600-4-84-038
      CHARACTERIZATION OF HAZARDOUS WASTE SITES
                 A METHODS MANUAL

VOLUME III.   AVAILABLE LABORATORY ANALYTICAL METHODS


                        by
               Russell "H. Plumb",  Jr.
  Lockheed Engineering Mamtgement  Services Company
             Las Vegas, Nevada 39114
            Contract dumber 68-03-3050
                  Prsjtct Officer

                 Werner F.  Beckert
            Quality Assurance Division
    Environmental Monitoring Systems Laboratory
            Las Vegas,  Nevada  89114
    ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
        OFFICE OF RESEARCH *MD DEVELOPMENT
       U.S. ENVIRONMENTAL  PROTECTION AGENCY
             LAS YE5AS.  NEVADA  "35114

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                                    NOTICE
     Although the research described in this  manual  has  been funded
in part by the U.S. Environmental  Protection  Agency through  Contract
69-03-3050 to the Lockheed Engineering and Management Services  Comoa
Las Vegas, Nevada, it has not been subjected  to Agency policy review
therefore does not necessarily reflect the views of the  Agency.   Men-
of trade names or commercial  products does not constitute endorsemem
recommendation for ?e.
                                       ii

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                                    FOREWORD


     This document is part of a multlvolume manual, entitled Characterization
of Hazardous Waste Sites--A Methods Manual, that is being prepared by the U.S.
Environmental Protection Agency.It is intended to serve a wide variety of
users as a source of Information on available methods for collection and
analysis of samples from hazardous waste disposal sites.

     Volume I.  Integrated Approach to Hazardous Waste Site Characterization
1 ncltides discussions on preliminary assessment, Initial data evaluation, ad-
minlstratlve procedures, offsite reconnaissance, site Inspection, chain of
custody, quality assurance, safety, and personnel training.  In addition,
considerations to be included in the development of a sampling strategy and
the selection of analytical methods are presented.

     Volume II.  Available Sampling Methods 1s dedicated to sampling proce-
dures and sampling information.It is a description of methods and materials
available to field investigators ^or most sampling situations that arise
during routine hazardous waste disposal site and hazardous spill investiga-
tions.

     This volume, Volume III.  Available Laboratory Analytical  Methods,
presents detailed methodology suitable for hazardous waste sample analysis.
The distinctions between this analytical compendium and existing contemporary
manuals are the scope of analytes to be potentially covered and the diversity
of sample matrices to be addressed.  Thus, while existing manuals provide
detailed guidance for 50 to 100 specific analytes (and will be used as primary
sources for these procedures), Appendix VIII of the Resource Conservation and
Recovery Act lists more than 350 substances of concern.  The purpose of this
compendium 1s to serve as a repository of available analytical  techniques for
these substances.  Also, existing manuals generally address a single sample
matrix whereas this compendium provides sample preparation guidance for as
many as five sample matrices.  This 1s consistent with the common source intent
of the multivolume manual and the complex nature of hazardous wastes that
may require collection of samples of various sample matrices during a single
sampling event.

     This volume 1s to be a dynamic document that will be updated and expanded
to reflect state-of-the-art methodology achieved by EPA, the regulated commu-
nity, and other members of ttre scientific community.  Thus, as new compounds
are manufactured or classified as hazardous, the analytical part of the compen-
dium veil! have to be sxoanded-  As analytical  procedures are developed or
evaluated, the contents of the compendium wiil  oe modified.

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     The user is cautioned,  however,  that not all  included  procedures  have
been used with all  sample types.   If  problems are  encountered  with  certain
sample types, or if there are any comments,  corrections,  suggested  additions,
or questions concerning the  material  contained in,  or  omitted  from  this  com-
pendium, the user is asked to direct  comments to:

                   Hazardous Waste Metnods Evaluation  Branch
                   Quality Assurance  Division
                   Environmental  Protection  Agency
                   P.O. Box 15027
                   Las Vegas, Nevada   89114

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                                ACKNOWLEDGEMENT
     The development of this analytical manual 1s the result of Initial
direction provided by the EPA-w1de Steering Committee.  The contributions
of the committee members and additional reviewers Identified below are
appreciated.
      Laboratory Committee
                                    Individual
                                  M. Birch
                                  D. Friedman
                                  F. Haeberer
                                  E. Meier
                                  T. Melggs
                                  J. Poppitl
                                  F. Richardson
                                  D. Weltzman
                 Organization
                     Region IV
                     OSW
                     OERR
                     EMSL-LV
                     NEIC
                     OSW
                     OSW
                     DOHS
      Additional Reviewers
                                  W.
                                  S.
                                  w.
                                  M.
                                  A.
                                  D.
                                  D.
                                  J.
                                  W.
                                  G.
Beckert
Bromberg
Budde
Dellarco
Galliart
Garnas
Gurka
Huang
Reynolds
Schweitzer
EMSL-LV
EMSL-RTP
EMSL-CIN
OMSQA
MCCC
NEIC
EMSL-LV
OMSQA
LEMSCO
EMSL-LY
     Assistance provided by the following Individuals was essential  to the
completion of this manual and is gratefully acknowledged:  H.  Kerfoot and
J.  Sherma for drafting some of the analytical  sections;  H. Kerfoot  and
J. Engels for proofing each of the analytical  sections; M.  Birch for
reviewing each of the manual jdrafts; and S. Brown,  L. Steele,  and D. Nidy
for word processing support.

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                               TABLE OF CONTENTS
                                                                         Page
Foreword	    111
Acknowledgement 	      v

Chapter I.  Introduction	    1-1

     A.  Background	    1-1
     B.  Objectives	    1-1
     C.  Organization	    1-4

Chapter II.  Phase Separation and Screening Procedures for
             Hazardous Waste Samples	   II-l

     A.  Introduction	   II-l
     B.  Storage and Handling of Hazardous Waste Samples	   II-l
     C.  Phase Separation of Hazardous Waste Samples	   11-3
         C.I  Phase Separation	   II-3
         C.2  Aliquot Preparation	   11-5
     D.  Inorganic Screening Procedures	   11-9
         D.I  Spot Test for Oxidants	   II-9
         D.2  Spot Test for Cyanide	. ,  .   II-12
         D.3  Spot Test for Sulfide	   11-16
         D.4  Alternate Spot Test for Sulfide	   11-18
         D.5  Spot Test for pH	   11-21
     E.  Organic Screening Procedures	   11-22
         E.I  Extract Preparation	   11-22
         E.2  Extract Analysis	   11-23

Chapter III.  Procedures for Organic Compounds. ... 	  III-l

     Section 1.  Volatile Organic Compounds 	  III-2

       J.I.I  Analysis of Solid Hazardous Waste Samples for Volatile
                Organic Compounds by Methanol Partitioning	111-21
       J.I.2  Analysis of Solid Hazardous Waste Samples for Volatile
                Organic Compounds by Polyethylene
                Glycol Partitioning 	  II1-25
       J.2.1  Analysis of Water Samples for Volatile Organic
                Compounds	111-31
       0.3.1  Analysis of Soil/Sediment Samples 	  111-36
       J.4.1  Analysis of Volatile Organic Compounds in
                Biological Tissue . . ,.-	, . ,  ,  !!!-AQ
                                      vii

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                    TABLE  OF  CONTENTS  (Continued)
  0.4.2  Analysis  of Biological  Tissue  Samples  for  Purgeable
           Organic Compounds	111-44
  J.5.1  Analysis  of Air Samples for Volatile Organics  	   111-46

Section 2.   Acid-Extractable  Organic Compounds  	   111-56

  J.I.I  Analysis  of Hazardous Waste Samples for
           Acid-Extractable Organic  Compounds	111-68
  J.2.1  Analysis  of Water Samples for
           Acid-Extractable Organic  Compounds	111-72
  J.3.1  Analysis  of Sediment Samples for  Acid-Extractable
           Organic Compounds  by  Hexane-Methanol Extraction  .  .  .   111-77
  J.3.2  Analysis  of Sediment Samples for  Acid-Extractable
           Organic Compounds  by  Methylene  Chloride  Extraction.  .   111-80
  J.4.1  Analysis  of Biological  Tissue  Samples  for  Acid-
           Extractable Organic Compounds by Methylene
           Chloride Extraction  	   111-85

Section 3.   Base/Neutral-Extractable Compounds  	   111-93

  J.I.I  Analysis  of Hazardous Wastes for  Base/Neutral  Compounds   III-105
  J.2.1  Analysis  of Methylene Chloride Extracts  of Aqueous
           Samples for Base/Neutral  Compounds	III-112
  J.3.1  Analysis  of Sediment Samples for  Base/Neutral  Compounds   111-118
  J.3.2  Analysis  of Methylene-Chloride Sediment
           Extracts for Base/Neutr?!  Compounds  ....,....   !!I-!2
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                    TABLE OF CONTENTS (Continued)
  H.I.I  Determination of Organophosphorus  Pesticides
           1n Hazardous Wastes (Pesticide Formulations)	   III-231
  H.2.1  Determination of Organophosphorus  Pesticides
           1n Water	III-235
  H.3.1  Determination of Organophosphorus  Pesticides  1n Son.  .   III-243
  H.4.1  Determination of Organophosphorus  Pesticides
           1n Fruits and Vegetables	III-244
  H.5.1  Determination of Organophosphorus  Pesticides  1n A1r  .  .   III-247

Section 6.  Methods for the Determination of
              Organonltrogen Pesticides	III-255

  1.1.1  Determination of Carbamates and Urea  Pesticides
           In Hazardous Waste Samples.   Reserved  	   III-267
  1.2.1  Determination of Carbamate and Urea Pesticides
           1n Industrial and Municipal  Wastewater	III-268
  1.3.1  Determination of Carbamate Pesticides 1n Soil  	   III-276
  1.3.2  Determination of Urea Herbicides In Soil	III-281
  1.4.1  Determination of Carbamate Pesticides 1n
           Fruits and Vegetables	111-284
  1.5.1  Determination of Carbamate Pesticides In A1r	III-2S3

Section 7.  Methods for the Determination of Chlorinated
              Phenoxy Add Herbicides	III-303

  F.I.I  Analysis of Solid Waste Samples for Chlorinated
           Herbicides by High Performance Liquid  Chromatography.   III-310
  F.2.1  Analysis of Water Samples for  Chlorinated Phenoxy
           Add Herbicides by Chloroform Extraction	III-312
  F.2.2  Analysis of Water Samples for  Chlorinated Phenoxy
           Add Herbicides by Ethyl Ether Extraction	111-315
  F.3.1  Analysis of Sediment Samples for Chlorinated  Phenoxy
           Add Herbicides by Acetone-Hexane Extraction	III-318
  F.3.2  Analysis of Soil Samples for Chlorinated Phenoxy
           Add Herbicides by High Performance Liquid
           Chromatography	111-322

Section 8.  Methods for the Determination of D1ox1n (TCDD)  .  .  .   III-327

  J.I.I  Analysis of Hazardous Waste Samples for  D1ox1n.
           Reserved	III-339
  J.2.1  Analysis of Water Samples for  D1ox1n	III-340
  J.2.2  Analysis of Water Samples for  TCDD	111-348
  J.3.1  Analysis of Methylene Chloride Extracts  of
           Sediment/Soil Samples for 2,3,7,8-TCDD	III-351
  J.2.2  Detsrwlnation of 2,3,7,8-TCDD  1n Methanol
           Extracts of Soil and Sediment 	   111-25^
                                  1x

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                         TABLE OF CONTENTS (Continued)
       J.4.1  Analysis of Hexane Extracts  of Biological
                Tissue for TCDO	111-364

     Section 9.   Methods for the Determination  of  Polycyclic
                   Aromatic Hydrocarbons	III-369

       1.1.1  Analysis of Hazardous  Waste  for Polycyclic
                Aromatic Hydrocarbons	111-381
       1.2.1  Analysis for Polycyclic Aromatic  Hydrocarbons
                in Water Samples	II1-386
       1.3.1  Analysis of Sediments  for Polycyclic
                Aromatic Hydrocarbons	111-395
       1.4.1  Analysis of Fish and Shellfish Tissue  for PAH  	   III-402
       1.4.2  Analysis of Plant Tissue for PAH	III-407
       1.5.1  Analysis of Air for Polycyclic Aromatic  Hydrocarbons.  .   III-414

Chapter IV.  Procedures for Inorganic Substances	    IV-1

     Section 10.  Elemental Analysis by Atomic  Absorption
                    Spectrometry	    IV-2

       J.I.I  Determination of Metals in LMB/LiF Fusion Pellets
                of Waste Samples	    IV-13
       J.2.1  Analysis of Water Samples for Metals	    IV-15
       J.3.1  Analysis of Soil/Sediment Samples for  Heavy  Metals.  .  .    IV-33-
       J.4.1  Analysis of Tissue Camples ror Heavy Metals  	    17-42
       J.5.1  Analysis of Air Samples for  Heavy Metals.  .......    IV-44

     Section 11.  Methods for the Determination of Mercury	    IV-51

       G.I.I  Analysis of Hazardous  Waste  Samples  for  Mercury.
                Reserved	    IV-57
       G.2.1  Analysis of Water Samples for Mercury  	  ....    IV-58
       G.3.1  Analysis of Sediment Samples for  Mercury	  ....    IV-60
       G.4.1  Analysis of Fish Tissue Samples for  Mercury	    IV-62
       G.5.1  Analysis of Solid-Phase Collectors from  Air
                Sampling for Mercury	    IV-64
       G.5.2  Sampling and Analysis  of Air Samples Using
                Liquid-Phase Collectors for Mercury  	    IV-66

     Section 12.  Methods for the Determination of Methyl  Mercury  .  .    IV-70

       G.I.I  Analysis of Hazardous  Waste  Samples  for
                Methyl Mercury.  Reserved  	    IV-73
       G.2.1  Analysis of Water Samples for Methyl Mercury	    IV-74

     Jec-ion 13.  Methods ror the Detern-;nation of Ar-jeni::.  .  .  .  .  .    TY-"T~>

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                    TABLE OF CONTENTS (Continued)

                                                                   Page
  F.I.I  Analysis of Hazardous Waste Samples
           for Arsenic.   Reserved	   IV-80
  F.2.1  Analysis of Aqueous Samples for Arsenic  	   IV-81
  F.2.2  Determination of Arsenic 1n Water Samples
           with the Graphite Furnace Technique  	   IV-83
  F.3.1  Determination of Arsenic 1n Sediment Samples	   IV-85

Section 14.  Methods for the Determination of Selenium  	   IV-88

  F.I.I  Analysis of Hazardous Waste Samples
           for Selenium.  Reserved 	   IV-91
  F.2.2  Analysis of Aqueous Samples for Selenium Using
           Hydride Generation	   IY-92
  F.3.1  Analysis of Sediment Samples for Selenium
           Using Graphite Furnace Techniques  	   IV-94
  F.3.2  Analysis of Sediment Samples for Selenium  Using
           Hydride Generation (AA) Techniques	   IV-96

Section 15.  Methods for the Determination of Trace Metals
               Using Inductively Coupled Plasma Atomic
               Emission Spectroscopy 	   IV-99

  6.1.1  ICAP Determination of Metals in LMB/L1F
           Fusion Pellets of Waste Samples 	   IV-111
  G.2,1  Determination of Metals in Aaueous Samples Using
           Inductively Coupled Plasma Atomic Emission
           Spectrometrlc Analysis	   IV-118

Section 16.  Methods for the Determination of Cyanide	   IV-126

  H.I.I  Analysis of Hazardous Waste Samples for  Cyanide.
           Reserved	   IV-130
  H.2.1  Analysis of Aqueous Samples for Cyanide  	   IV-131

Section 17.  Methods for the Determination of Sulflde	   IY-136

  G.I.I  Determination of Sulfide in Aqueous Phase
           Hazardous Waste Disposal  Site Samples  	   IY-141
  G.I.2  Determination of Sulflde in Solid Phase
           Hazardous Waste Disposal  Site Samples  	   IV-143
  G.2.1  Methylene Blue, Color1metr1c Determination
           of Sulfide in Aqueous Samples 	   IY-146
  6.2.2  Determination of Sulflde in Aqueous Samples
           by lodometric Titration 	   IV-149
  G.3.1  Determination of Sulfide in Sediment Samples
           'Jsina *he Methylene Blue ColoHmetrfc  Technique  .  .  .   IY-151
                                  xi

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                         TABLE OF CONTENTS (Continued)

                                                                         Page

     Section 18.   Methods for the Determination of Ammonia	    IV-156

       G.I.I  Analysis of Hazardous  Waste Samples
                for Ammonia.   Reserved	    IV-164
       G.2.1  Analysis for Ammonia in Aqueous Samples
                Using an Automated Phenate Procedure	    IV-165
       G.2.2  Automated o-Tolidine Colorimetric Analysis for
                Ammonia in Aqueous Samples	    IV-169
       G.2.3  Titrimetric or  Colorimetric Determination of
                Ammonia in Aqueous Samples	    IV-171
       G.3.1  Determination of Ammonia in Sediments
                Following Distillation	    IV-174
       G.4.1  Determination of Ammonia in Biological Tissue.
                Reserved	    IV-176
       G.5.1  Determination of Ammonia in Air Samples 	    IV-177

Chapter V.  Screening and General Sample Characterization
              Procedures	     V-l

     Section 19.   Methods for the Determination of Oxidants 	     V-2

       F.l.l  Oxidant Capacity of Hazardous Waste  Samples.  Reserved.     V-5
       F.2.1  Oxidant Capacity of Aqueous Samples  	     V-6
       F.3.1  Determination of Oxidant Capacity in Soil Samples.
                Reserved	     V-8
       F.4.1  Determination of Oxidant Capacity in
                Biological Tissue Samples.  Reserved	     V-9
       F.5.1  Determination of Oxidants in Air	     V-10

     Section 20.   Method for  the Determination of
                    Reductant Capacity	     V-15

         F.I  Determination of Reductant Capacity  	     Y-17

     Section 21.   Methods for the Determination of Acidity. .....     V-19

       F.l.l  Determination of Acidity in Hazardous Waste Samples.
                Reserved	     V-21
       F.2.1  Titrimetric Determination of Acidity in Aqueous
                Samples	     V-22

     Section 22.   Methods for the Determination of Alkalinity ....     V-24

       F.l.l  Determination of Alkalinity in Hazardous  Waste
                Samples.  Reserved	     V-26
       F.2.1  Titr^metric Determination of Alkalinity in
                Aqueous Samples 	     v-27

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                    TABLE OF CONTENTS (Continued)

                                                                    Page

Section 23.  Methods for the Determination of Percent
               Moisture and Percent Solids 	    V-29

    E.I  Percent Moisture Determination in Hazardous Waste
           Samples	    V-31
    E.2  Determination of Total Solids in Water	    V-32
    E.3  Total Solids Determination for Sediment Samples ....    V-34

Section 24.  Methods for the Determination of Conductivity .  . .    V-36

  6.1.1  Conductivity Measurements for Hazardous Wastes.
           Reserved	    V-38
  6.2.1  Measurement of Conductivity of Aqueous Samples	    V-39
                                 xiii

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                                   CHAPTER I

                                 INTRODUCTION
A.   BACKGROUND

     Existing regulations require generators of waste material  to determine
whether their waste products meet the established definition of hazardous
wastes [Section 3001 of the Resource Conservation and Recovery Act (RCRA), PL
94-580 (40 CFR Part 261)]* and are therefore subject to regulation.   Since
no single characteristic can reliably establish the hazardous nature of all
wastes, the protocol developed for this purpose requires the measurement of
five behavioral characteristics of the waste and analysis of the waste for
the presence of specific constituents.2

     The five behavioral characteristics are ignitability, corrosivity,
reactivity, EP toxicity (Teachability), and acute biological toxicity (Figure
1-1).  Guidance for using these procedures has been developed by the EPA
Office of Solid Waste and Emergency Response.2  \ny waste that exceeds the
specified limits established for these procedures is considered hazardous.

     A waste is also classified as hazardous if it contains any of the'spec'ffic
compounds listed in Subpart D or Appendix VIII of the RCRA regulations.*  Since
chemical  composition of a waste is an important part of the waste characteri-
zation protocol, the Environmental Protection Agency convened an Agency-wide
steering committee in August 1981 to assess the need for development of a
comprehensive manual to facilitate implementation of the analytical  require-
ments of the hazardous waste regulations.  The committee concluded that such a
hazardous waste manual was indeed necessary for the following reasons:

     1.  the number of specific compounds for which analysis may be  required
         exceeds the scope of existing analytical manuals (Appendix  VIII of
         RCRA lists 360 compounds),

     2.  hazardous waste analysis requires sampling and sample preparation
         guidance for complex and diverse sample matrices whereas most existing
         manuals only address a single matrix (usually water).

The purpose 1n preparing this hazardous waste manual is to provide a compen-
dium of methods 1n response to this regulatory need.

B.   OBJECTIVES

     This analytical compendium is Volume 3 of a broader compilation of informa-
tion on riazaroous *asta sampling and analysis that *s befng prepared by the

                                      1-1

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-------
Environmental Protection Agency, Environmental  Monitoring Systems Laboratory
1n Las Vegas.  The Individual volumes 1n this compilation are:

Volume 1.  Integrated Approach to Hazardous Waste Site Characterization

     This volume discusses the administrative and technical  factors that
     must be considered in planning and implementing a hazardous waste site
     Investigation.

Volume II.  Available Sampling Methods

     This volume provides situational guidance on the use of sampling proce-
     dures and equipment to obtain samples during hazardous  waste site
     investigations.

Volume III.  Compendium of Procedures for the Analysis of Hazardous Wastes

     This volume is a compilation of analytical  procedures of known perform-
     ance that are suitable for the analysis of hazardous waste samples.

     The objectives established by the steering committee for this analytical
compendium include:

     1.  serving as a common source of analytical methods available to the
         Hazardous Waste Program and other Individual  EPA Program Offices
         for use in their programs, as the need arises;

     2.  providing detailed guidance to the bench chemist 1n EPA and State
         Regulatory laboratories on the use of these procedures, Including
         sample handling and preparation;

     3.  serving as a planning document for EPA by identifying  areas requiring
         further analytical  research and development work.

One additional requirement established by the steering committee is that
each procedure must be accompanied by a statement of performance (precision
and accuracy).

     The compilation of analytical procedures 1n this volume obviously ad-
dresses the first two objectives.  Hazardous waste analytical needs, objective
three, are identified by:

     1.  absence of analytical methods for specific compounds listed in
         the regulations;

     2.  absence of sample preparation guidance for one  or more sample
         matrices;

     3.  absence of sufficient method performance data to Include a method or
         to rate 
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     4.  absence of necessary information on sample handling and sample
         preservation.

Since the analytical  needs are defined by the absence of essential  information,
a standardized format was developed for presenting  analytical  procedures  to
alert users to these .information gaps.

C.   ORGANIZATION

     This volume is divided into five Chapters.   Chapter I  identifies  the
objectives and structure of the document.  Chapter  II presents an approach  for
screening and pretreatment of hazardous waste samples.   Chapter III  and Chapter
IV present analytical protocols for organic compounds and inorganic  substances,
respectively, listed in RCRA Appendix VIII.  Chapter V  presents analytical
procedures for general  sample characterization.

     The guidance presented in Chapter II is based  on a protocol  developed  at
the National  Enforcement Investigation Center,  Denver,  Colorado.   It consists
of two aspects that must be considered when hazardous waste samples  are to  be
analyzed.  The first part of Chapter II describes an approach  to conduct  a
safety screening of hazardous waste samples.  The screening results  alert the
analyst to potential  hazards that may be encountered during the handling  and
analysis of such samples due to the presence of strong oxidants, strong re-
ductants, the evolution of potential1y toxic gases  such as  hydrogen  cyanide  or
hydrogen sulfide, or extreme pH values.  Appropriate precautions are recom-
mended that should be taken when such safety hazards and potential  analytical
interferences are identified.  In the second part of Chapter II, a protocol  for
fractionating a complex hazardous waste sample into its component phases  is
presented.  This approach allows independent a.nalyis of each  sample phase
according to the methods detailed in Chapters II! to V.

     Available analytical procedures for the determination  of  a wide range  of
sample constitutents and properties in as many as five sample  matrices are
presented in Chapters III, IV, and V for organic compounds, Inorganic  con-
stituents, and general  sample characteristics,  respectively.  The sample
matrices that have been addressed are 1) hazardous  wastes,  2)  water, 3) soil/
sediment, 4) biological tissue, and 5) air.  The distinction between the
hazardous waste category and the water or soil  category may, at times, only
be a matter of concentration but the higher concentrations  associated  with
hazardous waste samples may require special safety  precautions and special
sample handling (i.e. the use of glove boxes or regulated laboratories, smaller
aliquots, and/or special sample preparation methods).

     The criteria for the selection of analytical procedures for inclusion  in
this compendium are based on a combination of practical and analytical con-
siderations.  Ideally, each procedure selected 1) should have  been used in
numerous laboratories, 2) should have been evaluated for sensitivity,  accuracy,
and precision, and 3) should have been evaluated for analytical interferences.
Also, effective sample preservation, maximum storage time,  and sample prepara-
tion -^rocadurss *
-------
     These considerations Included 1)  an established regulatory need,  2)  the
existence of only one procedure, 3)  time and/or cost requirements,  or
4) concern over safety in the use of a procedure.   Another factor is that
many of the analytical techniques required for the analysis of hazardous
wastes are state-of-the-art developments, and sufficient data may not  have
been generated to fully evaluate the performance of each procedure.  Because
the analytical and practical  considerations are not always compatible, a
ranking system is being used to identify the present status of each  procedure
to the user.

     Minimum requirements for selection of a procedure are:   1) an established
need exists for a specific procedure,  2) single-laboratory precision and
accuracy data exist for the procedure, and 3) the range of applicability  of
the procedure has been defined.  Analytical procedures-that meet these require-
ments are listed as "available".  If multi-laboratory precision and  accuracy
data exist for a procedure (suggesting more extensive evaluation and use),
the procedure is listed as "evaluated".  The user is cautioned that  the intent
is to assemble procedures of known performance and that these procedures  may
not necessarily produce results with acceptable levels of precision  and accu-
racy.  Also, the ranking of each procedure is based on available data.  It is
expected that, as more information becomes available, some of the included
procedures will be upgraded in status  in future revisions of this compendium.

     The analytical methods in each section are presented in a format  that
follows the topical outline presented  below:

     A.  Scope
     B.  Sample Handling and Storage
     C.  Interferences
     0.  Safety
     E.  Apparatus
     F.  Reagents
     G.  Quality Control
     H.  Calibration
     I.  Daily Performance Tests
     J.  Analytical Procedures

         J.I.  Analysis of Hazardous Wastes
         J.2.  Analysis of Water Samples
         J.3.  Analysis of Solid-Phase Samples
         J.4.  Analysis of Biological  Tissue Samples
         J.5.  Analysis of Air Samples

     K.  Qualitative Identification
     L.  Calculations
     M.  References

     Sample handling and storage information (Subsection B)  for each analytical
section will be summarized in a flow diagram similar to that shown in  Figure
1-2.  When a particular method of samole ^and^'ng is inaDorooriate (i.e.  air
drying of sediment samples scheduled for analysis  of volatile organic  com-
pounds), that portion of tne diagram is deleted.  TMs approacfi visually

                                      1-5

-------
o\
        Suole
        Httrli
Miter or
Uecfcate
        ctitlnf

1
Filter
Ptper
Cart-
rlate



I Praserve
ph






i"iuri


Preserve


                |Eitract/l| trtract/  II Store I
                 Wiest || mtttt   |l  _   I
II 1


Anal/it

AMlya

tit net/ 1
OlOMt
                                    1
                                                 Mo
                                                Tr..t-
Met Star**
                                                 (p

                                                (city
                                                direct/
                                                 Digest
                                  I AMlyte I    I AMtyn I
        Cantlnr


        -**r-


        Sior*H
   IP
TotlcUy
        P res   Total     Total
                 COM.     COM.   OlssoUH
                 I* Air    In      COM. (      Nobility       feblUty
                          Miter   Mater         at pH S        at on S
                                                                          Slwdox. Soil or S*alw*t
                                                                  ItoMroous
                                                                    Mittet
(tr1yie 1   I Aiulyie I   [AMlyte  I
                     Total        Total        Total
                     Cone. In     Cone. In     COM. In      COM. In
                     Soil. Slutfej  Sail. Sludoe  Soil. Slufe   lloloejcal   COM. In
                    . SedlMnt     Sedlo*nt     S*4lMnt      Tissue      Maste
        S^oU
        Site
          Figure  1-2.   Schematic diagram developed to summarize sample handling  and storage  information.

-------
reinforces the need to use certain types of samples, or that certain methods
of sample handling may be mandatory.

     Diagrams of the sort presented in Figure 1-2 provided the analyst with
a general overview of the current state of knowledge regarding sample handling
and storage for various sample matrices.  However, as discussed earlier,  some
analytical procedures have been included based on practical  need rather than
purely analytical considerations.  For some of these procedures, information  on
storage time and sample preservation  is not available.   The analyst is urged
to use professional judgement when this occurs and to minimize sample handling
and storage time prior to analysis.   The absence of information in tables
similar to those indicated in Figure  1-2 identifies additional research and
development efforts needed to upgrade sample preparation techniques for each
analytical procedure.

     Some of the topical headings in  the procedure format do not apply to all
methods and have therefore been omitted where appropriate.  For example,  Daily
Performance Tests (I) and Qualitative Identification (K) usually refer to
GC/MS and GC procedures and are omitted from inorganic procedures sections.
However, within the Analytical Procedures Subsection (J), the matrices are
always presented in the order indicated.  When information is lacking for one
or more sample matrix, that subsection has been reserved.  This approach
was taken to allow inclusion of newly developed and reviewed methods as they
become available and to identify existing analytical needs.

     Within the Analytical Procedures subsection, detailed guidance describing
the 'use of each method is being presented under the following headings.

     J.I.  Analysis of Hazardous Wastes                  .

          J.I.I  Reference
          J.I.2  Method Summary
          J.I.3  Applicability
          J.I.4  Precision and Accuracy
          J.I.5  Sample Preparation
          J.I.6  Sample Analysis

Primary references are provided to allow users to confirm the appropriateness
or applicability of a procedure.  Also, by maintaining visible integrity
between a method and the original author, a greater participation in future
revision of this document is anticipated.

     The selected method of presenting analytical procedures resulted in  a
certain amount of intentional redundancy.  It is felt this approach will
provide continuity to the user while  achieving the objective of providing
guidance for the analysis of samples  of several matrix types.
                                      1-7

-------
                                 REFERENCES


1.    Environmental  Protection  Agency.   "Hazardous Waste Management System.
     Identification and Listing  of  Hazardous Wastes."  Federal Register Vol. 45
     (92):33084-33133.   May  19,  1980.

2.    Environmental  Protection  Agency.   "Test Methods for Evaluating Solid
     Waste.   Physical  Chemical Methods."  U.S. EPA, Office of Solid Waste and
     Emergency Response, Washington, D.C.  Report SW-846 (July 1982).
                                      1-8

-------
                                   CHAPTER  II

                 PHASE  SEPARATION AND SCREENING  PROCEDURES FOR
                            HAZARDOUS WASTE  SAMPLES
 A.    INTRODUCTION

      The  passage of  time  between  sample  collection and  sample analysis is a
 critical  consideration  in any  analytical protocol.   In  addition to keeping
 this  time as  short as possible, proper procedures must  be  instituted to
 maintain  sample integrity and  minimize sample contamination.  This is usually
 accomplished  by dividing  the original sample into representative subsamples
 or  aliquots based on the  number of specific analyses to be performed.  Each
 individual aliquot can  then be treated with the appropriate  preservative,
 digestion solution or extraction  solvent without fear of invalidating the use
 of  the  sample for the determination of a second analyte or loss of the analyte
 of  concern.

      Proper pretreatment  of hazardous waste samples  can serve an additional
 function.  Specifically,  the use  of screening techniques can identify poten-
'tial  health or safety problems associated with the handling  of samples.  This
 will  permit.the necessary precautions to be implemented.  For example, if
 samoles known to contain  cyanide  are to  be analyzed  for total metals, they-
 should  be handled in a  hood or pretreatea to remove  cyanide  to prevent prob-
 lems  associated with the  evolution of hydrogen cyan-ide  when  the samples are
 acidified.  Furthermore,  screening results can be used to judge the necessity
 of  performing more detailed and/or more  expensive analyses on the same sample.

      The  purpose of this  chapter  is to provide general  information for handling
 and storing hazardous waste samples in the laboratory and to provide guidance
 for implementing a standardized approach for pretreatment of hazardous waste
 samples.   The guidance  addresses  phase separation of complex, multi-phase
 samples and screening of  the resultant fractions for inorganic and organic
 constituents.

 B.    STORAGE  AND HANDLING OF HAZARDOUS WASTE SAMPLES

      This section summarizes general safety procedures  and good laboratory
 practices that should be  followed when handling hazardous waste samples in
 the laboratory.  Specific sample  handling requirements are presented with
 the appropriate analytical section in the following Chapters.

      Samples  of hazardous material should be stored at  4C in a refrigerator
 designed  -for  flammable  materials  or a specially designed cold room within a
 regulated laboratory, if  availaoie.  Samples inouia ^e  jtorea in the original
 shipping  container,  Also, the refrigerator or cold room should only be for

                                     II-l

-------
hazardous waste samples.  This precaution is necessary to prevent cross-con-
tamination of samples or standards.

     When transporting hazardous wastes within the laboratory, storage vessels
containing hazardous substances must be placed first in an unbreakable outer
container before being transported to laboratory work areas using good transfer
practices.  Plastic-coated glass bottles with polypropylene caps, which can
satisfy a 4-foot drop test, are currently available which can serve as both
the storage vessel  and the unbreakable outer container combined.   Contami-
nated materials which are transferred from work areas to disposal areas must
first be placed in a closed plastic  bag or other suitable impermeable and
sealed primary container.  The primary container must be placed in a durable
outer container before being transported.  The outer container must be labeled
with both the name of the hazardous  substance and the warning:  CAUTION -
HAZARDOUS SUBSTANCE.

     Sample containers should only be opened in a glove box or operational
fume hood by personnel wearing protective clothing.  The objective of this
procedure is to minimize exposure of the personnel to the samples.

     No more than one sample container is to be opened at any one time.  This
precaution will avoid cross-contamination of samples and potential synergistic
effects between components of different samples.

     When samples are not being jssd for extended periods, they are to be
placed back into the original containers, resealed and returned to storage.

     If stock quantities of hazardous reagents are used, they should be stored
in- a properly ventilated storage area that is secured at all  times.  The stor-
age area should be postad with ? s:gr bearing the ""egend:  CAUTION - HAZARDOUS
MATERIALS - AUTHORIZED PERSONS ONLY.  An inventory of stock quantities shall
be maintained by the laboratory Safety Officer.  The inventory records shall
include the quantities of materials  acquired and dates of acquisition and
disposition.

    " No solvents, sample, or other contaminated materials, are to be poured
into any sink.- Generally, concentrated sample should only come in contact
with disposable glassware, unless the sample attacks glass, in which case
polypropylene labware should be used.

     During sample preparation, contaminated glassware (glassware contacting
the sample) is separated from glassware for reagent handling.  Disposable
glassware, pipets, sample vials, etc., are placed in waste containers for
disposal.  Other glassware is rinsed thoroughly (at least 3 rinses) with an
appropirate solvent, the solvent placed in waste bottles and the glassware
washed with soap and water.

     Any contaminated glassware or equipment that cannot be safely cleaned
is to be disposed of as hazardous material.
     All -*tQrk rur^cas fw,nch *.3os, htxxls, *1ocrs, tc,^ shall  be covered with
stainless steel, plastic sheets, dry adsorbent plastic-backed paper or other
                                     II-2

-------
Impervious material.  The protective surfaces shall be examined for possible
contamination immediately after each procedure involving the hazardous mate-
rial has been completed.  Contaminated surfaces shall  be decontaminated or
disposed of as is appropriate.

C.   PHASE SEPARATION OF HAZARDOUS WASTE SAMPLES

1.   Phase Separation

     The separation of a sample into its component phases is accomplished by
     centrifugation based on a procedure developed by  the Environmental  Pro-
     tection Agency National Enforcement Investigation Center.   This separation
     procedure may yield as many as three distinct phases (Figure II-l).   These
     phases are 1) aqueous phase, 2) solid phase, and  3) non-aqueous phase(s).
     The details of this separation method are presented below.

     1.1  Reference

          U.S. Environmental Protection Agency, "Hazardous Waste Site Samples  -
          Phase Separation."  U.S. EPA, National  Enforcement Investigation
          Center, Denver, Colorado.  2 p.  (February,  1980).

     1.2  Summary

          Samples are centrifuged in their original containers  to achieve
          phase separation.  Individual phases are transferred  to separate
          containers for further processing.

   -  1.3  Apparatus

          Sample bottles, clear glass, screw-neck finish, wide-mouthed round
          jars.  (Kerr, A.C. No. 802; available from VWR Scientific, Catalog
          No. 16194-063).

          Teflon liners for sample bottles.

          International Centrifuge, Model FXD with explosion-proof motor  for
          Class 1, group D location, or equivalent.

          Automatic pipeting device, pipet-aid, or equivalent.

          Disposable pi pets, 10 and 25 ml.

          Polybags to fit sample bottles.

     1.4  Procedure

          Wipe the original sample container  to remove any outside contamina-
          tion.  Label  the bottle appropriately.   Place the bottle in a poly
          bag and seal  with a rubber band or  "twister".
                                     II-3

-------
                                     MuNf-phaM
                                      Sampla
                                     Cantrifuga
                                    t 2.0OO rpm
                                      Saparata
                   _L
                 1.0ml
               Attquot (aach)
               Mbcibility
                 Mad
r w/
LJ
Miscibla
Only w/ H,O




Aquaoui
PhaM
1
Saa Figura 1
for Prap.
MiMiMa or
Not MitciMa
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1
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Kari Fishar
Racult*
1

Mitcibla
Only w/ MaCI,




Nonaquaou*
Pttata
1
SM Figura 3
lor Prap.


It % H,O
U >60%
1
Aquaoui
PhaM
1
Saa Figura 1
for Prap.





If % H,O
te<60*
1
Nonaquaoui
Phaaa
1
Saa Figura 3
for Prap.

Figure  II-l.  Phase separation of  hazardous waste  samples.

-------
          Heigh the sample bottle and prepare a counterbalance  for  the
          centrifuge.

          Place the sample and counterbalance in the  centrifuge.  Centrifuge
          the sample for 30 minutes at 2000  RPM.   Stop  the  centrifuge and
          remove the sample.

          Mark the interface levels between  each phase  on  the sample bottle.
          Relative volumes may be estimated  by using  a  clean sample bottle
          calibrated in 10-ml  graduations.

          Using an automatic pipeting device and disposable pipets, transfer
          each distinct layer into a separate container.  Label all phases
          with the appropriate sample number.  Classify each liquid phase as
          either aqueous or nonaqueous based on sample  miscibility  with water.

          (Procedure:   Transfer 2 ml of each liquid phase  Into  separate test
          tubes.  Insert a 1-cm loosely packed plug of  glass wool followed
          by 1 cm of indicating silica gel.   Cap the  test  tube  and  let stand
          for 30 minutes.   Disappearance of  the blue  indicator  color indicates
          that water is a major portion of the phase.   Alternatively, use the
          Karl Fischer titration procedure).

2.   Aliquot Preparation

     The centrifugation process may yield as many as  three  distinct phases.
     These are the aqueous phase, the solid  phase, and  the  non-aqueous phase(s).
     The preparation of aliquots for screening and analysis of  each phase is
     summarized in the flow charts presented in Figures II-2, II-3, and II-4.

     2.1  Aqueous Phase

          Five aliquots should be set aside  for screening  of the  aqueous phase
          samples (Figure IJ-2).  The first  1-ml  aliquot  is to  be screened  for
          oxidant capacity, reductant capacity, cyanide,  sulfide, and pH.   A
          second 2-ml  aliquot is diluted to  200 ml with distilled water and
          analyzed (using procedures in Chapter V) for  acidity, alkalinity,
          conductivity, oxidant capacity, and reductant capacity.  Two separate
          1-ml aliquots are set aside for cyanide and sulfide analyses.  (These
          two aliquots can be omitted if the spot tests for these analytes  are
          negative).  If necessary, the sulfide aliquot may be  preserved by the
          addition of zinc acetate and the cyanide aliquot  may  be preserved by
          the addition of sodium hydroxide and refrigeration.   The  final 1-ml
          aliquot should be diluted to 100 ml with 2-percent nitric acid.   This
          solution is analyzed for mercury,  other metals,  and ammonia, if
          desired.

     2.2  Solid Phase

          A schematic outline of the suggested approach to  prepare  solid-phase
          nazaraous waste sample aVquots ror Anorganic screeninq or analysis
          is presented in Figure II-3.  The  sulfide and cyanide procedures

                                     II-5

-------
                                                 9-II
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pHd



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2g
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lOOml
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FHtar
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Oxidant
Spot Tart

Raductant
Spot Tart

Oxidant or
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Datarmination

Solid
Phasa
1
1
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ig
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I
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Spot Tost

Hlar 1 g
60m Aliquot


ig
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T 	 '
""^ Surflda
mmon Datarminaaon
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Dataii mnalion
	 Task Always Parformad by Lab
  I  Task Only Parformad by lab
Altar Positive Spot Test
1 Task Only Parformad by Lab
Whan Concuctivity is > 1.O000mhos
and pH is S 4 or > 9
=> Task performed by Lab whan Subsequent
Analysis is Requested by Ragion
ajSBBSi Task Not Parformad by Lab
:= Task May Not be Performed by Lab
Figure II-3.   Solid phase screening and sample preparation.

-------
I
00


1
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Residue
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             Figure II-4.  Preparation of nonaqueous phase samples for inorganic analysis.

-------
          simply require the setting aside of 1-g aliquots  to  be  analyzed  in
          the event the spot test is positive.   However,  there are  no  known
          effective preservatives for solid-phase samples.   A  fourth aliquot,
          2 g, is extracted with 200 ml  distilled water.  The  extract  is
          screened for the presence of oxidants  and reductants, then analyzed
          for oxidants and reductants, if necessary,  and  analyzed for  conduc-
          tivity, pH, acidity, alkalinity, and the anions of strong and weak
          acids, if desired.  A fifth aliquot is dried over ?2^S  for percent
          moisture detennination and subsequently fused with lithium metaborate
          and lithium fluoride prior to metals analysis.  A sixth aliquot  is
          digested with potassium persulfate and potassium  permanganate and
          analyzed for mercury.  The seventh indicated aliquot is extracted
          with 0.1 N HC1 and analyzed for ammonia.

     2.3  Nonaqueous Phase

          A schematic outline of the approach to prepare  nonaqueous phase
          hazardous waste samples for inorganic  analysis  is presented  in
          Figure 11-4.  The two indicated fractions are for total mercury  and
          total  metals.  The aliquot preparation techniques are the same as
          those provided for the preparation of  solid phase aliquots for the
          same analyses.

D.   INORGANIC SCREENING PROCEDURES

     Each sample phase separated from the original  sample using procedures
specified in Subsection C should be qualitatively analyzed  using  procedures
specified in this section.  Results of this testing will  provide  information
on the presence of strong oxidizing or reducing  agents, cyanide,  sulfide,  or
extreme pH conditions.  This information can be  used to classify  the waste
sample being analyzed as hazardous and to determine the most appropriate method
of sample handling.

     Samples should be subjected to qualitative  spot testing in the order  pre-
sented in the text.  For example, oxidizing spot tests should  be  performed
prior to CN spot tests which should be performed prior to pH tests. This
approach is suggested based on the relative order of importance of  the informa-
tion to be obtained and partially out of convenience.  By testing for  the
presence of oxidizing agents first, the tests for cyanide and  sulfide  can
be omitted if oxidizing reagents are present. Similarly, if oxidizing agents
are absent, it would be beneficial to confirm the abence  of cyanide or sulfide
prior to adjusting the pH of the sample.

Qualitative Screening Procedures for Hazardous Waste Samples

1.   Spot Test for Oxidants in Aqueous-Phase Samples and  Aqueous  Extracts  of
     Solid-Phase Samples from Hazardous Waste Disposal  Sites
     ^
     1.1  Puroose

          The objective of this test is to detect the presence of strong
          oxidizing agents such as chlorine ana  cnicnne  uioxiae.   Results

                                     U-9

-------
     are expressed as an equivalent amount of chlorine and are used to
     determine the necessity of performing spot tests for cyanide and/or
     sulfide.
     This text can be performed on undiluted HWDS aqueous-phase and
     water extracts of HWDS solid phase samples.
1.2  Procedure Summary
     A drop of sample is placed on potassium iodide-starch test paper
     which has been previously moistened with concentrated acetic acid;
     a blue color indicates the presence of oxidants.
1.3  Safety Considerations
     This test should be performed in a hood.
1.4  Appartus
     1)   Small white disposable weighing dishes.
     2)   100-yl Eppendorf pipet and tips.
1.5  Reagents
     1)   Kl-starch test paper.
     2)   Glacial acetic acid, cone.
     3)   1.0% w/v chloramine-T.
1.6  Detailed Procedure
     1)   Place a strip of Kl-starch paper in the weighing dish.
     2)   Moisten the paper with a drop of acetic acid.
     3)   Transfer 100 yl (2 drops) of the sample from the pH aliquot or the
          aqueous extract from the solid-phase sample onto the test paper.
     4)   Wait approximately 5 minutes for color development.
     5)   A blue color will appear if an oxidant is present.   Compare to a
          blank prepared from distilled water.
1.7  Test Results
     1)   Record whether a positive or negative test was obtained.
     2)   If a positive test is obtained, determine the oxidant strength by
          applying tne ox'aant quantIT^cation procedure.
                               11-10

-------
     3)   If a positive test result is obtained with an aqueous-phase sam-
          ple, it is very unlikely that sulfide or cyanide will  exist in
          the sample.  Therefore,  these spot tests (sulfide and  cyanide)
          do not have to be performed.

     4)   If oxidant test results  were negative, proceed with the test for
          cyanide.

1.8  Quality Control Requirements

     1)   Check the spot test daily by testing 50 yl of the 1.0-percent
          chloramine-T solution.   A positive test should be obtained if
          the test  is proceeding  properly.   A negative test indicates
          something is wrong and  steps are  to be taken to find the problem.

     2)   Do not perform the test  on any samples until a positive test is
          obtained  for the chloramine-T solution.

     3)   Record the positive standard check on the bench sheet.
                               11-11

-------
2.   Spot Test for Cyanide in Aqueous-Phase and  Solid-Phase Hazardous  Waste
     Disposal  Site Samples

     2.1  Purpose

          The  object of the test  is  to quickly determine whether cyanide  is
          present in the sample.   If present,  precautions must  be taken  (such
          as cyanide removal  or working in a hood/glove box)  to ensure that
          acidification of the sample does not create  hazardous conditions  for
          laboratory personnel.

     2.2  Procedure Summary

          Cyanide is reacted with chloramine-T in  a  buffered  solution  to  pro-
          duce cyanogen chloride.  The cyanide is  quantified  based on  the
          intensity of the red-blue  color that develops when  cyanogen  chloride
          is mixed with pyridine-barbituric acid.

          This procedure permits  a rapid screening for the presence of cya-
          nide above 60 ppb in aqueous samples (3-drop sample size) and  10
          ug/g in solid samples (1-g sample size).   The method  will detect
          cyanide in many of the  common metallic cyanide complexes. However,
          platinum, gold, and cobalt cyanide are not detected as cyanide.

     2.3  Safety Considerations

          Because of the volatility  and hazards  associated with cyanide,  all
          sample handling'should  be  done in a  well-vented hood  or glove box.
          If cyanides are found to be oresent, all  subsequent processing  of
          the  sample should also  be  completed  in a hood or glove box.

     2.4  Interferences

          1)   Thiocyanate produces  the same reaction  in the  spot test as
               cyanide.  These two substances  can  be distinguished by  removing
               the cyanide by reaction with formaldelhyde and quantifying the
               remaining thiocyanate with the  chloramine-T procedure.

          2)   A highly reduced sample interferes  by consuming  chloramine-T.
               Additional chloramine-T should  be added as necessary.

          3)   Aldehydes in excess of 0.5 mg/1 interfere by converting cyanide
               to cyanohydrin.

     2.5  Appartus

          1)   Plastic disposable 2-ml  conical beakers.

          2)   Disposable capillary  pipets and rubber  bulb.

          3)   Rudoer, 2-noie stoppers r'or che test  tuoes.


                                    11-12

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     4)   Teflon connecting tube, 1 mm I.D.

     5)   Disposable, long-stem eye droppers.

     6)   Tygon rubber tubing, 1/4" I.D.

     7)   Compressed nitrogen and regulator.

     8)   Heating block capable of maintaining 75  5C with hole openings
          for the test tubes.

2.6  Reagents
     (All reagents to be made from ACS grade  chemicals.)

     1)   Pyridine-barbituric acid reagent:   place 3 g  barbituric  acid  in
          a 100-ml volumetric flask.  Rinse the sides of the flask with a
          minimum amount of distilled water.   Add 15 ml  pyridine and  mix.
          Add 3 ml cone. HC1  and mix.  Add additional distilled water and
          stir until the barbituric acid  dissolves.   Dilute  to volume with
          distilled water.

     2)   Chloramine-T solution:  dissolve 1.0 g of white, water-soluble
          chloramine-T in 100 ml distilled watar and refrigerate until
          ready to use.  Prepare fresh weekly.

     3)   Phosphate buffer:  dissolve 138 g NaH2P04*H20 in distilled  water
          and dilute to 1 liter.  Refrigerate  solution  after preparation.

     4)   Stock cyanide solution, 1000 mg/1 CN:  dissolve 2.51 g KCN  and .
          2 g KOH in 1000 ml  distilled water,

     5)   Cyanide spiking solution:  add  20 ml 1.25-N NaOH solution to  a
          100-ml  volumetric flask.   Pipette 5.0 ml  stock cyanide solution
          to the flask and  dilute to volume with distilled water.   Store
          solution in the dark.   The cyanide  concentration is 50 mg/1 CN.

     6)   Magnesium chloride  solution: weigh  510 g  MgCl2*6^0 into 1000 ml
          volumetric flask, dissolve and  dilute to volume with distilled
          water.

     7)   Hydrochloric acid:   dilute 50 ml concentrated HC1  with 50 ml
          distilled water;  and 0.1 g aluminum  metal.

     8)   Sodium hydroxide  solution:  add 500  ml  distilled water to 40  g
          NaOH in a beaker, mix  and cool.  Transfer  the NaOH solution to
          a 1-liter volumetric flask and  dilute to volume.

     9)   Methyl  violet "indicator:   dissolve  1 g methyl  violet in  100 ml
          ethyl  alcohol.
                               11-13

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2.7  Detailed Procedure

     Liquid Samples

     1)   Place 0.25 ml (approximately 5 drops)  of sample in a disposable
          beaker.

     2)   Add one drop phosphate buffer and mix.   Check the resultant pH
          with a pH indicator strip.   If the pH  is above 8, continue
          adding buffer until a pH of approximately 8 is obtained.

     3)   Add 4 drops chloramine-T reagent  and mix.

     4)   Add 4 drops pyridine-barbituric acid reagent and mix again.
          Allow 8 minutes for full  color development.

     5)   The appearance of a pink to red color  indicates the presence of
          1.0 mg/1 or more of cyanide.  In  comparison, a faint yellow
          color should develop in a cyanide-free  blank.

     Solid Samples

     1)   One gram of sample is weighed into a disposable test tube to
          which 1 ml MgCl2-solution is added; a  small  glass rod is  used to
          disperse the sample.  The apparatus is  shown in Figure II-5.

   ,  2)   One drop methyl violet indicator  is added to the sample tube.
          The apparatus is assembled as shown in  Figure II-5 and.l  ml NaOH
          is added to tube No. 2.

     3)   Nitrogen is bubbled through the tubes  at the rate of 2 bubbles
          per second for 25 minutes.   Temperature of the heating block
          is 75  5C.

     4)   Four drops (200 ul) 6-N aluminum-treated HC1 are added to the top
          of the dropper with the apparatus at an angle such, that tubing
          from the nitrogen supply can be attached to the dropper before
          the acid hits the sample.  The solution should turn yellow or the
          violet coloration should be absent.  If not, more acid must be
          added.

     5)   One ml MgCl2-solution should be analyzed as a blank solution.

     6)   Transfer 250 ul of clear solution from  tube No. 2 to a 2-ml
          conical  beaker.  Add 100 ul phosphate  buffer solution.

     7)   Add 4 drops chloramine-T reagent  and mix.

     8)   Add 4 drops of pyridine-barbituric acid reagent and wait  8
          minutes.  A faint oink to violet  color  indicates the presence
          of cyanide.            


                               11-14

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           Ph Test Strip
                              Teflon Connecting Tube






Sai
s,
u


nple

Tube#1

*N  *
Heating Block
                                          Tube #2
    Figure II-5.  Appar:tj rcr cyanide and sulfide spot tests.
2.8  Test Results

     Record the test results.  If a positive cyanide test is obtained,
     quantify the cyanide concentration using the procedure in Chapter
     IV, Section 16.

2.9  Quality Control Requirements

     1)   Check the performance of the spot test daily by-analyzing the
          cyanide spiking solution.  If a positive test is not obtained,
          measures must be taken to identify and correct the problem.

     2)   Perform sample spot test only after a positive test is obtained
          for Step 1 above.

     3)   Record the positive standard check.
                               11-15

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3.   Spot Test for Sulfide'in Aqueous-Phase Hazardous Waste Disposal  Site
     Samples

     3.1  Purpose

          The objective of the test is to quickly determine whether sulfide,
          that may interfere with other analyses or create a hazard during
          sample processing, is present in the sample.

     3.2  Procedure Summary

          One drop of the sample is placed on a lead acetate test paper
          previously moistened with an acetic acid solution.  The presence of
          sulfide is indicated by a darkening of the paper.  The method detec-
          tion limit is approximately 4 mg/1.

          If the aqueous sample is highly colored or turbid or the sample is
          solid, then the Alternate Spot Test Procedure for Sulfide (paragraph
          4) should be employed to check for sulfide.

     3.3  Safety Considerations

          This procedure should be carried out in a hood or glove box.

     3.4  Interferences

          None.

     3.5  Apparatus

          1)   Disposable capillary.pipets and bulbs.

          2)   Small white weighing dish.

     3.6  Reagents

          1)   Lead-acetate test paper [Fisher Scientific (Catalogue  No.
               14-862)].

          2)   Acetate buffer:  dissolve 410 g sodium acetate trihydrate
               (NaCpHTO?, 3H20) in 500 ml  water.  Add glacial  acetic  acid
               to pH 4.5.

     3.7  Detailed Procedure

          1)   Place a strip of the lead-acetate test paper in a white  weighing
               dish.

          2)   Wet the paper with 2 or 3 drops of the acetate buffer.

          3)   Add ore drop :arDla,  A darkeninq of *;he *sst 'lane1"
                                    11-16

-------
     the presence of sulfide.  Not less than 4 mg/1  sulfide can be
     detected.
3.8  Test Results
     Record the results.  If a positive result is obtained, sulfide can
     be quantified using the procedures given in Chapter IV, Section 17.
3.9  Quality Control Requirements
     1)   Check the performance of this spot test daily by performing the
          test on a 10 mg/1  sulfide standard.
     2)   A positive test must be obtained before analyzing samples.
     3)   Record the positive standard check with the sample results.
                               11-17

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4.   Alternate Spot Test Procedure for Sulfide in Highly Colored or Turbid
     Aqueous Samples or Solid-Phase Samples from Hazardous Waste Disposal
     Sites

     4.1  Purpose

          This method is for qualitatively determining the presence of sulfide
          when the principal  spot test cannot be used due to matrix inter-
          ference.

          The objective of the test is to quickly determine whether sulfide,
          that may interfere with other analyses or create a hazard during
          sample processing, is present in the sample.

     4.2  Procedure Summary

          This procedure allows for a quick screening of turbid aqueous samples
          or semi-solid samples such as soil  or sediment for the presence  of
          10 yg/g or greater of sulfide.  Nitrogen is bubbled through a heated
          sample mixed with MgCl2 and HC1.  H2$ is evolved into a collection
          media containing Cd(N(h)2  The presence of sulfide is indicated
          by the discoloration or a lead acetate test strip suspended above
          the sample.  Larger amounts of sulfide are indicated by the formation
          of a yellow precipitate in the collection medium.

     4.3  Interferences

          The treatments of acidification and gas-stripping release HCN and
          H2$ in a reasonably pure state.  However, cyanide and sulfide may
          react  in the collection medium to produce thiocyanate that can inter-
          fere in the test.

          The method detects all common metal sulfides except those of copper.

     4.4  Apparatus

          1)   Disposable 1 x 7 cm test tubes.

          2)   Rubber, 2-hole stoppers for the test tube.

          3)   Teflon connecting tube, 1 mm 1.0.

          4)   Lead-acetate test strips.

          5)   Disposable, long-stem eye droppers.

          6)   Tygon rubber tubing, 1/4" I.D.

          7)   Compressed nitrogen and regulator.

          3}   .-Seating blocK  :apable  :f ,7iaint a living 75  - cC *ith hole
               openings for the test tubes.

                                    11-18

-------
4.5  Reagents

     (All reagents are to be prepared with ACS grade chemicals.)

     1)   Magnesium chloride solution:  weigh 510 g  of MgCl 2*6^0  into  a
          1000-ml  volumetric flask, dissolve  and  dilute to  volume  with
          distilled water.

     2)   Hydrochloric acid:  dilute 50 ml concentrated HC1  with 50  ml
          distilled water; add 0.1 g aluminum metal.

     3)   Cadmium nitrate solution:  dissolve 30.9 g of Cd(N03)*4H20
          in distilled water and dilute to 100 ml.

     4)   Sodium hydroxide solution:  add 500 ml  distilled  water to  40  g
          NaOH in a beaker, mix and cool.  Transfer  the NaOH solution to
          a 1-liter volumetric flask and dilute to volume.

     5)   Methyl violet indicator:  dissolve  1 g  methyl  violet  in  100 ml
          ethyl alcohol.

     6)   Acetate buffer:  dissolve 410 g sodium  acetate trihydrate  in
          500 ml water.  Add glacial acetic acid  to  pH 4.5.

4.6  Detailed Procedure

     Aqueous. Samp! es

     1)   One ml of the aqueous sample is oioetted into a disposable test
          tube to which 1 ml MgCl? solution is added.   A small  glass rod is
          used to disperse the sample.  The necessary apparatus is shown in
          Figure II-5.

     2)   One drop methyl violet indicator is added  to tne  sample  tube  and
          a lead acetate test strip moistened with acetate  buffer  is sus-
          pended above the sample between the stopper edge  and  the lip  of
          the tube.

     3)   The apparatus is assembled as in Figure I1-5 with 1 ml Cd  (1^3)2
          solution and 1 ml NaOH in tube No.  2.

     4)   Nitrogen is bubbled through the tubes at the rate of  2 bubbles
          per second for 25 minutes.  Temperature of the heating block
          is 75  5C.

     5)   Four drops (200 yl) 6 N aluminum-treated HC1 is added to the  top
          of the dropper with the apparatus at an angle such that  tubing
          from the nitrogen supply can be attached to the dropper  before
          the acid hits the sample.  The solution should turn yellow or the
          violet coloration should be absent.  If not. more acid must be
          added.  One ml MgCl solution should be analyzed  as a blank
          solution.

                               11-19

-------
     Solid Samples

     1)    One gram of sample  is  weighed  into  a  disposable  test  tube  to
          which 1 ml  MgCl 2  solution  is added; a small  glass  rod  is used
          to disperse the sample.  The apparatus is  shown  in Figure  II-5.
                    \
     2)    One drop of methyl  violet  indicator  is  added to the  sample  tube
          and the lead-acetate  test  strip  moistened  with acetate  buffer  is
          suspended above  the sample between the  stopper edge  and the lip
          of the tube.

     3)    The apparatus is assembled as  in Figure II-5 with  1  ml
          solution in tube No.'  2.

     4)    Nitrogen is bubbled through the  tubes at the rate  of 2  bubbles
          per second for 25 minutes.  Temperature of the heating  block is
          75  5C.

     5)    Four drops (200  yl) 6 N  aluminum treated HC1 are added  to the
          top of the dropper with  the apparatus at an angle  such  that
          tubing from the  nitrogen supply  can  be  attached to the  dropper
          before the acid  hits  the sample.  The solution should turn
          yellow or the violet  coloration  should  be  absent.  If not,  more
          acid must be added.  One ml  MgCl2 solution should  be analyzed as
          a blank solution.

*.7  Test Results
                                     
   - Any darkening of the  lead-acetate test strip within 2 to  3 minutes
     indicates the presence of  suicide-  Yellow coloration in  tube. No. 2
     indicates higher sulfide concentrations.

     If a positive sulfide result  was obtained, sulfide can  be quantified
     using the procedures  in Chapter IV, Section  16.

4.8  Quality Control Requirements

     Check the performance of this spot  test daily by performing  the  test
     on a standard sulfide solution  (approximately 100 ppm).   A positive
     test must be obtained before  analyzing samples. Record positive
     standard test with the sample results.
                               11-20

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5.   Spot Test for pH in Aqueous Samples and  Aqueous  Extracts  of  Solid  Phase
     Samples from Hazardous Waste Disposal  Sites
     5.1  Purpose
          The objective of this test is to  determine  whether the  pH  of  any
          samples is extreme (<1 or >12) and  would  require  special care in
          handling and preparing the samples  for  further  analysis.
     5.2  Procedure Summary
          The pH of the sample is approximated  using  pH indicator paper.
     5.3  Sample Handling
          Samples should be analyzed as soon  as possible.   This test  should
          be performed in the hood.
     5.4  Apparatus
          1)   Disposable cups and caps.
          2)   pH test paper 0-14.
     5.5  Procedure
          Dip the pH test paper into the aqueous  phase sample.  Compare the
          resultant color of the test paper with  the  chart  on  the package.
     5.6  Test Results
          1)   Record the observed pH of the  sample.
          2)   If the pH of the sample is less  than 8, acidity of the sample
               can be determined using procedures in  Chapter V, Section 21.
          3)   If the pH of the sample is greater than 5, alkalinity  of the
               sample can be determined using procedures  in Chapter  V,
               Section 22.
                                    11-21

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E.   ORGANIC SCREENING PROCEDURES

     The following procedure is presented as a screening technique for hazard-
ous waste disposal site samples suspected of being highly contaminated with
organic compounds.  Each of the sample phases prepared in Subsection C.I. can
be screened using GC/FID because of the wide linear range of the flame ioni-
zation detector and its relative insensitivity to water vapor.   However, the
user is cautioned that the presence of a peak in the sample chromatogram is
only presumptive evidence for a specific component since one or more components
may co-elute at identical retention times under the stated analytical  condi-
tions.  The intent of the procedure is not to quantify specific compounds but
to identify those samples and/or sample phases that may require more time-
consuming and detailed analysis.

1.   Extract Preparation

     Separate the original sample into component phases by centrifugation
     as described in Subsection C.I.  Prepare an extract of each phase using
     the appropriate guidance given below.

     1.1  Direct Aqueous Screening

          Analyze 1- to 5-ul aliquots of each liquid phase.  Dilute samples
          as necessary to obtain appropriately scaled responses.

          Screen the samples and compare, by GC retention time, unknown sample
          peaks to known standards.  Prepare a iist of probable components
          for each sample.

     1.2  Aqueous-Phase Extraction

          Transfer a 10-ml aliquot of the aqueous phase to a 17-ml vial con-
          taining 2 g NaCl.  Add 2 ml hexane.  Cap with a Teflon-lined screw
          cap and shake for 1 minute.  Allow the layers to separate and trans-
          fer 1 ml of the hexane layer into a 2-ml crimp-seal  vial for analysis.

     1.3  Nonaqueous Liquid Extraction

          Dilute 1.0 ml of nonaqueous phase sample to 10 ml with hexane.  Add
          1 g anhydrous sodium sulfate and mix to dry the solvent.  If the
          sample phase is soluble in hexane, transfer 1.0 ml of the diluted
          sample to a clean vial and add 9.0 mi  hexane.  Transfer 1.0 ml of
          the second dilution to a 2-ml crimp-seal vial for analysis.   Save
          the dilutions until the analyses are completed.

          If the sample phase is not soluble in hexane, repeat the procedure
          using acetone as the solvent.

     1.4  Solid and Sediment Extraction

          Cheat che iolubi'i-'-y -f  -.he  :o1;d by placing '  i of samc'e 4n i
          17-ml vial and adding 10 ml acetone.  If more than 50 percent of

                                     11-22

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     the solid dissolves, proceed with (a).   Otherwise, extract the sample
     as described in (b).

     a)   Acetone-Soluble Samples

          Dilute 1 ml of the acetone-solid suspension (if more than 50
          percent of the solid has dissolved) to 10 ml  with acetone and
          analyze.

     b)   Acetone-Insoluble Samples

          Mix 5 g sample with 10 g granular anhydrous sodium sulfate.
          Place the mixture into a prerinsed cellulose  thimble and place
          the thimble into a micro-Soxhlet extractor (Kontes K292000,  or
          equivalent).   Extract the sample for 3 hours  (at least 15 cycles)
          with acetone.

     Transfer the extract to a vial and dilute to 25 ml with acetone.
     Transfer 1.0 ml to a 2-ml crimp-seal  vial  for analysis.  Save the
     extract in a glass vial with a Teflon-lined cap until the analysis
     is complete.

Extract Analysis

Sample extracts are screened by GC/FID.  Screening of hexane and acetone
extracts is performed by injecting 1 to 2 yl of extract onto a 300-cm  x
2-mm-I.D. glass column packed with 6 percent OV-101 on  60/80 mesh GC-Q
or equivalent.  The temperature should be programmed to increase from
3QC 10 230C at a rate of 6 to 8/minute.  A maximum run time of 75
minutes should be sufficient to observe most compounds  of significance.

Dilute and/or concentrate the extracts, as necessary, to obtain appro-
priately scaled responses.

Volatile organic compounds at concentrations above 1 mg/1 can be deter-
mined by direct aqueous injection gas chromatography.  Lower concentra-
tions, based on the ratio of sample volume to extraction solvent volume,
should be detectable in the other sample phases.
                               11-23

-------
                                  REFERENCES
1.    U.S. Environmental  Protection  Agency.   "Hazardous Waste  Site Samples -
     Phase Separation."   National  Enforcement Investigation Center,  U.S.  EPA,
     Denver, Colorado.  9 p.   (February,  1980).

2.    U.S. Environmental  Protection  Agency.   "Laboratory Preparation  Procedures
     for Analytical  Screening of Hazardous  Waste Site Samples."   National
     Enforcement Investigations Center, U.S. EPAj Denver,  Colorado.   9 p.
     (No date).

3.    American Society for Testing and Materials.  "Measuring  Volatile Organic
     Matter in Water by Aqueous Injection Gas Chromatography."   Method D-2908.
     1983 Annual Book of ASTM Standards.   Section 11.  Philadelphia,
    .Pennsylvania.  (1983)
                                    11-24

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III.   PROCEDURES FOR ORGANIC COMPOUNDS

-------
                                   SECTION 1

                          VOLATILE ORGANIC COMPOUNDS
A.  SCOPE
     Volatile organic compounds are characterized  by  high  vapor  pressure.   This
common property is used to isolate the compounds from the  sample matrix  prior
to GC/MS identification and measurement.   Detailed methods are presented in
Subsection J for the determination of volatile  organic compounds in  hazardous
wastes, municipal and industrial  discharges,  ground and surface  water, sedi-
ments, biological tissue, and air.  Method detection  limits  for  these compounds
using multi-component, screening  procedures will vary with sample  matrix and
sample size.

B.   SAMPLE HANDLING AND STORAGE

     The same physical property that is used  to isolate volatile compounds
from the sample matrix can create potential problems  when  trying to  maintain
sample integrity prior to analysis.  Because  of the volatility of  the analytes,
samples should be collected in as quiescent a manner  as possible,  and the
sample bottles should be comoletely Billed (no  head spaca).   These steps are
intended to minimize the loss of  volatile constituents through degassing.
Also, the sample bottle should remain hermetically sealed  until  the  time of
analysis.

     All samples must be iced or refrigerated from the time  of collection
until the time of analysis (Figure 1).  This  step  should also counter the
tendency for these compounds to vaporize.  If the  sample source  is known to
contain residual chlorine, sodium thiosulfate should  be added as a preser-
vative to the sample bottles just prior to sample  collection. Addition  of  10
mg/40 ml is sufficient to neutralize 5 ppm chlorine.   If necessary,  a separate
sample aliquot can be analyzed for total  chlorine  content  using  a  field  kit.
A stoichiometric quantity of sodium thiosulfate plus  a 10-percent  excess can
then be added to neutralize the chlorine.! The completely filled  (no head
space) and sealed sample bottle should be shaken vigorously  for  1  minute to
ensure uniform distribution of the preservative.   There are  no known chemical
preservatives for solid-phase samples due to  the uncertainty of  attaining homo-
geneous mixing.

     Evidence suggests that certain aromatic  compounds, such as  benzene,
toluene, and ethyl benzene, are susceptible to  rapid  biological  degradation.2
Refrigeration alone w-5"P not orovide ^deauate *amole  observation  *cr these
compounds in all types of waters.  Therefore, when these compounds are of
interest, a separata sample should be collected for analyses and preserved

                                     III-2

-------
SAMPll
NATRII
           UATEM ON
           LEACHATE
SAMPLE
PROCESSING
PURPOSE    TOTAL CONttNTRATIOR    TOTAL  CONCENTRATION
               IN HASTE             IN MATER
SAMPLE
CONTAINER
SAMPLE
PRESERVATIVE
STORAGE
TIME
SAMPLE
SIZE
 14 d
25




SLUDGE. SOIL
OR SCUINCNT


MET
SI OH AGE


PURGE


ANALYZE






DDT
STORAGE





1
FROZEN
STORAGE

T;)TAl
LONCEMRATION IN
SID INC NT

0
BIOLOGICAL
TISSUE


FROZEN
STORAGE


I>URGE
ANALYZE


TOTAL
TISSUE
ONUNTRAT10N


CO
Air


TENAX
CARTRIDGE


PURGE


ANALYZE
TOTAL
NCENTRATIOI
IN AIR
                            14
1-10 g
            CARTRIDGE


              -tt'C



             4 UCEKS



             VARIABLE
      Figure  1.   Handling and  sample  storage  Information  for  volatile organic  analysis.

-------
with hydrochloric acid.  It is suggested that  a  500-ml  sample  be collected  in  a
clean glass container (no head space).   The pH of  this  sample  should  be  adjusted
to about 2 with 1:1 HC1.   (This sample  should  be stirred to  ensure  adequate mix-
ing of the preservative,  but in such a  manner  as to  prevent  or minimize  degassing
of sample constituents.)   The preserved sample should be quiescently  transferred
to a clean 25-ml  sample bottle and  hermetically  sealed  to exclude all  trapped
air bubbles.  If residual chlorine  is known or suspected to  be present,  sodium
thiosulfate should also be added to the sample.

     Soil, sediment, or sludge samples  should  be stored in a field-wet condition
with refrigeration.  Drying or freezing are not  recommended  as storage techniques
since volatile constituents may be  lost during the drying or thawing  cycles.

     Organic vapors collected from  ambient air on  Tenax GC cartridges  are
stable and can be quantitatively recovered from  the  cartridge  sampler up to 4
weeks after sampling when the cartridges are tightly enclosed  in cartridge
holders and placed in a second container that  can  be sealed, protected from
light, and stored at -20C.34,5

     All samples must be analyzed within 6 to  28 days of collection, 16
depending on the sample matrix (Fiaure  1).

C.   INTERFERENCES

     The majority of contamination  problems encountered in volatile organic
analyses occur due to either impurities in the purge gas or  organic compounds
outgassing from the plumbing ahead  of the trap.  The analytical system must be
demonstrated to be free from contamination under the conditions of  the analysis   
by running laboratory  reagent blanks,   me use of  non-TFE plastic tubing,
non-TFE thread sealants, or flow controllers with  rubber components in the
purging device should be avoided.

     Samples.can be contaminated by diffusion  of volatile organics  (partic-
ularly fluorocarbons and methylene  chloride) through the septum seal  into the
sample container during shipment and storage.  A field reagent blank  con-
sisting of reagent water and treated by the sampling and sample processing
protocol used can serve as a check  on such contamination.

     Contamination by carry-over can occur whenever high-level and  low-level
samples are sequentially analyzed.    In  order to  reduce carry-over,  the purging
device and sample syringe must be rinsed with  reagent water  between sample
analyses.  Whenever a  sample is encountered that shows an unexpectedly high
concentration of volatile organics, it  should  be followed by an analysis of
reagent water to check for possible cross-contamination.  For  samples containing
large amounts of water-soluble materials, suspended solids,  high-boiling com-
pounds or high levels  of purgeable compounds,  it may be necessary to wash out
the  purging device with a detergent solution,  rinse it with  distilled water,
and  then dry it  in a 105C-oven between analyses.   The trap  and other parts, of
the  system are also subject to contamination;  therefore, frequent bakeout and
purging of  tne entire  jyscem ;nay oe  require'.;.
                                     III-4

-------
     Purging may cause some samples to foam excessively.   Any foam that  passes
through the gas transfer lines and enters the sorbent trap can have a  negative
effect on resultant sample analyses due to deactivation of the trap or the
introduction of nonvolatile artifacts.7  These problems can be countered by
the use of surfactants such as silicone antifoaming agents, the application  of
heat to dissipate the foam where possible, or cleaning of the analytical
system when necessary.

D.   SAFETY

     The following chemicals covered by this method have  been tentatively
classified as known or suspected carcinogens to humans or other mammals:
benzene, carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl
chloride.  Primary standards of these hazardous compounds should be prepared
in a functional fume hood.  A NIOSH/MESA-approved toxic gas respirator should
be worn when the analyst handles samples containing high  concentrations  of
these compounds.

     The toxicity or carcinogenicity of each reagent used in this method has
not been precisely defined; however, each chemical  compound should be  treated
as a potential health hazard.  From this viewpoint, exposure to these  chem-
icals must be reduced to the lowest possible level  by whatever means avail-
able.  Each laboratory is responsible for maintaining a current awareness file
of OSHA regulations regarding the safe handling of the chemicals specified in
this method.  A reference file of material data handling  sheets should also  be
made available to all personnel  involved in the chemical  analysis.  Additional
references to laboratory safety are available and have been identified for the
information of the analyst.8.3,19

E.   APPARATUS

1.   Sampling equipment, for discrete sampling.

     1.1  Vial - 25 ml capacity or larger, equipped with  a screw cap with hole
          in center (Pierce No.  13075, or equivalent).  Wash with detergent,
          rinse with tap water and distilled water, and dry at 105C before  use.

     1.2  Septum - Teflon-faced silicone (Pierce No. 12722 or equivalent).
          Wash with detergent, rinse with tap water and distilled water, and
          dry at 105C for 1 hour before use.

2.   Purge-and-Trap device - The purge-and-trap device consists of three
     separate pieces:  the sample purger, the trap, and the desorber.

     2.1  The sample purger must be designed to accept 5  ml-samples with a
          water column at least 3 cm deep.  The head space between the water
          column and the trap must have a total volume of less than 15 ml.
          The ourae qas must oass through the water column as finely divided
          bubbles with buobie diameters of less :nan j .Tin jt the origin. The
          purge nas must be introduced no more than 5 mm  from the base of the
          water column.  The sample purger illustrated in Figure i meecs cnese
          design criteria.

                                     III-5

-------
      Optional
      Foam
      Trap
  xit 1/4 in.
   O. D.

14 mm.
    O. D.

Inlet  1/4 in.
   O. D.
        1 /4 in.
       O. O. Exit
          Sample Inlet
          2 Way Syringe Valve

          17 cm. 20 Gauge Syringe Needle

         '6 mm. O. D. Rubber Septum

       10 mm. O.D.
       10.mm. Glass Frit
       Medium Porosity
                                          Inlet
                                       1/4 in. O. D.
                             ^1/16 in. O.D.
                                Stainless Steel
                                                        13X Molecular
                                                         Sieve Purge
                                                          Gas Filter
                                   Purge Gas.
                                  Flow Control
                      Figure 2.   Purging device.
2.2  The trap must be at least 25 cm  long and have an  inside diameter of
     at least 0.25 cm.  The trap must be packed to contain the following
     minimum lengths of adsorbents:   1.0 cm of methyl  silicone-coated
     packing (Subsection F.3.2), 15 cm of 2,6-diphenylene oxide polymer
     (Subsection F.3.1), and 8 cm of  silica gel (Subsection F.3.3),  The
     minimum specifications for the trap are illustrated  in Figure 3.
                                  III-6

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               Packing Procedure
                 Contraction
             Smm
             8cm
            15 cm
             1cm
            Smm
Glass
Wool
Grade IS
Silica Gel
Tenax
                   Trap Inlet
3% OV-1
 Glass
 Wool
Compression
 Fitting Nut
and Ferrules


 14ft. 7 n /Foot
 Resistance Wire
 Wrapped Solid


 Thermocouple/
   Controller
    Sensor
                             Tubing 25 cm.
                             0.105 in. I. D.
                             0 125 in. O.D.
                             Stainiess Steel
    Figure 3.  Trap packings and construction to include desorb capability.


     2.3  "ITie desorber must be capable  of  rapidly heating the trap tc 130C.
          The polymer section of the  trap  should not be heated higher thai
          180C and the temperature of  the remaining sections should not
          exceed 220C.  The desorber design  illustrated in Figure 3 meets
          these criteria.

     2.4  The purge-and-trap device may be assembled as a separate unit or be
          coupled to a gas chromatograph as illustrated in Figures 4 and 5.

3.   Vacuum extraction equipment for  the recovery of volatile organic com-
     pounds from sediments and biological  tissue.  A diagram of the apparatus
     for vacuum extraction and cryogenic concentration of volatile organic
     compounds from sediments and fish  tissue is shown in Figure 6.  The
     vacuum extractor can be assembled  from materials normally available in
     the laboratory.  The low pressure  necessary for extraction is supplied by
     a vacuum pump capable of producing a  10~3-torr vacuum and a flow rate
     of 25 1/min.  The concentrator traps  (25-ml Tekmar purging tubes, or
     equivalent) are used for condensing the  volatile vapors and transferring
     the extract to the purge-and-trap  device.   The concentrator trap is
     connected to the transfer lines  of the vacuum extractor with 1/8-inch
     compression Sittings and graphite  farruies.  The cransfar iir.es ure mace
     of glass-lined 1/4 inch O.D, stainless steel tubinq.  Gas-tiqht valves
     vVl-V'4), Hupro 8-48KT, are connected  with  compression fittings and
     graphite ferrules.  The 125-irl septum vial  containing the sample aliquot
                                      III-7

-------
    13X Motacuiai
     Swira Filter
                                         bqu*4
                                         lnr*ctton
                                          Port
                                                  Option*! 4-Port Column
                                                  Salocnon Varw
                                                               Column Own
                                                 (I I~I P	  Confirmatory Column
                                                -L4J-L  i >T.D~
                                                             ^ Analytical Column
                                                 -     Trip    (  Ott ]   Haat
                                                       22 "C    V     J   Control
                                                  Purging
                                                  O*viet
                                                          Not*.
                                                          All lin*t bnwn
                                                          trap and GC
                                                          nouM IM rwtd
                                                          toBO C
 Figure  4.    Schematic of purge-and-trap device  -  purge  mode.
      Prvtiur*
     A?ulator
      Purge
     G*
 Purg*       I
s*F.o*    [
Control       ^^\ t
    13X Mot*>cul*f -
    S*v Ftlt*tr
                                                               Column Qv*n


                                                            ^. Confirmatory Column

                                                               To Otctof

                                                            .> Anatyticjl Coi
-------
                  Velva 1
                      Sampla
                                                  Valve 4
Vacuum
 Pump
                              Concentrator
                                Trap
                         Figure 6.   Vacuum extractor.
     is connected to the system with a  one-hole  rubber stopper pierced  with
     the 1/4-inch O.D.  tubing.   A liquid nitrogen cold trap is placed between
     the vacuum pump and concentrator trap in order to prevent condensation  of
     pump oil  vapors in the concentrator trap.  The helium line and  pressure
     gauge are connected at a  junction  in the transfer line between  sample and
     V2 and are used primarily to test  ihe apparatus for 1-aaks.  The helium
     used is 99.999 percent pure, and normally,  because of the small quantities
     used, it  is not necessary to purify che gas further..  If  it is  desired,
     however,  a-gas filter trap can be  added to  the helium line to ensure
     purity.  An ultrasonic vibrator is used to  agitate the sample during extrac-
     tion.

4.   Gas chromatograph/mass spectrometer system.

     4.1  Gas  chromatograph -  An analytical  system complete with a tempera-
          ture-programmable gas chromatograph suitable for on-column injection
          and  all required accessories  including syringes, analytical columns,
          and  gases.

     4.2  Column - 1.8  m long  x 0.25 cm I.D. stainless steel or glass,  packed
          with 1 percent SP-1000 on Carbopack B  (60/80 mesh),  or equivalent.
          This column was used  to develop the method performance statements  in
          Subsection J.2.1.4.   Capillary columns can be used,  as long as the
          criteria of Subsection G are  met.

     4.3  Mass spectrometer -  Capable of scanning from 20 to 260 amu every 7
          seconds or less, utilizing 70 volts (nominal) electron energy in the
          electron impact ionization mode and producing a mass spectrum which
          meets all  the criteria 1n Table 1  when 50 ng of 4-bromofluorobenzene
          (BFB) is injected through the GC inlet.

     4.4  oC/HS interface - .-my GC-oo-MS ;ntsr*ace that jives  acceptable cal-
          ibration ooints at 50 ng per  injection for each of the parameters  of
          interest and  achieves all  acceptaoie performance criteria  (see

                                     III-9

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                     TABLE 1.   BFB KEY ION ABUNDANCE CRITERIA*!


                 Mass                       Ion  Abundance  Criteria
                  50                 15  to  40% of  mass  95
                  75                 50  to  70% of  mass  95
                  95                 Base peak, 100% relative  abundance
                  96                 5 to 9% of mass 95
                 173                 <1% of mass 95
                 174                 >50% of mass  95
                 175                 5 to 9% of mass. 174
                 176                 >50% of mass  95
                 177                 5 to 9% of mass 176
          ======================3==========3============================.
          Subsection I)  may be  used.   GC-to-MS  interfaces constructed of
          all-glass  or glass-lined materials  are  recommended.  Glass can be
          deactivated by silanizing with  dichlorodimethylsilane.

     4.5  Data system -  A computer system must  be  interfaced to the mass
          spectrometer that allows the continuous  acquisition and  storage on
          machine-readable media  of all mass  spectra obtained throughout the
          duration of the chromatographic program.  The computer must have
          software that  allows  searching  any  GC/MS data file for fons Of a
          specific mass  and plotting  sucn ion aoundances versus time or scan
          number.  This  type of plot  is defined as an Extracted Ion Current
          Profile  (EICP).   Software must  also be  available that allows
          integrating the abundance in any  EICP between specified  time or scan
          number 1imits.

 5.   Syringes  - 5  ml  glass hypodermic with  Luerlok tip (2 each), compatible
     with the  purging device.

 6.   Micro syringes  - 25 ul, 0.15 mm  I.D. needle.

 7.   Syringe valve - 2-way, with  Luer ends  (3 each), compatible with the
     purging device.

 8.   Syringe - 5 ml,  gas-tight  with shut-off  valve.

 9.   Bottle -  15 ml,  screw-cap, with  Teflon cap liner.

10.   Balance - analytical, capable of accurately weighing 0.0001 g.
                                     TII-10

-------
11.  Sample transfer implements - A series  of implements  are  suggested  for the
     rapid transfer of hazardous waste aliquots  from  sample containers  to
     laboratory glassware.   The intent is to provide  a  simple and  quick
     transfer process that  avoids or minimizes the  loss of volatile compon-
     ents.  Liquids of low to moderate viscosity may  be transferred using
     conventional  laboratory pipets.  Non-tacky  solids may be transferred
     using conventional  laboratory spatulas.  Spoon-shaped porcelain  spatulas
     (Coors No. 60478, or equivalent) are useful  in that  they have a  measure-
     able bowl-volume.  Samples having a desired approximate  volume can thus
     be obtained.   Transfer of tacky or non-tacky and semi-solids  may be
     simplified using the implements described below.

