum




      at the end of each work day to  allow overnight purging of the  system is




      also good practice.



          3.1.2  Detectors - The analyst must thoroughly acquaint himself with




      the descriptive and theoretical information on  the detectors employed.



      Review of technical papers and manufacturer's instructions on a specific



      detector is  necessary  to become familiar with its use and  limitations.



          3.1.2.1   Electron  Capture Detector  (EC)  - The electron  capture



      detector is  extremely  sensitive to  electronegative  functional groups,




      such  as halides, conjugated  carbonyls,  nitriles, nitrates,  and  organo-




      metallics.   It  is  virtually  insensitive to  hydrocarbons, amines,  alco-



      hols  and ketones  (10)  (11).  The  selective  sensitivity  of  halides makes




      this  detector particularly valuable for the determination  or  organo-



      chlorine pesticides.   It  is  capable of detecting picogram  (10"    gram)




      quantities  of many organochlorine pesticides.   Organophosphorus pesti-



      cides containing nitro- groups  are  also detected, although with much



      less  sensitivity.



           Electron capture detectors  may be of parallel plate and concentric
       tube or concentral design and employ one of two ionization sources:



       tritium (H )  or radioactive nickel (Ni  ).   The tritium detector has



       temperature limit of 225 C (It should not be operated above 210 C.)

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                                                                           11
which makes it susceptible to a buildup of high boiling contaminants which
reduce its sensitivity and require frequent clean-up.   The nickel detector,
on the other h.ind, can be  operated or baked out up to 400 C,  to reduce
contamination and cleaning problems.
    3.1.2.2  Microcoulometric Titration Detector (MC)  - The microcoulo-
metric detector (12) is selective for halogen containing compounds, ex-
cept fluorides, when used with the halogen cell.  Under optimum conditions,
this detector is capable of detecting 5-20 ng of organochlorine pesticides.
Although the sensitivity of this detector is not as great as that of elec-
tron capture, the high degree of specificity makes it a very valuable in-
strument for qualitative identification as well as for minimizing sample
clean-up.   Under the proper oxidative-reductive conditions, the  system can
be made specific for sulfur, phosphorus, and nitrogen compounds.
     3.1.2.3  Electrolytic  Conductivity Detector  (BCD) - The electrolytic
conductivity  detector  has  a  sensitivity  2  to 3 times  greater  than  the
microcoulometric  system.   Although perhaps  slightly less  selective than
the  MC,  it is,  nonetheless,  effective for  qualitative identification,  and
cleanup  appears to  be  less of  a problem  (13).   Use of tne electrolytic
conductivity  detector in  the reductive mode with a platinum catalyst is
recommended when  determining halogen compounds.   If the oxidative  mode is
used,  a  scrubber must be  employed to remove SO ,  which  also responds to
 the  detector.
     3.1.2.4  Flame  Photometric Detector (FPD)  - The  flame photometric
 detector is selective for sulphur and phosphorus (14).   With  the use of
 a dual head,  the detector is capable of simultaneously measuring both sulfur
 and phosphorus as well as a normal flame ionization response.  Using a
 single head,  either sulfur or phosphorus and  normal  flame ionization

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12
       response is measured.  The characteristic optical emissions of sulfur



       and phosphorus are measured using filters with transmission at 394 mg




       (sulfur) and 526 m\i  (phosphorus).  The FPD is capable of detecting sub-



       nanogram quantities of both sulfur (4 x 10    gram) and phosphorus



       (10'U gram).




           3.1.3  Columns - A well-prepared column is essential to an acceptable



       gas chromatographic  analysis.  The most advanced gas chromatographic in-



       strumentation  available  is no better than the column used with it.  A



       well-conditioned efficient column is a must.  Column packings may be pre-



       pared by the analyst or  purchased already prepared from a supply house



       that specializes in  this  service.  The packing materials and  column di-



       mensions selected by EPA are specified under the individual methods in



       Part II.



            3.1.3.1  Preparation of Column Packing  - The analyst who  prepares his



       own packing  must develop a highly refined technique to produce consistently



       good efficient columns.   Particular  care should be taken to accurately



       measure  loadings,  uniformly distribute the  liquid phase, and  to preserve



       the  structure  of the fragile solid support.  Improved column  efficiency is



       obtained when  the  solid  support  is dried at 100  C overnight prior to



       coating.   Several  methods may be employed to coat  the packing material:



       slurry  (15), filtration  (16), and frontal analysis  (17).



            The slurry technique consists of dissolving  a weighed  amount  of  the



        liquid phase in an appropriate  solvent  in a beaker and  slowly pouring



        the weighed solid  support into  it with  constant  stirring.   The beaker



        is immersed to the level of the solvent  in  a hot water  bath and  gently



        stirred until  the  bulk of the  solvent has evaporated.   Extra care must



        be exercised to minimize crushing of the solid support. The  filtration

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                                                                       13





technique consists of mixing the solid support  with the solution  of



liquid phase as above then carefully pouring into a Buchner funnel




fitted with a filter flask.  The excess solution is removed by vacuum.




The frontal analysis technique consists of passing the solution through a



column of the solid support until the effluent from the column is of




the same composition as the original solution.  The analyst should



select the method that provides the best results for him.  Drying of



the coated support may be  accomplished by spreading on a tray in a



convection or vacuum oven  at 100-120 C, with a rotary evaporator (18)



or with  a fluidized bed drier  (19).  The latter is recommended.



      3.1.3.2  Column Material  and Dimensions - The column should be con-



structed of borosilicate glass.  The most useful length of column  is



about 6  ft. with  a diameter of 1/4  in. O.D. to  1/8 in. O.D., depending



on the detector employed and the volume of  sample  injected.  Electron



capture  detectors of  parallel  plate design  and  the Tracor concentral



design perform well with either 1/4 in. or  1/8  in. columns, while  the




Varian-Aerograph  concentric tube design operates best  with a  1/8 in.



column.   A 1/4  in.  column  for  the microcoulometric detection  system is




 recommended.



      3.1.3.3  Packing the  Column -  It is  important that  the  column be



 packed to a uniform density not so  compact  as to cause unnecessary back



 pressure and not  so loose as to create voids  during use.   Care should



 be exercised so as not to crush the packing.   Column tubing should be



 rinsed with solvent,  eg.  chloroform, and  dried prior to packing.



 Columns are filled through a  funnel connected by flexible tubing to one



 end.  The other end of straight or coiled tubing is plugged with about



 1/2  in. of silanized glass wool and filled with the aid of gentle vibra-

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14




       tion or  tapping.  A mild vacuum may also be applied to the plugged end.




       When filled the open end is also plugged with silanized glass wool.  In




       a  similar manner, one-half of a "U" shaped column is filled and then the



       other  and the  ends are plugged with silanized glass wool.



            3.1.3.4   Column Conditioning - Proper thermal conditioning is



       essential to eliminate column bleed and to provide acceptable gas




       chromatographic analyses.  A number of procedures may be  used for  this



       purpose.  The  procedure described below is used by the Analytical



       Quality  Control  Laboratory with excellent  results:



        Install  the packed  column  in the oven.  Do not connect the  column  to



        the detector.  However, gas  flow through  the  detector should be main-




        tained.   This  can be  done  using the diluent gas  line or,  in dual column



       ovens, by connecting  an unpacked column to the detector.  Heat the oven



        to near  the maximum recommended temperature for  the  liquid  phase without



        gas flow for  2 hours.   Reduce the oven temperature to  approximately 40 C



       below  the maximum recommended temperature and allow  temperature to equili-




       brate  for a  minimum of 30  minutes still without  flow.  Then adjust the



        carrier  gas  flow  to about  SO ml per minute  for a 1/4 inch column  and



        about  25 ml  per  minute for a 1/8  inch column.   (Caution—bleed off of



        liquid phase will occur if not  fully  temperature equilibrated.)   After



        one hour,  increase the temperature  to about  20 C above normal  operating



        temperature  with gas  flow for 24-48 hours.   (Do not  exceed maximum recom-



        mended operating temperature.)   Cool  down and connect column to the detector



        system,  then raise to normal operating temperature.   Columns prepared and




        conditioned in this manner should yield good chromatograms with no further




        treatment.



              3.1.3.S  Optimizing Operating Conditions - The analyst must determine




        the optimum conditions for obtaining  the best results for  the compounds

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                                                                          15





under study.  Standard mixtures of interest should be chromatographed to




determine retention times and maximum resolution that can be  achieved.



Parameters such as gas flow, temperature,  column length  and diameter,



as well as the electronics and detector performance,  must be  evaluated



and adjusted as required to achieve the desired results.   Other important




requirements for attaining optimum operating conditions  include a clean




injection block and a leak-free pneumatic system.  The instrument must be




operated within the linear range of the detector.  The recorder gain and



damping adjustments must be optimized.  Regulated electric line current



may be required for the electronic system.  Detailed instructions for



carrying out these operations are given in the manufacturer's instrument



manuals.



     Optimum detector sensitivity for each :.iethod is defined as the



minimum acceptable response to a designated amount of a selected compound.



The detector response for other compounds that may be determined by the




method relative to the selected compound  are listed  in each method.




     Continued optimum performance is maintained by  following the routine




maintenance and check program given in the instrument manuals.  Frequent




checks on the  injection block, oven,  and  detector temperatures should be




made.  To avoid the risk of system or detector  contamination from impurities




in gas cylinders,  replace  the  cylinders when the pressure  reaches 200 psi.



Use of molecular  sieve gas-filter  driers  on all  gases is  recommended.



     Column performance  is  monitored  by observing daily  response to  a



selected  standard mixture  and  comparing it  to  the  response obtained  under



previously  established  conditions.   Changes in  elution pattern,  relative




proportions of peaks,  and  peak  geometry are signs of a deteriorating column,




when all  other parts  of  the system are properly maintained.  Columns should




be replaced when  deterioration is  observed.

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16
         3.2  Injection into the Gas Chromatograph




         3.2.1  Loading Syringe and Measuring Volume Injected - The analyst




     must develop the ability to make accurate and reproducible injections




     into the gas chromatograph.  Several techniques may be used.  The



     analyst should select the one that provides the best results for him.




     One technique, preferred by some analysts is as follows:



     Wet syringe needle and barrel with solvent solution of the standard or



     sample to be injected and expel all air bubbles.  Draw the entire quan-



     tity of solution into the calibrated barrel and note volume.  Inject



     into the chromatograph rapidly and withdraw syringe immediately.  Then



     partially withdraw plunger and note volume remaining.  Determine volume



     injected by subtracting  this volume from  the original volume.   It is



     important that the syringe be thoroughly  cleaned  after each  injection.




     Usually, several solvent  rinses are adequate.



          3.2.2   Injection of  Standard  Solutions - The  concentration  of stan-



     dard solutions  should be such  that  the injection  volume  of the  standard




      is approximately the same as  that of  the  sample.








          3.3  Qualitative Analysis  -  Qualitative  identification of an unknown



      component  is  made by matching  the retention  time  (R )  of the unknown



      with that  of a standard obtained under identical  conditions.  The R



      is the time lapsed from injection (time zero)  to the peak maximum.   The



      absolute and relative retention time  (RRt) are commonly recorded.  The



      RR  is defined as the R  (component)   * Rt (reference compound).  When



      solvent response is observed, as with an electron capture, the leading



      edge of the solvent peak is considered time zero.  When no solvent



      response is observed, as with a microcoulometric detector, time zero




      is electrically or manually marked immediately after  injection.

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                                                                          17



    3.3.1  Confirmatory Identification - The analyst must be aware that



a single gas chromatographic determination does not provide unequivocal




identification of an unknown component.   The retention time and peak



geometry must be matched on two or more  unlike columns.   Co-injection



of the sample with a standard of the suspected compound will assist in



confirming the qualitative identification.  Clean-up and separation



techniques such as thin-layer and column chromatography also help to




make the qualitative assignment.  Identification of multicomponent



pesticides requires not only matching of all retention times and



overall peak geometry, but also the correct number and relative propor-




tion of each peak - a so called "fingerprint" of the pesticide.  Further



corroboration using infrared spectroscopy and/or mass spectrometry



should be obtained whenever possible.








    3.4  Quantitative Analysis  - The  quantity of compound present  is



proportional  to the area of the peak  and  can be used  to  determine  the




concentration of  the  components in  a  sample.  The  area measurement is




usually  preferred;  however, peak height measurement may  be  more  accurate



when  sharp  narrow peaks  occur.  Peak  area may  be measured  by electronic



integrator,  disc  integrator,  or by  planimeter  or may  be  calculated by



taking  the  peak height  x peak width at  half height.   The planimeter,



 although less precise than the other techniques,  is  recommended for



measuring the area  of unsymmetrical peaks that do  not originate at the



 original baseline.   To improve precision, measure  area several times  and



 take  the average  value.



     3.4.1  Unresolved Peaks - To  quantitate peaks  that are not completely



 resolved, inject  standards of the suspected compounds mixed in the same



 ratio as they occur in the sample and giving response equivalent to  that

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18
       of the  sample.  An  alternative method is to draw a  line perpendicular

       from  the baseline to  the  low point of the valley between the peaks.

       Shoulders on  larger peaks may be measured, although not accurately, by

       using as t.he  baseline a line drawn to conform  to the  shape of the major

       peak.   Resolution may sometimes be accomplished usinvj different G.C.

       columns and/or by preliminary separation using thin-layer and column

       chromatography.

            3.4.2   Standard  Calibration

            3.4.2.1   Absolute Calibration  (20)  -  Using the absolute method,

       pesticide  concentrations are  determined by  direct  comparison to  a single

       standard when the  injection volume  and  response are very  close  to that

       of the sample.  The concentration of pesticide in  the sample is  calcu-

       lated as follows:

                         micrograms/liter =    fv'1)     {\i \

                          A  =  ng std
                                std area

                          B  =  sample aliquot area

                          Vi =  volume of extract injected

                          Vt =  volume of total  extract  (yl)

                          Vs =  volume of water extracted (ml)

            3.4.2.2  Relative Calibration  (Internal Standardization)  (20) - A

       relative calibration curve is prepared by simultaneously chromatograph-

       ing 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 mater-

       ial  is then  added  to  the sample and the mixture chromatographed.  The

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                                                                             19
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:
                  micrograms/liter  =  Rwy* Ws

                  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
and  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
      c)   approximates concentration  of  unknown
      d)   structurally similar to unknown.
      3.4.3   Linear range  - Accurate  quantitative  analysis depends  upon a
 linear  relationship between concentration and  detector response.  The
 closer  the linear relation the more accurate the  analysis.  The analysis
 range of a detector is defined as the ratio of the largest to  the smallest
 concentration within  which the detector  is  linear.

 4.   Column Chromatography
     4.1  Adsorbents  - A  variety  of adsorbents  are used in the  various
 pesticide methods to  remove  interferences and to  separate individual

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20
      pesticides.  These  are  usually purchased preactivated  from the manu-




      facturer.  The  adsorptivity of the  adsorbent  is checked by determining




      the  elution  pattern of  specified  dyes  and/or  of given  pesticides.   Re-



      coveries  of  the pesticides must be  determined prior  to using the  adsor-




      bent for  the analysis of samples.




           4.1.1   Florisil -  Florisil preactivated  by the  manufacturer  at 1200




      F is used.   Prior to use,  the Florisil is  heated  for at  least  S hours



      (overnight  is  convenient)  at  130  C.  Although the adsorbent  tends to yellow



      when stored  in this manner for several days,  it remains  satisfactory for



      use.



           4.2  Packing the Column  - To pack the column, slowly pour the adsor-



      bent into the column while vigorously tapping it.  This  will  assist in



      providing a uniform packing and minimize  :hinneling during elution.



           4.3  Eluting the Column  - Liquids should be  poured  slowly down the




      inside wall  of the column to avoid disturbing the surface of the adsor-




      bent.  Mixing of solvents above the adsorbent is  minimized by adding



      succeeding solvents just as the last of the previous solvent reaches the



      adsorbent surface.  However,  the surface of the adsorbent must not be



      allowed to run dry, since introduction of air may cause  channeling and



      reduce the efficiency of separation.



           4.3.1  Pre-elution - Prior to addition of the sample, the column is



      pre-eluted with the solvent prescribed by the procedure.  This is done to



      remove trapped air  and trace contaminants that may interfere with the



      analysis.   It may be necessary to  tap the column to free all of  the trapped



      air.