     11.1 Implement for transfer of non-tacky semi-solids.  A 3-ml glass
          hypodermic syringe is modified.   The plunger  is removed  and the
          normally closed end of the barrel  is cut  away.  To  use this imple-
          ment, the plunger is replaced flush with  the cut-away, open end.
          The device is pressed into a semi-solid sample, thereby  forcing the
          plunger out of the barrel.  When  the plunger has been displaced by a
          volume equal to the approximate sample volume desired, the  syringe
          is withdrawn and  the semi-solid plug is transferred to a tared
          vessel by displacing the material  with the  plunger.

     11.2 Implement for the transfer of tacky semi-solids and solids.   This
          approach will  be  useful  for the transfer  of some tacky or tarry
          materials.  Glass tubing of approximately 1 cm  I.D.  is cut  into short
          sections having a desired approximate  volume  (i.e.,  1 ml =  1.0 cm I.D.
          x 1.3 cm length).  To obtain a desired volume of sample  take  a tared
          tubing section of that volume, and using  a  Teflon-coated laboratory
          spatula, press a  portion of tarry sample  into the tubing section.
          The sample-filled tubing section  is then  placed directly into a
          centrifuge tube containing PEG (see Subsection  J.I.2); the  centrifuge
          tube and PEG are  weighed before the sample  is added.

     11.3 Implement for the transfer of viscous  liquids.  This device is fash-
          ioned by cutting  the constricted  end from a 5-ml graduated  pipet.
          The large-bore pipet thereby obtained  is  used in conjunction  with a
          conventional laboratory pipetting aid, preferably of the syringe
          type.  This implement allows convenient transfer and approximate
          volumetric measurement of some viscous liquids.

F.   REAGENTS

1.   Reagent water - Reagent water is defined as a  water  in which  an  inter-
     ferent is not observed at the method detection limit of  the parameters of
     Interest.

     1.1  Reagent  water may be generated by passing tap water through a carbon
          filter bed containing approximately 1  Ib. of activated carbon (Calgon
          Corp., Filtrasorb-300, or equivalent).
                                     III-ll

-------
     1.2  A water purification  system  (Millipore  Super-Q, or equivalent) may be
          used  to generate  reagent water.

     1.3  Reagent water may also  be  prepared  by boiling water  for  15 minutes.
          Subsequently, while maintaining  the temperature at 90C, bubble a
          contaminant-free  inert  gas through  the  water for  1 hour.  While
          still  hot,  transfer the water  to a  narrow-mouth screw cap bottle and
          seal  with a Teflon-lined septum  and cap.

2.   Sodium thiosulfate - (ACS) Granular.

3.   Trap materials

     3.1  2,6-Diphenylene oxide polymer  -  Tenax  (60/80 mesh),  chromatographic
          grade, or equivalent.

     3.2  Methyl silicone packing -  3% OV-1 on Chromosorb-W (60/80 mesh), or
          equivalent.

     3.3  Silica gel, (35/60 mesh) Davison Chemical  grade-15,  or equivalent.

4.   Methanol - Pesticide quality, or  equivalent.

5.   Stock standard solutions - Stock  standard solutions may be prepared  from
     pure standard materials or purchased  as  certified solutions.   Prepare
     stock standard solutions in  methanol  using  assayed  liquids or gasses as
     appropriate.  Because of the toxicity of some  of the organohalides,
     primary dilutions of these materials  should  be prepared In a  hood.   A
     NIOSH/MESA-approved toxic  gas  respirator should be  used when  the  analyst
     handles potentially hazardous  amounts of these materials.

     5.1  Place 9.8 ml of methanol  into  a  10-ml  ground glass stoppered vol-
          umetric flask.  Allow the  flask  to  stand, unstoppered, for  about  10
          minutes or until  all  alcohol-wetted surfaces have dried. Weigh the
          flask to the nearest  0.1 mg.

     5.2  Add the assayed reference  material  as  described  below:

          5.2.1  Liquids - Using  a 100-ul  syringe,  immediately add 2  drops  of
                 assayed reference material to the  flask,  then reweigh.   The
                 liquid must fall directly into  the alcohol without contacting
                 the neck of the flask.

          5.2.2  Gases - To prepare standards for any of the four halocarbons
                 that  boil below 30C (bromomethane, chloroethane, chloro-
                 methane, and vinyl  chloride), fill a 5-ml  valved  gas-tight
                 syringe with the reference standard to  the 5.0-ml mark.
                 Lower the needle to  5 mm above the methanol  meniscus.  Slowly
                 introduce the "eference standard above  the surface of the
                 liquid,   fhe neavy gas rapiaiy dissolves   in  cne methanol.
                                      T T T ,1 O

-------
5.3  Reweigh, dilute to volume, stopper, then mix by inverting  the  flask
     several  times.  Calculate the concentration in micrograms  per  micro-
     liter from the net gain in weight.   When compound purity  is  assayed
     to be 96 percent or greater, the weight  may be used  without  correc-
     tion to calculate the concentration of the stock standard.   Com-
     mercially prepared stock standards  may be used at any  concentration
     if they are certified by the manufacturer or by an independent source.

5.4  Transfer the stock standard solution into a Teflon-sealed  screw-cap
     bottle.   Store, with minimal headspace,  at -10 to -20C and  protect
     from light.

5.5  Prepare fresh standards weekly for  the four gases and  2-chloro-
     ethylvinyl ether.  All  other standards must be replaced after  1
     month, or sooner if comparison with check standards  indicates  a
     problem.

Secondary dilution standards - Using stock standard solutions,  prepare
secondary dilution standards in methanol that contain the compounds of
interest, either singly or mixed together.  The secondary dilution  stan-
dards should be prepared at  concentrations such that the  aqueous  cali-
bration standards prepared in Subsections H.3 or H.4 will bracket the
working range of the analytical system.12  Secondary dilution  standards
should be stored with minimal neadspace  and should be checked  frequently
for signs of degradation or evaporation, especially just  prior  to pre-
paring calibration standards from them.   Standards should be discarded  if
evaporation losses exceed 5 percent of the initial volume.   Quality
control check standards that can he-used to determine the accuracy  of
calibration standards will be available  from the U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.

Surrogate standard spiking solution - Select  a minimum of three surrogate
compounds from Table 2.  Prepare stock standard solutions for  each  sur-
rogate standard in methanol  as described in Subsection F  5. Prepare a
surrogate standard spiking solution from these stock standards  at a
concentration of 150 pg/10 ml in water.   Store the spiking  solution at
4C in Teflon-sealed glass containers with a  minimum of headspace.   The
solutions should be checked frequently for stability.  They should  be
replaced after 6 months.  The addition of 10 yl of this solution  to 5 ml  '
of sample or standard is equivalent to a concentration of 30 yg/1 of each
surrogate standard.  Surrogate standard  spiking solutions,  appropriate
for use with this method, will be available from the U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
                                111-13

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             TABLE 2.  SUGGESTED SURROGATE AND INTERNAL- STANDARDS3
                                Retention Time        Primary       Secondary
         Compound                   (Min.)              Ion            Ions


Surrogate Standards

Benzene-ds                         17.0                84
4-Bromofluorobenzene               28.3                95           174, 176
l,2-Dich1oroethane-d4              12.1
1,4-Difluorobenzene                19.6               114            63, 83
Ethylbenzene-d5                    26.4
Ethylbenzene-d^o                   26.4                98
FTuorobenzene                      18.4                96              70
Pentafluorobenzene                 23.5

Internal Standards

Bromochloromethane                  9.3               128          49, 130, 51
2-Bromo-l-chloropropane             nd                 77           79, 156
1,4-Dichlorobutane                  nd                 55            90, 92

aFor chromatographic conditions, see Tabie 3.
G.   QUALITY CONTROL

1.   A formal  quality control  program should be an integral  component  of the
     analytical  methods presented in this  section.  The  minimum  requirements
     for this  program consist  of an initial  demonstration of laboratory capa-
     bility, the analysis of spiked samples  as  a continuing  check  on method
     performance, and the analysis of blanks to demonstrate  that reagent
     interferences are under control.

2.   To demonstrate the ability to generate  data with  acceptable accuracy and
     precision,  the analyst must perform the following operations:

     2.1  For each parameter to be measured, select a  spike  concentration
          representative of the expected levels 1n the samples.   Using stock
          standards, prepare a quality control  check sample  concentrate in
          methanol 500 times more concentrated  than the  selected concentra-
          tions.  Quality control check sample  concentrates, appropriate for
          use  with this method, will  be available from the U.S.  Environmental
          Protection Agency, Environmental Monitoring  and Support  Laboratory,
          Cincinnati, Ohio 45268.
                                     111-14

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     2.2  Using a syringe, add 10 pi  of the check  sample  concentrate  and  10  yl
          of the surrogate standard  dosing  solution  (Subsection  F.7)  to each
          of a minimum of four 5-ml  aliquots of reagent water.   A  representa-
          tive wastewater may be used in place  of  the  reagent water,  but  one
          or more additional  aliquots must  be analyzed to determine background
          levels, and  the spike level  must  exceed  twice the  background level
          for the test to be  valid.   Analyze the aliquots according to the
          method beginning in Subsection J.2.1.

     2.3  Calculate the average percent recovery (R),  and the standard devi-
          ation of the percent recovery (s), for all parameters  and surrogate
          standards.  Wastewater background corrections must be  made  before  R
          and s calculations  are performed.

3.   The analyst must  calculate method performance criteria  for  each  of the
     surrogate standards.

     3.1  Calculate upper and lower  control limits for method performance for
          each surrogate standard, using the values  for R and s  calculated in
          Subsection G.2.3:
                    Upper Control  Limit (UCL)  = R  + 3 s
                    Lower Control  Limit (LCL)  *   - 3 s
          The UCL and LCL can be used to construct  control  charts^  that  are
          useful  in observing trends  in performance.

     2.2  For each surrogate standard, the ""aboratory -nust  deve^oo and  main-
          tain separate accuracy statements  of  laboratory performance for
          wastewater samples.  An accuracy statement  for the method  is  defined
          as R   s.  The accuracy statement  should  be developed by the  anal-
          ysis of four aliquots of wastewater as  described  in  Subsection
          G.2.2,  followed by the calculation of R and s.  Alternately,  the
          analyst may use four wastewater data  points gathered through  the
          requirement for continuing  quality control  in  Subsection G.4.   The
          accuracy statements should  be updated regularly.i3

4.   The laboratory is required to spike all  samples  with the  surrogate stan-
     dard spiking solution to monitor spike  recoveries.   If the recovery  for
     any surrogate standard does not  fall  within  the  method performance con-
     trol limits, the results should  be labeled as  suspect. The  laboratory
     should monitor the frequency of  qualified  data to insure  that it remains
     at or below  5 percent of total output.

5.   Each day, the analyst must demonstrate, through  the analysis of reagent
     water, that  interferences from the analytical  system are  under  control.

6.   It is recommended that the laboratory adopt  additional quality  assurance
     practices *or use with this method.  The soec-if'ic oracticss  that, are -nost
     productive depend upon the needs of the laboratory  and the nature  of the
     samples,  field duplicates may oe analyzed to  monitor  the prec'slon  of
     the sampling technique.  Whenever possible,  the  laboratory should  perform

                                     iil-i5

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     analysis of standard reference materials  and  participate  in  relevant  per-
     formance evaluation  studies.   Also,  any modifications of  the procedure  in
     response to changes  in  the  state-of-the-art should be evaluated  in terms
     of method performance.

H.   CALIBRATION

1.   Assemble a purge-and-trap device  that  meets the  specifications in Sub-
     section E.2.   Condition the trap  overnight at  180C by backflushing with
     an inert gas  flow of at least  20  ml/min.  Prior  to use, condition traps
     daily by backflushing for an additional 10 minutes at 180C.

2.   Connect the purge-and-trap  device to a gas chromatograph.  The gas chro-
     matograph must be operated  using  temperature  and flow rate parameters
     equivalent to those  in  Table 3.   Calibrate the purge-and-trap GC/MS
     system using  either  the internal  standard technique (Subsection  H.3)  or
     the external  standard technique (Subsection H.4).

3.   Internal standard calibration  procedure.  To  use this approach,  the
     analyst must  select  one or  more internal  standards that are  similar in
     analytical behavior  to  the  comoounds of interest.  The analyst must
     further demonstrate  that the measurement  of the  internal  standard is  not
     affected by method or matrix interferences.   Because of these limita-
     tions, no internal standard can be suggested  tnat is applicable  to all
     samples.  Due to their  generally  unique retention times,  bromochloro-
     methane, 2-bromo-l-chloropropane, and  1,4-dichlorobutane  have been used
     successfully  as internal standards.  Additional  compounds that may be
     used are listed in Table 2.

     3.1  Prepare  calibration standards dt  a minimum  of three  concentration
          levels for each parameter of interest.

     3.2  Prepare  a spiking  solution containing each  of the internal  standards
          using the procedures described  in Subsections F.5 and F.6.  It is
          recommended that the secondary  dilution  standard be  prepared at  a
          concentration of 15 yg/ml  for each internal standard compound.   The
          addition of 10  yl  of this  standard to 5.0 ml of sample  or calibra-
          tion standard would be equivalent to 30  yg/1.

     3.3  Analyze  each calibration  standard, according to Subsection  J, adding
          10 yl of internal  standard spiking solution directly to the syringe
          (Subsection J.2.1.5).  Tabulate the  area  response of the character-
          istic ions (Table  4) against concentration  for each  compound and
          internal standard  and  calculate response  factors (RF) for each
          compound using  Equation 1.


                            RF      (AsC1s)/(A1sCs)                         Eq. 1
                                     TIJ-16

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       TABLE 3.  CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
                      =========================================================
        Parameter
Retention Time
    (Min.)
  Column 1
    Method
Detection Limit
    (ug/D
Chloromethane
Bromomethane
Vinyl chloride
Chloroethane
Methyl ene chloride
Tri chl orof 1 uoromethane
1,1-Dichloroethene
1,1-Dichloroethane
Trans-1, 2-dichl oroethene
Chloroform
1,2-Di chloroethane
1 , 1 , 1-Tri chl oroethane
Carbon tetrachloride
Bromodi Chloromethane
1 ,2-Di chl oromethane
Trans-1 ,3-dichloropropene
Trichl oroethene
Benzene
Di bromochl oromethane
1,1 ,2-Tri chl oroethane
Ci s-1 ,3-dichl oropropene
2-Chloroethyl vinyl ether
Bromoform
1,1,2,2-Tetrachloroethane
Tetrachl oroethene
Toluene
Chlorobenzene
Ethyl benzene
1,3-Di chlorobenzene
1 ,2-Di chl orobenzene
1 ,4-Di chl orobenzene
2.3
3.1
3.8
4.6
6.4
8.3
9.0
10.1
10.8
11.4
12.1
13.4
13.7
14.3
15.7
15.9
Ib.o
17.0
17.1
17.2
17,2
18.6
19.8
22.1
22.2
23.5
24.6
26.4
nd
nd
nd
nd
nd
nd
nd
2.8
nd
2.8
4.7
1.6
1.6
2.8
3.3
2.8
2.2
6.0
5.0
1.9
4.4
3.1
5.0
nd
nd
4.7
6.9
4.1
6.0
6.0
7.2
nd
nd
nd
x==============================================================================

nd = not determined

Column Conditions:.  Carbopak B (60/80 mesh) coated with 1 percent SP-1000
packed in 1.8 m x 2-mm i.D.  giass column with hen urn jsrr-ier gas  at  2 -"! ow
rate of 30 ml/min.  Column temoerature is isothermal at 45C for  3 min, then
programmed at 8C per minute to 220 and neid for 15 mm.
                                     111-17

-------
            TABLE 4.  CHARACTERISTIC IONS FOR PURGEABLE ORGANICS
         Parameter
Primary
  Ion
        Secondary Ions
Chioromethane
Bromomethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorof1uoromethane
1,1-Dichloroethene
1,1-Dichloroethane
Trans-l,2-dichloroethene
Chloroform
1,2-Dichloroethane
1,1,1-Trichloroethane
Carbon tetrachloride
Bromodi chloromethane
1,2-Dichloropropane
Trans-l,3-dichloropropene
Trichloroethene
Benzene
Di bromochloromethane
1,1,2-Trichloroethane
Ci s-1,3-dichloropropene
2-Chloroethylvinyl ether
Bromoform
1,1,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
Chlorobenzene
Ethyl benzene
1,3-Di chlorobenzene
1,2-Dichlorobenzene
1,4-Dichlorobenzene
   50
   94
   62
   64
   84
  101
   96
   63
   96
   83
   98
   97
  117
  127
  112
   75
  130
   78
  127
   97
   75
  106
  173
  168
  164
   92
  112
  106
  146
  146
  146
             52
             96
             64
             66
         49, 51, 86
            103
           61, 98
    65, 83, 85, 98, 100
           61, 98
             85
        62, 64, 100
        99, 117, 119
          119, 121
        83, 35, 129
        63, .65, 114
             77
        95, 97, 132

       129, 208, 206
    83, 85, 99, 132, 134
             77
           63, 65
171, 175, 250, 252, 254, 256
   83, 85, 131, 133, 166
       129, 131, 166
             91
            114
             91
          148, 113
          148, 113
          148, 113
                                    111-18

-------
          where:

          AS   =   Area of response of the characteristic  ion  for the
                  analyte being measured
          Ais  =   Area response of the characteristic  ion for the  internal
                  standard.
          C.jS  *   Concentration of the internal  standard.
          Cs   *   Concentration of the analyte being measured.

          If the  RF value over the working range is a  constant  (<10%  RSD), the
          RF can  be assumed  to be invariant  and  the average RF  can  be used for
          calculations.  Alternatively, the  results can be used to  plot  a
          calibration curve  of response ratios,  AS/A-JS, vs RF.
     3.4  The working calibration curve  or RF  must  be verified on each working
          day by the measurement of one  or more  calibration  standards.   If the
          response for any parameter varies from the predicted response  by
          more than 10%,  the test must  be repeated using  a  fresh calibration
          standard.  Alternatively, a new calibration curve  must be prepared
          for that compound.

4.   External standard calibration procedure.

     4.1  Prepare calibration standards  at a minimum of  three concentration
          levels for each  parameter by carefully adding  20.0 yl of one or more
          secondary dilution  standards to 50,  250,  or 500  ml of reagent  water.
          A 25 yl  syringe  with a 0.15 mm I.D.  needle should  be used for  this
          operation.  On of  the external  standards should be at a concentra-
          tion near, but  above, the method detection limit (see Table 3) and
          the other concentrations should correspond to  the  expected range of
          concentrations  found in real samples or should define the working
          range of the GC/MS  system.  Aqueous  standards  may  be stored up to 24
          hours, if held  in sealed "vials with  zero  headspace.  If not so
          stored, they must be discarded after 1 hour.

     4.2  Analyze each calibration standard according to Subsection J and
          tabulate the area response of  the primary characteristic ion (see
          Table 4) against the concentration in  the standard.  The results can
          be used to prepare  a calibration curve for each  compound.  Alterna-
          tively, if the  ratio of response-to-concentration  (calibration
          factor) is a constant over the working range  (<10% relative stan-
          dard deviation,  RSD), linearity through the origin can be assumed
          and the average  ratio or calibration factor can  be used in place of
          a calibration curve.

     4.3  The working calibration curve  or calibration factor must be veri-
          fied on each working day by the measurement of one or more calibra-
          tion standards.   If the response for any  parameter varies from the
          predicted -espouse  by more than ilO%,  the test must be repeated
          using a fresh calibration standard.  Alternatively, a new calibra-
          tion curve or calibration factor tiust  be  prepared  *c- that
          parameter.
                                     111-19

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I.   DAILY GC/MS PERFORMANCE TESTS

1.   At the beginning of each day that  analyses  are to  be  performed, the GC/MS
     system must be checked  to see  if acceptable performance criteria are
     achieved for BFB.H  The performance  test must be  passed before any
     samples, blanks, or standards  are  analyzed, unless the instrument has met
     the DFTPP test described in Method 625  earlier in  the day.14

2.   These performance tests require the followtng instrumental parameters.

          Electron Energy:   70 Volts (nominal)
          Mass Range:       20 to 260
          Scan Time:         to give at  least 5 scans per peak but
                            not to  exceed  7  seconds per scan.

3.   At the beginning of each day,  inject  2  pi of BFB solution directly on
     column.  Alternately., add 2 ul of  BFB solution to  5.0 ml of reagent water
     or standard solution and analyze according  to Subsection J.2.  Obtain a
     background-corrected mass spectrum of BFB and verify that all the key ion
     criteria in Table 1 are achieved.   If all the criteria are not achieved,
     the mass spectrometer must be  retuned and the test repeated until all
     criteria are met.
                                     111-20

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J.  ANALYTICAL PROCEDURES

     1.1  Analysis of Solid Hazardous Waste Samples for Volatile Organic
          Compounds by Methanol  Partitioning
               Analytical Procedure:  available
               Sample Preparation:   available

          1.1.1  Reference/Title

                 U.S. Environmental  Protection Agency, "Method for Preparation
                 of Medium Concentration Hazardous Waste Samples."   U.S. EPA
                 Region IV, Athens,  Georgia,  p.  7. 1981.

          1.1.2  Method Summary

                 One gram aliquots  of soil, solid, aqueous liquid,  or non-
                 aqueous liquid  are  transferred to vials inside a chemical
                 carcinogen glovebox.  The samples are then removed from the
                 glovebox and diluted with methanol.  After mixing, an aliquot
                 of the methanol solution is added to reagent  water and the
                 resultant solution  is analyzed by the purge-and-trap GC/MS
                 technique.

          1.1.3  Applicability

                 This procedure  is  designed for the safe handling and prep-
                 aration of potentially hazardous samples  from hazardous waste
                 sites to be analyzed for organic chemicals including priority
                 pollutants.  The method is directed to contaminated soil
                 samples and waste  samples that may be solid,  aqueous liquid,
                 or non-aqueous  liquid and suspected to contain less than 10
                 percent of any  one  organic chemical component.  Method detec-
                 tion limits and retention times  for compounds quantitated by
                 this procedure  are  summarized in Table 3.

          1.1.4  Precision and Accuracy

                 This extraction and preparation  procedure was developed for
                 rapid and safe  handling of hazardous samples.  The design of
                 the method thus did not stress efficient  recoveries of all
                 components.  Rather, the procedure was designed for a mod-
                 erate recovery  of  a broad spectrum of organic chemicals.
                 Thus, analytical results may sometimes only reflect the mini-
                 mum amount of analyte in the sample.

          1.1.5  Sample Preparation

                 Place the oriainal  samcle container into  the  glovebox.
                 Additional items tnat snouia oe  in cne gioveoox inciuae 1}
                 :3libr.itad 2nd  *ared ?0-fi via1-; with cans, ?> ; soatula, 3)
                 a balance, 4) a capped vial containing 10 ml  of interference-
                 free methanol,  5) a capped vial  containing reagent water and

                                     111-21

-------
6) a medicine dropper.  The vial  of methanol  is  to be
used as a method blank.  (One method blank  should  be run
for each batch of 20 samples or less.)   Open  the sample
transportation can and remove the sample vial.   Note and
record the physical state and appearance of the  sample.  If
the sample bottle is broken, immediately repackage the
sample and terminate the analysis.

It is easy to contaminate a sample with purgeable  organic
materials.  Therefore, the following precautions must be
followed once sample processing has been initiated.

a.  There should be no solvent fumes or solvents,  other
    than the methanol  blank, inside the glovebox during the
    time the samples are being weighed  out.

b.  Tightly recap each sample container after weighing.
    Allow fumes from the first sample to be exhausted
    before opening the next sample.

Open the sample vial and mix the sample.  If  the sample is
a liquid, transfer a drop to the vial containing water to
determine if the sample is aqueous or non-aqueous.  Record
the result.  Transfer approximately 1 gram  (or  1 ml) of
sample to the tared calibrated vial. Wipe  the mouth of the
vial with tissue to remove any sample material and cap tne
vial tigntly.  Record the exact weight  of sample taken.
Reseal the original sample and replace  it in  the original
packing.

Remove the vials frcm the glovebox and  place  them  in an
efficient hood.  If the sample is liquid, dilute the sanple
as indicated in paragraph a.  If the sample is solid,
dilute the sample as indicated in paragraph b.

a.  Dilute the VOA sample to the 10 ml-mark with
    interference-free methanol.  Recap  the  sample  vial and
    shake.

b.  If the sample is soil or solid waste, add 10 ml  of
    interference-free methanol.  Recap  the sample  vial and
    shake.

Methylene chloride or other solvents should not  be used in
the hood when samples are being diluted.  Also,  the samples
should be stored in a solvent-free atmosphere at 4C prior
to purge-and-trap analysis.  Sample analyses  should be
completed within 14 days^ (Figure 1).
                  111-22

-------
1.1.6  Sample Analyses
         Transfer a  25-pl  aliquot  of  the methanol extract to 5 ml of
         reagent  water in  a  purge  device.   Process as a liquid
         sample (Subsection  J.2.).

         Adjust the  purge  gas  (helium)  flow rate to 40  3 ml/min.
         Attach the  trap inlet  to  the purging device, and set the
         device to purge.  Open the syringe valve located on the
         purging  device sample  introduction needle.

         Remove the  plunger  from a 5-ml syringe and attach a closed
         syringe  valve. Open the  sample or standard bottle which
         has  been allowed  to come  to  ambient temperature, and care-
         fully pour  the sample  into the syringe barrel to just short
         of overflowing.   Replace  the syringe plunger and compress
         the  sample.   Open the  syringe  valve and vent any residual
         air  while adjusting the sample volume to 5.0 ml.  Since
         this process  of taking an aliquot  destroys the validity of
         the  sample  for future  analysis, the analyst should fill a
         second syringe at this time.   The  second subsample can:

         1.  be used to prepare dilutions ^f ^analysis is required,

         2.  serve as  a backup  in  the event  the original subsample
             is lost,  or

         o.  serve as  a Jupllcate.

         Add  10.0 ill of the  surrogate spiking solution (Subsection
         F.7) and, if  applicable,  10.0  yl of the internal standard
         spiking  solution  (Subsection H.4.2) through the valve bore,
         then close  the valve.   The surrogate and internal  standards
         may  be mixed  and  added as single spiking solution.  Attach the
         syringe-syringe valve  assembly to  the syringe valve on the
         purging  device.   Open  the syringe  valves and inject the
         sample into the purging chamber.

         Close both  valves and  purge  the sample for 11.0  0.1
         minutes  at  ambient  temperature.

         After purging the sample, attach the trap to the chromato-
         graph, adjust the device to  the desorb mode, and begin the
         GC temperature program.   Concurrently, introduce the
         trapped  materials to the  GC  column by rapidly heating the
         trap to  180C while backflushing the trap with an inert gas
         between  20  and 60 ml/min  for 4 minutes.  If this rapid
         heating  requirement cannot be met,  the gas chromatographic
         column must De usea as a  secondary trap by cool Inn *t to
         30C (or subambient, if problems persist) Instead of the
         recommended initial temperature of 45"C.
                           111-23

-------
While the trapped material  is being  desorbed  into the  gas
chromatograph,  empty the purging  chamber  using  the  sample
introduction syringe.  Wash the chamber with  two 5-ml
flushes of reagent water.

After desorbing the sample  for 4  minutes, recondition  the
trap by returning the purge-and-trap device to  the  purge
mode.  Wait 15  seconds then close the syringe valve on the
purging device  to begin gas flow  through  the  trap.   Maintain
the trap temperature at 180C. Do not allow  the trap  tem-
perature to exceed 180C,  since the  sorption/ desorption is
adversely affected by heating the trap to higher temperatures,
After approximately 7 minutes turn off the trap heater and
open the syringe valve to  stop the gas flow through the
trap.  When cool, the trap  is ready  for the next sample.

If the response for any ion exceeds  the working range  of
the system, dilute the sample aliquot in  the  second syringe
(paragraph 1.1.6) with reagent water and  re-analyze.

If nothing is detected in  the methanol extract  of the  solid
phase sample, the process  can be  repeated using a larger
sample aliquot.

Calculate and report results as indicated in  Subsections K
and L.
                  111-24

-------
1.2  Analysis of Solid  Hazardous  Waste  Samples  for  Volatile
     Organic  Compounds  by Polyethylene  Glycol Partitioning
          Analytical  Procedure:   available
          Sample Preparation:  available

     1.2.1  Reference/Title

            Battelle  Laboratories,  "Manual  of Collaborators on Evaluation
            of Methods  for Analysis of  Hazardous Wastes."  Prepared under
            EPA Contract  68-03-3098.  Battelle  Columbus  Laboratories,
            Columbus, Ohio (1981).I6

     1.2.2  Method  Summary

            A portion of  solid waste is dispersed in  polyethylene qlycol
            (PEG) to  dissolve the purgeable organic constituents.1'  A
            portion of  the PEG solution is  combined with water in a spec-
            ially designed purging  chamber. An inert gas is then bubbled
            through the solution  at ambient temperature  and the purgeable
            components  are efficiently  transferred  from  the aqueous phase
            to the  vapor  phase.   The vapor  is swept through a sorbent
            column  where  the purgeable  components are trapped.  After
            purging is  completed, the sorbent column  is  heated and back-
            flushed with  inert gas  to desorb the purgeable components
            onto a  gas  chromatographic  column.  The gas  chromatographic
            column  is heated to elute the purgeable components which are
            detected  with a mass  spectrometer.!6

     1.2.3  Applicability

            This method covers the  determination of purgeable organic
            compounds in  a variety  of solid waste materials.  It is
            applicable  to nearly  all  types  of samples, regardless of
            water content, including aqueous sludges, caustic liquors,
            acid liquors, waste solvents, oily  wastes, tars, fibrous
            wastes, polymeric emulsions, filter cakes, spent carbon,
            spent catalysts, soils, and sediments.  The  method is based
            upon a  purge  and trap/gas chromatographic/mass spectrometric
            procedure.18

            The method  detection  limits for the procedure can be expected
            to vary with  the sample matrix. A  detailed  round-robin
            testing program is presently (1981-82)  underway to define
            these limits.

            This method shoul.d be restricted to use by or under the super-
            vision  of analysts experienced  in the use of purge-and-trap
            systems and gas chromatograph/mass  spectrometers and skilled
            
-------
1.2.4  Precision and Accuracy

       These variables  are presently being  evaluated.   Analytical
       precision and accuracy should be  similar to  the  limits  pre-
       sented in Subsection J.2.1.

1.2.5  Estimation of Total  Volatiles

       In order to avoid overloading the detector when  analyzing
       samples for volatile organic  compounds,  an approximate
       determination of total volatiles  is  made by  extracting  or
       dissolving a portion of sample with  n-hexadecane.   An aliquot
       of the n-hexadecane solution  is then analyzed by gas chro-
       matography.  The estimate  of  total volatile  content  is  based
       on the total area response for all components eluting prior
       to n-dodecane.  The estimated total  volatile content is
       calculated by applying a response factor obtained  for n-
       nonane to the total  area response defined above.

       The response factor and retention time for n-dodecane are
       determined by analyzing a  solution containing 0.20 mg/ml
       n-nonane and 0.20 mg/ml  n-dodecane in n-hexadecane.  Inject  a
       2-yl  aliquot of  this solution into a gas chromatographic
       system operated  under the  following  conditions:

         Column - 30 m  x 0.25 mm  I.D. DB-5  fused silica capillary
         column.

         Column Temperature - maintain the  initial  column tempera-
         ture at 20C for 4 minutes  and  then increase to  300C at
         8C per minute.  The final  column  temperature  is maintained
         for 10 minutes.

         Detector - Flame ionization detector.

       Determine the area response for n-nonane, and divide by 0.2
       to obtain the area response factor for n-nonane.  Record the
       retention time of n-dodecane.

       Add 1.0 gm of sample to 20 ml of  n-hexadecane and  2 ml  of  0.5
       M_ N32HP04 contained in a 50-ml glass centrifuge  tube.   Cap
       securely with a  Teflon-lined  screw cap.   Shake the mixture
       vigorously for 1 minute.  If  the  sample  does not disperse
       during the shaking process, sonify the mixture in  an ultra-
       sonic bath for 30 minutes. Allow the mixture to stand  until i
       clear supernatant is obtained.  Centrifuge if necessary to
       facilitate phase separation.

       Analyze a 2-ul aliquot of the n-hexane supernatant using the
       thromatograohic  
-------
       corresponding area of an n-hexadecane  blank.   Using the  area
       response factor determined  for n-nonane  in  paragraph  1.2.5,
       estimate the total volatile content  of the  sample  as  mg  of
       volatile components per gm  of  sample as  follows:


                 TVC  =     TARsample -   TAR  blank     x  20
                         n-Nonane  Area Response factor


       where:

             TVC  -  total volatile content of  sample in  mg/g

       TARsamp-]e  =  total area response  obtained  for the sample

       T"W*blank   =  total area response  obtained  for a blank.

       When using the purge-and-trap  GC/MS  analytical procedure, the
       total  quantity of volatile  components  injected should not
       exceed  approximately 10 yg. The volume  of  PEG solution  con-
       taining sample that is  analyzed is selected based  on  the GC
       screen  of the n-hexadecane  solution.

       a)   If  the total  volatile content  (TVC)  of  the sample, as
       determined in paragraph 1.2.5, is  1.0-mg/g  or  less, use  a
       200-yl  aliquot of the-PEG extract  prepared  according  to
       paragraph 1.2.6.

       b)   If  the TVC is greater than 1.0 mg/g, use an aliquot  of
       PEG extract that  contains approximately  10  yg.  The volume
       (in yl) of the aliquot  to be taken is  calculated by dividing
       200 by  the TVC.

       c)   If  the TVC is greater than 20  mg/g,  dilute a 200-yl  aliquot
       of  PEG  extract to 10 ml  with reagent PEG.   For this case,
       calculate the volume of the diluted  extract to be  taken  for
       analysis by dividing 10,000 by the TVC.

1.2.6  Preparation of Polyethylene Glycol (PEG) Solution

       To  a 50-ml  glass  centrifuge tube with  Teflon-lined cap,  add
       15  ml  of reagent  PEG using  a graduated pi pet.  Weigh  the cap-
       ped centrifuge tube and PEG on an  analytical balance  to  1 mg.

       Using  an appropriate implement (see  Subsection E.10),
       transfer 1 gm (10%) of sample to  the  PEG in the centrifuge
       tube.   Take care  not to touch  the  sample-transfer  implement
       to  che  PEG.
                                111-27

-------
       The transfer should  be  carried  out  in  such  a  fashion  that  the
       sample is  dissolved  in  or  submerged  in the  PEG  as expedi-
       tiously as possible.  This technique is intended to prevent
       loss of volatiles  from  the sample to a degree consistent with
       the requirements of  this method.17   After the sample  has
       been transferred,  recap the centrifuge tube and weigh on an
       analytical balance to 1 mg.  After  the sample weight  has been
       obtained,  add reagent PEG  to the 20-ml  mark on  the centrifuge
       tube and securely  recap the tube.

       Disperse the sample  by  vigorous agitation for 1 minute. The
       mixture may be agitated manually or  with the  aid of a vortex-
       mixer.  If the sample does not  disperse during  this process,
       sonify the mixture in an ultrasonic  bath for  30 minutes.
       Allow the  mixture  to stand until a clear supernatant  is
       obtained.   Centrifuge if necessary to  facilitate phase separa-
       tion.

       The supernatant solution may be stored for  future analytical
       needs.  If this is desired, transfer the solution to  a 10-ml
       screw cap  vial  with  Teflon cap  liner.   Store  at -10 to -20C,
       and protect from light.

1.2.7  GC/MS Analysis of  PEG Solution

       To the purging device,  add 5.3  ml of reagent water.   The add-
       ition is made using  a 5-ml -glass syringe equipped with a
       15-cm 20-22-gauge  needle.   The  needle  is inserted through  the
       sample inlet shown in Figur? 2.  The intsrnal diameter of  the
       14-gauge needle that forms the  sample  ^nlet will permit
       insertion  of a 20-gauge needle.

       Using a 25-vl  microsyringe equipped  with a  long needle,
       enrich the reagent water in the purging device with the ap-
       propriate  internal standards, surrogate standards, and BFB.
       Add the aliquot directly to the reagent water in the  purging
       device by  inserting  the needle  through the  sample inlet (see
       Figure 2).  When discharging the contents of the micro-
       syringe, be sure that the  needle is  well beneath the  surface
       of the water.

       Based on the calculations  in paragraph 1.2.5., take an
       appropriate aliquot  of  the PEG  solution described in  para-
       graph 1.2.6.  Use  a  microsyringe equipped with a long needle
       to measure and dispense the aliquot.   In order to load the
       PEG solution into  the microsyringe it  will be necessary to
       draw the solution  into  a disposable  glass pipet and dis-
       charge the viscous solution directly into the barrel  of the
       microsyringe.   Completely  fill  the barrel with PEG solution,
       and than ''nsert the  plunger ind ""owe*-  to ^he  .Jesired  -nark.
       Dispense the aliquot directly into the aqueous solution in
       the purging device by inserting che  neeale  througn the

                               111-28

-------
sample Inlet.  When discharging the contents  of the  micro-
syringe, be sure that the end of the needle is  well  below the
surface of the water.

Close the 2-way syringe valve at the sample'in-let.

Adjust the gas (helium) flow rate to 40  3 ml/min.   Attach
the trap inlet to the purging device, and set the device to
purge.  Open the syringe valve located on the purging device
sample introduction needle.

Close both valves and purge the sample for 11.0  0.1 minutes
at ambient temperature.

At the conclusion of the purge time, attach the trap to the
chromatograph, adjust the device to the desorb  mode, and
begin the GC temperature program.

Temperature - Isothermal at 45C for 3 minutes, then in-
creased at 8C per minute to 220C, and maintained at 220C
for 15 minutes.

Concurrently, introduce the trapped materials to the GC col-
umn by rapidly heating the trap to 180C while  backflushing
the trap with an inert gas between 20 and 60  ml/min  for 4
minutes.  If this rapid heating requirement cannot be met,
the gas chromatographic column must be used as  a secondary
trap by cooling it to 30C (or suDamoient, if proolems per-  
sist) instead of the recommended initial  temperature of 45C.

While the trapped material is being desorbed  into the gas
chromatograph, empty the purging chamber using  a glass
syringe equipped with a long needle.  Rinse the chamber with
two 5-inl flushes of reagent water.

(Between each use, wash the purging device with a detergent
solution, rinse it with distilled water, and  then dry it in
an oven at 105C.  It is convenient to have several  purging
devices available so that a clean unit 1s available  as
required.)

After desorbing the sample for 4 minutes, recondition the
trap by returning the purge-and-trap device to  the purge
mode.  Wait 15 seconds, then close the syringe  valve on the
purging device to begin gas flow through the  trap.   Maintain
the trap temperature at 180C.  Do not allow  the trap tem-
perature to exceed 180eC, since the sorption/desorption is
adversely affected by heatinq the trap to higher tempera-
tures.  After approximately 7 minutes, turn off the  crap
heater ind ooen the syringe valve to stoo the gas flow
through the trap.  When cool, the trap is ready for  the next
sample.

                         111-29

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If the response for any ion exceeds  the  working  range  of  the
system, repeat the analysis using  a  correspondingly  smaller
aliquot of the PEG solution described  in paragraph  1.2.6.

Calculate results as indicated in  Subsections  K  and'L.
                          111-30

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2.1  Analysis of Water Samples  for Volatile  Organic  Compounds
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     2.1.1  Reference/Title

            U.S. Environmental  Protection Agency,  "Purgeables  - Method 624."
            Federal  Register,  Vol.   44, No.  233:69532-69539. December 3,
            1979.18

     2.1.2  Method Summary

            An inert  gas  is bubbled through  a  5-ml water sample contained
            in a specially designed purging  chamber  at ambient temper-
            ature.  The purgeables  are efficiently transferred from the
            aqueous  phase to the  vapor phase.   The vapor is swept through
            a sorbent column where  the purgeables  are trapped.  After
            purging  is completed, the sorbent  column is heated and back-
            flushed with  the inert  gas to desorb the purgeables onto a
            gas chromatographic column.  The gas chromatograph is temper-
            ature programmed to separate the purgeables which  are then
            identified and quantified with a mass  spectrometer.^*^

     2.1.3  Applicability

            This method is appropriate for the determination of the
            listed volatile organic compounds  in municipal and industrial
            discharges, ground water, and surface  water samples.

     2.1.4  Precision and Accuracy

            The method was tested in 2 to 4  laboratories.*9  Reported
            method detection limits ^minimum concentration that can be
            measured  and  reported with 99 percent  confidence that the
            value is  greater than zero),20 average recoveries, and
            average  standard deviations for  the recovery data are pre-
            sented in Table 3  and Table 5.

     2.1.5  Sample Purging

            Table 3 summarizes the  recommended operating conditions for
            the gas chromatograph.   This table Includes retention times
            and method detection  limits that were  achieved under these
            conditions.  An example of the separations achieved by Column
            1 is shown in Figure  7. Other packed  columns or chromato-
            graphic conditions may  be used if  the  requirements of Sub-
            section G.2 are met.

            ^fter ?chiev?nn the key ''on abundance  criteria in Subsection
            H, calibrate  the system dally as aescnbea in ^uosection I.

-------
            TABLE 5.   ACCURACY AND PRECISION FOR  PURGEABLE ORGANICS



Parameter
Benzene
Bromodi chl oromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chl oroethane
Chloroform
Chl oromethane
Di bromochl oromethane
1,1-Dichloroethane
1,2-Dichl oroethane
1,1-Dichloroethene
Trans-1, 2-dichloroethene
1 ,2-Dichl oroorooane
Cis-l,3-dichlorooropene
Trans-1 ,3-di chl oropropene
Ethyl benzene
Methyl ene chloride
1 , 1 ,2 ,2-Tetrachl oroethane
Tetrachl oroethene
Toluene
1,1, 1-Trichl oroethane
1,1,2-Tri chl oroethane
Tri chl oroethane
Trichlorofluoromethane
Vinyl chloride
Reagent Water
Average
Percent
Recovery
89
97
94
90
91
94
67
90
91
5
83
102
74
90
94
95
91
109
82
81
97
96
92
102
106
59
103
Standard
Deviation
(%)
12
11
14
16
23
23
22
18
22
12
10
12
24
25
26
15
13
19
46
31
13
22
21
14
14
23
30
Wastewater
Average
Percent
Recovery
93
103
88
78
91
103
60
91
64
99
87
103
80
85
99
98
93
106
66
78 '
99
97
94
103
110
67
79
Standard
Deviation
(%)
24
31
12
15
33
24
23
26
28
17
21
. 27
32
35
30
' 20
16
28
66
31
26
25
36
19
22
48
22
Samples were spiked between 10 and  1000 yg/1.

  Column Conditions:  Carbopak B  (60/80 mesh)  coated with  1 percent SP-1000
  packed in a 1.8 m x 2-mm I.D. glass  column with helium carrier gas at a flow
  rate of 30 ml/min.  Column temperature is  isothermal  at  458C for 3 min.,
  then programmed at 8C per min. to 220 and  held  for  15  min.
                                     111-32

-------
Column 1% grMOOO on *upleacort

Program 48'C. 3 rnin . > p*> nwi to 220C

O*Mctor. MM SpocrranwMr
   I     I     I
   24*
I      I     I     I     I      I     I      I     I      I     I
     10    12    14    II    11     20 .   22    24    2    21
                                       Mmutn
Figure  7.   Gas chromatogram  of volatile organics by  purge and  trap.
                                     111-33

-------
       Adjust  the purge  gas  (helium)  flow  rate  to  40   3 ml/min.
       Attach  the trap inlet  to  the  purging device, and set the
       device  to purge.   Open the  syringe  valve located on the
       purging device sample  introduction  needle.

       Remove  the plunger from a 5-ml  syringe and  attach a closed
       syringe valve.  Open the  sample  or  standard bottle which has
       been allowed  to come to ambient  temperature, and carefully
       pour the sample into the  syringe barrel  to  just  short of
       overflowing.   Replace  the syringe plunger and compress the
       sample.  Open the syringe valve  and vent  any residual air
       while adjusting the sample  volume to 5.0 ml.  Since this
       process of taking an aliquot  destroys the validity of the
       sample  for future analysis, the  analyst  should fill a second
       syringe at this time.   The  second subsample can:

       1.   be  used to prepare dilutions if reanalysis is required,

       2.   serve as  a backup  in  the  event  the original  subsample is
           lost, or

       3.   serve as  a duplicate.

       Add 10.0 pi of che surrogate  spiking solution (Subsection F.7)
       and, if applicable, 10.0  yl of the  internal standard spiking
       solution (Subsection ri.4.2) through the  valve bore, then close
       the valve.' The surrogate and  internal standards may be mixed
       and added as  single spiking solution.

       Attach  the syringe - syringe  valve  assembly to the syringe
       valve on the  purging device.   Open  the syringe valves and
       inject  the sample into the  purging  chamber.

       Close both valves and  purge the  sample for  11.0   0.1 minutes
       at  ambient temperature.

2.1.6  Sample  Analysis

       When sample purging is complete, attach  the trap to the
       chromatograph, adjust  the device to the  desorb mode, and
       begin the GC  temperature  program.  Concurrently, introduce
       the trapped materials  to  the  GC'column by rapidly heating the
       trap to 180C while backflushing the trap with an inert gas
       between 20 and 60 ml/min  for  4 minutes.   If this rapid heat-
       ing requirement cannot be met, the  gas chromatographic column
       must be used  as a secondary trap by cooling it to 30C (or
       subambient, if problems persist) instead  of the  recommended
       initial temperature of 45C.

       '/hile the Crapped na^'-'al  ;s  beinn desorbed *ntc the gas
       chromatograph, empty the  purging chamber using the sample


                                111-34

-------
Introduction syringe.  Wash the chamber with  two  5-ml  flushes
of reagent water.

After desorbing the sample for 4 minutes,  recondition  the trap
by returning the purge-and-trap device  to  the purge mode.
Wait 15 seconds then close the syringe  valve  on the purging
device to begin gas flow through the  trap.  Maintain t^e trap
temperature at 180C.  Do not allow the trap  temperature to
exceed 180C, since the sorption/desorption is adversely
affected by heating the trap to higher  temperatures.   After
approximately 7 minutes, turn off the trap heater and  open the
syringe valve to stop the gas flow through the trap.   When
cool, the trap is ready for the next  sample.

If the response for any ion exceeds the working range  of the
system, dilute the sample aliquot in  the second syringe with
reagent water and reanalyze.

Calculate results as indicated in Subsections K and L.
                         111-35

-------
3.1  Analysis of Soil/Sediment  Samples
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     3.1.1  Reference/Title

            Hiatt, M.,  "Analyses  of Fish  and  Sediment  for  Volatile
            Priority  Pollutants."   Anal.  Chem. 3:1541-1543  (1981).21

     3.1.2  Method Summary

            This method is a modification of  that  used for Purgeable
            Organics  in Water.2  However, instead  of stripping the
            volatile  organic compounds  of interest from the  sample  matrix
            with an inert  gas,  the  compounds  are drawn off in a vacuum
            and condensed  in a  super-cooled trap.   The condensed vola-
            tile compounds are  then transferred to a conventional purge-
            and-trap  device and the sample  is processed as a water
            sample.  The sample is  vaporized  and swept through a sorbent
            column where the purgeables are trapped.   After  purging is
            completed,  the sorbent  column is  heated to desorb the purge-
            ables and the  sample  is backflushed onto a gas chromato-
            graphic column with an  inert  gas. The gas chromatograph is
            temperature-programmed  to separata the purgeables which are
            then identified and quantified with a  mass spectrometer.

     3.1.3  Applicability

            This method is appropriate  for  the determination of the
            listed volatile organic compounds in sediments.  Limited
            testing has shown  this  method to  produce better  compound
            recovery  than  conventional  purge-and-trap  or sample dilution
            techniques.21

     3.1.4  Precision and  Accuracy

            Data for the recovery of known  spikes  from a sediment matrix
            are presented  in Table  6.  The  average compound  recovery
            using the vacuum technique  was  96   7  percent  compared  to 82
            t 18 percent for direct purge and trap with a  diluted sample
            and 83  10 percent for direct  purge and trap  with thermal
            desorption.2l

     3.1.5  Sample Preparation

            The vacuum extractor  (Figure  6) must be airtight: and  free of
            moisture before an extraction can be started.  A clean  125-ml
            septum vial is connected, the vacuum pump  started, and  V2 to
            V4 are opened  to evacuate the apparatus.   The  elimination of
            ''ine condensation  is  iccomolished by warming the transfer
            lines while evacuating  the  system.  Heating tape is effective
            in creating even  cransfer 1;ne  temperatures and  can be  used

                                     111-36

-------
       TABLE 6.  SPIKE RECOVERY DATA FOR VOLATILE ORGANICS FROM SEDIMENT
                MATRIX USING THE VACUUM EXTRACTION TECHNIQUE*
       ====z======r=========================r================a=================

        Compound                      Percent  Recovery      Standard  Deviation
Chl oromethane
Bromomethane
Vinyl chloride
Chl oroethane
Methyl ene chloride
Tri chl orof 1 uoromethane
1,1 -Di chl oroethene
1 ,1-Di chl oroethane
Trans-1 ,2-dichl oroethene
Chloroform
1,2-Di chl oroethane
1,1,1-Tri chl oroethane
Carbon tetrachloride
Acrylonitrile
Bromodi chl oromethane
1 ,2-Di chl oromethane
Trans-1, 3-dichl oropropene
Trichl oroethene
Benzene
Di bromochl oromethane
1 , 1 , 2-Tri chl oroethane
Ci s-1 ,3-di chl oropropene
Bromoform
Tetrachl oroethene
1 , 1 ,2,2-Tetrachl oroethane
Toluene
Chlorobenzene
Ethyl benzene
98
86
108
106
Sample
80
82
101
92
102
96
106
100
89
96
96
31
98
94
98
98
32
90
104
98
102
101
97
22
24
35
27
Contaminated
8
9
7
10
11
17
11
13
8
8
4
6
6
4
10.
5
7
9
13
8
4
5
5
:a=a=a=====================:===a=a===============s=z===:============== ====== = = = = =
*After Hyatt21
            continuously during extraction.   The vacuum extractor is
            pressurized with helium by closing  V3 and  opening  VI
            (Figure 6).  The apparatus is then  leak-tested  by  applying
            soapy water on all  connections and  making  the appropriate
            adjustments when leaks are located.  When  the apparatus  is
            airtight, close VI  and open V3.   Heat the  transfer lines and
            concentrator trap" for 5 minutes  to  eliminate any contamina-
            tion from previous  extracts.   The system is now ready for
            sample extraction.

            Weigh out 10 g of sediment sample and transfer  to  a 125-ml
            septum vial.

                                     111-37

-------
       To begin the  extraction  process, close V2  (V3 and V4 remain
       open),  cool the  concentrator trap with a liquid nitrogen
       bath, and replace  the  empty 125-ml vial with the sample
       vial.   Disconnect  the  vacuum source by closing V3.  Open V2
       to permit vapors from  the  sample vial to reach the concen-
       trator  trap.   The  sample vial, containing  10 g of sediment
       sample, is then  immersed in the ultrasonic water bath.  The
       equilibrium temperature  generated in the ultrasonic bath is
       50C.   Therefore,  the  bath is  initia-lly filled with water at
       50C and that temperature  is maintained by continuous ultra-
       sonic operation.

       After 5 minutes  of ultrasonic  agitation, the vacuum source is
       connected by  opening V3  (Figure 6).  The lower pressure
       hastens the transfer of  volatile compounds from the sample to
       the super-cooled concentrator  trap.  After 15 minutes of
       vacuum, close V3 and open  VI to fill the system with helium
       until atmospheric  pressure is  obtained.  Close VI and V2 to
       isolate the concentrate.   The  sample extraction is now com-
       plete and the concentrate  is ready for transfer to a purge-
       and-trap device.  The  concentrate can be held in the liquid
       nitrogen bath for  up to  an hour prior to analysis.

3.1.6  Sample  Analysis

       Disconnect the sample  concentrator trap from the vacuum
       extractor and connect  it to the purge-and-trap device.  Some
       outgassing.may be  observed when the sample extract is melted;
       therefore, the axtract should  be kept frozen until the
       concentrator  trap  is attached  to the purge-and-trap device.
       Warm the concentrator  trap walls to loosen the extract and
       allow the ring of  ice  formed during condensation to drop to
       the bottom of the  trap.  To this partially melted extract,
       add 5 ml of distilled, deionized water containing the in-
       ternal  standard.  Continue processing the sample using a
       modified purge-and-trap  procedure.  Place the concentrator
       trap in an ice water bath  and  purge for 5 minutes.  Then
       immerse the concentrator trap  in a 55C water bath and purge
       for an  additional  7 minutes.   This modification is intended
       to standardize sample  handling procedures  in order to produce
       reproducible  purging efficiencies.

       At the  conclusion  of the purge time, attach the trap to the
       chromatograph, adjust  the  device to the desorb mode, and
       begin the GC  temperature program.  Concurrently, introduce
       the trapped materials  to the GC column by  rapidly heating the
       trap to 180C while backflushing the trap with an inert gas
       between 20 and 60  ml/min for 4 minutes.  If this rapid heat-
       ting requirement cannot  be met, the gas chromatographic
       column  nust ba used as 3 secondary two by -oolinq *t to 30C
       (or subambient,  if problems persist) instead of the recom-
       mended  initial temperature of  453C.

                               111-38

-------
While the trapped material  is being  desorbed  into the gas
chromatograph, empty the purging  chamber  using the  sample
introduction syringe.  Wash the chamber with  two 5-ml flushes
of reagent water.

After desorbing the sample  for 4  minutes, recondition the trap
by returning the purge-and-trap device to the purge mode.
Wait 15 seconds, then close the syringe valve on the purging
device to begin gas flow through  the trap.  Maintain the trap
temperature at 180C.  Do not allow  the trap  temperature to
exceed 180C, since the sorption/desorption is adversely
affected by heating the trap to higher temperatures.  After
approximately 7 minutes, turn off the trap heater and open the
syringe valve to stop the gas flow through the trap.  When
cool, the trap is ready for the next sample.

If the response for any ion exceeds  the working range of the
system, dilute the sample aliquot in the  second syringe with
reagent water and reanalyze.

Calculate results as indicated in Subsections K and L.

-------
4.1  Analysis of Volatile Organic  Compounds  in  Biological  Tissue
          Analytical  Procedure:  evaluated
          Sample Preparation:   available

     4.1.1  Reference/Title

            Hiatt,  M.,  "Analysis of Fish  and Sediment  for  Volatile
            Priority  Pollutants."   Anal.  Chem.  3:1541-1543  (1981).21

     4.1.2  Method  Summary

            This method is a modification of that  used  for Purgeable
            Organics  in Water.2 However, instead  of stripping the
            volatile  organic compounds  of interest from the sample matrix
            with an inert gas,  the compounds are volatilized  in a vacuum
            system  and  condensed in a super-cooled trap.   The condensed
            volatile  compounds  are then transferred to  a conventional
            purge-and-trap device  and the sample is processed as a water
            sample.  The sample is vaporized and swept  through a sorbent
            column  where the purgeables are  trapped.   After purging  is
            completed,  the sorbent column is heated to  desorb the purge-
            ables and the sample is backflushed onto a  gas chromato-
            graphic column with an Inert  gas.   The gas  chromatograph  is
            temperature-programmed to separate  the purgeables which are
            then identified and quantified with a  mass  spectrometer.

     4.1.3  Applicability

            This method is appropriate  for the  determination  of the
            listed  volatile organic compounds in fish  tissue.  Limited
            testing has shown  this method to produce better compound
            recovery  than conventional  purge-and-trao  or sample dilution
            techniques.21

     4.1.4  Precision and Accuracy

            Data for  the recovery  of known spikes  from a fish tissue
            matrix  are  presented in Table 7. The  average  compound
            recovery  using the  vacuum technique was 76  20 percent
            compared  to 44  16 percent for  direct purge and  trap with a
            diluted sample and  64   12  percent  for direct  purge and trap
            with thermal  desorption.21

     4.1.5  Sample  Preparation

            The vacuum  extractor (Figure  6)  must be airtight  and free of
            moisture  before an  extraction can be started.   A  clean 125-ml
            septum  vial is connected, the vacuum pump  started, and V2 and
            VA are  ooened to evacuate the ^ooaratus,   The  elimination of
            line condensation  is accomplished by warming the  transfer
            lines while evacuating the  system.  Heating tape  J;s af'ecflve
            in creating even transfer line temperatures and can be used

-------
     TABLE 7.  SPIKE RECOVERY DATA FOR VOLATILE ORGANICS  FROM FISH  TISSUE
                    USING THE VACUUM EXTRACTION TECHNIQUE*
    ;==========================================================================
        Compound
Percent Recovery
Standard Deviation
Chi oromethane
Bromomethane
Vinyl chloride
Chi oroethane
Methyl ene chloride
Trichlorofluoromethane
1,1-Dichloroethene
1,1-Di chl oroethane
Trans-l,2-dichloroethene
Chloroform
1 ,2-Dichl oroethane
1,1,1-Tri chl oroethane
Carbon tetrachloride
Bromodi chl oromethane
1,2-Dichloropropane
1,2-Dichloropropane
Trans-l,3-dichloropropene
Tricnl oroetnene
Benzene
Di bromochl oromethane
1 ,1 ,2-Tri chl oroethane
Ci s-1 ,3-dichl oropropene
Bromoform
Tetrachl oroethene
1 , 1 ,2 ,2-Tetrachl oroethane
Toluene
Chlorobenzene
Ethyl benzene
85
' 126
64
69
Sample
99
74
90
86
107
92
92
91
64
54
54
52
65
57
56
56
54
ND
NO
61
ND
64
ND
22
75
11
22
Contaminated
2
8
6
9
31
5
8
9
11
7
7
9
11
10
9
7
9
-
-
10
-
15
-
*After Hyatt21
            continuously during extraction.   The vacuum extractor is
            pressurized with helium by closing V3 and  opening  VI
            (Figure 6).  The apparatus is then leak-tested  by  applying
            soapy water on all  connections and making  the appropriate
            adjustments when leaks are located.  When  the apparatus  is
            airtight, close V-l  and open V3.   Heat the  transfer lines and
            concentrator trap for 5 minutes  to eliminate any contamina-
            tion from previous  extracts.   The system is now ready for
            sample extraction.

            Weign out 10 g of homogenized fish t'ssue  and transfer to a
            125-ml septum vial.
                
                                     111-41

-------
     .  To  begin the  extraction process, close V2 (V3 and V4 remain
       open), cool the concentrator trap with a liquid nitrogen
       bath, and  replace the empty 125-ml vial with the sample
       vial.  Disconnect the vacuum source by closing V3  Open V2
       to  permit  vapors from the sample vial to reach the concen-
       trator trap.   The sample vial, containing the fish tissue, is
       then  immersed in the ultrasonic water bath.  The equilibrium
       temperature generated in the ultrasonic bath is 50C.  There-
       fore, the  bath is initially filled with water at 50C and
       that  temperature is maintained by continuous ultrasonic
       operation.

       After 5 minutes of ultrasonic agitation, the vacuum source is
       connected  by  opening V3.  The lower pressure hastens the
       transfer of volatile compounds from the sample to the super-
       cooled concentrator trap.  After applying a vacuum for 15
       minutes, close V3 and open VI to fill the system with helium
       until atmospheric pressure is obtained.  Close VI and V2 to
       isolate the concentrate.  The sample extraction is now com-
       plete and  the concentrate is ready for transfer to a purge-
       and-trap device.  The concentrate can be held in the liquid
       nitrogen bath for up to an hour prior to analysis.

4.1.6  Sample  Analysis

       Disconnect the sample concentrator trap from the vacuum ex-
       tractor and connect it to the purge-and-trap device.  Some
       outgassing may be observed when the sample extract is melted;
       therefore, the extract should be keot frozen until the con-
       centrator  trap is attached to the purge-and-trap device.
       Warm  the concentrator trap walls to loosen the extract .and
       allow the  ring of ice formed during condensation to drop to
       the bottom of the trap.  To this partially melted extract,
       add 5 ml of distilled, deionized water containing the in-
       ternal  standard.  Continue processing the sample using a
       modified purge-and-trap procedure.  Place the concentrator
       trap  in an ice water bath and purge for 5 minutes.  Then 
       immerse the concentrator trap in a 55C water bath and purge
       for an  additional 7 minutes.  This modification is intended
       to standardize sample handling procedures in order to produce
       reproducible  purging efficiencies.

       At the  conclusion of the purge time, attach the trap to the
       chromatograph, adjust the device to the desorb mode, and
       begin the  GC  temperature program.  Concurrently,  introduce
       the trapped materials to the GC column by rapidly heating the
       trap  to 180  while backflushing the trap with an  inert gas
       between 20 and 60 ml/min for 4 minutes.   If this  rapid heat-
       ing requirement cannot be met, the gas chromatographic column
       must  be used  as a secondary trao by cooling it to 30C (or
       suoambient,   if prodiems persist;  instead of the recommenGea
       initial  temperature  of  45C,

                                111-42

-------
While the trapped material  is being desorbed  into  the  gas
chromatograph, empty the purging chamber using  the sample
introduction syringe.  Wash the chamber with  two 5-ml  flushes
of reagent water.

After desorbing the sample  for 4 minutes-, recondition  the  trap
by returning the purge-and-trap device  to the purge mode.
Wait 15 seconds, then close the syringe valve on the purging
device to begin gas flow through the trap.  Maintain the trap
temperature at 180C.  Do not allow the trap  temperature to
exceed 180C, since the sorption/desorption is  adversely
affected by heating the trap to higher  temperatures.   After
approximately 7 minutes, turn off the trap heater  and  open the
syringe valve to stop the gas flow through the  trap.   When
cool, the trap is ready for the next sample.

If the response for any ion exceeds the working range  of the
system, dilute the sample aliquot in the second syringe with
reagent water and reanalyze.

Calculate results as indicated in Subsections K and L.
                         111-43

-------
4.2  Analysis of Biological  Tissue Samples  for Purgeable Organic
     Compounds
          Analytical  Procedure:   available
          Sample Preparation:   available

     4.2.1  Reference/Title

            U.S. Environmental  Protection Agency,  "Extraction and Analysis
            of Priority Pollutants in Biological  Tissue."   Method PPB 10/80.
            U.S.  EPA, S&A Division, Region IV.   Laboratory Services  Branch,
            Athens, Georgia,  p.  7.  (1980).22

     4.2.2  Method Summary

            A 1-gram aliquot of homogenized fish  tissue is diluted with
            organic-free water.   The volatile compounds are stripped  from
            the sample at 55C and collected  in a  purge-and-trap  appa-
            ratus.  The compounds are backflushed  from the trap,  identi-
            fied and quantified using computerized GC/MS methodology.

     4.2.3  Applicability

            The limit of detection for chis methoa is usually dependent
            upon the level of interferences rather than instrumental
            limitations.  Where interferences are  not a problem,  the
            limit of detection for most compounds  analyzed by GC/MS is 2
            mg/kg (wet weight basis).

            This method is recommended for use only by experienced
            residue analysts or under the close supervision of such
            qualified persons.

     4.2.4  Precision and Accuracy

            This information is not presently available.

     4.2.5  Sample Preparation

            The fish must be ground in an area free of volatile organic
            compounds.  The preferred procedure is to usa a blender and
            blend equal amounts of dry ice with the fish.

             If the sample is very large, a food processor or meat grinder
            is used.

             Immediately after homogenizing the fish sample, weigh 1 g
            into a screw cap tube lined with aluminum foil.  Store in a
            freezer until analyzed on GC/MS for volatile organic
            compounds.

            Add 5 mi of organic-free water already spiked with the sur-
             rogate spike to the sample.  Replace the cap and shake the

                                      111-44

-------
       contents until  the solids are dispersed throughout  the
       water.

       Immediately place the tube on the purge-and-trap apparatus
       and heat at 55C for 12 minutes  while  purging.

4.2.6  Sample  Analysis

       The volatiles are trapped on a 24"  Tenax trap and backflushed
       onto the GC column at 180C for  4 minutes while  the column  is
       held at room temperature (50C).  The  GC is then programmed
       to 210C at 8C/min and held for 11 minutes.

       The volatile compounds are identified  and quantified by  the
       MS computer system.

       Calculate results as indicated in Subsections K  and L.
                                111-45

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5.1  Analysis of Air Samples  for Volatile  Organics
          Analytical  Procedure:   available
          Sample Preparation:   available

     5.1.1  Reference/Title

            Erickson, M.  D.,  C.  M.  Sparacino,  M.  K.  Alsup,  M.  T.  Giguere,
            R. W.  Handy,  and  E.  D.  Pellizzari, "Preliminary Study on
            Toxic  Chemicals in Environmental and'Human  Samples:   Part  II.
            Protocols for Environmental and  Human Sampling  and Analysis."
            EPA Contract  No.  68-01-3849.   Research  Triangle Institute,
            Research Triangle Park,  North  Carolina.   Prepared  for U.S.
            EPA, Office of Research  and Development, Washington,  D.-C.
            p. 304, (1981).

     5.1.2  Method Summary

            Recovery of volatile organics  from Tenax GC is  accomplished
            by thermal  desorption and  purging  witn  helium into a  liquid
            nitrogen cooled nickel  capillary trap4,5,23 ancj ^nen  intro-
            ducing the vapors into  a high  resolution gas chromatographic
            glass  column  where the  constitutents  are separated from
            each other.5>24  Characterization  and quantification  of the
            constituents  in the sample are accomplished by  mass spec-
            trometry either by measuring the intensity  of the  total :on
            current signal or by mass  fragmentography,5

     5.1.3  Applicability

            The linear range for the analysis  of  volatile organic com-
            pounds is a function of  the breakthrough volume of each
            specific compound trapped  on the Tenax  GC sampling cartridge
            and of the inherent limits of  detection  of  the  mass spec-
            trometer for each organic  compound.5,25   The breakthrough
            volume is defined as that  point  at which 50 percent of a
            discrete sample introduced into  a  sampling  cartridge  is lost.
            Although the  identity of a compound  is  not  known during
            ambient air sampling (therefore  its  breakthrough volume is
            also unknown), the compound can  still  be quantified after
            GC/MS/COMP identification  once the breakthrough volume has
            subsequently  been established.  The  breakthrough volumes for
            some volatile organics,  and verified  by  a previously  de-
            scribed technique,3.5 are  shown  in Table 8.5t23,26 The
            linear range  for quantisation  using  glass capillary columns
            on a gas chromatograph/mass spectrometer/computer  (GC/MS/COMP)
            is generally  three orders  of magnitude.3 Table 9  lists the
            overall detection limits for some  examples  of volatile
            organics.25
                                     111-45

-------
    TABLE 8.  TENAX GC BREAKTHROUGH VOLUMES FOR TARGET COMPOUNDS (LITERS)

                                               Temperature (C)
       Compound                 BP       50     60     70     80     90    100
Chloroform
Carbon tetrachloride
1,2-Dichloroethane
1,1,1-Trichloroethane
Tetrachl oroethyl ene
Trichl oroethyl ene
Chlorobenzene
61
77
83
75
121
87
132
56
45
71
31
481
120
1989
41
36
55
24
356
89
871
32
28
41
20
261
67
631
24
21
31
16
. 192
51
459
17
17
24
12
141
37
332
13
13
19
9
104
28
241
BP = Boiling Point in C.
      TABLE 9.  OVERALL THEORETICAL SENSITIVITY OF HIGH-RESOLUTION GAS
             CHROMATOGRAPHY/MASS SPECTROMETRY/COMPUTER ANALYSIS
                       FOR VOLATILE ORGANIC POLLUTANTS
                                            Estimated Detection  Limit3
              Compound                      ng/m^               ppt
Chloroform
Carbon tetrachloride
1,2-Dichloroethane
1,1,1-Trichloroethane
Tet rachl oroethyl ene
Tri chl oroethyl ene
Chlorobenzene
Benzene
200
250
32
66
2.5
10
2.10
100
420
400
8.15
12.45
0.38
1.92
0.47
210
      aLimits are calculated, on the basis of the breakthrough
       volume for 2.2 g of Tenax GC (at 70F) capillary column
       performance and sensitivity of the mass spectrometer to that
       comoound in the mass fraamentoaraohv mode of most intense ion.

-------
5.1.4  Precision and Accuracy

       The reproducibility of  this  method  has  been  determined  to
       range from 10 to 30 percent  of the  relative  standard  devia-
       tion for different substances  when  replicate sampling car-
       tridges are examined.24,25,26,27 jne inherent  analytical
       errors are a function of  several  factors:   1)  the  ability to
       accurately determine the  breakthrough volume and  its  relation
       to field sampling conditions for each of the organic compounds
       identified; 2) the accurate  measurement of the ambient  air
       volume sampled; 3) the  percent recovery of the organic  from
       the sampling cartridge  after a period of storage;  4) the
       reproducibility of thermal  desorption for  a  compound from the
       sampling cartridge and  its  introduction into the  analytical
       system; 5) the accuracy of  determining  the relative molar
       response ratios between the  identified  substance  and the
       external standard used  for  calibrating  the analytical system;
       6) the reproducibility  of transmitting  the sample  through the
       high resolution gas chromatographic column;  and 7) the
       day-to-day reliability  of the  MS/COMP system.3-5,23-28

       The accuracy of analysis  is  generally 30  percent  but depends
       on the chemical and physical nature of the compound.3>5

5.1.5  Sampling Equipment

       a)  Personnel Monitor Pump

           A personnel monitor pump (MSA Co. - Model  C-2QO}  is ifsed
           for sample collection.   Flow rates  are adjusted  to   0.05
           1/min for an 8-hour collection  period.  Flows  are ad-
           justed such that a  total volume of 0.024 m3 air  is
           sampled for a given collection  period.

       b)  Sampling Cartridges

           The sampling cartridges are prepared  by packing  a  10 cm  x
           1.5 cm  I.D. glass tube containing 8 cm of  35/60  mesh Tenax
           GC with glass wool  in the  ends  to provide  support.5*28
           Sampling cartridges with longer Tenax  flow paths  can be
           used to achieve larger breakthrough volumes.25  Virgin
           Tenax  (or material  to be recycled) is  extracted  in  a
           Soxhlet apparatus for a minimum of 18 hours each with
           methanol and n-pentane prior to preparation of sampling
           cartridges.5*"  After purification of the Tenax GC
           sorbent and drying in a vacuum oven at 120C for 3  to  5
           hours  at 28 inches of water, the Tenax is  sieved to
           provide a  35/60 particle size range.   The  sieving and  all
           further sampling cartridge preparation steps should be
           conauctea  .r.  j ''^earr'   ~oom,  Camping cartridges  are
           then oreoared  and conditioned at 270C with helium  flow
                                111-48

-------
           at 30 ml/min for  30 minutes.   The conditioned cartridges
           are transferred to Kimax  (2.5  cm x 150 cm) culture
           tubes, immediately sealed  using Teflon-lined caps, and
           cooled.   This procedure is  performed in order to avoid
           recontamination of the sorbent bed.5

5.1.6  Sample Analysis

       The instrumental conditions for the analysis of volatile
       organics on the  sorbent Tenax  GC sampling cartridge are shown
       in Table 10.  The thermal desorption chamber and the six port
       Valco valve are  maintained at  270 and 240, respectively.
       The jet separator is  maintained at 240.  The mass spectrom-
       eter is set to scan the mass  range from approximately 20 to
       350.  The helium purge gas through the desorption chamber is
       adjusted to 15-20 ml/min.  The nickel capillary trap on the
       inlet manifold is cooled with  liquid nitrogen.  In a typical
       thermal desorption cycle, a sampling cartridge is placed in
       the preheated desorption chamber and the helium gas is chan-
       neled through the cartridge to purge the vapors into the
       liquid nitrogen  capillary trap (the inert activity efficiency
       of the trap has  been  shown in  a previous study).24,27 After the
       desorption has been completed,  the six-port valve is rotated
       and the temperature at the capillary loop is rapidly raised
       (greater than 10/min); the carrier gas cnen sweeps the vapors
       onto the high resolution GC column.  The glass capillary
       column is temperature programmed from ambient to 240C at
       4C/min and held at  che upper limit  r'or a minimum of 10 min-
       utes.  After all the  components have eluted, the column is
       cooled to ambient temperature and  the next sample is pro-
       cessed.5

       The standard can be  added as  an internal standard during
       sampling.  Since, however, the volume of air taken to produce
       a given sample is accurately  known, it is also possible and
       more practical to use an external  standard where the standard
       is introduced into the cartridge prior to its analysis.  Two
       standards, hexafluorobenzene  and octafluorotoluene, are used
       for the purpose of calculating RMR's  (Relative Molar Re-
       sponses).  It has been determined  that the retention times
       for these two compounds are such that they elute from the
       glass capillary column (SE-30) at  a temperature and retention
       time which does not  interfere with the analysis of unknown
       compounds in ambient  air samples.

       Since the volume .of air taken to produce a given sample is
       accurately known and  an external standard is added to the
       sample, then the weight per cartridge and hence the concen-
       tration of the jnxnown can je Jeterminea.   The approach for
       quantitating ambient  air pollutants  requires that the RMR be
       determined for each  constituent of interest during the
       analysis of field samples.   Every  sixth cartridge is a

                                111-49

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     1.1  The characteristic ions  for each  compound  of  interest must  have
          their maxima  in  the same or within  one  scan of each other.

     1.2  The retention time must  fall  within 30 seconds of the retention
          time of the authentic  compound.

     1.3  The relative  peak heights of the  three  characteristic ions  in the
          EICP's must fall  within  20 percent of  the relative intensities of
          these ions  in a  reference mass  spectrum.   The reference mass spectrum
          can be obtained  from a standard analyzed in the GC/MS system or from
          a reference library.

2.   Structural isomers that have  very similar mass  spectra and less  than 30
     seconds difference in retention time can be  explicitly identified only if
     the resolution between authentic isomers in  a standard mix is acceptable.
     Acceptable resolution is achieved if the baseline  to valley height
     between the isomers is less than 25  percent  of  the sum of the two peak
     heights.  Otherwise,  structural  isomers  are  identified as isomeric pairs.

L.   CALCULATIONS

1.   When a compound  has been identified, the quantitation of that parameter
     should be based  on the Intsgratsd abundance  from the E!C? of the first
     listed characteristic ion given in Table 4.   If the sample produces an
     interference for the  primary  ion, use  a  secondary  characteristic ion to
     quantitate.  Quantitation may be performed using the external or internal
     standard techniques.

2.   If the external  standard calibration procedure  is  used, calculate the
     concentration of the  parameter being measured from the area of the
     characteristic ion using the  calibration curve  or  calibration factor in
     Subsection H.3.2.   In order to eliminate the need  to obtain complete
     calibration curves for each compound for whicn  quantitative information
     is desired, use  the method  of relative molar response (RMR) fac-
     tors. 26t27,28  Successful use of this  method requires information on
     the exact amount of standard  added and the relationship of RMR (unknown)
     to the RMR (standards).  The  method of calculations is as follows:
               (1)
                                            Astd/Molesstd
                    A    system response,  height  or  area determined by
                          integration  or triangulation.

3.   The value of RMR is determined from at least  six inaepenaent ana-
     lyses. -^  Linearity over the dynamic range  and an 'ntercept of z
     has been previously described.^

-------
               ,  %
               (2)   RMRunk/std   =
                    A  =  system response,  as  above
                    g  =  number of  grams present
                  GMW  *  gram molecular weight .
               ,  %            Aunk'GMWunk-9   std
               (3)   Sunk   '
                             Astd.6MWstd.RMRunk/std


4.   If the internal  standard calibration  procedure  is  used, calculate the
     concentration in the sample using  the response  factor  (RF) determined  in
     Subsection H.4.3 and Equation  2.


                   Concentration yg/l   =   (^Cis)/(Ais)(RF)               Eq.  2


     where:

             As   =  Area of the characteristic  ion  for the parameter or
                     surrogate standard to be  measured

             ATS  =  Area ^ the characteristic  ion  for the internal standard

             C-jS  *  Concentration  of the  internal standard.


5.   If a sample is analyzed, and the ratio of the internal standard to sur-
     rogate standard is more than 20 percent higher  than that observed for  the
     calibration solution, these results  indicate that  the  internal standard
     in question was present as a sample component prior to spiking.  When
     this condition is observed, then an alternate calculation is  employed  in
     which the response factors (RF) are determined  based on the surrogate
     standards rather than the internal standards normally  used.   Calculations
     are carried out using Eq. 2 with the  exception  that the concentration  and
     area response of the surrogate standard are substituted for the internal
     standard.

6.   Report results in micrograms per liter.  The results for cis- and
     trans-l,3-dichloropropene should be  reported as total  1,3-dichloropropene
     (Storet No. 34561, CAS No. 542-75-6).  When duplicate  and spiked samples
     are analyzed, report all data  obtained with sample results.

7.   If any of the surrogate standard recoveries fall outside the  control
     limits which were established  as directed -'n Subsection G.3,  data for  all
     parameters in that sample must be  labeled as suspect.'

-------
                                  REFERENCES


1.   Analytical  Sciences Division.  "Master Scheme for the Analysis of Organic
     Compounds in Water.  Interim Protocols."   Chemistry and Life Sciences
     Group, Research Triangle Institute.   Research Triangle Park, North
     Carolina.  Prepared for Environmental  Research Laboratory, U.S.
     Environmental  Protection Agency, Athens,  Georgia.  1980.

2.   Bellar, T.  A., and J. J. Lichtenberg.   "Semi-Automated Headspace Analysis
     of Drinking Waters and Industrial  Waters  for Purgeable Volatile Organic
     Compounds," Measurement of Organic Pollutants in Water and Wastewater,
     C. E. Van Hall, editor, American Society  for Testing and  Materials,
     Philadelphia,  Pennsylvania.  Special  Technical Publication 686.  1978.

3.   Erickson, M. D, C. M. Sparacino, M.  K. Alsup, M. T. Giguere, R. W. Handy
     and E. D. Pellizzari.  "Preliminary  Study on Toxic Chemicals in Environ-
     mental and  Human Samples.  Part II.   Protocols for Environmental  and
     Human Sampling and Analysis."  EPA Contract No.  68-01-3849.   Research
     Triangle Institute, Research Triangle  Park, North Carolina.   Prepared for
     U.S.  Environmental Protection Agency,  Office of Research  and Development,
     Washington, D. C.   p. 304, 1981.

4.   Pellizzari, E. D.   Development of Method  for Carcinogenic Vapor Analysis
     in Ambient  Atmospheres.  Publication No.  EPA-650/2-74-121, Contract No.
     68-02-1228, p. 148, July, 1974.

5.   Pellizzari, E. 0.   Development of Analytical  Techniques for Measuring
     Ambient Atmospheric Carcinogenic Vapors.   Puoiication No. EPA-600/
     2-75-075, Contract No. 68-02-1228, p.  187,  November, 1975.

6.   "Preservation  and  Maximum Holding Time for the Priority Pollutants."
     U.S.  Environmental Protection Agency,  Environmental Monitoring and
     Support Laboratory, Cincinnati, Ohio 45268.  In preparation.

7.   Rose, M. E. and B. N. Colby.  "Reduction  in Sample Foaming and Purge and
     Trap  Gas Chromatography/Mass Spectrometry Analysis."  Anal.  Chem.  51:
     2176-2180.   1979.

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

 9.  "OSHA Safety and Health Standards, General Industry."  (29CFR1910),
     Occupational Safety and .Health Administration, OSHA 22-6.  (Revised,
     January, 1976).

10.  "Safetv in  Academic Chemistry Laboratories."   American Chemical Society
     Publication, Committee on Chemicai .Safety, 3rd edition.  197S.

-------
11.   Budde, W.  L., and J.  W.  Eichelberger.   "Performance Tests  for the
     Evaluation of Computerized  Gas  Chromatography/Mass  Spectrometry Equipment
     and Laboratories".   EPA-600/4-80-025,  U.S.  Environmental  Protection
     Agency, Environmental  Monitoring  and  Support  Laboratory,  Cincinnati,
     Ohio.   45268.  p. 16.   April  1980.

12.   Bellar, T. A., and  J.  J.  Lichtenberg.   "Determining Volatile Organics  at
     Micrograms per Liter Levels by  Gas  Chromatography." Journal  American Water
     Works  Association.  6_ pp.  739-744.   1974.

13.   U.S. Environmental  Protection Agency.   "Handbook  of Analytical  Quality
     Control in Water and  Wastewater Laboratories."   EPA 600/4-79-019,
     U.S.  Environmental  Monitoring  and  Support  Laboratory,  Cincinnati,  Ohio.
     45268.  March 1979.

14.   Eichelberger, J. W.,  L.  E.  Harris,  and W.  L.  Budde.  "Reference Compound
     to Calibrate Ion Abundance  Measurement in  Gas Chromatography - Mass
     Spectrometry Systems."  Analytical  Chemistry, 7, 995-1000.   1979.

15.   U.S. Environmental  Protection Agency.   "Method  for Preparation of Medium
     Concentration Hazardous  Waste Samples."  U.S. Environmental  Protection
     Agency,  Region IV,  Athens, Georgia,   p.  7.  May, 1981.

16.   Battelle Laboratories.  "Manual of  Collaborators  on Evaluation of Methods
     for Analysis of Hazardous Wastes."   Prepared  under EPA  Contract
     68-03-3098.  Battelle Columbus  Laboratories,  Columbus,  Ohio.  1981.

17.   Ligon, Jr., W. V.,  ana ri. Grace.   '?oiy\ethylene  glycol)  as  a Diluent
     for Preparation of Standards for Volatile-Drganics  in Water."  Analytical
     Chemistry, 53:920-921.  1981.

18.   U.S. Environmental  Protection Agency.   "Purgeables  - Method  624."
     Federal Register.  44 No. 233:69532-69539.   December 3,,, 1979.

19.   Kleopfer, R. D.  "Priority Pollutant Methodology Quality  Assurance
     Review."  U.S. Environmental  Protection Agency,  Region VII, Kansas City,
     Kansas.  Seminar for Analytical Methods for Priority Pollutants, Norfolk,
     Virginia.  January 17-18, 1980.  U.S.  Environmental Protection Agency,
     Office of Water Programs, Effluent  Guidelines Division, Washington, D. C.
     20460.

20.   U.S. Environmental  Protection Agency.  "Analytically Determined Method
     Detection Limits for Priority Pollutant Methodology as  Method Performance
     Criteria."  U.S. Environmental  Protection Agency, Environmental Monitoring
     and Support Laboratory, Cincinnati, Ohio.   In preparation.

21.   Hiatt, M.  "Analysis of Fish and Sediment for Volatile  Priority
     Pollutants."  Anal. Chem. 53:1541-1543.  1981.
                                     111-54

-------
22.  U.S.  Environmental  Protection Agency.   "Extraction and Analysis  of
     Priority Pollutants in Biological  Tissue."   Method PPB 10/80.   U.S.
     Environmental  Protection Agency,  SAA Division,  Region  IV,  Laboratory
     Services Branch,  Athens, Georgia,   p.  7.

23.  Pellizzari,  E.  D.,  J.  E. Bunch,  B.  H.  Carpenter and E. Sawicki.
     "Collection  and Analysis of Trace  Organic Vapor Pollutants in  Ambient
     Atmospheres.   Technique for Evaluating Concentration of Vapors by  Sorgent
     Media." Environ.   Sci. Technol.   9:  pp. 552-555.   1975.

24.  Pellizzari,  E.  D.   The Measurement  of  Carcinogenic Vapors  in Ambient
     Atmospheres.   Publication No. EPA-600-7-77-055, Contract No. 68-02-1228.
     p. 288.  June,  1977.

25.  Pellizzari,  E.  D.   "Analysis of  Organic Air Pollutants by  Gas
     Chromatography  and Mass Spectroscopy."  EPA-600/2-77-100.   p.  114.

26.  Pellizzari,  E.  D.   "Analysis of  Organic Air Pollutants by  Gas
     Chromatography  and Mass Spectroscopy."  EPA-600/2-79-057.   p.  243.

27.  Pellizzari,  E.  D.   "Ambient Air  Carcinogenic Vapors Improved Sampling and
     Analytical  Techniques  and Field  Studies."  EPA-600/2-79-0081.   p.  340.
     May,  1979.