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                                                                           21
     4.3.2  Introduction and Elution of Sample - The sample is  intro-
duced just as the last of the pre-eluting solvent reaches the surface
of the adsorbent.  The sample container is then rinsed with a few ml
of the solvent and the rinse added to the column.  Just as the last of
this solvent reaches the surface a small volume of eluting solvent is
used to rinse down the internal wall of the column.  Then the remaining
eluting solvent  is added.  Successive eluting solvents are added in a
similar manner.
     4.3.3   Eluate Composition  - Since variations  in  elution pattern may
occur  from  tir.  to time,  it  is  necessary  to demonstrate  that the eluate
composition is proper for a given  analyses.   This  can be done by eluting
standard  pesticides  from the column and/or by using the  activity test
given  in  the "Official  Methods  of Analysis of the  AOAC"  (3).

 S.  Thin-Layer Chroroatography
     5.1  Equipment - Special equipment required for preparing layers  and
 carrying out thin-layer chromatography is listed in the appendix.   Layers
 prepared in the laboratory or purchased precoated layers may be used.   The
 adsorbent is usually less tightly bound to the plate when prepared in  the
 laboratory and is thus somewhat easier to scrape for subsequent elution
 and recovery of the  pesticides.
     S.2  Layer Preparation - Layers of desired thickness are prepared  by
making a  homogeneous slurry of adsorbent in water.  The  slurry  is  poured
 into the  applicator  and the gate is quickly opened and the applicator  is
smoothly  and rapidly passed over an aligning  tray  holding the  glass plates.
The layers are allowed to stand at room temperature for  a time, then  activated

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22
      in an oven,  and stored in a desiccator for future  use.   Layers  stored



      longer than  one week should be reactivated before  use.   Prior  to  acti-




      vation, marks should be made on the layer to define the spotting  line and



      the upper limit of solvent development in order to minimize  exposure to



      the atmosphere during the spotting operation.



           5.3  Preparation of Developing Chamber - The  developing solvent is



      added to the chamber, and two chromatography paper wicks,  one  on  each of



      the long sides of the chamber, are placed so that  the entire side is




      covered and the bottom edge contacts the solvent.   The chamber is closed,



      shaken, and allowed to equilibrate.  It is important that  the  chamber be




      protected from drafts and large temperature changes.



           5.4  Spotting the Layer - Standards are spotted in the center and  at




      least at one edge of the layer.  The standards and samples should be dis-



      solved in the same solvent and spotted in the same volume.  Utmost care



      must be exercised to keep the spot small  (less than 10 mm diameter).  A



      gentle stream of clean dry air or nitrogen may be  applied over the spot to



      facilitate  close boundary evaporation, but  this gas flow exposure should



      be  kept to  a minimum.



            5.5  Developing  the Layer - The  spotted  layer is placed in  the pre-




      equilibrated chamber  so  that  the bottom  edge  is in contact with  the



      developing  solvent  and  the  lid is  replaced.   When  the  developing solvent



      reaches  the upper  reference  mark,  the layer is  removed from the  chamber and



      allowed  to  air-dry  at room temperature.



            5.6  Visualizing and Sectioning  the Layer  -  After development, the




      portion  of  the layer containing  the standards is  sprayed  evenly  with a

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                                                                           23



chromogenic agent.  Hie sprayed area is allowed to thoroughly dry and,




where required, is further treated by exposure to short wave UV light




or some other means.  The location of each standard pesticide is marked.



     5.6.1  The distance of travel for pesticides present in the unknown



samples and recovery test standards will be, respectively, the same as



those of the sprayed standards.  Using this information, the vertical



zone for each sample is divided into horizontal sections depending on the




pesticides being determined.



     5.7  Pesticide Removal from the TLC Plates - With the aid of a sharp



pointed object, the silica gel sections of interest are individually



ruled off.  With the aid of a mild vacuum, the silica gel, first from the



periphery of the section and then from the center of the section is col-



lected.  It is convenient to use a medicine dropper plugged at the tip



with filtering grade glass wool (Figure 1).  The pesticides are eluted



quantitatively into a  K-D ampul with a selected solvent  to an appropriate



volume  for  gas chromatographic  analysis.

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24
      PART II  -  METHODS  OF ANALYSIS






      A.   METHOD FOR ORGANOCHLORINE  PESTICIDES








      1.   Scope  and Application



          1.1  This method covers  the determination of various  organochlorine



      pesticides, including some pesticidal  degradation products  and related



      compounds.  Such compounds are composed of carbon, hydrogen,  and chlor-



      ine, but may also contain oxygen, sulfur, phosphorus or nitrogen.



          1.2 The following compounds may be determined individually by this



      method:   BHC, lindane, heptachlor, aldrin, heptachlor epoxide, dieldrin,



      endrin, Perthane, DDE, ODD, DDT, methr>v.ychlor, endosulfan,  f-chlordane



      and sulphenone.  Under favorable circumstances, Strobane, toxaphene,




      kelthane,  chlordane  (tech.) and others may also be determined.



          1.3  When organochlorine pesticides exist as complex mixtures, the



      individual compounds may be difficult to distinguish.  High, low, or



      otherwise unreliable results may be obtained through misidentification




      and/or one compound  obscuring  another of  lesser  concentration.   Provisions



      incorporated  in this method are  intended  to  minimize the occurrence of  such



      interferences.








      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.   This  method is  recommended



      for use only by experienced residue analysts or under  the  close  supervision

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                                                                          25





of such qualified persons.   Specifically,  the procedure describes  the



use of an effective co-solvent for efficient sample extraction;  provides,




through use of thin-layer,  column chromatography,  and liquid-liquid par-




tition methods for the elimation of non-pesticide  interferences, and



the pre-separation of pesticide mixtures.   Identification is made  by



selective gas chromatographic separations  through  the use of two or more




unlike columns.  Detection and measurement is accomplished by electron



capture, microcoulometric or electrolytic  conductivity gas chromatography.



Techniques for confirming qualitative identifications are suggested.  Re-



sults are reported in micrograms per liter without correction for recovery



data; but, such data is to be included in  the report.








3.  Significance



    3.1  The  extensive and widespread use of persistent organochlorine



pesticides has resulted in their presence in all parts of our environment.




Their occurrence  in surface waters throughout the  country is common.  Such



common  occurrence  coupled with the toxic nature of these materials  is cause



for concern.   The  known  lethal effects of these substances  to fish  and



wildlife  and  the  unknown long term consequences to humans make  it  imperative



that we identify  and quantitate  the pesticides present  in the environment.



Effective  evaluation  and control programs require  such  information.



     3.2  Because  of the  concept  of biological concentration, we need  to



detect  minute quantities  (low nanogram amounts) of pesticides in water.



The method presented  here  is  capable of detecting  these  small quantities.








 4.  Interference



     4.1  Solvents, reagents,  glassware, and other sample processing hardware

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26
      may  yield  discrete  artifacts  and/or elevated baselines causing mis-



      interpretation  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  is  required.   (Refer to Part 1,



      Sections  1.4  and 1.5)




          4.2  Sample treatment  required  to remove non-pesticide materials which



      cause interference  may result in  the  loss of certain  organochlorine



      pesticides.  Methods for eliminating  or  minimizing  interferences are de-



      scribed below in the section  on Clean-up and Separation  Procedures  (5.2).



      It  is beyond  the scope of  this  method to describe procedures  for overcoming



      all  of the possible interferences that may  be  encountered, particularly,



      in  highly contaminated water and  wastewater.



          4.3  Polychlorinated Biphenyls  -  Special  attention is  called  to



      industrial plasticizers and hydraulic fluids  such as  the chlorinated bi-



      phenyls (PCB's, Aroclors ) which  are a  potential source of interference in



      pesticide analysis.  Chlorinated  biphenyls  containing 4 to 8 chlorine  atoms



      per molecule have been reported in extracts of birds, fish,  mussels and



      water.  Possible interferences from these compounds are indicated by un-



      resolved peaks  (shoulders and nongaussian peaks), slight discrepancies




      in  retention times, and peaks which elute later than p.p'-DDT.  With some



      dependence upon  the relative concentrations, a number of chlorinated



      biphenyl  isomers may  interfere with  the determination of DDE, ODD and DDT



      isomers.   The  authenticity of  the DDT identification may be determined by



      treating  the extvact  with  alcoholic  KOH which converts  DDT to DDE  (21).
        Tradename  of the  Monsanto  Company,  St.  Louis,  Missouri

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                                                                           27



Particularly severe PCB interference will require special  separation




procedures, such as those of Reynolds (22), Armour and Burke (23),  and



Mulhern, et al (24).