28.  Pellizzari,  E.  D.,  J.  E. Bunch,  R.  E.  Berkley and J. McRae.
     "Determination  of Trace Hazardous  Organic Vapor Pollutants in  Ambient
     Atmospheres  by  Gas  Chromatography/Mass Spectrometry/Computer."  Anal.
     Chem.  48:803-807.   1976.

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                                   SECTION 2

                       ACID-EXTRACTABLE ORGANIC COMPOUNDS
A.   SCOPE
     Acid-extractable organic compounds can be measured using the analytical
procedures presented in Subsection J.   These compounds are extracted from the
initial sample matrix under acidic conditions and identified/quantified using
GC/MS procedures.


B.   SAMPLE HANDLING AND STORAGE

     Conventional water sampling practices should be followed except that the
bottle should not be prerinsed with sample oefore cai'ec'ion.   Grsb  samoles
should be collected in glass containers and refrigerated immediately.   Com-
posite samples should preferably be collected in glass containers and,  if
possible, refrigerated during the period of compositing.  ATI  automatic
sampling equipment must be free of Tygon and other potential  sources of
organic contamination.  When necessary, *he equipment :hould  be prerinsed with
hexane prior to use.

     Water samples should be iced or refrigerated from the,time of collection
until extraction.!  Chemical preservatives should not be used in the field
unless more than 24 hours will elapse  before sample delivery  to the  lab-
oratory.  If the samples cannot be extracted within 48 hours  of collection,
the recommended method of sample preservation is as follows:

1.   If the sample contains residual chlorine, add 35 mg of sodium thiosulfate
     per 1 ppm of free chlorine per liter of sample.1

2.   Adjust the pH of the water sample to pH of 7 to 10 using sodium hydroxide
     or sulfuric add.  Record the volume of acid or base used.1

     All water samples must be extracted within 7 days and completely analyzed
     within 30 days of collection (Figure 1).

     Sediment and soil samples may be  stored field-moist (refrigerated)
frozen, or dried.  The effective storage period is not known.
                                     111-56

-------
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Hazardou;
Hastes






1 Analyze 1 I Analyze
P. rpwe Total Total "Dissolved" Total Cone. Total Cone. Total Cone. Total Cone. Total
Cone. Cone. Cone. In Nobility Mobility In Soil. In Soil. In Soil. In Biological Cone. I
In Art In Mater Hater at pH S at pl S Sediment Sediment Sediment Tissue Waste
Container 666 C
S*ple
Preser-
vative 4*C 4C 4"C 4"C 4'c
Storage '
Tlae 7d 7d 7d
Staple
Si'e 11 11 11 Ju 9 - 50 9 |0 g la
   Mgure  1.   Sample handling and storage Information for samples scheduled for acid-extractable analysis.

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 C.    INTERFERENCES

      Solvents,  reagents,  glassware,  and  other  sample  processing  hardware may
 yield discrete  artifacts  and/or  elevated baselines causing misinterpretation
 of  chromatograms.   All  of these  materials must be demonstrated to be free from
 interferences under the conditions of  the analysis by  running method blanks.
 Specific selection of  reagents and purification of solvents by distillation in
 all-glass systems  may  be  required.

      Analytical  interferences associated with  samples  will vary  considerably
 from source to  source,  depending on  the  diversity of the industrial complex,
 municipality, or system being sampled.   Matrix interferences may be caused by
 components that  are coextracted  from the sample but are not normally of inter-
 est.  The most  common  such components  are petroleum-derived naphthenes, high-
 molecular-weight polymeric components, and.long-chain  components such as waxes
 and triglycerides.  The extent of such matrix  interferences will vary consid-
 erably from source to  source.  A cleanup procedure using gel permeation
 chromatography  has been incorporated into the  method  for certain cases to
 remove long-chain  and  high-molecular-weight material.  No cleanup procedure is
 available for the  removal  of naphthenes. When such matrix interferences are
 present, the sample extract is diluted arid  the method  detection  limit is
 increased proportionately.  Many of  the  matrix interferences are solvent-
 sxtractabla nonvolatile components whicn necessitate the -ncre frequent clean-
 ing of the GC  injection port and the more frequent removal of the injection
 end of the GC capillary column.

	Interferences due to fish oil in  tissue samples can be eliminated by
 acetonitrile partitioning.2 The ultrasonic probe used to assist in the
 partitioning process must be scrupulously cleaned between samples.  The pro-
 cedure consists  of rinsing the probe with solvent (acetonitrile) into the
 sample, removing residue  on the  probe  with  a wet tissue, rinsing the probe
 with methylene  chloride,  and sonicating  in  hexane for  3 to 4 minutes on 50
 percent pulse.


 D.    SAFETY

      The toxicity  or carcinogenicity of  each reagent  used in this method has
 not been precisely defined; however, each chemical compound should be treated
 as  a potential  health  hazard.  From  this viewpoint, exposure to  these chem-
 icals must be minimized by whatever  means available.   The laboratory is
 responsible for maintaining a current  awareness file  of OSHA regulations
 regarding the  safe handling of the chemicals specified in this method.  A
 reference file  of  material data  handling sheets should also be made available
 to  all personnel involved in the chemical analysis.   All operations Involving
 the use of methylene chloride, including the extraction of the waste sample,
 filtration of the  extract, and concentration of the extract, must be performed
 in  a fume hood.   Care  should be  taken  to avoid skin contact with methylene
 chloride.
                                      i 1 4- ,/

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E.   APPARATUS
 1.  Oven, drying.
 2.  Desiccator.
 3.  Crucibles, porcelain, squat  form,  Size  2  or  equivalent.
 4.  Sonicator Cell  Disruptor - Heat  Systems - Ultrasonics,  Inc., with  a  3/4 in.
     high gain probe,  375 watt or equivalent.
 5.  Beakers, 400-ml.
 6.  Separatory funnels  - 250 ml, 500 ml,  and  2 1  with  Teflon  stopcocks.
 7.  Drying tubes -  180  mm x 25 mm.
 8.  Glass wool,  Pyrex.
 9.  Furnace, muffle.
10.  Buchner funnels.
11.  Filtrator -  Fisher-9-789 or  equivalent.
12.  Drying column  - Twenty (20)  mm  I.D. pyrex chromatographic column equipped
     with coarse  glass frit or glass  wool  plug.
13.  Kuderna-Danish  (K-D) apparatus.
     13.1 Concentrator tube - Ten (10)  ml, graduated  (Kontes K-570050-1025, or
          equivalent).  Calibration must be checked.    Ground  glass stopper
          (size 19/22  joint) is used  to prevent evaporation of extracts.
     13.2 Evaporative  flask - Five hundred (500)  ml  (Kontes K-57001-0500, or
          equivalent).  Attach to concentrator tube with springs (Kontes
          K-662750-0012).
     13.3 Snyder  column  - Three-ball  macro (Kontes K-503000-0232, or
          equivalent).
     13.4 Snyder  column  - Two-ball micro (Kontes  K-569002-0219, or
          equivalent).
     13.5 Boiling chips  - Extracted,  approximately 10/40 mesh.
14.  Water bath - Heated, with concentric  ring cover, capable  of temperature
     control  (2n.
15.  Pins, ipproximately 35 cm x  ?5 cm-x 6 cm.
                                     111-59

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16.   Gas chromatograph -  Analytical  system  complete with  chromatographic
     column suitable  for  on-column  injection and all  required accessories
     including flame  ionization  detector  and electron capture detector, column
     supplies, recorders, gases,  and  syringes.

17.   Acid column and  analytical  conditions  - Supelcoport  (100/120) coated with
     1% SP-1240 DA, packed in  a  10  cm x 2 mm I.D. pyrex glass column.  Use ultra
     pure nitrogen carrier gas at a flow  rate of-30 ml/min.  Column tempera-
     ture held at 80C for 2 minutes  programmed to 290C  at 8C/min., and held
     at 290C for 16  minutes.

18.   Gas chromatograph/mass spectrometer, Finnigan 4000 and ..INCOS 2300 Com-
     puter Data System -  Capable  of scanning from 35  to 450 a.m.u. every 7
     seconds or less  at 70 V (nominal) and  producing  a recognizable mass
     spectrum at unit resolution  from 50  ng of DFTPP  when the sample is intro-
     duced through the GC inlet.  The mass  spectrometer must be  interfaced
     with a gas chromatograph  equipped with an injector system designed for
     splitless. injection  and glass  capillary columns  or an injector system
     designed for on-column injection with  all-glass  packed columns.  All
     sections of the  transfer  lines must  be glass or  glass-^ined and must be
     deactivated (use Sylon-CT,  Supelco,  Inc., or equivalent, to deactivate).

     NOTE:  Systems utilizing  a  jet separator for the GC  effluent are recom-
     mended since membrane separator: may lose 'ansitivity *or light molecules
     and glass frit separators may  inhibit  the. elution of polynuclear aro-
     matics.  Any of  these separators may be used provided that  it gives
     recognizable mass spectra and  scceotable calibration ooints at the limit
     of detection specified for  each  individual compound.

     A computer system must be interfaced to the mass spectrometer to allow
     acquisition of continuous mass scans for the duration of the chromato-
     graphic program.  The computer system  should also be equipped with mass
     storage devices  for  saving  all  data  from GC/MS runs.  There must be
     computer software available  to allow searching any GC/MS run for specific
     ions and plotting the intensity  of the ions with respect to time or scan
     number.  The ability to integrate the  area under any specific ion plot
     peak is essential for quantification.

19.   Extracting equipment.

     19.1 ADT tissumizer  (Tekmar SDT 182EN  or equivalent).

     19.2 Centrifuge  (IEC CU-5000 or equivalent).

     19.3 Screw-capped centrifuge bottles,  200 ml (Scientific Products C4144)
          with TFE-lined  screw caps.

     19.* Peakers 'or beaked  - 200 nil  or eouivalent.

     19.5 Glass jyringe - 53 il  squipped  with i  ISO Tit x  "5 mm T.D. TFE tube.
                                     111-60

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20.  Gel permeation chromatography cleanup.

     20.1 Chromatography column - 500 mm x 19 mm I.D.  (Scientific  Products
          C-4670-106 or equivalent).

     20.2 Bio-Beads S-X3, 200/400 mesh (Bio-Rad Laboratories  152-2750).

     20.3 Glass wool.

     20.4 Graduated cylinders - 100 ml.

     20.5 GPC Autoprep or equivalent  (Analytical  Biochemistry Labs,  Inc.  1002
          or equivalent with 25-mrn I.D.  column containing 50  to  60 g of  Bio-
          Beads S-X3) (Optional).

21.  Continuous Liquid-Liquid Extractors - Teflon- or  glass-connecting joints
     and stopcocks, no lubrication.  (Hershberg-Wolf Extractor - Ace Glass
     Co., Vineland, New Jersey.  P/N 6841-10, or equivalent).

F.   REAGENTS

1.   Sodium sulfate, granular anhydrous  and reagent grade.  Rinse  with
     methyiene cnioriae (20 mi/g) dna condition at 500C for  a minimum of 2
     hours.  Cool  in a desiccator and store in a glass bottle.

2.   Hexane - Pesticide quality, distilled in glass.

o.   rtcetone - Pesticide quality, distilled in glass.   .

4.   Methyiene chloride - Pesticide quality, distilled in glass.

5.   Petroleum ether - Pesticide quality, distilled .in glass.

6.   Diethyl ether - Preserved with 2% ethanol, pesticide quality  and dis-
     tilled in glass.  The ether must be free of peroxides  as indicated  by EM
     Quant Test Strips (EM Laboratories, Inc., 500 Executive  Blvd.,  Elmsford,
     New York. 10523).

7.   Florisil - PR grade (60/100 mesh).

8.   Methanol - Pesticide quality, distilled in glass.

9.   Sodium hydroxide solution, 50%,  extracted 3 times with methyiene
     chloride.

10.  Sodium hydroxide (ACS),-ION in distilled water.

11.  Sodium hydroxide (ACS), 6N in distilled water.

12.  Sulfuric acid (ACS), 6N in distilled water.

13.  Hydrochloric  acid (ACS), concentrated, 12N.

                                     111-61

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14.   Stock Standards  - Prepare  stock  standards  from  EPA  Priority "Pollutants
     Kits containing  the  preanalyzed  compounds  or  from alternative  commercial
     sources.  Dissolve 0.100 g of  assayed  reference material  in pesticide-
     quality isooctane or other appropriate solvent  and  dilute to volume  in a
     100-ml  ground glass  stoppered  volumetric flask.  The  concentration of
     this solution is 1.00 yg/ul.   Transfer the stock solution to a 15-ml
     Teflon-lined screw cap vial, store  in  a refrigerator, and check  fre-
     quently for signs of degradation or evaporation, especially just prior to
     preparing working standards.

15.   Calibration Standards - Prepare  calibration standards that contain the
     compounds of interest, either  singly or mixed together.   The standards
     should be prepared at concentrations that  will  completely bracket the
     working range of the chromatographic system (two or more  orders  of magni-
     tude are suggested).  For  example,  if  the  limit of  detection can be  cal-
     culated as 20 ng injected, prepare  standards  at 10  ug/ml, 100  yg/ml,
     1,000 wg/ml, etc. so that  injections of 1  to  5  yl of  the  calibration
     standards will  define the  linearity of the detector in the working range.

16.   Surrogate Standards  Addition - Surrogate standards  should be stable
     isotope derivatives  of compounds that  will  aid  in the monitoring of  the
     analysis procedures.  "Tie  number snd composition of surrogate  standards
     will, to some extent, be dependent  on  compound  avaiiaoility.   Where
     possible the stable isotope-labeled compounds  (which  are  mass  resolved
     from the unlabeled compound) will be utilized to simplify calculations in
     the measurement  of recovery.   The surrogate standards should be  added to
     the chilled ?olid sample via injection of  an  appropriate  volume  of the
     surrogate standard mix.  [The  sample sh'ould be  laced  with a quantity of
     surrogate standard which is equivalent to  5 ppm (e.g., 250 yg/50 g
     sample)].  The surrogate spike should  be added  to the solid samp^ in
     aliquots of the total volume to  be  added  (e.g., five  20-ul injections
     from a 100-yl syringe) while manually  stirring  the  material with a clean
     glass stirring rod to facilitate dispersion of  surrogate  compounds.  It
     is important that the sample and sample container are chilled  during the
     surrogate standard addition.   Surrogate standard addition should be
     accomplished as quickly as possible to minimize the loss  of the  most
     volatile analytes covered  by this method.   Upon surrogate standard
     addition, the isolation/cleanup  procedure  can be initiated.

     It is recognized that uniform  distribution of surrogate standards to a
     solid phase sample will not be obtained by this procedure.  Also, it must
     be recognized that the surrogates will not mirror the properties of  organic
     compounds which have been  associated with  soil  or sediment samples for
     decades.  Nevertheless, data generated from surrogate standards  will
     serve to exemplify a "best" case recovery  for the sample  in question, and
     will provide QA/QC data for every sample.

G.   QUALITY CONTROL

     Before processing any samples, demonstrate through  the analysis  of  a
method blank that all glassware and reagents are interference-free.  Eacn time
a set of  samples is extracted or there is a change in  reagents, a method  blank

                                     111-62.

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should be processed as a safeguard against chronic laboratory contamination.
Field replicates should be collected and analyzed to determine the precision
of the sampling technique.  Laboratory replicates should be analyzed to deter-
mine the precision of the analysis.  Fortified samples should be analyzed to
determine the accuracy (recovery) of the analysis.  Field blanks should be
analyzed to check for contamination introduced during sampling and transpor-
tation.

H.   CALIBRATION

     Prepare calibration standards that contain the compounds of interest,
either singly or mixed together.  The standards should be prepared at concen-
trations that will bracket the working range of the chromatographic system
(two or more orders of magnitude are suggested).  Assemble the necessary gas
chromatographic apparatus and establish operating parameters equivalent to
those required.  By injecting calibration standards, establish the linear
range of the analytical system and demonstrate that the analytical system
meets the detection limits requirements specified in Table 1.  Characteristic
ions for compounds in the acid-extractable fraction are summarized in Table 2.
If the sample gives peak areas above the working range, dilute and reanalyze.

     Internal standard method - The internal standard approach is acceptable
for all of the semivolatile organics.  The uti^'zatfon of the internal
standard method requires the periodic determination of response factors (RF)
that are defined in Equation H-l.

                  RF  =  (AsCis)/(AisCs)                                Eq. H-l

where:

        As   =  the integrated area or peak height of the characteristic
                ion for the analyte standard

        ATS  =  the integrated-area or peak height of the characteristic
                ion for the internal standard

        Cis  *  the amount (pg) of the internal standard

        Cs   =  the amount (pg) of the analyte standard.

     The relative response ratio for the analytes should be known for at least
two concentration values - 20 ng injected to approximate 10 pg/1 and 200 ng
injected to approximate the 100-pg/l level (assuming 1 ml final volume and a
2-pl injection).  Those compounds that do not respond at either of these
levels may be run at concentrations appropriate to their response.

     The response factor (RF} should be determined over all concentration
ranges for the standards (Cs) that are being determined.  [Generally, the
amount of 'nternal standard idded *o 
-------
          TABLE  1.   GAS  CHROMATOGRAPHY OF ACID-EXTRACTABLE COMPOUNDS
                        Storet   Retention time(a)
 Compound                 No.         (minutes)        Limit of detection^)
2-Chlorophenol
2-Nitrophenol
Phenol
2, 4-Dimethyl phenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
4-Chloro-3-methyl-
phenol
2,4-Oinitrophenol
2-Methyl-4,6-dinitro-
phenol
Pentachlorophenol
4-Nitrophenol
34586
34591
34694
34606
34601
34621
34452
34616
34657
29094
34646
==================
5.9
6.4
8.0
9.4
9.8
11.8
13.2
1-.5
-.5.:
17-5
20.3
=====
50
50
50
50
50
50
50
500
500 
C~C
50
==================
25
25
25
25
25
25
25
250
250
25
25
(a)l.8 m glass column (6.4 mm  O.D.  x  2 mm  I.D.) packed with  1 percent SP-1240
   DA coated on 100/120 mesh Supelcoport.   Carrier  gas:  helium at  30 ml per
   min.  Temperature program:   2 min  isothermal at  70, then 8 per min to
   200C. If desired, capillary or  SCOT  columns may be used.

(b)This is a minimum level  at  which the  entire analytical  system must give
   mass spectral  confirmation. (Nanograms  injected is based on a 2-yl
   injection of a 1-liter sample that has  been extracted and concentrated
   to a volume of 1.0 ml.)
Once this calibration curve has been determined,  it  should  be  verified daily
by injecting at least one standard  solution  containing  internal  standard.   If
significant drift has occurred, a new calibration curve must be  constructed.
To quantify, add the internal  standard *:o  *>e  ~oncantratad  samole  extract,  no
more than a few minutes before injecting into  the GC/MS system to  minimize the
possibility of losses due to evaporation,  adsorption, or chemical  reaction.
Calculate the concentration by using the previous equation  using the


                                     II1-64

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          TABLE 2.  CHARACTERISTIC IONS OF ACID-EXTRACTABLE COMPOUNDS

                                        Characteristic ions
 Compound
Electron Impact
Chemical  lonization
    (methane)
2-Chl orophenol
2-Nitrophenol
Phenol
2, 4-Dimethyl phenol
2, 4-Di chl orophenol
2,4 ,6-Tri chl orophenol
4-Chloro-3-methyl phenol
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
Pentachl orophenol
4-Nitrophenol
Phenanthrene (d-l)(a)
========= ==================
128
139
94
122
162
196
142
184
198
266
65
188
========
64
65
65
107
164
198
107
63
182
264
139
94
130
109
66
121
98
200
144
154
77
268
109
80
129
140
95
123
163
197
143
185
199
267
140
189
:==========
131
168
123
151
165
199
171
213
227
265
168
217
157
122
135
163
167
201
183
225
239
269
122
==============
(a) Suggested internal  standard.
appropriate response factor taken from the calibration curve.   Either deute-
rated or fluorinated compounds can be used as internal  standards  and surrogate
standards.  Phenol-de, pentafluorophenol, 2-perfluoromethyl  phenol,  and
2-fluorophenol have been suggested as appropriate internal  standards or
surrogates for the acid compounds.  The compounds used as  internal  standards
must be different from the surrogate standards.


I.   DAILY INSTRUMENT CALIBRATION

     At the beginning of each day, the mass calibration of the GC/MS system
must be checked and adjusted,.if necessary, to meet  specifications,  using
decafluorotriphenylphosphine (DFTPP). Each day acid-extractable compounds are
measured, the column performance specification with  pentachlorophenol  must be
met.  DFTPP can be mixed in solution with the pentachlorophenol to complete two
specifications with one injection, if desired.

     To perform the mass calibration test of the GC/MS system, the following
Instrumental  parameters are required:

                  Electron energy - 70 V (nominal)
                  Mass range - 35 to 450 amu
                  Scan time - 7 seconds or less
                  Source temperature - ^3G-OOQ0C
                                     II!-65

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GC/MS system calibration - Evaluate the system performance  each  day  that  it  is
to be used for the analysis of samples  or blanks  by  examining  the  mass  spec-
trum of DFTPP.  Inject a solution containing 50 ng of DFTPP and  check to
ensure that performance criteria  listed in Table  3 are met.   If  the  system
performance criteria are not met, the analyst must retune the  spectrometer  and
repeat the performance check.  The performance criteria must be  met  before  any
samples or standards may be analyzed.

     Column performance is evaluated by injecting 50 ng of  pentachlorophenol
into the instrument.  The tailing factor for the  resultant  peak, as  calculated
in Figure 2, must be less than five for the performance to  be  considered
acceptable.
              TABLE 3.  DFTPP KEY IONS AND ION ABUNDANCE  CRITERIA
        ==============================================================
         Mass               Ion Abundance Criteria
         51          30 to 60 percent of mass  198

         68          Less than 2 percent of mass  69

         70          Less than 2 percent of mass  69

        127          40 to 60 percent of mass  198

        197          Less than 1 percent of mass  198

        198          Base peak, 100 percent relative abundance

        199          5 to 9 percent of mass 198

        275          10 to 30 percent of mass  198

        365          Greater than 1 percent of mass 198

        441          Present but less than mass 443

        442          Greater than 40 percent of mass 198

        443          17 to 23 percent of mass  442
 ======3======================================================================
                                     111-66

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               Tailing Factor =
BC
AB
Example Calculation:  Peak Height = OE = 100 mm
                   10% Peak Height = BD = 10 mm
                   Peak Width at 10% Peak Height = AC = 23 mm
                     AB =11 mm
                     BC - 12 mm
                   Therefore: Tailing Factor =  = 1.1
       Figure 2.   Tailing factor  calculation.
                         111-67

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J.  ANALYTICAL PROCEDURES

     1.1  Analysis of Hazardous Waste Samples for Acid-Extractable Organic
          Compounds.
               Analytical Procedure:   available
               Sample Preparation:   available

          1.1.1  Reference

                 U.S. Environmental  Protection Agency,  "Method for Preparation
                 of Medium Concentration Hazardous Waste .Samples."  U.S.  EPA,
                 Region IV, Athens,  Georgia,  May, 1981.

          1.1.2  Method Summary

                 Approximately one-gram aliquots of soil,  solid,  aqueous
                 liquid, or non-aqueous liquid are transferred to vials inside
                 a chemical carcinogen glove  box.  The  acidified  samples  are
                 then extracted with  methylene chloride.  The methylene chlor-
                 ide extract is screened by GC/FIQ and,  based on  the screening
                 results, the sample extracts are appropriately concentrated
                 and analyzed with  a GC/MS system.

          1.1.3  Applicability

                 This procedure is  designed for the safe handling and prepar-
                 ation of potentially hazardous samples  from hazardous waste
                 sites for analysis  for organic acid-extractable  compounds.
                 The method is directed to contaminated  soil  samples and  waste
                 samples that may be solid, aqueous liquid,  or non-aqueous
                 liquid and suspected to contain less than 10% of any single
                 organic chemical component.   The method is  not designed  for
                 use with samples expected to contain less than 10 ppm of
                 specific acid-extractable compounds.  This  type  of sample,
                 such as sediment samples taken from leachate streams, should
                 be analyzed using a method for sediment/soil samples (Sub-
                 section J.3.1 or J.3.2).

          1.1.4  Precision and Accuracy

                 These extraction and preparation procedures were developed
                 for rapid and safe handling of hazardous waste samples.   The
                 design of the method thus did not stress efficient recoveries
                 of all components.   Rather,  the procedure was designed for
                 moderate recovery of a broad spectrum  of organic acids.   The
                 results of the analyses thus may sometimes  reflect only  the
                 minimum amount of the constituent present in the sample.

                 The procedure is designed to allow detection limits as low  as
                 10 pom for orgamc acld-extractaoie compounds.  Some samples,
                 however, iiay contain high concentrations of chemicals that
                 interfere with the analysis of other components  at low levels.

                                     TII-68

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                 The detection limits  in those cases may  be  significantly
                 higher.   Percent  recovery and standard deviation  information
                 on the use of this method in a single laboratory  is  presented
                 in Table 4.
      TABLE 4.  RECOVERY DATA FROM SOIL BY REGION IV  MEDIUM  CONCENTRATION
                           HAZARDOUS WASTE METHOD3
                                                          r================:
    Acid Fraction            Cone,  yg/gm       Avg.  %  Rec.        Std.  Dev.*
2-Chlorophenol
2-Nitrophenol
Phenol
2 ,4-Dimethyl phenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
4-Chloro-m-cresol
2,4-Dinitrophenol
4,6-Dinitro-2-cresol
Pentachlorophenol
4-Nitropnenol
20
20
20
20
20
20
20
160
40
40
130 .
89
87
85
89
92
89
89
52
87
88
31
3.4
4.1
5.0
4.1
4.9
5.2
5.0
3.3
3.4
2.8
5.0
:=========================
* -  one standard deviation based on three trials.
          1.1.5  Sample Preparation

                 Place the sample container into  the  glovebox.   Additional
                 items that should be in the glovebox include  (1) calibrated
                 and tared 20-ml  vials with caps, (2) a  spatula,  (3)  a
                 balance, (4) a capped vial  containing 10 ml of  interference-
                 free methanol, (5) a vial  of water,  and (6) a medicine
                 dropper.  The vial of methanol is  to be used  as  a method
                 blank (One method blank should be  run for  each  batch of 20
                 samples or less).  Open the sample transportation can and
                 remove the sample vial.  Note and  record the  physical state
                 and appearance of the sample. If  the sample  bottle  is
                 broken, immediately repackage the  sample and  terminate the
                 analysis.

                 Open the sample vial  and mix the sample.   If  the sample is i
                 liquid, transfer one drop to a vial  containing  water to
                 determine whether the sample is  aqueous or non-aqueous.

                 Record the results based on the  degree  of  mixing of  the
                 sample with the *ater.
                                     111-69

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       Transfer  approximately  1 gram  (or  1 ml) of the sample to a
       calibrated and tared 20-ml vial.   Wipe the mouth of the vial
       with  tissue to remove any excess sample material.  Cap the
       vial.   Record the exact weight of  sample taken..  Reseal the
       original  sample  and replace  it in  the original packaging.

       Proceed with a methylene chloride  extraction of the organic
       acid  compounds in the sample based on the miscibility of the
       original  sample  with water.  Follow paragraph  (a) for an
       aqueous sample,  paragraph (b) for  a non-aqueous sample, and
       paragraph (c) for a solid phase sample.

       a.   If  the sample has been classified as aqueous, dilute the
           sample with  10 ml of methylene chloride.   Cap the vial and
           shake the sample for 2 minutes.  Add 2 grams of anhydrous
           sodium sulfate to the vial to  absorb the water.  Shake the
           sample.

       b.   If  the sample has been classified as non-aqueous, dilute
           the sample to a final volume of 10 ml with methylene
           chloride.  Cap the  vial  and mix for 2 -inutes.  Add 1
           gram  of anhydrous sodium sulfate to absorb any water that
           may be present.  Shake the sample.

       c.   If  the sample has been classified as a solid, add 10 ml of
           methylene chloride.  Cap the sample and shake for 1 hour
           on  a  wrist action shaker.  Add 1 gram of anhydrous sodium
           sulfate to the sample and  thoroughly mix.

1.1.6  The extract should be screened by  GC/FID using the column
       indicated in Subsection E.   Prior  to use, standardize the
       GC/FID  detector  for full scale response to 40  ng/yl of
       dig-phenanthrene.

       If  the  response  of any  sample  component is greater than 25%
       of  the  dio-phenanthrene response,  analyze the  10-ml ex-
       tracts  prepared  in paragraph 1.1.5 by GC/MS (paragraph 1.1.7).

       If  the  sample extract does not produce a reponse that is
       greater than 25% of the dlO-phenanthrene response, concen-
       trate the extract under a gentle stream of nitrogen to a
       final volume of  1 ml and analyze by GC/MS (paragraph 1.1.7).

       GC/MS Analysis of the Acid Fraction.

1.1.7  At  the  beginning of each day that  analyses are to be per-
       formed, inject 50 ng of pentachlorophenol either separately or
       as  part of a standard mixture  that may also contain 50 ng of
       OFTPP,  *nto  the  ^nstrument.   """he tailing *actor for penta-
       chlorophenol, calculated as  indicated in Figure 2, should be
       less than 5.
                             f  ^ 
                              -/U

-------
Establish instrument operating conditions equivalent  to  those
provided below:

  Mass Spectrometer

  Mass range                   m/e 41-475
  Scan time                    7 seconds  or less
  Electron energy              70 eV
  Source temperature           280-300C
  Start acquisition            0.1 min. after stopping flow
  Column Conditions

  1.8 m glass column (6.4 mm 0.0.,  2 mm I.D.)  packed  with  3%
  SP-2250 coated on 100/120 mesh Supelcoport;  carrier gas:
  helium at 30 ml/min.   Temperature program:   isothermal for
  4 minutes at 50C, then increasing at 8/min to 270C, and
  hold at 270C for 30 minutes.   If desired, capillary or
  SCOT columns may be used in place of the packed column.

Program the GC/MS to operate in  the Extracted  Ion Current
Profile (EICP) mode, and colleci EICP's for the three char-
acteristic ions listed in Table  2 for each compound being
quantitated.  Operating in this  mode, calibrate the system
response for each compound using either the internal  or
external standard procedure.

If the internal standard approach is being used, the  standard
is not to be added to the sample extract until  immediately
before injection into the instrument.  Mix the extract thor-
oughly before withdrawing an aliquot for analysis.  Inject 2
to 5 yl of the sample extract using the solvent flush tech-
nique.

If external calibration is employed, record the volume of
extract and standard solution injected to the  nearest 0.05
pi.  If the response for any ion exceeds the linear range  of
the system, dilute the extract and  reanalyze.

When the extracts are not being  used for analysis, they
should be stored in vials capped with unpierced septa, in the
dark, and at 4C.

Proceed to Subsection K for qualitative identification
criteria and calculation of the  results.
                    111-71

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2.1  Analysis of Water Samples for Acid-Extractale Organic  Compounds
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     2.1.1  Reference

            U.S. Environmental Protection Agency,  "Semi-Volatiles  Determina-
            tion."  Method 625.   Federal  Register  44  No.  233:   69540-69551.
            December 3, 1979.

     2.1.2  Method Summary

            The pH of a 1-liter water sample is adjusted  to 2  or less and
            the sample 1s extracted with methylene chloride.   Following
            concentration of the extract, it is analyzed  on a  calibrated
            GC/MS.  Qualitative Identification is  performed using  the
            retention time and the relative abundance of  three charac-
            teristic ions.  Quantitative analysis  is  performed using
            internal  standard  techniques with a single characteristic ion.

     2.1.3  Applicability

            This method is applicable to the determination  of  those
            organic acids listed in Table 5 when they occur in aqueous
            samples such as municipal and industrial  discharges.   The
            method is designed to be used to meet  the monitoring re-
            quirements of the  National  Pollutants  Discharge Elimination
            System (NPDES). Method detection limits  and  characteristic
            ions for compounds measured by this procedure are  summarized
            in Tables 2 and 5.

            The method should  be restricted to use by, or under the
            direct supervision of, analysts experienced in  the operation
            of gas chromatograph/mass spectrometers and skilled in the
            interpretation of  mass spectra.

     2.1.4  Precision and Accuracy

            Average recovery data and method detection limits  based on
            the analysis of spiked reagent water by a single laboratory
            are presented in Table 5.  Analytical  recovery data for
            wastewater and reagent water samples are  presented in
            Table 6.

     2.1.5  Sample Extraction

            Samples may be extracted using separatory funnel  techniques
            or with a continuous extractor.  Where emulsions prevent the
                                     111-72

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    TABLE 5.   METHOD DETECTION LIMITS FOR ACID-EXTRACTABLE ORGANIC COMPOUNDS
                   IN REAGENT WATER ANALYZED BY METHOD 6254.5
====================================
Spike
Compounds ug
4-Chl oro-3-methyl phenol a
2, 4-Dichl orophenol3
2, 4-Dimethyl phenol3
2,4-Dinitrophenola
2-Methyl -4 ,6-di ni trophenol a
4-Nitrophenola
Pentachl orophenol3
Phenol3
2, 4, 6-Trichl orophenol3
BHC
BHC
4, 4 '-DDE
Endosulfan sul^ate
======
Level
/I
10
10
10
40
40
10
10
10
10
6
6
10
7
Average %
Recovery
71
60
57
94
77
52
87
28
64
69
56
69
79
MDL
ug/i
3.0
2.7
2.7
42
24
2.4
3.6
1.5
2.7
4.2
3.1
5.6
5.6

aMDL based on 8 aliquots of reagent water
         TABLE 6.   PRECISION AND ACCURACY DATA FOR THE DETERMINATION
                       OF ACID-EXTRACTABLE COMPOUNDS6
==========================================================
       Parameter
   Reagent Water
AverageStandard
Percent    Deviation
Recovery*     %
                                                            Wastewater
Average   Sta naa rd
Percent   Deviation
Recovery*     %
4-Chl oro-3-methyl phenol
2-Chlorophenol
2 , 4-Dichl orophenol
2, 4-Dimethyl phenol
2,4-Dinitrophenol
2-Methyl -4 ,6-di ni trophenol
4-Ni trophenol
2-Ni trophenol
Pentachl orophenol
Phenol
96
80
86
71
89
87
65
95
87
61
16
22
24
19
22
34
33
22
24
11
2,4,6-Trichlorophenol 91 22
*c= ==============================================
99
71
84
72
92
102
59
87
84
54
19
23
23
16
40
23
46
22
22
24
80 24
==============================
*Soikes ranged from 20 to 2,500 ug/liter.
                                     111-73

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       use of the separatory funnel  technique,  the continuous
       extractor technique is recommended (paragraph  2.1.6).

       Mark the water meniscus on the side of a 1-liter sample bottle
       for later determination of the volume  extracted.   Pour  the
       entire sample into a 2-liter  separatory  funnel.   Adjust the  pH
       of the sample with hydrochloric acid to  a pH of  2 or  less.
       Thoroughly mix the sample and measure  the pH to  ensure  that
       it is 2 or less.

       Add 60 ml methylene chloride  to the original sample bottle.
       Cap the bottle and shake for  30 seconds  to rinse  the  con-
       tainer.  Transfer the solvent into the separatory funnel and
       extract the sample by shaking for  2 minutes with  periodic
       venting to release excess vapor pressure.

       Allow the solvent layer to separate from the water phase for
       a minimum of 10 minutes.  If  the emulsion interface between
       layers is more than one-thinj the  size of the  solvent layer,
       mechanical techniques such as stirring,  filtration of the
       emulsion through glass wool,  or cantrifugation should be
       attempted.  Collect the methylene  chloride extract in a
       250 ml ^r1 enmeyer -''ask.  If  the emulsion  cannot  be broken
       or the amount of solvent recovered is  less than  80 percent
       (after correcting for water soluDility)  of that  initially
       added, the sample, solvent, and emulsion should  be trans-
       ferred into a continuous extractor to  complete the extraction
       process I paragraph 2.1.6).

       Add a second 60-ml portion of methylene  chloride  to the
       original sample container. Rinse  the  container  and transfer
       the solvent to the separatory funnel.  Extract the sample for
       an additional  2 minutes.  Add the  solvent  layer  to the  first
       extract in the Erlenmeyer flask.

       Repeat the extraction process a third  time with  a  final 60-ml
       portion of methylene chloride.   Combine  the extracts  in the
       Erlenmeyer flask.  (The sample  can be  discarded  or retained
       for extraction of base/neutral  compounds following pH adjust-
       ment).

       Pour the combined extracts through a drying column containing
       7 to 10 cm of anhydrous sodium sulfate,  and collect it  in a
       500-ml K-D flask equipped with a 10 ml concentrator tube.
       Rinse the Erlenmeyer flask with 20 to  40 ml  of methylene
       chloride.  Pour the rinse through  the  drying column and
       combine with the sample extract.   Proceed  to paragraph  2.1.7

2.1.5  Continuous Samole Extraction.

       Place 100 to 150 ml  of mettry^ene chloride  in the  extractor
       and 200 to 500 ml of methylene chloride  in the distilling

-------
       flask.  Add the aqueous  sample (pH  2  or  less) to the extrac-
       tor.   Add distilled  water as  necessary to operate the
       apparatus and extract  for 24  hours.   Remove  the distilling
       flask and pour the contents through a drying column contain-
       ing 7 to 10 cm of anhydrous sodium  sulfate.  Collect the
       extract in a 500-ml  K-D  evaporator  flask equipped with a
       graduated 10-ml  concentrator  tube.

2.1.7  Sample Extract Concentration

       Add 1 or 2 clean boiling chips to the 500-ml K-D flask and
       attach a three-ball  macro-Snyder column.  Prewet the Snyder
       column by adding about 1 ml of methylene chloride through the
       top of the column.   Place the K-D apparatus  on a warm water
       bath  (60 to 65C) so that the concentrator tube is partially
       immersed in the water  and the entire  lower rounded surface of
       the flask is bathed  with water vapor.  Adjust the vertical
       position of the apparatus and the water temperature as required
       in order to complete the concentration process in 15 to 20
       minutes.  At the proper  rate  of distillation, the balls of the
       column actively chatter  but the chambers do  not flood.  When
       the liquid has reached an apparent volume of 1 ml, remove the
       K-D apparatus and allow  the solvent co drain for ^t 1-,aast 10
       minutes while cooling.  Remove the  Snyder column and Hnse
       the flask and its lower  joint into the-concentrator tube with
       1 to  2 ml  of methylene chloride.  A 5-ml syringe is recom-
       mended for this operation.

       Add a clean boiling  chip and  attach a two-ball micro-Snyder
       column to the concentrator tube.  Prewet the column by adding
       about 0.5 ml  methylene chloride through the  top of the col-
       umn.   Place the K-D  apparatus on a.warm water bath (60 to
       65C) so that the concentrator tube is partially immersed in
       the water.  Adjust the vertical position of  the apparatus and
       the water temperature  as necessary to complete the concen-
       tration process  in 5 to  10 minutes.  At the  proper rate of
       distillation, the balls  of the column actively chatter but
       the chambers  do not  flood.  When the liquid  reaches an
       apparent volume of approximately 0.5 ml, remove the K-D
       apparatus  from the water bath and allow the  solvent to drain
       and cool for at  least  10 minutes.  Remove the micro-Snyder
       column and rinse its lower joint into the concentrator tube
       with  approximately 0.2 ml  of  methylene chloride.  Adjust the
       final  volume to  1.0  ml,  seal,  and label as the acid fraction.

       Determine the original sample volume by refilling the sample
       container to  the meniscus  mark  and transferring the liquid to
       a 1,000-ml graduated cylinder.  Record the sample volume to
       the nearest 5 ml -
                                ill-75

-------
2.1.8  GC/MS Analysis  of the Acid  Fraction

       Establish  instrument  operating  conditions  equivalent  to  those
       provided below:
         Mass Spectrometer

         Mass -range
         Scan time
         Electron energy
         Source temperature
         Start acquisition

         Column Conditions
               me/41-475
               1 second or less
               70 eV
               280-300C
               0.1 min after stopping flow
         1.8 m glass column (6.4 mm O.D.,  2 mm I.D.)  packed  with  1%
         SP-1240 DA coated on 100/120 mesh Supelcoport;  carrier gas:
         helium at 30 ml /min.  Temperature program:   isothermal for
         4 min at 50C, then increasing at 8C/min to 270C, and
         hold at 270C for 30 min.   If desired,  capillary  or SCOT
         columns may be used.

       At the beginning of eacn aay tnat acia  rraction analyses are
       to be performed, inject 50 ng of pentachlorophenol , either
       separately or as part of a standard mixture tnat  may  also
       contain 50 ng of DFTPP, into the instrument.   "Die tailing
       factor for pentachlorophenol, calculated  as indicated in
       Figure 2, inouid oe less than 5.

       Program the GC/MS to operate in the Extracted  Ion Current
       Profile (EICP) mode, and collect EICP's for the three char-
       acteristic ions listed in Table 2 for each compound being
       quantitated.  Operating in this mode, calibrate the system
       response for each compound using either the internal  or
       external standard procedure.

       When the internal  standard procedure is being  used, the
       standards should not be added to the sample extracts  until
       immediately before injection Into the instrument.  Mix the
       extract thoroughly before withdrawing an  aliquot  for  anal-
       ysis.  Inject 2 to 5 pi of the sample extract. The preferred
       method is the solvent-flush technique.

       When the extracts are not being used for analysis,  they
       should be stored in vials with unpierced  septa in the dark at
       4C.

       Calculate the concentration of sample constituents  as
in  uasecfions
                                  anc ..
                                111-76

-------
3.1  Analysis of Sediment Samples for Acid-Extractable  Organic  Compounds
     by Hexane-Methanol  Extraction
          Analytical  Procedure:   available
          Sample Preparation:   available

     3.1.1  Reference/Title

            U.S. Environmental  Protection Agency,  "Extraction and  Analysis  of
            Priority Pollutants in Sediment."   U.S.  EPA,  Region IV,  SiA
            Division, Athens,  Georgia.  Method PPS-9/80,  p.  7,  (1980).

     3.1.2  Method Summary

            A 30-gram sample of field-moist sediment is mixed with anhy-
            drous sodium sulfate and extracted with  40  percent  hexane  in
            methanol  using an  ultrasonic probe.   Extraneous  compounds  are
            removed by extraction under basic  conditions.  The  extract
            containing acid-extractable compounds  is then dried and  con-
            centrated.  The extracts are screened  on GC/FID  and analyzed
            on GC/MS if peaks  are noted on the FID chromatogram.

     3.1.3  Applicability

            This method covers the determination of  oriority pollutants
            in soils and sediment.

            The limit of detection for this method is usually dependent
            upon the level of  interferences rather than instrumental
            limitations.  Where interferences  are  not a problem, the
            limit of detection for most compounds  analyzed by GC/MS  is
            1,000 ug/kg.

            This method 1s recommended for use only  by  experienced
            residue analysts or under the close supervision  of  such
            qualified persons.

     3.1.4  Precision and Accuracy

            Information is not available at this time.

     3.1.5  Sample Preparation

            Decant and discard the water layer over  the sediment.  Mix
            samples thoroughly,  especially composited samples.   Discard
            any foreign objects such as sticks,  leaves, and  rocks.

            Weigh 30 grams of sample into a 400-ml beaker and add  30
            grams of anhydrous sodium sulfate.  Mix  well  and allow to  dry
            to a sandy texture.

            Immediately after weighing the sample  for extraction,  weigh
            separate 5- to 10-grams sliqucts of the  partially-dried

                                     111-77'

-------
       sediment into a tared  crucible.   Determine  the  percent solids
       by  drying overnight  at 103.   Allow  to  cool  in  a  desiccator
       for half an hour before weighing.   If percent volatile solids
       is  to  be determined, place  the oven-dried sample  into  a
       muffle furnace and heat to  550C  for 60 minutes.   Allow  to
       cool  in a desiccator before weighing.   Discard  when  finished.

       Begin  the extraction process  by adding  100  ml of  40-percent
       hexane 1n methanol to  the sample.  Adjust the pH  of  the
       mixture to 2.  Place an ultrasonic probe approximately 1/2"
       below  the surface of the solvent  but above  the  sediment  layer
       and sonicate the sample for 3 minutes at full power  with
       pulse  set at 50 percent. Decant  the solvent into a  Buchner
       funnel.

       Repeat the extraction  two more times using  100  ml  of 40-
       percent hexane in methanol  for each  extraction.   Prior to the
       sonicatlon step, check the  pH of  the sample suspension and
       adjust to pH 2, as necessary. Collect  the  extracts  in the
       Buchner funnel and concentrate to a  volume  of 100 ml.

3.1.6  Acid Extract Cleanup

       Transfer the extract to a 500-ml  separatory funnel containing
       250 ml of distilled  water and 25  ml  of  saturated  sodium
       sulfate solution. Adjust the pH  to  about 11.   Shake the
       separatory funnel for  2 minutes.

       Drain  the water layer  into  a  beaker  and discard the  hexane
       layer.  Return the aqueous  phase  to  the separatory funnel and
       add 25 ml of hexane.  Check the pH and  adjust to  11  if
       necessary.  Shake for  2 minutes.  Discard the hexane layer.

       Adjust the pH of the aqueous  layer to 2. Extract with three
       separate 25-ml portions of  methylene chloride and combine the
       extracts in a clean  250-ml  separatory funnel.   Wash  the
       extract with two 100-ml portions  of  acidified water  (pH  <2).

       Pass the methylene chloride extract  through a drying column
       packed with 7 to 10  cm of anhydrous  sodium  sulfate and 2 to
       3 cm of acid-washed  glass wool.   The glass  wool should also
       be solvent-rinsed with methanol,  acetone, and methylene
       chloride prior to use.

       Concentrate the extract to  a  volume  of  approximately 6 ml
       using a K-D apparatus.  Rinse the Snyder column and  the
       concentrator tube with a small volume of methylene chloride
       and reduce the extract to a final volume of 1 ml  under a
       stream of dry nitrogen.  Transfer the extract to  a GC  vial
       and label.  If the sample will not  be analyzed  Immediately,
       store at 4C.
                                i i
                                  1-78

-------
3.1.7  Sample Analysis by Gas Chromatography/Flame  lonization
       Screening.

       Determine the FID response of the  analytical  system  to  100  ng
       pentachlorophenol.

       Screen the acid extract on GC/FID  to  determine  whether  GO/MS
       analysis  is necessary.  If any of  the peaks  in  the sample
       extract produce a response that is greater than 100  ng  pent-
       achlorophenol , calculate the  concentration of the largest
       peak.

       (a)  If the concentration is  greater  than 1,000 yg/kg (dry
            weight basis), analyze the extract  using GC/MS.

       (b)  If the concentration is  less  than 1,000 yg/kg,  report
            the  sample concentration as <1,000  yg/kg.

       (c)  If all  peaks in the sample extract  are  less than that
            produced by 100 ng pentachlorophenol, record the minimum
            detection limit for the  sample.

       (d)  Analyze ail blanks and spikes,   xecora  ^ne precision and
            accuracy data.
                                111-79

-------
3.2  Analysis of Sediment Samples for Acid-Extractable Organic Compounds
     by Methylene Chloride Extraction
          Analytical Procedure:   available
          Sample Preparation:   available

     3.2.1  Reference

            Jacobs Engineering Group, "Manual  of Methods for the Analysis
            of Hazardous Wastes."  Contract Report 68-03-2569 Task 8008
            prepared for EPA Environmental Monitoring Systems Laboratory -
            Las Vegas, Nevada.  Jacobs Engineering Group,  Pasadena,
            California (1981).

            Method Summary

            A 50-g sample of soil or sediment  is extracted with methylene
            chloride using wet residual  waste/solvent techniques.   Aided
            by a high speed homogenizer, samples are extracted at pH 2  to
            Isolate the organic acid compounds.   The extract is cleaned
            up using gel  permeation chromatography prior to analysis.

     3.2.3  Applicability

            This method is suitaoie for che determination  of acid-
            extractable compounds in solid-phase samples such as soil  and
            sediment.  Method detection limits will  vary with sample size
            and co-extracted interferences.

     3.2.4  Precision and Accuracy

            No explicit information is available at this time.  However,
            analytical performance should be similar to that reported for
            acld-extractable compounds in water (Subsection J.2.1).

     3.2.5  Sample Preparation

            Thoroughly mix the sample by homogenizing 1t in the original
            sample bottle.  Weigh into a 200-ml  centrifuge bottle a  50-g
            aliquot or an appropriate weight based on screening analysis.
            Add surrogate standards and mix the aliquot to be analyzed.

            Adjust the pH of the sample with hydrochloric  acid to a  pH  of
            2 or less.  The add should be added slowly and with constant
            mixing to minimize foaming of the  sample.  Mix briefly with a
            homogenizer to ensure uniform sample pH.

            Add 60 ml of methylene chloride to the sample  bottle and
            homogenize briefly.   Rinse the homogenizer with a minimum of
            water and then with 5 to 10 ml of  methylene chloride.   Addi-
            tional liiethyiene ;h1or"'de ?ay be jdded 'intil *he total "Mould
            1n the centrifuge bottle is near the top.
                                     TII-80

-------
       Centrifuge the samples  for 15 minutes.   The  mixture  will
       separate into an aqueous  layer over the  methylene  chloride
       extract.  A solid cake  or emulsion may form  at  the water-
       methyl ene chloride interface.   If  the emulsion  interface
       between layers is more  than one-half the size of the solvent
       layer,  a smaller sample size should be used  to  complete the
       phase separation.

       Withdraw the organic extract from  the centrifuge bottle with
       a 50-ml  glass syringe that has been equipped with  a  150-mrn x
       5-mm I.D. TFE tube.  Discharge the  extract  into  a 300-ml beaker.

       Repeat  the sample extraction procedure a second time with a
       60-ml portion of methylene chloride.  Combine the  extracts.

       Perform a third extraction with a  final  60-ml portion of
       methylene chloride and  combine the extracts.

3.2.6  Sample  Extract Drying

       Pour the combined extract resulting from the extraction
       procedure through a  drying column  containing 7 to  10 cm of
       organics-free anhydrous sodium sulfate.   Collect the dried
       extract in a 500-ml  K-D flasK  equipped with  a 10-mi  concen-
       trator tube.

       Wash the flask that  original-ly contained the extract and the
       drying  tube three times with 30-ml  portions  of methylene
       chloride.  Add these washes to uhe sample  extract  in the K-D
       flask.

3.2.7  Sample  Extract Concentration

       Add one or two clean boiling chips to the  flask and  attach a
       three-ball  macro-Snyder column. Prewet  the  column by adding
       approximately 1 ml  of the extracting solvent (methylene
       chloride) through the top of the column.   Place the  apparatus
       in a 60 to 65C water bath so the  concentrator tube  is par-
       tially  immersed in the  water and the lower rounded surface of
       the flask is bathed  with  water vapor.  Adjust the  apparatus
       as necessary to complete  concentration to  approximately 10 ml
       in 15 minutes.  (At  the proper rate of distillation, the
       balls of the column  will  chatter but the chambers  will not
       flood.)

       Remove  the Snyder column, and  rinse the  flask and  its lower
       joint into the concentrator tube with 1  to 2 ml of methylene
       chloride.
                                HI-81

-------
       Fit the concentrator tube with  a  modified  macro-Snyder
       column.  Organic-free nitrogen  is employed to  reduce  the
       volume of the extract to approximately 5 ml.   Wash  the
       concentration tube  with  two 0.2-ml  volumes of  methylene
       chloride.

       Adjust the final  extract volume to 5 ml  for subsequent  internal
       standard addition and GC/MS analysis. If the extract  obtained
       above is "clean", then a final  extract volume  of  1  ml is
       required.

3.2.8  Gel Permeation Cleanup

       Determine the residue weight of the concentrated  sample
       extract by placing  a 1-ml  aliquot on a tared aluminum foil
       pan, allowing the solvent to evaporate,  and reweighing the
       pan.  These results are  used to determine  the  volume  of
       extract to be applied to the column for cleanup.  The volume
       of extract applied  to the column  should  not exceed  the capa-
       city of the column, approximately 200 mg.   If  the residue
       weight is on the  order of 1 to  5  mg, cleanup by gel permea-
       tion can, in many cases, be avoided.

       Transfer 5 ml of  the GPC calibration solution  to  the  Bio-
       Beads S-X3 column.   Drain the column into  a 100ml  graduated
       centrifuge tube until  the liquid  is just above the  surface of
       the GPC packing.  Wash the calibration solution on  the column
       with several 1-ml aliquots of methylene  chloride.   Elute the
       columns with 200-ml aliquots of methylene  chloride  and col-
       lect 10-ml fractions.

       Analyze the fractions for bis-[2-ethylhexyl]phthalate and
       pentachlorophenol by GC/FID on  a  1% SP-1240 DA column.
       Determine the corn  oil elution  pattern by  evaporation of each
       fraction to dryness followed by gravimetric determination of
       the residue.  Plot  the concentration of  each component  in
       each fraction versus the total  eluent volume.

       The first fractions of the elution volume  that represent an
       approximate 85% removal  of the  corn oil  and 85% recovery of
       the bis-(2-ethylhexyl)phthalate can be discarded.   Collect
       the fractions that  elute up to  a  retention volume represented
       by 50 ml after the  elution of pentachlorophenol.  (Typical
       procedures are to discard the first 60 ml, to  collect the
       next 110 ml, and  to wash the column with 250 ml of  methylene
       chloride between  'samples.)

       Select 3  'olume if  'amole extract 'based on the -esidue
       weight determination) that will not overload the  column.
       Apply an aliquot  (i to 4 ml; of the extract to the  column and
       drain the column  until the sample is just  above the surface


                                111-82

-------
of the GPC packing.  Wash the extract  onto the column  with
several  1-ml  portions of methylene chloride.   Elute  the
column with 200-ml  portions of methylene  chloride.

Collect the first 60 ml  of eluent  in a 100-ml  graduate
cylinder, pass the next  110 ml of  eluent  through  a drying
column containing 6 cm of anhydrous sodium sulfate and
collect it in a 500-ml K-D flask equipped with a  10-ml concen-
trator tube.   Rinse the  drying column  with three  25-ml por-
tions of methylene chloride.

Add one or two clean boiling chips to  the flask and  attach a
three-ball macro-Snyder  column.  Prewet the column by  adding
approximately 1 ml  of methylene chloride  through  the top of
the column.  Place the apparatus in a  60  to 65C  water bath
so that the concentrator tube is partially immersed  in the
water, and the lower rounded surface of the flask is bathed
with water vapor.  Adjust the apparatus to complete  concen-
tration to approximately 10 ml in  15 minutes.   (At the proper
rate of distillation, the balls of the column  will chatter
but the chambers will not flood.)

Semove the Snyder column, and rinse the -"ask  2nd 't.s  * awer
joint into the concentrator tube with  1 to 2 ml of methylene
chloride.

Fit the concentrator tube with a modified macro-Snyder col-
umn.  Organic-free nitrogen is employed to reduce the  volume
of the extract to approximately 5  ml or 1 ml  (but not  below
0.5 ml).  Wash the concentrator tube with two  0.2-ml volumes
of methylene chloride.

Adjust the final extract volume to 5 ml or 1 ml for  subse-
quent internal standard  addition and GC/MS analysis.  If the
extract obtained above is "clean", then a final extract
volume of 1 ml is required.

If the extract is to be  stored before  GC/MS analysis,  trans-
fer the extract to an appropriately-sized serum vial equipped
with a Teflon-lined rubber septum  and  crimp cap.  The  extract
volume should be scored  on this vial,  and appropriate  sample
identification must be affixed.  Store the extracts  in the
dark at 4C.

It is possible that samples which  contain high concentrations
of extractable organic compounds will  not be amenable  for
concentration to 5 ml.  For extracts of this type the  final
volume after concentration should  be adjusted  to  a minimal
volume *hat permits extract samoling with a Tncro-syHnqe.
Obvious remedies will likely include either starting with
                         Iii-83

-------
smaller sample size or concentration to  a  volume  greater than
5 ml.

Transfer the cleaned, concentrated  extract to  a 6-ml  serum
TFE capped bottle and store at  4C  for 6C/MS analysis.

Qualitatively identify specific compounds  in the  sample
extract and calculate their concentrations as  indicated in
Subsection K.
                         111-84

-------
4.1  Analysis of Biological  Tissue Samples for Acid-Extractable  Organic
     Compounds by Methylene  Chloride Extraction
          Analytical  Procedure:   available
          Sample Preparation:   available

    4.1.1   Reference

            U.S. Environmental  Protection Agency,  "Extraction and Analyses
            of Priority Pollutants in Biological Tissue."   U.S.  EPA S4A
           'Division, Region IV,  Laboratory Services Branch,  Athens,  Georgia.
            Method PPB 10/80.   p. 7, (1980).2

     4.1.2  Method Summary

            A 10-g sample of homogenized fish tissue is mixed with 40 g
            of sodium sulfate,  and extracted which  methylene  chloride
            using an  ultrasonic  probe.  The samples are filtered,  con-
            centrated to 10  ml  or less,  cleaned up  with acetonitrile
            partitioning, and  concentrated to 1 ml.  The extract is
            screened  using gas  chromatography and  quantified  using mass
            spectrometry.

     4.1.3  Applicability

            The limit of detection for this method  1s usually dependent
            upon the  level of  interferences rather  than instrumental
            limitations.   Where  interferences are  not a problem,  the
            limit of  detection  for most compounds  analyzed  by GC/MS is
            2 mg/kg (wet.weight  basis).                        	.

            The method is recommended for use only  by experienced residue
            analysts  or under  the close  supervision of such qualified
            persons.

     4.1.4  Estimates of Precision and Accuracy

            No information is  presently available.   However,  analytical
            performance should  be similar to that  reported  for Subsection
            J.2.1.

     4.1.5  Sample Extraction

            Blend equal amounts  of fish  tissue and  dry ice.  If  a large
            sample is being  processed, a food processor or  meat  grinder
            may be convenient.

            Weigh 10  g of homogenous sample into a  400-ml beaker and  mix
            with 40 g of sodium  sulfate.  Ensure that the sample is
            thoroughly dry.
                                     111-85

-------
Add 100 ml methylene chloride to the tissue mixture.   Place
an ultrasonic probe in the mixture and sonicate at 50 percent
pulse for 3 minutes.  Transfer the methylene chloride phase to
a 500-ml K-D flask.

Repeat the methylene chloride sonication/extraction of the
tissue sample with a second 100-ml  portion of methylene
chloride.  Combine the extracts in a K-D flask.  Extract the
sample residue a third time with 100 ml  methylene chloride.
Combine the extracts.

NOTE:  The probe should be carefully cleaned between  samples
as indicated in Subsection C (Interferences).

Add a clean boiling chip to the K-D flask and attach  a three-
ball  macro-Snyder column.  Place the K-D apparatus on a water
bath and concentrate the extract to 10 ml.

Quantitatively transfer the concentrated extract to a 125-ml
separatory funnel.  Add enough hexane to bring the final
volume to approximately 15 ml.  Extract the sample four times
by shaking vigorously with 30-ml oortions of hexane-saturated
acetonitrile for one minute.

Combine and transfer the acetonitrile phases to a 1-liter
separatory funnel and add 650 ml of distilled water and 40 ml
of saturated sodium chloride solution. ~MTx "thoroughly for 30
to 45 seconds.  Adjust tne pri of tne aqueous pnase to 2 and
extract with two 100-ml portions of methylene chloride.
Shake the sample vigorously for 15 to 30 seconds during each
extraction.

Combine the methylene chloride extracts in a 1-liter  separ-
atory funnel and wash with two 100-ml portions of distilled
water.  Discard the water layer and pour the methylene
chloride layer through a drying column containing 7 to 10 cm
of anhydrous sodium sulfate and 2 to 3 cm of glass wool.
Collect the extract in a 500 ml K-D flask equipped with a 100-
ml ampul.  Rinse the separatory funnel and drying column with
three 10-ml portions of methylene chloride.  Add the  rinsings
to the K-D flask.

Attach a three-ball macro-Snyder column and place the K-D
apparatus in a hot water bath (60-65C).  Concentrate the
extract to 6 to 10 ml.  Use a stream of dry nitrogen  to
concentrate the extract to 1 ml.

Transfer the extract to a GC vial and label as the acid-
extractaoie fraction of che semi-volatile compounds.   Tins
extract is now readv for analysis.
                         111-86

-------
4.1.6   Analysis by Gas Chromatopraphy

        The acid-extractable compounds are screened with GC/FID using
        the column Indicated in Subsection E to  determine whether
        GC/MS analyses are necessary.   Relative  retention times and
        limits of'detection are summarized in Tables 1  and 5.   A
        representative chromatogram is presented in Figure 3.

        Determine the FID response of  100 ng pentachlorophenol.  If
        any peaks in the sample extract produce  a greater instrument
        response than pentachlorophenol, calculate the  concentration
        of the largest peak:

        (a)  If the calculated concentration of  the sample component
             is greater than 2 mg/kg (wet weight basis), analyze the
             extract by GC/MS.

        (b)  If the calculated concentration of  the sample component
             is less than 2 mg/kg, report the concentration as  less
             than 2 mg/kg.

        (c)  If all sample peaks in the chromatograms are less  than
             the pentachlorophenol peak, "ecord  the minimum detection
             limit for the sample.

        Analyze all blanks and spikes.  Record the pertinent pre-
        cision and accuracy data with  the sample information.

 4.1.7  Gas Chromatography/Mass Spectroscopy Analysis for cne Acia-
        Extractable Compounds

        At the beginning of each day that acid extractable compound
        analyses are to be performed,  inject 50  no pentachlorophenol,
        either separately or as part of a standard mixture that may
        also contain 50 no DFTPP.   The tailing factor for pentachloro-
        phenol, calculated as shown in Figure 2, should be less
        than 5.

        Establish chromatographic conditions equivalent to those
        listed in Table 1.  Included in this table is information on
        estimated retention times and  sensitivities that can be
        achieved by this method.  An example of  the pre-separation
        achieved by. the column is shown in Figure 3.

        Program the GC/MS to operate in the Extracted Ion Current
        Profile (EICP) mode, and collect EICP spectra for the three
        characteristic ions listed in  Table 2, page 111-65 for  each
        compound being quantified.  Operating in this node,  calibrate
        the system response for each compound by using  the internal
        standard procedure.
                                UI-87

-------
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                                 O v *  
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                                                     2-chlorophenol

                                                          2-nitrophenol
      phenol



2,4-dimethylphenol
                                                   4-chloro-3-methylpheno!
2,4-dinitrophenol

     =^*
     2-methyl-4.6-dinitrophenol


      pentachlorophenol
                                               4-nitrophenol
                                     1  00 >
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-------
            If the internal  standard approach  is  used,  the  standards
            should not  be added to the  sample  extracts  until  immediately
            before injection into the instrument.   Mix  the  spiked extract
            thoroughly.   Inject 2 to 5  pi  of the  extract  using the
            solvent-flush technique.

            If the instrument response  for any ion  exceeds  the linear
            range of the system, dilute the extract  as  necessary and
            reanalyze.

            Qualitative identification  and quantitative calculations are
            completed as indicated in Subsection  K.  When the extracts
            are not being used for analysis, store  them in  vials capped
            with unpierced septa in the dark at 4C.

K.   QUALITATIVE IDENTIFICATION

     To qualitatively identify a compound, obtain an Extracted Ion  Current
Profile (EICP) for the  primary ion and  the two other ions listed in Table 2.
The criteria below must be met for a qualitative  identification.

1.   The characteristic ions for each compound must  have  their maxima in the
     same or within one scan of each other.

2.   The retention time for the experimental mass spectrum  must be  within 30
     seconds of the retention time.of the  authentic  compound.

3.   The ratios of the  three EICP oeak  heiahts must  agree within 20% with the
     ratios of the relative intensities for these ions  in a reference mass
     spectrum.  The reference mass spectrum can be  obtained from either a
     standard analyzed  through the GC/MS system or  from a reference library.

4.   Structural isomers that have very  similar mass  spectra can be  explicitly
     identified only if the resolution  between the  isomers  in a standard mix
     is acceptable.  Acceptable resolution is  achieved  if the valley height
     between isomers is less than 25% of the sum  of the two peak heights.
     Otherwise, structural  isomers are  identified as isomeric pairs.

     For samples that contain an inordinate number  of interferences, the
     chemical ionization (CI) mass spectrum may make identification easier.
     Characteristic CI  ions for most of the compounds are given in  Table 2.
     The use of chemical ionization MS  to  support El MS is  encouraged but not
     required.

L.   CALCULATIONS

1.   Acid-Extractable Compounds in Water.

     When a comoound has been identified,  the  ouantification of that compound
     will be based on the integrated area  from tne  specific ion plot of the
     first listed characteristic ion in Table  2.


                                     111-39

-------
     If the sample  produces  an  Interference  for the  first  listed  ion,  use  a
     secondary  ion  to  quantify.   Quantification can  be accomplished by the
     internal standard method.
     Internal  standard  -  By  adding  a  constant  known  amount  of  internal
     standard  (C^s  in yg)  to every  sample  extract, the concentration o
     (C0)  in yg/1  in the  sample  is  calculated  using  Equation L-l.

                                (As)(Cis)
                      C0   =    	                            Eq.  L-l
                              (Ais)  (RF)  (Y0)
     where:
            V0  =  the volume  of the original  sample  in  liters;  the  other
                   terms  are defined in text  above  (Eq.  H-l).

     Report all  results to  two significant  figures.   Report  results  in  v9/l
     without correction for recovery data.  When  duplicate and spiked samples
     are analyzed, all  data obtained should be reported.

     In order to minimize unnecessary GC/MS analysis  of  method blanks and
     field blanks, the f;eld blank  Tiay be  screened  on a  FTD/GC equipped with
     an SP-1240 DA column.

2.   Acid-Extractable. Compounds in  Solid-Phase Samples  (Soil/Sediment).

     2.1  Percent Dry Solids


             gm of dried  sample
             	  x   100   =   % dry solids
              gm of wet sample
     2.2  Percent Volatile Solids


           gm of dried sample - gm ignited sample
                                                   x 100 = % volatile  solid
                     gm of wet sample


     2.3  Concentration of acid-extractable compounds


                                            C   0
          Cone. Acid Cmods (wet weight)   =  	
                                              F



                                     111-90

-------
                                   C   D
Cone. Acid Cmpds (dry weight)  =   	
                                    F-S
where:
      C  =  concentration of acid compound in sample extract,  pg/1
      D  =  final  volume of sample extract, 1
      F  =  wet weight of sample initially extracted, g
      S  =  percent solids of initial  sample.
                           111-91

-------
                                  REFERENCES
1.   U.S. Environmental  Protection Agency.  "Base/Neutrals, Acids, and
     Pesticides - Method 625."   Federal  Register Vol.  44:   No.  233:
     69540-69552. December 3, 1979.

2.   U.S. Environmental  Protection Agency.  "Extraction and Analysis of
     Priority Pollutants in Biological  Tissue."   Method PPB 10/80.  U.S. EPA,
     Region IV, S&A Division, Laboratory Services Branch,  Athens, Georgia.
     p. 7, (1980.).

3.   U.S. Environmental  Protection Agency.  "Method for Preparation of Medium
     Concentration Hazardous Waste Samples."  U.S. EPA, Region  IV, Athens,
     Georgia,  p. 8, May 1981.

4.   Glaser, J. A.,  D.  L. Foerst, G.  D. McKee,  'S. Quave and W.  L. Budde.
     "Theory and Application of Method  Detection Limit.  A new  Performance
     Criterion for Chemical Analysis."   U.S. Environmental Protection Agency,
     Environmental Monitoring and Support Laboratory,  Cincinnati, Ohio.
     September 1981.

5.   Glaser, J. A., 0. L. Foerst, G. 0.  McKse, S. Ouave and W.  L. Budde.
     "Trace Analyses for Wastewaters."   ES&T  .!: 1426-1435 (1981).

6.   U.S. Environmental  Protection Agency.  "IFB WA-81-H017."   U.S. EPA,
     Washington, D.C.  July 6,  1981.
                                      
7.   U.S. Environmental  Protection Agency.  "Extraction and Analysis of
     Priority Pollutants in Sediment."   U.S. EPA, Region IV, S&A Division,
     Athens, Georgia.  Method PPS 9/80,  p. 7, (1980).

8.   Jacobs Engineering Group.   "Manual  of Methods for the Analysis of
     Hazardous Wastes."   Contract Report 68-03-2569 Task 8008 prepared for EPA
     Environmental Monitoring Systems  Laboratory-Las Vegas, Nevada.  Jacobs
     Engineering Group, Pasadena, California (1981).

9.   U.S. Environmental  Protection Agency.  "Extraction and Analysis of
     Priority Pollutants in Biological  Tissue."   U.S.  EPA, Region IV, S&A
     Division, Laboratory Services Branch, Athens, Georgia.  Method PPB 10/80
     p. 7, (1980).

-------
                                  SECTION 3

                      BASE/NEUTRAL-EXTRACTABLE COMPOUNDS
A.   SCOPE
     Base/neutral compounds in the semi-volatile fraction of the priority
pollutants (Table 1) can be quantified using the analytical  procedures
presented in Subsection J.  These compounds are solvent-extractable under
alkaline conditions and identified/quantified using GC/MS procedures.
                      TABLE 1.  BASE-NEUTRAL EXTRACTABLES
          ComDOund
Storet No.
Comnound
Storet Mo.
 Acenaphthene                  34205
 Acenaphthylene                34200
 Anthracene                    34220
 Benzo[a]anthracene            34526
 Benzo[b]fluoranthene          34230
 Benzo[k]fluoranthene          34242
 Benzo[a]pyrene                34247
 Benzo[ghi]pery1ene            34521
 Benzidine                     39120
 Bis[2-chloroethyl]ether       34273
 Bis[2-chloroethoxy]methane    34278
 Bis[2-ethylhexyl]phthalate    39100
 Bis[2-chloroisopropyl]ether   34283
 4-Bromophenyl phenyl ether    34636
 Butyl benzyl phthalate        34292
 2-Chloronaphthalene           34581
 4-Chlorophenyl phenyl ether   34641
 Chrysene                      34320
 Dibenzo[ah]anthracene         34556
 Di-n-butyl phthalate          39110
 1,3-Dichlorobenzene           34566
 1,4-Dichlorobenzene          " 34571
 1,2-Dichlorobenzene           34536
                               34631
            Diethyl  phthalate           '  34336
            Dimethyl  phthalate            34341
            2,d-0initrotoluene            34611
            2,6-Dinitrotoluene            346?6
            Di-n-octyl  phthalate          34596
            1,2-Diphenylhydrazine         34346
            Fluoranthene                  34376
            Fluorene                      34381
            Hexachlorobenzene             39700
            Hexachlorobutadiene           34391
            Hexachloroethane              34396
            Hexachlorocyclopentadiene     34386
            Indeno[l,2,3-cd]pyrene        34403
            Isophorone                    34408
            Naphthalene                 .  39250
            Nitrobenzene                  34447
            N-Nitrosodimethylamine        34438
            N-Ni trosodi-n-propylami ne     34428
            N-Nitrosodiphenylamine        34433
            Phenanthrene                  34461
            Pyrene                        34469
            2,3,7,8-Tetrachloro-          34675
             dibenzo-p-dioxin
            1.2,4-Trichlorobenzene        34551
                                                                ===============
                                     111-93

-------
B.   SAMPLE HANDLING AND STORAGE

     Conventional  water sampling practices  should  be  followed  except  that  the
bottle should not  be prerinsed with  sample  before  collection.   Grab samples
should be collected in glass containers and refrigerated immediately.   Com-
posite samples should preferably be  collected  in glass  containers"  and,  if
possible, refrigerated during the period of compositing.   All  automatic
sampling equipment must be free of Tygon and other potential sources  of
organic contamination.  When necessary, the equipment should be prerinsed  with
hexane prior to use.

     Water samples should be iced or refrigerated  from the time of collection
until extraction.1  Chemical preservatives  should  not be used  in the  field
unless more than 24 hours will elapse before sample delivery to the labora-
tory.  If the samples cannot be extracted within 48 hours of collection, the
recommended method of sample preservation is as follows:

1.   If the sample contains residual  chlorine,  add 35 mg of sodium thiosulfate
     per 1 ppm of free chlorine per  liter of sample.1

2.   Adjust the pH of the water sample to a pH  of  7 to 10 using sodium
     hydroxide or sulfuric acid.  Record the volume of acid or base used.1

     VP water samples must be extracted within 7  days  and completely analyzed
within 30 days of collection (Figure 1).

     Sediment and soil samples may be stored field-moist (refrigerated),
frozen, or dried.   The effective storage period is not known (Figure  1).

C.   INTERFERENCES

     Solvents, reagents, glassware,  and other  sample  processing hardware may
yield discrete artifacts and/or elevated baselines causing misinterpretation
of chromatograms.   All of these materials must be  demonstrated to  be  free  from
interferences under the conditions of the analysis by running  method  blanks.
Specific selection of reagents and purification of solvents by distillation in
all-glass systems may be required.

     Analytical interferences associated with  samples will vary considerably
from source to source, depending on  the diversity  of  the Industrial complex,
municipality, or system being sampled.  Matrix Interferences may be caused by
components that are coextracted from the sample but are not normally  of
Interest.  The most common such components are petroleum-derived naphthenes,
high-molecular-weight polymeric components, and long-chain components such as
waxes and triglycerides.  The extent of such matrix interferences  will  vary
considerably from source to source.   A cleanup procedure using gel permeation
chromatography has been incorporated into the  method for certain cases  to
remove long-chain and high-molecular-weight material.  No cleanup  procedure 1s
available for the removal of  naphthenes.  When such matrix Interferences are
present, the samole extract is diluted and the method detection limit is
Increased proportionately.  Many of the matrix interferences  are ^olvent-
extractable nonvolatile components which necessitate the more  frequent

                                     in-94

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cleaning of the GC injection port and the more frequent removal  of the
injection end of the GC capillary column.

     Interference due to fish oil in tissue samples can be eliminated by
acetonitrile partitioning,2 but the ultrasonic probe used to assist in the
partitioning process must be scrupulously cleaned between samples.  The
procedure consists of rinsing the probe with solvent (acetonitrile) into the
sample, removing residue on the probe with a wet Kimwipe, rinsing the probe
with methylene chloride, and sonicating in hexane for 3-4 minutes on 50%
pulse.

     The recommended analytical procedure may not have sufficient resolution
to differentiate between certain isomeric pairs.  These are anthracene and
phenanthrene, chrysene and benzo(a)anthracene, and benzo(b)fluoranthene and
benzo(k)fluoranthene.  The GC retention time and mass spectral  data are not
sufficiently unique to make an unambiguous distinction between these com-
pounds.  Alternative techniques should be used to identify and quantify these
specific compounds.


D.   SAFETY

     The toxicity or carcinogenicity of each reagent used in this method has
not been precisely defined; however, -?ach x;hemical  compound should be treated
as a potential health hazard.  From this viewpoint, exposure to these chem-
icals must be minimized by whatever means available.  The laboratory manager
is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method.  A
reference file of .-natsrlal data handling sheets should also be made available
to all  personnel involved *n the chemical analysis.  All  operations involving
the use of methylene chloride, including the extraction of the waste sample,
filtration of the extract, and concentration of the extract, must be performed
in a fume hood.  Care should be taken to avoid skin contact with methylene
chloride.


E.   APPARATUS

1.   Class I Biological safety cabinet (Glovebox) suitable for handling
     chemical carcinogens, as described on page 123 of "Laboratory Safety
     Monograph, A Supplement to the NIH Guidelines for Recombinant DNA
     Research," U.S. Department of Health, Education and Welfare, PHS, NIH,
     January 1979.  The cabinet should have an interchange panel for intro-
     ducing materials, a retaining tray to catch spills, and a static pressure
     gauge.*

2.   Spatula.  Stainless stee.l or Teflon.  Fisher Scientific catalog No.
     14-375-10 or equivalent.
*Kewaunee, Inc. model SH-3704-MS-X, or equivalent is acceptable.


                                     111-96

-------
3.   Balance capable of weighing 100 grams  to the^nearest 0.01  gram.
4.   Vials,  specimen,  screw cap, approximately 20  ml.   Use Teflon liner.
     Calibrate at 10 ml by pipetting 10 ml  of solvent  into the  tube  and
     marking the bottom of the meniscus.
5.   Vials and caps, 2 ml  for GC auto-samplers.
6.   Disposable pipets, Pasteur.
7.   Test tube rack.
8.   Oven, drying.
9.   Desiccator.
10.  Crucibles, porcelain, squat form,  Size 2 or equivalent.
11.  Sonlcator Cell  Disrupter - Heat Systems - Ultrasonics,  Inc., with a
     3/4"-high gain probe, 375 watt or  equivalent.
12.  Beakers, 400 ml.
13.  Separatory funnels -  120 ml, 250 ml,  500 ml,  and  2 1 with  Teflon
     stopcocks.
14.  Pyrex glass wool.
15.  Furnace, muffle.  -
16.  Biichner funnels.
17.  Kuderna-Danish (K-D)  apparatus.
     17.1  Concentrator tube - 10 ml, graduated (Kontes K-570040-1025 or
           equivalent).
     17.2  Evaporative flask - 500 ml (Kontes K-570001-0500  or  equivalent).
     17.3  Snyder column - three-ball macro (Kontes  K-50300-0121 or
           equivalent).
     17.4  Snyder column - Two-ball micro  (Kontes  K-569002-0219, or
           equivalent).
18.  Boiling chips - Beryl saddles (Fisher, 91915) crushed.
19.  Water bath - Heated,  with concentric  ring cover,  capable of temperature
     control (2'C).
                                     111-97

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20.  Pans, approximately 35 cm x 25 cm x 6 cm.

21.  Filter paper - Whatman 41, ashless.

22.  Vacuum filtration apparatus (Fisher 9-788)  or 500-ml  suction filtration
     flasks.

23.  Vacuum pump.

24.  Drying columns - 25 mm x 200 mm packed with 4 cm of glass wool.

25.  Florisil  columns - Pyrex, 400 mm x 25 mm O.D. with  Teflon stopcock,  but
     without glass frit.

26.  Food Processor - (Hobart food processor -  8181D or  equivalent).

27.  Gas chromatograph - analytical  system complete with gas  chromatograph
     suitable for on-column injection and all  required accessories  including
     flame ionization detector and electron capture detector,  column  supplies,
     recorders, gases, and syringes.

     27.1  Column 1 - for base/neutral  and pesticides, a 6-ft (1.83 m)  glass
           column (6 mm 0.0.  x 2 mm I.D.) packed with 3% SP-2250  coated on
           100/120 mesh Supelcoport, or equivalent.  This  column  was  used to
           generate tne precision ana accuracy  cata summarized in Table 5.

     27.2  Column 2 and analytical conditions  -  Chromosorb W  (100/120 mesh),
           coated with 3% OV-17 packed  fn a 1.83 m x 2 mm  I.D.  Pyrex  glass
         -  column.  Use ultra pure nitrogen at  a flow rate of 30  ml/min.
           Column temperature is held at 30"C  for 2 min.,  programmed-to 290
           at 8e/min., and held at 290"C for 16  min.

28.  Mass spectrometer - capable of scanning from 35 to  450 a.m.u.  every  7
     seconds or less at 70 V  (nominal)  and producing an  acceptable  mass
     spectrum at unit resolution from 50 ng of  DFTPP when  the sample  is
     introduced through the GC inlet.  The mass  spectrometer  must be  inter-
     faced with a gas chromatograph equipped with an injector system  designed
     for splitless injection  and glass  capillary columns or an Injector system
     designed for on-column injection with all-glass packed columns.  All
     sections of the transfer lines must be glass or glass-lined  and  must be
     deactivated (use Sylon-CT, Supelco, Inc.,  or equivalent,  to  deactivate).

     NOTE:  Systems utilizing a jet separator  for the GC effluent are recom-
     mended since membrane separators may lose  sensitivity for light  molecules
     and glass frit separators may inhibit the  elution of  polynuclear aromat-
     tics.  Any of these separators may be used  provided that it  gives
     recognizable mass spectra and acceptable  calibration  points  at the limit
     of detection specified for each individual  compound.

     A computer system must be interfaced to the mass spectrometer  to allow
     continuous acquisition of -nass :cafis *or  *.he luraf'on of the chromato-
     graphic program.  There  must be computer  software available  to allow

                                     III-9R

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     searching any GC/MS run for specific Ions and plotting the Intensity of
     the ions with respect to time or scan number.  The ability to integrate
     the area under any specific ion plot peak is essential for quantification.

29.  Extracting equipment

     29.1  ADT tissumizer (Tekmar SDT 182EN or equivalent).

     29.2  Centrifuge (IEC CU-5000 or equivalent).

     2^.3  Screw-capped centrifuge bottles, 200 ml (Scientific Products C4144)
           with TFE-lined screw caps.

     29.4  Fleakers (or beakers) - 300 ml or equivalent.

     2<>.5  Glass syringe - 50 ml equipped with a 150 mm x 5 mm I.D.  TFE tube.

30.  Continuous liquid-liquid extractors - Teflon or glass connecting joints
     and stopcocks, no lubrication.  (Hershberg-Wolf Extractor - Ace Glass
     Co., Vineland, New Jersey, P/N 6841-1D, or equivalent).

31.  Gel permeation chromatography cleanup equipment.

     31.1  Chromatography column - 500 mm x 19 rnm T.D.  'Scientific Products
           C-4670-106 or equivalent).

     31.2  Bio-Beads S-X3, 200/400 mesh (Bio-Rad Laboratories 152-2750).

     31.3  Glass vool.

     31.4  Graduated cylinders - 100 ml.

     31.5  GCP Autoprep or equivalent (Analytical Biochemistry Labs, Inc.  1002
           or equivalent with 25 mm I.D. column containing 50 to 60  g of  Bio-
           Beads S-X3).  (Optional)


F.   REAGENTS

1.   Sodium sulfate, anhydrous, reagent grade - heated  2 hours at 500C,
     cooled in a desiccator for 4 hours, and stored in  a glass bottle.

2.   Methylene chloride - pesticide residue analysis grade or equivalent.

3.   Hexane - pesticide residue analysis grade or equivalent.

4.   Reagent water.  Water purified by passage through  activated charcoal  or
     equivalent.  When aliquots of this water are analyzed using the pro-
     cedure, the Impurities measured shall  be below the detection limits  based
     on 3 1-gram samole aliquot.
                                     ni-99

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5.   Methanol,  pesticide residue analysis grade,  free of purgeable  organics.
     (Open a fresh bottle and check  It by adding  100  pi  to  5 ml  of  organic-
     free water.   Analyze on the GC/MS system using the  purge-and-trap
     technique.)

6.   Acetone -  pesticide quality and distilled in glass.

7.   Petroleum  ether - pesticide quality and distilled in glass.

8.   Diethyl ether - preserved with  2% ethanol, pesticide quality and dis-
     tilled 1n  glass.  NOTE:  Ether  must be free  of peroxides  as indicated by
     EM Quant Test Strips (test strips are available  from EM Laboratories,
     Inc., 500  Executive Blvd., Elmsford, New York   10523).

9.   FTorisIl - PR grade (60/100 mesh).

10.  Sodium hydroxide (ACS), 6 N in  distilled water.

11.  Sodium hydroxide (ACS), 10 N 1n distilled water.

12.  Sodium hydroxide (ACS), 50% in  distilled water.   Extracted  3 times with
     methylene  chloride.

13.  Hydrochloric acid (ACS), concentrated ;12 ;j).

14.  Sulfuric add (ACS), 6 N in distilled water.

15.  Stock standards - Prepare stock standard solutions  at  a concentration of
     1.00 ug/ul.   For example, dissolve 0.100 3 of  assayed  reference material
     in pesticide-quality i-^ooctane  or other acpropr^ate solvent and dilute to
     volume in  a 100-ml ground glass stoppered volumetric flask.  The stock
     solution is transferred to 15-ml  Teflon-lined  screw cap vials, stored in
     a refrigerator, and checked frequently for signs of degradation or
     evaporation, especially just prior to preparing  working standards from
     them.  Protect PNA standards from light.

16.  Calibration standards - Prepare calibration  standards  that  contain the
     compounds  of Interest, either singly or mixed  together.   The standards
     should be  prepared at concentrations that will  completely bracket the
     working range of the chromatographic system  (two or more  orders of mag-
     nitude are suggested).  For example, if the  limit of detection can be
     calculated as 20 ng Injected, prepare standards  at  10  ug/ml, 100 pg/ml,
     1000 ug/ml, etc. so that Injections of 1-5 ul  of the calibration stan-
     dards will define the linearity of the detector  in  the working range.