     4.4  Phthalate Esters - These compounds, widely used  as plasticizers,



respond to the electron capture detector and are 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.  The presence of phthalate esters is implicated in samples




that respond to electron capture but not to the microcoulometric or electro-



lytic conductivity halogen detectors or to the flame photometric detector,



since these materials are not detected by the latter three detectors.



     4.5  Organophosphorus Pesticides - A number of organophosphorus



pesticides, notably those containing a nitro group, eg,  parathion, also



respond to the electron capture detector and may interfere with the deter-



mination of the organochlorine pesticides.  The presence of such compounds



is  indicated  in samples which respond to both the electron capture  and



flame photometric  detectors but not  to the microcoulometric halogen detector.








5.  Method  for Analysis Using Electron Capture Gas  Chromatography



    S.I  Extraction of Sample



    5.1.1   The size of sample taken  for  extraction  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  help  to  determine the  sample  size required  as well  as  the final



volume  to  which  the extract needs to be  concentrated.   A  1-liter  sample is

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28
      usually taken for electron capture  analysis.   The  extract  should not be
      concentrated further than required  to  meet  the sensitivity dictated by the
      purpose for the analysis.  Each  time a set  of samples  is extracted, an
      aliquot of solvent equivalent to that  used  for extraction  is  carried through
      the entire procedure to provide  a method blank. To  assist in interpretation
      of results, the pH of the sample is taken prior to extraction. When the  volume
      of the sample permits, one set of duplicates and one do ;ed sample  should also
      be analyzed as a quality control check.
          S.I.2  A measured volume (1-liter) of sample is  drained into  a 2-liter
      separatory funnel equipped with a Teflon stopcock, and extracted  with  60 ml
      of 15% ethyl ether in hexane by shaking vigorously for two minutes.  The
      sample container is rinsed with each aliquot of extracting solvent prior to
      extraction of the sample.
          S.I.3  The mixed  solvent is allowed to separate from the water;  the water
      is then drawn into the original sample container or into a second 2-liter
      separatory  funnel.  The  organic  layer  is passed through a small column of
      anhydrous  sodium sulfate topped with  a pledget of cotton  (previously rinsed
      with  hexane) and collected  in a  500 ml Kuderna Danish  flask  equipped with a
      10 ml ampul.  The  extraction is repeated and  the  solvent treated as above.
      Approximately  35 ml of sodium sulfate saturated water  is  then added to the
      sample and a third extraction is completed with 60  ml  of hexane (not hexane-
      ethyl ether).   This  solvent, too,  is  passed  through the sulfate column  and
       collected in the flask.   The column is rinsed with  several small portions  of
      hexane and this solvent is recovered  in the collection flask containing the
       combined extracts.  The extract is concentrated in  the Kuderna-Danish evapora-
       tor as described in Part I, Section 2.3.1.
           5.1.4  Concentration of Extract - The  final concentration volume  for
       samples of high pesticide content  (eg. pesticide plant wastewater samples)

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                                                                          29




is adjusted as necessary.   Samples containing small quantities of




pesticides (low nanogram amounts, eg.  most surface water samples)  are



concentrated to 1 ml.  The volume of the initial K-D concentrate is  5 to



6 ml.  This is reduced to 1 ml in a warm water bath (70 C)  with a gentle




draft of clean dry air or nitrogen.  The internal wall of the ampul  is




rinsed several times during this operation.  Initial gas chromatographic



analysis is made on this volume.  Up to 10 pi of extract are injected.



If insufficient pesticide is present for detection at this volume and



greater sensitivity is required, the extract is concentrated further to



a minimum volume of 0.2 ml in the manner described above.  The extract



volume is reduced below the volume sought so that the internal wall  of



the  ampul may be rinsed.  (See Part I, Section 2.3.2)   (The volume should



never be reduced below 0.1 ml).  Repeat this operation  three times,  exer-



cising great care to prevent the extract  from going to  dryness.



     5.2  Clean-up and Separation Procedures



     5.2.1  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) will indicate



whether clean-up  is  required.  When these  interfere with measurement  of the



pesticides, proceed  as directed  below.  Whether  required for quantitative



analysis  or not,  all  extracts  should be subjected  to  these  procedures,



subsequent  to  the initial  analysis  and  rechromatographed  for qualitative



corroboration  of the results.   Another  clean-up  technique,  acetonitrile



partition,  although  not  ordinarily  required for surface water  extracts,  is




sometimes  useful  for cleaning up high  organic  wastewater samples.   Refer to

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30
     the FDA methods manual (1) for this procedure.



          S.2.2  Florisil Column Adsorption Chromatography - The sample extract



     previously concentrated to 1 ml or less is diluted to 10 ml.  A 15 g charge



     of activated Florisil is placed in a column over a small layer (one-half



     inch) of anhydrous granular sodium sulfate.  After tapping the Florisil into



     the column, about a three-fourths inch layer of granular sodium sulfate is



     added to the top.  The column, after cooling, is pre-eluted with about 75 ml



     of hexane.  The pre-eluate is discarded, and just prior to exposure of the



     sulfate  layer  to  air, the sample extract is quantitatively transferred into



     the column by  decantation and subsequent hexane washings.  The elution rate



     is adjusted to about  5 ml per minute with  two eluates collected separately



     in 500 ml K-D  apparatus equipped with  10 ml ampuls.  The first elution is




     performed with 200 ml of  6% ethyl ether in hexane, and the second elution with



     200 ml of 15%  ethyl  ether in hexane.   The  K-D apparatus containing the



     eluates  are connected to  three ball Snyder columns and the solvents are



     evaporated  as  described in Part  I, Section 2.3.  The concentrated extract



     may be analyzed directly  by injecting  suitable aliquots from  the K-D  ampuls



     into  the gas  chromatograph.   If  the residues  are high  in total organics, they



     may be  further cleaned  up and  separated by thin-layer  chromatography  prior



     to gas chromatographic  analyses.



           5.2.2.1   Eluate Composition -  If the  Florisil has been properly  acti-



      vated and  stored, and if  the  reagents  are  carefully  prepared, the  following



      eluate compositions  will  be obtained  when  the pesticides  are  present. The



      first eluate  (6% ethyl  ether  in  hexane) will  contain:

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                                                                          31
lindane               DDE                              methoxychlor




BHC                   ODD                              toxaphene



kelthane              DDT                              Strobane




aldrin                Perthane                         chlordane  (y  6 tech)



heptachlor            heptachlor epoxide               endosulfan I



The second eluate (15% ethyl ether in hexane) will contain:



             dieldrin                 endosulfan II




             endrin                   lindane (possible trace of total)



                                      kelthane (possible trace of total)








Pol/chlorinated biphenyls are recovered in the first eluate and phthalate



esters  in the second eluate.



     5.2.3  Thin-Layer Chromatography - The sample extract (5,1.4) or the



eluates  from the Florisil clean-up (5.2.2) may be subjected to thin-layer



chromatography according to the procedure described below.  (Refer to Part I,




Section  4.3 for general discussion and conditions for preparation of thin-



layers).  Silica gel G layers, 250 u thick, are employed.



     5.2.3.1  Spotting the Extract - The extract volume should be adjusted



so  that  no more than 100 ul must be spotted in order to retain adequate GC




sensitivity in the TLC eluates when they are  reduced to the minimum volume of



0.2 ml.   Up to 100 yl of the  eluate is then spotted on the thin-layer.



     5.2.3.2  Spotting of Standards - A mixture of reference standard



pesticides is spotted at the  center and at one edge of the layer, in 10-20 ug



amounts of each pesticide,  to confirm the separation of the individual



pesticides by visual observation.   It is convenient to use a  100 ng/yl mixture

-------
32
      of encrin, lindane, ODD and DDT.  Standards for recovery and instrumental



      measurement are spotted in the 20 to 100 nanogram range and handled just




      as a sample would be treated in the subsequent steps of the procedure.




           5.2.3.3  Developing the Layer - The layer is developed with carbon



      tetrachloride.  When the solvent front reaches the upper reference mark




       (10 cm), the  layer is  removed from the chamber and allowed to air dry at




       room temperature.  The r"3veloping solvent should be checked frequently  for




       contamination and changed  as necessary.



           5.2.3.4  Visualizing  and Sectioning the Layer - The portions of  the



       layer  containing the samples are covered with a glass plate or cardboard.



       The portion of  the layer containing the standards is sprayed evenly with a



       fairly heavy  coat of Rhodamine  B  (0.1 mg/ml in ethanol).  The sprayed area



       is allowed to thoroughly dry  (about 5 minutes) and then is exposed to and



       viewed under  short wave UV light.  The pesticides show up as quenched areas



       (dark) on  a fluorescent background.  Mark the location of each pesticide.



       From  this  information, the vertical zone  for each sample is divided into five




       horizontal sections.   The  sections are identified with Roman numerals as



       shown  in Figure 2.   Examples of respective  Rf and Rr values for  various



       pesticides are  listed  in Table  1.



            5.2.3.5   Removal  of Pesticide  From  the TLC  Layer  - Using  the  spotting



       template as  a ruler,  and with  the  aid  of a  sharp pointed object, the  silica



       gel  sections  of interest  are individually ruled  off.   With  the aid of a mild



       vacuum, the  silica gel,  first from the periphery  of the section  and then



       from the center of the section, is  drawn into  a  medicine  dropper which  is



       plugged at the tip with  filtering grade  glass wool  (Figure  1).   The pes-



       ticides adsorbed on this  silica gel are  eluted quantitatively into a  10 ml

-------
                                                                          33



K-D ampul with successive small washes of ethyl ether-petroleum ether (1+1)



to a total volume of 5 to 10 ml.  The ampul is glass stoppered and the con-




tents are retained for gas chromatographic analysis.  Prior to gas chromato-



graphic analysis, the extracts are concentrated as required.  Refer to 5.1.4.




     5.3  Gas Liquid Chromatography




     5.3.1  Reasonably positive identification of a pesticide is obtained



by corroborating the results using, at least two different types of gas




chromatographic columns.  To achieve this, a relatively less polar packing



[5% OV-17 on Gas Chrom Q  (80-100 mesh)] and a more polar packing  [5% QF-1




(FS-1265) plus 3% DC-200 on Gas Chrom Q (80-100 mesh)] are employed.  Other



packings reconmended  for  this purpose are  3% OV-101 on Gas Chrom Q  (80-



100 mesh) and 3% OV-210 on Gas  Chrom Q  (80-100 mesh).  Packings and packed



columns  can be obtained from commercial sources or may be prepared in the



laboratory.



     5.3.2  Preparation of Columns - To prepare the column packings, dis-



solve 5  g of OV-17 in 225 ml of methylene  chloride  - chloroform (1+1) in



a 500 ml beaker  and add 95 g of Gas Chrom  Q.  Similarly, dissolve 5 g of



QF-1 plus 3 g of DC-200 in methylene  chloride - chloroform  (1+1)  and add



92 g of  Gas Chrom Q.  Dissolve  3  g of OV-101  in chloroform  and add  to 97 g



of Gas Chrom Q.  Dissolve 3 g  of  OV-210 in acetone  and add  to 97  g  of Gas



Chrom Q.  Proceed as  described in Part  I,  Section 3.1.3.1.



     5.3.2.1  Columns of  borosilicate glass,  6  ft.  long x  1/4 in. O.D.



 (5/32 in. I.D.)  or  1/8  in. O.D.  (1/16 in.  I.D.) are packed  and conditioned



 according to  directions in  Part I, Sections  3.1.3.3 through 3.1.3.5.  The



 column o.ven operating temperature is  approximately  210 C  for the  OV-17



 column,  185  C for the QF-l/DC-200 column,  190 C for the OV-101  column,  and



 185  C  for the OV-210  column.   The operating conditions are optimized for

-------
34
     the individual instrument as described in Part I. Section 3.1.3.5.  Opera-
     ting conditions are considered acceptable for an electron capture system
     when the response to 0.3 ng of aldrin is at least 50% of full scale while
     operating within the linear range of the detector,
          5.3.3  Sample Measurement - The volume of sample extract (5.14), the
     Florisil eluates, or the TLC eluates is noted and suitable aliquots
      (5-10  jil) are  analyzed by gas chromatography, employing at least  two columns
     of varying  polarity  for  identification  and quantitation.  Standards are
      injected frequently, as  a check  on  the  stability of the operating conditions.
      Gas chromatograms  of several standard pesticides are shown in Figures  3,  4,
      5, and 6.   The elution order, as well  as  elution ratios  for  various pesticides
      in Table 2, are provided only as a  guide.   It  is the responsibility of the
      analyst to  develop his own  identification keys  to  fit the  chosen  operating
      conditions  of the  instrument.
           5.4  Confirmatory Evidence - The  qualitative  identification  of a
      pesticide should be confirmed using infrared spectroscopy or mass spec-
      troscopy whenever the  instrumentation  is available and/or the quantity of
      the compound permits.   If this  is not  possible, gas chromatographic analysis
      using additional unlike columns and other selective detectors is  recommended.
      Lack of response to the flame photometric detector is negative evidence
      which supports the identification of organochlorine compounds.   Determination
      of the p-values of an unknown pesticide in several solvent systems will
      assist in confirming the identification (25).
           5.5  Calculation of Results - The pesticide concentrations are deter-
      mined using the absolute or the relative calibration procedure described in
      Part I, Section 3.4.2.

-------
                                                                           35
    5.6  Reporting Results - Report results  in micrograms per  liter with-
out correction for recovery data.   The percent recoveries of known pesti-
cides added to samples or to distilled water as well  as  the  step  in the
procedure where they were added must also be reported.   The  recoveries of
several organochlorine pesticides  from natural waters during collaborative
testing are listed in Table 3.  The precision of the  method  within the
designated range varies with the concentration as shown  in Table  4.
    5.6.1  If a sample is reported negative for a given  pesticide,  the
minimum detectable limit for that compound should also be reported.   If
favorable conditions prevail and ultimate sensitivity is required by the
purpose for the analysis, sample response of  less than  two  times  the de-
tector noise  level (N) should be reported as negative.   For sample  response
at two times  the detector noise level, list the result  as presumptive.
Responses of  greater than 2N should be quantified if possible.  In  cases
of questionable identification, the analyst should qualify the reported
resUiC to insure the subsequent misinterpretation will not occur.

6.   Method  for Analysis  Using Microcoulometric or Electrolytic Conductivity
     Gas  Chromatography
     6.1  Extraction of Sample
     6.1.1   The size of sample taken for  extraction is dependent on the
type of  sample and the sensitivity required  for  the  purpose at hand.  Back-
ground information on  the pesticide levels previously detected at a given
sampling site will help  to  determine  the sample  size required as well as the
final volume  to which  the extract  needs  to be concentrated.   A 3-liter  sample is
usually  taken for  microcoulometric analysis.   Since  the conductivity

-------
36
     detector is 2 to 3 times more sensitive than the microcoulometric




     detector, less than 3 liters may be required when using the conductivity



     detector.  If such isthe case, the volume of extracting solvents and other



     reagents are correspondingly decreased.




         6.1.2  A measured volume (3 liters) of sample is drained into a 4-liter




     separatory funnel equipped with a Teflon stopcock, and extracted with 150 ml



     of 15% ethyl ether in hexane by shaking vigorously for two minutes.  The



     sample container is rinsed with each aliquot of extracting solvent prior to



     extraction of the sample.