17.  Surrogate  standards - Surrogate standards should be stable  isotope
     derivatives of compounds -which  will aid 1n the monitoring of the
     analysis procedures.  The number and composition of surrogate  standards
     will to some extent be dependent on compound availability.  Where pos-
     sible the  stable-"!sotope-labeled compounds  (which are  TWSS  resolved  from
                                    III-100

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     the unlabeled compound) will be utilized ^to simplify calculations in the
     measurement of recovery.  The surrogate standards should be added to
     the chilled solid sample via injection of an appropriate volume of the
     surrogate standard mix.  [The sample should be spiked with a quantity of
     surrogate standard which is equivalent to 5 ppm (e.g., 250 ug/50 g
     sample).]  The surrogate spike should be added to the solid sample in
     aliquots of the total volume to be added (e.g., five 20-ul injections
     from a 100 jil syringe) while manually stirring the material with a clean
     glass stirring rod to facilitate dispersion of surrogate compounds.  It
     is important that the sample and sample container are chilled during the
     surrogate standard addition.  Surrogate standard addition should be
     accomplished as quickly as possible to minimize the loss of the most
     volatile analytes quantitated with this method.  Upon surrogate standard
     addition, the isolation/cleanup procedure can be initiated.

     It is recognized that uniform distribution of surrogate standards in the
     sample will not be obtained via this procedure.  Also, it is recognized
     that the surrogates will not mirror the properties of organic compounds
     which have been associated with soil or sediment samples for decades.
     Nevertheless, data generated from surrogate standards will serve to
     exemplify a "best" case recovery for the sample in question, and will
     provide QA/QC data for every sample.

G.   QUALITY CONTROL

   .  Before processing any samples, demonstrate through the analysis of a
method blank that all  glassware and reagents are interference-free.   Each time
a set of samples is extracted or there is a change in reagents, a method blank
should ba procsssed as a safeguard against chronic laboratory contamination.

     Standard quality assurance practices should be used to document the
performance of this method.  Field replicates should be collected and analyzed
to determine the precision of the sampling technique.  Laboratory replicates
should be analyzed to determine the precision of the analysis.  Fortified
samples should be analyzed to determine the accuracy (recovery) of the
analysis.  Field blanks should be analyzed to check for contamination intro-
duced during sampling and transportation.

     Five percent of the samples (1 of 20), or one sample each time  a set of
samples is prepared, whichever frequency is higher, should be blanks of the
reagents, water and solvents.  Contaminants shall  be below the detection limits
based on a 1-gram (1-ml) sample aliquot.

     Five percent of the samples (1 of 20), or one sample each time  a set of
samples is prepared, whichever frequency is higher, should be spiked, prepared,
and analyzed in duplicate.

H.   CALIBRATION

     Prepare calibration standards that contain the compounds of interest,
either singly or mixed together.  The standards should be prepared at concen-
trations tnat will bracket tne working range of the -ftromatograpfrfc  .system

                                    III-101

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(two or more orders of magnitude are suggested).   If the limit of detection
can be calculated as 20 ng injected, for example,  prepare standards at 1
ug/ml, 10 ug/ml, 100 wg/ml, etc. so that injections of 1-5 ul  of the calibra-
tion standards will define the linearity of the detector in the working
range.

     Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated.  By injecting calibration
standards, establish the linear range of the analytical system and demonstrate
that the analytical system meets the detection requirements.   If the sample
gives peak areas above the working range, dilute and reanalyze.

     Internal standard method - The internal  standard approach is acceptable
for all of the semivolatile organics.  The utilization of the internal  stan-
dard method requires the periodic determination of response factors (RF) that
are defined in Equation 1.


                            RF  =  (AsC1s)/(AisCs)                        Eq.  1


where:

       As   =  the integrated area or peak height of the characteristic ion
               for the contaminant being measured

       A^s    the integrated area or peak height of the characteristic ion
              for the internal standard

       Cfs    the amount (jig) of the internal  standard

       Cs   *  the amount (ug) of the contaminant.

     The relative response ratio for the analytes should be known for at least
two concentration values - 20 ng injected to approximate 10 ug/1  and 200 ng
injected to approximate the 100-ug/1 level (assuming 1 ml final volume  and a
2-ul injection).  Those compounds that do not respond at either of these
levels may be run at concentrations appropriate to their response.

     The response factor (RF) should be determined over all  concentration
ranges for the standards (Cs) that are being determined.  [Generally, the
amount of internal standard added to each extract is the same (20 ug) so that
C-is remains constant.]  This should be done by preparing a calibration  curve
where the response factor (RF) is plotted against the standard concentration
(Cs), using a minimum of three concentrations over the range  of interest.
Once this calibration curve has been determined,  1t should be verified daily
by injecting at least one standard solution containing internal standard.  If
significant drift has occurred, a new calibration curve must be constructed.
To quantify, add the internal standard to the concentrated sample extract no
more than a few iirinutes before Injecting Into the GC/MS system to minimize the
possibility of losses due to evaporation, adsorption, or chemical  reaction.
                                    III-102

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Calculate the concentration by using the previous equations with the approp-
riate response factor taken from the calibration curve.  Either deuterated or
fluorinated compounds can be used as Internal standards and surrogate
standards.  Napththalene-dg, anthracene-dig, pyridine-ds, aniline-ds,
nitrobenzene-dc, l-fluoronaphthalene, 2-ftuoronaphthalene, 2-fluorobiphenyl,
2,2'-difluorobiphenyl, and 1,2,3,4,5-pentafluorobiphenyl  have been used or
suggested as appropriate Internal standards/surrogates for the base/neutral
compounds.  Compounds used as Internal  standards are not to be used as surrogate
standards.  The Internal standard must be different from the surrogate standards.

     External standard method - The external standard method can also be used
at the discretion of the analyst.  Prepare a master calibration curve using a
minimum of three standard solutions of each of the compounds that are to be
measured.  Plot concentrations versus integrated areas or peak heights (selected
characteristic ion for GC/MS).  One point on each curve should approach the
limit of detection.  After the master set of Instrument calibration curves has
been established, the curves should be verified daily by Injecting at least one
standard solution.  If significant drift has occurred, a new calibration curve
must be constructed.

I.   DAILY GC/MS PERFORMANCE TESTS

     At the beginning of each day, the mass calibration of the GC/MS system
must be cnecxed ind Adjusted, if necessary, to meet DFTPP specifications.
Additionally, each day base/neutral compounds are to be analyzed, the column
performance specifications with benzidine must be met.  DFTPP can be -nixed -'n
solution with benzidine to complete the two specifications with one Injection,
if desired.  The performance criteria must be met before any samples or
standards are analyzed.

     Evaluate the analytical system performance each day that It 1s to be used
for the analysis of samples or blanks by examining the mass spectrum of DFTPP.
The following instrumental conditions are required to perform the mass cali-
bration test of a GC/MS system:

          Electron Energy - 70 volts (nominal)
          Mass Range      - 35-450 amu
          Scan Time       - 7 seconds or less

Inject a solution containing 50 ng DFTPP and check to ensure that established
performance criteria listed in Table 2 have been satisfied.  If the system
performance criteria are not met, retune the spectrometer and repeat the
performance check.

     Column performance 1s evaluated by injecting 100 ng of benzidine into the
Instrument.  The tailing factor for the resultant peak, as calculated in
Figure 2, must be less than 3-for the performance to be considered acceptable.
The user is cautioned that some problems may be encountered due to the oxida-
tion of benzidine.  However, benzidine has been specified for this purpose.1
Also, tailing -factor criteria have not been established *or any other ral-J-
bration material to be used with this fraction (base/neutral compounds).


                                    III-103

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TABLE 2.  OFTPP  KEY  IONS  AND  ION ABUNDANCE CRITERIA

Mass
        Ion Abundance Criteria
 51
 68
 70
 127
 197
 198
 199
 275
 365
 441
 442
30 to 60 percent of mass 198
Less than 2 percent of mass 69
Less than 2 percent of mass 69
40 to 60 percent of mass 198
Less than 1 percent of mass 198
Base peak, 100 percent relative abundance
5 to 9 percent of mass 198
10 to 30 percent of mass 198
Greater than 1 percent of mass 198
Present but less than mass 443
Greater than 40 percent of mass 198
              Tailing Factor = -
                           AB

Example Calculation: Peak Height = DE = 100 mm
                 10% Peak Height - BO = 10 mm
                 Peak Width at 10% Peak Height = AC = 23 mm
                   AB = 11 mm
                   BC = 12 mm
                                       12
                 Therefore: Tailing Factor = - - ". 1


       Figure 2.   Tailing factor calculation.
                       !!1-104

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J.   ANALYTICAL PROCEDURES

     1.1  Analysis of Hazardous Wastes for Base/Neutral  Compounds
               Analytical  Procedure:   available
               Sample Preparation:   available

          1.1.1  Reference

                 U.S. Environmental  Protection Agency,  "Method for Preparation
                 of Medium Concentration Hazardous Waste Samples."  U.S.  EPA,
                 Region IV, Athens,  Georgia.  May 1981.3

          1.1.2  Method Summary

                 Approximately one-gram allquots of soil, solid,  aqueous
                 liquid or non-aqueous liquid are transferred to  vials inside
                 a chemical carcinogen glove box.  The  samples are then
                 extracted with methylene chloride.  The methylene chloride
                 extract is screened by GC/FID using the appropriate base/
                 neutral column.  Based on initial screening "esults,  the
                 sample extracts are appropriately concentrated and analyzed
                 with a GC/MS system.

          1.1.3  Applicability

                 This procedure is designed for the safe handling jnd prepar-
                 ation of potentially hazardous samples  from hazardous waste
                 sites for analysis  of organic chemicals Including priority
                 pollutants.  The method is directed to  contaminated soil
                 samples and waste samples that may be  solid, aqueous liquid,
                 or non-aqueous liquid and suspected to  contain less than 10%
                 of any one organic  chemical component.   The method is not
                 designed for samples expected to contain less than 10 ppm of
                 base/neutrals and acids, such as many  sediment samples taken
                 from leachate streams.  This type sample should  be analyzed
                 using a method for  sediment/soil samples (Subsection J.3.1
                 or J.3.2.

          1.1.4  Precision and Accuracy

                 These extraction and preparation procedures were developed
                 for rapid and safe  handling of hazardous samples.  The design
                 of the methods thus did not stress efficient recoveries  of
                 all components.  Rather, the procedures were designed for
                 moderate recovery of a broad spectrum  of organic chemicals.
                 The results of the  analyses thus may sometimes reflect only
                 a minimum of. the amount present In the  sample.

                 The procedure is designed to allow detection limits as low as
                 10 ppm for base/neutrals.  Some samples, however, may contain
                 high concentrations of chemicals that  interfere  with the


                                    III-105

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                 analysis of other components  at low  levels.  The  detection
                 limits 1n those cases may  be  significantly higher.   Percent
                 recovery and standard deviation Information  on  the  use  of
                 this procedure 1n a single laboratory  is  presented  in
                 Table 3.

             TABLE 3.  RECOVERY DATA FROM SOIL BY REGION  IV MEDIUM
                    CONCENTRATION HAZARDOUS WASTE METHOD3
Base/Neutral
Fraction
Cone.
ug/gm
Avg.
% Rec.
Std.
Dev.*
1,3-Dichl orobenzene                  20              92                 8.2
Bis[2-chloro1sopropyl] ether         20              83                 2.8
1, 2, 4-Tr1chl orobenzene               20              94                 2.8
Naphthalene                          20              90                 2.9
2-Chloronaphthalene                  20              78                10
Dimethyl phthalate                   20              79                 2.9
4-Chlorophenyl  phenyl  ether          20              93                 1.4
Di ethyl phthalate                    20              83                 2.1
4-Bromophenyl  phenyl  sther           45              ?1     -            0.47
Dibutyl phthalate                    20              99                 8.6
Butyl benzyl phthalate               20              "5                 9.3
Chrysene                             20              90                13
Di-n-octyl phthalate         '        20              53                11
Benzo[a] pyrene                      40              92                25
BenzoCghi] perylene                  20              89                30
Acenaphthylene                       20              83                 4.1
1,2-Di phenyl hydrazlne               20              85                 9.8
Phenanthrene                         20              85                 3.4
Fluoranthene                         20              85                 4.1
Benzldine                            20              47                 4.1
Benzanthracene                       20              86                 4.3
Ideno[l,2,3-cd] pyrene               30              83                 4.1
*  one standard deviation based on three  trials.

          1.1.5  Sample Preparation
                 Place the sample container Into the glovebox.   Additional
                 Items that should be 1n the glovebox Include  (1)  calibrated,
                 tared 20-ml vials with caps, (2)  a spatula,  (3)  a balance,
                 (4) a capped vial containing 10 ml  of Interference-free
                 methanol, (5) a vial of water,  and (6)  an eye  dropper.   The
                 vial of methanol 1s to be used  as a method blank.   (One
                 method blank should be run for  each batch of 20 samples  or
                 less.)  Open the sample transoortatlon  can and remove  the
                 sample container.  Note and record the physical  state  and
                 appearance of *he -amole,  !f the samole  bottle 1s broken,
                 Immediately repackage the sample and terminate the analysis.

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       Open the sample bottle and mix the sample.   If the sample  is
       a liquid, transfer one drop to a  vial  containing water to
       determine whether the sample is aqueous or  non-aqueous.
       Record the result.  Transfer approximately  1 gram (or 1 ml)
       of the sample to a calibrated and tared 20-ml  vial.   Wipe  the
       mouth of the vial  with tissue to  remove any excess sample
       material.  Cap the vial.   Record  the exact  weight of sample
       taken.  Reseal  the original  sample and replace it in the
       original packaging.

       Proceed with a methylene  chloride extraction of the base/
       neutral  compounds  in the  sample based on the miscibility of
       the original sample with  water.  Follow paragraph (a)  for  an
       aqueous  sample, paragraph (b) for a non-aqueous sample, and
       paragraph (c) for a solid sample.

       a.  If the sample  has been determined to be aqueous, dilute
           the sample with 10 ml of methylene chloride.   Cap the
           vial and shake the sample for two minutes.  Add  2  grams
           of anhydrous sodium sulfate to the vial to absorb all
           water.  Shake  the sample.

       b.  If the sample  was determined  to be non-aqueous,  dilute
           the  sample to  5 final volume  of 10 ml with methylene
           chloride.  Cap the vial  and mix for two minutes.  Add  one
           gram of anhydrous sodium sulfate to absorb all water.
           Shake the sample.

       c.  If the sample  is a solid matrix, add 10 ml methylene
           chloride.  Cap the sample and shake for one hour on a
           wrist-action shaker.   Add one gram of anhydrous  sodium
           sulfate to the sample and thoroughly mix.

       Withdraw a sample of the  organic  extract with a syringe for
       analysis as described in  paragraph 1.1.6.

1.1.6  Screening of Base/Neutral Extracts

       The base/neutral extracts should  be screened by GC/FID using
       appropriate columns.  If  packed columns are used, the
       extracts must be screened on both base/neutral and acid GC
       columns  specified  in Method 625.*

       Using the GC analytical conditions given in Reference 1,
       analyze the base/neutral  extracts by GC/FID.  Standardize  the
       GC/FIO for full-scale response with 40 ng/ul of djQ-phen-
       anthrene for .the base/neutrals.

       If the response of any component  is greater than 25% of the
       resoonse of the di^-phenanthrene, analyze the extract (para-
       graph 1.1.5) by GCVMS. In some cases 1t may be necessary  to
       dilute the extract prior  to analysis.   If no "ssponse  exceeds

                          III-107

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       25% of the response of the d^o-phenanthrene,  concentrate  the
       extract under a gentle stream of nitrogen  to  1  ml  and  perform
       the GC/MS analysis.

1.1.7  GC/MS Analysis of the Base/Neutral  Fraction

       Establish Instrument operating conditions  equivalent to those
       provided below:

       Mass Spectrometer

       Mass Range                m/e 41-475
       Scan Time                 7 seconds or less
       Electron Energy           70 eV
       Source Temperature        280-300eC
       Start Acquisition         0.1 min after  stopping
                                 flow.

       Column Conditions

       Column:  1.8 m glass (6.44 mm O.D.  x 2 mm  I.D.), packed with 3%
       SP-2250 coated on 100/120  mesh Supelcoport.   carrier gas:
       helium at 30 ml/min.  Temperature program:  isothermal for 4
       nin it SO'C, then increasing at 8*/m1n to  270C, and hold at
       270C for 30 min.  If desired,  capillary or SCOT columns  may
       be used.

       Program the GC/MS to operate in the Extracted Ion  Current
       Profile (EICP)  mode, and collect EICP's  for the three  char-
       acteristic ions listed in  Table 4 for eacn compound oeing
       quantltated.  Operating in this mode, calibrate cne system
       response for each compound using either  the internal or
       external standard procedure.

       If the internal standard approach 1s being used, the analyst
       should not add the standard to the  sample  extracts until
       immediately before Injection into the instrument.  Mix the
       extract thoroughly before  withdrawing an aliquot for
       analysis.  Inject 2 to 5 ul  of the  sample  extract.  The
       solvent-flush technique 1s preferred.

       If external calibration is employed, record the volume of
       extract and standard solution injected to  the nearest
       0.05 ill.  If the response  for any ion exceeds the  linear
       range of the system, dilute the extract  and reanalyze.

       When the extracts are not  being used for analyses, they should
       be stored in vials with unpierced septa  in the  dark at 4C.

       Proceed to Subsection K for qualitative  Identification
       criteria and calculation of the results.
                          III-108

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TABLE 4.  CHARACTERISTIC IONS FOR BASE/NEUTRAL EXTRACTABLES
                                   Characteristic  Ions
Compound
1,3-Dichl orobenzene
1 ,4-Di chl orobenzene
Hexachi oroethane
Bi s[2-chl oroethyl ]ether
1 ,2-Dichl orobenzene
Bis[2-chloroisopropyl]-
ether
N-Ni trosoai -n-propy i ami ne
Isophorone
Nitrobenzene
Hexachi orobutadi ene
1 ,2,4-Trichlorobenzene
Naphthalene
B1 s[2-chl oroethoxy]-
me thane
Hexachi orocycl opentadi ene
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2,6-Dinitrotoluene
Fluorene
Electron Impact
146
146
117
63
146
45
130
32
77
225
180
126
83
237
162
152
154
63
. 165
166
148
148
201
83
148
77
42
95
123
223
182
129
95
235
164
151
153
194
63
165
113
113
199
95
113
79
101
138
65
227
145
127
123
272
127
153
152
164
121
167
Chemical lonization
(methane)
146
146
199
63
146
77
--
129
124
223
181
129
65
235
163
152
154
151
83
166
148
148
201
107
148
135

157
152
225
182
157
107
237
191
153
155
163
211
167
150
150
203
109
150
137

175
164
227
209
169
137
239
203
181
183
164
223
195
                                                        (continued)
                          III-109

-------
TABLE 4.  (Continued)
Characteristic Ions
Compound
4-Chl orophenyl phenyl ether
2,4-Dinitrotoluene
1,2-Di phenyl hydrazine*
Di ethyl phthalate
N-Ni trosodi phenyl ami ne**
Hexachl orobenzene
4-Bromophenyl phenyl ether
Phenanthrene
Antnracene
Oibutyl pnthalate
Fluoranthene
Pyrene
Benzldine
Butyl benzyl phthalate
B1s[2-ethylhexyl]-
phthalate
Chrysene
Benzo[a]anthracene
3,3-Dichlorobenzidine
D1-n-octyl phthalate
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Electron Impact
204
165
77
149
169
284
248
178
173
149
202
202
184
149
149
228
228
252
149
252
252
206
89
93
177
168
142
250
179
179
150
101
101
92
91
167
226
229
254

253
253
141
163
105
150
167
249
141
176
175
104
100
100
185

279
229
226
126
--
125
125
Chemical lonization
(methane)
--
183
185
177
169
284
249
i78
173
149
203
203
185
149
149
228
228
--

252
252

211
213
223
178
286
251
179
173
205
231
221
213
299
--
229
229

--
253
253
--
223
225
251
198
288
277
207
207
279
243
243
225
327
--
257
257
--
--
281
281
(continued)
       III-110

-------
ssaasaasaaasaasaaaaasaaaaaasa
TABLE 4.  (Continued)
   SSSBSSBSBaBBas-=SS = SSSBaS8a =

                Characteristic Ions
Compound
Benzo[a]pyrene
Indeno[l ,2 ,3-cd]pyrene
Dibenzo[ah]anthracene
Benzo[ghi]perylene
N-Ni trosodimethyl ami ne
Bis[ch1oromethy1]ether
2,3,7,8-Tetrachlorodibenzo-
Electron Impact
252
276
278
276
42
45
__
253
136
139
138
74
49
322
125
277
279
277
44
51
320
Chemical lonization
(methane)
252 253
276 277
278 279
276 277
...

59
281
305
307
305
--
--
 .
  p-dioxin
                                168
           94
80
 Oeuterated anthracene
  [d-10]***
=====================================================
  *Detected as azooenzene
 **Detected as diphenylanrine
***Suggested internal standard
189    217
                                                     ==========================
                                    III-lll

-------
2.1  Analysis of Methylene Chloride Extracts of Aqueous Samples  for
     Base/Neutral Compounds
          Analytical  Procedure:   available
          Sample Preparation:   available

     2.1.1  Reference

            U.S. Environmental  Protection Agency,  "Semi-Yolatiles
            Determination." Method 625.   Federal Register  44
            No. 233:69540-69551.   December 3,  1979.1       ~

     2.1.2  Method Summary

            The pH of a one-liter water  sample is  adjusted to  a  pH  of  11
            or greater and the  sample is extracted with methylene chlor-
            ide.  Following extract concentration, the  sample  is analyzed
            on a calibrated GC/MS.

     2.1.3  Applicability

            This method is applicable to the determination of  those
            compounds listed 1n Table 1  when they  occur in aqueous
            samples such as -nunicipal  and industrial  discnarges.  The
            method is designed  to be used to meet  the monitoring require-
            ments of the National  Pollutant Discharge Elimination System
            (NPDES).

            TM3 method should  be restricted to use by,  or under the
            supervision of, analysts experienced 1n the operation of gas
            chromatograph/mass  spectrometers and skilled in the  inter-
            pretation of mass spectra.

     2.1.4  Precision and Accuracy

            Precision and accuracy performance data for this procedure,
            based on the analysis  of reagent water and  wastewater in a
            single laboratory,  are summarized in Table  5.

     2.1.5  Separatory Funnel Sample Extraction

            Samples may be extracted by  separatory funnel  techniques or
            with a continuous extractor.   Where emulsions  prevent accept-
            able solvent recovery with the separatory funnel technique,
            the continuous extraction technique is recommended.

            Mark the water meniscus on the side of a  one-liter sample
            bottle for later determination of  the  volume extracted.  Pour
            the entire sample Into a 2-1  separatory funnel.  Adjust the
            pH if khe samel? with 6 N MaOH to  11 or greater,   Thorouahlv
            mix the sample and measure the pH  to ensure that it  1s  11 or
            greater.
                                    m-112

-------
 TABLE 5.   ACCURACY AND PRECISION FOR BASE/NEUTRAL EXTRACTABLES
r====z================s:=s==z===================================
                            Reagent Water       	Hastewater
Parameter
Acenaphthene
Acenaphthylene
Aldrin
Anthracene
Benzo[a]anthracene
Benzo[b]f 1 uoranthene
Benzo[k]f 1 uoranthene
Benzo[ghi]perylene
Benzo[a]pyrene
Benzidine
Butyl benzyl phthalate
Beta-BHC
Delta-BHC
Bi s[2-chl oroethoxylmethane
Bi s[2-chl oroethyl ]ether
3ix[2-chloroisopropy1 ]ether
Bi s[2-ethyl hexyl ]phthal ate

-------
                             TABLE 5.  (Continued)
===============================================================================
                                    Reagent Water              Wastewater	
                                 AverageStandardAverageStandard
Parameter	% Recovery  Deviation %  % Recovery  Deviation %
Indeno[l ,2 ,3-cd]pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propyl ami
N-Nitrosodiphenylamine
PCB-1221
PCB-1254
Phenanthrene
Pyrene
1 ,2 ,4-Trichl orobenzene
65
75
6
72
ne 68
84
77
80
84
86
64
37
33
32
31
39
24
11
13
14
15
16
===================
81
77
75
82
76
86
__,
_..
76
80
69
43
42
35
54
45
31
--
__
22
23
26
Enrichment concentrations ranged from 5 to 2400 ug/1
            Add 60 ml  methylene chloride to the original  sample  bottle.
            Cap the bottle and shake for 30 seconds to rinse the container.
            Transfer the solvent into the separatory funnel  and  extract
            tne sample by shaking for two minutes with periodic  venting  to
            release excess vapor pressure.

            Allow the organic layer to separate from the water phase for
            a minimum of 10 minutes.  If the emulsion interface  between
            layers is more than one-third the size of the solvent layer,
            mechanical techniques such as stirring, filtration of the
            emulsion through glass wool, or centrifugation should be
            attempted.  Collect the methylene chloride extract in * 250-ml
            Erlenmeyer flask.  If the emulsion can not be broken and/or
            the amount of solvent recovered is less than 80% (after cor-
            recting for water solubility) of that Initially added, the
            sample, solvent, and emulsion should be transferred  into a
            continuous extractor to complete the extraction process
            (paragraph 2.1.6).

            Add a second 60-ml portion of methylene chloride to  the
            original  sample container.  Rinse the container and  transfer
            the solvent to the sample in the separatory funnel.   Extract
            for an additional two minutes and combine extracts 1n an
            Erlenmeyer flask.

            Repeat the extraction process a third time with a final 60 ml
            portion of methylene chloride.  Combine the extracts 1n the
            Erlenmeyer flask.  (The sample can be discarded or retained
            for extraction of the acidic organic compounds following ?K
            adjustment.)
                                    TTT.IId

-------
       Pour the combined extracts through a drying  column  containing
       7 to 10 cm of anhydrous sodium sulfate,  and  collect it  in  a
       500-ml K-D flask equipped with a 10-ml  concentrator tube.
       Rinse the Erlenmeyer flask with 20 to 40 ml  of methylene
       chloride.  Pour the rinse through the drying column and combine
       with the sample extract.   Proceed to paragraph 2.1.7.

2.1.6  Continuous Sample Extraction

       Place 100 to 150 ml of methylene chloride  in the  extractor
       and 200 to 500 ml  of methylene chloride  in the distilling
       flask.  Add the aqueous sample (pH 11 or greater) to the
       extractor.  Add distilled water as necessary to operate the
       extractor and extract for 24 hours.   Remove  the distilling
       flask and pour the contents through  a drying column contain-
       ing 7 to 10 cm of anhydrous sodium sulfate.   Collect the
       extract in a 500-ml K-D evaporator flask and label  as the
       base/neutral fraction.

2.1.7  Sample Extract Concentration

       Equip the K-D flask with a 10-ml  concentrator tube.  Add 1 *o
       2 clean boiling chips to the flask and  attach a three-ball
       macro-Snyder column.  Pr-swet the Snyder column by adding
       about 1 ml of methylene chloride through the top.   Place the
       K-D apparatus on a warn water bath (50  to  65*C) so  that the
       concentrator tube is partially immersed  in the water and the
       entire lower rounded surface of the flask  is bathed with
       water*vapor.  Adjust the vertical  position of the aoparatus
       and the water temperature as required in order to complete
       the concentration process in 15 to 20 minutes.  At  the  proper
       rate of distillation, the balls of the  column actively
       chatter but the.chambers do not flood.   When the  liquid has
       reached an apparent volume of 1 ml,  remove the K-D  apparatus
       and allow the solvent to drain for at least  10 minutes  while
       cooling.  Remove the Snyder column and  rinse the  flask  and
       its lower joint into the concentrator tube with 1 to 2  ml  of
       methylene chloride.  A 5-ml syringe is  recommended  for  this
       operation.

       Add a clean boiling chip and attach a.two-ball micro-Snyder
       column to the concentrator tube.   Prewet the column by  adding
       about 0.5 ml methylene chloride through  the  top.  Place the
       K-D apparatus on a warm water bath (60  to  65*C) so  that the
       concentrator tube is partially Immersed in the water.   Adjust
       the vertical position of the apparatus  and the water temper-
       ature as necessary to complete the concentration  process in 5
       to 10 minutes.  At the proper rate of distillation,  the balls
       of the column actively chatter but the  chambers do  not  flood.
       When the liquid caches an aooarent volume of aooroximately
       0.5 ml, remove the K-D apparatus from the  water bath and
       aV.ow the solvent to drain and cool  for  at least  10 minutes.

                               III-115

-------
       Remove the micro-Snyder column and rinse  its  lower  joint  into
       the concentrator tube with  approximately  0.2  ml  of  methylene
       chloride.   Adjust the final  volume to 1.0 ml, seal, and label
       as the base/neutral  fraction.

       Determine  the original  sample  volume by refilling the  sample
       container  to the meniscus mark and transferring  the liquid  to
       a 1,000-ml graduated cyclinder.  Record the  sample  volume to
       the nearest 5 ml.

2.1.8  GC/MS Analysis of the Base/Neutral  Fraction

       At the beginning of each day that base/neutral analyses are
       to be performed, inject 100 ng of benzidine,  either separ-
       ately or as part of a standard mixture that  may  also contain
       50- ng of DFTPP, into the instrument.  The tailing factor  for
       benzidine, calculated as indicated in Figure 2,  should be
       less than  3.

       Establish  instrument operating conditions equivalent to those
       provided below:

       Mass Spectrometer

             Mass Range              m/e 41-475
             Scan Time               7 seconds or less
             Electron Energy         70 eV
             Jource Temperature      230-200*0
             Start Acquisition       0.1 min after  stooping flow.

       Column Conditions

       Column:   1.8 m glass column (6.4 mm O.D.  x 2 mm  I.D.), packed  with
       3% SP-2250 coated on 100/120 mesh SupeTcoport.  Carrier  gas:
       helium at 30 ml/min.  Temperature program:  isothermal for  4
       min at 50'C, then increasing at 8C/min to ?70C,  and  hold
       at 270eC  for 30 minutes.  If desired, capillary  or  SCOT
       columns may be  used.

       Program the GC/MS to operate in the Extracted Ion  Current
       Profile (EICP) mode, and collect EICP's for  the  three  char-
       acteristic Ions listed in Table 4 for each compound being
       quantitated.  Operating  in this mode, calibrate  the system
       response  for each compound using either the  internal or
       external  standard procedure.

       If the internal  standard approach is being used,, the analyst
       should not add  the standard to the  sample extracts  until
        immediately before Injection 
-------
If external calibration is employed, record the volume of
extract and standard solution injected to the nearest
0.05 ul.  If the response for any ion exceeds the linear
range of the system, dilute the extract and reanalyze.

When the extracts are not being used for analyses, tfiey
should be stored in vials with unpierced septa in the dark  at
4'C.

Proceed to Subsection K for qualitative identification
criteria and calculation of the results.
                        III-117

-------
3.1  Analysis of Sediment Samples for Base/Neutral  Compounds
          Analytical  Procedure:   available
          Sample Preparation:   available

     3.1.1  Reference/Title

            U.S. Environmental  Protection Agency,  "Extraction  and Analysis
            of Priority Pollutants in Sediments."   PPS-9/80, U.S. EPA,
            Region IV, S&A Division,  Athens,  Georgia.   7  pp. (1980).4

     3.1.2  Method Summary

            A 30-gram sample is mixed with anhydrous sodium  sulfate  and
            extracted with 1:1  acetone:hexane using an  ultrasonic probe.
            The base/neutral extract 1s washed with water to remove  the
            acetone,  dried, and concentrated.  The  extracts  are  screened
            on GC/FID and analyzed on GC/MS if peaks are  noted on the FID
            chromatogram.

     3.1.3  Applicability

            This method covers  the determination of priority pollutants
            in soils  and sediment.  The limit of detection for this
            method is usually dependent upon the level  of interferences
            rather than instrumental  limitations.   Where  interferences
            are not a problem,  the limit of detection fo^ most compounds
            analyzed by GC/MS is 1,000 ug/kg.

            This method is recommended for use only by  experienced
            residue analysts or under the close supervision  of such
            qualified persons.

     3.1.4  Precision and Accuracy

            This information is not presently available.

     3.1.5  Sample Preparation

            Decant and discard the water layer over the sediment. Mix
            samples thoroughly, especially composited samples.  Discard
            any foreign objects such as sticks, leaves and rocks.

            Weigh 30 g of sample Into a 400-ml beaker and add  30 g of
            anhydrous sodium sulfate.  Mix well and allow to dry to  a
            sandy texture.

            Immediately after.weighing the sample for extraction, weigh
            5 to 10 g of the partially-dried sediment into a tared cruci-
            ble.  Determine the percent solids by drying overnight at
            103'C to 105C.  Allow ;o cool In a desiccator for half  an
            hour before weighing.  If percent volatile solids  are to be
                                    TIT-118

-------
       determined, place the oven-dried sample into a muffle furnace
       and ignite at 550DC for 60-minutes.   Allow to cool  in a
       desiccator before weighing.

       Add 100 ml of 1:1 acetone:hexane to  the sampTe-sodium sulfate
       mixture.  Place a sonification probe about 1 cm below the
       surface of the solvent but above the sediment layer.   Sonicate
       for 3 minutes at full power  with pulse set at 50%.   Decant the
       solvent into a Biichner funnel.  Add  100 ml of 1:1 acetone:
       hexane.  Sonicate for 3 minutes at full  power with  the pulse
       set at 50%.  Decant the solvent into the Buchner funnel.   Add
       a third 100-ml  portion of 1:1 acetone:hexane to the residual
       sample.  Sonicate for 3 minutes at full  power with  a 50%
       pulse.

       Pour the entire sample into  the Biichner funnel and  rinse  with
       hexane.

       Concentrate the B/N extract  to about 100 ml.

3.1.6  Extract Cleanup

       Transfer the extract to a 500-ml separatory funnel  containing
       250 ml of distilled water and 25 ml  of saturated sodium sul-
       fate solution.   Chake the separatory funnel  for 2 minutes.

       Drain the water layer into a clean beaker and the hexane
       layer into a clean 250-ml separatory funnel.

       Transfer the ^atsr *ntc the  500-nil separatory funnel  and
       re-extract with 25 ml of methylene chloride by shaking the
       separatory funnel for 2 minutes.  Combine extracts.

       Repeat the process by adding an additional 25 ml of methylene
       chloride to the separatory funnel  and extracting for  an
       additional 2 minutes.  Transfer the  solvent phase to the
       250-ml separatory funnel  containing  the  first two extracts.

       Wash the extracts with 2 x 100 ml  of distilled water.  Pass
       the solvent extract through  a drying column  packed  with 7 to
       10 cm of organic-free anhydrous sodium sulfate and  an inch of
       glass wool, solvent-rinsed with methyl alcohol, acetone,  and
       hexane.

       Concentrate the extract to 10 ml with a  K-D  apparatus on  a
       steam bath.  Remove the concentrator tube and concentrate the
       extract to 1 ml with nitrogen.

       Remove one-half of the extract and place in  a GC vial.
       Dilute to 1 ml  with methylene chloride.   This is the  B/N
       fraction of the sample,  '.ibel the volume as ? ml.
                               III-119

-------
3.1.7  Gas Chromatography/Flame lonization Screening of the
       Base/Neutral Extract

       Calculate the FID response of the Instrument to a 50-ng
       Injection of hexachlorobenzene (HCB).   {The GC/MS requires
       about 50 ng HCB to give a complete mass spectra.)  Screen the
       prepared base/neutral  extract on GC/FID.

       Calculate the concentration of the sample peak  that produces
       the greatest Instrument response above that observed for the
       HCB standard.

       If the concentration 1s less than 1,000 ug/kg,  report the
       sample concentration as <1,000 ug/kg.

       If the concentration is greater than 1,000 ug/kg, analyze the
       base/neutral extract by GC/MS (paragraph 3.1.8).

       If the sample peaks 1n the chromatogram are less than the
       responses produced by the HCB standard, record the sample
       concentrations as less than the minimum detection limit.

       Analyze all reolicate samples, blanks, and spiked samples in
       a similar fashion.  Record the variability, spike recovery,
       and blank analysis information in a QC log book.

3.1.8  Gas Chromatography/Mass Spectroscopy Analyses of the
       Base/Neutral Extracts

       At the beginning of each day that base/neutral  analyses are
       to be performed, inject 100 nanograms  of benzidine either
       separately or as part of a standard mixture that may also
       contain 50 ng of DFTPP.  Calculate the tailing factor as
       shown 1n Figure 2 and discussed elsewhere.5  The tailing
       factor for benzidine should be less than 3.

       Establish chromatographic conditions equivalent to those
       presented 1n Table 6.  Included 1n these tables are estimated
       retention times and sensitivities that can be achieved by
       this method.  An example of pre-separation achieved by this
       column is shown 1n Figure 3.

       Establish the GC/MS operating conditions Indicated below:

            Mass Range             m/e 41-475
            Scan Time              7 seconds or less
            Electron Energy        70 eV
            Source Temperature     280-300eC
            Start Acquisition      0.1 minute after stopping flow.

       Program the GC/MS to operate  in the Extracted Ion Current
       Profile uICP) mode, and collect ZIC?  for the thj-ee  ions

                                III-120

-------
    TABLE 6.  GAS CHROMATOGRAPHY OF BASE/NEUTRAL EXTRACTABLES
==============s==========s:=============================================

                        Retention Time (fl)
Compound                   (minute)          Limit of Detection
1,3-Dichlorobenzene
1,4-Dichl orobenzene
Hexachloroethane
Bi s[2-chl oroethyl ]ether
1 ,2-Dichl orobenzene
81 s[2-chl oroi sopropyl ]ether
N-Ni trosodi-n-propyl ami ne
Nitrobenzene
Hexachlorobutadiene
1 ,2 ,4-Trichl orobenzene
Isophorone
Naphthalene
Bis[2-chloroethoxy]me thane
Hexachl orocycl opentadiene
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2,6-Dinitrotoluene
Fluorene
4-Chlorophenyl phenyl ether
2,4-Dinitrotoluene
1 ,2-Di phenyt hydrsrrine'0'
Di ethyl phthalate
N-NitrosodiphenyTaminefd)
Hexachl orobenzene
4-Bromophenyl phenyl ether
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Benzidine
Butyl benzyl phthalate
Bi s[2-ethyl hexyl ]phthal ate
Chrysene
BenzoFalanthracene
3,3'-Dichlorobenz1dine
Di-n-octyl phthalate
Benzo[b]f 1 uoranthene
Benzo[k]fl uoranthene
Benzo[a]pyrene
7.4
7.8
8.4
8.4
8.4
9.3

11.1
11.4
11.6
11.9
12.1
12.2
13.9
15.9
17.4
17.8
13.3
18.7
19.3
19.5
19.8
20.1
20.1
20.5
21.0
21.2
22.8
22.8
24.7
26.5
27.3
28.8
29.9
30.6
31.5
31.5
32.2
32.5
34.9
34.9
36.4
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
                                                           (continued)
                             III-121

-------
                             TABLE 6.   (Continued")
       8=======================================================================
                                Retention Time  (a)
        Compound                   (minute)          Limit of Detection  vb)
Indeno[l,2,3-cd]pyrene
Di benzo[ah]anthracene
Benzo[ghi]perylene
N-Nitrosodimethyl amine
2 ,3 ,7 ,8-Tetrachl orodibenzo-p-
dioxin
42.7
43.2
45.1
50
50
50
25
25
25
(a)Co1umn:   1.8 m glass (6.4  mm  O.D.  x  2 mm  I.D.), packed, with 3% SP-2250 coated
   on 100/120 mesh Supelcoport.   Carrier gas:  helium at  30 ml per min.
   Temperature program:   isothermal for 4 minutes at 50"C, then 8C per min to
   270eC;  hold at 270C for 30 minutes.  If  desired, capillary or SCOT columns
   may be  used.
(b'This is a minimum level  at which the entire analytical system must give
   mass spectral confirmation.   (Nanograms injected is based on a 2-ul
   injection of a 1-1  sample  that has been extracted and  concentrated to a
   .-olume  of 1.0 ml.)
(c)[)etected as azobenzene.
.^Detected as diphenyl amine.
            listed in Table 4  for each  compound  being measured.  Opera-
            ting in this mode,  calibrate  the  system  response for each
            compound by using  either the  internal  or external standard
            procedure.

            If the internal standard approach is used,  the  standards
            should not be added to the  sample extracts  until immediately
            before injection into the instrument.  Mix  the  spiked extract
            thoroughly.  Inject 2 to 5  ul  of  the sample extract.  The
            solvent-flush technique is  the preferred procedure.

            If the external calibration approach is  used, record the
            volume of extract  injected  to the nearest 0.05  ul.

            If the instrument  response  for any ion exceeds  the linear
            range of the system, dilute the extract  as  necessary and
            reanalyze.

            Qualitative identification  criteria  and  calculations are
            described in Subsection K.  When  the extracts are not being
            used for analysis,  store them in  vials with unpierced septa
            in the dark at 4*C.
                                      HI-122

-------
                                                 21-111
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                             Hxachlorocyclop*fitad>n
                         . 1.3 - Dichlorobcnzaf*
                           i 1.4-Dichlorobnzen
                                     1.2-Oichlorobnz*n * Haxachlorocthan*

                          Bi* Banzo(a)pyran
                                                                      Oi-n-octyl Phthalata
                           Indenod ,2.3-cd)pyrn


                           Oibanzola. h)anthracen

                              Bnzo(ghi)pryl*n
? ' ?


* "  w
2 o X
                                                                                                  0 91
                                                                                                 o
                                                                                                 w
                                                                                                 -J
                                                                                                 O
                                                                                                 o
                                                                                                 O

-------
3.2  Analysis of Methylene Chloride Sediment Extracts  for Base/Neutral
     Compounds
          Analytical  Procedure:   available
          Sample Preparation:   available

     3.2.1  Reference

            Jacobs Engineering Group, "Manual  of Methods for the Analyses
            of Hazardous Wastes."  Contract Report 68-03-2569 prepared  for
            Environmental Protection Agency, Environmental  Monitoring
            Systems Laboratory,  Las Vegas, Nevada.   Jacobs  Engineering
            Group, Pasadena,  California.  1981.6

     3.2.2  Method Summary

            A 50-g sample of soil/sediment is extracted with methylene
            chloride using wet residual  waste/solvent  techniques.   Aided
            by a high-speed homogenizer, samples are extracted at  pH  11
            to isolate the base/neutral  compounds.   The extract is
            cleaned up using  gel  permeation chromatography.

     3.2.3  Applicability

            The procedure is  for use with solid-phase  samples such as soil
            and sediment.  The detection limit of the  procedure will  be
            influenced by sample size, co-extracted materials, and sample
            cleanup.

     3.2.4  Precision and Accuracy

            This information  is not presently available.

     3.2.5  Sample Preparation

            Thoroughly mix the sample by homogenizing  it in the original
            sample bottle.  Weigh into a 200-ml  centifuge bottle a 50-g
            aliquot or an appropriate weight based on  screening analysis.
            Add surrogate standards and  mix the  aliquot to  be analyzed.

            Adjust the pH of the sample with 10  N sodium hydroxide to a
            pH of 11 or greater.   Mix briefly with the homogeni-zer to
            ensure uniform sample pH.

            Add 60 ml of methylene chloride to the sample bottle and
            homogenize briefly.   Rinse the homogenizer off with a  minimum
            of water and then with 5 to 10 ml  of methylene chloride.
            Additional methylene chloride may be added until  the liquid
            surface 1n the centrifuge bottle is  close  to the top.

            Centrifuge the sample for 15 minutes.  The mixture wiil
            separate into an aqueous "layer over  the methyl *>ne chloride
            extract.  A solid cake or emulsion may form at the water-
            methyl ene chloride interface.  If the emulsion interface

                                    III-124

-------
       between layers is more than one-half the size of the solvent
       layer, a smaller sample size should be used to complete the
       phase separation.  Withdraw the organic extract from the
       centrifuge bottle with a 50-ml  glass syringe that has been
       equipped with a 150-mm x 5-mm I.D.  TFE tube.  Discharge the
       extract into a 300-ml  beaker.

       Repeat the sample extraction procedure a second time with a
       60-ml portion of methylene chloride.  Combine the extracts.

       Perform a third extraction with a final 60-ml portion of
       methylene chloride and combine  the  extracts.

3.2.6  Sample Extract Drying

       Pour the combined extract resulting from the extraction proce-
       dure through a drying column containing 7 to 10 cm of organic-
       free anhydrous sodium sulfate..   Collect the dried extract in a
       500-ml Kuderna-Danish flask equipped with a 10-ml  concentrator
       tube.

       Wash the flask that originally  contained the extract and the
       drying tube three times with 30-ml  aliquots of methylene chlo-
       ride.  Add these washes to the  sample extract in the Kuderna-
       Danish flask.

3.2.7  Sample Extract Concentration

       Add one or two clean boiling chips  to the flask anoV attach a
       three-bal"  siacro-Snyder column.  P'-swet the column by adding
       approximately 1 ml of the extracting solvent through the top
       of the column.  Place the apparatus in a 60 to 65"C water
       bath so that the concentrator tube  is partially immersed in
       the water and the lower rounded surface of the flask is
       bathed with water vapor.  Adjust the apparatus as necessary
       to complete concentration to approximately 10 ml  in 15
       minutes.  (At the proper rate of distillation, the balls of
       the column will chatter but the chambers will not flood.)

       Remove the Snyder column, and rinse the flask and its lower
       joint into the concentrator tube with 1 to 2 ml  of methylene
       chloride employed in the extraction.

       Fit the concentrator tube with  a modified macro-Snyder column.
       Organic-free nitrogen is employed to reduce the volume of the
       extract to approximately 5 ml or 1  ml  (but not below 0.5 ml).

       Wash the concentrator tube with two 0.2-ml volumes of methylene
       chloride.

       Adjust the final  sxtract volume to  S TI"! or 1 ml  eor subse-
       quent internal standard addition and GC/MS analysis.  If the
                               111-125

-------
       extract obtained above 1s "clean,"  then a final  extract
       volume of 1 ml  1s required.

3.2.8  Gel Permeation Cleanup

       Determine the residue weight of the concentrated sample
       extract by placing a 1-ml aliquot on a tared aluminum foil
       pan, allowing the solvent to evaporate, and reweighing the
       pan.  These results are used to determine the volume of
       extract to be applied to the column for cleanup.  The volume
       of extract applied to the column should not exceed the
       capacity of the column, approximately 200 mg.  If the residue
       weight is on the order of 1  to 5 mg, cleanup by gel  permea-
       tion can, 1n many cases, be avoided.

       Transfer 5 ml of the GPC calibration solution to the Bio-
       Beads S-X3 column."  Drain the column Into a 100-ml graduated
       centrifuge tube until the liquid is just above the surface of
       the GPC packing.  Wash the calibration solution on the column
       with several 1-ml aliquots of methylene chloride.  Elute the
       columns with 200-ml aliquots of methylene chloride and
       collect 10-ml fractions.

       'Analyze the fractions for bis[2-ethylhexyl]phthalate and
       pentachlorcphenol by GC/FID on a 1 percent SP-1240 DA column.
       Determine the corn oil elution pattern by evaporation of each
       fraction to dryness followed by gravimetric determination of
       *he residue.  Plot the concentration of each component in
       each fraction versus the total eluant volume.

       The first fractions of the eluant that represent an approximate
       85 percent  removal of the corn oil and 85 percent recovery
       of the bis[2-ethylhexyl]phthalate can be discarded.  Collect
       the fractions that elute up to a retention volume represented
       by 50 ml after the elution of pentachlorophenol.  (Typical
       procedures  are to discard the first 60 ml, to collect the
       next 110 ml, and to wash the column with 250 ml of methylene
       chloride between samples.)

       Select a volume  of sample extract (based on  the residue
       weight determination) that will not overload the column.
       Apply an aliquot (1 to 4 ml) of the extract  to  the column and
       drain the column until the sample 1s just above the surface
       of the GPC  packing.  Wash the extract  onto the  column with
       several 1-ml portions of methylene chloride.  Elute the
       column with 200-ml aliquots of methylene chloride.

       Collect the first 60 ml  of eluant 1n a 100-ml graduate
       cylinder  ,nd nass  the  next 110 ml of eluant  through a drying
       column containing 6  cm of anhydrous soaium suifate and
             et  In  a 500-inl  Kuderna-Oanish  flask equipped with  10-ml
                                II I-i.26

-------
concentrator tube.  Rinse the drying column with three 25-ml
portions of methylene chloride.

Add one or two clean boiling chips to the flask and attach a
three-ball macro-Snyder column.  Prewet the column by adding
approximately 1 ml of the extracting sol vent, through the top
of the column.  Place the apparatus in a 60 to 65C water
bath so that the concentrator tube is partially immersed in
the water, and the lower rounded surface of the flask is
bathed with water vapor.  Adjust the apparatus to complete
concentration to approximately 10 ml  in 15 minutes.  (At the
proper rate of distillation, the balls of the column will
chatter but the chambers will not flood.)

Remove the Snyder column, and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml  of the
solvent employed in the extraction.

Fit the concentrator tube with a modified macro-Snyder
column.  Organic-free nitrogen is employed to reduce the
volume of the extract to approximately 5 ml or 1 ml (but not
below 0.5 ml).  If the extract obtained above is "clean,"
then a final  extract volume of 1 ml is required.  After the
desired volume '">as been reached, wash the Snyder column joint
and the concentrator tube with two 0.2-ml volumes of the
extracting solvent.  Adjust the final extract volume to 5 ml
or 1 ml for subsequent internal standard addition and GC/MS
analysis.

If the extract is to be stored before GC/MS analysis, trans-
fer the extract to an appropriately sized serum vial  equipped
with a Teflon-lined rubber septum and crimp cap.  The extract
volume should be scored on this vial, and appropriate sample
identification must be affixed to the vial.  Store the
extract in the dark at 4C.

It is possible that samples which contain high concentrations
of extractable organic compounds will not be amenable for
concentration to 5 ml.  For extracts of this type, the final
volume after concentration should be adjusted to a minimal
volume which results in an extract viscosity that allows sam
pllng with a micro-syringe.  Obvious remedies will likely include
either starting with smaller sample size or concentration to  a
volume greater than 5 ml.

Qualitative Identification criteria and calculations are
described in Subsection K.
                        III-127

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4.1  Analysis of Base/Neutral  Compounds in Biological  Tissue
          Analytical  Procedure:   available
          Sample Preparation:   available

     4.1.1  Reference/Title

            U.S. Environmental  Protection Agency,  "Extraction and Analyses
            of Priority Pollutants in Biological  Tissue."  Method 10/80.
            U.S.  EPA, S&A Division,  Region IV,  Laboratory Services  Branch,
            Athens, Georgia,   p.  7.   (1<580).2

     4.1.?  Method Summary

            A 10-g sample of homogenized fish tissue is  mixed with 40  g
            of sodium sulfate and extracted with  methylene chloride  using
            an ultrasonic probe.   The sample is  filtered,  concentrated
            to 10 ml  or less,  cleaned up using acetonitrile partitioning,
            and concentrated to 1 ml.  The extract is  screened using gas
            chromatography and quantified using  mass spectrometry.

     4.1.3  Applicability

            The limit of detection for this method is  usually dependent
            upon the  level  of interferences rather than  instrumental
            limitations.  Where interferences are  not  a  problem,  the
            limit of  detection for most compounds  analysed Dy GC/MS  is
           -2 mg/kg (wet weight basis).

            The method is recommended for use only by  experienced residue
            analysts  or under the close supervision of such qualified
            persons.

     4.1.4  Estimates of Precision and Accuracy

            No information is presently available.

     4.1.5  Sample Extraction

            Blend equal amounts of fish tissue and dry ice.  If a large
            sample is being processed, a food processor  or meat grinder
            may be convenient.

            Weigh 10  g of homogeneous sample into  a 400-ml  beaker and  mix
            with 40 g of sodium sulfate.  Ensure  that  the  sample  is
            thoroughly dry.

            Add 100 ml of methylene chloride to  the tissue mixture.
            Place an  ultrasonic probe in the mixture and sonicate at 50
            percent pulse for 3 minutes.  Transfer the methylene chloride
            phase to  a 500-ml  K-D flask.
                                    III-128

-------
       Repeat the methylene chloride/sonication extraction of the
       tissue sample with a second 100-ml portion of methylene
       chloride.  Combine the extracts in the K-D flask and extract
       the residue a third time with 100 ml methylene chloride.
       Combine the extracts.

       NOTE:  The probe should be carefully cleaned between each
       sample as indicated in Subsection C (Interferences).

       Add a clean boiling chip to the K-D flask and attach a
       three-ball macro-Snyder column.  Place the K-D apparatus on a
       water bath and concentrate the extract to 10 ml.

       Quantitatively transfer the concentrated extract to a 125-ml
       separatory funnel.  Add enough hexane to bring the final
       volume to approximately 15 ml.  Extract the sample four times
       by shaking vigorously for 1 minute with 30-ml portions of
       hexane-saturated acetonitrile.

       Combine and transfer the acetom'trile phases to a one-liter sep-
       aratory funnel and add 650 ml of distilled water and 40 ml of
       saturated sodium chloride solution.  Mix thoroughly for 30 to
       45 seconds.  Adjust the oH of the aqueous phase to 12 and ex-
       tract with two iOO-inl portions of .methylene :n!oride.  Shane tne
       sample vigorously for 15 to 30 seconds during each extraction.

       The residual aqueous sample phase may be retained for extrac-
       tion of acidic compounds or discarded.

       Combine the methylene chloride extracts in a one-liter separa-
       tory funnel and wash with two 100-ml portions of distilled
       water.  Discard the water layer and pour the methylene chloride
       layer through a drying column packed with 7 to 10 cm of organic-
       free anhydrous sodium sulfate and one inch of glass wool.  Col-
       lect the extract in a 500-ml  K-D flask equipped with a 100-ml
       ampul.  Rinse the separatory funnel and drying column with
       three 10-ml portions of methylene chloride.  Add the rinsings
       to the K-D flask.

       Attach a three-ball macro-Snyder column and place the K-D
       apparatus in a hot water bath (60-65*C).  Concentrate the ex-
       tract to 6 to 10 ml.  Use a stream of dry nitrogen to concen-
       trate the extract to 1 ml.

       Transfer the extract to a GC vial and label as the base/
       neutral fraction of the semi-volatile compounds.  This
       extract is now ready for analysis.

4.1.6  Analysis by Gas Chromatography

       The base/neutral  extracts are screened on GC/FID using the
       appropriate column to determine whether GC/MS analyses are

                               III-129

-------
       necessary.  Relative retention times and limits of detection
       are  summarized  in Table 6; a representative chromatogram is
       presented  in Figure 3.

       Calculate  the FID response of 50 ng hexachlorobenzene (HCB)
       for  base/neutral compounds.  (The GC/MS requires approxi-
       mately  50  ng HCB to give a complete mass spectrum.)

       If any  sample peaks produce a greater response than hexa-
       chlorobenzene,  calculate the concentration of the largest
       peak.

       a.  If  the calculated  concentration of the sample component
           is  greater  than 2  mg/kg (wet weight basis), analyze the
           extract by  GC/MS.

       b.  If  the calculated  concentration of the sample component
           is  less than 2 mg/kg, report the concentration as less
           than 2 mg/kg.

       c.  If  all  sample peaks  in the chromatogram are less than the
           hexachlorobenzene  peak, record the minimum method
           detection  limit for  the sample.

       Analyze all blanks and spikes and record the pertinent
       precision and  accuracy data with the sample Information.

4.1.7  Gas  Chromatography/Mass  Spectroscopy Analysis of the Base/
       Neutral Extracts

       At the beginning  of each day that base/neutral analyses are
       to be performed,  inject  100 ng of benzidine either separately
       or as part of  a standard mixture that may also contain 50 ng
       DFTPP.   The tailing factor for benzidlne, calculated as shown
       in Figure 2, should be less than 3.

       Establish chromatographic conditions equivalent to those
       presented in Table 6.   Included  in  this  table is information
       on estimated retention times and sensitivities that can be
       achieved by this  method. An example of  the pre-separation
       achieved by this  column  is shown in Figure 3.

       Establish the GC/MS operating conditions  Indicated below:

            Mass Range            m/e  41-475
            Scan Time             7  seconds or  less
            Electron Energy        70  eV
            Source Temperature    280-300"C
            Start Acquisition     0.1  minute  after  stopping  flow.

       Program the GC/MS to  ooerate  in the Extracted  Ion  Current
       Profile (EICP) mode,  and collect EICP  for the  tnree

                               IH-130

-------
            characteristic ions listed in Table 4 for each compound being
            measured.   Operating in this mode,  calibrate the system
            response for each compound by using either the internal  or
            external  standard procedure.

            If the internal  standard approach is used, the standards
            should not be added to the sample extracts until immediately
            before injection into the instrument.  Mix the spiked  extract
            thoroughly.   Inject 2 to 5 yl of the sample extract.   The
            solvent-flush technique is the preferred  procedure.

            If the external  calibration approach is used,  record the
            volume of extract injected to the nearest 0.05 yl.

            If the instrument response for any  ion exceeds the  linear
            range of the system, dilute the extract as necessary and
            reanalyze.

            Qualitative identification criteria and quantitative calcula-
            tions are described in Subsection K.  When the extracts
            are not being used ^or analyses, store then in vials with
            unpierced septa  in the dark at 4C.


K.   QUALITATIVE AND QUANTITATIVE DETERMINATION

     To qualitatively identify a compound', obtain an  Extracted  Ion Current
profile (EICP^ for the primary ion and the two  other  ions  listed in Table 4.
The criteria below must  be met for a qualitative identification.

1.   The characteristic  ions for each compound  must have their  maxima  in  the
     same or within one  scan of each other.

2.   The retention time  for  the experimental mass spectrum must be within 30
     seconds of the retention time of the authentic compound.

3.   The ratios of the three EICP peak heights  must agree  within 20%  with  the
     ratios of the relative  intensities for these ions in  a reference  mass
     spectrum.  The reference mass spectrum can be obtained from either a
     standard analyzed through the GC/MS system or from a  reference library.

4.   Structural isomers  that have very similar  mass spectra can be explicitly
     identified only if the  resolution between  the isomers in a standard  mix
     is acceptable.  Acceptable resolution is achieved if  the valley height
     between isomers is  less than 25 percent of the sum of the  two peak heights.
     Otherwise, structural  isomers are identified as  isomeric pairs.

     In samples that contain an inordinate number of  interferences, the
     chemical  ionization (CD mass spectrum may make  identification easier.
     In Table 4, characteristic CI ions for most; of cne compounds  are  given.
     The ':se o-f7 chemical ''onization MS to support EI/MS is encouraged  but not


                                    !! 1-1.31

-------
required.  When a compound has been identified,  the quantification  of
that compound will  be based on the integrated area from the specific  ion
plot of the first listed characteristic ion in Table 4.  If the  sample
produces an interference for the first listed ion, use a secondary  ion  to
quantify.  Quantification can be done by the external  or internal
standard method.

Internal standard - By adding a constant known amount  of internal  stan-
dard (Cis in yg)  to every sample extract, the concentration of contam-
inant (C0) in yg/1  in the sample is calculated using Equation  2.


                             (As)(C1s)
                  C0  =   	'                            Eq. 2
                          (Ais) (RF) (V0)
where:

       V0  =  the volume (in liters) or mass (in grams)  of the original
              sample; the other terms are defined in text  (Subsection  H).

External  standard - The concentration of the unknown is  calculated  from
the slope and intercept of the calibration curve.  The unknown
concentration is determined using Equation 3.


                                       (A)(Vt)
                    ug/1  =  ng/ml   =  	                      Eq.  3
                                       (Vi)(Vs)
where:

   '     A   =  mass of compound from the calibration curve (ng)

        V-j  =  volume of extract injected (ul)

        Vt  =  volume of total  extract (ul)

        Vs  =  sample volume (ml) or sample  mass (g) extracted.

Report all results in yg/1  to two significant figures without correction
for recovery data.  When duplicate and spiked samples are analyzed,  all
data obtained should be reported.
                               III-132

-------
                                     REFERENCES


1.   U.S.  Environmental  Protection Agency.   "Base/Neutrals, Acids, and
     Pesticides - Method 625."   Federal  Register Vol. 44:  No. 233
     69540-69552. December 3,  1979.

2.   U.S.  Environmental  Protection Agency.   "Extraction  and Analyses of
     Priority Pollutants in Biological  Tissue."  Method  10/80.  U.S.
     Environmental  Protection  Agency, S&A Division,  Region IV, Laboratory
     Services Branch, Athens,  Georgia.   7 p.  (1980.).

3.   U.S.  Environmental  Protection Agency.   "Method  for  Preparation of Medium
     Concentration Hazardous Waste Samples."   U.S. Environmental Protection
     Agency, Region IV,  Athens,   Georgia.   May 1981.

4.   U.S.  Environmental  Protection Agency.   "Extraction  and Analysis of
     Priority Pollutants in Sediments."  PPS-9/80, U.S.  Environmental
     Protection Agency,  Region IV, S&A  Division, Athens, Georgia.  7 p.
     (1980).

5.   Eichelberger,  J. W., L. E.  Harris  and  W.  L. Budde.  "Reference Compound
     to Calibrate Ion Abundance  Measurement in Gas Chromatography - Mass
     Soectrometry Systems." Anal. Chem. Vol.  47:995-1000 (1975).

6.   Jacobs Engineering  Group.   "Manual  of  Methods for the Analysis of
     Hazardous Wastes."   Contract Report 68-03-2569  prepared  for Environmental
     Protection Agency,  Environmental Monitoring Systems Laboratory, Las
     Vegas, Nevada.  Jacobs Engineering  Group, Pasadena, California.  (1981).
                                    III-133

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                                   SECTION 4

                    PESTICIDES AND POLYCHLORINATED BIPHENYLS
A.   SCOPE

     The analytical procedures provided 1n Subsection J  of this Section cover
the determination of chlorinated pesticides and PCBs in  hazardous  waste,  waste
oil, water, soil/sediment, biological  tissue,  and air samples.   The compounds
of interest are extracted, the extracts are concentrated,  and  the  compounds  are
identified/quantified using GC or GC/MS procedures.


B.   SAMPLE HANDLING AND STORAGE

     Conventional water sampling practices should be followed  except that the
bottle should not be prerinsed with sample prior to  collection.  Grab samples
should be collected in glass containers and refrigerated immediately.  Com-
posite samples should preferably be collected  in glass containers  and,  if
possible, refrigerated during the period of compositing.  All  automatic
sampling equipment must be free of Tygon and other potential sources of
organic contamination.  Wh'en necessary, the equipment should be prerinsed with
hexane prior to use.

     Water samples must be iced or refrigerated at 4*C from the time of
collection until extraction.  If the samples will  not be extracted within 72
hours of collection, they should be adjusted to a pH range of  5.0  to 9.0  with
sodium hydroxide or sulfuric acid, as  appropriate.  Record the volume of  acid
or base used.  If aldrin is to be determined,  add sodium thiosulfate when
residual chlorine is present.  EPA Methods 330.4 and 330.5 may be  used to
measure chlorine residual.1  Field test kits are also available for this
purpose.

     Plastic bottles must not be used  since they are known to  introduce
interferences and absorb pesticides.  The sample size is dictated  by the
sensitivity required for a particular  purpose  and the detection system to be
employed.  Sufficient sample should be collected to  permit the analysis of
duplicate and spiked analyses.  Breakage of glass sample bottles can be
minimized by shipping them in expanded polystyrene containers  molded to fit
the bottles.

     Ml samples must be extracted within 7 days of  collection and the
extracts completely analyzed within 40 days or extraction^ (Figure i).
                                    HT-134

-------
Satole 1 Air I Hater or
Matrix I 1 teachate

nut
Sample Paper
Pro- or
cess Ing Tenax
Cart-
ridge



| Slwdoe. Soil or Sedlwnt
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\ \ \
r | Preserve Centri-
1
, * ,,
1 Slortl Preserve

Extract]! Extract I 1 Store


*- I Analyze 11 Analyie I Extract/
7* 1 II 1 Ole*t
-
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Ha f Uet Storage 1 Or
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ent


di 	
I ^
*
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lox 1 loxlctty | |
lelt* 1 	 . 	
Anal


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Extract

I Analyie j
L -- -  *
Analyie 1
lolog- Haiardousl
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I i
y frwen Frtutn
age. Storage Storage Store
I
act Extract Extract | Extract 1

jr/e Analyte Analyte 1 | Analyte

Purpose Total Cone. Total "Dissolved* Total Cone. Total Cone. Total Cone. Total Total
In Air Cone. Cone. In Nobility Mobility In Sedlwnt. In Seotccnt. In Sedlxnt. Cone. In Cone. In
In Hater Hater at pM S *t pH S Sludge. Soil. Sludge. Soil. Sludge. Soil. Tissue HasU
S*plt
Container foil. 6 6 G 6 G G 6 6
Sample
Preser-
vative 4t 4*C 4*C
Storage
T1o 74 74 74
S*"p1t *
Site la1 it 11 II 10 g 10 g 10 g 10 g 1 ! I fl
Figure 1.  Handling and sample  storage Information  for  pesticide  and  PCB  samples.

-------
     Sediment/soil samples should also be stored 1n glass containers.   To
prevent sample contamination from sample bottle caps or cap liners,  the
containers should be sealed with either Teflon or acetone/hexane-washed
heavy-duty aluminum foil.  Sediment samples may be stored in a field-moist,
air-dried, or frozen condition if a total sediment concentration is  to be
determined.  When operational  procedures such as the EP Toxicity Test  are to
be performed, the samples should be stored in a field-moist condition.

     Air can be sampled for PCB analysis using polyurethane cartridges  as the
collection medium.3  Place a P-4000 pump and sampling cartridge on a tripod
or other support so that the intake is at least 1 meter above the ground. A
shelter (such as an umbrella)  should be used to protect the sampler  in  the
event of precipitation.  Operate the sampler for a minimum of 4 hours.   A
nominal flow of 3.8 1/min. will provide a total sampled volume of approxi-
mately 1 m^ in 4 hours.  This  volume should not exceed the breakthrough
volumes of the cartridges for the compounds to be analyzed.  Package and
remove the collected samples from the sampling area.

     All polyurethane plugs should be kept in hexane-rinsed aluminum foil and
individual glass containers before and after sampling to minimize possible
contamination.

     Oil samples snould oe collected in glass containers with a volume  of at
least 20 ml and equipped with  Teflon-lined screw caps.4  Prior to sample
collection, the containers should be cleaned in the following manner:

a) -  Wash all sample bottles and seals in detergent solution.  Rinse with tap
     *ater ind l^en distilled  water.  Allow the bottles and seals to dry in  a
     contaminant-free area.  Rinse the seals with pestle:de-grade hexane ind
     allow to air dry.

b)   Heat the sample bottles to 400*C for 15 to 20 minutes or rinse  with
     pesticide grade acetone or hexane and allow to air dry.

c)   Store the clean bottles inverted or sealed until used.

     Oil samples should be stored in a cool, dry, dark area until analyzed.
Storage times in excess of 4 weeks are not recommended for unknown or
undefined sample matrices.*


C.   INTERFERENCES

1.   Method interferences may be caused by contaminants in solvents, reagents,
     glassware, and other sample processing hardware that lead to discrete
     artifacts and/or elevated baselines in gas chromatograms.  All  of these
     materials must be routinely demonstrated to be free from interferences
     under the conditions of the analysis by running laboratory reagent
     blanks.
                                    III-136

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     1.1  Glassware must be scrupulously cleaned.5   Clean  all  glassware  as
          soon as possible after use by rinsing  with the last  solvent  used  in
          it.  This should be followed by detergent-washing with  hot water,
          and rinses with tap water and distilled water.   After the  glassware
          has dried, heat in a muffle furnace at 400C  for 15  to  30  minutes.
          Some thermally stable materials,  such  as  PCBs, may not  be  elimi-
          nated by this treatment.   Solvent rinses  with acetone and  pesticide-
          quality hexane may be substituted for  the muffle furnace heating.
          Thorough rinsing with such solvents usually eliminates  PCB inter-
          ference.  After drying and cooling, glassware should be sealed  and
          stored in a clean environment to  prevent  any  accumulation  of dust or
          other contaminants.  Store inverted or capped with aluminum  foil.

     1.2  The use of high-purity reagents and solvents  helps to minimize
          interference problems. When necessary, purification of solvents by
          distillation in all-glass systems may  be  required.

2.   Interferences by phthalate esters can  pose  a major problem in pesticide
     analysis when using the electron capture detector. These compounds  gener-
     ally appear in the chromatogram as large late  eluting peaks, especially in
     the 15- and 50-percent fractions from  Florisil  column cleanup.  Common
     flexible plastics contain varying amounts of phthalates that are  easily
     extracted or leached during laboratory operations. Cross-contamination of
     clean glassware, especially solvent-wetted  surfaces,  routinely  occurs
     when plastics are handled during extraction steps. Interferences from
     phthalates can best be minimized by avoiding the use  of plastics  in  the
     laboratory.  Exhaustive cleanup of reagents and glassware may be  required
     to eliminate background phthalate contamination.?   The  interferences
     from phthalate esters can be avoided by using  a microcoulometric  or
     electrolytic conductivity detector.

3.   Matrix interferences may be caused by  contaminants that are  coextracted
     from the sample.  The extent of matrix interferences  will vary  consider-
     ably from source to source, depending  upon  the nature and diversity  of
     the industrial complex or municipality being sampled.  The cleanup
     procedures in Subsection J can be used to overcome many of these  inter-
     ferences, but unique samples may require additional cleanup  approaches to
     achieve the MDL listed in Table 1.9

4.   Interference from fish oil can be eliminated by acetonitrile parti-
     tioning. 8  However, the ultrasonic probe must  be scrupulously cleaned
     between samples.  The procedure is:

     1)  Rinse the probe with solvent into  the sample.
     2)  Remove residue on the probe with a wet  tissue.
     3)  Rinse the probe with methylene chloride.
     4)  Sonicate with hexane for 3 to 4 minutes on 50  percent pulse.

5.   Special attention is called to industrial plasticizers and hydraulic
     fluids such as the chlorinated biphenyls which are potential sources of
     interference in pesticide analysis.10   Chlorinated biphenyls containing


                                    111-137

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         TABLE 1.  METHOD DETECTION LIMITS FOR ORGANOCHLORINE
           PESTICIDES AND PCBs9 BY ECD GAS CHROMATOGRAPHY

                                               Method
              Parameter                 Detection Limit,  ug/1
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma- BHC
Chlordane
4,4'-DDD
4,4'-DDE
4, 4 '-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan Sulfate
Endrin
Endrln aldehyde
Heptachlor
Heptachlor Epoxide
Toxaphene
OC3-1016
PC8-1221
PCB-1232
PCB-1242
PC8-1248
PCB-1254
0.004
 0.003
0.006
0.009
0.004
0.014
0.011
0.004
0.012
0.002
0.014
0.004
0.066
0.006
0.023
0.003
0.083
0.024
nd
nd
nd
0.065
nd
nd
PCB-1260 nd
================================ ====================
         nd * not determined
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 unresolved peaks (shoulders and non-gaussian
peaks) slight discrepancies in retention times,  and peaks that elute
later than p,p'-DDT.  A number of chlorinated biphenyl  Isomers may
Interfere with the determination  of DDE, ODD, and DDT Isomers.  The
purity of compounds associated with specific peaks 1n the DDT chromato-
gram should be periodically examined by repeating the chromatographic
analysis after conversion of DDT  to DDE with KOH in ethanol.   Particu-
lar1;-' savere *nter*erencss will require a more detailed examination of
the sample extract.
                               111-138

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6.   The electron capture detector responds to a wide variety of organic
     compounds.  It is likely that some compounds will  be encountered as
     Interferences during GC-EC analysis.  Periodic mass spectrometric
     analyses on composited and/or selected samples will provide for positive
     identification of specific compounds and interferents.


D.   SAFETY

1.   The toxicity or carcinogenicity of each reagent used in this method has
     not been precisely defined; however, each chemical  compound should be
     treated as a potential health hazard.  From this viewpoint, exposure to
     these chemicals must be reduced to the lowest possible  level  by whatever
     means available.  The laboratory 1s responsible for maintaining a current
     awareness file of OSHA regulations regarding the safe handling of the
     chemicals specified in this method.  A reference file of material data
     handling sheets should also be made available to all personnel  involved
     in sampling and analysis of samples containing these compounds.  Addi-
     tional references on laboratory safety have been identifiedll-13 for
     the information of the analyst.

2.   The following parameters covered by this method have been tentatively
     classified as known, or suspected, human or mammalian carcinogens:
     4,4'-DDT, 4,4i-DDD, and the PC8s.  Primary standards of these toxic
     compounds should be prepared 1n a glovebox and/or containment laboratory.

3.   Diethyl  ether should be monitored regularly to determine the peroxide
     content.  Under no circumstances should diethyl  ether be used with a
     peroxide content in excess of 50 ppm as an explosion eculd result.
     Peroxide test strips manufactured by EM Laboratories (available from
     Scientific Products Co., Cat. No. P1126-8, and other suppliers) are
     recommended for this test.  Procedures for removal  of peroxides from
     diethyl  ether are included in the instructions supplied with  the per-
     oxide test kit.
E.   APPARATUS

1.   Sample Transfer Implements - Implements are required to  transfer  portions
     of solid, semi-solid,  and liquid wastes from sample  containers  to  lab-
     oratory glassware.   The transfer must be accomplished rapidly  to  avoid
     loss of volatile components during the transfer  step.  Liquids  of  low to
     moderate viscosity may be transferred using conventional  laboratory
     pipets.  Non-tacky solids may be transferred using conventional labora-
     tory spatulas.   Spoon-shaped porcelain spatulas  (Coors No. 60478,  or
     equivalent)  are useful  in that they have a  measureable bowl-volume.
     Samples having  a desired approximate volume can  thus be  obtained.  Trans-
     fer of tacky or non-tacky solids and semi-solids may be  simplified using
     the imolements  described below.   A modified pipet suitable for  transfer
     of some viscous liquids is also  described.
                                    III-139

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     1.1  Implement for transfer of non-tacky semi-solids  -  A  3-ml  glass
          hypodermic syringe is  modified.   The plunger is  removed  and  the
          normally closed end of the barrel  is cut away.   To use this
          implement, the plunger is replaced flush with the  cut-away,  open
          end.  The device is pressed into a semi-solid sample, thereby
          forcing the plunger out of the  barrel.   When the plunger has been
          displaced by a volume  equal  to  the approximate sample volume
          desired, the syringe is withdrawn  and the semi-solid plug is
          transferred to a tared vessel by displacing  the  material  with the
          plunger.

     1.2  Implement for the transfer of tacky semi-solids  and  solids - This
          approach will be useful  for the  transfer of  some tacky or tarry
          materials.  Glass tubing of approximately 1  cm diameter  is cut into
          short sections having  a desired  approximate  volume (i.e.,  1  ml = 1.0
          cm I.D. x 1.3 cm length).  To obtain a  desired volume of sample, take
          a weighed tubing section of that volume, and, using  a Teflon-coated
          laboratory spatula, press a portion of  tarry sample  into the tubing
          section.  The sample-filled tubing section is then placed directly
          into a centrifuge tube containing  polyethylene glycol (PEG); the
          centrifuge tube and PEG are weighed before the sample is  added.

     1.3  Implement for the transfer of viscous liquids -  This device  is
          fashioned oy cutting the constricted end from a  5-ml graduatsd
          pi pet.  The large-bore pi pet thereby obtained is used in conjunction
          with a conventional laboratory  pipetting aid, preferably of  the
          syringe type.  This implement allows convenient  transfer and approx-
          imate volumetric measurement of  some viscous liquids.

2.   Class I Biological Safety Cabinet (glovebox)  suitable for handling
     chemical  carcinogens.  The  cabinet should have an interchange  panel for
     introducing materials, a retaining tray to catch  spills,  and  a static
     pressure gauge {Kewaunee, Inc. - Model  SH-3704-MS-X,  or equivalent).

3.   Water bath - Heated, with concentric  ring cover,  capable  of temperature
     control (2C).  The bath should be  used in  a hood.

4.   Balance - Analytical, capable of accurately weighing  0.0001 g.

5.   Gas chromatograph - An analytical system complete with  chromatographi.c
     column suitable for on-column injection and  all required  accessories
     Including syringes, analytical columns, gases, electron capture detector,
     and strip-chart recorder.  A data system is  recommended for measuring
     peak areas.

     5.1  Column 1.  Supelcoport (100/120  mesh) coated with  1.5% SP-2250,
          1.95% SP-2401, packed  1n a 1.8 m x 4-mm I.D. Pyrex glass column.  Use
          argon 95%/methane 5%,  carrier gas  at a  flow  rate of  60 ml/min.
          Column temperature Isothermal at 200*C.   This column was used to
          deveiop cne method performancs  stataments "Ms tad 
-------
     5.2  Column 2.  1.8 m long x 4 mm I.D.  glass columns,  packed with 3?  OV-1
          on Supelcoport (100/120 mesh)  or equivalent.

     5.3  Column Conditioning.   Proper thermal  conditioning is  essential to
          minimize column bleed and to provide  acceptable gas chroma tographic
          analysis.  A number of procedures  may be used for this  purpose such
          as the following.  Install the packed column  in the oven.   Do not
          connect the column to the detector.   However, gas flow  through the
          detector should be maintained.   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  tem-
          perature for the liquid phase  without gas flow for 2  hours.   Reduce
          the oven temperature to approximately 4QC below  the  maximum recom-
          mended temperature and allow temperature to equilibrate for  a
          minimum of 30 minutes without flow.   Then adjust  the  carrier gas
          flow to about 50 ml/min for a  6.4-mm  (1/4-inch) column  and  about 25
          ml/min for a 3.2-mrn (1/8-inch)  column (Caution:  NOTE 1).   After 1
          hour, increase the temperature  to  about 20C  above normal operating
          temperature for 24 to 48 hours  while  maintaining  the  gas flow.   (Do
          not exceed the maximum recommended operating  temperature.)   Cool
          down and connect the column to  the detector system, then raise it to
          the normal operating temperature.   Columns prepared and conditioned
          1n this manner should yield good chromatograms with no  further
          treatment.

          NOTE 1:  Caution - Bleed-off of liquid phase  will  occur if  the
          liquid phase is not fully temperature-equilibrated.

6.   Gas Chromatographic Detectors

     6.1  Electron Capture Detector (ECD).  The electron capture  detector  is
          capable of responding to picogram  quantities  of chlorinated  hydro-
          carbons, but the practical sensitivity of the detector  is only 30
          to 50 times greater than microcoulometry.   An ECD was used  to
          develop the method performance  statements provided in Tables 9 and
          10 of Subsection J.2.2.

     6.2  Microcoulometric Detector (NOTE 2).   The microcoulometric titri-
          metric detection system is the  most specific  detector for chlori-
          nated hydrocarbons in general  use. This titration-cell  detector is
          capable of responding to low nanogram quantitities for  most  of these
          materials under oxidative conditions, while almost entirely Insensi-
          tive to other organics.   Under  optimum conditions it  is capable  of
          measuring 5 to 20 ng in a reproducible manner, depending on  the
          chlorine content of the individual compound and its Chromatographic
          qualities.  However,  relatively large samples must be processed  in
          order to approach the sensitivity  of  the electron capture detector
          described above (Subsection E.I).
          NOTE 2:   electrolytic  conauc-civrcy jetectors  that  are  2  *:o  3
          mor sensitive than microcoulometric  detectors,  but  less selective,
          may be used as a substitute.

                                    III-141

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7.   Gas Chromatograph/Mass Spectrometer,  Finnigan  3200  and INCOS  2300  Data
     System.
8.   Glassware
     8.1  Separatory funnels - 2,000  ml, 500 ml,  and 250 ml,  with  Teflon  stopcock,
     8.2  Drying column - Chromatographlc  column, approximately 400 mm  long  x
          19 mm I.D., with coarse frit.
     8.3  Chromatographlc column - Pyrex,  400 mm  long x  22  mm I.D., with  coarse
          fritted plate and Teflon stopcock  (Kontes  K-42054 or equivalent).
9.   Kuderna-Danish concentrator fitted with graduated evaporative concen-
     trator tube.  Available from the Kontes Glass Co.,  each  component  bearing
     the following stock numbers:
     9.1  Flask, 250 ml, Stock No. K-570001
     9.2  Snyder column, 3-ball, Stock No. K-503000
     9.3  Steel springs, 1/2 1n.,  Stock No.  K-662750
     9.4  Concentrator tubes,  10 ml,  Size  1025, Stock No. K-570050
10.  Desiccator.
11.  Crucibles, porcelain, squat form, Size  2.
12.  Omni or Sorvall mixer with  chamber of approximately 400  ml.
13.  Filter tube, 180 mm x 25  mm.
14.  Pans, approximately 35 cm x 25 cm x 6 cm.
15.  Oven, drying.
16.  Muffle furnace.
17.  Pyrex glass wool, pre-extracted  with methylene  chloride  in a  Soxhlet
     extractor.
18.  Disposable pipets.
19.  Magnetic Stirrer and 5/8-1n.  Teflon-coated stirring bars.
20.  Graduated centrifuge tubes, 15 ml with  glass stoppers.
21.  tfoiumeiric rlasxs.
22.  Micro-syringes, 10 ui for GLC injection and  100 ul  for preparation of
     standard solutions.
                                   ni-142

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23.  Graduated plpets, 2 and 10 ml.
24.  Heating plate.
25.  Vortex genie.
26.  Boiling chips,  approximately 10/40 mesh.   Heat to  400eC  for  30 minutes or
     Soxhlet-extract with methylene  chloride.
27.  Spatula.  Stainless steel  or Teflon.   Fisher Scientific,  Catalog  No.
     14-375-10, or equivalent.
28.  Vials, specimen, Teflon-lined screw cap,  approximately 20 ml.  Cali-
     brate at 10 ml  by pipetting 10  ml  of  solvent into  the  tube and marking
     the bottom of the meniscus.
29.  Beakers, 400 ml.
30.  Biichner Funnels, 9 cm.
31.  Filter Paper, Whatman No.  41, ashless.
32.  Vacuum filtration apparatus (Fisher 9-788)  or 500-ml  suction filtration
     flasks.
33.  Drying column,  25 mm x 200 mm packed  with 7 to 10  cm  sodium  sulfate and
     4 cm glass wool.
34.  Vials, 2 ml.
35.  Sonicator Cell  Disrupter,  Model W-375 both  high gain,  3/4-in.  probe
     (Heat Systems - Ultrasonics, Inc.  or  equivalent).
36.  Food Processor (Hobart Model 8181D or equivalent).
37.  Rotary vacuum evaporator.
38.  Centrifuge Tubes, graduated, 12 to 15 ml.
39.  N-evap apparatus (or equivalent) for  evaporation of solvent  under a
     gentle nitrogen stream.
40.  Extractor, Soxhlet, 125, 250, or 500  ml.
41.  Cleanup microcolumn, Chromaflex column,  size 22, 20 cm x 7 mm  (Kontes)
     or equivalent.
42.  Pasteur pipettes (23 cmT.
F.   REAGENTS
1.   Reagent watsr - Reagent *ater is defined  as water  in  which an  interferent
                                    III-143

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     is not observed at the method detection  limit of each  parameter  of
     interest.
2.   Sodium hydroxide solution (ION)  -  (ACS).   Dissolve  40  g  NaOH  in  reagent
     water and dilute to 100 ml.
3.   Sodium thiosulfate - (ACS).   Granular.
4.   Sulfuric acid solution (1+1)  - (ACS).  Slowly,  add  50  ml ^$04 (sp.
     gr. 1.84) to 50 ml of reagent water.
5.   Acetone, pesticide quality or equivalent.
6.   Hexane, pesticide quality or  equivalent.
7.   Isooctane, pesticide quality  or equivalent.
8.   Methylene chloride, pesticide quality  or equivalent.
9.   Acetonitrile.
10.  Benzene.
11.  Petroleum ether, pesticide quality or  equivalent.
12.  Acetone-hexane, 1:1.
13.  Methylene chloride - hexane,  15% v/v.
14.  Ethyl ether, pesticide quality or equivalent, redistilled  in  glass,  if
     necessary.
     14.1 Must be free of peroxides as indicated  by EM Laboratories Quant test
          strips (available from Scientific Products Co., Cat.  No. P1126-8,
          and other suppliers).
     14.2 Procedures recommended for removal  of peroxides are provided with
          the test strips.  After  cleanup,  20 ml  ethyl alcohol  preservative
          must be added to each liter of ether.
15.  Sodium sulfate (ACS) granular, anhydrous.  Purify by heating  at  400*C for
     4 hours  in a shallow tray.  Pre-rinse  or Soxhlet extract with methylene
     chloride.
16.  Sodium sulfate solution, saturated.
17.  Florist! - PR grade (60/100 mesh); purchase  activated  at 1250*F  and store
     1n glass containers with glass stoppers  or foil-lined  screw caps.  Before
     use. activate each batch at least 16 hours at 130"C in a foil-covered
     glass container.