         6.1.3  The mixed solvent is allowed to separate from the water and this



     water  is drawn off into  the original container or into a second  4-liter



     separatory funnel.  The  organic layer  is passed through a  small  column of




     anhydrous  sodium sulfate topped with a pledget of cotton (previously rinsed



     with hexane)  and collected  in  a 600 ml tall  form beaker.   The  extraction  is



     repeated and  the  solvent treated  as above.   Approximately  100  ml of sodium




     sulfate saturated  water is  then  added  to  the sample and a  third extraction



      is completed  with 150 ml of hexane (not  hexane-ethyl  ether).   This solvent



      too,  after separation, is passed through the column of sodium  sulfate.   The



      column is then rinsed with several small portions of hexane and this solvent



      is recovered in the collection beaker  containing the combined  extracts.   The



      contents of the beaker are partially  evaporated to  about  300 ml in a water



      bath at 70 C applying no air or vacuum and quantitatively transferred to a



      500 ml K-D evaporator equipped with a 10 ml receiver ampul. The extract is



      concentrated in the K-D evaporator as described in Part I, Section 2.3.1.




          6.1.4  Concentration of Extract - The final concentration volume of



      sample extract is adjusted as necessary according to 5.1.4.  The use of

-------
                                                                         37




a "keeper" is recommended when concentrating below 0.3 ml.   Two milli-




grams of "keeper" is placed in the concentrated extract through syringe




addition of 100 pi of 20 yg/pl of Nujol in hexane.  This "keeper" will not



interfere with mocrocoulometric detection and will prevent  major residue



losses in this exhaustive evaporation step.  Because of interference




possibilities, it is not advisable to use a "keeper" in extracts to be



analyzed by electron capture.



     6.2  Clean-up and Separation Procedures



     6.2.1  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) will indicate



whether clean-up is required.  When these interfere with measurement of the



pesticides, proceed as directed below.  Whether required for quantitative analysis



or not, all extracts should be subjected to these procedures, subsequent to



the  initial analysis and rechromatographed  for qualitative corroboration of




the  results.  Another clean-up technique, acetonitrile partition, although



not  ordinarily required  for surface water extracts, is sometimes useful for



cleaning  up high organic wastewater samples.  Refer to the FDA methods manual



 (1)  for this  procedure.



      6.2.2   Florisil Column Adsorption Chromatography  -  Refer  to 5.2.2.



      6.2.3  Thin-Layer  Chromatography -  Refer to  S.2.3.



      6.3  Gas Liquid Chromatography



      6.3.1   Reasonably  positive  identification of a pesticide  is obtained



 by  corroborating the results  using,  at least  two  different  types of gas



 chromatographic  columns.   Refer  to 5.3.1.

-------
38
              6.3.2   Preparation  of Columns  -  Refer to  5.3.2.  Columns of boro-



         silicate  glass,  6  ft.  long x  1/4  in.  O.D. are  employed.  The operating




         conditions  are optimized for  the  individual  instrument  as described  in




         Part  I, Section 3.1.3.5.   Conditions  are considered optimum when the



         response  to 15 ng  and  30 ng of aldrin is at  least  50% of full scale  for



         the electrolytic conductivity and microcoulometric detectors, respec-




         tively, while operating  within the linear range.



              6.3.3  Sample Measurement -  The  volume  of sample  extract  (6,1.4),



         the Florisil eluates,  or the  TLC  eluates is  noted  and  suitable  aliquots



         (20-100 yl) are analyzed by gas chromatography, employing  at  least two



         columns of varying polarity for identification and quantitation.   Stan-



         dards are injected frequently, as a check on the stability of the operating



         conditions.  Gas chromatograms of several standard pesticides are shown



         in Figures 3, 4, 5, and 6.  The elution order as well as the elution




         ratios for various pesticides are provided in Table 2, only as a guide.



         It is the responsibility  of the  analyst to develop his own identification




         keys to  fit  the chosen  operating conditions of the instrument.




              6.4  Confirmatory  Evidence  - Refer to 5.4



              6.5  Calculation of Results  - The  pesticide  concentrations are



         determined  using  the  absolute or the relative calibration procedure




         described  in Part  I,  Section  3.4.2.



              6.6  Reporting Results  - Refer  to  5.6.

-------
                           TABLE 1
     SOME Rf AND Rr VALUES OF PESTICIDES DEVELOPED WITH CC1
                                                           4

                    ON SILICA GEL G THIN LAYER
Pesticide
Dieldrin
Endrin
Heptachlor Epoxide
Lindane
DDD
Y-Chlordane
Heptachlor
DDT
DDE
Aldrin
Rf Value
0.17
0.20
0.29
0.37
0.54
0.55
0.67
0.68
0.72
0.73
Rr Value
0.33
0.37
0.52
0.69
1.00
1.02
1.24
1.26
1.33
1.35
Section

II

III


IV


Rf  *  distance traveled by the compound divided by  the  distance


       traveled by the solvent front.


Rr  •  distance traveled by the compound divided by  the  distance


       traveled by standard p,p'-DDD.

-------
                                        TABLE 2

          RETENTION TIMES OF ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Liquid Phase1
Column Temp.
Pesticide
«-BHC
Lindane
Heptachlor
Aldrin
Kelthane
Heptachlor Epoxide
Y-Chlordane
Endosulfan I
p,p'-DDE
Dieldrin
Endrin
o,p'-DDT
Endosulfan 11
p,p'-DDD
p,p'-DDT
Methoxychlor
Aldrin (Minutes
Absolute)
3% DC-200
+
5% QF-1
200 C
RRt3
0.40
0.51
0.80
1.00
1.19
1.38
1.53
1.77
1.93
2.10
2.43
2.62
2.62
2.68
3.41
5.26
3.76
$%
OV-17
200 C
RRt3
0.45
0.6'
0.79
1.00
1.52
1.58
1.82
2.00
2.67
2.54
3.21
3.97
3.97
4.13
5.19
11.17
3.84
3%
OV-101
17S C
RRt3
0.33
0.42
0.76
1.00
1.12
1.30
1.55
1.70
2.18
2.08
2.33
3.02
2.45
2.'94
3.97
6.88
.•» f •
£.V-»
1%
OV-210
160 C
RRt3
0.54
0.75
0.82
1.00
2.46
2.16
2.12
2.89
2.91
3.65
4.46
4.04
5.96
5.61
6.28
13.52
2.28
Relative
Sensitivity
to EC Detector


1.0
1.0
1.0
1.0
0.1
0.5
0,5
0.4
0.5
0.5
0.3
0.1
0.3
0.1
0.2
0.1

All columns glass, 6 ft. long x 4 mm ID, solid support Gas-Chrom Q (80/100 mesh),
nitrogen carrier flow 80 ml/rain.

Sensitivity factors relative to aldrin.

Retention times relative to aldrin.

-------
                          TABLE 3




   Recovery of Organochlorine Pesticides  from Natural Waters
Pesticide
Aldrin

Lindane

Dieldrin

DDT

Added Level
ng/liter
IS
110
10
100
20
125
40
200
Recovery
Without
Cleanup, %
69
72
97
73
108
85
101
77
Added Level
ng/liter
25
100
15
85
25
130
30
185
a
Recovery
With
Cleanup, %
68
65
94
70
70
65
118
71
isil  column  clean-up used.

-------
                                 TABLE 4




   Precision of Method for Organochlorine Pesticides in Natural Waters
Pesticide

Aldrin



Lindane



Dieldrin


DDT


Pretreatment

No
Cleanup
Cleanup

No
Cleanup
Cleanup

No
Cleanup
Cleanup

No
Cleanup
Cleanup

Mean Recovery
ng/ liter
10.42
79.00
17.00
64.54
9.67
72.91
14.04
59.08
21.54
105.83
17.52
84.29
40.30
154.87
35.54
132.08
Precision,
ST
4.86
32.01
9.13
27.16
5.28
26.23
8.73
27.49
18.16
30.41
10.44
34.45
15.96
38.80
22.62
49.83
a
ng/liter
so
2.59
20.19
3.48C
8.02°
3.47
11.49
S.20
7.75
17.92
21.84
5.10°
16.79°
13.42
24.02
22.50
25.31
ZS_,  =  Overall precision, and



 S.  =  Single-Operator precision.
 Use of Florisil column cleanup prior to analysis
 S0  <  ST/2

-------
                   FIGURE  I


        SILICA GEL COLLECTION ASSEMBLY
        AIR FLOW                /—EYE DROPPER
       «-
VACUUM



             HOSE        ^GLASS WOOL






    SILICA GEL COLLECTION  ASSEMBLY

-------
                       FIGURE 2

     DIAGRAM OF  DESIGNATION OF TLC SECTIONS

           IN  THE CLEANUP AND  SEPARATION

               ON SILICA  GEL PLATES
10.0 cm
 8.0cm
      SECTION
        I
 6.0cm
 3.8cm
       1.0 cm