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     17.1 Florlsil from different batches or sources may vary in adsorptive
          capacity.  To standardize the amount of Florisil  which is  used,  the
          use of lauric acid valued is suggested.  The referenced procedure
          determines the adsorption from hexane solution of lauric acid (mg)
          per gram Florisil.  The amount of Florisil  to be  used for  each  col-
          umn is calculated by dividing this factor into 110 and multiplying
          by 20 grams.

     17.2 Before using any cleanup procedure, the analyst must process a
          series of calibration standards through the procedure to validate
          elution patterns and demonstrate the absence of interferences from
          the reagents.

18.  Neutral alumina, Hoelm, activity Grade I deactivated with 5 percent  water.

19.  Alumina, basic, 60 mesh, Alfa Products, or equivalent.   Adjust  to
     Brockmann activity IV by adding 6 percent (w/w)  distilled water to the
     adsorbent in the flask, stoppering, and shaking well.   Allow to equili-
     brate for at least 15 hours before use.  Discard after 2 weeks.

20.  Nitrogen gas, dry, purified.

21.  Polyurethane foam plugs for air sampling.

     21.1 Cutting of Air Sampling Cartridges

          Polyurethane foam (PUF) plugs are cut from 3-in.  (7.6 cm)  sheet
          stock of upholstery material  (polyether type,  density 0.0225
          Q/cm3) using a 24-mm U.D.) stainless steel  cutting die.   This  die
          is turned in a drill-press while a stream of water is directed  on it
          to provide cooling.

     21.2 Cleanup of Air Sampling Cartridges

          PUF plugs are pre-cleaned by Soxhlet extraction,  either singly  or in
          batches, as desired.

          21.2.1 Extract with 5 percent diethyl  ether in ji-hexane (glass-
                 distilled, pesticide quality or equivalent)  for 100 cycles.

          21.2.2 Analyze an extract from a representative sample of  each  batch
                 of plugs using procedures in Subsection J.5.1.5.  Soxhlet
                 extract the plugs twice for 50 cycles each,  concentrate  the
                 combined extracts, and analyze for possible  background con-
                 tamination.

          21.2.3 Dry the plugs under vacuum at 758C.

     21.3 Loading of Air Sampling Cartridges

          21.3.1 Using  forceps,  carefully place  the UF  plug  into  the
                 hexane-rinsed glass  sampling cartridge.

                                    III-145

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                 NOTE 3:  It is Important not to touch the cleaned PUF with
                 bare hands.

          21.3.2 Wrap the sampling cartridge in hexane-rinsed aluminum foil
                 and store in glass jars until  ready for.use.

22.  Mercury, triple distilled.

23.  Copper powder, activated.

24.  Stock standard solutions (1.00 ug/ul)  - Stock standard solutions can be
     prepared from pure standard materials  or purchased as certified
     solutions.

25.  Standards, analytically pure, obtainable from the Pesticides and Indus-
     trial Chemicals Repository, U.S.  Environmental  Protection Agency, MD-8,
     Research Triangle Park, North Carolina 27711.  Telephone (919)  541-3951.

     25.1 Prepare stock standard solutions  by accurately weighing 0.0100  grams
          of pure material.   Dissolve  the material in isooctane,  dilute to
          volume in a 10-ml  volumetric flask.  Larger volumes can be used at
          the convenience of the analyst.  If compound purity is  certified at
          96% or greater, the weight can be used without correction  co
          calculate the concentration  of the stock standard.   Commercially
          prepared stock standards can be used at any concentration  if they
         . are certified by the manufacturer or by ati independent  source.

     25.2 Transfer the stock standard  soiutions into Teflon-sealed screw-cap
          bottles.  Store at 4*C and protect from light.  Stock standard
          solutions should be checked  frequently for signs of degradation or
          evaporation, especially just prior to preparing calibration stan-
          dards from them.  Quality control check standards that  can be used
          to determine the accuracy of calibration standards will be available
          from the U.S. Environmental  Protection Agency, Environmental Moni-
          toring and Support Laboratory, in Cincinnati.

     25.3 Stock standard solutions must be  replaced after 6 months,  or sooner
          1f comparison with check standards indicates a problem.

G.   QUALITY CONTROL

1.   The minimum requirements of a formal quality control  program consist of
     an initial demonstration of laboratory capability and the analysis of
     spiked samples as a continuing check on performance.   The laboratory
     should maintain performance records to define the quality of data that
     are generated.

     1.1  Before performing any analyses, the analyst must demonstrate the
          ability to generate dcceptaole accuracy and precision with this
          method.  This ability is established as described in Subsection G.2.
                                    III-146

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     1.2  In recognition of the rapid advances that are occurring in chroma-
          tography, the analyst is permitted certain options to improve the
          separations or lower the cost of measurements.  Each time such
          modifications are made to the method, the analyst is required to
          repeat the procedure in Subsection G.2.

     1.3  The laboratory must spike and analyze a minimum of 10 percent of all
          samples to monitor laboratory performance.  This procedure is
          described in Subsection G.4.  Five percent (1 of 20) or each time a
          set of samples is prepared (whichever is more frequent), a blank of
          the reagents, water, and solvents should be prepared and analyzed.
          Contaminants shall be below the detection limits based on a 1-gram
          (1-ml) sample aliquot.

2.   The ability to generate data of acceptable accuracy and precision must be
     demonstrated by performing the following operations:

     2,1  For each compound to be measured, select a spike concentration
          representative of the expected levels in the samples.  Using stock
          standard solutions, prepare a quality control check sample con-
          centrate in acetone 1,000 times more concentrated than the selected
          concentrations.  Quality control check sample concentrates, appro-
          priate for use with this method, are available from the U.S.
          Environmental ~otection Agency, Environmental Monitoring and
          Support Laooratory, Cincinnati, Ohio 45268.

     2.2  Using a pipet, add 1.00 ml  of the check sample concentrate to each
          of a minimum of four 1,000-ml aliquots of reagent water.  A repre-
          sentative wastewater may be-used in place of the reagent water, but
          one or inore additional diiquots must oe analyzed to determine b2c!;-
          ground levels, and the spike level must exceed twice the background
          level for the test to be valid.  Analyze the aliquots according to
          the appropriate method in Subsection J.

     2.3  Calculate the average percent recovery, (R), and the standard devia-
          tion of the percent recovery(s).  Wastewater background corrections
          must be made before R and s calculations are performed.

     2.4  Using the appropriate data from Table 2, determine the recovery and
          single-operator precision expected for the method and compare these
          results to the values calculated in Subsection G.2.3.  If the data
          are not comparable, the analyst must review potential problem areas
          and repeat the test.

3.   The analyst must calculate method performance criteria and define the
     analytical performance for each spike concentration and compound being
     measured.
                                    III-147

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             TABLE 2.   SINGLE-OPERATOR ACCURACY AND PRECISION FOR
                    PESTICIDES AND PCBs ANALYZED BY 6C/ECD
;a=============================================================================
Parameter
AT dri n
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma-BHC
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan !
Endosuifan II
Endosulfan Sulfate
Endrin
Endrin aldehyde
Heptachlor
rieptacnior Lpoxiae
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Average
Percent
Recovery
89
89
88
86
97
93
92
89
92
95
96
37
99
95
87
	 8&
93
95
94
96
88
92
90
92
91
Standard
Deviation
%
2.5
2.0
1.3
3.4
3.3
4.1
1.9
2.2
3.2
2.8
2.9
2.4
4.1
2.1
2.1
-3.3
1.4
3.8
1.8
4.2
2.4
2.0
1.6
3.3
5.5
Spike
Range
(ug/1)
2.0
1.0
2.0
2.0
1.0
20
6.0
3.0
8.0
3.0
3.0
5.0
15
5.0
12
1.0
2.0
200
25
55-100
110
28-56
40
40
80
Number
of
Analyses
15
15
15
15
15
21
15
15
15
15
12
14
15
12
11
12
15
18
12
12
12
12
12
18
18
Matrix
Types
3
3
3
3
3
4
3
3
3
2
2
3
3
2
2
2
3
3
2
2
2
2
2
3
3
aaaaaaasaaaaasaaaaaaaaasBaaaaaaaaaaasaaaaaaaBaaaaaaaaaaaaaaaaaaaaaaaaaaaaasaaa
     3.1  Calculate upper and lower control  limits  for method performance:

          Upper Control  Limit (UCL)  =  R +  3  s
          Lower Control  Limit (LCD  *  R -  3  s

          where R and s  are calculated as  detailed  in Subsection G.2.  The
          UCL and LCL can be used to construct  control charts15 that are
          useful in observing trends in performance.
                                    III-148

-------
     3.2  The laboratory must develop and maintain separate accuracy state-
          ments of laboratory performance for wastewater samples.   An accuracy
          statement for the method is defined as R  s.   The accuracy state-
          ment should be developed by the analysis of four aliquots of waste-
          water as described in Subsection G.2.2 followed by the calculation
          of R and s.  Alternately, the analyst may use  four wastewater data
          points gathered through the requirement for continuing quality con-
          trol in Subsection G.4.  The accuracy statements should  be updated
          regularly.15

4.   The laboratory is required to collect a portion of  their samples in
     duplicate to monitor spike recoveries.  The frequency of spike-sample
     analysis must be at least 10 percent of all samples, or one sample per
     month, whichever is greater.  One aliquot of the sample must  be spiked
     and analyzed as described in Subsection G.2.  If the recovery for a
     particular parameter does not fall within the. control limits  for method
     performance, the results reported for that parameter in all samples
     processed as part of the same set must be qualified as described in
     Subsection M.5.  The laboratory should monitor the  frequency  of data so
     qualified to ensure that it remains at or below 5 percent.

5.   Before processing any samples, the analyst should demonstrate, through
     the analysis of a 1-liter aliquot of reagent water, that all  glassware
     and <"?aqent interferences are under control.  Each  time a set of samples
     is extracted or there 1s a change in reagents, a lacoratory reagent oianK
     should be processed as a safeguard against laboratory contamination.

6.   It is recommended that the laboratory adopt additional quality assurance
     practices for use with this method.  The specific practices that are most
     productive depend upon the needs of the laooratory  ana che  ,iatjre of >tne
     samples.  Field duplicates may be analyzed to monitor the precision of
     the sampling technique.  When doubt exists over the identification of a
     particular peak in a chromatogram, independent confirmatory techniques
     such as gas chromatography using a minimum of two columns of  different
     polarity, specific element detectors, or mass spectrometry must be used.
     Whenever possible, the laboratory should perform analysis of  standard
     reference materials and participate in relevant performance evaluation
     studies.


H.   CALIBRATION

1.   Establish gas chromatographic operating parameters  which produce reten-
     tion times equivalent to those indicated 1n Table 3.  The gas chromato-
     graphic system may be calibrated using the external standard  technique
     (Subsection H.2.) or the internal standard technique (Subsection H.3.).

2.   External standard calibration procedure:
                                    III-149

-------
         TABLE 3.  CHROMATOGRAPHIC RETENTION TIMES AND METHOD DETECTION
                    LIMITS FOR SELECTED PESTICIDES AND PCBs
Parameter
                                     Retention Time
                                          (min)
Column 1
            Column 2
                   Method
                Detection Limit
                     ug/l
Alpha-BHC
Gamma-BHC
Beta-BHC
Heptachlor
Delta-BHC
Aldrin
Heptachlor epoxide
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
4,4'-ODD
Endosusfan II
4,4'-DDT
Endrin aldehyde
Endosulfan sulfate
Chlordane
Toxaphene
PCB-101S
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
  1.35
   .70
  1.90
  2.00
  2.15
    40
    50
    50
    13
    45
              1,
              2.
   82
   13
  7.
  Q
 6.55
  .83
  .00
 9.40
11.82
14.22
   mr
   Tir
   mr
   mr
   mr
   mr
   mr
   mr
   mr
 1.97
 3.35
 2.20
 4.10
 5.00
 6.20
 7.15
 7.23
 8.10
 9.08
 R.28
11.75
 9.30
10.70
   mr
   mr
   mr
   mr
   mr
   mr
   mr
   mr
   mr
0.003
0.004
0.006
0.003
0.009
0.004
0.083
0.014
0.004
0.002
0.006
0.011
0.004
0.012
0.023
0.066
0.014
0.24
  nd
  nd
  nd
0.065
  nd
  nd
  nd
                                                                     ==========
Column 1 conditions:  Supelcoport (100/120 mesh)  coated with  1.5% SP-2250/
     1.95% SP-2401 packed in a 1.8-m x 4-mm I.D.  glass  column with 5%
     methane/95% argon carrier gas at a flow rate of 60 ml/min.   Column
     temperature Isothermal  at 200"C, except for  PCB-1016  through PCB-1248,
     which should be measured at 160C.

Column 2 conditions:  Supelcoport (100/120 mesh)  coated with  35  OV-1  packed
     in a 1.8-m long x 4-mm I.D. glass column with 5% methane/95% argon
     carrier gas at a flow rate of 60 ml/min.  Column temperature, iso-
     thermal at 200eC, for th pesticides; 140*C  for PCB-1221 and 1232;
     170eC for PCB-1016 and 1242 to 1268.

mr - Multiple peak response.  See Figures  2 thru  10.

nd - Not determined.

-------
       Column: 1.5% SP-2250 +
               1.95% SP-2401 on Supeicoport
       Temperature: 200 C.
       Detector:  Electron Capture
           Retention Time - Minutes
Figure 2.   Gas  chromatogram of chlordane.
                  III-151

-------
                         Column: 1.5% SP-2250 -
                                 1.95% SP-2401 on Supelcoport
                         Temperature: 200 C.
                         Detector:  Electron Capture
i     i    i    i
    6        10
 i    I
14
 i
18
^    I    i     T
22       26
           Retention Time  Minutes
    Figure 3.  Gas chromatogram  of toxaphene.
                     III-152

-------
 Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
 Temperature: 160 C.
 Detector: Electron Capture
    i        i     *             J     *
2        6        10       14        18

               Retention Time - Minutes
22
 Figure 4.   Gas  chromatogram of PCB-1016.
                  III-153

-------
Column:  1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature:  160 C.
Detector: Electron Capture
I     I
I     I    I    I     I    I    1     I
    10  .     14        18       22

Retention Time  Minutes
 Figure  5.   Gas chromatogram of PCB-1221.
                  Ai.i-J.3t

-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160 C.
Detector: Electron Capture
                10
til
     14
 I     T
18
22
                  Retention Time  Minutes
  Figure 6.  Gas chromatogram of PCB-1232.
i     i
    24
                  III-155

-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160C.
Detector: Electron Capture
I    I    I     I     I     I     I
6         10        14        18

      Retention Time  Minutes
                                                  22
     Figure  7.   Gas chromatogram of PCB-1242.
                      III-156

-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature.  160C.
Detector: Electron Capture
              Retention Time - Minutes
      Figure  8.   Gas chromatogram  of PCS-1248.
                                                        26
                       III-157

-------
  Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
  Temperature: 200 C.
  Detector:  Electron Capture
     I          1      i     I      i    T    '     I    '
2          6          10         14        18        22
                Retention Time - Minutes
    Figure  9.   Gas chromatogram of PCB-1254.
                     III-158

-------
   Column:  1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
   Temperature:  200 C.
   Detector: Electron Capture
i
2
6
 i
10
                       I
          14       18

Retention Time  Minutes
22
 I    T
26
     Figure 10.   Gas chromatogram of  PCB-1260.
                       III-159

-------
2.1  Prepare calibration standards at a minimum of three concentration
     levels for each parameter of interest by adding volumes of one or
     more stock standards to a volumetric flask and diluting to volume
     with isooctane.  One of the external standards should be at a
     concentration near, but above, the method detection limit, and the
     other concentrations should correspond to the expected range of
     concentrations found in real samples or should define the working
     range of the detector.

2.2  Using injections of 2 to 5 ul of each calibration standard, tabulate
     peak height or area responses against the mass injected.  The
     results can be used to prepare a calibration curve for each com-
     pound.  Alternatively,  if the ratio of response to amount injected
     (calibration factor) is a constant over the working range (<10%
     relative standard deviation, RSD), linearity through the origin can
     be assumed and the average ratio or calibration factor can be used
     in place of a calibration curve.

2.3  The working calibration curve or calibration factor must be verified
     on each working day by the measurement of one or more calibration
     standards.  If the response for any parameter varies from the pre-
     dicted response by more than 10%, the test must be repeated using a
     fresh calibration standard.  Alternatively, a new calibration curve
     or calibration factor must be prepared for that ccmoound.

Internal Standard Calibration Procedure.

3.1  To use this approach, the analyst must select one or more internal .
     standards that are similar in analytical behavior to the compounds
     of interest.  The analyst must further demonstrate tnat the mea-
     surement of the internal standard is not affected by method or
     matrix interferences.  In addition, the Internal standard should
     elute close to the analyte under analysis (6 min).  Because of
     these limitations, no internal standard can be suggested that is
     applicable to all samples.

3.2  Prepare calibration standards at a minimum of three concentration
     levels for each parameter of interest by adding volumes of one or
     more stock standards to a volumetric flask.  To each calibration
     standard, add a known constant amount of one or more internal stan-
     dards, and dilute to volume with isooctane.  One of the standards
     should be at a concentration near, but above, the method detection
     limit and the other concentrations should correspond to the expected
     range of concentrations found 1n real samples or should define the
     working range of the detector.

3.3  Using Injections of 2 to 5 ul of each calibration standard, tabulate
     peak height or area responses against concentration for each com-
     pound and Internal standard, and calculate response factors (RF) for
     ach comoound using Eauation 1:

                            RF    (ASCSV(A1SCS)                   Ea.

                               III-160

-------
          where:

                  As   =  response for the parameter to be measured
                 Ais   =  response for the Internal standard
                  C-(S  =  concentration of the internal standard, (ug/1)
                  Cs   *  concentration of the parameter to be measured,
                          (ug/1).

          If the RF value over the working range is a constant (<1Q% RSD),  the
          RF can be assumed to be invariant, and the average RF can be used
          for calculations.  Alternatively, the results can be used to plot a
          calibration curve of response ratios, As/Ais, vs. Cs.

     3.4  The working calibration curve or RF must be verified on each working
          day by the measurement of one or more calibration standards.  If  the
          response for any parameter varies from the predicted response by
          more than 10%, the test must be repeated using a fresh calibration
          standard.  Alternatively, a new calibration curve must be prepared
          for that compound.

I.   DAILY GC/MS PERFORMANCE TESTS

     At the beginning of each day, the mass calibration of the GC/MS system
must be checked and adjusted, if necessary, ;o meet DFTPP specifications.
Additionally, each day pesticides or PCBs are to be analyzed, the column
performance specifications with benzidine must be met.   DFTPP can be mixed  in
solution w.ith benzidine to complete the two performance tests with one
injection, if desired.
                                               
     Evaluate the analytical system performance each day that it is to be used
for the analyses of samples or blanks by examining the  mass spectrum of DFTPP.
The following instrumental conditions are required to perform the mass cali-
bration test of a GC/MS system:

               Electron Energy - 70 volts (nominal)
               Mass Range - 35-450 atnu
               Scan Time - 7 seconds or less
               Source Temperature - 280-300*C

Inject a solution containing 50 ng DFTPP and check to ensure that established
performance criteria listed in Table 4 have been satisfied.  If the system
performance criteria are not met, the analyst must retune the spectrometer  and
repeat the performance check.

     The performance criteria must be met before any samples or standards may
be analyzed.

     Column performance is evaluated by injecting 100 ng of benzidine into  the
Instrument.  The tailing factor for the resultant peak, as calculated in
Figure ii, must oe less cnan ^ r'or ;ne per-onnance k.o be considered accsnt-
able.
                                    III-161

-------
   TABLE 4.  DFTPP KEY IONS AND ION ABUNDANCE  CRITERIA
Mass                  Ion Abundance Criteria

 51           30 to 60 percent of mass 198
 68           Less than 2 percent of mass 69
 70           Less than 2 percent of mass 69
 127          40 to 60 percent of mass 198
 197          Less than 1 percent of mass 198
 198'          Base peak, 100 percent relative  abundance
 139          3 :s } percen* of mass 198
 275          10 to 30 percent of mass 198
 365          Greater than 1 percent of mass 198
 441          Present but less than mass 443
 442          Greater than 40 percent of mass  198
 443          17 to 23 percent of mass 442
                         III-162

-------
                             BC
               Tailing Factor =- 
                             AB
Example Calculation:
Peak Height = DE = 100 mm
10% Peak Height = BD = 10 mm
Peak Width at 10% Peak Height = AC = 23 mm
  AB = 11 mm
  BC = 12 mm
Therefore: Tailing Factor =  = 1.1
       Figure 11.  Tailing factor calculation.
                         III-163

-------
J.   ANALYTICAL PROCEDURES

     1.1  Analysis of Hazardous Waste Samples for Pesticides
               Analytical Procedure:  available
               Sample Preparation:  available

          1.1.1  Reference/Title

                 U.S. Environmental  Protection Agency, "Method for Preparation
                 of Medium Concentration Hazardous Waste Samples," U.S.  EPA,
                 Region IV, Athens,  Georgia.  (1981).16

          1.1.2  Method Summary

                 Approximately 1-gram aliquots of soil, solid, aqueous liquid,
                 or non-aqueous liquid are transferred to viails inside a
                 chemical carcinogen glovebox.  The samples are then removed
                 from the enclosure  for dilution with hexane.   Half of the
                 hexane extract is cleaned up for GC/EC identification and
                 quantification.  The other half of the solution can be  used
                 for TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin).

          1.1.3  Applicability

                 This procedure is designed for the safe handling and prepara-
                 tion of potentially hazardous samples from hazardous waste
                 sites.  The method  is directed to contaminated soil samples
                 and waste samples that may be solid, aqueous  liquid, or non-
                 aqueous i-'quid, and suspected to contain ls?. *han 10 percent
                 of any one organic  chemical component.  The procedure has an
                 approximate detection limit of 0.1 ppm for pesticides.

          1.1.4  Precision and Accuracy

                 These extraction and preparation procedures were developed
                 for rapid and safe  handling of hazardous samples.  The  design
                 of the methods thus did not stress efficient  recoveries of
                 all components.  Rather, the procedures were  designed for
                 moderate recovery of a broad spectrum of organic chemicals.
                 The results of the  analyses thus may sometimes reflect  only
                 the minimum of the  amount present in the sample.16

                 The procedure has been used in one laboratory.  Pesticide
                 recovery data expressed as percent recovery and standard
                 deviations are summarized in Table 5.

          1.1.5  Sample Preparation

                 Place the original  sample container into the  glovebox.
                 Additional 'terns that should be 1n the rjlovebox -include (l\
                 calibrated and tared 20-ml vials with caps, (2) a spatula,


                                    III-164

-------
             TABLE 5.   RECOVERY DATA FROM SOIL BY REGION IV  MEDIUM '
                   CONCENTRATION HAZARDOUS WASTE  METHOD
===============================================================================
Pesticide Fraction
Dieldrin
p.p'-DDT
p.p'-DDE
p.p'-DDD
Alpha-endosulfan
Beta-endosulfan
Endosulfan sulfate
Endrin
Heptachlor
Heptachlor epoxide
ilnha-BHC
Gamma-BHC
Beta-BHC
Delta-BHC
Cone.
ug/g
0.04
0.10
0.04
0.10
0.04
0.08
0.20
0.04
0.04
0.04
0.02
0.04
0.04
0.04
Avg.
% Rec.
99
97
99
98
99
98
97
97
101
98
102
100
101
101
Std.
Dev.*
1
1.5
0
0
0.5
0
1
1
i.5
1
1
1.5
1.5
1.5
S=======;=========================================================

* -  one standard deviation based on two trials.
                 (3) a balance, (4) a capped vial containing 10 ml  of
                 Interference-free methanol, (5) a vial  containing  distilled
                 water, and (6) a medicine dropper.  The vial of methanol  is
                 to be used as a method blank.   (One method blank should be
                 run for each batch of 20 samples or less.) .Open the sample
                 transportation can and remove  the sample vial.  Note and
                 record the physical state and  appearance of the sample.  If
                 the sample bottle is broken, immediately repackage the sample
                 and terminate the analysis.
                                    !!1-165

-------
       Open the sample  vial  and mix  the sample.  .If  the sample  is  a
       liquid,  transfer one  drop to  a  vial  containing  water  to
       determine whether the sample  is aqueous  or  non-aqueous.
       Record the result.

       Transfer approximately 1 gram (or 1  ml)  of  the  sample to each
       of the calibrated vials.  Wipe  the mouth  of the vials with
       tissue to remove any  sample material.  Cap  the  vials. Record
       the exact weights of  sample taken.   Reseal  the  sample and
       replace it in the original packaging.

       Proceed with  a hexane extraction of  the  pesticide-containing
       sample based  on  the miscibility of the original  sample with
       water.  Follow paragraph (a)  for an  aqueous sample, paragraph
       (b) for a non-aqueous sample,  and paragraph (c)  for a soil  or
       solid-phase sample.

       a.  If the sample was determined to  be aqueous,  add 10 ml of
           hexane to the sample vial.   Cap  and  shake for 2 minutes.
           Add 2 grams  of anhydrous  sodium  sulfate to  each vial  to
           adsorb all water.  Shake  the sample.

       b.  If the sample was determined to  be non-aqueous, dilute
           the sample to a final  volume of  10 .Til with  hexane.   ^dd 1
           gram of anhydrous sodium  sulfate to  adsorb  any water in
           the sample.   Shake the sample.

       c.  If the sample is  a solid  matrix  such  as soil, sediment  or
           sludge, add  10 ml of hexane.  Cao the samole and  shake
           for 1 hour on a wrist action shaker.  Add 1  gram  of
           anhydrous sodium  sulfate  to the  sample  and  thoroughly
           mix.

1.1.6  Extract Analysis

       Process 5 ml  of  the hexane solution  through one of the
       cleanup techniques specified  in Method 608,17 such as
       acetonitrile  partitioning (Subsection J.I.2.7,  p. III-175).
       Perform GC/EC analysis as specified.

       Retain the other 5 ml of hexane solution  for  TCDD analysis
       following Method 613  (Section 8).I7
                          III-166

-------
1.2  Analysis of Transformer Fluid and Waste Oil  for  Polychlorinated
     Biphenyls
          Analytical  Procedure:   available
          Sample Preparation:   available

     1.2.1  Reference

            U.S. Environmental  Protection Agency,  "The  Analysis  of
            Polychlorinated Biphenyls  in Transformer  Fluid  and Waste
            Oil," U.S.   EPA, Office of Research  and Development,
            Environmental  Monitoring and Support Laboratory, Cincinnati,
            Ohio.  36 p.  February 1981.4

     1.2.2  Method Summary

            The sample  is  diluted on a weight/volume  basis  with  hexane
            so that the concentration  of each PCB  isomer  is within the
            analytical  limits  of the gas chromatographic  (GO system
            (0.01 to  10 ng/nl).   The diluted sample is  injected  into a GC
            for separation of  the PCB  isomers.   Measurement is accom-
            plished with a halogen-specific  detector  that maximizes
            baseline  stability and minimizes interferences  normally
            encountered with other detectors. The electron capture
            detector  (ECD) can  normally be substituted  for  the halogen-
            specific  detector  when samples contain dichloro- through
            decachlorobiphenyls,  or when the sample matrix  does  not
            interfere with PCB  analysis.  A  mass  spectrometer operating
            *n the selected ion  monitoring mode  of data acquisition may
            also be used as an  alternative detector -*hen  ''CS levels ire
            sufficiently high  and the  PCS m/e ranges  are  free from
            interferences.  Several  cleanup  techniques  (acid cleanup,
            Fluorisil  cleanup,  alumina cleanup,  silica  gel  cleanup, gel
            permeation  cleanup,  acetonitrile partitioning)  are provided
            for samples containing interferences.  However, even
            exhaustive  use of  these procedures may not  remove all inter-
            ferences  from  all waste oil  samples.

            The concentrations  of PCBs are calculated on  a mg/kg basis
            using commerical mixtures  of PCBs as  standards.  The analysis
            time, not including  data reduction,  is approximately 35
            minutes per sample.

     1.2.3  Applicability

            This 1s a gas  chromatography (GC) method  applicable to the
            determination  of commercial  mixtures of polychlorinated bi-
            phenyls (PCBs) in .transformer fluids  and  certain other
            hydrocarbon-based waste oils.  The method can be used to
            analyze waste  oils  for Individual PCB  isomers or complex
            mixtures  of chlonnateo oiphenyls  from nonochlorobipheny'1
            through decachlorobiohenyl-only  if they have  been previously


                                    111-167

-------
       Identified by other  methods1^  or  by  knowledge  of  the  sample
       history.   The method detection limits  are  dependent upon  the
       complexity of the  sample  matrix and  the  ability of the
       analyst to properly  maintain the  analytical  system.   Using a
       carefully optimized  instrument, this method  has been  shown to
       be useful  for the  determination of commercial  PCB mixtures
       spiked into transformer  fluid  over a range of  5.0 to  500
       mg/kg.  Based upon a statistical  calculation at 5 mg/kg for a
       simple oil  matrix, the method  detection  limit  for Aroclors
       1221,  1242, 1254,  and 1260 is  1 mg/kg.   The  method detection
       limit  (MOL) is defined as the  minimum  concentration of a
       substance that can be measured and reported  with  99 percent
       confidence that the  value is above zero.

       This method is restricted to use  by  or under the  supervision
       of analysts experienced  in the use of  gas  chromatography  and
       in the interpretation of gas chromatograms.

1.2.4  Precision and Accuracy

       Separate accuracy statements of laboratory performance should
       be maintained for the analysis of waste  oil  and similar sam-
       ple matrices.  Single-operator precision and recovery data
       ror this method are  presented  in  Tables  6  through 3.

1.2.5  Sample Preparation

       Because of the unique sample matrix, PCB concentrations are
       determined by diluting  the sample with solvent and analyzing
       the resultant mixture directly,  in  order co ansurs  cnai  cne
       diluted sample is within the analytical  range  of  the  instru-
       ment,  the approximate PCB concentration of the sample may be
       determined by x-ray  fluorescence, microcoulometry, density
       measurements, or by  analyzing  a very dilute  mixture  of  the
       sample (10,000:1) according to paragraph 1.2.6.

       Based on the estimated PCB concentration of  the  sample,
       continue processing  the sample according to  either  (a),  (b),
       (c), or (d), as appropriate.

            a.  For samples in the 0- to 100-mg/kg  range,  dilute
                100:1 with  hexane.  Pipet  1.0 ml  of sample  into  a
                100-ml volumetric flask using a 1.0-ml  Mohr pipet.
                For viscous samples,  1t may be necessary to cut  the
                capillary tip off the pipet.   Dilute  the sample  to
                volume with hexane.   Stopper  and mix.  Analyze
                according to paragraph 1.2.6.

                Using the same pipet, deliver 1.0 ml  of sample  into
                a tared  beaker weighed to 0.001 g.  Reweigh the
                beaker to sO.OOI g co determine  cne mjignt of
                used  ^n  preparing the diluted sample.

                               III-168

-------
       TABLE 6.  ACCURACY AND PRECISION DATA FOR THE GC ANALYSIS OF PCBs
                        IN SPIKED MOTOR OIL  SAMPLES4
Detector*
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
MED
ECD
HED
ECD
HED
^CD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
Method
Cleanup
None
None
None
None
1.2.7.1
1.2.7.1
1.2.7.1
1.2.7.1
1.2.7.2
1.2.7.2
1.2.7.2
1.2.7.2
" ,2.7.2
i'.i'j'.i
1.2.7,3
1.2.7.3
1.2.7.4
1 2 7,4
1.2.7.4
1.2.7.4
1.2.7.5
1.2.7.5
1.2.7.5
1.2.7.5
1.2.7.6
1.2.7.6
1.2.7.6
ECD 1.2.7.6
Spike
mg/kg
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
31.1
30.3
31.1
HED = Hall Electrolytic
ECD * Electron Cai
Aroclor
Spike
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
35SSSE3SS3
Detector
Avg.
Cone.
Found
mg/kg
28.2
26. 7b
27.2
23.9
28.4
25. 4&
28.1
24.3
 30.7
27. 3b
30.9
31.0
30.3
28.913
29.8
30.8
29.4
26. 4b
29.4
23.6
31.9
23. 4b
33.6
30.9
34.4
23. 4b
29.1
(Precision)
Rel. Std.
Deviation
%
4.2
5.7
2.0
2.2
11.5
6.1
8.0
7.8
2.4
10.2
3.6
8.6
8,6
5.0
4.7
6.5
5.8
5.3
5.2
4.5
8.5
3.0
9.2
5.5
3.8
4.4
4.2
27.0 4.6

(Accuracy)
Percent
Recovered
93.1
88.1
87.5
76.8
93.7
83.8
90.3
78.1
101
90.1
99.4
99.7
100
95.4
95.8
99.0
97.0
87.1
94.5
75.9
105
77.2
108
99.4
107
77.2
96.7
Number
of
Dilutions
5
3
5
3
3
3
3
3
4
4
4
4
3
3
3
3
3
3
3
3
3
2
3
3
4
4
4
86.7 4

iture Detector
t>Severe interference problems in elution area of 1242.   Measurement based
 upon only 3 of the 10 normally resolved major peaks.   Cleanup techniques
 1n paragraph 1.2.7 did not Improve the quality of the  1242 chromatogram.
 If this were an unknown sample, it would be impossible to qualitatively
 Identify the presence of Aroclor 1242 using ECD.   The  HED provided an
 
-------
TABLE 7.  ACCURACY AND PRECISION DATA FOR THE GC ANALYSIS OF PCBs
              IN WASTE TRANSFORMER FLUID SAMPLES*
(Precision)
Method 1260 Avg.D Rei . std. (Accuracy) Number
Dilution Clean- Spike Cone. Deviation Percent of
Sample Ratio Detector* up mg/kg Found % Recovered Dilutions
A
A
A
A
A
A
A
A
A
A
- A
A
A
. A
B
B
B
B
C
C
C
C
100:1 ECD None
100:1 HED None
100:1 ECD 1.2.7.1 ~
100:1 HED 1.2.7.1
100:1 ECD 1.2 7.2 
100:1 HED 1.2.7.2 
100:1 ECD 1.2.7.3 
100:1 HED 1.2.7.3 
100:1 ECD 1.2.7.4 --
100:1 HED 1.2.7.4 
1.00:1 ECD 1.2,7.5
100:1 HED 1.2.7.5
100:1 ECD None 27.0
100:1 HED None 27.0
1000:1 ECD None
1000:1 HED None
1000:1 ECD None 455
1000:1 HED None 455
1000:1 ECD None
1000:1 HED None
1000:1 ECD None 300
1000:1 HED None 300
22.6
27.0
22.8
29.7
22.4
28.2
22.7
27.8
20.9
30.2
23.8
28.6
45.0
55.2
452
471
875
916
284
300
607
686
3.6
1.7
2.5
1.4
1.0
2.2
1.3
2.8
--
...
0.3
4.1
3.3
1.5
0.8
1.2
0.5
2.0
1.2
1.4
3.6
3.9
7E
7E
7E
7
3E
3^
3^
3^
1
1
7E
7t
91 7E
102 7E
7E
-- 7
96 7E
99 7E
7
7
104 7E
114 7
==============================================================::==================
* HED *
ECD =
A - dark
Hall Electrolytic Detector
Electron Capture Detector
waste oil









B - black waste oil with suspended solids
C - clear waste oil
D - all
E - dupl
samples contained Aroclor 1260
icate analyses made at each dil

ution




                             III-170

-------
         TABLE 8.  ACCURACY AND PRECISION AND LIMIT OF DETECTION DATA
                RESULTS OF ANALYSES OF SHELL TRANSFORMER FLUID
                    SPIKED WITH PCB AT 5.0 AND 27 mg/kg4
Aroclor
Spike
(rag/kg)
Number of
Analyses
Avg.
(mg/kg)
Standard
Deviation
% Recovery
MDL*
(mg/kg)
 1221
 1242
 1254
 1260
 1221
 1242
 1254
 1260
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
                            Electron Capture Method
                               (100:1 Dilution)
 7
14
 7
14
 6
 7
 6
 7
7.5
3.8
4.1
4.7
                                          0.43
                                          0.18
                                          0.08
                                          0.18
                                  Hall  Method
                               (100:1 Dilution)
7.5
5.9
5.8
5.4
                                          0.23
                                          0.17
                                          0.16
                                          0.10
150
 76
 82
 94
150
118
116
108
1.4
0.5
0.2
0.5
0.7
0.5
0.5
0.3
                  Shell  Transformer Oil  +27 ppm Aroclor 1260
                               (100:1 Dilution)

                                                  Rel.  Std.
Method
(mg/kg)
vuir.ber of
Analyses
avq.
(mg/kg)
Deviation
% Recovery
Electron Capture     27
Hall  700-A           27
                               14
                                7
                              24.0
                              28.3
                               .70
                              2.1
                                    89
                                   105
===============================================================================
3MDL = Method Detection Limit at 99 percent confidence that the value is
       above zero.
       NOTE:  At these values it would be impossible to identify Aroclor
       patterns with any degree of confidence.   1 mg/kg appears to be a
       reasonable MDL.
      where:

              MDL
      t(n-1..99)
                                   t(n-l,.99)
           the method detection limit
           the students'  t value appropriate for a 99 percent
           confidence level  and a standard deviation estimate
           with i-1. degrees  of freedom
           standard deviation of the rephcate analyses.
                                    III-171

-------
             b.  Alternatively,  weigh  approximately 1.0  g  of  sample
                0.001  g into a volumetric  flask  and  dilute  to
                100 ml  in hexane.   Store  the  diluted  sample  in  a
                narrow-mouth  bottle with  a  Teflon-lined screw cap.
                Analyze according  to  paragraph  1.2.6.

             c.  For samples above  100 mg/kg PCS,  dilute 1000:1  with
                hexane.  Pipet 0.10 ml  of sample  into a 100-ml  vol-
                umetric flask using a 0.10-ml Mohr pipette.   Dilute
                to volume with hexane,  stopper  and mix.   Analyze
                according to  paragraph 1.2.6.

                Using the same pi pet, transfer  0.10 ml  of sample
                Into a  tared  beaker weighed to    0.001  g.  Reweigh
                the beaker to determine the weight of sample used in
                preparing the diluted sample.

             d.  Alternatively,  weigh  approximately 0.1    0.0001 g of
                sample  into a 100-ml  volumetric flask and dilute to
                volume  with hexane.  Store  the  diluted  sample in a
                narrow-mouth  bottle with  a  Teflon-lined screw cap.
                Analyze the diluted sample  according  to paragraph
                1.2.6.

1.2.6  Sample Analyses

       Analyze the sample by  injecting the  hexane mixture into  the
       GC using.auto injectors or  the solvent flush technique.19
       The 2C should be tssnoerature-orogrammable, eauiooed -for
       on-column injection, capable of accepting  6.4  mm 0.0. glass
       columns,  and equipped  with  one of  the  following  detectors:

      (a)  A halogen-specific detector to eliminate interferences
           causing misidentification  or false-positive  values due to
           non-organohalides  that  commonly  coelute with the  PCBs.
           Electrolytic conductivity  detectors  such as  the Hall
           Model 700-A have been shown to provide the necessary
           sensitivity and stability.  Other  halogen-specific detec-
           tors, including older model electrolytic conductivity
           detectors and microcoulometric titration,  may  be  used.
           However, the stability, sensitivity, and response time of
           these detectors may raise  the  method detection limit and
           adversely affect  peak resolution.

      (b)  Semi-specific detectors such as  ECD  may be substituted
           for halogen-specific detectors when  sample chromato-
           graphic patterns  closely match those of the  standards.
           Either acid cleanup or  Florisil  slurry (paragraph 1.2.7)
           cleanup techniques should  be routinely incorporated  into
           the analytical scheme orior to sample  injection when
           these detectors are used.
                               III-172

-------
The GC columns and conditions listed below are recom-
mended for the analysis of PCB mixtures in oil.   If these
columns and conditions are not adequate, the analyst may
vary the column parameters to improve separations.   The
columns and conditions selected must be capable of  ade-
quately resolving the PCBs in the various Aroclor mix-
tures so that each Aroclor is identifiable through  isomer
pattern recognition (Figures 4 through 10).

Capillary columns and the required specialized injection
techniques are acceptable alternatives to the recommended
packed columns.  Due to problems associated with the use
of capillary columns the analyst must demonstrate that
the entire system will produce acceptable results by
performing the operations described in Subsection G.

Recommended primary analytical column:  Glass, 6.4  mm
O.D. (2 mm I. D.), 6 ft (180 cm) long, packed with Gas-
Chrom Q (100/120 mesh) coated with 3% OV-1.

Carrier gas:  40 to 60 ml/min (helium, nitrogen or  10%
methane in argon).

Temperature program:  120"C isothermal for 2 minutes,
6'C/minute to 220JC, and noid at 220"C until all
compounds elute.

Isothermal Operation:

Aroclor 1221, 1232, or Cl]_ chrough 014 isomers - 140
to 150'C.

Aroclor 1016, 1242, 1254, 1260, 1262, 1268,  or C13
through Clio isomers 170 to 200C.

Recommended confirmatory column:  Glass tubing 6.4  mm O.D.,
2 mm I.D., 6 ft (180 cm) long, packed with Gas Chrom Q
100/120 mesh coated with 1.5% OV-17 + 1.95% OV-210.

Carrier gas:  40 to 60 ml/min (helium, nitrogen or  10%
methane in argon).

Column temperatures:  Aroclor 1221, 1232, or Clj
through Cl4 Isomers - 170 to 180C.
Aroclor 1016, 1242, 1254, 1260, 1262, 1268, or C13
through Clio isomers - 200*C.

If the resulting chromatograms show evidence of column
flooding or non-linear responses due to excess sample,
dilute tne sample as necessary (paragraph 1.2.5) using
hexane as solvent.

                        III-173

-------
Determine whether or not PCBs are present in the sample
by comparing the sample chromatogram to that obtained
with a PCB locator mixture.  Proceed as indicated below:

(a)  If a series of peaks in the sample match some of the
     retention times of PCBs in the PCB locator mixture,
     attempt to identify the source by comparing chromat-
     ograms of each standard prepared from commercial
     mixtures of PCBs.

(b)  If a 1000:1 or higher dilution ratio sample was
     analyzed and no measurable PCB peaks were detected/
     analyze an aliquot of sample diluted 100:1.

(c)  Samples diluted 100:1 and analyzed by electron
     capture GC consistently produce results that are 10
     to 20 percent lower than the true value due to
     quenching of the detector response by high-boiling
     hydrocarbons coeluting with the PCBs.  The degree of
     error is matrix dependent and is not predictable for
     samples of unknown origin.  As the PCB concen-
     tration approaches 20 percent of a control level,
     e.g. 50 mg/kg, the analyst must routinely reanalyze
     a duplicate 'spiked sample to determine the Actual
     recovery.  Spike the duplicate or diluted sample at
     twice.tne apparent concentration of the original
     sample and correct the results accordingly.

(d)  If PCB interference problems are encountered or **
     PCB ratios do not match the standards, proceed to
     paragraph 1.2.7 to clean up the sample prior to
     further analysis,  analyze the samples using alter-
     nate columns, or use GC/MS analytical techniques to
     verify whether or not the non-representative
     patterns are due to PCBs.

Quantitative GC/MS techniques may be used in place of GC
analysis; the recommended approach is selected ion moni-
toring.  The GC/MS system must have a program that
supports this method of data acquisition.  The program
must be capable of monitoring a minimum of eight ions,
and it is desirable for the system to have the ability to
change the ions monitored as a function of time.  For PCB
measurements, several sets of ions may be used depending
on the objectives of the study and the data system capa-
bilities. The alternatives are as follows:

     Single ions for             154, 188, 222,
     sensitivity                 256, 292, 326,
                                 360, 394
                        III-174

-------
                Short mass ranges which     154-156,  188-192,
                may give enhanced sensi-     222-226,  256-260,
                tivity depending on the     290-295,  322-328,
                data system capabilities     356-364,  390-398

                Single ions that give       190,  224, 260,  294,
                decreased sensitivity       330,  362, 394
                but are selective for
                levels of chlorinationl?

           The data system must have the  capability of integrating
           the abundances of the selected ions  between specified
           limits,  and relating integrated abundances to concentra-
           tions using the calibration  procedures described in this
           method.

1.2.7  Sample Cleanup Procedures

       Several tested cleanup techniques  are described below.
       Depending on the past experience of the  analyst with the
       particular sample matrix being analyzed  and the complexity of
       the sample,  one or all  of the following  techniques may be
       required to  seoarate any PCBs that may be  present from the
       interferences.

1.2.7.1  Acid Cleanup

         (a)   Place 5,0 ml  of concentrated sulfuric acid into a
              40-ml narrow-moutn screw  cap Don!a.  Add 10.0 ml of
              the diluted sample.  Seal the bottle with a Teflon-
              lined screw cap and shake for 1 minute.

         (b)   Allow the phases to separate, transfer  the sample
              (upper phase) to a clean  narrow-mouth screw-cap
              bottle.  Seal with a Teflon-lined cap.   Analyze
              according to paragraph 1.2.6.

         (c)   If the sample is highly contaminated, a second or
              third acid cleanup may be employed.

              NOTE  4:  This cleanup technique was tested over a
              period of about 6 months  using both electron  capture
              and electrolytic conductivity detectors.  Care was
              taken to exclude any samples that formed an emulsion
              with  the acid.  The sample was withdrawn well  above the
              sample-acid interface. Under these conditions no
              adverse effects associated  with column  performance and
              detector sensitivity to PCBs were noted.  This
              cleanuo technique could adversely affect the  chromato-
              graphic column  performance  for future ^c*d-degradable
              samel 9$.
                               III-175

-------
1.2.7.2  norfsll  Column Cleanup

         (a)  Variances between batches  of Florisll  may affect the
              elution volume of the various PCBs.  For  this  reason,
              the volume of solvent required to completely elute  all
              of the PCBs must be verified by the  analyst,,   The
              weight of Florisil  can then be adjusted accordingly.

         (b)  Place a 20.0-g charge of Florisil, activated at  130C,
              into a Chromaflex column.   Settle the  Florisil by
              tapping the column.  Add about 1 cm  of anhydrous
              sodium sulfate to the top  of the Florisil.   Pre-elute
              the column with 70 to 80 ml  of hexane.  Just before
              the exposure of the sodium sulfate layer  to air, stop
              the flow.  Discard the eluate.  Add  2.0 ml  of  the
              undiluted sample to the column with  a  2-ml  Mohr  pipet.
              (For viscous samples, cut  the capillary tip off  the
              pipet.)  Add 225 ml of hexane to the column.

         (c)  Carefully wash down the inner wall of  the column with
              a small amount of the hexane prior to  adding the total
              volume.  Collect and discard the first 25.0 ml.

         (d)  Tollect exactly 200 ml of  hexane eluate in a 200-ml
              volumetric flask.  All of  cne PCBs should be in  :nis
              fraction.  Using the same  pipet as in  (b),  deliver
              2.0 ml of sample into a tared 10 ml  oeaker weighed  to
              0.001 g.  Reweigh the beaker to determine the weight
              of the sample diluted to 200 ml.

         (e)  Analyze the sample according to paragraph 1.2.6.

1.2.7.3  Alumina Column Cleanup

         (a)  Adjust the activity of the alumina by  heating  to 200C
              for 2 to 4 hours.  When cool, add 3% water (wt:wt)  and
              mix until uniform.  Store  in a tightly sealed  bottle.
              Allow the deactivated alumina to equilibrate with room
              air at least 1/2 hour before use.  Reactivate  weekly.

         (b)  Variances between batches  of alumina may  affect  the
              elution volume of the various PCBs.   For  this  reason,
              the volume of solvent required to completely elute  all
              of the PCBs must be verified by the  analyst.  The
              weight of alumina can then be adjusted accordingly.

         (c)  Place a 50,0-g charge of alumina Into a Chromaflex
              column.  Settle the alumina by tapping.  Add about
              1 cm of anhydrous  sodium sulfate.  Pre-elute the
              column with 70 to 80 ml of hexane.  Just  before
                               III-176

-------
              exposure of the sodium sulfate layer to air,  stop the
              flow.   Discard the  eluate.

         (d)   Add 2.5 ml  of the undiluted sample  to the column  with
              a 5-ml  Mohr pipet.   (For viscous  samples, cut the
              capillary end off the pipet.)   Add  300 ml of  hexane  to
              the column.  Carefully wash down  the inner walls  of
              the column with a small  volume of hexane prior to
              adding  the total  volume. Collect and discard the 0-
              to 50-ml fraction.

         (e)   Collect exactly 250 ml  of the  hexane in a 250-ml  vol-
              umetric flask.  All  of the  PCBs should be in  this
              fraction.  Using the same pipet as  in (d), deliver 2.5
              ml of sample into a tared 10-ml beaker weighed to
              0.001  g.  Reweigh  the beaker  to  determine weight of
              sample  diluted to 250 ml.  Analyze  the sample
              according to paragraph 1.2.6.

1.2.7.4  Silica Gel Column Cleanup

         (a)   Activate silica gel  at 135C overnight.  Variances
              between batches of  silica gel  may affect the  elution
              volume  of the various PCBs. For  this reason, tne
              volume  of solvent required  to  comoletely elute all of
              the PCBs must be verified by the  analyst.  The weight
              of silica gel can then be adjusted  accordingly.

         (b)   Place a 25-g charge of activated  silica gel  into  a
              Chromaflex column.   Settle  the silica gel by  tapping
              the column.  Add about 1 cm of anhydrous sodium
              sulfate to the top  of the silica  gel.

         (c)   Pre-elute the column with about 70  to 80 ml of hexane.
              Discard the eluate.   Just before  the exposure of  the
              sodium  sulfate layer to air, stop the flow.

         (d)   Add 2.0 ml  of the undiluted sample  to the column  with
              a 2-ml.  Mohr pipet.   (For viscous  samples, cut the
              capillary tip off the pipet.)

         (e)   Wash down the inner wall of the column with 5 ml  of
              hexane. Elute the PCBs with 195 ml  of 10 percent  (V/V)
              diethyl ether in hexane.   Collect  exactly 200  ml of the
              eluate  in a 200-ml  volumetric  flask.  All of  the  PCBs
              should  be in this fraction.

         (f)   Using the same pi pet as in  (d), deliver 2.0 ml of
              sample  Into * tared 10-ml beaker  (0.001 g).   Reweigh
              to determine the weignt of  sample diluted co  200  ,nl.
              Analyze the sample  according to oaragraoh 1.2.6.
                               TII-17?

-------
1.2.7.5  Gel Permeation Cleanup

         (a)  Set up and calibrate the gel  permeation cnromatograph
              with an SX-3 column according to the instruction
              manual.  Use 15 percent (V/V) methylene chloride in
              cyclohexane as the mobil  phase.   Place 1.0 ml  of sample
              into a 100-ml  volumetric flask,  using a 1-ml  Mohr
              pipet.  (For viscous samples, cut the capillary tip
              off the pipet.)

         (b)  Dilute the sample to volume,  using 15 percent (V/V)
              methylene chloride in cyclohexane.

         (c)  Using the same pipet as in (a) deliver 1.0 m'l  of
              sample into a tared 10-ml  beaker weighed to 0.001  g.
              Reweigh the beaker to determine  the weight of sample
              used in (a).

         (d)  As an alternative to (a)  and  (b), weigh approximately
              1 gram 0.001  g of sample  and dilute to 100.0 ml  in
              15 percent (V/V) methylene chloride in cyclohexane.

         (e)  Inject 5.0 ml  of the diluted  sample into the  instru-
              ment.  Col'ect the fraction containing the C'h
              through Cl IQ PCBs (see operator's manual)  in  a K-0
              flask equipped with a 10-ml ampule.

         (f)  Concentrate the (PCB) fraction down to less'than 5  ml.
              using K-D evaporative concentration techniaues.

         (g)  Dilute to 5.0 ml with hexane, then analyze according
              to paragraph 1.2.6.  Be sure  to  use 100 ml  as the
              dilution volume for the final calculation.

1.2.7.6  Acetonitrile Partitioning

         (a)  Place 10.0 ml  of the previously  diluted sample into a
              125-ml separatory funnel  with enough hexane to bring
              the final volume to 15 ml. Extract the sample four
              times by shaking vigorously for  1 minute with 30-ml
              portions of hexane-saturated  acetonltrile.

         (b)  Combine and transfer the acetonltrile phases  to a
              1-Hter separatory funnel  and add 650 ml of distilled
              water and 40 ml of saturated  sodium chloride  solution.
              Mix thoroughly for 30 to 35 seconds.  Extract with  two
              100-ml portions of hexane  by  vigorously shaking about
              15 seconds.

         (c)  Combine the hexane extracts in a 1-1 separatory funnel
              and wash with two iOO-mi  portions of disclllea *dier.


                               III-178

-------
              Discard the water layer and pour the hexane layer
              through a 7 to 10 cm anhydrous  sodium sulfate  column
              into a 500-ml  K-D flask equipped with a 10-ml  ampule.
              Rinse the separatory funnel  and column with three 10-
              ml  portions of hexane.

         (d)  Concentrate the extracts to 6 to 10 ml  in  the  K-D
              evaporator in  a hot water bath, then adjust the volume
              to  10.0 ml. Be sure to use the correct dilution vol-
              ume for the final calculation.

         (e)  Analyze according to paragraph  1.2.6.
1.2.7.7  Florisil  Slurry Cleanup

         (a)  Place 10 ml of the diluted sample into a 20-ml
              narrow-mouth screw cap container.  Add 0.25  g  of  Flor-
              isil.  Seal with a Teflon-lined screw cap and  shake
              for 1 minute.

         (b)  Allow the Florisil  to settle,  then decant the  treated
              solution into a second container.  Analyze according
              to paragrapn i.2.6.
                               III-179

-------
2.1  Analysis of Water Samples for Pesticides/Polychlorinated Biphenyls
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     2.1.1  Reference/Title

            "Organochlorine Pesticides and PCB's - Method 608,"  Federal
            Register, Vol. 44, No. 233.  69501-69509, December 3,
            1979.20

     2.1.2  Method Summary

            A measured volume of sample, approximately 1 liter,  is
            solvent-extracted with methylene chloride using a separatory
            funnel.  The methylene chloride extract is dried, exchanged
            to hexane, and concentrated  to a final  volume of 10  ml  or
            less.  Gas chromatographic conditions are described  which
            permit the separation, identification, and quantification of
            the chemical compounds in the extract by electron capture gas
            chromatography.21

            A Florisil column cleanup procedure and an elemental  sulfur
            removal procedure are also described to aid in the elimina-
            tion of interferences that may be encountared.

     2.1.3  Applicability

            This method is suitable for the determination of the com-
            pouncs "-istcd in Tab1es 1 and 2 when present in municipal and
            industrial discharges.  The method detection limits  are diso
            presented in Tables 1 and 3.  However, actual detection
            limits will vary with the sample size chosen, the extent of
            extract concentration, the types of interferences present,
            and the nature of the original sample matrix.

            This method should only be used by or under the supervision
            of analysts experienced in the use of gas chromatography and
            in the interpretation of gas chromatograms.  Each analyst
            must demonstrate the ability to generate acceptable  results
            with this method using the procedure described in Subsection
            G.2.

     2.1.4  Precision and Accuracy

            This procedure was used by a single laboratory to analyze
            triplicate  spiked wastewater samples on separate days.  The
            average recovery and calculated standard deviation for each
            compound  is presented in Table 2 (p. 111-148).

            This method "ias been tested *or "Hnearly of sm'ke recovery
            from reagent water and has been demonstrated to be appiicaole
            over ;he  concentration range  rrom l x MOL 'jp to 1000 x MDL

                                    III-180

-------
       with the following exceptions:   chlordane recovery at 4 x MDL
       was low (60%); toxaphene recovery was demonstrated linear
       over the range of 10 x MDL to 1000 x MDL.21

       NOTE:  The Environmental Protection Agency 1s 1n the process
       of conducting an Inter!aboratory study to fully define the
       performance of this method (1982).

2.1.5  Sample Preparation

       Mark the water meniscus on the  side of the sample bottle for
       later determination of sample volume.  Pour the entire sample
       Into a 2-1 Her separatory funnel.

       Add 60 ml  methylene chloride to the sample bottle, seal, and
       shake 30 seconds to rinse the Inner surface.  Transfer the
       solvent to the separatory funnel  and extract the sample by
       shaking the funnel for 2 minutes with periodic venting to
       release excess pressure.  Allow the organic layer to separate
       from the water phase for a minimum of 10 minutes.  If the
       emulsion Interface between layers is more than one-third the
       volume of the solvent layer, the analyst must employ mechan-
       ical techniques to complete the phase separation.  The
       optimum technique depends upon  the sample, but may Include
       stirring,  filtration of the emulsion through glass wool,
       centrifugation, or other physical methods.  Collect the meth-
       ylene chloride extract 1n a 250-ml Erlenmeyer flask.

       Add a second 60-ml volume of methylene chloride to the sample
       bottle and repeat the extraction procedure a second time,
       combining the extracts in the Erlenmeyer flask.  Perform a
       third extraction in the same manner.

       Assemble a Kuderna-Danish (K-D) concentrator by attaching a
       10-ml concentrator tube to a 500-ml  evaporative flask.  Other
       concentration devices or techniques may be used in place of
       the Kuderna-Danish 1f the requirements of Subsection G.2.  are
       met.

       Pour the combined extract through a drying column containing
       about 10 cm of anhydrous sodium sulfate, and collect the
       extract 1n the K-D concentrator.   Rinse the Erlenmeyer flask
       and column with 20 to 30 ml of  methylene chloride to complete
       the quantitative transfer.

       Add 1 or 2 clean boiling chips  to the evaporative flask and
       attach a three-baTl Snyder column.  Pre-wet the Snyder column
       by adding about 1 ml methylene  chloride to the top.  Place
       the K-D apparatus on a hot *ater bath !60 to 55*C) o *,hat
       the concentrator tube is partially Immersed in the hot water
       and the entire lower rounded surface of the flask Is bathed
       with hot vapor.  Adjust the vertical position of the

                               III-181

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       apparatus and the water temperature as required to complete
       the concentration in 15 to 20 minutes.  At the proper rate of
       distillation the balls of the column will  actively chatter
       but the chambers will  not flood with condensed solvent.   When
       the apparent volume of liquid reaches 1 ml, remove the K-D
       apparatus and allow it to drain and cool  for at least 10
       minutes.

       Increase the temperature of the hot water bath to about  80C.
       Momentarily remove the Snyder column, add 50 ml of hexane and
       a new boiling chip and reattach the Snyder column.  Pre-wet
       the column by adding about 1 ml  of hexane to the top.  Con-
       centrate the solvent extract as before.  The elapsed time of
       concentration should be 5 to 10 minutes.   When the apparent
       volume of liquid reaches 1 ml, remove the K-D apparatus  and
       allow it to drain and cool at least 10 minutes.

       Remove the Snyder column and rinse the flask and its lower
       joint into the concentrator tube with 1 to 2 ml of methylene
       chloride.  A 5-ml syringe is recommended for this operation.
       Stopper the concentrator tube and store refrigerated if
       further processing will not be performed immediately.  If the
       extracts will be stored longer than two days, they should be
       transferred to Teflon-sealed screw-cap bottles.  If the
       sample extract requires no further cleanup, proceed with gas
       chromatographic analysis.  If the sample requires cleanup,
       proceed to paragraph 2.1.6.
              ns the or^'gi"3.!  isnol^ "oluins bv *"9f'''''ir'|n ths
       bottle to the mark and  transferring the liquid  to a  1,000-ml
       graduated cylinder.  Record the sample volume to the nearest
       5 ml.

2.1.6  Sample Cleanup and Separation

2.1.6.1  Cleanup procedures may not be necessary for a relatively
         clean sample matrix.   The cleanup procedures  recommended in
         this method have been used for the analysis of various
         clean waters and industrial effluents.  If particular cir-
         cumstances demand the use of an alternative cleanup procedure,
         the analyst must determine the elution profile and demon-
         strate that the recovery of each compound of  interest is no
         less than 85 percent.  The Florisil  column allows  for a select
         fractionation of the  compounds and will  eliminate  polar
         materials.  Elemental sulfur interferes with  the electron
         capture gas chromatography of certain pesticides,  but can be
         removed by the techniques described below.

2.1.6.2  Florisil  Column Cleanup

         (a)    Add a weight of Florisil (nominally 21  g) predeter-
               mined by calibration (Subsection H.4. and H.5.K  to

                               III-182

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               a chromatographic column.   Settle the Florisil  by
               tapping the column.   Add sodium sulfate  to the  top
               of the Florisil  to form a  layer 1 to 2 cm deep.  Add
               60 ml of hexane  to wet and rinse the sodium sulfate
               and Florisil. Just prior  to exposure of the sodium
               sulfate to air,  stop the elution-of the  hexane  by
               closing the stopcock on the chromatography column.
               Discard the eluate.

         (b)   Adjust the sample extract  volume to 10 ml  and
               transfer it from the K-D concentrator tube to the
               Florisil column.  Rinse the tube twice with 1 to
               2 ml hexane, adding each rinse to the column.

         (c)   Place a 500-ml K-D flask and clean concentrator tube
               under the chromatography column.  Drain  the column
               into the flask  until the sodium sulfate  layer is
               nearly exposed.   Elute the column with 200 ml of 6%
               ethyl ether in hexane (v/v) (Fraction 1) using  a drip
               rate of about 5  ml/min.  Remove the K-D  flask and set
               aside for later  concentration.  Elute the  column
               again, using 200 ml  of IS% ethyl ether in  hexane (v/v)
               (Fraction 2), into a second K-D flask.  Perform the
               ;hird aiution using 200 ml of SOS ethyl  ether 1-n
               hexane (v/v) (Fraction 3).  The distribution patterns
               for the pesticides and ?CBs are shown in Table  9.

         (d)   Concentrate the  eluates by standard K-D  techniques
               Subsection J.2.1.5), substituting hexane  for the
               glassware rinses and increasing the water  batn  to aoout
               85*C.  Adjust final  volume to 10 ml  with hexane,
               Analyze by gas chromatography.

2.1.6.3  Elemental sulfur will  usually elute entirely in  Fraction 1
         of the Florisil column cleanup.   To remove sulfur inter-
         ference from this fraction or the original  extract, pipet
         1.00 ml of the concentrated extract into a clean concen-
         trator tube or Teflon-sealed vial.  Add 1 to 3 drops  of
         mercury and seal.*4 Agitate the contents of the vial for
         15 to 30 seconds.  Prolonged shaking (2 hours) may be re-
         quired.  If so, this may be accomplished with  a  reciprocal
         shaker.  Alternatively, activated copper powder  may be used
         for sulfur removal.22   Analyze by gas chromatography.

2.1.7  Sample Analysis by Gas Chromatography

       Table 3 summarizes the recommended operating conditions for
       the gas chromatograph.   This table includes retention times
       and method detection limits that were obtained under these
       conditions.  Ixamcl^s cf the seoarations achieved  bv column 1
                               III-183

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   TABLE 9.  DISTRIBUTION OF CHLORINATED PESTICIDES AND PCBs
              INTO FLORISIL COLUMN FRACTIONS20
======3===============================================::=======

                            Percent Recovery by Fraction
    Parameter
Fraction 1
Fraction 2
Fraction 3
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma- BHC
Chlordane
4, 4 '-ODD
4, 4 '-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
^ndn'n aldehyde
Heptachlor
Heptachlcr epoxide
Toxaphene
PCB-1016
CS-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
100
100
97
98
100
100
99
98
100
0 100
37 64
0 7
0 0
4 96
0 68
100
100
96
97
97 	
95
97
103
90
95











91
106

26










                          :============== =====
                                                        ====:==
Eluant composition by fraction:
Fraction 1-6% ethyl ether in hexane
Fraction 2 - 15% ethyl  ether 1n  hexane
Fraction 3 - 50% ethyl  ether In  hexane
    are shown in Figures 2 to 10.   Other packed columns,  chro-
    ma tographic conditions, or detectors may be used if the
    requirements of Subsection G.2. are met.   Capillary (open-
    tubular) columns may also be used if the relative standard
    deviations of responses for replicate injections are  demon-
    strated to be less than 6% and the requirements of Subsection
    G.2. are met.

    Calibrate the system daily as  described in Subsection H.
                            III-184

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If the internal standard approach 1s bei/ig used,  the Internal
standard must be added to the sample extract and  mixed thor-
oughly immediately before injection into the instrument.

Inject 2 to 5 ul of the sample extract using the  solvent-
flush technique.  Smaller (1.0 pi) volumes can be injected if
automatic devices are emp^yed.  Record the volume Injected
to the nearest 0.05 pi, tne total extract volume, and the
resulting peak size in area or peak height units.

The width of the retention time window used to make Identi-
fications should be based upon measurements of actual  reten-
tion time variations of standards over the course of a day.
Three times the standard deviation of a retention time for a
compound can be used to calculate a suggested window size;
however, the experience of the analyst should weigh heavily
in the interpretation of chromatograms.

If the response for the peak exceeds the working  range of the
system, dilute the extract and re-analyze.

If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required using
one of the methods presented in Subsection J.I.2.7.

Calculate the concentrations of identified sample constitu-
ents as indicated in Subsection K.
                        III-185

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2.2  Analysis of Water Samples for Organochlorlne Pesticides/
     PolychloMnated Biphenyls
          Analytical Procedure:   evaluated
          Sample Preparation:   available

     2.2.1  Reference

            American Society for Testing and Materials,  "Tentative Method
            of Test for Organochlorine  in Water."  Method  D  3086-72T,  D-19
            Water pp. 519-534  (1980), American  Society  for Testing and
            Materials, Philadelphia, Pennsylvania.1^

     2.2.2  Method Summary

            The method offers  several analytical  alternatives,  dependent
            on the nature and  extent of Interferences and  the complexity
            of the pesticide mixtures found.  It  is recommended for  use
            only by experienced residue chemists  or under  the close
            supervision of such qualified persons.  Specifically, the
            procedure describes the use of an effective  co-solvent for
            efficient sample extraction and provides, through use of
            thin-layer and column chromatography, methods  for the
            elimination of non-pesticide Interferences  and the  pre-
            separation of pesticide mixtures.   Identification is by
            selective gas chromatographic separations chrougn the use  of
            two or more unlike columns.   Detection  and measurement are
            accomplished by electron capture  and  microcoulometric or
            electrolytic conductivity gas chromatography.  Results are
            reported in nanograms per liter without correction  for
            recovery data but  such data  should  be included wr;h  the  data,

     2.2.3  Applicability

            This method covers the determination  of various  chlorinated
            hydrocarbon pesticides, Including some  pesticidal degradation
            products and related compounds, in  water.   Such  compounds  are
            composed of carbon,  hydrogen, and chlorine,  but  may also
            contain oxygen, sulfur, phosphorus, or nitrogen.

            The following compounds may be determined Individually by
            this method; BHC,  lindane,  heptachlor,  aldrin, heptachlor
            epoxide, dleldrin, endrin,  Perthane,  ODE, ODD, DDT, meth-
            oxychlor, endosulfan, gamma-chlordane and Sulphenone.  Under
            ideal  circumstances,  Strobane, toxaphene, Kelthane, chlordane
            (technical grade), and others may also be determined.

            When chlorinated hydrocarbons exist as  complex mixtures, the
            individual compounds may be difficult to distinguish.  High,
            low, or otherwise  unreliable results  may be  obtained through
            misidentification, or one compound  obscuring another of
                                    III-186

-------
       lesser concentration, or both.  Provisions Incorporated 1n
       this method are Intended to minimize the occurrence of such
       Interferences.

2.2.4  Precision and Accuracy

       The precision of the method in paragraph 2.2 within the desig-
       nated range varies with the determined concentration as shown
       1n Table 10.  Additional data on the recoveries observed with
       this method are presented in Table 11.

2.2.5  Sample Extraction

       The size of the sample taken for extraction is dependent on
       the type of sample, the detection system employed,  and the
       sensitivity required.  Background information on the pesti-
       cide 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.
       The extract should not be concentrated further than required
       to meet the sensitivity dictated by the purpose for the anal-
       ysis.  Each time a set of samples is extracted, an  aliquot of
       solvent equivalent to that used for extraction is carried
       through tne 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 dosed sample should
       also be analyzed as a quality control  check.
                                         
       If the extract Is to be analyzed by microcoulometric tecn-
       niques, follow paragraph (a).  If the  extract is to be
       analyzed by electron capture analysis, follow the instruc-
       tions in paragraph (b).

       (a)  Microcoulometric Detection:

            1.  Place 3 liters of sample 1n a 4-liter separatory
                funnel, equipped with a TFE-fluorocarbon stop-
                cock, and extract with 150 ml of a mixture of ethyl
                ether 1n hexane (15+85) by shaking vigorously for 2
                minutes.  Rinse the sample container with  each
                aliquot of extracting solvent prior to extraction of
                the sample.
                               III-187

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      TABLE 10.  PRECISION OF METHOD FOR DETERMINATION OF
        ORGANOCHLORINE PESTICIDES IN NATURAL WATERSlO
Pesticide
Aldrin



Lindane



Dieldrin



DDT



Pretreatment
no cleanup

cleanup^

no cleanup

cleanupb

no cleanup

cleanupb

flo cleanup

cleanupb

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
ng/liter*
Sr So
4.86 2.59
32.06 20.19
9.13 3.48
27.16 8.02
5.28 3.47
26.23 11.49
8.73 5.20
27.49 7.75
18.16 17.92
30.41 21.84
10.44 5.10
34.45 16.79
15.96 13.42
38.80 24.02
22.62 22.50
49.83 25.31
aS.. = overall orecision, and  .
 S0 = single-operator precision.
bllse of Florisil column cleanup prior to analysis,
         2.  Allow the mixed solvent to separate from the water,
             and draw off the water into the original  container
             (1f it is of approximate size of the sample being
             extracted) or into a second 4-liter separatory fun-
             nel.  Pass the organic layer through a small column
             of anhydrous sodium sulfate topped with a pledget of
             cotton (previously rinsed with hexane) and collect
             in a 600-ml  tall-form beaker.  Repeat the extraction
             and treat the solvent as above.  Add approximately
             100 ml of sodium-sulfate-saturated water to the
             sample and complete a third extraction with 150 ml
             of hexane (not hexane-ethyl ether).  Pass this sol-
             vent, too, after separation, through the column of
             sodium sulfate.  Rinse the column with several small
             portions of hexane.  Blow out this solvent and recover
             *n the collection beaker containing the combined
             extracts.  Partially evaporate tne contents of tfie
                            II1-188

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   TABLE 11.  RECOVERY OF ORGANOCHLORINE PESTICIDES FROM NATURAL WATERS10
SBasassssaaasaaaaaaasasassaaaaaaaasaaaaaaasaaasaaBassaasssssaaaasaasaazaasaaaaar.
Pesticide
Aldrin
Llndane
Dleldrln
DDT
Added
Level
ng/liter
15
110
10
100
20
125
40
200
=============
Recovery,
without
Cleanup,
percent
69
72
97
73
108
85
101
77
Added
Level
ng/l1ter
25
100
15
85
25
130
30
185
Recovery,
with
Cleanup,
percent
68
65
94
70
70
65
118
71
                     beaker to aoout 300 ml  In a *ater bath 3t 70C,
                     applying no air or vacuum, and transfer to a 500-ml
                     Kuderna-Danish (K-D) evaporator equipped with a
                     10-ml  receiver ampule.

                     Hdjust che ;"'inal. axtrsct /olunje f^r tsmplss ?f Mgh
                     pesticide content (for  example, pesticide plant  waste
                     water samples), as necessary.   Concentrate samples
                     containing small  quantities of pesticides (low nano-
                     gram amounts, for example, most surface water samples)
                     to 1 ml.

                     Mount a three-ball Snyder column to the top of the
                     flask and evaporate solvent in a steam bath.  After
                     no more solvent actively distills, remove the
                     assembled K-D evaporator from  the bath and allow to
                     cool.   Disconnect the ampule and concentrate the
                     volume of the extract to 1 ml  in a warm water bath
                     (70*C) with a gentle draft of  clean dry air.  Make
                     an Initial gas chromatographic analysis on this
                     volume,  as described in paragraph 2.2.7.  If Insuf-
                     ficient pesticide Is present for quantitation at
                     this volume and greater sensitivity is required,
                     concentrate the extract further 1n accordance with
                     paragraph 2.2.7.
                                    III-189

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     5.  Interferences in the form of distinct peaks or high
         background,  or both, in the Initial  gas  chromato-
         graphic analysis, along with the physical  character-
         istics of the extract (color,  cloudiness,  and
         viscosity) will  indicate whether cleanup is re-
         quired.  When these  conditions interfere with measure-
         ment of the  pesticides, proceed in accordance with
         paragraph 2.2.6.   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.

(b)   Electron Capture Detection:

     1.  Drain 1 liter of sample into a 2-liter separatory
         funnel, equipped with a TFE-fluorocarbon stopcock,
         and extract  with  60  ml  of a mixture  of ethyl  ether
         and hexane (15+85) by shaking  vigorously for 2
         minutes.  Rinse  the  sample container with  each
         aliquot of extracting solvent  prior  to extraction of
         the sample.

     2.  Allow the mixed  solvent to saoarate  from the water
         and draw the water into the original  sample
         container (if 1t is  of the aoproximate si^e  of  the
         sample" being extracted) or into a  second 1-liter
         separatory funnel.   Pass the organic layer through  a
         small  column of  anhydrous sodium suTfate topped with
         a  plug- of cotton  (previously rinsea  witn nexane;  and
         collect in a- 250-ml  beaker.  Repeat  the  extraction
         and treat the solvent as above.   Add approximately
         35 ml  of sodium-sulfate-saturated  water  to  the
         sample and complete  a third extraction with  60  ml of
         hexane (not  hexane-ethyl  ether).   Pass this  solvent
         through the  sodium sulfate column  and collect in  the
         beaker.   Rinse the column with several small  portions
         of hexane, blow  out  the solvent and  recover  it  in the
         collection beaker containing the combined  extracts.

     3.  Adjust the final  extract volume for  samples  of  high
         pesticide content (for example,  pesticide  plant waste
         water samples),  as necessary.   Concentrate samples
         containing small  quantities of pesticides  (low  nano-
         gram amounts,  for example,  most surface  water samples)
         to 1 ml.

     4.  Mount a three-ball Snyder column to  the  top  of  the
         flask and evaporate  solvent in a steam bath.  After
         no more solvent  actively distills, remove  the
         assemolea K-0  evaporator rrom  ;ne  oath ana  allow  :o
                        III-190

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                cool.  Disconnect the ampule and concentrate the.
                volume of the extract to 1  ml  1n a warm water bath
                (70eC) with a gentle draft of clean dry air.  Make
                an Initial  gas chromatographic analysis of this
                volume as described in paragraph 2.2.7.  If insuf-
                ficient pesticide is present for quantisation at
                this volume and/or greater sensitivity is required,
                concentrate the extract further in accordance with
                paragraph 2.2.7.

            5.  Interferences 1n the form of distinct peaks or high
                background, or both, 1n the initial  gas chromato-
                graphic analyses, along with the physical  character-
                istics of the extract (color,  cloudiness,  and
                viscosity)  will Indicate whether cleanup is re-
                quired.  When these factors Interfere with measure-
                ment of the pesticides, proceed 1n accordance with
                paragraph 2.2.6.  Whether required for quantitative
                analysis or not, all extracts should be subjected to
                these procedures, subsequent to the  initial  anal-
                ysis, and rechromatographed for qualitative corrob-
                oration of the results.

2.2.6  Sample Cleanup and Separation Procedures

       A sample that contains interferences such as  oil  or wax-like
       materials and other organic matter may be treated in several
       ways:, column chromatography23, thin-layer chromatog-
       rapny^.-S^ or Coiumn cnromatography followed by thin-i?yer
       Chromatography.  These procedures not only remove many of
       the oily and waxy substances that prevent distinct  and
       specific gas chromatograms, but pre-separate  mixtures of
       pesticides, thereby aiding the analyst in interpretation of
       subsequent gas chromatograms.

       (a)  Florisll Column Adsorption Chromatography:

            1.  Dilute the sample extract previously concentrated
                to 1 ml to  10 ml with hexane.   Place 15 g  of
                activated Florisil, that has been stored in an
                airtight container at 130*C, in a column over a
                small layer [13 mm (1/2 in.)] of anhydrous gran-
                ular sodium sulfate.  After tapping  the Florisil
                Into the column, add a 2-cm (3/4-1n) layer of gran-
                ular sodium sulfate to the  top.  After cooling,  pre-
                elute the column with approximately 75 ml  of
                hexane. .Discard the pre-eluate, and just  prior to
                exposure of the sulfate layer to air, quantitatively
                transfer the sample extract into the column by
                aecamcation and subsequent  hexane washings.   *d.iust
                the elution rate to-approximately 5  ml/min for two
                eluates collected separately in the  300-iiil  X-D

                               III-191

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         apparatus  equipped  with  10-ml  ampules.   Perform  the
         first elutlon  with  200 ml  of  a mixture of ethyl
         ether 1n hexane  (6+94) and the second elutlon with
         200  ml  of  a  mixture of ethyl  ether  1n hexane
         (15+85).  Connect the K-D  apparatus containing the
         eluates to three-ball Snyder  columns and evaporate
         the  solvents in  accordance with  paragraph 2.2.5.D.4.

     2.   Eluate Composition.  If  the Florisil has been prop-
         erly activated23 and stored,  and if the  reagents
         have been  carefully prepared,  the following eluate
         compositions will be obtained when  the pesticides
         are  present.  The first  eluate (ethyl ether in
         hexane (6+94)) will  contain lindane, BHC, Kelthane,
         aldrin, heptachlor,  DDE, ODD,  DDT,  Perthane, hep-
         tachlor epoxide, methoxychlor, toxaphene, Strobane,
         chlordane  (gamma and tech), and  endosulfan I.  The
         second eluate  (ethyl ether in  hexane (15+85)) will
         contain dieldrin, endrin,  endosulfan II, lindane,
         and  Kelthane.

     3.   Standard pesticide  mixtures should  be used fre-
         quently to demonstrate the effectiveness of the
         r'on'sil to  characterize the  aluate composition  and
         provide quantitative recovery.   The concentrated
         extract may  be analyzed  directly by injecting
         suitable aliquots from the K-D ampule into the gas
         chromatograph.   If  the residues  are high in total
         ornanics,  further cleanup  prior  to  gas chromato-
         graphic analysis may oe  necessary.

(b)   Thin-Layer Chromatography (TLC):

     1.   The  sample extract  [paragraph 2.2.5(a) or 2.2.5  (b)]
         or the Florisll  treated  sample extracts  [paragraph
         2.2.6 (a)] may be further  purified  by TLC to separate
         pesticides and remove interfering substances.

     2.   Layer Preparation - Prepare layers  of silica gel
         0.25 mm thick  on 20-cm x 20-cm (8 x 8-1n) glass
         plates. Prepare a  homogeneous slurry by shaking 30 g
         of silica  gel  in approximately 60 ml of  water.
         With the aid of  a variable thickness applicator,
         immediately  spread  the slurry over  five  plates held
         on a mounting  board. Allow the  layers to stand  for
         5 minutes  then activate  them  in  an  oven  for 60
         minutes at 110'C and store 1n a  desiccator for
         future use.  Reactivate  layers stored longer than 1
         week before  use.  Just prior  to  use, make marks  15
         and  115 mm above the bottom edge of the  layer to
         define che spotting ".ine and  the point at .rtiich  the
         solvent front  has moved  100 mm.

                             III-192

-------
3.  Developing Solvent - Add the developing solvent,
    carbon tetrachloride, to the developing chamber to a
    depth of 10 mm.  Place two filter paper wicks, one
    on each side of the chamber, so that one end con-
    tacts the solvent.  After the I1d is in place, allow
    the chamber to equilibrate for 1 hour.  It is
    Important that the chamber be protected from drafts
    and large temperature changes.

4.  Sample Spotting - Carefully evaporate the sample
    extracts contained 1n K-0 ampules [paragraph
    2.2.6 (a)3, or extracts retained [paragraph 2.2.5 (a)
    or 2.2.5 (b)], 1n a bath of warm water at 40"C with a
    fine draft of clean air to an appropriate volume for
    spotting (not less than 200 ul) (NOTE 5).  Adjust
    the final sample volume so that no more than 100 ul
    must be spotted 1n order to maintain adequate gas
    chromatographlc sensitivity when analyzing the
    subsequent eluates.  Never concentrate the eluates
    below 100 ul.  Very carefully wash down the Internal
    wall of the ampule with small portions of ethyl
    ether and evaporate by the same technique to
    approximately 100 yl.  Carry out this step three
    times.  Then adjust the volume 
-------
               the  layer 1s in contact with the developing  solvent,
               and  replace the lid.  When the developing solvent
               reaches  the upper reference line (100 mm),, remove
               the  layer from the chamber and allow it to air dry
               at room  temperature.

           7.  Visualization and Sectioning of Layer - After
               development, evenly spray the portion of the layer
               containing the standards (from 10 to 20 ug)  with a
               fairly heavy coat of Rhodamine B (0.1 mg/ml  in
               ethanol).  Allow the sprayed area to thoroughly dry
               (approximately 5 minutes) and then expose it to and
               view 1t  under a short-wave UY light.  The pesticides
               appear as quenched areas (dark) on a fluorescent
               background.  Mark the location of each pesticide.

           8.  The  distance of travel for pesticides present in the
               unknown  samples and recovery test standards  will be
               the  same as those of the standards.  From this
               Information, divide the vertical zone for each
               sample into five horizontal sections.  Identify the
               sections with Roman numerals.

               Examoles of respective Rf and Rr values for  various
               pesticides are listed in Facie 12.

           9.  Pesticide Removal from the TLC Plates - Using tne
               spotting template as a ruler and with the aid of a
               sharp pointed object, individually rule off  the
               silica gel sections or  interest.  Kith tne JIG of a
               mild vacuum draw the silica gel, first from  the
               periphery of the section and then from the center of
               the  section, Into a medicine dropper that is plugged
               close to the tip with filtering-grade glass  wool.
               Quantitatively elute the pesticides adsorbed on this
               silica gel into a 10-ml K-D ampule with successive
               small washes of ethyl ether-petroleum ether  (1+1) to
               a total  volume of 5 to 10 ml.  Plug the ampule with
               a glass  stopper and retain the contents for  the
               determinative  steps 1n paragraph 2.2.7.

2.2.7  Sample Analysis

       Obtain reasonable positive  Identification of a pesticide by
       corroborating the results  using  a minimum of two chromato-
       graphlc columns of  different  polarity.  Columns  recommended
       for this purpose  and  their  approximate operating temperatures
       are:  5 percent OV-17  on Gas Chrom Q  (80  to  100 mesh)-210eC,
       5 percent  QF-1 plus  3  percent  DC-200 on Gas Chrom  Q  (80  to
       100 mesh)-185C,  3  percent  OY-101 on Gas  Chrom Q  (80  to  100
       mesn)-i90'iC,  ana  3  percent-V-10 on 3as  Chrom Q  (3G  to  100
                               III-194

-------
   TABLE 12.  SOME Rf AND Rr VALUES OF PESTICIDES DEVELOPED WITH CCL4
                        ON SILICA GEL THIN LAYER PLATE
a=a=================z======z==========================================


Pesticides and Metabolites        Rf Value9     Rr Value5     Section
Dieldrin
Endrin
Heptachlor epoxide
Lindane
ODD
Gamma-Chlordane
Heptachlor
DDT
DDE
Al dri n
0.17
0.20
0.29
0.37
0.54
0.55
0.67
0.68
0.72
0.73
0.33
0.37
0.52
0.69
1.00
1.02
1.24
1.26
1.33
1.35 .