SPOTTING 0-,
    LINE "
           ZONE FOR;
           SPOT 1 SPOT 2 SPOT 3
      SECTION

        XE
       SECTION
        HI
       SECTION
        n
       SECTION
         I
              I
                            •SOLVENT  LINE
 .7.8 ALDRIN
 •7.5 DDE
 • 7.3 DDT

 • 6.5 HEPTACHLOR
--5.5 X'CHLOROANE
--5.2 ODD

--4.2 LINDANE
                               - 3.4 HEPTACHLOR  EPOXIDE
                               2.4ENDRIN , DIELDRIN
                     I
                         I
                                        I
                                               J_
       SPOT  1234

              1.0 cm
           8  9   10   II  12

-------
            LINDANE
                         FIGURE 3
           ELECTRON CAPTURE  GAS CHROMATOGRAM
                 OF PESTICIDE STANDARDS
                          HEPTACHLOR
                           EPOXIDE
                                 p.p'-DDE
"V
                                                         p.p'-ODT
           I
I
4
I
I
I
12
                                      I
                   6        8        10
                RETENTION TIME IN MINUTES
           (Chart  speed one-half inch per minute)
Column Packing- 3%DC-200+5%QF-1  on  Gas Chrom Q (80/100 Mesh)
Carrier Gas-  Nitrogen at 80 ml/min.
Column Temperature-  200°C
                                                                 14

-------
      LINDANE
             FIGURE 4
ELECTRON CAPTURE GAS CHROMATOGRAM
      OF PESTICIDE  STANDARDS
                HEPTACHLOR
                 EPOXIDE
I
0        2        4        6        8        10
                    RETENTION TIME IN MINUTES
               (Chart speed one-half inch per minute)
 Column Picking- 3% OV-101 on Gas Chrom Q (80/100 Mesh]
 Carrier Gas- Nitrogen at 80m!/min.
 Column Temperature - 175°C
                              12

-------
LINDANE
                FIGURE 5
ELECTRON CAPTURE GAS CHROMATOGRAM
        OF PESTICIDE  STANDARDS
HEPTACHCLOR
           HEPTACHLOR
             EPOXIDE
                                                 p.p'-DDT
02      46       8     10      12      14
                       RETENTION TIME IN  MINUTES
                (Chart speed one-half  inch inch  per  minute]
Column Packing- 5%OV-17 on Gas Chrom  Q (60/80 Mesh)
Carrier Gas - Nitrogen at  80ml/min.
Column Temperature- 2QO°C
                                                 16
                                      18

-------
        INDANE + HEPTACHLOR
                                               FIGURE  6
                                ELECTRON CAPTURE GAS CHROMATOGRAM
                                       OF PESTICIDE STANDARDS
                      HEPTACHLOR
                        EPOXIDE
                               p.p'-DDE
                                                               p,p'-DDT
I
0
T
 2
T
                      I         I         I
                      6         8        10
                  RETENTION TIME IN MINUTES
             (Chart speed one-half inch  per minute]
Column Packing- 3% OV-210 on Gas  Chrom Q (80/100 Mesh)
Carrier Gas- Nitrogen at 80 ml/min.
Column Temperature- 160°C
T
12
 I
14

-------
REFERENCES CITED:

(1)  "Pesticide Analytical Manual", U.S. Department of Health, Education,
     and Welfare, Food and Drug Administration, Washington, D.C.,
     Volumes I and II, 1968.

(2)  "Guide to the Chemicals Used in Crop Protection", Canada Department
     of Agriculture, Catalog Number A-43-1093, Queen's Printer, Ottawa
     Canada, 5th Edition, 1968.

(3)  "Official Methods of Analysis of the Association of Official
     Agricultural Chemists", Association of Official Agricultural
     Chemists, Washington, D.C., 20044, 10th Edition, 1965.

(4)  "Tentative Recommended Practice for General Gas Chromatography
     Procedures", E-260-65T, Part 30, ASTM Standards - General Test
     Methods, p. 806, May, 1968.

(S)  "Control of Chemical Analyses in Water Pollution Laboratories",
     Environmental Protection Agency, Analytical Quality Control Laboratory,
     1014  Broadway, Cincinnati, Ohio  45202.   (In Press)

(6)  "Standard Methods of Sampling Industrial  Water", D-S10-68, Part 23,
     ASTM  Standards - Water; Atmospheric Analysis, p. 3, November, 1970.

(7)  Gannon, H. and Bigger, J.H., Journal of Economic Entomology, 51,
     No. 1,  1  (1958).

(8)  Hendrick, R.D. et al, Journal of Economic Entomology, 59, No. 6
     1388  (1966).

(9)  Eichelberger, J.W.  and Lichtenberg, J.J., "Persistence of Pesticides
     in River Water", U.S. Department of the Interior, FWQA,  DWQR, AQCL,
     Cincinnati, Ohio, April 1970  (Accepted for publication by Environmental
     Science and Technology).

(10)  Lovelock, J.E. and  Lipsky, S.R., Journal  of  the American Chemical
     Society.  82_, 431  (1960).

(11)  Lovelock, J.E.,  Analytical Chemistry,  33_, 162  (1961)

(12)  Challacombe, J.A. and McNulty, J.A.,  "Applications  of the Micro-
     coulometric Titrating System  as a  Detector in Gas Chromatography of
     Pesticide  Residues", Residue  Reviews,  5_,  57  (1964).

(13)  Coulson,  D.M., Journal  of Gas  Chromatography, 4_,  285  (1966)

(14)   Brody,  S.  and  Chancy, J.,  Journal  of Gas  Chromatography, £,  42  (1966)

(15)   Purnell,  H.,  "Gas Chromatography", Wiley, New York, 1962, p.  240.

-------
(16)   Homing,  E.G.,  Moscatelli,  E.A., and Sweeley,  C.C.',"QleW.ulIrtd,.,
      751, London (1959) .

(17)   Smith, E.D., Analytical Chemistry, 32, 1049 (1960).

(18)   Burke, J.A., Journal of the Association of Official  Analytical
      Chemists. 413, 1037 (1965) ,

(19)   Kruppa, R.F., Henley, R.S., and Smead, D.L., Analytical Chemistry,
      39, 851 (1967).

(20)   McNair, H.M. and Bonelli, E.J., "Basic Gas Chromatography",
      Varian-Aerograph, Walnut Creek, California 94598, 1965.

(21)   Schafer, M.L., Busch, K.A., and Campbell, J.E., Journal of Dairy
      Science. XLVI, No. 10, 1025 (1963).

(22)   Reynolds,  L.M., Bulletin of Environmental Contamination S
      Toxicology, 4_, No. 3, 128  (1969) .

(23)  Armour, J.A., and Burke, J.A.,  Journal of the Association of
      Official Analytical  Chemists.  5_3_,  761  (1970).

(24)  Bagley, G.E., Reichel, W.L., Cromartie, E.,
             Journal of the Association of Official Analytical Chemists,
      S3,  251  (1970).

(25)  Beroza. M.  and Bowman, M.C., Analytical Chemistry,  37, 291  (1965).

ADDITIONAL RECOMMENDED REFERENCES:

(26)  Littlewood,  A.B.,  "Gas Chromatography - Principles, Techniques  and
      Applications", Academic  Press,  New York,  1962.

(27)  Noebels, H.J., Wall, R.F.,  and Brenner, N., "Gas  Chromatography",
      Second and Third  International Symposium  Held Under the Auspices
      of the Analysis  Instrumentation Division  of the  Instrument  Society
      of America,  June  1959  and  June 1961.

 (28)  Heftmann,  E.  ed.  "Chromatography", Reinhold Publishing Corporation,
      New York  1961.

 (29)  Stahl, E.  ed.,  'Thin-Layer Chromatography,  A  Laboratory Handbook",
       (Springer-Verlag,  Berlin)  Academic Press,  Inc.,  Publishers, New
      York, 2nd  edition,  1969.