II


III


IV


B==============================================================================
aRr - Distance traveled V/ the ^omcound divided by the distance traveled
      by the solvent front.
^R  - Distance traveled by the compound divided by the distance traveled
      by the distance traveled by standard p,p'-DDD.


            mesh)-185C.  The analyst must determine  the optimum condi-
            tions to obtain the best results for compounds under study
            with the available equipment.   Chromatograph standard mix-
            tures of interest to determine the required separation with
            maximum resolution.  Evaluate  parameters  such as gas flow,
            temperature, column length and diameter,  as well as detector
            performance and alter as required to achieve the desired
            results.

            Final Extract Concentration

            If necessary to meet the sensitivity requirements for the
            purpose at hand, concentrate the sample extract contained in
            a graduated K-D receiver ampule from the  appropriate extrac-
            tion or purification step [paragraph 2.2.5 (a), 2.2.5 (b), or
            2-2.5 ?b)l *o ""sss than 1 ml 1n a beaker  of warm water (60eC).
            Continue to carefully evaporate the solvent with a fine
            stream of dried and filtered air to no less than 0,3 ml.

                                    in-195

-------
Intermittently wash down the internal  wall  of the tube with
hexane during this final concentration step and adjust the
volume with hexane to an appropriate level  (from 0.3 ml  to
0.5 ml for micro-coulometric determination  or from 0.5 to 1.0
ml for electron capture determination).

If more exhaustive evaporation of the  extract is required to
achieve the necessary sensitivity for  microcoulometric
detection, the use of "keeper" is strongly recommended.
Place 2 mg of "keeper" in the concentrated extract by syringe
addition of 100 ul of 20 ug/ul light mineral  oil in hexane.
This "keeper" will not interfere with  microcoulometric detec-
tion and will prevent major residue losses  in this exhaustive
evaporation step.

Sample Analysis - Note the volume of the sample extracts and
analyze suitable aliquots (from 20 to  100 ul  for microcoulo-
metric detection and from 5 to 10 ul for electron capture
detection) by gas chromatography, employing at least two
columns for identification and quantification.  Frequently
inject standards to ensure optimum operating conditions.  Gas
chromatograms of several standard pesticides are shown in
Figures 12, 13, 14, and 15.  The elution order as well as
elution ratios for various pesticides  are provided in Table
13 us u guide only.  It Is the responsibility of the analyst
to develop his own identification keys to fit the chosen
operating conditions of the instrument.  Calculate the
concentration of identified sample constituents as indicated
in Subsection K.
                        III-196

-------
        Linda ne
           Heptachlor
                   [ Heptachlor
                     Epoxide
             Aldrin
                         p.p'-DDE
               UU
                                              p.p'-DDT
                   I
                   4
I
6
 I
10
I
12
 I
14
                   Retention Time in Minutes
              (Chart speed one-half inch per minute)


     Column Packing- 3% QC-200 + 5% Qf-1 on Gas Chrom Q (80/100 Mesh)
     Carrier Gas- Nitrogen at 80 ml/min
     Column Temperature- 200C
Figure  12.   Electron  capture gas  chromatogram  of pesticide  standards
                                 III-197

-------
1 
0
1
2
1
4
1
6
1
8
1
10
1
12
                     Retention Time in Minutes
                (Chart speed one-half inch per minute)

            Column Packing- 3% OV-101 on Gas Chrom Q (80/100 Mesh)
            Carrier Gas- Nitrogen at 80 ml/min
            Column Temperature- 175C
Figure 13.   Electron capture gas chromatogram of pesticide  standards,
                                III-198

-------
           Lindane
            Heptachlor
                     jHeptachlor
                       Epoxide
                                                        p.p-DDT
    0      2     46     8     10    12     14    16

                        [Retention Time in Minutes]
                    Chart speed one-half inch per minute

     Column Packing-5% OV/17 on Gas Chrom Q [60/80 Mesh]
     Carrier Gas-Nitrogen at 80 ml/min.
     Column Temperature 200C
18
Figure 14.  Electron capture gas chromatogram of pesticide standards,
                                III-199

-------
           Lindan*  Hptchlor
                       Hepuchlor
                 I Atdrin   Epoxide
                                                        p.p'-DDT
                        I
                        4
I
6
                                     8
 I
10
 I
12
 I
14
                          Retention Time in Minutes
                     (Chart speed one-half inch per minute)

          Column Packing- 3% OV-210 on Gas Chrom B (80/100 Mesh)
          Carrier Gas- Nitrogen at 80 ml/min
          Column Temperature- 180C
Figure  15.   Electron capture gas  chromatogram  of pesticide  standards.
                                  III-200

-------
  TABLE 13.  RETENTION TIMES OF ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
      Liquid
      Phase3
   Column
Temperature
                        ==========================
5 Percent
 DC-200 +
3 Percent  5 Percent  3 Percent  3 Percent
  QF-1       OV-17      OV-101     OV-210
  200 C
200'C
175*C
160C
 Relative
Sensitivity
   to EC
 Detectorb
   Pesticide
 absolute)
  RRtc
           RRtc
Alpha-BHC
Lindane
Heptachlor
Aldrin

-------
3.1  Analysis of Sediment Samples for Pesticides/Polychlorinated
     Biphenyls
          Analytical  Procedure:   available
          Sample Preparation:   available

     3.1.1  Reference/Title

            Thompson, J. F., "Analysis of Pesticide Residues in Human and
            Environmental  Samples,"  U.S. Environmental  Protection Agency
            Pesticide and Toxic Substances Effects Laboratory.   Research
            Triangle  Park, North Carolina.  197426

     3.1.2  Method Summary

            A sediment is partially dried and extracted by column elution
            with a mixture of 1:1 acetone/hexane.   The  extract  1s washed
            with water to remove the acetone and then the  pesticides  are
            extracted from the water with 15 percent methylene  chloride
            1n hexane.  The extract is dehydrated, concentrated to a
            suitable  volume, subjected to Florisil partitioning, desul-
            furized,  if necessary, and analyzed by gas  chromatography.

     3.1.3  Applicability

     3.1.4  Precision and Accuracy

            Many pesticides and PCBs can be recovered at the 85-104 per-
            cent level based on duplicate analyses. However, compounds
            iucn as r.eptacr.ior, vhlorcbenz'ilate, and arganophosphates are
            degraded  by the sulfur cleanup procedure and effectively
            lost.26

     3.1.5  Sample Preparation

            Decant and discard the water layer over the sediment.  Mix
            the sediment to obtain as homogeneous  a sample as possible
            and transfer to a pan to partially air dry  for about 3 days
            at ambient temperatures.

            NOTE 6:  Drying time varies considerably depending  on soil
            type ana  drying conditions.  Sandy soil will sufficiently dry
            in 1 day, whereas muck requires, at least,  3 days.   Silt  and
            muck sediments are sufficiently dry when the surface starts
            to split, but there should be no dry spots. Moisture content
            will be 50 to 80 percent at this point.

            Weigh 50  g of the partially dried sample Into  a 400-ml Omni
            mixer chamber.  Add 50 g of anhydrous  sodium sulfate and  mix
            well with a large spatula.  Allow to stand  with occasional
              Erring   "or -ipprox-! mats'* y \ sour.
             > V t i
                                    III-202

-------
NOTE 7:  If the final calculations will  be made on a dry-
weight basis, it is necessary at this point to initiate the
test for percent total solids on the sample being extracted
for pesticide evaluation.  Immediately after weighing the
50-g sample for extraction, weigh approximately 5 g of the
partially dried sediment into a tared crucible.  Determine
the percent solids by placing the sample in a drying oven
overnight at 103 to 105C.   Cool the sample, desiccate, and
weigh the residue.  Repeat  the process until a constant
weight is obtained.

Attach the 400-ml chamber to an Omni or Sorvall mixer and
blend for about 20 seconds.  The sample should be fairly
free-flowing at this point.

Carefully transfer the sample to a chromatographic column.
Rinse the mixer chamber with small portions of hexane, adding
the rinsings to the column.

Elute the column with 250 ml of 1:1 acetone-hexane at a flow
rate of 3 to 5 ml/min into  a 400-ml beaker.

Concentrate tne sample extract to about 100 ml under a
nitrogen stream and at a temperature no higher than 55C.
Transfer to a 500-ml separatory funnel containing 300 ml of
distilled water and 25 ml of saturated sodium sulfate sol-
ution.  Shake the separatory funnel for 2 minutes.

Drain the water layer into  a clean beaker and the hexane
layer into a clean, 250-ml  separatory funnel.

Transfer the water layer back into the 500-ml separatory
funnel and re-extract with  20 ml of 25 percent methylene
chloride in hexane, again shaking the separatory funnel for 2
minutes.  Allow the layers  to separate.   Discard the water
layer and combine the solvent extracts in the 250-ml separ-
atory funnel.

Wash the combined solvent extract by shaking with 100 ml of
distilled water for 30 seconds.  Discard the wash water and
rewash the extract with an  additional 10 ml of distilled
water, again discarding the wash water.

Attach a 10-ml evaporator concentrator tube to a 250-ml
Kuderna-Danish flask and place under a filter comprised of a
small wad of glass wool and approximately 1.2 cm of anhy-
drous sodium sulfate in a filter tube.

Pass che solvent extract through the drying filter into the
K-0 flask and rinse with three portions of approximately
5 ml each of nexane.     	
                        III-203

-------
       Attach a Snyder column to the top joint of a K-D flask,
       immerse tube in an 80"C water bath, and concentrate extract
       to 5 ml.

       Remove the tube and rinse the joint with a small volume of
       hexane.  The sample is now ready for Florisil  partitioning.

3.1.6  Sample Cleanup with Florisil  Columns

       Prepare a Florisil chromatographic column containing 10 cm
       (after settling) of activated Florisil  topped with 1 cm of
       anhydrous, granular sodium sulfate.  A  small  wad of glass
       wool, pre-extracted with hexane, is placed at the bottom of
       the column to retain the Florisil.

       NOTE 8:  If the oven is of sufficient size,  the columns may
       be prepacked and stored 1n the oven, for withdrawal  of
       columns a few minutes before  use.

       The amount of Florisil  needed for  proper elution should be
       determined for each lot of Florisil.

       Place a 500-ml  Erlenmeyer flask under the column and prewet
       the packing with hexane (40 to 50  ml, or a sufficient volume
       to completely cover the sodium sulfate  layer).

       NOTE 9:  From this point and  through the elution process,  the
       solvent level  should never be allowed to go below the top  of
       tr.e :oaium sulfats layer.   If air  Is 'ntrcducsd,  channel :nc
       may occur that results  in reduced  column efficiency.

       Assemble two more K-D apparatus but with 500-ml  flasks and
       position the flask of one assembly under the Florisil  column.
       However, at this point, use 25-ml  graduated evaporator con-
       centrator tubes instead of the 10-ml  size used  previously.

       Using a 5-ml  Mohr or a  long disposable  pipet,  immediately
       transfer the extract from the evaporator tube onto  the column
       and permit 1t to percolate through.   Rinse tube with two
       successive 5-ml  portions of hexane,  carefully transferring
       each portion to the column with the pipet.

       NOTE 10:   Use of the Mohr or  disposable pipet to  deliver the
       extract directly onto the column precludes the  need to rinse
       down sides of the column.

       Commence elution with 200 ml  of 6  percent diethyl  ether in
       petroleum ether (Fraction 1).   The elution rate should be
       approximately 5 ml  per  minute.   When the last of the eluting
       jolvent reaches 3 point spproxlmatelv 3 im *rom t.he  too
       of the sodium sulfate layer,  place the  second 500-ml  Kuderna-
       Oanisn assemoly under the column and continue slutlon with

                               III-204

-------
       200 ml of 15 percent diethyl ether In petroleum ether (Frac-
       tion 2).  Place both Kuderna-Dam'sh evaporator assemblies in
       a water bath and concentrate extracts to approximately 20 ml.

       NOTE 11:  If there is reason to suspect the presence of
       malathion in the sample, have a third 500-ml K-D assembly
       ready.  At the end of the 15 percent fraction elution, add
       200 ml of 50 percent diethyl ether in petroleum ether
       (Fraction 3), evaporating the eluate in the same manner.

       Remove K-D assemblies from the bath, cool, and rinse the
       T-joint between the tube and flask with a little petroleum
     .  ether.  Finally, dilute both extracts to exactly 25 ml and
       proceed with the GLC determinative step.

3.1.7  Sample Analysis by Gas Chromatography

       Inject 5 ul  of each fraction extract into the gas chromato-
       graph (electron capture mode) primarily to determine whether
       the extracts will  require further adjustment by dilution or
       concentration.

       When aoDrooria-te dilution adjustments have been made in the
       extracts and the column oven ~.z set it trie <-equired tempera-
       ture, the relative retention values of the peaks on the
       chromatograms should be calculated.  Wien these values are .
       compared with the values in Table 3 for the appropriate
       column, the operator should be able to make tentative com-
       pouna identifications,  ''icrocoulcmetry ard/or "H.C may be
       required for positive confirmation of some of the tentatively
       identified chlorinated compounds, whereas flame photometric
       detectors (FPD) may be utilized for confirmation of the
       organophosphate compounds.

       An analytical problem that must be considered when sediment
       samples are analyzed for chlorinated hydrocarbon pesticides
       is sulfur interference.  Elemental sulfur is encountered in
       most sediment samples, marine algae, and some industrial
       wastes.  The solubility of sulfur in various solvents is very
       similar to the organochlorine and organophosphate pesticides;
       therefore, the sulfur interference follows along with the
       pesticides through the normal extraction and cleanup tech-
       niques.  The sulfur will be quite evident in gas chromato-
       grams obtained from electron capture detectors, flame photo-
       metric detectors operated in the sulfur or phosphorus mode,
       and Coulson electrolytic conductivity detectors.  If the gas
       chromatograph is operated at the normal conditions for
       pesticide analysis, the sulfur interference can completely
       mask the region from the solvent peak through aldrin.

       One techniaue eliminates sulfur by the formation of copper
       sulfide on the surface of the copper.  There ?re two critical

                               II1-205

-------
steps that must be followed to remove all the sulfur:  (a)
the copper must be highly reactive; therefore, all  oxides
must be removed so that the copper has a shiny, bright
appearance; and (b) the sample extract must be vigorously
agitated with the reactive copper for at least 1 minute.

It will probably be necessary to treat both the 6 and
15 percent Florisil eluates with copper if sulfur crystal-
lizes out upon concentration of the 6 percent eluate.

Certain pesticides such as the organophosphates, chloro-
benzilate, and heptachlor can be degraded by this technique.
However, these pesticides are not likely to be found in
routine sediment samples because they are readily degraded in
the aquatic environment.

If the presence of sulfur is indicated by an exploratory
injection of the final extract concentrate (presumably 5  ul)
into the gas chromatograph, proceed with removal as follows:

(a)  Under a nitrogen stream at ambient temperature, con-
     centrate the extract in the concentrator tube to exactly
     1.0 ml.

(b)  If" the sulfur concentration is sucn that crystalization
     occurs, carefully transfer, by syringe, 500 ul of the
     supernatant extract (or a lesser volume if tne sulfur
     deposit is too heavy) into a glass-stoppered,  12 ml
     graduated conical centrifuge tube.  Add 500 ul of iso-
     octane.  AUQ approximate"!,/ 2 g of bright copper powder,
     stopper, and mix vigorously 1 minute on a vortex genie
     mixer.

     MOTE 12:  The copper powder, as received from the
     supplier, must be treated for removal of surface oxides
     with 6 N[ HN03.  After about 30 seconds of exposure,
     decant acid and rinse several times with distilled water
     and finally with acetone.  Dry under a nitrogen stream.

(c)  Carefully transfer 500 ul of the supernatant-treated
     extract into a 10-ml graduated concentrator tube. An
     exploratory injection into the gas chromatograph at  this
     point will provide Information as to whether further
     quantitative dilution of the extract 1s required.
                        II1-206

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3.2  Analysis of Sediment Samples for Pesticides/Polychlorinated
     Biphenyls
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     3.2.1  Reference/Title

            Environment Canada,  "Analytical  Methods Manual,"  Inland
            Waters Directorate,  Water Quality Branch,  Ottawa,  Ontario,
            Canada.  (1974).27

     3.2.2  Method Summary

            Sediment is extracted with acetonitrile and the chlorinated
            hydrocarbons are partitioned into petroleum ether.   The ether
            extract is cleaned up on a Florisil  column and separated into
            four fractions for subsequent GLC analysis.

     3.2.3  Applicability

            This method has been used to quantify the  following chlor-
            inated hydrocarbons  and PCBs in  sediment samples.   The  values
            1n parentheses are the lowest level  of detection  (in ppm).

            Lindane              (0.001)     tndrin               (O.C1)
            Heptachlor           (0.001)     p,p'.-Methoxychlor    (0.05)
            Heptachlor epoxide   (0.001)     Alpha-endosulfan      (0.01)
            Aldrin               (0.001)     Beta-endosulfan       (0.01)
            Disldr-'n             (0.001)     Cis-chlordane       (0.005)
            p,p'-ODD             iO.OOl)     Trans-chlcrdane      (C.OOS)
            p.p'-DDT             (0.001)     Aroclor 1248        (0.100)
            p.p'-ODE             (0.001)     Aroclor 1254        (0.100)
            o,p'-DDT             (0.001)     Aroclor 1260        (0.100)

     3.2.4  Precision and Accuracy

            Recovery studies with the method demonstrated over 95%  recovery
            for all compounds listed in paragraph 3.2.3 in the 10-ng
            range.  The overall  recovery including Florisil column  frac-
            tionation is approximately 90% in the 10 to 50 ng  range. The
            accuracy of the procedure has yet to be determined.28

     3.2.5  Sample Preparation

            Transfer a 10-g dry-weight equivalent of sediment  into  the
            glass jar of a Waring blender with a Bakelite top.   (Do not
            use a rubber or p-lastic top.)  Add 120 ml  of acetonitrile and
            blend at medium-high speed for 15 minutes.  Allow  solid
            particles to settle somewhat.  Pour the acetonitrile extract,
            which may contain some suspended particles, into  an All inn
            filter tube containing prewashed celite covering  the win
            glass,

                                    II1-207

-------
       NOTE  13:   If  the  residue  In the Allihn filter tube becomes
       excessive,  it should be scraped out with a spoon-type spatula
       and combined  with the material in the blender before the
       second  blending and extraction.

       (a)   To the residue in the blender, add another 120 ml of
            acetonitrile and 40  ml of distilled water and blend for
            10 minutes.   Filter  as before.

       (b)   Pour  60  ml of acetonitrile into the blender and blend
            the homogenate for 10 minutes.  Transfer all the resi-
            due,  if  necessary, with 2 to 20 ml acetonitrile, into
            the Allihn tube and  filter.  Apply sufficient; suction to
            recover  as much solvent as possible.

       (c)   Quantitatively transfer the acetonitrile extract to a
            1-1 funnel with several petroleum ether washes of the
            filter flask. Adjust the aqueous content of the extract
            to approximately 20  percent with distilled water.
            Extract  the  resulting mixture with 150 ml of petroleum
            ether and twice with 75 ml petroleum ether.

       (d)   Wash  the combined petroleum ether extracts with approx-
            imately  200  ml distilled water.  Discard water washing
            and pass the organic extract, under suction or witn air
            pressure, through an anhydrous sodium sulfate (10 to 15
            g) column using a 500-ml round-bottomed flask; as a
            receiver.

       (e)   In a  rotary  evaporator, concentrate the contents in tne
            500-ml flask to 2 or 3 ml.  (Do not let contents go dry
            and do not use a water bath temperature over 40C;
            otherwise, there may be a possible loss of pesticides
            and PCBs.)

3.2.6  Sample Extract Cleanup

       Transfer the  concentrated petroleum ether extract with a
       clean disposable  pipet onto a 30-g column (to determine the
       exact amount  of Florisll  required, follow the guidance pro-
       vided with the water analysis procedure) with 1 cm of anhy-
       drous sodium  sulfate on  the top of the Florisll.  Use a 300-ml
       round-bottomed flask as  a receiver.

       Allow the  extract to enter  the Florisil column just  to the
       sodium sulfate layer.  Rinse  the round-bottomed flask with 2
       or 3 ml of petroleum ether  and transfer the  rinsing  with the
       same disposable  pipet  onto  the column.  Let  the rinsing
       solvent again drain  just to the  sodium sulfate layer.  Rinse
       the "ound-bottomed flask again with 2 or 3 ml of petroleum
       ether ana transfer the rinsing onto  tne column.
                               II1-208

-------
       Again rinse the round-bottomed flask,  this  time with 20  to
       30 ml  petroleum ether.   Carefully  pour the  petroleum ether
       onto the column so that the sodium sulfate  layer  is not  dis-
       turbed.   Elute the column  with a total  of 200 ml  (including
       the above rinsings) of  petroleum ether.

       Concentrate eluate with a  rotary evaporator to 1  to 2 ml and
       transfer, with benzene  rinsings, to a  10-ml  volumetric flask.
       Make up  to 10 ml  with benzene for  GLC  examination (Frac-
       tion 1).

       Change the receiver and elute column with 200 ml  of 6-percent
       diethyl  ether containing 2 percent ethanol.  Concentrate
       eluate to 1 to 2 ml on  a rotary evaporator.  Transfer to a
       10-ml  volumetric flask. Rinse round-bottomed flask with
       benzene  and add to volumetric flask.  Dilute to volume with
       benzene.  This fraction, Fraction  2, is  now ready for GLC
       analysis.

       With a third 300-ml round-bottomed flask as receiver, elute
       the column with 200 ml  of  15-percent ether  in petroleum
       ether.  Concentrate to  10  to 20 mi  witn  a  rotary  evaporator.
       Add 50 to 60 ml of benzene and concentrate  to 1 to 2 ml.
       Make up  to 10 ml  with benzene in a volumetric flask
       (Fraction 3).

       Elute the column with 200  ml  chloroform or  200 ml  50-percent
       diethyl  ether in petroleum ether.   Collect  th^ eluate in a-
       round-bottomed flasic and concentrate to 2  to 3 ml  on a  rotary
       evaporator.  Add 50- to 60-ml  portion  of benzene  and evaporate
       to 2 to  3 ml.  Add a second 50- to 60-ml portion  of benzene
       and evaporate to 2 to 3 ml.  Transfer  the concentrate to a
       10-ml  volumetric flask. Rinse the round-bottomed flask with
       benzene  and add the rinsing to the volumetric flask.  This
       fraction is now ready for  GLC analysis (Fraction  4).

3.2.7  Sample Analysis

       The petroleum ether fraction (Fraction l) contains PCBs,
       heptachlor, aldrin, p,p'-ODE, and  alpha-BHC.

       The 6-percent diethyl ether petroleum  ether fraction
       (Fraction 2) contains lindane, heptachlor epoxide, p,p'-DDT,
       p,p'-DDD, methoxychlor, o,p'-DDT,  cis-chlordanes, and
       trans-chlordanes.

       The 15-percent diethyl  ether petroleum fraction (Fraction 3)
       contains dieldrin, alpha-endosulfan, and endrin.

       The last fraction (4) contains beta-andosulfan.

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Extracts may have to be cleaned up for sulfur interference.
Follow procedures given In Sediment Subsection  J.3.1.6.

Analyze extracts by gas chromatography using suggested
instrument operating conditions.   Further concentration
and dilution may be necessary to produce on-scale GLC peaks.
Procedures for confirmation of identity are  the same as  for
water extracts (Subsection K).
                        II1-210

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4.1  Analysis of Biological Tissue for Pesticides/Polychlorinated
     Biphenyls
          Analytical  Procedure:  available
          Sample Preparation:  available

     4.1.1  Reference/Title

            U.S. Environmental  Protection Agency, "Extraction and Analysis
            of Priority Pollutants in Biological  Tissue,"  U.S.  EPA, S&A
            Division, Region IV, Laboratory Services Branch,  Athens,
            Georgia.   Method PPB 10/80.  7 p., 1980.8

     4.1.2  Method Summary

            A 10-gram aliquot of homogenized fish tissue is mixed with
            40 grams  of anhydrous sodium sulfate, and extracted with
            petroleum ether using an ultrasonic probe.   The samples are
            filtered, concentrated to 10 ml  or less, cleaned  up with
            acetonitrile partitioning and concentrated to  1 ml.  The
            extract is analyzed by gas chromatography.

     4.1.3  Applicability

            The limit of detection for this method is usually dependent
            upon the  level of interferences rather than instrumental
            limitations.  Where interferences are not a problem, the
            limit of  detection for most compounds analyzed by GC/MS is 2
            mg/kg (wet weight basis).

            This method is recommended for use only by experienced residue
            analysts  or under the close supervision of such qualified  persons.

     4.1.4  Precision and Accuracy

            Information is not  presently available.

     4.1.5  Sample Preparation

            Blend equal amounts of fish tissue and dry ice.  The pre-
            ferred procedure is to use a blender  but a  food processor  or
            meat grinder may be more appropriate  with large samples.

            Weigh 10  g of homogenized sample into a 400-ml  beaker.  Mix
            with 40 g anhydrous sodium sulfate until the sample is
            thoroughly mixed.

            Add 100 ml petrol.eum ether to the sample.  Using  an ultra-
            sonic probe, sonicate the sample at 50% pulse  for 3 minutes.
            Allow the layers to separate and decant the solvent through a
            Bucnner funnel filtration system.

            Add a second 100-ml  portion of petroleum ether to the tissue

                                    III-211

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       residue in the beaker.  Repeat the extraction process and add
       the filtered solvent layer to the first extract.

       Extract the sample with a third 100-ml  portion of solvent.
       Filter the entire sample through the Buchner funnel  filtra-
       tion system and combine the petroleum ether extracts.

       Quantitatively transfer the extract to a K-D flask equipped
       with a 10-ml concentrator tube.  Add a boiling chip to the
       flask and attach a three-ball Snyder column.  Place the K-D
       apparatus on the water bath and concentrate the extract to
       10 ml.

4.1.6  Sample Extract Cleanup (Acetonitrile Partitioning)

       This procedure is used to isolate fats and oils from the
       sample extracts.  It should be noted that not all pesticides
       are quantitatively recovered by this procedure.  The analyst
       must be aware of this and demonstrate the efficiency of the
       partitioning for specific pesticides.

       Quantitatively transfer the previously concentrated extract
       to a 125-ml separatory funnel with enough hexane to bring the
       final volume to 15 ml.  Extract the sample four Vnes Ky
       shaking vigorously for 1 minute with 30-ml portions of
       hexane-saturated acatonitrile.

       Combine and transfer the acetonitrile phases to a 1-liter
       saparatory  c'jnne! and add 650 ml of distilled wate- *nd 40 nl
       of saturated sodium chloride solution.   Mix thoroughly for 30
       to 45 seconds.  Extract with two 100-ml portions of hexane by
       vigorously shaking about 15 seconds.

       Combine the hexane extracts in a 1-liter separatory funnel
       and wash with two 100-ml portions of distilled water.
       Discard the water layer and pour the hexane layer through a
       drying column containing 7 to 10 cm sodium sulfate and 2 cm
       glass wool  into a 500-ml K-D flask equipped with a 100-ml
       ampule.  Rinse the separatory funnel and column with three
       10-ml portions of hexane.

       Concentrate the extracts to 6 to 10 ml  in the K-D evaporator
       'in a hot water bath.  Concentrate the extract to 1 ml using
       the nitrogen blowdown technique.  Transfer the extract to a
       GC vial and label.  The extract is ready for analysis.

4.1.7  Gas Chromatography/Electron Capture Analysis of Pesticide
       Extracts

       \  1:50  iilutlon will provide adeauate detection limits for
       most samples.  Calculate the concentration of sample constitu-
       ents as indicated ii Subsection K.

                               III-212

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5.1  Sampling and Analysis of Air for Polychlorinated  Biphenyls
          Analytical  Procedure:   available
          Sample Preparation:  available

     5.1.1  Reference

            Lewis, R. G., "Procedures for Sampling  and Analysis  of
            Polychlorinated Biphenyls in the Vicinities of  Hazardous
            Waste Disposal Sites," Advanced Analysis Techniques  Branch,
            Environmental Monitoring Systems Laboratory,  Research
            Triangle  Park, North Carolina.  14  p. March 16,  1982.3

     5.1.2  Method Summary

            Polyurethane foam plugs from air samplers  are Soxhlet-extracted
            with a mixed solvent consisting of  5 percent  ether in hexane.
            The extract is reduced in volume and passed through  an  alumina
            cleanup column.  PCBs in the extract are quantified  using
            GC/ECD.  Analyte confirmation is conducted by GC/MS  analysis of
            composited or selected samples.

     5.1.3  Applicability

            Several different parameters involved in both the sampling
            and analytical steps of this method collectively determine
            tne sensitivity with which each PCS isomer is detected.  As
            the volume of air sample collected  is increased, the
            sensitivity increases proportionately within  limits  set by:
            (.a) *,!".e ~-?t3nticn f*5).
            Sample recoveries for individual  PCB mixtures generally fall
            within the range of 90 to 110% but  recoveries ranging from 75
            to 115% are considered acceptable.  More volatile components
            give lower recoveries such as 40 to 60% to the early eluted
            components of Aroclor 1242.   Overall recoveries  for  Aroclors
            1242, 1254 and 1260 have been found to  be  96%, 95% and  109%,
            respectively.

     5.1.5  Sample Extraction -

            After sampling, the foam plug in the glass cartridge must be
            wrapped ^n hexane-rinsed aluminum foil  until  analysis.
                                    III-213

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       (a)  Remove the foam plug with forceps and place  into
           Soxhlet extractors.

       (b)  Extract with an appropriate volume of 5  percent
           ether in hexane for  50 to 100  cycles  in  the  extractor.

       (c)  Remove the boiling flask to a  rotary  evaporator and
           reduce the solvent volume to approximately 5 ml.
           (CAUTION:  Do not let sample go dry.)

       (d)  Transfer the concentrate to a  graduated  centrifuge tube
           with rinsing and adjust the volume to exactly  10 ml.

       Reduce the volume in the centrifuge tube  to  just below 1 ml
       by careful evaporation under a gentle stream of  nitrogen at
       room temperature.

5.1.6  Alumina Cleanup

       Place a small plug of pre-extracted glass wool in  the
       Chromaflex column and wash with 10 ml  of  hexane, which is
       then discarded.

       Pack the column with 10  cm of Woelm activity grade IV
       alumina.

       Transfer the sample from the centrifuge tube to  the top of
       the column; rinse the tube three times with  successive 1-ml
       *or*::ons of ^-Hexane. adding and eluting  >ach rinse into the
       column.     ~

       Add 15 ml of n-hexane and elute the column,  collecting  the
       eluate in a 15~-ml centrifuge tube.

       Adjust the final volume  of the eluate by  nitrogen  evaporation
       to 10 ml for gas chromatographic analysis.

5.1.7  GC Analysis

       Determine PCBs on 180-cm x 4-mrn I.D. glass columns packed
       with 1.5 percent OV-17/1.95 percent OV-210 or 4  percent
       SE-30/6 percent OV-210.   The nitrogen flow should  be 65 to
       85 ml/minute; column temperature,  200C.

       Inject 5 ul, or other appropriate  volume, of the sample
       extract or cleanup column eluate into the gas chromatograph.

       Record chromatograms and measure retention times relative to
       p,p'-DDE or other suitable reference standard.
                               III-214

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            Compare the retention time  of each major  GC  peak  against
            those of the corresponding  primary Arochlor  standard.

            Quantify PCB mixtures by comparison  of  the total  heights  or
            areas of GC peaks with the  corresponding  peaks  in the
            best-matching standard.   Use Aroclor 1242 for early-eluting
            and either Aroclor 1254  or  Aroclor 1260,  as  appropriate,  for
            late-el uting PCBs.


K.   CALCULATIONS

1.   The concentration of pesticides and PCBs  in a  sample 1s  determined by
     calculating peak areas for a sample extract, either by disk integrator,
     planimeter, triangulation, or digital  Integrators,  and comparing the area
     to a standard calibration curve.   The standard curve 1s  a  plot of area
     response produced by known quantities of  the Individual  pesticides or
     PCBs under identical analytical  conditions  versus amount or concentration
     of the compound.  The calibration  curve should be prepared daily.
     Pesticide and PCB concentrations in the sample extracts may also be
     determined by direct comparison to a single standard when  the injection
     volume and peak area are very close to that of the  sample.

2.   The concentration of individual  pesticides  and/or PCBs in  the original
     sample matrix is calculated using  tne appropriate equation identified
     below:

     2.1  If the external standard calibration procedure is used, calculate
          the amount of material injected from the  peak  response using the
          calibration curve or calibration factor in  3ubsecticn H.C 2.  "^e
          concentration in the sample can be calculated  from  Equation ?:

                                     (A) (Vt)
             Concentration, yg/1  =  -                            Eq. 2
                                     (V) (V)
          where:

                  A   =  Amount of material  Injected,  in  nanograms
                  V-j  *  Volume of extract Injected  (ul)
                  Vt  *  Volume of total  extract (ul)
                  Vs  =  Volume of water  extracted (ml).

     2.2  If the internal standard calibration procedure  was  used, calculate
          the concentration in the sample using the  response  factor  (RF)
          determined in Subsection H.3.2  and Equation  3.

                                            (As (Is)
                    Concentration, ug/1   - -               Eq. 3
                                    III-215

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          where:

                  As   *   Response  for  the  parameter  to be measured
                 A-js   =   Response  for  the  internal standard
                  Is   =   Amount  of Internal  standard added to each extract
                         (ug)
                  Ys   =   Volume  of water extracted,  in liters.


          When it is  apparent  that two or  more PCS (Aroclor) mixtures  are
          present, the Webb  and  McCall  procedure^ may be used to identify
          and quantify the Aroclors.

     2.3  The chlorinated hydrocarbon  pesticide  or PCB concentration of
          sediment can be calculated as:

                                                         (A) (B) (C)
          Chlorinated hydrocarbons ug/kg (wet weight)
          Chlorinated hydrocarbons  ug/kg  (dry  weight)
                                                          (E)  (F)  (G)

                                                            (A)  (B)  (C)
                                                          (E)  (F)  (G)  (K)

          where:

                  A  =  nanograms  standard  injected  into GC
                  B  =  peak  height  (or  area)  produced  by -sample Injection
                  C  =  rinal  volume of  sample extract, ml
                  E  =  peak  height  (or  area)  produced  by  standard  injection
                        A
                  F  =  wet weight of sediment sample initially extracted, g
                  G    volume of  extract injected to produce  B, ml
                 IS  =  sediment percent solids as a decimal fraction.

2.   For multicomponent mixtures (chlordane,  toxaphene  and PCBs), match
     retention times of peaks in the standards with  peaks  in the sample.
     Quantitate every identifiable peak  unless interferences with  individual
     peaks persist after cleanup.  Add peak height or peak area of  each
     identified peak in the chromatogram.   Calculate as total  response in the
     sample versus total response  in the standard.

3.   Report results in micrograms  per liter without  correction for  recovery
     data.  When duplicate and spiked samples are analyzed, report  all data
     obtained with the sample results.

4.   For samples processed as part of a  set where the laboratory spiked  sample
     recovery falls outside of the control  limits in Subsection G.3.,  data for
     the affected parameters  must  be labeled as suspect.
                                    TII-216

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If a sample is reported as negative for a given pesticide,  also report
the minimum method detectable limit for that compound.   Report sample
response of less than three times the detector noise level  (N) as neg-
ative.   For sample response at three times the detector noise  level, list
the result as presumptive.  Quantify responses of greater than 3 N if
possible.  In cases of questionable identification,  qualify the reported
result to ensure that subsequent misinterpretation will not occur.
                               III-217

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                                  REFERENCES


1.   U.S. Environmental  Protection Agency.  "Methods 330.4  (Titrimetric,
     DPD-FAS)  and 330.5  (Spectrophotometric,  DPD)  for Chlorine, Total
     Residual."  Methods for Chemical  Analysis  of  Water and Wastes.  EPA
     600/4-79-020, U.S.  Environmental  Protection Agency, Environmental
     Monitoring and Support Laboratory,  Cincinnati, Ohio.   (1979).

2.   U.S. Environmental  Protection Agency.  "Preservation and Maximum Holding
     Time for  the Priority Pollutants."   U.S. Environmental Protection Agency,
     Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.  (In
     Preparation).

3.   Lewis, R. G.  "Procedures for Sampling and Analysis of Polychlorinated
     Biphenyls in the Vicinities  of Hazardous Waste Disposal Sites."  Advanced
     Analysis  Techniques Branch,  Environmental  Monitoring Systems Laboratory,
     Research  Triangle Park, North Carolina.  14 p.  March  16, 1982.

4.   U.S. Environmental  Protection Agency.  "The Analysis of Polychlorinated
     Biphenyls in Transformer Fluid and  Waste Oil."  U.S. Environmental
     Protection Agency,  Office of Research and  Development, Environmental
     Monitoring and Support Laboratory,  Cincinnati, Ohio.   (1981).

5.   American  Society for Testing and  Materials.   "Standard Practice for
     Preparation of Sample Containers  and for Preservation."  ASTM Annual Book
     of Standards, Part 31, D3694.  American  Society for Testing and
     Materials, Philadelphia, Pennsylvania.   679 p. .(1980).

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

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

8.   U.S. Environmental  Protection Agency.  "Extraction and Analysis of
     Priority  Pollutants in Biological Tissue." U.S. Environmental Protection
     Agency, SAA Division, Region IV,  Laboratory Services Branch, Athens,
     Georgia.   Method PPb 10/80.   7 p. (1980).

9.   U.S. Environmental  Protection Agency.  "Analytically Determined Method
     Detection Limits for Priority Pollutant  Methodology as Method Performance
     Criteria."  U.S. Environmental Protection  Agency, Environmental
     Monitoring and Support Laboratory,  Cincinnati, Ohio.   (In Preparation).

10.  American  Society for Testing and  Materials.   "Tentative Method of Test
     for Organochlorine in Water."  Method D  3086-72T, D-19 Water, pp. 519-534,
     {1980).  ^me^can Society *or Testing and  Materials, Philadelohia,
     Pennsylvania.                    	


                                    III-218

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

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

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

14.  Mills, P. A.  "Variation of Florisil Activity:  Simple Method for
     Measuring Absorbent Capacity and its use in Standardizing Florisil
     Columns," Journal of the Association of Official  Analytical Chemists,
     51:29 (196W-;

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

16.  U.S. Environmental Protection Agency.  "Method for Preparation of Medium
     Concentration Hazardous Vaste Samoles."  U.S. Environmental Protection
     Agency, Region IV, Athens, Georgia. (1981).

17.  U.S. Environmental Protection Agency.  "Guidelines Establishing Test
     Procedures for the Analysis of Pollutants; Proposed Regulations."
     Federal Regirrtar ^ No, ?33:69*64-69575.  Decembers, 1979.	

18.  Eichelberger, J. W., L. E. Harris and W. L. Budde.  "Analysis of the
     Polychlorinated Biphenyl Problem.  Application of Gas Chromatography-
     Mass Spectrometry with Computer Controlled Repetitive Data Acquisition
     from Selected Specific Ions." Anal. Chem 46:227-232(1974).

19.  White, L. D., D. G. Taylor, P. A. Mauer, and R. E. Kupel.  "Convenient
     Optimized Method for the Analysis of Selected Solvent Vapors in the
     Industrial Atmosphere." AIHA Journal 3jh pp. 225-232 (1970).

20.  U.S. Environmental Protection Agency.  "Organochlorine Pesticides and
     PCB's - Method 608."  Federal Register 44 No. 233:69501-69509.
     December 3, 1979.

21.  "Development and Application of Test Procedures for Specific Organic
     Toxic Substances in Wastewaters.  Category 10-Pesticides and PCB's."
     Report for EPA Contract 68-03-2606 (In Preparation).

22.  "Manual of Analytical Methods for the Analysis of Pesticides in Human and
     Environmental  Samples."  U.S. EPA, Health Effects Research Laboratory,
     Research Wangle ''ark. North Carolina, EPA Report 600/8-80-038, Section
     11, B, p. 6.  (1980).
                                    III-219

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23.  "Official  Methods of Analysis  of  the Association of Official Agricultural
     Chemists," Association of  Official  Agricultural Chemists, Washington,
     D. C. 20044, 10th Edition,  1965,  p. 382.

24.  Smith, D.  and J.  W.  Eichelberger.   JWPFA, Vol. 37, 1965, p. 77.

25.  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.

26.  Thompson,  J. F.  "Analysis of  Pesticide Residues in Human and Environ-
     mental Samples."   U.S. Environmental Protection Agency, Pesticide and
     Toxic Substances Effects Laboratory.   Research Triangle Park, North
     Carolina.   (1974).

27.  Environment Canada.   "Analytical  Methods Manual."   Inland Waters
     Directorate, Water Quality Branch.  Ottawa,  Ontario, Canada (1974).
                                    III-220

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                                   SECTION 5

          METHODS FOR THE DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES


A.   SCOPE

     The analytical procedures provided in Subsection H of this Section cover
the determination of organophosphorus (OP) pesticides in pesticide formulations
(hazardous waste) (Subsection H.I.), water (Subsection H.2.),  soil (Subsection
H.3.), biological tissues (Subsection H.4.), vegetable and fruit tissues (Sub-
section H.4.) and air (Subsection H.5.).  All of the determinations are based
on gas chromatography (GC).   Several do not require extensive  cleanup because
of the selectivity of the flame photometric detector.  The analytical method
for human tissues determines alkyl  phosphate metabolites rather than the parent
pesticides, which are usually not present in these samples in  significant
amounts.

B.   SAMPLE HANDLING AND STORAGE

1.   Sampling

     See Section 4, Subsection B of this Chapter for general  information c:i
     pesticide sampling that is applicable to OP compounds. General  and spec-
     ific sampling procedures have been discussed in detail for human and
     environmental samples in Chapter 8 of the EPA Quality Control Manual! and
     for food samples in Sections 140-142 of the U.S. Food and Drug Administra-
     tion Pesticide Analytical Manual (FDA PAM).2

     Analytical results can  be no more valid than the samples  or sampling scheme
     used.  Samples should truly represent the component being examined and
     should be compatible with the goals of the analysis.   The location of sam-
     pling sites, the sampling technique, the number of samples, and the frequency
     and duration of sampling should be such that the analytical  results can be
     evaluated in a statistically satisfactory manner.  The size and number  of
     samples should enable replication of analyses and confirmation of any
     residues found.3

     OP pesticides are collected from air using high volume air samplers.
     Collection media have included polyurethane foam-granular sorbent combina-
     tion filter pads, glass fiber-Poropak composite pads, and polyurethane
     foam plugs.  The use of polyurethane cartridges for collection of PCBs  from
     air? *- iiscusse-i *n Section 4, Subsection B of this Chapter.  Section  8A
     of the EPA PAM4 contains methods and equipment for anwnent 
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     Most recovery studies to date have been made for organochlorines and PCBs
     present in air.

     Acceptable methods for sampling liquids, emulsions, suspensions, wettable
     powders, and dusts are described in detail  in Official  Methods  of Analysis
     of the Association of Official  Analytical  Chemists, 13th Edition (1980).

2.   Sample Handling

     Water samples containing high concentrations of suspended matter or sedi-
     ment may require special  handling such as  decantation,  filtration,  or
     centrifugation, after which the water and  sediment can  be analyzed sep-
     arately, if so desired.

     Plant or animal samples,  either frozen or  unfrozen, are frequently ground
     with sodium sulfate to bind the tissue moisture before  extraction.   Appro-
     priate manuals or publications  should be consulted for  specific details
     for preparing plant or animal  tissues.1.2,4

     It is generally undesirable to  dry soil  samples beyond  the  air-dry  state
     before extraction because of possible decomposition or  irreversible adsorp-
     tion of pesticide residues on  soil  surfaces.   However,  soil  samples should
     not be too wet because of particle aggregation, which can result in
     extraction aiffaculties.   ~hey  should be in a damn,  friable  condition
     before extraction, with  a moisture content  of about 5 to 15  percent,for
     sandy soils and 10 to 30  percent for loams.   Satisfactory extraction  from
     muck soils can be made with moisture levels as high as  85 percent.  Mois-
     ture content should be determined on a separate portion  of  soil  so  that
     results -ran be reported  on a dry-weight basis.

     Sediment samples should  be in the same condition as  soils prior to
     extraction.  Excess water should be removed by decantation,  filtration,
     or centrifugation, and air-drying,  if necessary,  until  the  sediment is
     friable.

3.   Storage

     3.1  Temperature

          Samples other than  water should ordinarily be stored in a  freezer,
          preferably below 0*C.   Even then,  physical  and  chemical changes  may
          occur 1n either the  sample or in the  residues sought.   Extended
          storage in freezers  can cause moisture to migrate  to the surface of
          the sample and then  to the freezer coils, slowly desiccating the
          sample.  This effect may be of importance if water  content affects
          the subsequent analysis and can affect the calculated  residue  concen-
          tration.  Water samples should be stored slightly  above freezing to
          avoid rupture of the containers as a  result of freezing.

     3.2  Tfme

          Samples should be analyzed as quickly  as possible  after collection,

                                    III-222

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          before physical  and chemical  changes  occur.   If  prolonged  storage  is
          required, it may be preferable to extract  the samples,  and store the
          extracts at a low temperature.

          When feasible, studies of the stability of residues  in  samples  or  ex-
          tracts,  with time and temperature of  storage, should be carried out
          with representative pesticides and substrates.   When there is  doubt
          about the stability of residues in storage,  spiked control  samples or
          extracts should be held under the same conditions as the samples or
          extracts.  OP pesticides degrade more quickly than organochlorine
          pesticides and should be analyzed within 4 days  of sampling.

     3.2  Light

          Light degrades many pesticides; therefore  it is  advisable  to protect
          the samples and any solutions or extracts  from needless exposure.  In
          the case of air sampling, the collection apparatus should  be shielded
          from light during sample collection.

     3.3  Containers

          Avoid plastic containers, or plastic-lined caps, unless made of Teflon
          or other inert plastic which does not interfere  with the analytical
          method.   Aluminum foi1 liners for caos may tear; hence  it  is preferable
          to use Teflon sheeting or liners.  If polyethylene or other plastic
          storage containers are used,  tests should  be made to determine  whether
          there is any analytical  interference  due to  the  containers being used;
          laboratories fre-quently have experienced such interferences.

          If cans are used, they should first be cneckea to demtnctrats  ths  ab-
          sence of materials, such as oil films, lacquers, or  rosin  from  sol-
          dered joints, that could interfere with analyses.

          Glass containers should be used for water  or liquid  samples and. should
          be thoroughly cleaned and rinsed with one  or more suitable solvents
          such as acetone, Isopropanol, or hexane, and dried before  use.  Pesti-
          cides can migrate to the walls of a container and be adsorbed;  hence
          even a glass container, after the water sample is poured out,  should
          be rinsed with solvent if the extraction is  not  made in the container
          Itself.

          In summary, any type of container or  wrapping material  should  be checked
          before use for possible interferences in the analytical  method  and at
          the limit of detection employed in the analysis. Sample handling  and
          storage information is summarized in  Figure  1.

C.   INTERFERENCES

     Solvents and reagents should be free of substances that interfere with
analysis sr -leqrade the samole.  To obtain low  background  levels  and avoid
spurious peaks arising from solvent impurities, it is  usually  necessary  to
employ ipecially purified or distilled-in-glass solvents.  Solvents  should be

                                    111-223

-------
I
ro
ro
-p.
>*rw


IMitCI


	
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~-




C 	 |

Pm

,..,

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1M

l><

km

*n*



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fra*tt*w*


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E Kit Ml
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               Figure 1.   Handling  and  sample  sto>age infomu,tion for organophosphorus compounds.

-------
checked by using them in the amounts and manner described  in  the method  without
a sample being present, concentrating them as  described  in the  method, and  then
testing with the particular detector prescribed.   Similar  precautions must  be
taken with reagents to make sure that they will  not  cause  undue interference.
Cleanup procedures described in the various methods  will usually eliminate
Interferences from the samples.  Use of the phosphorus-selective GC  detector
such as the flame photometric or alkali flame  ionization detector should reduce
problems caused by interferences and reduce the amount of  extract cleanup
required.

D.   SAFETY

     Precaution when using diazopentane reagent:   Because  of  the demonstrated
carcinogenicity and skin irritating characteristics, do  not allow the nitroso-
gyanidine or the diazoalkane to come in contact with the skin.   Wear dispos-
able vinyl gloves and safety goggles while handling.Avoid breathing vapors.
Working inside a glove box, if possible, is strongly recommended. Do not use
ground-glass-stoppered bottles or bottles with visible interior etching. Avoid
strong light.

E.   APPARATUS

1.   Gas chromatograph aquipped with thermal conductivity  or  flame ionization
     detector and recorder for formulation analysis. The  roil owing  parameters
     are typical:  glass column, 4 ft (1.2 meters) x 3-4 mm I.O., 3-10 percent
     liquid phase, coated on 80/100 mesh acid-washed  and  silanized diatomite
     (e.g., Gas Chrom Q, Anabrom ABS), injector temperature 190C, helium
     (carrier qas^ flow rate 80 ml/min, detector temperature  175C.

2.   Erlenmeyer flask, 50 ml.

3.   Bottle, 4 oz, with Vinylite-lined screw top.

4.   Chromatographic-column, glass, 400 mm x 20 mm.

5.   Volumetric flask, 50ml.

6.   Blender, high speed.

7.   Gas chromatograph fitted with a flame photometric detector with 526 nm
     phosphorus filter (thermionic detection may be  substituted for  the  FPD)
     for residue analysis.  GC columns, borosilicate glass, 1.8 m x  4 mm I.D.,
     packed with 1.5% OV-17/1.95% OV-210, and  5% OV-210, both coated on  Gas-
     Chrom Q, 80/100 mesh.  The following are  typical operating parameters
     used:  column, 165 to 200C; nitrogen carrier gas flow,  70 to 80 ml/min;
     hydrogen flow, 50 to 100 ml/min; oxygen content of  air flow, 0.2 to 0.4
     of the hydrogen flow; total air flow, 1.5 times the hydrogen flow;
     injector block, 225eC; FPD detector 175 to 225C (thermionic detector,
     250Ch transfer line, 235C.  A switching valve between the column and
     FPD allows solvent venting and prevents flame oiowout when Injectors  ire
     made.  Other columns and conditions that  have been  used  for determination
     of OP pesticide residues with the FPD detector  include:  a) 1.2 m x

                                    111-225

-------
     2 mm I.D. 2% DECS column, 180C,  60 ml/min helium carrier gas  flow;  b)
     1.2 m x 2 mm I.D. 2% OV-101 column, 200'C, 30  to 60 ml/miri He  flow;  and  c)
     76.2 x 2 mm I.D., 4% SE-30/6.5% OV-210 column,  200eC,  60  ml/min  He  flow.

8.   Chromaflex columns, size 22,  7  mm I.D. x  200 mm,  Kontes 420100 or
     equivalent.

9.   Chromaflex column, size 241,  22 mm I.D. x 300  mm, Kontes  420530  or
     equivalent.

10.  Rinco evaporator, rotating, such  as Scientific  Glass Apparatus Co.  E-5500,
     E-5500-1 or equivalent, with  appropriate  stand.

11.  Variac or comparable voltage  control  regulator.

12.  Water bath for operation at 35'C.

13.  Vacuum source of 125 mm Hg, optimally.

14.  Kuderna-Danish evaporators, 250 ml, 500 ml.

15.  Centrifuge tubes, conical,  15 ml,  graduated, Corning No.  8082  with  Teflon-
     lined plastic screw caps, thread  finish 415-15,  Corning 9998 or  equivalent.

16.  Tubes, culture, screw caps  with Teflon liner,  16  x 125 mm,  Corning  9326  or
     equivalent.

17.  Evaporative concentrator tubes, 10 ml, graduated from 0.1  to 10.0 ml, size
     1D25 iit.h outer joint T 19/22.  
-------
26.  IEC centrifuge, Model EXD, explosion proof, or equivalent, suitable for
     operation at 2,000 rpm.
27.  Culture tubes, glass, 16 ran x 150 tart.
28.  Pipet, 0.1 ml" capacity, graduated in 0.01 ml units.
29.  Pipets, assorted capacities, to be used in combination with appropriate
     volumetric flasks for preparation of standard solutions.
30.  Bottles, reagent, narrow mouth, 1 oz. capacity, with polyseal  screw caps
     (A. H. Thomas 2203-C bottles and 2849-E caps or equivalent).
31.  Nitrogen evaporator with water bath maintained at 40C (Organomation
     Associates N-Evap or equivalent).
32.  Exhaust hood with minimum draft of 150 linear feet per minute.
33.  Extractor, Soxhlet, 1000, 500, and 250 ml.
34.  Separatory funnel, 500 ml, and 1 1, with Teflon stopcocks.
35.  Suchner filtration apparatus.
36.  Chromatoflo chromatography column, 25 cm x 9 mm I.D. ,  Pierce  No.  29020, or
     equivalent, equipped with a Teflon mesh support membrane,  Pierce  No.  29268
     or equivalent, lower end plate, adapter, and 500-ml solvent reservoir
     ;Ace '!o. 5321-10 ^
F.   REAGENTS
     (Solvents should be distilled-in-glass, pesticide grade.)
1.   Acetone.
2.   Acetonitrile.
3.   Benzene.
4.   Contaminant- free water.  To 1500 ml  of distilled water 1n  a 2-1  separatory
     funnel add 100 ml methylene chloride, stopper,  and shake vigorously  for  2  
     minutes.  Allow the phases to separate, discard the solvent layer, and
     repeat the extraction with another 100-ml  portion of methylene chloride.
     Drain the double-extracted water Into a glass stoppered bottle for storage,
     withdrawing 500 ml  to serve as a reagent blank  with each set of  samples.
5.   Diethyl ether, containing 2 percent ethanol .
6.   Hexane.
7.   Hydrochloric acid,  reagent grade, approximately 37 percent.
8.   Isopropanol.
                                    111-227

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9.   Keeper solution, 1 percent paraffin oil,  USP grade,  in hexane.
10.  Keeper solution, polyethylene glycol-acetone (5/95 v/v).
11.  Methanol.
12.  Methylene chloride.
13.  Anion exchange resin, Amberlite CG-400 AR or equivalent,  100/200 mesh
     (available from Mallinckrodt 3345 and other vendors),  in  the chloride
     form, or BioRad AG1 x 8 or equivalent, 100/200 mesh  (available  from
     BioRad Laboratories, Richmond, California and other  vendors in  the
     chloride form).
14.  Carborundum chips, fine.  Purify by Soxhlet extracting with methylene
     chloride for ca. 60 discharge cycles.
15.  Diazopentane reagent - Preparation:
     a.   Dissolve 2.3 g KOH in 2.3 ml distilled water in a 125 ml Erlenmeyer
          flask.  When solution is complete, cool in a freezer for 30 minutes.
     b.   Add 25 ml cold diethyl ether, cover flask mouth with foil, and
          cool  in a -13C freezer for 15 -ninutss.
     c.   In a  very high draft hood, add 2.1 g N-amyl-fT-nitrp-N-nitroso-
          guanidine to the flask in small  portions over a perio'd" of  a few
          minutes, swirling the flask vigorously after each addition.
     d.   Decant the ether layer into a 1-oz.  reagent bottle fitted  with a
          Teflon-lined screw cap.  This may be stored at -20C for periods up
          to a  week.
16.  Glass wool, preextracted with methanol, acetone, and methylene  chloride to
     remove any contaminants.
17.  N-amyl-N'-nitro-N-nitrosoguanidine (available from Aldrich Chemical Co.).
18.  Pesticide  reference standards, analytical grade.
19.  Petroleum  ether.
20.  Potassium  hydroxide, pellets, AR grade.
21.  Silica gel, Woelm, activity grade  I  (available from ICN Pharmaceuticals,
     Inc.  and other vendors), activated at  130C for 48 hours and stored 1n a
     desiccator.
22.  Silica gel, Woelm, deactivated.  Activate for 48 hours at 175*C before
     usa.  "rppare  *inal deactivated material  by adding 1.0 ml of water to
     5.0  g silica  gel  1n a vial with a  Teflon-lined screw cap.  Cap  cigntiy a

                                    III-228

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     mix on the Roto-Rack for 2 hours at ca 50 rpm.  Discard deactivated silica
     gel after 5 days.

     NOTE:  It 1s recommended that the amount of silica gel activated at 175C
     be restricted to the quantity needed for Immediate deactivation.

23.  Sodium sulfate, granular, anhydrous.  Purify by Soxhlet extracting with
     methylene chloride for car 60 discharge cycles.

G.   CALIBRATION

     Calibration or linearity plots are obtained on varying ratios of the
pesticide primary standard and the internal standard.  The mixtures (four are
sufficient) are prepared so that they contain a constant amount of internal
standard and the range of the pesticide concentration is varied so that it
covers one-half to two times that which is to be used for the sample analysis.
To be more explicit, the linearity range should cover 10 to 50 ug of pesticide
per injected volume and the concentration of pesticide in a sample solution
prepared for analysis should contain 5 to 10 mg pesticide per milliliter of
solution.  Linearity curves are plots of peak area ratio versus concentration
in micrograms per volume injected.  The analysis of formulation samples is per-
formed by the addition of the same amount of internal standard to a specified
volume of sample solution (obtained after dilution or extraction of the formu-
lation with a suitable solvent) and the concentration determined either by
comparing the peak height to a standard curve or by comparing the peak height
ratio of the analyte to the internal standard in the sample to the analyte/
Sterna! standard ratio in the calibration standard.

     The use of a calibration curve for quantitation is not very satisfactory
when high precision is required.  Calibration curves having the identical
slope cannot be reproduced exactly from day to day and often not even within
an 8-hour work day.  However, high precision and accuracy can be achieved by
using a pesticide standard solution and replication of solution injections for
both the standard and sample solutions according to the injection sequence
described in the following example:

     The formulation solution or extract is diluted with the appropriate sol-
vent containing a known concentration of the internal standard to give a 1-
percent solution (10 mg/ml or 10 pg/ul) of the pesticide.  Simultaneously, two
solutions are prepared containing 5 mg/ml  and 10 mg/ml  of the pesticide
standard and the exact same amount of Internal  standard.

     Identify these as standard solution A and standard solution B, respec-
tively.  Set the gas chromatographic conditions as predetermined for the
specific pesticide, and using standard solution B, determine the appropriate
attenuation setting and injection portion to yield a minimum peak height for
the internal standard of 50 percent full-scale recorder deflection.  Inject
aoorooriate portions of standard solution B until  a consistent response is
obtained (three consecutive injections giv'ng -esocnse -.itios Mithin ? oercent
of each other).  The response ratio for each injection is calculated as
follows:


                                    III-229

-------
                            peak height (area) of pesticide
     response ratio  =  		
                        peak height (area) of internal  standard
     The average of the last three response ratios is used to calculate the
standard response factor in the formula below and the pesticide content of the
first three samples of a series.  Three portions of standard solution A are
injected and the response ratios are calculated and also must be within 2
percent.  The response factor for each standard is calculated by the following
formulas:


                               concentration (mg/ml) x purity of standard
response factor standard A  =  		
                                    average response ratio standard A


                               concentration (mg/ml) x purity of standard
response factor standard B  =
                                    average response ratio standard B
     The response factors should be within 2 percent of each other.  If they
are not, new standard solutions should be prepared and the response factor
again determined.  Continued discrepancies of the standard response factors
indicate a lack of linearity, and this condition must be resolved before
meaningful and valid analysis of samples can be conducted.

     ""^o portions ;f each sarcole^extract ire then injected.  These response' "
ratios should also be within 2 percent of eacn otner.  If cnis precision
limit is not met, two more portions of the solution are -injected.  Failure
to meet the 2 percent specification with the second pair of injections
indicates instrumental difficulties which must be corrected before proceeding
with the analysis.

     After every six samples, standard solution B is reinjected in duplicate.
The average of these response ratios should be within 2 percent of the
preceding average response ratio obtained with this standard solution.
Failure to meet these specifications indicates instrument drift which must
either be corrected or compensated for by more frequent measurement of
standard solution B than specified above.  The frequency of standard measure-
ment should be adjusted depending on the degree of drift observed.
                                    III-230

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H.   ANALYTICAL PROCEDURES

1.   Determination of Organophosphorus Pesticides in Hazardous Wastes
     (Pesticide Formulations)
          Analytical Procedure:  available
          Sample Preparation:  available

     1.1  Reference

          Zweig, G. and J. Sherma, "Gas Chromatographic Analysis," Vol.  6 of
          "Analytical Methods for Pesticides and Plant Growth Regulators."
          Ch. 4, p. 107, Academic Press, New York (1972).6

     1.2  Method Summary

          The sample is dissolved in or extracted with an appropriate solvent,
          internal standard is added,  and the analyte is determined by gas
          chromatography with thermal  conductivity or flame ionization
          detection.

     1.3  Applicability

          The method is ami 'cable to serosols,  solid samples, liquid formula-
          tions, and liquid solutions containing OP pesticides.

     1.4  Precision and Accuracy

          Accuracy ind sreclsion (relative standard deviation) values within
          1 to 2 percent are possible using internal  standardisation arvd j
          reference standard of known purity.

     1.5  Sample Preparation

          Typical examples of product formulations include aerosols, baits,
          dust concentrates, wettable powders,  granules, emulsifiable
          concentrates, ultralow-volume concentrates,  technical  materials,
          water-miscible liquids, water-soluble  concentrates, oil  solutions,
          emulsions, and suspensions.   Some general procedures for sample
          preparation prior to gas chromatography, which include separation  of
          the pesticide from the formulation ingredients or merely a dilution
          of the sample with a suitable solvent, are discussed in this section.
          These procedures are intended to be used only as guides and may not
          be applicable in all cases.

          1.5.1  Aerosols

                 Weigh the container and contents, cool in a dry-ice chamber or
                 other satisfactory cooler such  as a refrigerator freezing
                 compartment, for approximately  0.5 hour.   Punch a very small
                 hole 1n the top of the container, and allow ;  to s-;and ;n  i
                 hood at room temperature while  the propellant gas escapes.


                                    HI-231

-------
       After the propel!ant has volatilized,  carefully cut off the
       top of the container and gently warm near a  steam bath  to
       boil off the remainder of the volatile solvents.  Finally,
       warm on the steam bath until  all  the solvent is expelled.
       Cool to room temperature, and weigh the container with  the
       nonvolatile material.  Transfer the nonvolatile material  to
       a suitable container and retain for analysis.   Rinse the
       aerosol container with ether, dry,  and weigh.   The difference
       between these weights represents the weight  of nonvolatile
       material in the aerosol.  Weigh the top of the container
       which had previously been removed,  add this  weight to that
       of the empty and dried container,  and  subtract their combined
       weight from the original gross weight  of the aerosol to
       obtain the net content.  Calculate  the percentage of nonvol-
       atile residue obtained from the aerosol.

       Accurately weigh a portion of the nonvolatile,  well  mixed
       residual concentrate equivalent to 0.500 gm  of pesticide into
       a 50-ml flask (final concentration  to  be 10  mg/ml),  add the
       internal standard to the flask, and dilute to  volume with
       solvent.

1.5.2  Wettable Powders, Dust Concentrates, Baits,  and Granules

       The clays and carriers used in these formulation types  are
       hignly sorptive and as such have a tendency  to bind or  retain
       the' pesticides so that their complete  removal  may be a  prob-
       lem.  Although a pesticide may be equally soluble in a
       numner sf solvents, it does not mean that ?H  the solvents
       will extract the pesticide quantitatively from"the highly
       sorptive carriers.  The Inability to efficiently extract the
       pesticide from the carrier with any one solvent is more
       evident on aged formulation samples.  A solvent that will
       penetrate or wet the carrier will  generally  be the most
       efficient for this purpose.  For example, acetonitrile  or
       methanol are used to extract organophosphate pesticides from
       fresh and aged formulations more efficiently than hexane or
       Isooctane, although this group of compounds  shows good
       solubility in all of the solvents.

       Extraction of pesticides from carriers is generally performed
       1n a number of ways.  They are all  quite simple, give compar-
       able results, and require little actual work time, and  the
       main difference lies 1n the time necessary to complete  the
       extraction.  In all cases, a sample equivalent to 2.0 gm of
       the pesticide 1s taken for analysis.  In the first method of
       extraction, the sample to be extracted 1s placed into a 4-
       ounce screw cap bottle with a Yinyllte liner,  50 ml  of  the
       appropriate solvent 1s added, the cap  Is screwed tightly,
       and *he samole 1s shaken on a mechanical shaker for 30  to
       60 minutes.  After filtration, a portion of  the filtrate
       necessary  to produce z final concentration of '.0 -ng/ml  is

                          III-232

-------
       .taken and mixed with the appropriate predetermined volume of
       internal standard.

       In the second method of extraction, the same formulation sample
       size (equivalent to 2.00 gm of pesticide)  is transferred to
       an empty 20-mm x 400-mm chromatographic column,  and the extrac-
       tion solvent is percolated through the column at a drop rate
       of 1 to 2 drops per second until  100 ml of the effluent
       is collected.  A portion of the effluent to give a final
       concentration of 10 mg/ml  is taken and mixed with the appro-
       priate predetermined volume of internal standard as described
       earlier.

       Some analysts prefer to use a Soxhlet extraction.  This re-
       quires a much longer extraction period (minimum  of 4 hours)
       and the use of heat, which may cause destruction of heat-
       sensitive compounds.  Sample size, volume  of extraction, and
       dilutions are the same as described for the chromatographic
       column method in the previous paragraph (second  method).

1.5.3  Emulsifiable Concentrates, Oil Solutions,  Ultra-low Volume
       Concentrates, Water-Miscible Liquids, and  Water-Soluble
       Concentrates.

       These types of formulations are the easiest to prepare for
       gas chromatographic analysis.  A sample size equivalent to
       0.500 gm of the pesticide is transferred to a 50-ml  volumetric
       ^Msk '-^'lal concentration to be 10 mq/ml K the  aoorooriate
       volume of internal  standard is added to tne flasx, ana cr,e   *
       sample diluted to volume with the proper solvent.  The
       solvent may be the  same as that used for solid formulations
       analysis.  However, it should be completely miscible with
       the formulation ingredients, eluted rapidly from the gas
       chromatographic column, and the instrument should return to
       a flat baseline before the peaks  to be measured  elute.
       Acetonitrile is a very good solvent for emulsifiable concen-
       trates, water concentrates, and water-miscible concentrates.
       Hexane is preferable for oil solutions.

1.5.4-  Suspensions and Emulsions

       These mixtures are  generally obtained by the dilution of
       wettable powders or emulsifiable  concentrates with water and
       contain low concentrations of the pesticide (below 1 percent)
       1n contrast to the  preceding formulations  which  generally
       have very high concentrations (5-95 percent).  A much larger
       sample size is necessary,  larger  volumes of water-immiscible
       solvents are required for the extraction,  and a  concentration
       step is necessary.   Susoensions are either filtered  or centri-
       fuged to remove the solids whicn  are then  extracted  as described
       above, and the aqueous phase is extracted  several  times with a
       water-immiscible solvent (one volume of aqueous  phase to two

-------
                 volumes of solvent).   The solvent extracts for both the aque-
                 ous and solid phase are combined, a keeper added (3 drops of
                 a 5-percent solution  of polyethylene glycol  in acetone) and
                 the solution is taken to dryness with a rotary vacuum evapo-
                 rator.  The appropriate amount of internal standard is added
                 and the solution diluted with the predetermined solvent to
                 give a concentration  of 5 to 10 mg/ml pesticide.

                 Emulsions are diluted with a small  volume of concentrated
                 salt solution (10- to 20-percent solution) and extracted with
                 two volumes of a water-immiscible solvent using gentle to
                 moderate shaking.  Concentration and final sample prepara-
                 tion are accomplished as described above for suspensions.

     1.6  Sample Analysis

          Inject the sample and standards using conditions that will  elute the
          analyte and internal standard with similar retention times, but with
          peaks that are narrow and completely resolved.  For complex samples,
          the selective flame photometric or thermionic detector might be more
          advantageous than the thermionic or flame ionization detector, which
          is generally used for formulation analysis.  Examples of internal
          standards and GC columns and temperatures for some OP pesticides are
          shown below:
                                                            Column Conditions
Pesticide
Retention
Time
(minutes)
Internal
Standard
Retention
Time
(minutes)
Temperature
Substrate (C)
Dimethoate          2.9
Oisyston            3.4
Fenitrothion        2.8
Malathion           7.0
Malathion           2.5
Methyl parathion    1.9
Methyl-parathion    2.17
Parathion           3.9
Parathion           3.17
Phorate             2.5
Phorate             2.5
Trithion            3.6
Dibutyl  sebacate     5.9
Aldrin               5.8
Dibutyl  sebacate     5.5
Dimethoate           3.4
Dibutyl  sebacate     4.5
Dieldrin             5.1
Dibutyl  sebacate     5.2
Dieldrin'            7.0
Dibutyl  sebacate     5.5
Benzyl benzoate      3.5
Lindane              3.5
Heptachlor           1.0
5% DC-550     170
10% SE-30     205
5% DC-550     165
5% OV-22      180
0.25% DC-200  160
10% SE-30     208
0.25% DC-550  160
10% SE-30     195
0.25% DC-550  160
5% DC--550     165
5% DC-550     155
10% SE-30     170
          Calculate organophosphorus concentrations as indicated in Sub-
          section L
                                    III-234

-------
Determination of Organophosphorus Pesticides in Water
     Analytical Procedure:  available
     Sample Preparation:  available

2.1  Reference

     Sherma, J. and M. Beroza, "Manual of Analytical Methods for the
     Analysis of Pesticides in Humans and Environmental  Samples,"
     EPA-600/8-80-038, Section IDA (June, 1980).*

2.2  Method Summary

     Compounds are extracted from water with methylene chloride, and
     the extract volume is reduced at low pressure and temperature in an
     evaporative concentrator.  Compounds are separated into groups on
     a column of deactivated silica gel by elution with solvents of
     increasing polarity.  Figure 2 shows that OP pesticides appear in
     Fractions II, III and IV.  These compounds are determined by GC with
     a P-selective flame photometric detector (FPD).

2.3  Applicability

     Water samples were analyzed for the thirty-eight OP pesticides listed
     in Table 1 at concentrations ranging from i.6 to 400 ppb.

2.4.  Precision and Accuracy

     Thirty-one ?.f the 38 organophosphorus compounds were recovered in
     the 80+ percent range ana six between ou ana 79 percsnt [~s?. Tab! =
     1).  Reproducible and satisfactory recoveries were not achieved for
     carbophenoxon, disulfoton, methamidophos, monocrotophos, and
     oxydemeton methyl.  Of these five compounds, excellent extraction
     efficiency was observed for carbophenoxon and disulfoton, but
     complete loss was experienced on the silica gel column.  Six  com-
     pounds were partially recovered in the 0 to 60 percent range.  Of
     the 7 OP compounds yielding total recoveries of less than 80 per-
     cent, six of these gave over 90 percent extraction recovery, but
     losses occurred during silica gel chromatography (see Table 1).

2.5  Sample Extraction and Concentration

     Transfer 500 ml of water to a 1-liter separatory funnel and add 10 g
     of anhydrous sodium sulfate and 50 ml of methylene chloride.

     Shake vigorously for 2 minutes and allow a sufficient length of
     time for complete phase separation.

     NOTE 1.  If the expected pesticide concentration is extremely low,
     i.e., under Q.(M ug/1 , it may be advisable to increase the initial
     sample size to 1000 to 2000 ml.  in this case, the volume of T.ethvlene
     chloride should be 'ncreased to 75 ml and the separatory funnel size
     to 2 or 3 1.

                               III-235

-------
                           500 ml Water
Discard <
              AQ
   xtract
with 2x50 ml
   MeCl
                         Percolate through
                         anhydrous granular
                              N32S04
                          Concentrate to
                              ^ 4 ml
                          Concentrate to
                           Pass Through
                         silica gel column
                         1 gm deactivated
                          with 20% water
                                              Add hexane

10 ml
hexane
Fraction I
16 OGC's
PCB's


15 ml
60% benzene
in hexane
Fraction II
23
16
2
OGC's
OGP's
Carbamates
(partial )


15 ml
5% CH3CN
in benzene
Fraction III
8 OGC's
12 OGP's
7 Carbamates


15 ml
25% acetone
in MeCl?
Fraction IV
7 OGP's
              Figure 2.  Scheme for water analysis.
                             III-236

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       TABLE 1.  RECOVERIES OF 38 ORGANOPHOSPHORUS COMPOUNDS FROM WATER
===============================================================================

                                               Recoveries in Percent
Compound
Azinphos methyl (Guthion)
Carbophenothion (Trithion)
Carbophenoxon
Chlorpyrifos (Dursban)
Cruf ornate (Ruelene)
DEF
D1az1non
Dlazoxon
Dichlofenthion (YC-13)
Dicrotophos (Bidrin)
Dfmethoate (Cygon)
Dioxathion (Delnav)
Disulfoton (Di-Syston)
EPN
Ethlon
Ethoprop (Prophos)
Fenltrothion (Sumithion) *
Fenthion (Baytex)
"onofoc !Dyfonats)
Leptophos (Phosvel)
Malaoxon
Ma lath ion
Methamidophos (Monitor)
Mevlnphos (Phosdrin)
Monocrotophos (Azodrin)
Maled (Dibrom)
Oxydemeton methyl
(Metasysotox R)
Paraoxon ethyl
Paraoxon methyl
Parathion ethyl
Parathion methyl
Phencapthon
Phorate (Thimet)
Phosalone (Zolone)
Phosmet (Imidan)
Phosphamidon (Dlmecron)
Ronnel
Ronnoxon
Cone.
(pob)
320
48
80
4
90
24
20
10
1.6
120
24
28
2.6
60
20
2
12
12
20
200
80
4
200
6
72
56

300
40
36
16
16
60
1.3
400
220
. 80
4
120
======
Extraction
Only
78
99
94
99
80
102
108
92
102
17
40
103
92
99
100
97
99
93
98
107
104
100
5
69
0
92

67
99
98
101
99
99
98
102
82
43
100
94
===============

Silica Ge
Elution
I II

93

87




102


72

96
94

84
76
78
91










99
93
98
56
91


96
1 Partition!
Fraction
III IV
88



58
90
104
72

15
60
17



96




50
78

32 33

45


90
93





85
43

92
:===== =================
ng only
Total
88
93
0
87
58
90
104
72
102
15
60
89
0
96
94
96
84
76
78
31
50
78
0
65
0
45

0
90
93
99
93
98
56
91
85
43
96
92
                                    III-237

-------
NOTE 2.  To avoid troublesome caking of the sodium sulfate at the
bottom of the funnel, shaking should be conducted instantly after
adding the sodium sulfate.

NOTE 3.  A reagent blank of 500 ml  of the preextracted water should
be carried through all  procedural  steps in exactly the same manner
as the sample(s).

Place a small wad of glass wool at  the bottom of a 25 x 300 mm
Chromaflex column and add a 2 inch  depth of anhydrous sodium sulfate.
Position the tip of the column over a Kuderna-Danish assembly con-
sisting of a 250 ml K-D flask attached to a 10 ml evaporative concen-
trator tube containing two or three carborundum chips and 5 to 10
drops of keeper solution.

Drain the lower layer (methylene chloride phase) from the separatory
funnel through the sodium sulfate column, taking care to avoid the
transfer of any of the aqueous phase.

Add 50 ml more of methylene chloride to the aqueous phase in the
funnel.  Stopper and repeat the 2-minute shaking, phase separation,
and draining of the organic layer through the sodium sulfate column
into the K-D flask.

NOTE 4.  It is not uncommon with highly contaminated water samples
to encounter persistent and sometimes severe emulsion problems at the
methylene chloride-water interface.  When this occurs, for example,
in the extraction of some wastewater samples containing high surfac-
tant concantrations, "'t is inadvisable to oass the methylene chloride
phases through the sodium sulfate because the aqueous emulsion tends
to clog the column and make filtration difficult.  A good way to cope
with an emulsion is to pack a filter tube (A. H. Thomas 4797-N15 or
equivalent) with a 25 mm thick pre-washed glass wool pad and pass the
extract containing the emulsion through this filter into a 400 ml
beaker, applying air pressure if necessary.  If the emulsion persists
on the second methylene chloride extraction, this treatment is re-
peated.  The glass wool pad is then rinsed with 25 ml of methylene
chloride, collecting the extract and the washing on the surface of
the filtrate.  Repeat the process with a second glass wool filter.

Connect the K-D flask to the rotary evaporator and incline the
assembly to an angle approximately 20 from the vertical, with the
concentrator tube about half immersed in a water bath previously
adjusted to 35C.  Turn on the rotator, adjusting the speed to a slow
spin.  Switch off the bath heat and apply vacuum to the evaporator
at a pressure of ca. 125 mm of Hg.

NOTE 5.  The recommended adjustments of temperature, vacuum, and the
pitch of the assembly should result in a steady boiling action with
no bumping.  The -Ditch should be such that no extract condensate
collects in the lower part of the K-D flask (paragraph 2.8, NOTE i).
                          111-238

-------
     Continue evaporation until  the extract is condensed to ca. 4 ml,
     remove the assembly from the water bath, and rinse down the walls
     of the flask with 4 ml  of hexane delivered with a disposable
     pi pet.

     Disconnect the concentrator tube from the K-D flask, rinsing the
     joint with ca. 2 ml of  hexane delivered with a disposable pipet.

     Place the tube under a  gentle stream of nitrogen at ambient tempera-
     ture and concentrate the extract to ca. 0.5 ml.

     NOTE 6.  Under no circumstances should air be used for the blowdown
     as certain organophosphorus compounds may not survive the oxidative
     effects.

2.6  Silica Gel Fractionation and Cleanup

     Before starting the following steps, place 10 drops of the paraffin
     oil-hexane keeper solution  in the two 15-ml centrifuge tubes intended
     as the receivers for the eluates of Fractions III and IV.

     Prepare a silica gel column as follows:

     a. Lightly plug a size  22 Chromaflex column with a smal"1  wad of
        preextracted glass wool.

     b. Add 1.0 g of deactivated silica gel, tapping firmly to settle,
        then too with 1 inch of  anhydrous sodium sulfate and again tap
        firmly.

     c. Pass 10 ml of hexane through the column as a prewash,  discarding
        the eluate.

     When the last of the prewash hexane just reaches the top surface  of
     the sodium sulfate, quickly place a 15-ml  conical centrifuge tube
     under the column, and using  a disposable pipet, carefully transfer
     the 0.5 ml of sample extract to the column.  After the extract has
     entered the column, rinse the walls of the centrifuge tube with  1.0
     ml of hexane, and, using the same disposable pipet, transfer this
     washing increment to the column.  Repeat this 1.0-ml hexane wash
     twice more and finally  add  6.5 ml hexane to the column.  The result-
     ing 10 ml total effluent is Fraction I.

     NOTE 7.  There must be  no interruption of the procedure during this
     step.  Extreme care should  be taken to apply the sample to the column
     at the precise moment the last of the hexane prewash reaches the  top
     surface of the column.

     NOTE 8.  Faultless technique is required in this step to avoid any
     losses, particuiariy auring the transfer of *he 0.5 ml  concentrated
     extract and the first rinse.All the pesticide extracted from tne
     original  sample is concentrated in this very minisc-jle extract.   The

                               III-239

-------
     loss  of  one  drop may  Introduce a recovery error of 20 percent or
     more.

     Immediately  position  another  15-ml centrifuge tube under the column
     and pass 15  ml  of  the benzene/hexane  (60/40 v/v) elutlng solution
     through  the  column.   This 1s  the Fraction II eluate.

     Elute the column a third time using 25 ml of acetonitrile/benzene
     solution (5/95  v/v).  This eluate  is  Fraction III.

     A fourth elution fraction is  necessary if there is reason to suspect
     the presence of crufomate, dicrotophos, dimethoate, mevinphos,
     phosphamidon, or the  oxygen analogs of diazlnon and malathion.  The
     elution  solution is 15 ml of  acetone/methylene chloride (25/75 v/v).
     This  is  Fraction IV.

     Place the eluates  under a gentle nitrogen stream at ambient
     temperature  and concentrate as follows:

     a. Concentrate  Fraction I and II to ca. 3.0 ml, rinse down the tube
        sidewalls with  ca. 1.5 ml  hexane,  and adjust the volume to exactly
        5.0 ml with  hexane.  Cap the tubes tightly and mix on the Vortex
        mixer for 1  minute.

     b. Concentrate  Fractions III  and IV to 0.3 ml, rinse tube sidewalls
        with  hexane, and dilute back to exactly 5.0 ml with hexane.

     NOTE  9.   Fractions III and IV contain eluant solvents that may inter-
     fere  in  the  GC  determination, whereas those solvents in Fractions I
     and II would create no sucn prooiems. for  em's reason, "-actions *:!
     and IV are reduced to a lower volume  to increase the removal of the
     original solvents.

     Fractions II and  III  may contain carbamates as well as organophos-
     phorus compounds.   Gas chromatography of organophosphorus compounds
     by flame photometric  detection  1s  conducted on the eluates adjusted
     to 5.0 ml.

2.7  Sample Analysis

     For multlresidue  analysis  of  samples  with  unknown pesticidal
     contamination,  two GC columns yielding divergent compound elution
     patterns will aid  confirmation.  Two  such  columns are 5 percent
     OV-210 and 1.5  percent OV-17/1.95  percent  OV-210.  Sensitivity levels
     for the FPD detector  should be  carefully established before  starting
     chromatographic determination.  The majority of water samples will
     contain extremely low pesticide concentrations, and, therefore, an
     insensitive GC  system will  severely  handicap the analysis.   When  flow
     and temperature parameters  are  optimum, baseline noise should not
     exceed 2.5 percent of full  scale and  injection of 2.5 ug  of  parathion
     should result in  a peax  of  at least  50 percent  if  *un seal a.


                               II1-240

-------
     The majority of the halogenated pesticides will  be found in Frac-
     tions I and II, with a few of the more polar compounds  in  Fraction
     III.  Most of the organophosphorus compounds will  be in Fractions  II
     and III, none in Fraction I,  and a very few in  Fraction IV.   Carba-
     mates are eluted in Fractions II and III (Table 1).

     A number of organophosphorus  compounds chromatographed  with  the  FPD
     detector require considerable column preconditioning by repetitive
     injection of standards of relatively high concentrtion  before
     attempting quantification. Failure to carefully monitor linearity
     of response may result in erroneous quantitative values.

     A typiical gas chromatogram of silica gel column Fraction  II is
     shown in Figure 3.

2.8  Miscellaneous Notes

     1.  The recommended operation of the concentrator  is unusual for
     pesticide analysis.  Customarily, solvent evaporation is achieved
     by immersing the concentrator tube in a water bath at a higher
     temperature .than the boiling  point of the solvent, or the  flask  is
     attached to a conventional rotary evaporator.  The system  used
     achieves two ^oortant objectives-,  the extract is exposed to a
     maximum temperature of less than 35"C to minimize  degradation if
     heat labile compounds; and the concentrated extract is  confined  to
     one,container, thereby eliminating need for a transfer.  Using the
     temperature and vacuum levels specified, 100 ml  of methylene chlo-
     -*de extract can be reduced to 5 ml in ca. 20 minutes in this
     apparatus.

     2.  The activity and performance of deactivated silica  gel  changes
     in a matter of days.  It is desirable to deactivate only the amount
     required for a 2- or 3-day period.  Continuous  storage  of  activated
     silica gel at 175C may result in a shift of the compound  elution
     pattern of deactivated columns prepared from this  adsorbent. The
     quantity of silica gel activated should be limited to a 1-week
     supply.

     3.  Recoveries of OP pesticides were found, in  general, to be far
     better when methylene chloride-extracted water  rather than unextrac-
     ted distilled water was used  as the spiking substrate to evaluate
     this procedure.  Therefore, unextracted distilled  water was  used for
     all recovery studies.  As a further test, a sample of water was
     obtained a few hundred yards  downstream from the outfall of a large
     chemical manufacturing plant  and was fortified  with a mixture of
     pesticides and analyzed using the extraction and silica gel  fractiona-
     tion steps.  No extraneous peaks were observed  with the flame photo-
     metric detector, indicating applicability of the method to aqueous
     environmental water samples.
                               TII-241

-------
I
ro
ro
                                                                          32
         40
             Figure 3.  Four organophosphorus compounds eluted in Fraction II,

                                            OV-17/1.95% OV-210.
GC column 1.5%

-------
3.   Determination of Organophosphorus Pesticides 1n Soil
          Analytical  Procedure:   available
          Sample Preparation:  available

     3.1  Reference

          Carey, A.E., J. A. Gowen,  H. Tai,  W.  6. Mitchell  and 6.  B.   Wiersma,
          "Pesticide  Residue Levels  in Soils and Crops from 37 States,  1972  -
          National Soils Monitoring  Program  (IV)."  Pest.  Monit. J.  12(4):
          209 (1979).7

     3.2  Method Summary

          Pesticides  are extracted from soil with hexane-isopropanol,  and
          extracts are analyzed  without cleanup by GC with  a phosphorus-
          selective detector.

     3.3  Applicability

          DEF, diazinon, malathion,  ethyl  parathion, methyl  parathion,  trith-
          ion, and ronnel were determined  in cropland soils  at minimum detec-
          table levels of 0.01-0.03  ppm.  The method should be applicable to
          other OP pesticides with similar polarities.

     3.4  Accuracy, and Precision

          Pesticide recovery from soil was close to 100 percent and  ranged from
          30 to 100 percent.

     3.5  Sample Preparation

          Moisten a 100-g subsample  of soil  from a thoroughly mixed  field
          sample with 25 ml  distilled water. Extract with  200 ml  of  hexane/
          isopropanol (3/1 v/v)  by shaking for  4 hours on  a  mechanical  mixer or
          shaker.  Remove the isopropanol  with  three distilled water  washes.
          Dry the hexane extract through a small  column of  anhydrous  sodium
          sulfate.  Store the sample extract at low temperature prior to  GC
          analysis.

     3.6  Sample Analysis

          Analyze samples on a gas chromatograph equipped with a flame  photo-
          metric or thermionic detector.  Identify compounds based on  elution
          characteristics on at  least two  columns with differing polarity.
          Confirm residues by use of alternate  selective detectors (e.g.,
          Dohrman microcoulometric or electrolytic conductivity) or by GC/MS.1
          Carry standards through the analytical  procedure  to monitor recovery.
          Residue concentrations can be corrected for recovery if  desired.
          Kesults of  joil sample analyses  are usually converted to a  dry-weight
          basis.  To  determine moisture content,  weigh a separate  portion of soi i
          taicen for extraction,  heat in an even overnight, at 105 to 110'C. cool
          in a desiccator, and reweigh.

                                    i11-243

-------
4.   Determination of Organophosphorus Pesticides in Fruits  and Vegetables
          Analytical  Procedure:   available
          Sample Preparation:   available

     4.1  References

          Luke, M. A., J. E.  Froberg and H.  T.  Masumoto, -"Extraction  and
          Cleanup of  Organochlorine, Organophosphate,  Organonitrogen,
          and Hydrocarbon Pesticides in Produce for Determination  by  Gas-
          Liquid Chromatography."  J.  Assoc.  Off.  Anal. Chem.  55(5):1020
          (1975).H

          Luke, M. A., J. E.  Froberg,  6. M.  Doose and H. T.  Masumoto,
          "Improved Multiresidue Gas Chromatographic Determination  of
          Organophosphorus, Organonitrogen,  and Organohalogen  Pesticides
          in Produce  Using Flame Photometric  and Electrolytic  Conductivity
          Detectors."  J. Assoc. Off.  Anal.  Chem.  64(5):1187 (1981).12

     4.2  Method Summary

          Samples are extracted with acetone  and partitioned with methyl-
          ene chloride and petroleum ether to remove water.  The methylene
          chloride is removed with a Kuderna-Danish evaporator and  the
          resultant petroleum ether extract  is  analyzed by GC  using an
          FPD detector.

     4.3  Applicability

          The method  is applicable to nonionlc  OP pesticides and metabol-
          ites present in fruit or vegetaoie  matrices  at concantratlcri
          levels of ca 0.01 to 5 ppm.

     4.4  Precision and Accuracy

          Table 2 lists recovery data for 44  OP compounds from a variety
          of produce  samples.   Recoveries are 1n the range from 88  to
          118 percent.  ReproducibiHty of the  method has not  been  reported.

     4.5  Sample Preparation

          Chop or blend fruits and vegetables and mix thoroughly.   Weigh
          100 g of chopped or blended sample  into a high-speed blender
          jar, add 200 ml acetone, and blend for 2 minutes at  high  speed.
          Do not add  Celite.   Filter with suction through a  12 cm Biichner
          funnel fitted with  sharkskin paper, and collect the  extract  in
          a 500 ml suction flask.

          Place 80 ml of sample in a 1-1 Her separatory funnel,, add 100
          ml petroleum ether  and 100 ml  methylene chloride,  and shake  for
          1 minute.  Transfer the lower aqueous phase into a second 1-liter
          separator/  funnel.   Dry the upper  organic layer 1n *,he f
-------
                 TABLE 2
Compound
RECOVERIES OF OP PESTICIDES
                    asaaaaa:

                    Recovery
                       ====================================================
Fortification
 Level (ppm)
                                      Sampl e
Azinphos-ethyl
Carbophenothion sulfone
Chlorfenvinphos
Chlorpyrifos
Chlorthiophos
DDVP
OEF
Demeton-S-sulfone
Dial if or
Dicrotophos
Dimethoate oxygen analog
EPN
Fenamiphos
Fenitrothion
Fensulfothion
Fenthion
Fonofos
Leptophos
Malathion oxygen analog
Mephosfolan
Methidathion
Methyl carbophenothion
Naled
Oxydemeton-methyl
Oxydemeton-methyl sulfone
Parathion oxygen analog
Phenthoate
Phorate sulfone
Phorate sulf oxide
Phosalone
Phosmet
Phoxim
Phoxim oxygen analog
Profenofos
Prometryn
Pyrazophos
Ronnel
Sulprofos
Sulprofos sulfone
Sulprofos sulf oxide
Tetrachlorvinphos
Thionazin
Triazoohos
Trichlorfon
-:== ===-=::aaaa = a=aasasasaaasBa
1.00
1.94
0.324
0.10
0.0918
0.105
0.69
5.7
1.32
0.094
1.51
1.05
0.436
1.00
1.0
0.14
1.00
0.102
1.52
0.17
0.862
0.56
3.79
0.12
0.113
1.50
0.0117
0.105
0.114
1.80
0.25
0.10
0.10
0.10
0.108
0.66
0.10
0.105
0.10
0.105
1.08
0.608
0.134
0.110
============
102
116
97
99
95
90
105
115
115
105
90
105
97
88
107
97
92
113
112
106
93
118
97
88
103
105
110
93
116
92
108
110
105
102
104
107
104
105
105
106
113
100
106
no
===================
tomato
cucumber
bell pepper
green beans
tomato
tomato
lettuce
pepper
potato
green beans
grapes
green beans
bell pepper
blueberry
rutabaga
green beans
parsley
potato
potato
. tomato
orange
tomato
strawberry
grapes
grapes
tomato
tomato
lettuce
lettuce
grapes
tomato
tomato
tomato
tomato
cucumber
tomato
pears
green beans
green beans
green beans
green beans
tomato
tomato
orange

                                IH-245

-------
     by passing through a 4-cm layer of Na2SC>4 supported on washed
     glass wool in a 10-cm funnel  and collect the solution in a 500-ml
     Kuderna-Danish concentrator.   To the separatory funnel with the
     aqueous phase, add 7 g NaCl  and shake for 30 seconds until  most of
     the salt is dissolved.  Add 100 ml of methylene chloride, shake for
     1 minute, and dry the lower organic phase through the sane
     column.  Extract the aqueous  phase with an additional 100 ml
     methylene chloride and dry as above.  Rinse the N32S04 with ca.
     50 ml of methylene chloride.

     Attach a Snyder column to the K-D concentrator and start evapo-
     ration slowly by placing only the receiver tube into steam.
     After 100 to 150 ml  have evaporated, expose the concentrator to
     more steam.  Concentrate to 1 ml, add 100 ml  of petroleum
     ether to the concentrator, and reconcentrate the solution to
     1 ml.  Repeat the reconcentration with 50 ml  of petroleum ether
     and then with 20 ml  of acetone.

4.6  Sample Analysis

     Cool and adjust the volume of the concentrated extract to 7 ml.
     Inject ca. 2 ul into a gas chromatograph equipped with an FPD and
     a 2-percent DEGS, 2-percent OV-101, or 4-percent SE30/6.5-percent
     OV-"10 column.
                               111-246

-------
5.   Determination of Organophosphorus Pesticides in Air
          Analytical  Procedure:   available
          Sample Preparation:   available

     5.1  Reference

          Sherma, J.  and M. Beroza,  "Manual  of Analytical  Methods for the
          Analysis of Humans and Environmental  Samples."  EPA-600/ 8-80-038,
          Section 8B  (June, 1980).4

     5.2  Method Summary

          The sampling medium, such  as polyurethane foam or composite filter
          pad, is Soxhlet extracted  with hexane/diethyl  ether (95/5 v/v).   OP
          pesticides  are measured by direct  GC with an FPO detector.

     5.3  Applicability

          The method  is suitable for quantifying OP pesticides in ambient  air
          at ultratrace levels (ng/m3 and pg/m3).  The analytical  scheme  pre-
          supposes collection  of. samples by  a  high-volume  air sampler on  poly-
          urethane foam or on  Tenax  GC sorbent.

     5.4  Precision and Accuracy

          The collection efficiencies of OP  pesticides on  polyurethane/granular
          sorbent combination  media  are shown  in Table 3.   Essentially no
          differences are observed among sorbent systems for the  pesticides
          studied. Collection efficiencies  using the IRCO high  -clurre :anpler
          .are shown in Table 4 for filter pads  and  in Table 5 for a tandem pair
          of polyurethane foam plugs.

     5.5  Extraction  of the Sampling Module

          Place the sampling medium  in a Soxhlet extractor, handling  with
          forceps rather than  hands.

          NOTE 10. After sampling,  glass fiber filters  and foam  plugs should
          have been wrapped in aluminum foil until  analysis.   Use plugs and
          filters carried to the field along with those  employed  for  sampling
          as controls.

          Extract with an appropriate volume of n-hexane/acetone/diethyl ether
          (47/47/6 v/v) for 16 to 24 hours at  4 cycles per hour for the large
          Soxhlets and 8 to 12 hours at 8 cycles per hour  for the smaller
          Soxhlets.

          NOTE 11. As examples, extract large  foam plugs  in 1000-ml  Soxhlet
          extractors  with a total of 300 to 750 ml  of solvent, and smaller
          plugs and filters in 500 ml  Soxhlets  *ith 20fi  to 350 ml.
                                    111-247

-------
         TABLE 3.  HIGH-VOLUME COLLECTION EFFICIENCIES OF PESTICIDES  ON
                       FOAM/GRANULAR SORBENT COMBINATIONS
                         % Collection on Foam/Sorbent Combinations
                               After 24 hours at 225 1/minute
           Calc. Air         Chromosorb
              Cone.   Foam      102      Porapak R   XAD-20  Tenax GC   Florisil
Pesticide   (ng/m3)   Alone   (20/40)     (50/80)   (16/20)   (60/80)    (16/30)
Diazinon
Methyl
Parathion
Ethyl
Parathion
Malathion
3.0-30.0
1.8-18.0
3.6-36
0.9-9.3
===========
63
91
96
37
========
72
82
85
88
=======
59
72
72
78
71
80
81
89
76
87
86
91
72
83
83
81
               TABLE 4.  ERCO SAMPLER COLLECTION EFFICIENCIES AT
                            183 1/MINUTE FOR 2 HOURS
===============================================================================
                         Calculated Air Concentration     Collection Efficiency
  Compound                        (ug/m3)                          (%)


Methyl parathion                0.02 - 150                         105

Diazinon                        0.11 - 2.2                          93

ChlorpyHfos                  .  0.02 - 0.22                         77
                                                   :=========::======== = = :
                                    111-248

-------
       TABLE 5.   AVERAGE COLLECTION EFFICIENCIES ON POLYURETHANE  FOAM FOR
          ORGANOPHOSPHORUS PESTICIDES AT 225 1/MINUTE AND 184 1/MINUTE
===================
Pesticide
Diazinon


Methyl parathion


Malathion


Parathion


:==========================================:
Air Calculated
Volume Air Concentration
(m3) (ng/m3) % Collected
326
265
265
326
265
265
326
265
265
326
265
255
30.7
18.9
3.8
18.4
11.3
2.3
36.8
22.6
4.5
9.2
5.7
1.1
70.4
91.0
75.5
73.6
73.3
71.9
87.2
76.6
81.2
84.8
70.3
65.8
Statistical Data
n
5
6
6
5
5
4
5
5
4
5
5
4
sigma
3.97
16.19
14.40
4.56
6.52
4.12
33.40
12.47
14.68
4.15
4.70
3.75
==============================================================================
          Remove the boiling flask to a rotary evaporator and reduce the
          solvent volume to approximately 5 ml.

          Transfer the concentrate to a 15-ml  graduated centrifuge tube with
          rinsing.

     5.6  Sample Analysis

          Adjust the final  volume in the centrifuge tube as  required.

          Inject 5 ul  directly without cleanup into the gas  chromatograph
          equipped with an  FPD detector.  Typical  GC conditions  for determina-
          tion are as follows:  183 cm x 4 mm  I.D.  glass column  packed with  1.5
          percent OV-17/1.95 percent OV-210 and/or 4 percent SE-30/6 percent
          OV-210 on 80 to 100 mesh Gas Chrom Q;  column, 200C; injection port,
          215C; nitrogen carrier gas, 60 to 85 ml/minute; P-mode FPD, 200C.

          Record chromatograms under the above parameters and measure retention
          times relative to aldrin or another  suitable reference standard.

          Compare the relative retention time  of each component  of interest
          against those of  the corresponding primary standard.

          Quantify peaks in the usual  way, i.e., by measuring pea* neignts  to
          the nearest HOT when the basa width ss  
-------
          Confirm results as required by combined GC/MS or some other
          appropriate procedure.
I.    CALCULATIONS
1.    Extract Quantification
     The organophosphorus pesticide concentration of the sample extract is
     calculated based on the method of standardization.
     la.  Internal Standardization
          By adding a constant known amount of internal standard (C-js in yg)
          to every sample extract, the concentrtion of contaminant (C0) in
          ug/1 in the extract is calculated as:
                                            (As)(C1s)
                                     Co  -  	
                                            (A1s) (RF)
          where   As  =  response for the parameter being measured
                 C-jS  =  concentration of the internal standard
                 Ais  =  response for the internal standard
                  RF  =  calculated response factor.

     Ib.  External Standardization
          The concentration of the unknown in the extract is calculated from
          the slope and intercept of the calibration curve.  The extract con-
          centration is calculated as:
                                            (A)(Vt)
                                            (Vi)(Vs)
          where   C0  =  extract concentration
                   A  =  mass of compound from the calibration curve (ng)
                  Vi  =  volume of extract injected (pi)
                  Vt  -  volume of total extract (yl)
                  Vs  *  volume of water extracted (ml).
          Report ^11 results in ug/l to two significant figures without
          correction for recovery data.  When duplicate and spiked samples
          are  analyzed, all data obtained should be reoorted.
                                    III-250

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2.   Sample Concentration
     From the concentration of pesticide in the extract,  calculate the con-
     centration in the original  sample.
     2.1  Formulations
                                   (A)(B)
          Concentration (mg/g)  =  	
                                    (C)
          A  *  concentration in extract (mg/ml)
          B  =  volume of extract (ml)
          C  *  sample mass (g).
     2.2  Water Samples
          Concentration (mg/liter)   =
                                        (C)
          A  =  concentration in extract (wg/ml)
          B  *  volume of extract (ml)
          C  *  sample volume (ml).
     1.3  Soi'i Dumpies
          2.3.1  Dry Basis
                                          (A)(B)(100-D)
                 Concentration (ng/g)  =  	
                                           (C)    (100)
                 A  -  concentration in  hexane  extract  (ng/ml)
                 B  =  final  volume  of hexane extract  (ml)
                 C  *  wet mass  of  sample (g)
                 D  =  percent moisture  of sample.
          2.3.2  Wet Basis
                                           (A)(B)
                 Concentration (ng//g)   =  	
                                            (C)
                 A  =  concentration in  hexane  extract
                 B  =  final  volume  of hexane extract
                 C  *  sample mass  (wet).
                                    III-251

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2.4  Air Samples
     Concentration (ng/m3)  =
                               (0(1000)
     A    concentration in extract (ng/1)
     B  =  extract volume (ml)
     C  =  sample volume (m3).
2.5  Fruits and Vegetables
                              (A)(B)
     Concentration (ng/g)  =  	
                               (C)
     A  =  concentration in final  extract  (yg/ml)
     B  =  final  volume of extract  (ml)
     C  =  sample mass (g).
                               III-252

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                                   REFERENCES


1.   Sherma, J.  "Manual  of Analytical  Quality Control  for Pesticides  1n  Human
     and Environmental Samples."  EPA-600/2-81-059  (April, 1981).

2.   McMahon, B. M. and L. D. Sayer,  editors.  "Pesticide Analytical Manual  -
     Volume I:  Methods Which Detect  Multiple Residues."   United States FDA
     (September, 1982).

3.   "Guidelines on Sampling and Statistical  Methodologies for Ambient Pesticide
     Monitoring."  Federal Working Group on Pest Management,  Washington,  D.C.
     (October, 1974).

4.   Sherma, J. and M. Beroza.  "Manual  of Analytical  Methods for  the  Analysis
     of Pesticides in Humans and Environmental  Samples."   EPA-600/8-80-038
     (June, 1980).

5.   Lewis, R. G.  "Procedures for Sampling and Analysis  of PCBs in  the Vicinities
     of Hazardous Waste Disposal Sites."  Advanced  Analysis Techniques Branch,
     Environmental Monitoring Systems Laboratory, Research Triangle  Park, North
     Carolina.  14 p.  (March 16, 1982).

6.   Zweig, G. and J. Sherma.  "Gas Chromatographic Analysis," Volume  6 of
     "Analytical Methods for Pesticides  and Plant lirowtn  Regulators."  Chapter
     4, p. 107, Academic Press, New York (1972).

7.   Carey, A. ., -j. A.  aowen, n. 7
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12.  Luke, M.  A.,  J.  E.  Froberg,  G. M. Doose and H. T. Masumoto.  "Improved
     Multiresidue  Gas Chromatographic Determination of Organophosphorus,
     Organonitrogen,  and Organohalogen Pesticides  in Produce, Using Flame
     Photometric and  Electrolytic Conductivity Detectors."  J. Assoc. Off.
     Anal.  Chem.  64(5):1187  (1981).
                                    III-254

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                                   SECTION 6

          METHODS FOR THE DETERMINATION OF ORGANONITROGEN PESTICIDES
A.   SCOPE

     The analytical procedures provided in Subsection I of this section cover
the determination of carbamate, urea, and related pesticides in water (Sub-
section I.2.). soil (Subsection I.3.), vegetable and fruit tissues (Subsection
1.4.) and air (Subsection I.5.).  The water and crop analyses are based on high-
performance liquid chromatography with UV and fluorescence detection, respec-
tively.  Gas chromatography with N-mode electrolytic conductivity detection is
the determinative procedure for soil and air analyses, and HPLC is also used for
soil analysis.  All analyses involve solvent extraction of residues from the
sample, followed in certain cases by solvent partitioning and column chromato-
nraohy cleanuo steps and derivatization prior to detection and determination.

     Compounds covered in this section are all broadly classified as organo-
nitrogen pesticides.

J.   SAMPLE HANDLING AND STORAGE

     See Subsection B of Section 5 (Organophosphorus Pesticides) in this Chap-
ter for general  information on pesticide sampling and storage that is appli-
cable to carbamate and related compounds.

     Sampling techniques and sample preservation are important criteria in any
monitoring program.  Although sampling and analysis are distinct parts of the
program, they are interdependent since each controls certain aspects of the
other.  An analytical result can be no more valid than the samples or sampling
scheme used.  Good sampling procedures should be practiced, and the following
precautions should be taken to ensure that the samples received and analyzed
represent the field situation.

     Samples should be analyzed as soon as possible after collection to
avoid any biological or chemical alteration of the pesticides; in the interim
the sample should be frozen, or, in the case of water, stored at a low tempera-
ture (about 4C).  Since many of the N-methylcarbamates are prone to hydrolysis
at the pH of natural waters, the addition of acid (to about pH 2) is recom-
mended.  Since photochemical changes are also possible, the samples should be
stored in the dark to minimize these possibilities.

     Frequently, samples cannot be analyzed as quickly as desired.  Hence, :t
may be desirable to extract the samoles upon receipt (or in the field) and
store the extracts pending analysis.  All  samples must be extracted within

                                    111-255

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7 days and completely analyzed within 40 days of extraction.  The stabil-
ity of a pesticide during any type of storage is questionable, and it is often
beneficial to examine the stability of a standard or field-incurred residue in
a sample or extract Under various conditions of time, temperature, and in differ-
ent substrates.  The stability of carbofuran and carbaryl  appears to be more
dependent on the substrate than on the storage temperature.   On the other hand,
methomyl stability is related to the storage temperature,  and it appears to be
most stable under freezing conditions.  Ethylenebisdithiocarbamate (EBDC) fun-
gicides tend to decompose immediately after maceration of  substrates, and
storage by freezing is preferred over refrigeration.  Chloropropham (CIPC)  and
propham (IPC) show significant losses during storage.

     Clean glass bottles with Teflon- or aluminum-lined tops should be used
for water samples.  Polyethylene bags or glass jars are recommended for other
substrates.  The bottle must not be prerinsed with the water sample before
collection.  Automatic sampling equipment must be as free  as possible of plas-
tic and other potential sources of contamination.  When composite water samples
are collected, it is advisable to refrigerate the sample during the compositing
period.

     Phenylurea herbicides are relatively stable and do not  undergo rapid
chemical or biological degradation.  Thus,  water and sediment samples collected
from field sites can be shipped in glass containers without  special treatment.
On arrival at cne iaooratory, water samples T,ay be filtersd  -hrough jlass '-wol
to remove solid material and then stored in a refrigerator at 4"ct  If the
analysis cannot be carried out within 2 weeks, the sample  should be frozen  at
-20"C.  Sediment samples should be frozen at -20*C on arrival at the labora-
tory, and prior to analysis should be thawed, filtered to  remove excess water,
ana a'ir-ur'iea.

     Plant and animal samples are stored at -40C until analysis.  A storage
stability study on the ground or chopped sample is recommended by fortifying
untreated check samples.

C.   INTERFERENCES

     Method interferences may be caused by contaminants in solvents, reagents,
glassware, and other sample processing apparatus that lead to discrete arti-
facts or elevated baselines in chromatograms.  All reagents  and apparatus must
be routinely demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks.

     Clean all glassware* as soon as possible after use by thoroughly rinsing
with the last solvent used in it.  Follow by washing with  hot water and deter-
gent and thorough rinsing with tap and reagent-grade water.   Drain dry, and
heat 1n an oven or muffle furnace at 400C for 15 to 30 minutes.  Do not heat
volumetric glassware.  Thermally stable materials such as  PCBs might not be
eliminated by this treatment.  Thorough rinsing with acetone and pesticide-
quality hexane may be substituted for the heating.  After  drying and cooling,
seal ana store glassware *n 3 clean environment to prevent accumulation o*  dust
or other contaminants.  Store inverted or capped with aluminum foil.


                                    III-256

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     Matrix interferences may be caused by contaminants that are coextracted
from the sample, and will vary in extent depending on the nature of the
sample and its source.  The cleanup procedures in the methods will usually
overcome these interferences, but unique samples may require additional cleanup
approaches.

0.   SAFETY

     The toxicity or carcinogenicity of each reagent used in the methods in
this section has not been completely defined.  However, each chemical  compound
must be treated as a potential health hazard.  Exposure to chemicals must be
reduced to the lowest possible level by all means available.  Each laboratory
1s responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of all chemicals specified in the methods.2t3

E.   APPARATUS

1.   Equipment for discrete or composite sampling of water:

     1.1  Grab-sample bottle, amber borosilicate or flint glass, 1 quart or
          1 liter volume, fitted with screw caps lined with TFE fluorocarbon,
          or aluminum foil if the sample is not corrosive.  If amber bottles
          are not available, protect samples from light.  Wash the container
          ana cap iliner, nnse *ith acstone Dr methylene chloride, ?nd Hry
          before use to minimize contamination.

     1.2  Automatic sampler (optional), incorporating glass sample containers
      	for-collection of a minimum of 250 ml.  Refrigerate (4*C) and protect
          sample containers "rein "ight d'jf'rig tempos i^ng.  !* the samoler uses
          a peristaltic pump, a minimum length of compressible silicone rubber
          tubing may be used.  Before use, rinse the tubing thoroughly with
          methanol, followed by repeated rinses with reagent-grade water to
          minimize the potential  for contamination of the sample.  An integra-
          ting flow meter is required to collect flow-proportional composites.

2.   Analytical balance, capable of weighing to 0.0001 g.

3.   Water bath, heated, with concentric cover, capable of temperature
     control (2*C).

4.   Beakers, 2000 ml.

5.   Gas chromatograph, Tracer Model 560 or equivalent, equipped with a
     1inearized63Ni  electron capture detector, or a Model 700 Hal.l electrol-
     ytic conductivity detector operated in the reductive mode with a nickel
     wire catalyst (Ventron), strontium hydroxide scrubber, and isopropanol/
     water (15/85 v/v) conductivity solvent.  Detector used depends on the
     requirements of the method.

6.   GC column, 1.3 m x 2 m !.D., glass, 
-------
      with normal  carrier gas  flow.  Alternative  columns  include  1  percent
      OV-17 and the  mixed phase  0.5  percent OV-210 + 0.65 percent OV-17, both
      on Ultra-Bond.   A temperature  program of  115 to  1758C at  lO'/minute can be
      used for complex samples,  in place of Isothermal operation at 170C.  This
      column is to be  used when  the  Hall electrolytic  conductivity  detector is
      specified.

 7.    GC column,  1.8 m x 4 mm  I.D.,  glass, containing  3 percent (w/w) OV-225
      on 80 to 100 mesh Chromosorb W(HP) or 3.6 percent (w/w) OV-101/ 5.5
      percent (w/w)  OV-210 on  acid-washed, dimethyldichlorosilane-treated
      Chromosorb  W.  This column is  to be used when the electron capture detec-
      tor is specified.

 8.    Liquid chromatograph, high performance analytical system  complete with
      high pressure  syringes or  sample injection  loop, analytical column,
      gradient elution system, detector, and strip-chart  recorder or mini-
      computer data-handling system.  The Installation of a guard column is
      also recommended.   Typical  modules Include  an Altex Model 322 MP
      programmable gradient system,  Valco Model 16 AS-7000 automatic sampler
      with 10-ul  injection loop, Spectra Physics  Model 4000 Microprocessor/
      printer plotter, and 7 cm  x 2.1 mm I.D. guard column containing 25 to
      37 urn Co-Pell  ODS (Whatman).

 *?.    MPLC column, ^0  ~-n x 4 mm  !.0., stainless steel, packed with  u-Bondaoak
      Cis (10 urn)  (Waters Associates); 30 cm x 4.6 mm  I.O., stainless steel,
      packed *1th Spherisorb ODS (5  '^m) (Phase Separations Ltd.); or 25 cm
      x  4.6 mm.I.D., stainless steel, containing  Zorbax C-8 (6  urn)  (DuPont).
	 All  of these are chemically bonded reversed-phase columns.

 10.   HPLC fluorescence detector, Perkin-Elmer Moaei s5Q-iQLC or equivalent,
      with 20-iil  cell.  This detector requires the following apparatus:

      10.1 Carbamate hydrolysis  chamber -  column bath (18x18x13 cm) from
           Model  5360  Barber-Colman  gas chromatograph  with Model 700-113
           proportional  temperature  controller (RFL Industries, Inc., Boonton,
           NJ) containing 3 m  x  0.48 mm I.D. No.  321 stainless  steel tubing
           (Tubesales, Forest  Park,  GA).

      10.2 Sodium hydroxide and  reaction solution reservoirs -  60 cm x 25 mm
           I.D. glass  columns  with Teflon fittings (Glenco Scientific, Inc.,
           Houston,  TX).   Pressurize reservoirs with nitrogen.  Connect 6 m
           x 0.5  mm  Teflon restriction coil from  reservoir to 15 cm x 0.18 mm
           I.D. ss tubing.  Connect  ss tubing to  0.74  mm  I.D. ss reaction tee
           (No. ZVT-062,  Valco).

      10.3 Connecting  tubing - No. 304 ss tubing  to connect Injector, columns,
           and first tee.

 11.   HPLC UV detector, capable  of monitoring absorbance  at 240 nm, 254 nm, and
      280 nm.
                                     III-258

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12.  Chromatography. columns, 400 mm x 19 mm I.D.,  with  coarse  fritted  disc at
     the bottom and TFE-fluorocarbon stopcock  (Kontes K-420540-0224).
13.  Cleanup columns, Chromaflex size 22, 20 cm x  7  mm  (Kontes K-420100-
     0022), and Chromaflex 30 cm x 22 mm I.D.  (size  233) with  coarse porosity
     frit and Yaribor stopcock (size 2)  (Kontes K-420540-9042).
14.  Desiccator.
15.  Rotary evaporator.
16.  Extractors, Soxhlet, 1000,  500, and 250 ml.
17.  Swinny filter holder, 13 mm filter  size (Millipore No. XX3001200).
18.  Filter paper, Whatman No. 1 and No. 42, and S&S 597.
19.  Miltex filters,  5 urn, 13 mm diameter,  white,  plain (Millipore No.
     LSWP 01300).
20.  Filtration apparatus, as needed to  filter solvents prior  to HPLC.
21.  Flasks, Stanmeyer, 500 11!  and 1000 ml.
22.  Flasks, 2000 art, 1000 ml, 500 ml, and  250 ml  round bottom, with I 24/40
     joints.
:2.  ""isks, volumetric. !0 Til.  1000 ml, and 25 ml  (actinic).  "
24.  Separatory funnels, 2000 ml,  500 ml, and  250  ml, with TFE-fluorocarbon
     stopcock and ground glass or TFE stoppers.
25.  Homogenlzer, Polytron Model  PT 10-35 or equivalent, with  PT 35K generator
     containing knives (Brlnkmann Instruments) and four-sided  glass quart jar
     (Tropicana Products, Inc.,  Bradenton,  FL).
26.  Pipets, volumetric, assorted sizes.
27.  Wrist-action shaker.
28.  Centrifuge tube, 15 ml.
29.  Vacuum adapter I 24/40.
30.  Vacuum filtration apparatus,  with 12-cm Buchner funnel and 500-ml
     filter flask.
31.  Vials, 10 to 15  ml, amber glass, with  TFE-fluorocarbon lined screw caps.
                                    !!!-259

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F.   REAGENTS

     (Solvents should be pesticide quality, distilled-in-glass or HPLC grade
     for mobile phases.)

1.   Glacial acetic acid.

2.   Acetone

3.   Acetonitrile

4.   Benzene

5.   Carbon-Celite chromatography mixture.   Mix Nuchar S-N and silanized
     Celite (4/1 w/w).  Test the adsorbent  with a freshly prepared mixed
     carbamate solution (carbaryl, methiocarb, methiocarb sulfoxide, methomyl )
     prepared in methanol at 5 yg of each compound per ml.  Pipet 5 ml  of this
     solution into a 250-ml  round-bottom flask and 5 ml  into a 25-ml actinic
     volumetric flask.  Dilute the solution in the volumetric flask to 25 ml
     with methanol and use as HPLC reference standard.  Evaporate the standard
     solution in the round-bottom flask to.dryness with a vacuum rotary evap-
     orator as described in  Subsection H.4.1.5.  Dissolve the carbamate residue
     in 10 ml of methylene chloride.  Transfer the solution to a prepared
     adsorbent column and elute as described in Subsection H.4.1.7.  After evap-
     oration of the eluate in the round-bottom flask, dissolve the residue in
     25 Til of methanol.  Filter 5 to 8 ml of this solution through a Swinny
     filter holder, as described in Subsection H.4.1.7.  Quantify recovery of the
     carbamates using HPLC.   Nuchar: S-N is  satisfactory if recovery is  >^9
6.   Silanized Celite 545.  Slurry 150 g of Celite 545 (Johns-Manville)  *itn
     1 liter of 6 M hydrochloric acid in a 2-liter beaker,  cover with a  watch
     glass, and stir magnetically while boiling it for 10 minutes.   Cool,
     slurry, filter, and wash with ultrapure water until  the filtrate is
     neutral.  Wash Celite with 500 ml of methanol followed by 500  ml of
     methylene chloride, then air dry in a hood on a watch  glass to remove
     solvent.  Transfer Celite to a 1-liter Erlenmeyer flask with ground-glass
     joint.  Heat in the unstoppered flask at 120C overnight, and  then  cool
     the flask in a desiccator.  Place the flask in a hood  and carefully pipet
     3 ml of dichlorodimethylsilane (Pierce Chemical  Co.) onto the  Celite.
     Stopper, mix well, and allow to remain at room temperature for 4 hours.
     Add 500 ml of methanol to the flask, mix, and let stand 15 minutes.
     Filter the silanized Celite and wash with isopropanol  until neutral.   Air
     dry in a hood to remove isopropanol.  Dry the silanized Celite at 105C
     for 2 hours, cool  in a desiccator, and store in a glass-stoppered con-
     tainer.  Test Celite for total silanization by placing 1  g in  50 ml of
     water and 1 g in 20 ml of toluene saturated with methyl red/toluene solu-
     tion.  If Celite neither floats on water nor appears yellow with the
     methyl red solution, repeat the silanization to cover  active sites.

7.   Charcoal, Nuchar S-N.  Slurry 100 g of Nucnar 5-N vFisner} *ith 70C oil
     of HC1 , ".ove** with a watch glass, and boil for one hour with constant

                                    III-260

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      stirring.  Add 700 ml  of  distilled water, stir, and boil for 30 minutes.
      Cool,  slurry, filter,  and wash with ultrapure water until neutral.  Wash
      with 500 ml of methanol followed by 500 ml of methylene chloride and air
      dry in a hood to remove solvent.  Dry at 120'C for 4 hours, cool in a
      desiccator, and store  in  a glass-stoppered container.

 8.    Ethyl  ether, free of peroxides as indicated by EM Quant strips (Scien-
      tific  Products Co. No. P1126-8).  Procedures recommended for removal of
      peroxides are provided with the test strips.  After cleanup, 20 ml of
      ethanol preservative must be added to each liter of ether.

 9.    Florisil-PR grade (60/100 mesh).  Purchase Florisil activated at 1250F
      and store it in the dark  in a glass container with a ground-glass stopper
      or foil-lined screw cap.  Before use, activate each batch at least 16
      hours  at 130C in a foil-covered glass container.

 10.   Hexane

 11.   Hydrochloric acid aqueous solution, 6M.

 12.   Isooctane

 13.   Isopropanol

 14.   Methylene chloride

 15.   Methanol
     
 16.   Nitrogen gas, ary, pun flea.

'17.   Pentafluorobenzyl (PFB) bromide reagent, 1 percent (v/v); prepare by
      dissolving 1 ml of reagent (Pierce Chemical  Co., Rockford, IL, No. 58220)
      or alpha-bromo-2,3,4,5,6-pentafluoroto1uene (Aldrich Chemical Co.,
      Milwaukee, WI) in 100 ml of acetone in a low actinic volumetric flask.
      Prepare fresh every 2  to 3 weeks.  Caution:   the reagent is a strong
      lachrymator.

 18.   Petroleum ether

 19.   pH indicator paper.

 20.   Potassium carbonate, analytical  reagent grade.

 21.   Potassium hydroxide, ACS reagent grade.

 22.   Reaction solution for HPLC fluorescence detection of carbamate insecti-
      cides.  Weigh 500 mg of -phthalaldehyde,  transfer to a 1-liter volumetric
      flask, add 10 ml of methanol, and swirl to dissolve.  Add ca. 500 ml  of
      0,05 M sodium tetraborate solution and 1.0 ml  of 2-mercaptoethanol  and
      dilute to volume with 0.05 M sodium teiraborate solution.

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23.  Silica gel, grade 950 (Davison Chemical  Co.,  Baltimore,  MD),  deactivated
     by adding 1.5 percent (w/w)  distilled water and mixing for 2  hours.   Store
     in a tightly-stoppered container in a desiccator.

24.  Sodium chloride, ACS reagent grade.

25.  Sodium hydrogen carbonate,  ACS reagent grade.

26.  Sodium hydroxide solution,  0.05 M, prepared in degassed  ultrapure water.

27.  Sodium sulfate, ACS, granular, anhydrous.   Heat-treat in a shallow tray  at
     400*C for at least 4 hours  to remove phthalates and other interfering
     organic substances.  Alternatively, heat for  16 hours at 450  to  5008C in  a
     shallow tray, or Soxhlet-extract with methylene chloride for  48  hours.

28.  Sodium tetraborate solution, 0.05 M, prepared  in degassed ultrapure  water.

29.  Sulfuric acid, concentrated, ACS reagent grade.

30.  Analytical  standards.  Dilute stock standards  with  methanol or
     acetonitrile to give 1 ug/ml or as needed.  Store  solutions in actinic
     glassware,  and in the refrigerator.  Most  carbamate standards stored in
     this manner are stable for  several months.  However,  methiocarb  sulfone
     and sulfoxide degrade within hours and days,  respectively,  even  *ith these
     precautions.

31.  Stock standard solutions, 1.00 ug/ul.  Accurately weigh  ca. 0.0100 g
     of pure material, dissolve  in pesticide-quality acetonitrile  or
     ...atnancl, ..nti ri'uts 'o  -clime ~'r, ~ ^O-^l  "olumetr^'c  f1ask.   '..aroer
     volumes may be used, if necessary.  If the  compound purity is certified
     at 96 percent or greater, the weight can be used without correction  to
     calculate solution concentrations.  Commercial  stock  standards certified
     by the manufacturer can also be used.  Transfer the solutions into
     TFE-fluorocarbon-sealed screw-cap vials  and store at  4C protected from
     light.  Frequently check stock standard solutions  for degradation or
     evaporation, especially just prior to preparing calibration standards
     from them.   Replace stock standards after  6 months, or sooner if
     comparison  with check standards indicates  a change  in concentration.

32.  Tetradecane

33.  Toluene

34.  Reagent-grade water, tested for the absence of interferences  at  the
     method detection limit of each compound of  interest.

35.  Water, ultrapure, prepared using the Milli-Q  water  purification  system
     (Millipore  Corp.).  Water and solvents used for HPLC  mobile phases are
     degassed by placing them in a glass bottle, applying  vacuum and  slowly
     stirring with i magnetic stirrer for 5 minutes.
                                    III-262

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G.   QUALITY CONTROL

1.   The minimum requirements of this program consist of an initial demonstra-
     tion of laboratory capability and the analysis of spiked samples as a
     continuing check on performance.  The laboratory should maintain perform-
     ance records to define the quality of data that are generated.

     Before performing any analyses, the analyst must demonstrate the ability
     to generate data of acceptable accuracy and precision with this method.
     This ability is established as described in Subsection G.2.  Each time
     the analytical  method is modified, in response to state-of-the-art ad-
     vances, the analyst is required to repeat the procedure in Subsection G.2.

     The laboratory should spike and analyze a minimum of 10 percent of all
     samples to monitor continuing laboratory performance as described in
     Subsection G.4.

2.   To establish the ability to generate data of acceptable accuracy and
     precision, the analyst must perform the following operations:

     Select a representative spike concentration for each compound  to be meas-
     ured.  Using stock standards, prepare a quality control  check  sample
     concentrate in acetomtriie or methanol 1000 times more concentratea than
     the selected concentrations.

     Using a pipet, add 1.00"ml  of the check sample concentrate to  each of a
     minimum of four 1000-ml  aliquots of reagent water.   A representative
     waszewaxer may -e ussa ,r, piacs of ;ne reagent .vaisr-, jut jne  jr nore
     additional aliquots must be analyzed to determine background levels, and
     the spike level must exceed twice the background level  for the test to be
     valid.  Analyze the aliquots  according to the method, beginning with
     Subsection 1.2.1.5.

     Calculate the average percent recovery (R), and the standard deviation of
     the percent recovery (s), for the results.   Wastewater background correc-
     tions must be made before R and s calculations are performed.

     Table 2 (p. III-270) provides single-operator recovery and precision data
     for most of the carbamate and urea pesticides.  Similar results should be
     expected from reagent water for all  compounds listed with the  method.
     Compare these results to the  values calculated.  If the data are not
     comparable, review potential  problem areas  and repeat the test.

3.   The analyst should calculate  method performance criteria and define the
     performance of the laboratory for each spike concentration and compound
     being measured.

     Calculate upper and lower control limits for method performance as
     foi1ows:

          Upper Control Limit (UCL) = -R-+ 3 s
          Lower Control Limit (LCL)  R - 3 s

                                    III-263

-------
     where R and s are calculated as above.   The UCL and LCL can be used to
     construct control charts' that are useful  in observing trends  in per-
     formance.

     Separate accuracy statements of laboratory performance should  be developed
     and maintained for wastewater samples.   An accuracy statement  for the
     method is defined as R  s.   The accuracy statement should be  developed by
     the analysis of four aliquots of wastewater, as described in Subsection
     6.2, followed by the calculation of R and s.  Alternatively, the analyst
     may use four wastewater data points gathered through the requirement for
     continuing quality control.   The accuracy statements should be updated
     regularly.?

4.   The laboratory is required to collect in duplicate a portion of their
     samples to monitor spike recoveries.  The frequency of spiked-sample
     analysis should be at least 10 percent  of all  samples or one spiked sample
     per month, whichever is greater.  One aliquot  of the sample must be spiked
     and analyzed as described in Subsection G.2  If the recovery for a par-
     ticular parameter does not fall  within  the control  limits for  method
     performance, the results reported for that compound in all  samples pro-
     cessed as part of the same set must be  qualified as described  in Sub-
     section J.  The laboratory should monitor the  frequency of data so
     qualified to ensure that it  remains at  or below 5 percent.

5.   Before processing any samples, the analyst must demonstrate through the
     analysis of a^l-liter aliquot of reagent water that ail  glassware and
     reagent interferences are under control.  Each time a set of samples is
     extracted or there is a change in reagents, a  laboratory reagent blank
     mus": oe processed as d safeguard against: laooratory ^an^umir.aL'ion.

6.   It is recommended that the laboratory adopt additional  quality assurance
     practices for use with this  method.  The specific practices that are most
     productive depend upon the needs of the laboratory and the nature of the
     samples.  Field duplicates may be analyzed to  monitor the precision of the
     sampling technique.  When doubt exists  over the identification of a peak
     on the chromatogram, confirmatory techniques such as chromatography with a
     dissimilar column, or ratio  of absorbance at two or more wavelengths may
     be used.  Whenever possible, the laboratory should perform analysis of
     quality control materials and participate in relevant performance evalu-
     ation studies.

H.   CALIBRATION

     Establish HPLC operating parameters equivalent to those indicated in
     Table 1 (p. III-269).  The HPLC system  may be  calibrated using either, the
     external standard technique or the internal standard technique.

1.   External Standard Calibration Procedure

     For eacn compound of interest, prepare  calibration standards it i minimum
     of three concentration levels by adding accurately measured volumes of one
     or more stocx standards to a volumetric f'ask  and diluting to  volume with

                                    III-264

-------
acetonitrile or methanol.  One of the external standards should be repre-
sentative of a concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of concentrations
expected 1n the sample concentrates or should define the working range of
the detector.

Using  Injections of 10 pi of each calibration standard, tabulate peak
height or area responses against the mass injected.  The results can be
used to prepare a calibration curve for each compound.  Alternatively, the
ratio of the response to the mass injected, defined as the calibration
factor (CF), may be calculated for each compound at each standard concen-
tration.  If the relative standard deviation of the calibration factor is
less than 10 percent over the working range, the average calibration fac-
tor can be used in place of a calibration curve.  The working calibration
curve or calibration factor must be verified on each working shift by the
measurement of one or more calibration standards.  If the response for any
compound varies from the predicted response by more than 10 percent, the
test must be repeated using a fresh calibration standard.  Alternatively,
a new calibration curve or calibration factor must be prepared for that
compound.

Internal Standard Calibration Procedure

To use this approach, the analyst must select one or more internal  stand-
ards similar in analytical behavior to the compounds of interest.  The
analyst must further demonstrate that the measurement of the internal
standard is not affected by method or matrix interferences.  Due to these
V* mi tat"! ens, n 'iter^al  standard annl1'cable to all  samples can be sug-
gested.

Prepare calibration standards at a minimum of three concentration levels
for each compound of interest by adding volumes of one or more stock
standards to a volumetric flask.  To each calibration standard, add a
known constant amount of one or more internal  standards, and dilute to
volume with acetonitrile or methanol.  One of the standards should be
representative of a concentration near,  but above, the method detection
limit.  The other concentrations should correspond to the range of con-
centrations expected in the sample concentrates or should define the
working range of the detector.

Using Injections of 10 ul of each calibration standard, tabulate the peak
height or area responses  against the concentration for each compound and
Internal standard.  Calculate response factors (RF)  for each compound as
fol1ows:

                    RF  =  (AsC1s)/(A1s  Cs)

where:

     As   =  response for the compound to be measured
     AiS  -  response for the Internal  standard


                               III-255

-------
     C-js  =  concentration of the Internal  standard in ug/1
     Cs   =  concentration of the compound to be measured in ug/1.

If the RF value over the working range is constant, less than 10 percent
relative standard deviation, the RF can be assumed to be invariant,  and
the average RF may be used for calculations.  Alternatively, the results
may be used to plot a calibration curve of response ratios,  As/A-js,
against RF.

The working calibration curve or'RF must be verified on each working shift
by the measurement of one or more calibration standards.  If the response
for any parameter varies from the predicted response by more than 10
percent, the test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve must be prepared for  that compound.
                                III-266

-------
I.   ANALYTICAL PROCEDURES

     1.1   Determination of Carbamates  and Urea  Pesticides  in Hazardous Waste
          Samples.   Reserved.
                                    m-267

-------
2.1  Determination of Carbamate and Urea Pesticides in Industrial  and Municipal
     Wastewater
          Analytical  Procedure:   available
          Sample Preparation:   available

     2.1.1  Reference

            Pressley, T. A.  and J.  E.  Longbottom,  "The Determination  of  Carba-
            mate and Urea Pesticides in  Industrial  and Municipal Wastewater  -
            Method 632."  Report No. EPA-600/4-82-014, Environmental  Monitoring
            and Support Laboratory, Office of Research and  Development,  U.S.
            EPA, Cincinnati, Ohio (February,  1982).4

  "   2.1.2  Method Summary

            A measured volume of sample, approximately 1  liter,  is solvent-
            extracted with methylene chloride in a  separatory  funnel.  The
            extract is dried and concentrated to a  volume of 10  ml  or less.   If
            necessary, the extract  is  cleaned up on a  Florisili column.   The
            extract or column eluate is  analyzed by reversed-phase HPLC  with UV
            detection.

     2.1.3  Applicabilty

            The method covers the determination of  the compounds listed  in
            Table 1 plus the Bellowing:   aminocarb, carbofuran,  fenuron,
            fenuron-trichloroacetate (fenuron-TCA), monuron-trichloro-
            acetate (monuron-TCA),  Siduron, and Swep.   Neither monuron and
            Ticnuron-TCA nor  fenuron and  fenuron-TCA are distinguished by the
            method; results  ror tnese  pairs are reported  as monuron ana  renurcn,
            respectively. Detection limits range  from 0.003 to  11 ug/1
            (Table 1).  The  detection  limit is defined as the  lowest  concen-
            tration of pesticide that can be  measured  and reported with  99
            percent confidence that the  value is above zero.   The  values
            listed in Table  1 were  obtained using  reagent or river water.

     2.1.4  Precision and Accuracy

            Single-laboratory recovery and precision (standard deviation) data
            are listed in Table 2 for 15 pesticides that  were  determined with
            this method using water from five sources.

     2.1.5  Sample Extraction

            Mark the water meniscus on the side of  the sample  bottle  for later
            determination of sample volume.   Pour  the  entire sample into a
            2-liter separatory funnel.

            Add 60 ml methylene chloride to the sample bottle, seal,  and shake
            30 seconds to rinse the inner walls.  Transfer  the solvent to the
            separatory funnei ana extract cne sample by shaking  the runnel  ffor


                                    III-268

-------
        TABLE 1.  CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
     ==========================
  Parameter
Mobile
Phase*
Retention
Time (Min)
    UV
Wavelength
   (nm)
  Method
 Detection
Limit (ng/1)
Mexacarbate
Propoxur
Monuron
Carbaryl
Propham

Di uron
Linuron
Methiocarb
Chlorpropham
Barban
Neburon
Me thorny!
Carbaryl
Diuron
Linuron

Propoxur
Carbofuran
Fl uorometuron

Oxamyl
  A
  A
  A
  A
  A

  A
  A"
  A
  A
  A
  A

  3
  B
  8
  B
  B
  C
  C
  8.7
 14.3
 14.3
 17.0
 17.2

 19.5
 21.0
 21.4
 21.8
 22.3
 24.3

  2.0
  6.5
 14.1
 15.5
 17.9

  i. 7
  3.5
  3.6

  3.2
   254
   280
   254
   280
   254

   254
   254
   254
   254
   254
   254

   280
   254
   280
   254
   254

   230
   280
   254

   254
  0.52
  0.11
  0.003
  0.02
  0.07
0.
0.
0.
   .009
   .009
   .02
  0.03
  0.05
  0.012

  0.11
  6.9
  O.D2
  0.009
  0.009
  3.2
 11.1

  9.2
aaa===========================================================================
*Mobile Phase:

A = Methanol /I percent acetic acid, programmed linearly from 5 to 95 percent
    methanol at 2.0 ml/min flow rate and at ambient temperature.
B = Acetonitrile/water, orogrammed linearly from 10 to 100 percent acetonitrile
    1n 30 min at a flow rate of 2.0 ml/min.
C * 50 percent acetonitrile in water at a flow rate of 2.0 ml/min.
D  35 percent methanol 1n water at a flow rate of 2.0 ml/min.
Column: u Bondapak Cia (10 urn) packed in a 30 cm long x 4 mm I.D.  stainless
steel column, with a Whatmann Co. PELL ODS (30-38 urn) guard column,  7 cm
long x 4 mm I.D.

-------
TABLE 2. SINGLE-OPERATOR
Sample Spike
Parameter Type* (ug/1)
Fl uorometuron


Propoxur



Oxamyl


Me thorny!



Diuron

4


Linuron




Carbofuran

Barban
Carbaryl
Chlorpropham
Methiocarb
Mexacarbate
Monuron
Neburon
Propham
====================
*  Sample type
1 * Reagent water
1
2
4
1
3
4
5
1
2
2
1
3
2
2
1
*
2
2
5
1
2
2
2
5
1
4
5
5
5
5
5
c
5
5
50
50
1724
550
2200
550
0.5
100
53
1080
100
30660
100
1960
10
SCO
10
400
0.05
10
1000
10
210
0.05
37
148
0.3
0.1
0.2
0.2
4.0
0.05
0.05
0.3
ACCURACY AND PRECISION
=======================
Average
No. of Percent
Analyses Recovery
7
7
7
7
3
7
5
7
7
7
4
4
7
7
4
4
7
7
5
4
d
7
7
5
7
7
5
5
5
5
5
5
5
5
93 9
80.0
99
94.5
105
87.2
93
87
84.9
89.8
74.4
48.2
91.8
94.4
89.8
<6.1
90.6
35.7
98"
95.0
?2.2
93.0
103
99
87.8
99.3
98
101
95
95
96
97
96
88
= = = = = === = ===== = = = = =.= = = = = = = = = = = = = = = =====s = :s = =








1
Standard
Deviation
(I)
7.0
7.2
11.6
1.7
3.0
7.3
6.0
8.4
5.5
2.7
2.4
2.8
2.8
1.9
1.0
5.0
2.5
3.2
4.7
3.4
5.1
1.5
4.6 '
4.7
2.7
1.4
4.1
4.1
3.9
2.6
3.5
1.7
6.6
5.9
==============


2 * Municipal wastewater
3 = Industrial process water, pesticide
4 = Industrial wastewater,
5 - 31ver yatsr

manufacturing


pesticide manufacturing




III-270

-------
 2 minutes  with  periodic  venting to release excess pressure.  Allow
 the  organic  layer to  separate from the water phase for a minimum of
 10 minutes.   If the emulsion interface between  layers is more than
 one  third  the volume  of  the solvent layer, the  analyst must employ
 mechanical techniques to complete the phase separation.  The opti-
 mum  technique depends upon the sample, but may  include stirring,
 filtration of the emulsion through glass wool,  centrifugation, or
 other  physical  methods.   Collect the methylene  chloride extract in a
 250-ml  Erlenmeyer flask.

 Add  a  second 60-ml volume of methylene chloride to the sample
 bottle and repeat the extraction procedure a second time, combining
 the  extracts in the Erlenmeyer flask.  Perform  a third extraction
 in the same  manner.

 It is  necessary to exchange the extract solvent to hexane if the
 Florisil cleanup procedure is to be used.  For  direct HPLC analysis,
 the  extract  solvent must be exchanged to a solvent (either methanol
 or acetonitrile) that is compatible with the mobile phase.  The
 analyst should  only exchange a portion of the extract to the HPLC
 solvent if there is a possibility that cleanup  may be necessary.

 Pass a measured fraction or all of the combined extract through a
 drying column containing about 10 cm of anhydrous sodium sulfate
 and  collect  the extract  in a 500-mi rouna-oottom fiasK.  rvinse :ne
. Er'enmeyer flask and  column with 20 to 30 ml of methylene chloride
 to complete  the quantitative transfer.

 Attach the 500-ml round-bottom flask containing the extract to the
 rotary evaporator ana parviai'y  immerse  ,n che  50C *aisr jawh.

 Concentrate  the extract  to approximately 5 ml in the rotary evap-
 orator at  a  temperature  of 50C.  Other concentration techniques
 may  oe used  if  che accuracy and precision requirement in the
 Quality Control  Subsection are met.

 Add  50 ml  of hexane,  methanol, or acetonitrile  to the round-bottom
 flask  and  concentrate the solvent extract as before.  When the
 apparent volume of liquid reaches approximately 5 ml, remove the
 500-ml  round-bottom flask from the rotary evaporator and transfer
 the  concentrated extract  to a 10-ml volumetric  flask.  Wash the
 round-bottom flask with  2 ml of solvent and add the wasmngs to
 the  extract.  Dilute  the extract to volume with solvent.

 Stopper the  volumetric flask and store it at 4C if further pro-
 cessing will not be performed immediately.  If  the extracts will
 be stored  longer than 2  days, they should be transferred to
 TFE-f1uorocarbon-sealed  screw-cap bottles.

 Determine  the original sample volume by refilling the sample bot-
 tle  to the mane  ana transrerring tne water ;o d *000-ini ^r
 cylinder.  ecord the samole volume to the nearest 5 ml.

                         III-271

-------
2.1.6  Cleanup and Separation

       Cleanup procedures may not be necessary for a relatively clean
       sample matrix.   The cleanup procedure recommended  in  this method
       has been used for the analysis of various industrial  and municipal
       effluents.   If particular circumstances demand the use of an
       alternative cleanup procedure, the analyst must determine the
       elution profile and demonstrate that  the recovery  of  each compound
       of interest for the cleanup procedure is no less than 85 percent.

       The following Florisi 1-column cleanup procedure has been demon-
       strated to  be applicable to the five  pesticides listed in Table 3.
       It should also  be applicable to the cleanup of extracts for the
       other carbamate and urea pesticides listed in paragraph 2.1.3.

       Add a weight of Florisil  (nominally 20 g), predetermined by
       lauric acid calibration (see below),  to a chromatographic column.
       Settle the  Florisil by tapping the column.  Add anhydrous sodium
       sulfate to  the  top of the Florisil  to form a layer 1  to 2 cm  deep.
       Add 60 ml of hexane to wet and rinse  the sodium sulfate and
       Florisil.   Just prior to exposure of  the sodium sulfate to air,
       stop the elution of the hexane by closing the stopcock on the
       chromatography  column.   Discard the eluate.

       Adjust the  sample extract volume to 10 mi  with  nexane  ana transfer
       it from the volumetric flask to the Florisil  column.   Rinse the
       flask twice with 1 to 2 ml  hexane,  adding each  rinse  to tne column.

       Drain the column until  the sodium sulfate layer is nearly exposed.
       tlute tne column wren 00 .ni  of ^cnyi  etner/hexane ^20/50 .n]
       (Fraction 1) using a drip rate of ca.  5 ml/minute.  Place a 500-rnl
       round bottom flask under the column.   Elute  the column again  with
       200 ml of acetone/hexane (6/94 v/v) (Fraction 2) into  a second
       flask.  Perform a third elution using acetone/hexane  (15/85 v/v)
       (Fraction 3),  and a final  elution with 200 ml  of acetone/hexane
       (50/50 v/v) (Fraction 4), into separate flasks.  The  elution
       patterns for five of the pesticides are shown in Table 3.  Con-
       centrate the eluates to 10 ml  with a  rotary evaporator,  exchanging
       the solvent to  acetonitrile or methanol  as required.

       Florisil  from.different batches or sources may  vary in adsorptive
       capacity.   To standardize the amount  of Flonsil tnat  is  used, the
       use of the  lauric acid value is suggested.   With this  procedure5,
       the adsorption  from hexane solution of lauric acid, in mg per g of
       Florisil  is determined.   The amount of Florisil  to be  used for
       each column is  calculated by dividing this factor  into 110 and
       multiplying by  20.g.   Before using any cleanup  procedure,  the
       analyst must process a series of calibration  standards through the
       procedure to validate elution patterns and the  absence of inter-
       ference from the reagents.
                               III-272

-------
           TABLE 3.  RECOVERY FROM COLUMN CLEANUP PROCEDURE

                              	Percent Recovery by Fraction	
 Parameter                    No. 1      No.  2      No.  3      No.  4
Diuron
Linuron
Met homy!
Oxamyl
Propachlor
Florisil eluate compos i ton by
0
0
0
0
0
fraction
0
13
0
0
94

24
82
0
92
0

58
0
84
0
0

 Fraction 1  -  200 ml of 20 percent ethyl  ether in hexane
 Fraction 2  -  200 ml of 6 percent acetone in hexane
 Fraction 3  -  200 ml of 15 percent acetone in hexane
 Fraction 4  -  200 ml of 50 percent acetone in hexane


2.1.7  Sample Analysis by HPLC

       In Table 1, the recommended operating conditions for the liquid
       cnrcmaiograpn are jummar'zed.  Included in tm's taole ire estimated
       -etsntion times and method detection limits that can be achieved by
       chis metnoa.  An example of the 3epar2tions achieved by *his  column
       is shown in Figure 1.  Other HPLC columns, chromatographic condi-
       tions, or detectors may be used if the quality control  requirements
       Calibrate the system daily as described below.   The standards and
       extracts must be dissolved in a solvent (acetonitrile or methanol)
       compatible with the mobile ohase.

       If the internal  standard approach  is being used, add the internal
       standard to sample extracts immediately before  injection into the
       instrument.  Mix thoroughly.

       Inject 10 yl of the sample extract.   Record the volume of the
       extract injected to the nearest 0.05 vl , and the resulting peak
       size in area or peak height units.

       The width of the retention time window used to  make identifications
       should be based upon measurements  of actual retention time varia-
       tions of standards over the course  of a day. Three times the
       standard deviation of a retention  time can be used to calculate  a
       suggested window size for a compound.  However, the experience of
       the analyst should weigh heavily in  the interpretation of chro-
       matograms.

       If the response for the peak  exceeds the working range of the
       system, dilute cne dxtracz ana raanalyre.
                               iiI-273

-------
                    0       6       10       15      20

                          Retention Time (minutes)
c*g'jre l.  Mould rthromatoaram of urea herbicides on Column 1.  Compounds are:
       1) methomy!; 2) diuron; 3) linuron.   r'or conditions, see 7dDie A.
                                    III-274

-------
       If the measurement of the peak response  1s  prevented  by the  presence
       of Interferences,  further cleanup 1s  required.   Calculate  results
       according to Subsection J.

2.1.8  Confirmation of Residues

       When this method Is used to analyze unfamiliar  samples  for any or
       all  of the compounds to which  it is applicable,  compound identi-
       fication should be supported by at least one additional  qualitative
       technique, such as GC/MS.

       A similar procedure for the determination of phenylureas 1n  water
       was published by Farrington et a!.6  The sample  1s  extracted with
       methylene chloride, and determination is by HPLC on a 5-um Cjg
       reversed phase column developed with  methanol/0.6 percent  aqueous
       ammonia (60/40 v/v), with UV detection at 240 nm.  Recoveries  in
       excess of 95 percent and detection limits of 0.01 ug/ml were
       reported for residues of chlorbromuron,  chloroxuron,  diuron,
       linuron, metobromuron, monolinuron and monuron.
                               III-275

-------
3.1  Determination of Carbamate Pesticides in Soil
          Analytical  Procedure:  available
          Sample Preparation:  available

     3.1.1  Reference

            Hall, R.  C. and D.  E. Harris, "Direct Gas Chromatographic
            Determination of Carbamate Pesticides Using Carbowax 20M-
            Modified  Supports and the Electrolytic  Conductivity Detector."
            0. Chromatogr.  169:245(1979).8

     3.1.2  Method Summary

            A sample  is extracted with acetone/aqueous sodium chloride/
            aqueous sodium hydrogen carbonate solution and the extract is
            partitioned with benzene.  The solution is concentrated,
            cleaned up on a deactivated Florisil  column, and the pesti-
            cides in  the column eluate are determined by GC using a
            Carbowax  20M column and Hall  electrolytic conductivity
            detector.

     3.1.3  Applicability

            The 22 carbamate pesticides shown in Table 4 were determined
            .at 0.1 and 1.0 ppm levels.

     3.1.4  Precision and Accuracy

            As seen in Table 4, recoveries at ooth  concentration ;eveis
            ranged from 66 to 112 percent with an average of 39 percent,
            excluding 2-chloroallyl diethyldithiocarbamate (CDEC) and
            carbaryl  at 0.1 ppm.  The former compound was recovered to the
            extent of 33 percent, and the latter could not be determined
            because of low response.  Standard deviations for three trials
            ranged from 0 to 20 percent, with an average of 5 percent.

     3.1.5  Sample Preparation

            Air dry a 50-g soil sample and pass it  through a 20-mesh sieve.
            Thoroughly mix the soil and extract with 100 ml of extraction
            solution on a wrist-action shaker for 10 minutes.  The extraction
            solution is acetone/aqueous 2 percent sodium chloride + 1 percent
            sodium hydrogen carbonate (80/20 v/v).   Filter the extract through
            Whatman No. 1 filter paper, and rinse the filter cake with 100 ml
            of extraction solution.  Dilute the filtrate with an additional
            100 ml of extraction solution and extract three times with 50 ml
            of benzene.  Combine the extracts and dry on a sodium sulfate
            column (40g).  Elute any residual pesticide with an additional
            25 ml of benzene.  Add 1 ml of a 2-oercent tetradecane solution
            in benzene to the dried extract 10 serve as a  keeper".  Evaporate
            the  3xtr;ict *.o ca. 15 ml on 3 'otary avaoorator,  To determine
            recovery with this method, fortify samples at concentrations

                                    IIT-276

-------
     TABLE 4.
                      RECOVERY OF CARBAMATE PESTICIDES
                        FROM FORTIFIED SOIL
Compounds
                                            Recovery {%)
                                       0.1 pm       1.0 ppm
Aminocarb
Benthiocarb
Butyl ate
Bux
Carbaryl

Carbofuran
CDEC
Chlorpropham
Dimetilan
EPTC

2,3,5-Landrin
3,4,5-Landrin
Meobal
Methiocarb
Mexacarbate

Pebulate
Propham
Pyramate
SWEP
Terbutcl

Triallate
Vernolate
                                85  7*
                                82  20
                                95  3
                                88  6
                                 **
                                                    85  4
                                                    92  5
                                                    86  2
                                                    99  5
                                                    85  0
92  4
33  5
92  9
83  3
75  5
112 i 8
96  8
87  2
84  3
86  7
105  7
101 * i
99  5
85 i 8
QO t 6
92  9
87  2
saaaasaassaa:
86  5
66  3
80  5
71  2
87  3
96  4
98  4
92  5

85  1
94  3
04 ^ a
92  5
82  6
83 t 6
75  6
90  4
:aaaaas=aa=a=
    * Standard deviations for three detenrnnations.
   ** Determination precluded by insufficient reponse.
       of 0.1 and/or 1.0 ppm by addition of 2 ml  of a benzene solution
       of the pesticides.

       Transfer the solution quantitatively to a  ?5-ml  Kuderna-Oanish
       concentration tube, and reduce the volume  to 0.75-1.0 ml  on a
       rotary evaporator.  Transfer the concentrated sample quanti-
       tatively to a 200-mm x 9-mm column containing 1.5 g of Florisil
       (that has been deactivated by adding 10 percent water) and a top
       layer of 1 gram of sodium sulfate.  Elute  with 25 ml  of diethyl
       ether/benzene (25/75 v/v).

3.1.6  Sample Analysis

       Concentrate the column eluate to an appropriate volume (e.g.,
       2.5 mi for 0.1 ppm of pesticide)  and 'nject ca.  S ul  onto +.he
                               III-277

-------
       GC column.  Compare sample chromatograms with those of standards
       obtained under Identical  chromatographic conditions.   Represent-
       ative chromatograms of soil  extracts on the 3-percent OY-101/
       Carbowax 20 M column are  shown in Figures 2 and 3.   Extraneous
       peaks were not observed in the 1-ppm samples, but impurity peaks
       were present at 0.1 ppm.   These impurities interfered somewhat  with
       the determination of butyl ate, pebulate, EPIC, mexacarbate,  and
       vernolate on the OV-101 column.  Use of one of the  alternative
       columns or alternate temperature programs might improve analysis
       of these pesticides.  Calculate results acording to Subsection  J.

3.1.7  Confirmation of Residues

       Residues can be confirmed by GC/CI-MS under the following
       conditions:

       A Finnigan Model 3200 gas chromatograph-mass spectrometer
       equipped with a chemical-ionization  source and a Model  6100
       data system is used with  isobutane as the reaction  gas.
       Silanized glass columns (1.5 m x 2 mm I.D.) are operated with
       isobutane as the carrier  gas.  The carrier gas also serves as
       the reaction gas.  Source pressure is maintained at 550 urn.
       Column, source, transfer  and separator temperatures are 170,
       60*, 190*; ?nd ?2Q, "esDectivelv.  The electron energy is 82
       eV, and the emission current is *.Ji ..TA.
                               III-278

-------
          >
          -s  <
            I
          E  I
                        (a)
             -All
(b)
                                              I
                 0     I     4        0     5      4

                         Retention Time (minutes)
Figure 2.  Chromatograms of soil extract of carbamate pesticides separated
      on a 3-percent OY-101 on Ultra-Bond column.  Compounds are:
     1) pebulate, 2) 2,3,5-landrin, 3) 3,4,5-Landrin, 4) aminocarb
   5) benthiocarb. a) 10rng standard; b) extract of fortified soil.

   Operating conditions:  Helium at 25 ml/min is the carrier gas and
   hydrogen at 30 -nl/rain *s the ^action qas. The temperatures are:
  170*C, column; 180*C, Inlet; 200"C,  transrer tine; ema /20*C, rurnace.
                             111-279

-------
    1.CH
                                                   2
                                                          (b)
            024                    024

                          Retention Time (minutes)
Figure 3.  Chromatograms  of  soil extract of carbamate pesticides separated
       on a 3-percent OY-101 on Ultra-Bond column.  Compounds are:
                 1) propham, 2) pyramate, 3) mexacarbate.
             *)  10-nq standard: b) extract of fortified soil.

              Operating conditions ire  -nven under F

                                III-280

-------
3.2  Determination of Urea Herbicides in Soil
          Analytical  Procedure:   available
          Sample Preparation:  available

     3.2.1  Reference

            Farrington, D. S., R. G.  Hopkins, and 0.  H. A. Ruzicka,
            "Determination of Residues of Substituted Phenylurea Herbicides
            in Grain, Soil, and River Water by Use of Liquid Chromatography."
            Analyst 102:377 (1977).6

     3.2.2  Method Summary

            The phenylurea herbicides are extracted from soil  with meth-
            anol  and  are determined by HPLC on microparticulate Cjg-banded
            silica using a mixture of methanol, water, and ammonia as the
            mobile phase, and a UV absorption detector.

     3.2.3  Applicability

            Residues  of chlorbromuron, chlortoluron,  chloroxuron,  diuron,
            linuron,  metobromuron, monolinuron, and monuron were determined
            {n soil 2t a-concentration of 2 rag/kg fpom).  The lower limit.
            of detection was estimated co oe Q.I ppm.

     3.2.4  Precision and Accuracy

            The recoveries obtained with the method are shown in Table 5.
            The precision or cne .netncci ,i ^ncr.cn'cc-a  -,y ;r,e -inges :f-
            recovery  for five determinations of aach  compound.   The
           .recovery  for all compounds averaged 99.8  percent.

     3.2.5  Sample Preparation

            Air dry a soil sample and transfer 50 g into a 500-ml
            Erlenmeyer flask.  Add 100 ml  of methanol  and shake on a
            wrist-action shaker for 1 hour.  Filter the resulting slurry
            through Whatman No.  1 filter paper at reduced pressure.   Wash
            the flask with 50 ml of methanol  and add  the washings  to the
            filter funnel, leave for  4 minutes, and then apply vacuum.
            Repeat the wasning procedure with another JO ,m  of aietnanoi.

            Combine the extract  and washings  and remove the methanol  on
            a  rotary  evaporator with  a water bath at  55C.  Dissolve the
            residue in methylene chloride,  using a total  volume of 50 ml,
            and pass  the methylene chloride extract through a  column of
            anhydrous sodium sulfate  (50 g).   Wash the sodium  sulfate with
            50 ml  of  methylene chloride, combine the  extract and washings,
            and evaporate to dryness  at 55C  in a rotary evaporator.
            Cooi  the  flasK ana dda .0 ,nl  of  rnethanol, jwirl  *o dissolve
            the residue,  and filter through Whatman No.  42 filter  paper.


                                   TII-281

-------
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-------
    o
    1
    JD
    <
                                0.01
                                unit
                                  0.01
                                  unit
(a)
         0    10   20  30   40        0   10    20   30

                         Retention Time (minutes)
                                      40
Figure 4.  Typical chromatograms obtained from 5-iil Injections of son
extracts.  Compounds are.: 1) monuron, 2) monolinuron, 3) metobromuron,
    4) chlortoluron, 5) diuron, 6) Unuron, 7) chlorbromuron, and
       8) chloroxuron.  (a) unfortified; and (bj fortified with
                    \iron herbicides .t ? mq kq-1.
         Permaphase ODS column, 240 nm detection wavelength.
                               III-283

-------
4.1  Determination of Carbamate Pesticides in Fruits and Vegetables
          Analytical  Procedure:  evaluated
          Sample Preparation:  available

     4.1.1  References

            Krause, R. T., "Multiresidue Method for Determining
            N-Methylcarbamate Insecticides in Crops, Using High Perfor-
            mance Liquid Chromatography."  J. Assoc. Off. Anal. Chem.
            63(5):1114 (1980).13

            Krause, R. T. and M. August, "Applicability of a Carbamate
            Insecticide Multiresidue Method of Determining Additional
            Types of Pesticides in Fruits and Vegetables."  J.  Assoc.
            Off. Anal. Chem.   66(2):234 (1980)."

     4.1.2  Method Summary

            Residues are extracted from crops using methanol.   Coextrac-
            tants are removed by solvent partitioning and Chromatography.
            The carbamate residues are separated on a reversed-phase HPLC
            column eluted witn an acetonitrile/water gradient and detected
            using an in-line  post-column fluorometric labeling  technique.

     4.1.3  ApplicaDility

            The method is applicaole to the seven caroamates and four
            related carbamate. metabolites shown in Table 6, at  concentra-
            tion levels of 0.05 ppm or greater. ' The method was recently
            sxtsnaea tcj  induce pesticiues or otner clas3C-!: and -'afyir.rj
            polarity.1'*

     4.1.4  Precision and Accuracy

            Except for aldicarb sulfoxide, pesticide recovery from forti-
            fied samples averaged 99 percent at both 0.050 and  1.0 ppm,
            with standard deviations of 5.0 percent (n = 86) and 5.6
            percent (n = 87), respectively.  The average recoveries for
            the polar metabolite aldicarb sulfoxide were 55 and 57 at the
            respective concentration levels (Tables 6 and 7).   An inter-
            laboratory study of the method was conducted (Table 8).  The
            average recovery was 95 percent irange 37-101 percent) for
            duplicate analyses of grapes and potatoes fortified with
            carbaryl, methiocarb, and methomyl at levels ranging from
            0.050-15 ppm.

     4.1.5  Sample Extraction.

            Select the appropriate method of sample extraction  based on
            the water content of the original sample.  For high-moisture-
            content .samples  roi'iow paragraph i and  "or "*ow-Tioisture-
            content sameles follow oaragraph b.

                                    III-284

-------
TABLE 6.  CARBAMATE INSECTICIOE AND Mi TABOLIT i;LCOVERIES (PERCENT) THROUGH METHOD
                         AT 0.05 PPM iORTIFICATlON LEVEL3



(rop
Appl es
Carrots
Green beans
Green peppers
Lettuce
Oranges
Soybeans
 Strawberries
VTomutoes
00
^Av.
SO
Grand av.
Grand SO


Ald1-
carb
100
100
99
96
100
100
102
97
94

99
2.5
99e
5.0e

Aldi-
carb
sulfone
91
98
101
96

108
94

97
6.5





Oxamyl
94
93
96
93
97
92
95
98
91

94
2.4


a ^.Inijle determinations.
b Not jsed 1n
c Interference
av. or
due to
SD.
apparent

methomyl

1n crop














d Apparent crop coextractlve Interference.
e Aldicarb sul
foxide
not Included.

-------
TABLE 7.  CARBAMATE INSECTICIDE AND METABOLITE KECOVERIES (PERCENT) THROUGH METHOD
                         AT  1.0 PPM lORTIFICATiON LEVEL*











1-4
1 1
1
ro
ro






Aldi-
Crop carb
Apples 95
Carrots 100
Green beans 94
Green peppers 97
Lettuce 92
Onngt.s 98
So /beans 108
Strawberries 105
Tomatoes 97


Av. 98
SD 5.2
Grdmd av. 99e
Grand SD 5.6
==-=======
Aldi-
carb
sul f one
91
97
92
98
99
85
95
107
96


96
5.7


Aldi-
carb
sulf-
oxide
54
62
53
63
68
45
50
62
56


57
7.3




Bufen-
carb
93
99
102
97
99
98
105
109
98


100
4.7




Carb-
o-
fUi an
y'j
102
101
;)9
11)1
97
106
lil
100


Iu2
4.7


3-Hy-
droxy
Carbo-
furan
95
100
101
100
90
102
107
108
98


102
5.6




Methio-
carb
96
102
97
98
97
d
108
106
98


100
4.6


=========
Methio-
carb
sulf-
oxide
104
97
97
101
105
89
94
110
98


99
6.3




Meth-
omyl
92
99
b
102
100
85
95
106
95


97
6.5





Oxamyl
93
92
90
94
76C
89
95
103
94


94
4.3


a Si.igle determinations.


" Interference due to
c Nol used in av. or
apparent
sn.
methomyl

in ci

0,,.













d Apparent crop coextractive interference.

e Aldicarb sul f oxide
not included.

-------
       TABLE 8.  INTERLABORATORY COMPARISON OF HPLC METHOD FOR CARBAMATES
===============================================================================
                                   Grapes	           	Potatoes
Percent
Recovery
Carbamate/
metabolite
Carbaryl
Carbofuran
3-Hydroxy
carbofuran
Methiocarb
Met homy!
=====8=================
3 Interference present
"rfded,
ppm
8.0
1.0
0.05
0.05
0.05
=============

1
94
87
93
94
87

2
96
94
96
95
93

Added,
ppm
0.05
0.05
1.0
5.0
15.0

Percent
Recovery
1
a
101
99
99
94
=======
2
a
96
95
93
96
========
            a.   High-Moisture Products (greater than 75 percent water) - Add
            150 g of chopped sample and 300 ml  of .nethanol  to a homcgenirer jar
            and homogenize for 30 seconds at one-half speed (Polytron setting
            7)  and cnen for oO ^ecanas ^t ~ur  ;peed.  '-'acuu^-'ilter -.he
            hcmogenate through 3 12-cm nerforsted Buchner funnel  containing
            SharKSKin or 597 535 filter paper,  jollacting the filtrate 'i \
            500-ml filter flask.  Transfer a portion of filtrate equivalent to
            100 g sample to a 2-liter I 24/40 round-bottom flask.

            NOTE:  Volume of 100 g sample equals mi  water 'n 100 g samole, plus
            200 ml methanol, minus 10 ml  contraction factor.

            Add ultrapure water to the round-bottom flask to give a total of
            100 ml of water.

            Proceed to paragraph 4.1.6.

            b.   Dry or Low Moisture Products (e.g.,  grains) - Add 75 g of
            ground sample and 300 ml  of methanol  to  a homogenizer jar.  Homog-
            enize and filter as above in (a).  Transfer a portion of the
            filtrate equivalent :o 50 9 of jampla ;o a 2-t'ter 5 24/*0 round-
            bottom flask.

            NOTE:  Volume of 50 g sample equals ml  of water in 50 g sample plus
            200 ml of methanol.

            Add ultrapure water to the round-bottom flask to give a total of
            100 ml of water.   Add a small  star  magnetic stirrer to the flask.

            .'lacs a 250-inl  ~ tfr/~Q trap en the  "-liter -ryjnd-bottom *lask and
            attach to a rotary evaporator.  Apply vacuum slowly to minimize
                                    III-287

-------
       frothing.  After full vacuum is applied, slowly place the flask
       in a 35C water bath.  Concentrate the extract to 75 ml.

       Proceed to Subsection 4.1.6.

4.1.6  Cleanup of Extract by Solvent Partitioning

       Transfer the concentrated extract from the round-bottom flask
       to a 500-ml separatory funnel containing 15 g of sodium chloride.
       Shake the funnel until the salt is dissolved.  Wash the flask
       with three 25-ml portions of acetonitrile, transferring each
       to the separatory funnel, shake 30 seconds, and let the layers
       separate for 5 minutes.  Drain the aqueous phase into a 250-ml
       separatory funnel containing 50 ml of acetonitrile.  Shake 20
       seconds, let the layers separate, and discard the aqueous
       layer.

       Add 25 ml of 20-percent aqueous sodium chloride solution  to
       the acetonitrile in the 500-ml separatory funnel, shake 20
       seconds, let the layers separate, and transfer the .solution
       to the 250-ml separatory funnel.  Shake this funnel for 20
       seconds, let the layers separate, and discard the aqueous
       layers.

       Add 100 ml of petroleum ether to the 500-ml separatory
       funnel; shake 20 seconds, ''at *.fte Bayers separate, ;md oral n
       the.acetonitrile layer into a second 500-ml separatory funnel.
       Transfer the acetonitrile in the 250-mV funnel  to the first
       ^Or-Til ^'jr.ns! *hat tontairs rstr^leum ether-, shake 20 seconds.
       let the layers separate, ana transfer the acetomtrile to zne
       second 500-ml separatory funnel.  Add 10 ml of acetonitrile
       to the first funnel; shake, let the layers separate, and  trans-
       fer the acetonitrile to the second 500-ml funnel.  Discard the
       petroleum ether.

       Add 50 ml of 2-percent aqueous sodium chloride solution to the
       acetonitrile in the second 500-ml separatory funnel.  Extract
       the mixture successively with 100-, 25-, and 25-ml portions of
       methylene chloride, shaking each mixture for 20 seconds (shake
       the 25 ml portions gently).  Drain the lower methylene chloride/
       ac3toni*ri1-3 "* ayers through a 22-mrn T.D. column containing
       ca. 5 cm of anhydrous granular sodium sulfate.  Collect the
       eluate in a 1-liter 5 24/40 round-bottom flask and evaporate the
       solution to dryness with a rotary evaporator as described
       earlier.  Remove the flask from the evaporator immediately after
       the last traces of solution have evaporated and then add  10 ml of
       methylene chloride to the flask.

4.1.7  Chromatographic Cleanup

       Fit a 1-hole No. 5 ruboer stopper into the tip of & cnromatog-
       r-aphic tube  dth "an'bor stopcock, idd "5 ? ?4/^0 side arm

                               III-288

-------
       vacuum adaptor and 500 ml  f 24/40 round-bottom flask, open the
       stopcock, and connect the  apparatus to a vacuum line.  Place
       0.5 g of silanized Celite  545 in the tube, tamp, add 5 g of
       Nuchar S-N/silanized Celite 545 (1/4) mixture, and tamp again.
       Place a 1 to 2 cm glass wool  plug on top of the adsorbent.

       Prewash the column with 50 ml of toluene/acetonitrile (1:3,
       v/v), closing the stopcock when the solution is ca. 5 mm from
       the top of the glass wool.  Disconnect the vacuum, discard the
       eluate in the round-bottom flask, and reconnect the flask.

       Transfer the solution from paragraph 4.1.6 in 10 ml methylene
       chloride to the column and elute at 5 ml/min.  Wash the 1-liter
       round-bottom flask with 10 ml of methylene chloride and then with
       25 ml of the eluting solution.  Transfer each separately to the
       column and elute each to the  top of the glass wool  before
       adding the next solution.   Add 100 ml of eluting solution to
       the column and elute at 5  ml/min.  Turn off the stopcock when
       the top of the eluting solution reaches the top of the glass
       wool.

       Evaporate the solution in  the 500-ml round-bottom -Mask just
       to dryness on the vacuum evaporator as before.  Remove the
       """asx ~rom :r,s  evaporator  :nmealately -ntar ~Ti :he ""auid has
       evaporated.  Immediately pipet 5 ml  of methanol into tne flask
       to dissolve the ^asidue.  Pour the metnanol into a 10~-ni alass
       'syringe containing a Swinny filter holder with a 5-pm filter.
       Push the methanol solution through the filter with the plunger,
       rT! acting the 'Citrate n'n a.lO-ml  centrifuqe tube or other
       suitable container.  Approximately 4.5 .711 or fi"crate JHOUIO
       be recovered.  If the solution requires dilution, piper an
       aliquot into another container and dilute to volun.e as needed.

4.1.8  Sample Analysis

       Inject 10 vl of the methanol  sample solution onto the HPLC col-
       umn using the following parameters:   Adjust the mobile phase
       flow rate to 1.50  0.02 ml/minute at acetonitrile/water (1/1
       v/v).  Equilibrate the system at acetonitrile/water (12/88
       v/v) for 10 minutes, inject the sample, and begin a 30-minute
       linear gradient to acetonitrile/water (70/30 v/v),   Adjust the
       flow rate of the 0.05 M sodium hydroxide and reaction solution
       to 0.050  0.02 ml/minute  each.  Operate the column oven at 35C
       and the hydrolysis chamber at 100C.  Set the fluorescence
       detector excitation and emission wavelengths to 340 and 455
       nm, respectively, with slit widths 15 and 12 nm, respectively.
       Set the detector gain to "low" and time constant to 1 second.
       Adjust sensitivity so that 1  ng of carbofuran gives 50 percent
       full scale deflection on the  printer plotter set at attenu-
       ation :.
                               III-289

-------
            NOTE:  If the system will  not be used for several  days, re-
            place water mobile phase with methanol  and pump through the
            system, drain sodium hydroxide and reaction solutions from
            reservoirs, and wash reservoirs and associated tubing first
            with water and then methanol.  When starting up the system,
            change methanol  mobile phase to water,  and wash reaction
            reservoirs and associated tubing with water before adding
            reaction solutions.