 (30)  Randerath, K.,  "Thin-Layer Chromatography" (Verlag  Chemio,  CmbH,
      Weinheim/Bergstr.)  Academic Press, New York,  1964.

 (31)  Truter,  E.V.,  "Thin Film Chromatography", Interscience Publishers,
      Division of John Wiley and Sons, Inc., New York,  1963.

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                                                                              f?'
(32)   Smith.,  D.  and Eichelherger,  J.W.,  Journal  of Hater Pollution
      Control Federation.  37_,  77 (1965).

(33)   Mills,  P.A.,  Journal of the  Association of Official Analytical
      Chemists,  42, 734 (1959).

(34)   Johnson, L.,  ibid.,  45,  363  (1962).

(35)   Goerlitz,  D.F., "Methods for the Analysis  of Organic Substances
      in Water", Techniques of Water-Resources Investigations cf the
      U.S. Geological Survey,  Book 5, Chapter A2, (in review), 1970.

(36)   Boyle, H.W., Burttschell, R.H., and Rosen, A.A., "Infrared
      Identification of Chlorinated Insecticides in Tissues of
      Poisoned Fish", published in Organic Pesticides in the
      Environment, Advances in Chemistry Series-60, American
      Chemical Society, Washington, D.C., 1966.

(37)  Walker, K.C. and Beroza, M., Journal of the Association of
      Official Analytical Chemists, 46, 250  (1963).

(38)  Breidenbach, A.W.,  et al, "The  Identification and Measurement
      of Chlorinated Hydrocarbon  Pesticides  in Surface Waters",
      Publication  WP-22,  U.S. Department of  the  Interior, Federal
      Water  Pollution Control Administration, Washington, D.C.
      20242,  1966.

(39)  Teasley, J.I.  and Cox, W.S., "Determination  of Pesticides in
      Water  by Microcoulometric Gas Chromatography after Liquid-
      Liquid Extraction", Journal American Water Works  Association
      55_,  1093  (1963).

(40)  Lamar,  W.L., Goerlitz,  D.F., and  Law,  L.M.,  "Identification
      and  Measurement of  Chlorinated  Organic Pesticides in Water
      by Electron  Capture Gas Chromatography",  U.S. Geological
      Survey Water Supply Paper 1817-B, 1965.

(41)  "Guide to  The  Analysis  of Pesticide Residues",  U.S.  Department
      of Health, Education, and Welfare, Public Health  Service,
      Bureau of  State Services, Office  of Pesticides, Volumes  I and
      II,  1965.

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                               APPENDIX



7.   SPECIAL EQUIPMENT.  REAGENTS,  AND SOLVENTS

7.1  Equipment

7.1.1  Gas Chromatograph - Suitable gas chromatographs are available from

       many manufacturers.

7.1.2  Detectors

7.1.2.1  Electron Capture - Radioactive Source (tritium or nickel-63)

         a.  Concentric tube design

             Varian-Aerograph
             2700 Mitchell Drive
             Walnut Creek, California  94598

         b.  Unique concentral design

             Tracer, Inc.
             6500 Tracer  Lane
             Austin, Texas  78721

         c.  Parallel plate design

             Perkin-Elmer Corporation
             Norwalk, Connecticut  06852

             Also supplied by many other manufacturers.

7.1.2.2  Microcoulometric  (T-300-S)

         Dohrmann Instruments Company
         1062 Linda Vista Avenue
         Mountain View, California  94040

7.1.2.3  Electrolytic Conductivity

         Tracor, Inc.
         6500 Tracor Lane
         Austin, Texas  78721

7.1.2.4  Flame  Photometric

         Also from Tracor,  Inc.

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7.1.3  Recorder - 1 millivolt,  1 second full  scale potentioraetric  strip

       chart.   This type of recorder is supplied by many instrument

       manufacturers.

7.1.4  Kuderna-Danish Glassware

       Snyder Column - three ball (macro) and one ball (micro)

       Evaporative Flasks - 125 ml, 250 ml, and 500 ml

       Receiver Ampuls - 10 ml

       Ampul Caps

       Kontes Glass Company
       Vineland, New Jersey  08360

       Dohrmann  Instruments
       1062 Linda  Vista Avenue
       Mountain  View, California  94040

 7.1.5  Column Chroroatography -  Pyrex column  (I.D.  19 mm,  length 400 mm)

       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 Glass Company
       Vineland, New Jersey  08360

 7.1.6 Micro  Syringes -  (1,  5,  10,  25, 50, and  100 ul)

       Hamilton Company
       Post Office Box  307
       Whittier, California  90608

 7.1.7 Separatory Funnels -  two liter and four  liter funnels with Teflon

        stopcock,

        Pyrex  or Kimble  supplied through msny distributors.

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7.1.8  Thin-Layer Chromatography - Applicator,  aligning tray,  spotting

       template, developing chamber, and UV light source.

       Applied Science laboratories. Incorporated
       Post Office Box 440
       State College, Pennsylvania  16501

       Brinkmann Instruments, Incorporated
       Cantiague Road
       Westbury, New York  11590

       also - from many other suppliers

7.2  Standards, Reagents and Solven'r

7.2.1  Pesticide standards - highest available purity

       City Chemical Company
       132 West 22nd Street
       New York, New York  10011

       Applied  Science Laboratories, Incorporated
       Post Office Box 440
       State College, Pennsylvania   16501

       Environmental Protection Agency
       Perrine  Primate Research Branch
       P.O. Box 490
       Perrine, Florida   33157

       also -  from the manufacturer

 7.2.2  Florisil (60/100  mesh)  - purchased  activated  at  1200 F and stored

       at  130  C.

       Floridin Company
       2 Gateway  Center
       Pittsburgh, Pennsylvania   15222

 7.2.3  Sodium  sulfate  (A.C.S.)  -  granular, anhydrous

 7.2.4  Pyrex wool -  filtering  grade

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7.2.S  Solvents - hexane,  diethyl ether,  acetone,  benzene,  xylene,

       carbon tetrachloride,  acetonitrile, methylene chloride -

       high purity,  distilled in glass for pesticide analyses--

       either Nanograde type or purified in lab.

       Burdick and Jackson, Incorporated
       1953 South Harvey Street
       Muskegon, Michigan  49442

       Mallinckrodt Chemical Works
       2nd and Mallinckrodt Streets
       St. Louis, Missouri  63160

       Matheson Coleman and Bell
       Post Office Box 85
       East Rutherford, New Jersey  07073

7.2.6  Gas Chromatographic Column Materials

       Gas-Chrom Q  (80-100 mesh)

       Glass Wool  (silanized with dimethyldichlorosilane)

       OV-17

       OV-101

       OV-210

       UC-200  (12,500  centistokes)

       QF-1  (FS-1265)

       Tubing  (Pyrex 1/8  in.  and/or 1/4  in.  O.D.)

       Applied Science Laboratories,  Incorporated
       Post  Office  Box 440
       State Collage,  Pennsylvania  16501

       Ohio  Valley  Specialty Chemical, Inc.
       Marietta,  Ohio  45750

 7.2.7 Silica gel-G with  gypsum binder (No.  8076)

       Warner-Chilcott Laboratories
        Instruments Division
       200 South Garrard  Boulevard
       Richmond, California  94801

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    7.3  Sample  Collection Bottles and Shipping Containers

    7.3.1  One-Quart  Jars  - Standard 32 02. 63-400 flint (C-S020),

           FTK cap  P/0 C63-400)

           Cincinnati Container Co.
           2833  Spring Grove Avenue
           Cincinnati, Ohio  45225

    7.3.2  Teflon Insert for Bottle Cap - 2-7/16 in. diameter, 0.020 in.

           thick

           Cadillac Plastics
           3818  Red Bank Road
           Cincinnati, Ohio  45227

    7.3.3  Shipping Containers - Expanded polystyrene packer  for  one-quart

           jars

           Preferred Plastics Corp.
           Route 12
           North Grosvenordale, Connecticut

           Polystyrene packers are also available  for half-gallon and

           one-gallon bottles.
                       Mention of products  and manufacturers

                  is for identification  only and does not imply

                  endorsement by the  Water  Quality Office,

                  Environmental Protection  Agency.
6 «. V WnilJtttl NWIN OffKtt 197J-759-301/2113 K
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