            Tentatively identify residue peaks based on retention times
            (Table 9).  Measure peak areas or heights and determine
            residue amounts by comparison to areas  or heights  obtained
            from known amounts of appropriate reference material(s).
            Calculate results according to Subsection J.  To ensure valid
            measurement of residue amounts, sample  and standard peaks
            should aqree within 25 percent.  Chromatograph reference
            material is) Immediately after samples.   Figure 5 Illustrates
            the separation of the carbamates by HPLC.
     TABLE 9.  RETENTION DATA FOR 7 CARBAMATES AND * CARBAMATE METABOLITES,
                          USING ZORBAX C-8 HPLC COLUMN

                                                  Retention time relative
	Carbamate/metabol ite	y.s -arfrofuran	

      Aldicarb sulfoxide                                    0.33
      ^l-Hcarb sulfone         .                             0.40
      Oxamyl                                                J.-+4
      Methorny!                                              0.46
      3-Hydroxy carbofuran                                  0.60
      Methiocarb sulfoxide                                  0.64
      Aldicarb                                              0.83
      Carbofuran                                            1.00
      Carbaryl                                              1.06
      Methiocarb                                            1.26
      Bufencarba                                            1.44b
====================3=3==3=3===================================================
aMixture of 1-methyl butyl and 1-ethylpropyl phenyl N-methylcarbamates (3/1)
  with 70 percent meta, 20 percent para, and 4 percent ortho isomers.
bMajor peak.
     4.1.9  Confirmation of Residues

            An HPLC procedure reported by Lawrence1^ can serve to confirm
            the identity and amount of carbamate residues in food crops
            at 0.1 +o 0.3 nrm.  The carbamates are extracted from samoles
            with acetone and then partwonea into nexane/metnyiene
                                    III-290

-------
                         10        15        20
                          Retention Time (minutes)
25
30
Figure 5.  Chromatogram of carbamates and carbamate metabolites at 10 ng,
     using Zorbax C-8 column.   Compounds are:   1)  aldicarb sulfoxide;
  ?) ildicarb ulfone; 3)  rsxamyl:  4^  niethomyl:  5)  3-hvdroxy carbofuran;
    6) methiocarb sulfoxide;  7) alaicaro; 3}  w'aroofuran;  3} carbaryi,
                      '.Q)  -^ethiocarb: M) bufencarb.

                                 111-291

-------
chloride/water.  The organic extract is cleaned up by Florisil
chromatography (acetone/hexane, 15/85 v/v eluent) and the
eluate analyzed by HPLC with a silica gel column and UV absorp-
tion detector (254 nm).  The use of a different cleanup column,
different mode of separation (adsorption rather than reversed
phase), and a different type of detector assures that the re-
sults will be truly independent from those from the primary
method, a requirement for reliable confirmation.

A similar HPLC method for urea herbicides in vegetables was
also reported by Lawrence.16  Samples are extracted with acetone,
and the filtrate is partitioned with hexane/methylene chloride.
The organic phase is dried and concentrated for Florisil
column chromatography using acetone/hexane (15/85 and 50/50 v/v)
to elute the areas.  The column fractions are evaporated to
dryness and redissolved in isooctane for HPLC on a silica gel
column with isopropanol/isooctane (20/80 or 15/85 v/v) mobile
phase.  The compounds are detected by UV absorption at 254 nm.
Linuron, monuron, diuron, chlorbromuron, fluometuron, chloro-
xuron, and fenuron were determined in cabbage, corn, potato,
turnip, and wheat at 0.01-1.0 ppm with recoveries greater than
80 percent in most cases.
                        III-292

-------
5.1  Determination of Carbamate Pesticides in Air
          Analytical  Procedure:  available
          Sample Preparation:   available

     5.1.1  Reference

            Sherma, J.  and M.  Beroza, "Manual of Analytical  Methods for
            the Analysis of Pesticides in Humans and Environmental
            Samples." EPA-600/8-80-038, Sections 8A and 8B (June, 1980).9

     5.1.2  Method Summary

            The sampling medium, such as polyurethane foam or composite
            filter pad, is Soxhlet-extracted with hexane/diethyl  ether
            (95/5 v/v).  Carbamate pesticides are measured by direct
            GC with an  N-selective electrolytic conductivity detector
            or by electron capture GC after chemical derivatization
            with alpha-bromo-2,3,4,5,6-pentafluorotoluene.

     5.1.3  Applicabiity

            The method  is suitable for quantifying carbamate pesticides
            :i Ambient  air at  trace ''svels 'nq/r.3 '.nd -q/nv^.  The anal-
            ytical scneme presupposes collection OT samples  oy use of
            30!yursthane foam  oiugs or Tenax-oC adsorbent.^

     5.1.4  Precision and Accuracy

            almost no rsports  on tr.e coi'idcv^n =f-:u:3rcy ~T .aroamata
            pesticides  in air  nave oeen puoiisned.  A glass-fiber filter
            was found to have  a collection efficiency of 100 percent for
            carbaryl  at levels of 2 to 13 mg/m3 of air.iri

            Fenitrothion and aminocarb were collected on Tenax-GC adsor-
            bentH, and fourteen urea, carbamate, and thiocarbamate
            pesticides  on acetylcellulose or perch!orovinyl  filter papers
            (aerosols)  and on  silica gel or glass wool  or in traps con-
            taining acetone (vapors).12  The accuracy and precision of the
            method will depend mainly upon the collection efficiency of the
            sampling  medium *or the pesticide(s) of interest.

     5.1.5  Extraction  of the  Sampling Module

            Place the sampling medium in a Soxhlet extractor, handling
            with forceps rather than hands.

            NOTE:  After sampling, the glass-fiber filters and foam plugs
            should have been wrapped in aluminum foil until  analysis.
            Use oluqs and filters carried to the field  along with those
            employed  for sampling as controls.

-------
       Extract with an appropriate volume of _n-hexane/acetone/
       diethyl ether (47/47/6 v/v) for 16 to 24 hours at 4 cycles per
       hour for the large Soxhlets and 8 to 12 hours at 8 cycles per
       hour for the smaller Soxhlets.

       NOTE:  As examples, extract large foam plugs in 1000 ml Soxhlet
       extractors with a total  of 300 to 750 ml  of solvent, and smaller
       plugs and filters in 500-ml Soxhlets with 200 to 350 ml.

       Attach the boiling flask to a rotary evaporator and reduce
       the solvent volume to approximately 5 ml.

       Transfer the concentrate to a 15-ml  graduated centrifuge
       tube.  Rinse the boiling flask with solvent and add rinsing to
       concentrate.

       Adjust the final volume in the centrifuge tube as required.

5.1.6  Sample Analysis

       Inject yl of extract into the gas chromatograph containing a
       3-percent OV-101/Ultra Bond 20M. column and alectrolytic conduc-
       tivity detector.  Compare the relative retention time for each
       :cmponent of :ntsr*st against '.hose of the ccr*-?SDonding orimary
       standard.  Quantify peaks in the usual way, i.e., oy measuring
       peak heights to tne learest 
-------
Transfer the hydrolysis solution, by washing with 50 to 60 ml
of distilled water, into a 500-ml separatory funnel  and add
50 ml of methylene chloride.

Shake briefly and discard the methylene chloride.

Acidify to pH 2 or less with 0.3 to 0.5 ml  of 50-percent
sulfuric acid.

Extract the hydrolysis solution with two 50-ml portions of
benzene, and dry the benzene by suction filtration through a
10-gram sodium sulfate column into a 250-ml round-bottom
flask.

Evaporate the benzene to 1 to 2 ml on a rotary evaporator with
a water bath at 40C.

Transfer to a 15-ml centrifuge tube by rinsing with 5 to 6 ml
of acetone.

Add 25 ul  of 5-percent aqueous potassium carbonate and 100 yl
of 1-percent PFB bromide solution to the centrifuge tube.

Stopper, sna
-------
NOTE:  Determine the elution pattern of the PFB ether derivatives
of the carbamates of interest on the silica gel column under local
laboratory conditions.  A typical elution pattern is as follows:
PFB ether
derivative
                                Recovery, Percent8
 Fraction I
Fraction II
Fraction III
Propoxur
Carbofuran
3-Ketocarbofuran
Metmercapturon
Carbaryl
Mobam
   84-89
   97-100
   96-99
   93-97
   94-97

   12-15
    0-2
    0-3
                                        2-5
                                        2-4
                                       96-98
aObtained by comparing the peak areas of 5 samples passed through
 the silica gel columns with 3 samples not fractionated.
Concentrate the eluate fractions as needed and analyze by EC-6C on
the 3-percent OV-225 or 3.6-percent CV-1Q1/5.5-percent OV-210
column.  Relative retention times of PFB ether derivatives on the
two tslumns ire is *o11ows:
Derivative
     RRT
OY-101/OV-210*
                                                        RRT
PrQDOXUr
Carbofuran
3-Ketocarbofuran
Metmercapturon
Carbaryl
Mobam
0.43
0.64
1.15
1.26
1.38
1.48
0.41
0.63
1.13
1.28
1.31
1.31
^Relative to aldrin 2.7 minutes
^Relative to aldrin 7.9 minutes
Operating conditions:  injector temperature, ?05"C; column, 190C;
detector, 280*C; 5-percent methane-argon carrier gas flow rate, 50
ml/minute + 20 ml/minute purge for the OV-101/ OV-210 column, 30
ml/minute + 30 ml purge for OV-225; EC detector in pulsed mode with
electrometer settings of 55V, 90 jisec pulse rate,, 8 usec pulse
width, and 6.4 to 1.6 x 10~9 amp full-scale attenuation.

Quantify by comparison of peak areas against chromatograms of
derivatized standard carbamate phenols.  The standard derivatives
     synthesized  as follows:
                        III-296

-------
            React each carbamate phenol  with a tenfold molar excess of PFB
            bromide in acetone and a tenfold molar excess of methanolic
            potassium hydroxide.

            Reflux for 2 to 3 hours, cool, and remove the solvent on a
            rotary evaporator.

            Dissolve the product in benzene and wash the benzene twice
            with equal volumes of 0.1 M potassium carbonate.

            Dry the benzene, using suction, by passing it through a 10 to
            20 gram column of anhydrous  sodium sulfate.

            Remove the benzene on a rotary evaporator and recrystallize
            from hexane or methanol.

            Inject 5 ul or another appropriate volume of the sample extract
            into the gas chromatograph.

            Record chromatograms and measure retention times relative to
            aldrin or another suitable reference standard.   Calculate results
            according to Subsection J.

0.   CALCULATIONS

     Determine the concentration of individual compounds *n the simole.   If
the external standard calibration procedure is usea, calculate  the amount of
material injected 'from the peak response using the calibration  curve or cali-
bration factor in Subsection H.  The concentration in the sample can be
1.   Liquid Samples

                                                'A)(Vt>
                     Concentration, yg/1   =
                                               (VO(VS)

     where:

          A   =  Amount of material  injected, in ng
          V-i  *  Volume of extract injected in yl
          Vt  =  Volume of total  extract in yl
          Vs  =  Volume of water extracted in ml.

     If the internal  standard calibration procedure is used,  calculate the
     concentration in the sample using the response factor (RF)  determined in
     Subsection H.2.  as follows:

                                              (As)ds)
                    Concentration, yg/1   -    	
                                    III-297

-------
     where:
          As
          Ais
          Is
                  Response for the parameter to be measured
                  Response for the internal  standard
                  Amount of internal  standard added to each extract in yg
                  Volume of water extracted, in liters.
2.   Solid and Tissue Samples
               Concentration, yg/kg (wet weight)
                                                     (A)(Vt)  1000
     where:
               Concentration, yg/kg(dry weight)   =
                                                    (A)(Vt)  1000
             A  =
            Vt  =
             3  :
            %s  =

3.    Air Samples
                   amount of material  injected (from Calibration Curve), ng
                   volume of extract injected, yl
                   total  volume of extract, ul
                   ,-ret -veigni of campie extracted, u
                   percent solids in sample as a  decimal  fraction.
                        Concentration.,  mg/nP  =
     where:

             A  =  amount of material  injected (from Calibration Curve), ng
            V-j  =  volume of extract injected, yl
            Vt  =  total  volume of extract, ul
             V  =  volume of air filtered, m^.

     Report  results in appropriate units without  correction  for recovery
     data.  When duplicate and spiked  samples are analyzed,  report all  data
     obtained with the sample results.

     For samples processed as part of  a set where the laboratory spiked sample
     recovery falls outside of the control limits in Subsection G, data
     for the affected parameters must  be labeled  as suspect.
                                    III-298

-------
K.   CONFIRMATION

     Detailed discussion of general  and specific aspects of residue confirm-
ation is available.I?  In many cases, particularly where a high concentration of
contaminant is apparent, residues should or must be confirmed.   The most gener-
ally accepted method of confirmation of pesticide identity is on two or more GC
columns of different polarity, and/or with different detectors if heteroatoms
are present, or with different derivatives.  GC/MS of pesticide residues is one
of the best techniques for confirming identity.   TLC confirmation, once used
more extensively prior to the introduction of routine mass spectrometers, may
also be used, as may confirmation by jj-values, polarography,  spectrometry (IR,
UV, NMR), or via chemical derivatization.  In general, pesticide residues are
confirmed by analysis under different analytical  conditions (i.e., different
columns, detectors, derivatives, or methodologies) or by ancillary techniques.

     Numerous reports of analytical  methods for single residues or small
numbers of compounds have been published recently.  Some of these are
listed as references 18 to 30 at the end of this section.   Readers may find
these methods useful for determinations of carbamates and related compounds
in samples other than those covered  in this section or for confirmation of
results obtained by these methods.   References 21 and 24 are especially
pertinent because they provide retention data on various HPLC columns and
detection wavelengths and sensitivities for over 40 carbamate compounds.
                                    III-299

-------
                                   REFERENCES
     American Society for Testing and Materials.  "Standard Practice for
     Preparation of Sample Containers and for "Preservation."  American Society
     for Testing and Materials Annual Book of Standards, Part 31, D3694.
     American Society for Testing and Materials, Philadelphia, Pennsylvania.
     p. 679 (1980).

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

     American Chemical Society.  "Safety in Academic Chemical Laboratories."
     American Chemical Society Publication, Committee on Chemical Safety, 3rd
     edition.  (1979).

     Press! ey, T. A. and J. E. Longbottom.  "The Determination of Carbamate
     and Urea Pesticides in Municipal and Industrial Wastewater - Method 632."
     Report No. EPA-600/4-82-014 (February, 1982).

     American Society for Testing and Materials.  "Standardization of Florisil
     Column by Weight Adjustment Based on Adsorption of Laurie Acid."  ASTM
     book of Standards,  Part 31, D3086, Appendix X3.  American Society for
     Testing and Materials, Philaaelpnia, Pennsylvania,  p. ^55 J
6.   Farrington, D. S. , R. G. Hopkins, ana J. H.  A,  Ruzicka.   "Determination
     of Residues of Substituted Phenylurea Herbicides in Grain, Soil, and
     River Water by Use of Liquid Chromatography."  Analyst _10j!:377 (1977).

7.   'J.3. Environmental Protection Agency.  "Handbook for Analytical Quality
     Control in Water and Wastewater Laboratories."   EPA-600/4-79-019, U.S.
     Environmental Protection Agency, Environmental  Monitoring and Support
     Laboratory - Cincinnati, Ohio <*5268.  'March. 1979).

8.   Hall, R. C. and D. E. Harris.  "Direct Gas Chromatographic Determination
     of Carbamate Pesticides Using Carbowax 20M-Modified Supports and the
     Electrolytic Conductivity Detector."  J. Chromatogr. Jj>9:245 (1979).
                                                                     i
9.   Sherma, J. and M. Beroza.  "Manual of Analytical Methods for the Analysis
     of Pesticides in Humans and Environmental  Samples."  EPA-600/8-80-038,
     Sections 8A ana 3B.  (June, 1980).

10.  U.S. Department of Health, Education, and Welfare.  "Carbaryl".  Method
     S273, NIOSH Manual of Analytical Methods, 2nd  Edition,  Volume 3, Center
     for Disease Control, National Institute of Occupational  Safety and Health,
     Cincinnati, Ohio.  (April, 1977).

11.  Krzymien, M. E.  "Measurement of Atmospheric Fenitrothion and Aminocarb
     Concentrations Near the Spray Area."  Int. J. Environ.  Anal. Chem. 13:69
                                    II1-300

-------
12.  Aleksandrova, L. G. and M. A. KHsenko.   "Identification and Determination
     of some Urea, Carbamate, and Thiocarbamate Derivatives  in Air."   J.
     Chromatogr.  247^255 (1982).

13.  Krause, R. T.  "Multiresidue Method for Determining N-Methylcarbamate
     Insecticides in Crops,  Using High Performance Liquid Chromatography."
     J. Assoc. Off. Anal. Chem. 63(5):1114 (1980).

14.  Krause, R. T. and M. August.  "Applicability of a Carbamate  Insecticide
     Multiresidue Method for Determining Additional  Types of Pesticides  in
     Fruits and Vegetables."  J. Assoc. Off.  Anal.  Chem. 66(2):234  (1983).

15,  Lawrence, J. F.  "Direct Analysis of Some Carbamate Pesticides  in Foods by
     High Pressure Liquid Chromatography." J.  Agr.  Food Chem.  25:211 (1977).

16.  Lawrence, J. F.  "High  Pressure Liquid Chromatographic  Analysis  of  Urea
     Herbicides in Foods."  J. Assoc. Off. Anal.  Chem.  59:1066 (1976).

17.  Sherma, J.  "Manual of  Analytical Quality Control for Pesticides in  Human
     and Environmental Samples."  EPA-600/2-81-059 (April, 1981).

13.  Kirk!and, ]. J.  "Method for High-Speed  Liauid  Chromatograohic Analysis
     of Benomyl and/or Metabolite Residues in Cow rfil'rt,  Urine,  races,  ana
     T1ssue.:1  J. Agr. food Chem. 21_:i7i (1972;.

19.  Kirkland, J. J., R. F.  Holt, and H. L. Pease.  "Determination of Benomyl
     Residues *n SoiU and Plant Tissues by High-Speed Cation Exchange Liquid
     Chromatograpny. '  J. Agr. Fooa Chem. l_.~56  ,197C).

20.  Ernst, G. F., S. 0. Roder, G. H. Tjan, and J. T.  A.  Jansen.   "Thin  Layer
     Chromatographic Detection and Indirect Gas Chromatographic Determination
     of Three Carbamate Pesticides."  J. Assoc. Off.  Anal.   Chem.  58:1015
     (1975).                                                     ~~

21.  Sparacino, C. M. and J. W. Hines.  "High Performance Liquid  Chromatography
     of Carbamate Pesticides."  J. Chromatogr.  Sci.  ^4_:549  (1976).

22.  Lawrence, J. F. "Confirmation of Some Organonitrogen Herbicides  and  Fun-
     gicides by Chemical Derivatization and Gas Chromatography."  J.   Agr. Food
     Chem.  24:1236 (1976).

23.  Thean, J. E., W. G. Fong, D. R. Lorenz,  and  T.  L. Stephens.   "High  Pressure
     Liquid Chromatographic  Determination of  Methomyl  and Oxamyl  on Vegetable
     Crops."  J. Assoc. Off. Anal. Chem. 61_:15  (1978).

24.  Lawrence, J. F. and D.  Turton.   "High Performance Liquid Chromatographic
     Data for 166 Pesticides."  J. Chromatogr.  J.59_:207 (1978).

25.  Galoux, M., J.-C. Van Damme, A. Semes,  and  ^.  Potvin.   'Gas-Liquid
     Chromatographic Determination of Aldicarb, Aldicarb  Sulfoxide, and Aldicarb|
     Sulfone in Soils and Water Using a Hall  tleciroiytic  Cpnaucfivity Detec-cr."
     J. Chromatogr.  177_:245  (1979).

                                    III-301

-------
26.  Blaicher, G.,  W.  Pfannhauser and H.  Woidlch.   "Problems Encountered with
     the Routine Application of HPLC  to  the Analysis of Carbamate Pesticides."
     Chromatographia 13:438 (1980).

27.  Wehner, T. A.  and J.  N. Seiber.   "Analysis  of  N-Methylcarbamate  Insecticides
     and Related Compounds by Capillary  Gas Chromatography." J. High  Resolut.
     Chromatogr. Chromatogr. Commun.  :348  (1981).

28.  Mayer, W. J.  and  M.  S. Greenberg.   "Determination of Carbamate Pesticides
     by High Performance  Liquid Chromatography with Electrochemical Detection."
     J. Chromatogr. 208_:295 (1981).

29.  Gustafsson, K. H. and R. A. Thompson.  "High Pressure Liquid Chromatographic
     Determination  of  Fungicidal Dithiocarbamates." J. Agr. Food Chem. 29:729
     (1981).

30.  Grou, E., V.  Radulescu, and A.  Csuma.  "Direct  Determination of Some
     Carbamate Pesticides in Water and Soil by High Performance Liquid
     Chromatography."  J.  Chromatogr.   260:502  (1983).
                                    II1-302

-------
                                   SECTION 7

      METHODS FOR THE DETERMINATION OF CHLORINATED PHENOXY ACID HERBICIDES
A.   SCOPE

     Chlorophenoxyacetic adds such as 2,4-dichlorophenoxyacetic acid (2,4-D),
2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and silvex [2-(2.4,5-trichloro-
phenoxyjpropionic acid] are herbicides used for weed control.1  Each com-
pound may exist as a free acid or an ester.  In addition, the ester of each
herbicide is less stable than the free acid and may hydrolyze in aquatic
environments.2

     The analytical  procedure for these compounds consists of three steps.13
Residues are extracted into an organic solvent and esterifisd using boron
trifluoride (BF3).  The methyl esters are then extracted into benzene and
quantified using gas cnromatography.

B.   SAMPLE HANDLING AND STORAGE

     Water samples should be collected in an all-glass system.  The sample
snouid oe acidified *itn jui'-jr-'': ic*!d 'H?S04; *o "H <2 *ediately after
collection and stored at 4"C In the dar^,  Extraction of the samples snouio
begin within 12 hours of collection as the degradation of 2,4-D is rapid in
aqueous systems.3

     Sediment samples should be stored in glass containers.  Immediate extrac-
tion of samples is recommended to minimize the effects of sampTe degradation.
However, when necessary, sample freezing at -20C has been shown to prolong
the stability of 2,4-D.4

     All sample containers should be sealed with Teflon-lined screw caps.l  An
alternate method is to use pre-cleaned, heavy-duty aluminum foil to prevent the
sample from coming in contact with plastic caps and associated glue lining.
The aluminum foil may be cleaned by washing in acetone, followed by rinsing
with pesticide-grade hexane.3

     A flowchart for the processing of samples to be analyzed for chlorinated
phenoxy acid residues is presented in Figure 1.

C.   INTERFERENCES

     txtraneous .natter, especially  in highly colored water samoles, is a ooten-
*1a1 interference.  The cleanup procedure described here will usually eliminate
this source of interference.  Many organic compounds Jan -interfere with *he

                                    III-303

-------
SAmi AIR
wmii




FILTER
PAH* OR
HUM
CMTRIOGC


SAWIE
PROCESSING
D1GES1

1

ANALYZt







Purpose Total
Cone.
In Air
SMVLE
CONTAINEII
SMVLE 1
PRESERVATIVE
STORAGE
TIME
SAMPLE
SUE
1 WATER OR SlUOtt. SOI
1 UACHAH
1


11
FILTER MET STORAGE I
NO till AN 1
MEIO


EEP TOIICIU
RESERVE



1 fP EITRACT
| STORE lOIICITY
EITRACT 1


I ,
I EXTRACT iITRACT
KHALI tt [ ANALYZE


I ANALYZE ma.ni
Total Dissolved' Ti
Cone; Cone. In Nobility Kibtllty In
In Hater Hater at pH S at pH S
6 C 6 G
2*04 to >H 1 KjSOi to pH I 4'C 4*C
4*C 4*C
i< nmrt u hours <1H 
-------
analysis, however.  Boron trifluoride/methanol reagent is used  because 1t
reacts specifically with carboxylic acids, whereas diazomethane may react with
phenols and other organics with relatively active hydrogens.  All reagents must
be thoroughly checked and any interferences from this source eliminated.

     In natural sediment samples, benzene may elute humic substances which,
upon GLC Injection, remain in the glass sleeve liner due to their nonvolatile
nature.

     Where emulsions form at the solvent/water interface, the emulsion should
remain with the aqueous phase.   This allows the emulsion to be extracted
further with methylene chloride and prevents the sodium sulfate in the funnel
from becoming saturated with water.  Any amount of water in the extract could
inhibit esterification and result in decreased recoveries.

     Any compounds which are co-extracted from the sample and chromatograph
similarly to the compounds of interest are possible sources of interference.
Certain esters of the chlorophenoxy acids may cause mutual interference.
Phenols and chlorophenols may also Interfere.*

     Because the herbicides react readily with alkaline substances, loss  may
occur if there is contact with alkaline substances at any time except in  the
controlled alkaline hydrolysis step.  Glassware and glass wool should be  acid
rinsed to mimimize this possibility.A

D.   APPARATUS

      *!" :las3war? Ttust be washed in chromic acid, rinsed with dilute hydro-  . _.
chloric acid, followed by distiTiea *ater ana ir\er. .--nsea *f*h icstcne :nd
hexane.  Heat treatment is carried out at 300^ on all glassware sxceot volu-
metric flasks and pipettes.  Care must be taken to ensure that the glassware
is not alkaline.  Considerable loss at low herbicide concentrations can occur
due to the alkalinity of the glassware.

1.   Graduated centrifuge tubes, 15 ml, with ground-glass stoppers.

2.   Volumetric flasks (1.0 ml, 2.0 ml, 10 ml, 100 ml, 250 ml, and 2 1).

3.   Round-bottom flask, 300 ml.

4.   Erlenmeyer flask, 250 ml.

5.   Separatory funnels, 60 ml, 500 ml, and 21.  With TFE-fluorocarbon
     stopcocks and tapered ground-glass stoppers, Kontes or equivalent.

6.   Beakers, 100 ml, 200 ml.

7.   Flasks, 500 ml, flat-bottomed.

8.   Flasks, suction with ground-glass joints.

9.   Funnels, coarse, sintered glass with grouna-giass joints.

-------
10.  Funnels, powder, glass.

11.  Graduated cylinders,  50  ml,  and 1  liter.

12.  Watch glass.

13.  Pipettes, Pasteur, disposable,  140 mm long x 5  mm I.D.,  glass.

14.  Microsyringes, Hamilton, 10  ul  for injections.

15.  Evaporator concentrator, Kuderna-Danish,  250-ml  flask  and  5-ml  volumetric
     receiver, Kontes or equivalent.

16.  Snyder columns, three-ball macro,  one-ball  micro.

17.  Rotary evaporator.

18.  Gas chromatograph such as a  Varian 2800,  Microtec 220, or  equivalent.   It
     should be equipped with  an electron capture detector,  a  glass-lined
     injection port, and a recorder. The following  operating conditions  are
     recommended:  injection  temperature, 215C; oven  temperature, 185C;
     column temperature, 185*C; and  a carrier  gas flow of 70  ml/min  in  a  6.4
     mrn-O.D. column.

     Alternative operating conditions chat nave deen used are:   column  temper-
     ature, 200"C; injection  port, 230C: ana  ietector temoerature.  340C.
     Use 5 percent methane and 95 percent argon for  both  earner gas flow (40
     ml/min) and make-up gas  flow (20 ml/min).

19.  Chromatograpm'c coiumn:   the use of ;;vo :aiumns  s .uggestaa  "or  'asnfi-
     fication and confirmation.   One coiumn is packed.with  a  mixture of 1.5
     percent OV-17 and 1.95 percent  QF-1 on a  100/120-mesh  Gas  Chrom Q.   The
     second column is packed  with 5  percent OV-210 on  a 100/120-mesh Gas
     Chrom Q.

     Additional column packings that have been shown to be  useful  for separ-
     ating and quantifying chlorinated  phenoxy acetic  acids and herbicides
     are:

     11 percent OV-17 + QF-1, mixed  phase by weight, on 80/100-mesh  Gas Chrom
     Q, available from Applied Science.

     3 percent OY-17 on Chromosorb W, HP 80/100-mesh,  available from Applied
     Science.

20.  Chromatograph column, glass  U-tube, 2 m by 3.5  mm O.D.

21.  Column:  chromatographic (10 mm I.D. x 300 mm)  with  coarse frit and
     stopcock.  Reservoir at top  (28 mm I.D. x 150 mm).

22.  Sana oath, fiuidizea iTeCam  or  equivalent)  or water bath.


                                    III-306

-------
23.  Oven, capable of maintaining 300C.
24.  Ultrasonic homogenizer, such as the Sonicator Cell  Disrupter Model W-375
     with a solid disrupter form (No. 280-0.75).  This is available from Heat
     Systems-Ultrasonic, Inc., 38 East Mall, Plainview,  Long Island.
25.  Centrifuge tube heater, such as the Kontest Tube Heater block set at
     40C combined with a gentle stream of pure nitrogen gas for controlled
     evaporation.
26.  Sep-Pak, Cj8 reversed phase,  Waters Association.
27.  Steam bath.
28.  High-pressure liquid chromatograph (HPLC).
     28.1  Dual mobile phase.
     28.2  Gradient elution.
     28.3  5,000 psi back pressure.
     28.4  UV absorption detector,  284 nm.
     28.5  Recorder or integrating recorder.
29.  Mechanical shaker.
     All solvents and reagents must be of pesticide quality and checked before
use for purity by the gas chromatographic procedure.   Much time and effort can
be saved by selecting high-quality reagents that do not require extensive pur-
ification.  Some purification of reagents may oe necessary as outlined below.
If more rigorous treatment is indicated, the reagent should be obtained from
an alternative source.
1.   Benzene
2.   Chloroform
3.   Methanol
4.   Ethyl ether, reagent grade.  Redistill in glass after refluxing over
     granulated sodium-lead alloy for 4 hours.
5.   Hexane
6.   Methylene chloride
7.   Acetone
                                    I I 1-307

-------
 8.  1:1 Acetone:hexane
 9.  Aceton1tr1le
10.  Acetic add, glacial
11.  Sodium chloride,  reagent grade
12.  Ethanol,  USP or absolute
13.  Potassium hydroxide,  reagent grade.
14.  Potassium hydroxide solution:   dissolve 37  g  KOH  1n  distilled water  and
     dilute to 100 ml.
15.  Hydrochloric acid, analyzed reagent  grade or  better.
16.  Sulfuric  acid, cone., analyzed  reagent grade  or better.
17.  Sulfuric  add, 9 N.
18.  Boron trifluoride methanol  reagent,  14 percent boron  trifluoride  by
     weight (available from Analabs).
19.  Florisil  adsorbent -  60/100 mesn,  ractory-dctivatea  at 650C.   The
     F Tonsil  1s heated to 130C for 1  hour and  stored in  a desiccator prior
     to use.  Each oaten must be cneckea  ror activity  and  for  contamination.
20.  Sodium sulfate - ACS  grade  or better,  anhydrous.   The heat-treated
     material  is aiviaea,  ana one part  :s "uoei'.iG 'ieutr.il ;oaiura ^ul "ate"
     and stored at 130"C.   The other part is slurried  with snough 3ther to
     cover the crystals and acidified to  pH 4 by adding a  few  drops  of
     sulfuric  acid.
     To determine the pH,  a small quantity of slurry is removed, the ether
     evaporated, water is  added  to cover  the crystals, and the pH 1s measured
     with a pH meter.  The ether 1s  removed by vacuum  from the acidified
     sodium sulfate.  This fraction  1s  labelled  "acidified sodium sulfate" and
     stored at 130*C.
21.  Sodium sulfate solution: dissolve 50 ml anhydrous sodium sulfate 1n
     distilled water and dilute  to 1 liter.
22.  Acidified organic-free water:   add hexane  (50 ml) to distilled  water
     (5 1) stir for 4 hours with a magnetic stirrer at maximum speed.
     Transfer to a large separatory funnel  and  remove  the water layer  Into
     storage bottles.  Add cone. HC1 (2 ml/I).
23.  Celite filter aid:  keep silica gel  overnight at  650*C, homogenize with
     5 percent organic-free water for 2 hours prior to use.
?4.  Glass *ool:  filtering qrade,  acid washed.
                                    III-308

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25.  Analytical standards:  2,4,5-T; 2,4-D; 2,4,5-T methyl  ester; 2,4-D methyl
     ester; si 1 vex; si 1 vex methyl  ester; all  at least 98+ percent pure
     (available from Dow Chemical, Polyscience, or as EPA reference stan-
     dards).

26.  Stock herbicide solution:  dissolve 100 mg herbicide or methyl  ester  in
     60 ml ethyl  ether;  dilute to  100 ml in a volumetric flask with hexane.
     1.00 ml = 1.00 mg.

27.  Intermediate herbicide solution:  dilute 1.0 ml stock solution to 100 ml
     1n a volumetric flask with a  mixture of equal  volumes of ethyl  ether  and
     benzene. 1.00 ml = 10.0 ug.

28.  Standard solution for chromatography:  prepare final concentration of
     methyl ester standards in benzene solution according to the detector
     sensitivity and linearity.

29.  GLC columns and conditions.

     a)   McKinley and McCully's column3, 4% SE-30 and 6% QF-1 on 100/120
          mesh size Chromosorb W,  acid-washed and DMCS treated.

     b)   3% Dexil 300 GC on Chromosorb W, acid-washed, DMCS treated,  100/
          120 mesh size.

     c]   2% OV-1 on Chromosorb W, acid-washed, DMCS treated, 100/120  mesh
          size.

     d)   Chau-Wilkinson Column3,  4% OV-101/6% OV-210 on Chromosorb W, acid-
          *dsn8d, HOME treated, SO ts 100 ?,esh 1*s.

          Column:  Glass 1.8 m x 4-mm I.D.; packed with one of the aoove
          packing materials:
            Column Temperature:
            Injection Temperature:
            Detector Temperature:
            Attenuation:
            Chart Speed:
            Flow Rate:
            Detector:
            Recorder:
195'C
250C
275*C
16
1/2 inch/min
60 ml N2/min
Ni63 electron capture
1 millivolt strip chart
                                    111-309

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F.   ANALYTICAL PROCEDURES

     1.1  Analysis of Solid Waste Samples for Chlorinated Herbicides
          by High Performance Liquid Chromatography
               Analytical Procedure:  available
               Sample Preparation:   available

          1.1.1  Reference

                 Stephens, R. D., J. Nakao, and B.  P.  Simmons,  "Methods for the
                 Analysis of Hazardous Wastes."  California Department of
                 Health Services, Berkeley, California.  (1981).5

          1.1.2  Method Summary

                 Samples are acidified and extracted with dichloromethane.   In
                 the presence of interfering substances,  samples  are hydro!-
                 yzed in alcoholic potassium hydroxide,  diluted with water,
                 and washed with dichloromethane prior to acidification and
                 extraction with dichloromethane.  The dried extracts are then
                 diluted in methanol and injected into an HPLC  equipped with a
                 reverse-phase column and UY detector at 285 nm.

          1.1.3  Apolicaoili'y

                 This method is aopropr-ste for non-aqueous liquid industrial
                 wastes and agricultural chemicals.

          !.1.4  Recovery studies in which nine chloroohenoxv herbicides were
                 determined in extracts or tftrse separate soil  samples snowed
                 an average recovery of 92% (82.3-98.?%)  with an  average co-
                 efficient of variation of 9.2%.  The herbicides  used in this
                 study were 2,4-D,  2,4,5-T acid, 2,4-DB  acid, silvex, 2,4-D
                 isopropyl ester, 2,4-0 outyi ester, 2,4-0 outoxyethanol
                 ester, 2,4-D propylene glycol butyl ether ester, and 2,4-D
                 ethyl hexyl ester.

          1.1.5  Sample Preparation

                 Weigh 20.0 g sample into a 250-ml  beaker.  Add 50 ml 0.2 M
                 methanolic potassium hydroxide to the beaker.  Cover the
                 beaker with a watch glass and place on  a steam bath (reflux
                 temp.) for at least 1 hour.  Mix periodically to assure good
                 surface contact.  Proceed to paragraph  1.1.6.

          1.1.6  Sample Hydrolysis

                 Remove the beaker from the steam bath,  allow to  cool, and
                 transfer the sample (total volume of sample plus washings not
                 to exceed 50 ml) into a 500-ml seoaratorv funnel containing
                 250 ml water and 60 mi sodium cnionae-saturatea water.
                 T.xtract the :amols *hn?e times with 50  ml dichloromethane

                                    III-310

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       each.  Discard this extract (bottom layer).   Acidify the
       sample (pH <2) and extract three times again with 50.ml
       dichloromethane each.  Combine the extracts  from the acidi-
       fied sample and evaporate to dryness under a hood.   To speed
       the drying process, evaporate on the edge of a steam bath.

1.1.7  Extract Cleanup

       It is essential that all  non-soluble particulate matter above
       1.0 u be removed since the HPLC column is composed of 5  u
       particles and is easily plugged.  Apply one  of the following
       techniques for particulate removal.

1.1.7.1  Dissolve the dried sample in 2.0 ml of methanol.   Filter
         according to conventional  methods such as  Sweny-type
         filter.

1.1.7.2  Transfer the dried sample with 1.0 ml methanol onto a
         reversed phase Sep-Pak  and elute with methanol into a
         2-ml volumetric flask.

1.1.8  Sample Analysis

       Inject 10 nl sample  :nto  HPLC equilibrated ander the
       following conditions:

       Column:  C^, 5 u, 15 cm, reversed phase.

       Mooile pnase:  CC% icstonitr''!^ :roqr"rnmed *o "''ncrease to
                      30* acetonitr-'le over 3 minutes and 70*
                      dilute acetic acid (1%) programmed to
                      decrease to 20%.

                          1.0 minutes
                          8.0 minutes
                          2.0 ml/minutes
                          284 nm
                          1.0
                          0.5 cm/minutes
         Initial  hold
         Final  hold
         Flow rate
         Wavelength
         Attenuation
         Chart speed
1.1.9  Data Interpretation

       Tentatively identify peaks based on retention time and quan-
       titate by peak height or peak area.  Compare the peak  height
       or peak area to a standard which is known to be in the linear
       range of the instrument (Subsection H).
                          III-311

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2.1  Analysis of Water Samples for Chlorinated Phenoxy Acid Herbicides
     by Chloroform Extraction
          Analytical  Procedure:   evaluated
          Sample Preparation:  available

     2.1.1  Reference

            Environment Canada,  "Analytical  Methods Manual."  Inland
            Waters Directorate,  Water Quality Branch,  Ottawa,  Ontario,
            Canada, 1974.3

     2.1.2  Method Summary

            The water sample is acidified to pH 2 or less and  extracted
            with chloroform.  The solvent is dried, concentrated,  and
            replaced with a small amount of  methanol.   The  herbicide
            residues  are converted to their  methyl  esters using  boron
            trifluoride/methane! reagent. The methyl  esters are
            extracted into benzene;  the extract is  cleaned  up  and
            analyzed by gas liquid chromatography.

     2.1.3  Applicability

            This method includes the qualitative and quantitative  gas
            cnromatograpnic aetenm nation of 2.+-0, si"!vex,  2,4,5-7, and
            other phenoxy acid herbicides.

            The practical  limits of measurement utilizing electron
            capture detection are listed below for  some of  these herbi-
            cides as  netnyi estar-; ' n a ",-' ;ter atar  :-ample,

            1.  2,4-D    (0.010 ppb)
            2.  2,4,5-T  (0.010 ppb)
            3.  Silvex   (0.010 pob)
            4.  MCPA     (0.050 ppb)

            The absolute lower limit of detection will  vary with size and
            characteristics of the sample as well as analytical  condi-
            tions.

            Strict attention to protocol is  required on the part of the
            analyst to obtain reproducible and satisfactory recovery.  In
            the steps where solvents are evaporated, extreme care  must be
            exercised, especially when working with the methyl esters.  The
            extracts should never be taken to dryness  as the esters are
            extremely volatile.

     2.1.4  Precision and Accuracy

            Recovery studies at the 0.05-ppb level  showed that the
            recoveries "or 2,+-?> *ere consistently  35  oercent  or better.
                                    III-312

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       The recoveries for 2,4,5-T and si 1 vex were better than 90
       percent.  At the 1.0-ppb level recoveries of all  the herbicides
       were better than 95 percent.

2.1.5  Sample Extraction

       Acidify the water sample {1,000 ml) to approximately pH 2.0
       with concentrated sulfuric acid and transfer quantitatively
       to a 2,000-ml separatory funnel.

       Add 50 ml  chloroform to the separatory funnel  and shake
       the mixture thoroughly for 1 minute.  Occasionally emulsions
       are formed, but they can usually be broken by adding small
       portions of 2-propanol, acetone or a saturated sodium chlor-
       ide solution.

       Allow at least 5 minutes for complete separation of the
       layers and draw off the bottom chloroform layer into a clean
       500-ml separatory funnel.

       Extract the sample twice more and combine the extracts in the
       500-ml separatory funnel.  The chloroform extracts are then
       washed with 100 ml slightly scldic class-distilled xatar and
       the aqueous layer is removed.

       The combined chloroform extracts are dried "over acidified
       anhydrous sodium sulfate for about 10 minutes.  Caution:  The
       axtract ihculd "ot "W.ain 
-------
       About 5 ml  5% aqueous sodium sulfate  solution  is  added  to
       the tube and extraction  of  the  methyl  esters is carried out
       with two 2-ml portions of hexane.   The hexane  extract is
       concentrated to 1  ml  under  a stream of dry  nitrogen.

       The hexane  phase containing the methyl  esters  of  the phenoxy
       acid herbicides is passed through  a small column,  prepared by
       plugging a  disposable pipette with glass wool  and packing
       with 2 cm neutral  anhydrous sodium sulfate  over 2 cm
       Florisil.  The herbicide esters are eluted  with 10 ml of
       benzene.

       The benzene solution is  concentrated  to 0.5 ml under a  stream
       of dry nitrogen, quantitatively transferred to a  1-ml volu-
       metric flask and made up to volume.  This solution is now
       ready for GLC determination.

2.1.7  Sample Analysis

       Preliminary identification  is achieved via  electron capture
       GLC in which at least two different stationary phases of
       different polarity are employed (Subsection F).   The identity
       of the herbicide is based on the retention  time relative to
       aldrin (Subsection H).
                               III-314

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2.2  Analysis of Water Samples for Chlorinated Phenoxy Acid Herbicides
     by Ethyl Ether Extraction
          Analytical Procedure:   available
          Sample Preparation:   available

     2.2.1  Reference

            American Public Health Association, "Standard Methods  for the
            Examination of Water and Wastewater."   15th Ed.   American
            Public Health Association,  New York, New York.  1,134  p.
            (1980).

     2.2.2  Method Summary

            Chlorinated phenoxy acids and their esters are extracted  from
            an acidified water sample with ethyl ether.  The extracts are
            hydrolyzed and extraneous material  is  removed by a  solvent
            wash.  The acids resulting from the hydrolysis are  converted
            to methyl esters,  and the extract is cleaned up on  a micro-
            adsorption column.  The methyl  esters  are quantified using
            gas chromatography.

     2.2.3  Applicability

            This method is suitable for quantifying chlorophenoxy  acid
            herbicides in aqueous samples.   The detection ~! unit will
            depend on the initial sample size,  degree of extract concen-
            tration, and the presence of interferring substances.   If the
            extract from a t-Mter samnle *s concentrated to 2.00  ml  and
            5.0 ul  of concentrate is injected into a gas cnromatograpn
            equipped with an electron capture detector, reliable mea-
            surements of 50 ng/1 2,4-D, 10 ng/1 Si! vex, and 10  ng/1
            2,4,5-T are oossible.  Concentrating the extract to 0.5 ml
            permits the detection of approximately 10 ng/1  2,4-0,  2 ng/1
            Silvex, and 2 ng/1 2,4,5-T.

     2.2.4  Precision and Accuracy

            Information is not presently available.

     2. 2. "5  Sample Extraction

            Acidify 1 liter of aqueous  sample to pH 2 with concentrated
            sulfuric acid and  transfer  to a 2-liter separatory  funnel.
            Add 150 ml ethyl  ether to the separatory funnel  and shake
            vigorously for 1 minute.  Let the phases separate for  10
            minutes.  If emulsions form, drain  off the aqueous  layer,
            invert the funnel, and shake rapidly.
                 1:   Vent *he *unne1  freauentlv  to  orevent  excessive
            pressure  buildup.


                                    III-315

-------
       Collect the extract in a 250-ml  ground-glass stoppered
       Erlenmeyer flask containing 2 ml  KOH solution.   Repeat the
       extraction with two 50-ml  portions of ethyl  ether.   Combine
       the extracts in the Erlenmeyer flask.

2.2.6  Extract Hydrolysis

       Add 15 ml distilled water and a  small boiling stone to the
       flask.  Attach a three-ball Snyder column.  Remove  the ether
       on a steam bath and continue heating for a total of 60
       minutes.

       Transfer the concentrate to a 60-ml separatory funnel.  Ex-
       tract the sample with 20 ml ethyl  ether and discard the ether
       layer.  Repeat the ether extraction and again discard the
       ether layer.  The herbicides are  retained in the aqueous
       phase.

       Acidify with 2 ml cold (4C) 9M  sulfuric acid.   Extract once
       with 20 ml ethyl ether and twice  with 10 ml  ethyl ether.
       Collect the extracts in a 125-ml  Erlenmeyer flask containing
       0.5 g acidified anhydrous sodium  sulfate.  Let the  extract
       remain in contact wtih the sodium sulfate for at least 2
       lours.

2.2.7  Esterification

       Fit a Kuderna-Danish apparatus with a 5-ml volumetric re-
       ceiver.  Transfer the ether extract to the Kuderna-Danish 
       apparatus through a runnei plugged with 'jiass too;,  jse
       liberal washing of ether.  Crush  any hardened sodium julfate
       with a glass rod.  Before concentrating, add 0.5 ml benzene.

       Reduce the volume co 
-------
       stopper and shake vigorously for about 1  minute.   Let stand
       for 3 minutes to facilitate phase separation.

       NOTE 3:  Care must be taken to ensure that the tubes are
       tightly capped and remain so after introduction of the boron
       tri fluoride methanol  reagent.  The temperature should be
       about 50C for good yields.  The methylation is a very crit-
       ical step in the procedure.

2.2.8  Sample Cleanup and Analysis

       Pi pet the solvent layer from the receiver to the top of a
       small column prepared by plugging a disposable Pasteur pi pet
       with glass wool and packing with 2 cm sodium sulfate over
       1.5 cm Florisil adsorbent.  Collect the eluate in a 2.5-ml
       graduated centrifuge tube.  Complete the  transfer by repeat-
       edly rinsing the volumetric receiver with small quantities  of
       benzene until a final volume of 2.0 ml of eluate is obtained.
       Check calibration of centrifuge tubes to  ensure that the
       graduations are correct.

       Analyze the benzene extract by gas chromatography using at
       least two columns.  Injections of 5 to 10 pi  should be
       sufficient for this puroose.

       Inject methyl ster standards freauently  to ensure optimum
       operating conditions.  Always inject the  --ame volume.  Adjust
       the volume of sample extract with benzene, 1f necessary,, so
       that the heights of the peaks obtained are close to those of
       tne standards.  (If 3 portion of the extract solution was
       concentrated, the dilution factor 0 is iess cnan ,  ~ r: *ds
       diluted, the dilution factor exceeds 1.)

       Confirmation of residue identity can be achieved by trans-
       esterificationS, thin layer chromatography^, or mass
       spectroscopy.
                               III-317

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3.1  Analysis of Sediment Samples for Chlorinated Pheno^y Acid
     Herbicides by Acetone-Hexane Extraction
          Analytical  Procedure:   available
          Sample Preparation:   available

     3.1.1  References

            Walton, A., "Ocean Dumping Report 1.   Methods for Sampling
            and Analysis of Marine Sediments and  Dredged Materials."
            Department of Fisheries and Environment, Ottawa,  Ontario,
            Canada.  74 p.  (1978).6

            Peake, A. A., and H. S. Lesick, "Procedure for the Analysis
            of Phenoxy Acid Herbicides in Sediments."  Water  Quality
            Branch, Inland Waters Directorate, Calgary, Alberta, Canada
            9 p.  (No date).7

     3.1.2  Method Summary

            A 25-g sample of homogenized sediment is extracted with 1:1
            acetone/hexane under acidified conditions.  The extract is
            dried, exchanged into benzene, and esterified with boron
            trifluoride/methanol.  Extract cleanup is achieved with a
            3:!*ca nel column and ina'vsis *s comoletsd 'jslng a gas
            chromatograph equipped with an electron capture detector.

     3.1.3  Applicability

            The orocedure is suitable for use with sediment,  dredged
            material, and soil samples,  r.ie oetscticn ,-.mit  or ;ne
            procedure will be influenced z>y sample 51 ze, extraction
            efficiency, extract concentration, esterification efficiency,
            and the presence of interferences.

     3.1.4  Precision and Accuracy

            The procedure has been shown to produce greater than 90%
            recoveries with known standards.'  Recoveries from natural
            sediments with high organic matter and high sulfur content
            ranged from 68 to 106%.  The presence of organic matter can
            have  a negative effect on the recovery of chlorophenoxy acetic
            acids.

     3.1.5  Sample Extraction

            Weigh out a 25-g  dry-weight equivalent of homogenized
            sediment sample.  -Transfer the sample to a 250-ml beaker and
            add 10 ml acidified, organic-free water.  The resultant
            slurry should be  approximately 20 to 30% water,

            Thoroughiy mix  cne  sediment jiurry and carefully uc-.tiify  ;he
            samele with 4 ml  concentrated hydrochloric acid.   Add the

                                     III-318

-------
acid slowly with constant mixing to prevent mechanical loss
due to gas evolution.  Allow the mixture to stand 20 minutes
with occasional stirring.

Add 5 ml 1:1 acetone:hexane mixture to the acidified sediment
slurry.  Place the disruptor horn of an ultrasonic homog-
enizer approximately 2 cm into the sample.  Activate the
disruptor for 2 minutes in the pulsed mode at 35% duty cycle
with maximum output.  Allow the sediment to settle.

Prepare a slurry consisting of 1:1 acetone:hexane and celite.
Pour the slurry into a sintered glass funnel which is con-
nected to a suction flask.  Remove the solvent from the
celite filter bed by suction and discard the acetone/hexane.

Decant the supernatant solvent from the sample extraction
into the funnel and apply a vacuum to collect the extract in
the suction flask.  Retain the solids for a second extrac-
tion.

Add 75 ml 1:1 acetone:hexane to the original sediment sample.
Mix with the ultrasonic homogenizer, allow the sediment to
settle, ind 'i1tsr throuqh the celite ""Star bed as before.

Transfer the combined extract to a 500-ml separator^ funnel.
Add 100 ml acidified, organic-free water and shake for 1
minute.  Release the pressure frequently.  Allow the layers
to seoarate and transfer the aqueous layer back to the
suction flask.

Slowly pour the solvent layer through a glass powder funnel
plugged with glass wool and containing approximately 2 cm of
acidic sodium sulfate.  Collect the solvent in d 500-ml flat-
bottomed flask.

Return the aqueous layer from the suction flask to the sep-
aratory funnel.  Rinse the suction flask with 75 ml methylene
chloride and add the rinses to the aqueous phase in the
separatory funnel.  Shake for 1 minute and allow the layers
to separate.  If an emulsion persists, leave it with the
aqueous layer.  Pass the lower solvent layer through the
sodium sulfate funnel and collect in the 500-ml flat-bottomed
flask with the acetone:hexane extract.

Extract the aqueous phase with a second 75-ml portion of
methylene chloride.  Filter the methylene chloride phase
through the acidified Na2$04 funnel and combine with
previous extracts.   If necessary pour the combined extract
through a second dryinq column 'acidified Na^SOa) as
water can interfere with tne estenfication reaction ana
result >, "low -scoveries.

-------
3.1.6  Extract Esterification

       Transfer the extract to a Bu'chi  evaporator and reduce the
       volume to 2 to 5 ml.  Transfer the residue to a 15-ml  graduated
       centrifuge tube and evaporate to 0.5 ml.

       Add 1 ml  of benzene to the sample extract 1n the graduated
       centrifuge tube and shake.  Reduce the volume to 0.5 ml.
       Repeat the process of adding 1 ml  of benzene and reducing the
       volume to 0.5 ml until the extracted residue has been
       exchanged from methylene chloride to benzene.

       Add 0.2 ml 14-percent boron trifluoride methanol esterifica-
       tion reagent and shake for 1 minute.  Seal  tightly and place
       the tube in a water bath at 50C for 30 minutes.  (Care must
       be taken to ensure that the tubes are tightly capped and
       remain so after the introduction of the boron trifluoride/
       methanol  reagent.  The temperature should be about 50"C for
       good yields.  The methylation process is  a very critical  step
       in the procedure).

       Cool to room temperature and add 5 ml 5-percent sodium
       sulfate solution.  Shake for 1 minute and allow the layers to
       separate.  Withdraw the top solvent layer into a clean cen-
       triruge tuoe using a Pasteur pipet.

       Add 1 ml  benzene and jhaice.  Allow the layers to separate and
       transfer the top benzene layer to a clean centrifuge tube.
       Repeat the benzene extraction a second and a third time.
      ' Comoine ;he oenzene extncts.

       Evaporate the final  benzene extract to a  volume of 0.5 ml.
       Add 1 ml  hexane, shake, and reduce the volume to 0.5 ml.
       Repeat this process a second and a third  time to exchange the
       sample residue into hexane.

       Prepare a cleanup column by adding pre-heated silica gel  to a
       height of 75 mm in a disposable pipet. Tap the column while
       packing.   Add 1 cm of neutral anhydrous sodium sulfate to the
       top of the column.  Elute the column with approximately 30 ml
       of hexane and discard the eluant.

       Quantitatively transfer the 0.5-ml hexane concentrate to  the
       cleanup column.  Rinse the centrifuge tube with three 1-ml
       portions of hexane and add each rinsing to the cleanup col-
       umn.  Allow the column to elute until the hexane layer just
       recedes into the top of the sodium sulfate layer.

       Elute the column with 90 ml hexane.  Discard the eluant.
       Elute the column with 100 ml benzene.  Collect the solvent
       in j 500-nl Hat-bottomed "ask.  ^lute *.he ralumn with i
       second 100-ml  portion of benzene and combine with tne first

                               III-320

-------
       eluant.  Reduce the volume to approximately 5  ml  on a  Buchi
       evaporator.   Transfer the concentrate to a 10-ml  volumetric
       flask and dilute to volume with benzene.

3.1.7  Sample Analysis

       Preliminary identification is achieved via electron capture
       GLC in which at least two different stationary phases  of
       different polarity are employed.   The identity of the  herbi-
       cide is based on the retention time relative to aldrin.
       Calculate the concentration of sample constituents as
       indicated in Subsection H.  Methyl  ester standards should  be
       injected frequently to ensure optimum instrument operating
       conditions.

       Confirmation of identity can be achieved by transesterifica-
              thin  layer chromatographyS,  or mass spectroscopy.
                               III-321

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3.2  Analysis .of Soil  Samples for Chlorinated Phenoxy Acid Herbicides  by
     High Performance  Liquid Chromatography
          Analytical  Procedure:   available
          Sample Preparation:  available

     3.2.1  Reference

            Stephens,  R. D., J.  Nakao and B.  P.  Simmons,  "Methods for  the
            Analysis  of Hazardous Wastes."  California Department of
            Health Services.   Berkeley, California.   (1981).5

     3.2.2  Method Summary

            Samples are acidified and extracted  with  dlchloromethane.   In
            order to  Isolate the chlorophenoxy herbicides from  possible
            Interfering substances, sample extracts are hydrolyzed with
            alcoholic  potassium hydroxide, diluted with water,  and washed
            with dlchloromethane.  Herbicide residues are acidified and
            extracted  Into dlchloromethane.  The extracts are dried,
            diluted with methanol, and analyzed  by HPLC using a reversed-
            phase column and a UV detector at 285 nm.

     3.2.3  Applicability

            This method 1s suitable for quantitying chioropnenoxy add
            heroicides In soiid-onase \soi';)  samples.  The method detec-
            tion limit will depend on Initial sample  size, degree of
            extract concentration, and the presence of Interfering
            substances.

     3.2.4  Precision  ana Accuracy

            Recovery studies 1n which nine chlorophenoxy herbicides were
            determined in extracts of three separate  soil samples showed
            an average recovery of 92% (82.3-98.7%) with an average
            coefficient of variation of 9.2%. The herbicides included in
            the study were 2,4-D add, 2,4,5-T add,  2,4-DB acid, silvex,
            2,4-D isopropyl ester, 2,4-D butyl ester, 2,4-D butoxyethanol
            ester, 2,4-D propylene glycol butyl  ether ester, 2,4-D ethyl-
            hexyl ester.

     3.2.5  Sample Extraction

            Weigh 20.0 g homogenized sample Into a 250-ml Erlenmeyer
            flask, acidify with 5 ml 24 M sulfuric acid, and extract with
            50 ml dlchloromethane for at least 4 hours on a mechanical
            shaker.  Decant the dlchloromethane  into a 125-ml beaker.
            Use an additional 10 to 15 ml dichloromethane to rinse the
            flask and sample.  Add the wash to the Initial extract. Allow
            dichloromethane to evaporate at room temperature to dryness
            unaer a nooa.  To speea the drying process, evaporate on :he
            
-------
       interfering substances (pigments, oils, lipids, etc.), proceed
       directly to paragraph 3.2.7.  If the sample contains interfering
       substances, continue with the hydrolysis step, paragraph 3.2.6.

3.2.6  Extract Hydrolysis

       This step is designed to convert the chlorophenoxy esters to
       the sodium salts of their respective acids.  Since the acids
       are soluble in water, interfering organic compounds can then
       be removed with dichloromethane.  When using the hydrolysis
       step, both the quantitative and qualitative information on
       the chlorophenoxy esters is lost and the data must be
       reported as total chlorophenoxy acids.

       Add 50 ml 0.2 M methanolic potassium hydroxide to the beaker
       containing the sample residue.  Cover the beaker with a watch
       glass, place on a steam bath, and allow to reflux for at
       least 1 hour.

       Remove the beaker from the steam bath, allow to cool, and
       transfer the sample .{total volume of sample plus washings not
       to exceed 50 ml) into a 500-ml separatory funnel containing
       250 *ni jisfi'lsd -rater ind 30 ?.l sodium thloride-saturated
       water.  Extract the sample three times witn 50-ini portions of
       dichloromethane.  Discard the organic extracts.

       Acidify the aqueous phase in the separatory funnel to pH <2.
      " I/.trict *he amnle with three 50-ml  oortions of dichloro-
       methane.  Combine tne sxtracts  r'rom cne uciav.;"ea sample -ind
       evaporate to dryness in a nood.  Place the extract on the
       edge of a steam bath to speed the drying process.

3.2.7  Extract Cleanup

       It is essential that all non-soluble particulate matter above
       1.0 u be removed since the HPLC column is composed of 5 u
       particles and is easily plugged.  Select one of the following
       techniques for particulate removal.

      a. Dissolve the -iried residue in 2.0 ml methanol.  Filter
         according to conventional methods such as a 3weny-type
         filter.  Dilute the filtered sample to 2.0 ml.

      b. Transfer the dried residue with 1.0 ml methanol on to
         reversed phase Sep-Pak and elute with methanol into a
         2-ml volumetric flask.  Dilute to volume with methanol.

3.2.8  Sample Analysis

       Inject 10 ul of standara or sampie .nto d HPLC that "las been
       equilibrated :nder the *3T!owinq conditions:

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       Column:   CIQ, 5 u,  15  cm,  reversed-phase,  ultrasphere
       Mobile Phase:   30% acetonitrile programmed  to  Increase  to  80%
       acetonitrlle over 9 minutes  and 70%  dilute  acetic acid  (1%)
       programmed to decrease to 20%.

        .Initial  hold  *  1.0 minutes
         Final hold    -  8.0 minutes
         Flow rate     *  2.0 ml /minutes
         Wavelength    =  285 nm
         Attenuation     1.0
         Chart speed   =  0.5 cm/minutes

3.2.9  Data Interpretation

       Tentatively identify peaks based on  retention  time  and
       quantltate by peak height or peak area.   Compare the  peak
       height or peak area to a standard which  is  known to be  in  the
       linear range of the instrument.
                               III-324

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G.   CALCULATIONS

     Compare the peak height of a standard to the peak height of the sample  to
determine the amount of the herbicide injected.

     Calculate the concentration of herbicides in liquid samples as  follows:


                                        A x B x  C x D
                            P(ng/l)
                                          E x F x G

     where

          P  *. concentration of chlorinated phenoxy acid herbicides
          A  *  weight of herbicide standard injected
          B  *  peak height of sample, mm
          C  -  final extract volume, pi
          D  =  dilution factor
          E  *  peak height of standard,  mm
          F  s  extract volume injected,  pi
          ~  -  volume of samole extracted, ml.

     7he concentration of chlorinated phenoxy acid herbicides in sediment
     samples can be calculated as:
                            ? [wet weight)
                         P (dry weight)
                                          E x F x H


                                          A x B x C

                                       E x F x H x IS

where

     o  =  concentration of chlorinated phenoxy acid herbicides,  pg/kg
     A  =  weight in picograms of standard
     B    peak height (or area) of sample
     C  =  final  volume of sample extract, ml
     E  s  peak height (or area) of standard
     F  *  volume of extract required to produce B, pi
     H  *  wet weight of sediment initially extracted,  g
    IS    percent solids in sediment sample (expressed as a decimal
           fraction).
                                    111-325

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                                  REFERENCES


1.   American Public Health Association.   "Standard Methods for the Examination
     of Water and Wastewater Including Bottom Sediments  and Sludges."  15th
     Edition:  American Public Health  Association, New York, Mew York.  1,134
     p. (1980).

2.   Junk, 6. A., J. J. Richard,  J.  S. Fritz  and  H. J. Svec.   "Resin Sorption
     Methods for Monitoring Selected Contaminants in Water."   In:  "Identifica-
     tion and Analysis of Organic Pollutants  in Water.   L. H.  Keith (Ed.).
     Ann Arbor Science Publishers; Ann Arbor,  Michigan,   pp. 135-153 (1976).

3.   Environment Canada.  "Analytical  Methods Manual."   Inland Waters
     Directorate, Water Quality Branch; Ottawa, Ontario,  Canada (1974).

4.   Bristol, D.  "Effects of Storage  Conditions  on Residues of 2,4-D and
     2,4-DCP in Potatoes."  In:  "Accuracy in Trace Analysis:  Sampling,
     Sample Handling, and Analysis."  National Bureau of Standards Special
     Publication 422.  pp. 737-745  (1976).

5.   Stephens, R. D., J. Nakao and B.  P.  Simmons. "Methods for the Analysis
     of Hazardous Wastes."  California Department of Health Services,
     Berkeley, California.  (1981).

6,   Walton, A.  "Ocean Dumoinq Report 1.  Methods for Sampling and Analysis
     :f Marine Pediments and j-rsaged Material 3. '   Department of Fisheries and
     Environment; Ottawa, Ontario, Canada.  74 p. (1978).
          , V & -r,d H. S. '.ssick.   ""-ocedurs  for  the  ^nalvsis  of Phenoxy
     Acirt Heroiciaes in Sediments.*"   n'atar Guaiity orancn,  4,iiana Waters
     Directorate; Calgary, Alberta,  Canada.   9 p.   iNo aate).

8.   Yip, G.  "Confirmation of Chlorophenoxy Acid  Herbicide Residues  by
     Transesterification."  J. Assn. Gffic.  Anal.  Chem.  54:343-34 (1971).

9.   Chau, A. S. Y.  "Analysis of Chloriniated Hydrocarbon  Pesticides in
     Waters and Wastewaters.  Methods in Use in  Water Quality  Division Labora-
     tories."  Department of the Environment, Inland Waters Directorate;
     Ottawa, Ontario, Canada (1972).
                                    III-326

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                                   SECTION 8

                METHODS FOR THE DETERMINATION OF DIOXIN (TCDD)
A.   SCOPE
     This Section provides methods for the isolation and identification of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wastes and environmental
samples.  The general procedure consists of methylene chloride extraction,
extract cleanup, solvent-exchange into hexane, and quantification using GC/MS
methodology.

     Due to the highly toxic nature of this compound, the analyst is urged to
exercise extreme caution when handling the pure compound (standards) and
samples known or suspected to contain this material.  General  safety guidance
and protocols are provided in Subsection D.

B.   SAMPLE HANDLING AND STORAGE

     Aqueous samples should be collected in amber glass containers and  sealed
with' foil- or Teflon-lined screwcaps.  The container should not be prewashed  '
vit^ :amol^ prior to collection.  Also, all samojinq eouioment must be  as free
as possible of Tygon and other potential sources or jomannnation.

     The samples must be iced or refrigerated at 4C and protected from light
from the time of collection until extraction.  If the sample contains residual
chlorine, 80 mg/1 sodium thiosulfate snould be added to the sample.  Field
test kits are available to determine the need for use of a thiosulfate
preservative.  Aqueous samples should be extracted within 7 days of collection
and analyzed within 40 days of extraction.1

     Soil and sediment samples should be collected in glass containers  and
refrigerated until processing.  The maximum storage period is  not known;
therefore, it is recommended that these samples be orocessed within the same
time frame as water samples.

     Biological tissue samples should be wrapped in solvent-rinsed foil.
Samples should be frozen immediately after collection and remain frozen until
analysis is initiated.

     This information is summarized in Figure 1.
                                    III-327

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                                 82C-III
o
Ol

Q.
A
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O

Cat
U3
(T
O


01
 9 *  (">W>    V
        rt  f? tst  II    5
        i-  '| ,-,4  si    l
            1ft
        3
        ?
        3
        ?
        3
                            -4

                          i!
                  f.
                         r =
                         i  =
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                         I = S

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C.   INTERFERENCES

     Method interferences caused by contaminants in solvents,  reagents,  glass-
ware, and other hardware used in sample processing may lead to discrete  arti-
facts and/or elevated backgrounds for the ions being monitored.  All  of  these
materials must be routinely demonstrated to be free from interferences under
the conditions of the analysis by running laboratory reagent blanks.

     All  glassware must be scrupulously cleaned2 after each use.   Glassware
should be rinsed with the last solvent used in it, followed by detergent-wash-
ing with hot water and rinsing with tap water and distilled water.   Glassware
should then be drained dry and heated in a muffle furnace at 400C for 15 to
30 minutes.  (Some thermally stable materials, such as PCBs, may  not be  elimi-
nated by this treatment.)  Solvent rinses with acetone and pesticide-quality
hexane may be substituted for the muffle furnace heating.  Volumetric glassware
should not be heated in a muffle furnace.  After drying and cooling,  glassware
should be sealed or capped with aluminum foil and stored inverted in a clean
environment to prevent any accumulation of dust or other contaminants.

     The use of high-purity reagents and solvents helps to minimize interference
problems.  Purification of solvents by distillation in all-glass  systems may be
required.
     Matrix -'nt2rferenc3s ^av be caused by contaminants that are coextracted
from the sample.  The extent of matrix interferences nr<  t'ary cans', aeraoiy
from. sample to sample, (ieoenainq on ;he nature and Jiversity cf *he sample.
TCDD is often associated with interfering chlorinated compounds wmcn  are
present at concentrations several magnitudes higher than TCDD.  The suggested
:' ;=nuD orocsdures can overcome many of these interferences, but unique  samples
may reauire additional cleanup-"0 co eliminate ;ai':s ;os".""-vss :nc Achieve  ",he
method detection limit.

     Some other tetrachlorodibenzo-p-dioxin isomers may also interfere with  the
measurement of 2,3,7,8-TCDD.3-6  Capillary column 933 chromatography can be
used to achieve separation, but identification of some Isomers may not be
possible because of virtually identical mass fragmentation patterns.

D.   SAFETY

     The toxidty or carcinogenldty of each reagent used in this method has
not teen precisely defined.  Benzene and TCDD have been identified as
suspected human or mammalian carcinogens and the otner reagents snouid be
treated as potential health hazards.  From this viewpoint, exposure to these
chemicals must be reduced to the lowest possible level.

     A strict laboratory safety program for the handling of TCDD samples
should be developed that includes the following practices:

1.   Potential contamination of the laboratory should be minimized by  con-
     "lucfinq ill samole manioulations in a hood, glovebox, or secondary
     containment device.
                                    III-329

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2.   The effluents of sample splitters for the gas  chromatograph  and  roughing
     pumps on the GC/MS should pass through either  a  column  of  activated
     charcoal or be bubbled through a trap containing oil  or high-boiling
     alcohols.

3.   Liquid wastes should be dissolved in methanol  or ethanol and irradiated
     with ultraviolet light with wavelength greater than  290 nm for several
     days (use F 40 BL lamps or equivalent).   When  TCDD can  no  longer be
     detected, dispose of waste solutions as  appropriate.

     Dow Chemical USA has issued the following precautions (11/78) for the
safe handling of TCDD in the laboratory.   They are  as complete  as possible
based on available toxicological information  but necessarily general  in nature
since detailed, specific recommendations  can  only be  made for the particular
exposure and circumstances of each Individual  case.  Inquiries  about  specific
operations or uses may be addressed to the Dow Chemical Company,  Midland,
Michigan.

1.   Protective equipment including disposable plastic gloves,  apron  or lab
     coat, safety glasses, and a lab hood adequate  for radioactive work should
     always be used.

2.   Workers must be trained in the proper method of  removing contaminated
     gloves and clothing without contacting the exterior  surfaces.

3.   Thorough washing of hands ana rorearms after eacn inampuiation ana oefore
     oreans  (coffee, iuncn. end of snift; are .nanaatory.

4.   The work area should be posted with  signs and  isolated. All work
     surfaces should be covered with clastic-backed absorbent paper.   Separate
     glassware and toois snouiu oe prcviaea -cr  ;ne drs&.

5.   Waste cans should be lined with plastic bags.  Janitors must be  trained
     in safe handling of potentially hazardous wastes (one accidental case  of
     chloracne resulted from handling laboratory wastes  .n a routine  manner).
     It is a good practice to minimize the volume of  contaminated wastes  when
     possible.

6.   The following suggestions are offered for the  disposal  of  TCDD related
     wastes:  TCDD decomposes above 8008C.  Low-level waste such  as absorbent
     paper,  tissues, animal remains, and plastic gloves may be  burned in  a
     .good incinerator.  Gross Quantities  (milligrams) of  TCDD should  be
     packaged securely and disposed through commercial or governmental
     channels which are capable of handling high-level radioactive wastes or
     extremely toxic wastes.  Liquids should be placed in a disposable
     container and allowed to evaporate in a hood.   Residues can  then be
     handled as  described above.

7.   In the  event that contamination occurs, the following decontamination
     procedures  are recommended.
                                    III-330

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     7.1  Decontaminate personnel  with any mild soap  and  plenty  of  scrubbing
          action.

     7.2  Satisfactory cleaning of glassware,  tools,  and  surfaces may  be
          accomplished by rinsing  with Chlorothene  (Dow Chemical-Company),
          then washing with any detergent and  water.   Dish  water may be
          discharged to the sewer.  It is prudent to  minimize  solvent  wastes
          that may require special disposal through expensive  commercial
          services.

     7.3  Lab coats or other clothing worn in  TCDD  work may be laundered.
          Clothing should be collected in plastic bags.   Persons who convey
          the bags and launder the clothing should  be advised  of the potential
          hazard and trained in proper handling of  the clothes.  The clothing
          may be put into a washer without contact  if the launderer knows  the
          problem.  The washer should be run through  a cycle before being  used
          again for other clothing.

8.   A useful method of determining the cleanliness of work surfaces and tools
     is to wipe the surface with a piece of filter  paper.   Extraction  and
     analysis by gas chromatography can achieve a limit of  sensitivity of  0.1
     micrograms per wipe.  This procedure is provided in  Subsection L.  Less
     inan 1 ^9 T-DO per 
-------
          must be protected from light.   Containers must  be washed,  rinsed with
          acetone or methylene  chloride,  and  dried before use.
     1.2  Automatic sampler (optional).   Must incorporate glass  sample con-
          tainers for the collection  of  a minimum of  250  ml.  Sample containers
          must be kept refrigerated at 4C and protected  from light  during
          compositing.   If the  sampler uses a peristaltic pump,  the  length of
          compressible tubing (Tygon  or  silicone rubber)  used should be mini-
          mized.   Before use, the compressible tubing should be  thoroughly
          rinsed with methanol, followed by repeated  rinsings with distilled
          water to minimize potential  contamination of the sample.   An integra-
          ting flow meter is required to collect flow proportional composites.
     1.3  Freezer, for tissue samples.   Wipe  tests should be performed before
          and after use to eliminate  risk of  cross-contamination.
     1.4  Ice chest or refrigerator,  for water and soil/sediment samples.
2.   Safety
     2.1  Plastic gloves.  Inner gloves  of Viton or nitrile rubber,  covered
          by disposable surgical  gloves,  have been used.
     2.2  Aprons, polyethylene  or Saranex-laminated (Durafab P2110 or
          equivalent),
     2.3  Safety glasses, equipped with  side-shields.
     2.4  Ultraviolet lamp,  with wavelength greater than  290 nm.
3.   Samoie Preparation and Analysis
     3.1  pH paper, wide range.
     3.2  Funnel, glass.
     3.3  Erlenmeyer flasks, 250 ml and  125 ml.
     3.4  Round-bottom flasks,  500 ml  and 100 ml.
     3.5  Chromatographlc column, glass, 1 cm I.D. x  50 cm.
     3.6  Distillation receiver, 12 ml.
     3.7  Oven, capable of maintaining 200eC.
     3.8  Desiccator.
     3.9  Graduated Chromaflex sample tube, 2 ml (for water samples).
                                    III-332

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 3.10   Centrifuge (for soil/sediment  and  tissue  samples), with bottles and
       tubes.

 3.11   Extraction Apparatus

      3.11.1   Extraction jar,  250  ml  or larger;  shaker  or stirrer;  filter
              paper (Whatman No. 4 or equivalent), or:

      3.11.2   Soxhlet extraction apparatus, with thimbles, rotary evapor-
              ator, N-Evap analytical  concentrator.

      3.11.3   Chromatographic  column  (1 cm  I.D.  x 10 cm) and conical mini-
              vials, 2 ml, with Teflon-lined  septa, or:

      3.11.4   Glass macro-column,  2 cm O.D. x 23 cm, tapered 1 cm,  and
              5  ml  disposable  pipet.

      3.11.5   Blender, high-speed.

 3.12   Glass  tubing, 3 mm I.D. x 7 mm.

 3.13   Mortar and pestle,  glass.

 3.14   uiass  wooi,  ii^dmzsa.

.3.15   Separatory funnels, 125 ml  and 2000 Til, -yitn Teflon stopcocks.

 3.16   Concentrator tube,  Kuderna-Danish, 10  ml,  graduated (Kontes
       K-370050-1025, ;-r sqir '.t-'-nt}.  ^jl-'br^tion "lust be checked  at the
       volumes employed " the **st.   A nrouna gidss stopper is necessary
       to  prevent evaporation  of extracts.

 3.17   Evaporative *lssk,  Kuderna-Oanish, 500 ml  (Kontes 570001-0500 or
       equivalent).  Attach to concentrator tube with springs.

 3.18   Snyder column, Kuderna-Danish,  three-ball  macro  (Kontes K-503000-
       0121 or equivalent).

 3.19   Snyder column, Kuderna-Danish,  two-ball micro (Kontes K-569001-0219
       or  equivalent).

 3.20   Vials,  amber glass, 10  to 15 ml capacity,  with Teflon-lined
       screwcaps.

 3.21   Chromatography column,  30 cm x 1 cm  I.D.,  with coarse fritted disc
       at  bottom and Teflon stopcock.

 3.22   Chromatography column, 40 cm x 1 cm  I.D.,  with coarse fritted disc
       at  bottom and Teflon stopcock.

 *?.23   Boiling chips, 10/40 mesh.   Prior  to use,  neat to 4t)0"C for  50
       minutes or ioxniet-^xtract  *i c,*i .Tiethylene  "hlor-'de.

                               III-333

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     3.24  Water bath,  heated, with  concentric  ring cover.  The bath  should be
           used  1n a  hood  and capable of  2C temperature control.

     3.25  Gas chromatograph/mass  spectrometer  analytical system.

     3.25.1  Gas chromatograph.  An  analytical  system complete with all re-
             quired accessories  Including syringes and  gases.  The injection
             port must  be  designed for capillary columns.  Either split or
             splitless  Injection 1s  acceptable.

     3.25.2  Capillary  column, 50 m  x 0.25 mm I.D. glass coated with  SILAR-
             10C or equivalent). An equivalent column  must resolve 2,3,7,8-
             TCDD from  the other 21  TCDD  Isomers.

     3.25.3  Mass spectrometer.  Equipped with  a 70 volt (nominal) ion source
             and capable of acquiring 1on abundance data 1n real time for
             groups of  four or more  Ions  (Selected Ion  Monitoring or  SIM).

     3.25.4  GC/MS interface.  Any GC-to-MS  interface that gives acceptable
             calibration points  for  50 ng of TCDD per injection may be used.
             GC-to-MS interfaces constructed of all glass or  glass-lined
             materials  are recommended.   Glass  surfaces can be deactivated by
             silanizing with dichlorodimethylsilane.
     2.2S.5  Oata :ystsm.   \ computer  system  ~5ust  be  *nterfacsd to the
             spectrometer that allows  continuous acquisition  and  storage on
             machine-readaDle media  of al1  SIM  data obtained  throughout the
             duration of the chromatographic  program.  This output is defined
             as the Selected Ion Current Profile (SICP).  The computer must
             ^av*> software available that allows slotting the SICP and inte-
             grating the abundance or  any ;or. ,n ;ne  ilC? oetween specified
             time or scan number limits.  The capaoility  for  real -time analog
             output of the SICP 1s useful  but not  required.

     3.26  Balance, analytical.  Capable of accurately weighing 0.0001 g.

F.   REAGENTS

1.   Reagent water (defined as Interference-free at  the method detection
     limit of TCDD).

2.   Sodium hydroxide solution, 10 N.   Dissolve 400  g NaOH  in reagent water
     and dilute to 1 liter.  Wash the  solution  with methylene chloride and
     hexane before use.

3.   Sodium thiosulfate, granular.

4.   Sulfuric add, concentrated.

5.   Methylene chloride, pesticide quality or equivalent.

6.   Hexane, pesticide quality or equivalent.

                                    II 1-334

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7.   Benzene, pesticide quality or equivalent.

8.   Tetradecane, pesticide quality or equivalent.

9.   Sodium sulfate, anhydrous, granular (ACS).  Purify by heating at 400C
     for 4 hours in a shallow tray.

10.  Alumina, neutral, 80/200 mesh (Fisher Scientific Co., No.  A-540 or
    'equivalent).  Activate for 24 hours at 130C in a foil-covered glass
     container before use.

11.  Silica gel, 100/120 mesh, high purity grade.  (Fisher Scientific Co.,
     No. 5-679 or equivalent).

12.  Stock standard solutions (1.00 pg/yl).  Stock standard solutions can be
     prepared from materials of known purity or purchased as certified
     solutions.

     12.1  Prepare stock standard solutions of 2,3.7,8-TCDD (mol.  wt. 322),
           and either 37C14TCDD (mol. wt.  328) or I3C12-TCDD (mol.  wt. 332)
           in a glove box by accurately weighing about 0.0100 grams of pure
           material.  Dissolve the material in pesticide quality isooctane;
           dilute to volume in a 10-ml volumetric *lask.  If comoound ourity
           is certified at 96% or greater, cne weignt can oe used  witnout
           correction to calculate the concentration of tha itcck  standard.
           Commercially prepared stock standards can oe used at any concen-
           tration if they are certified by the manufacturer or an independent
           source.

     12.2  Transfer the stock standard solutions into Teflon-sealed :crew-cap
           bottles.  Store in a glove box  protected from light.  Stock stand-
           ard solutions should be checked frequently for signs of degradation
           or evaporation, especially just prior to preparing calibration
           standards or spiking solutions  from them.

     12.3  Stock standard solutions must be replaced after six  months, or
           sooner if comparison with check standards indicates  a problem.

13.  Internal standard spiking solutions (25 ng/ml).  Using stock  standard
     solution, prepare a spjkinq solution  by the appropriate dilution of
     either 13C12-TCDD or J/C14-TCDD in acetone.

14.  Sodium carbonate, anhydrous, powder.

6.   QUALITY CONTROL

     The minimum requirements'of a quality control  program consist of an
     initial  demonstration of laboratory capability and the analysis of spiked
     sameles as a continuing check on performance.   Performance records should
     be maintained to aeiine che quality jf generated aata.

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     Before performing any analyses,  the analyst  should  demonstrate  the  ability
     to generate acceptable accuracy  and precision  with  this method.  This can
     be accomplished by performing the following  operations:

1.   The analyst should demonstrate,  through  the  analysis  of a 1-liter aliquot
     of reagent water, that all  glassware and reagent Interferences  are  under
     control.   Each time a set of samples is  extracted or  there  is a change  in
     reagents, a laboratory reagent blank should  be processed to monitor pos-
     sible laboratory contamination.

2.   Using stock standard solution, prepare a quality control check  sample
     concentrate containing TCDD at a concentration of 15  ng/ml .  Using  a
     pipet, add 1.00 ml of the check  sample concentrate  to each  of a minimum of
     four 1,000-ml  aliquots of reagent water.  A  representative  waste water  may
     be used in place of the reagent  water, but one or more additional aliquots
     must be analyzed to determine background levels, and  the spike  level must
     exceed twice the background level  for the test to be  valid.  Analyze the
     aliquots according to the method given in Subsection  J.2.

3.   Calculate the  average percent recovery,  (R), and the  standard deviation
     of the percent recovery, (s), for the replicate analyses.   Vlastewater
     background corrections must be made before R and s  calculations are
     performed.

4.   Acceptable single-operator precision and accuracy data for  the  procedure
     ara:

                    Spike          Average Percent ______ Standard  Deviation
       TCDD         0.015       '        83.7                  5.1
       TCDD         0.150               82.2                  4.2

     If the calculated R and s values  are not comparable  to  those  provided
     above, the analyst must review potential  problem areas  and  repeat  the
     tests.

5.   The analyst must calculate method performance criteria  and  define  the
     performance of the laboratory for each  spike  concentration.   Calculate
     upper and lower control limits for method performance as follows:

                        Upper Control  Limit  (UCL)   =   R + 3  s
                        Lower Control  Limit  (LCD   =   R - 3  s

     where R and s are the values  calculated  in paragraph 3  above.   The UCL
     and the LCL can be used to construct control  charts7 that are useful in
     observing performance trends.

6.   The laboratory should develop and maintain separate  accuracy  statements
     of 1aboratory oerfomancs *or ach samole matrix.  An accuracy statement
     for the method is defined as  R   s and  snoula oe developed  cased on the


                                    III-336

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      analysis  of  four  aliquots  as described  in paragraphs 2 and 3.  These
      accuracy  statements  should be updated regularly.7

 7.    Spiked  samples  should be prepared and analyzed at a frequency of 10% of
      the total  sample  loading,  or one sample per month, whichever percentage
      is greater.  The  spiked samples must be analyzed as indicated in paragraphs
      2 and 3.   If the  TCDD recovery does not fall within the control limits for
      method  performance,  the TCDD results reported for all samples processed as
      part of the  same  set must  be qualified  as suspect.  The frequency of
      generation of suspect data should be monitored and corrective action
      should  be taken if the frequency exceeds 5 percent.

      Additional quality assurance practices  should be adopted by the laboratory
 as  needed.   Examples are  the analysis of field duplicates to monitor the pre-
 cision of the  sampling techniques, the analysis of standard reference materials,
 or  participation  in  relevant performance evaluation studies.

      In recognition  of the rapid advances that are occurring in chromatography,
 it  is realized that  procedural  modifications may be implemented to improve
 separations  or lower analytical costs.  Each time analytical methods are modi-
 fied, the ability to generate data of acceptable quality must be demonstrated.

 H.    CALIBRATION

      Establish gas chromatoqraphic conditions ror the GC/MS system equivalant
 to  those  indicated 'n  ^abla 1.  The GC/MS system ,-nust be calibrated using the
.internal standard technique.  By injecting calibration stanaaras, establish ion
 response factors  for TCDD vs. an internal standard (either ^C^-TCDD or
 -;C'.tt-"C3D),   !.?e Tubsecticn -1.2*1,7 for GC/MS Specific ion monitoring condi-
 tions for both mgh-resolution  and iow-resoiuc'ion ,,iass ^pecirometr'

 1.    Using stock  standards, prepare GC/MS calibration standard solutions in
      hexane  or tetradecane that will allow measurement of relative response
      factors of at least  three  concentration ratios of "TCDD to -internal
      standard.  Each solution must be prepared to contain the internal
      standard  at  a concentration of 25 ng/ml.  If- significant interferences
      are contributed by the internal standard at m/e 320 and 322, the
      concentration should be reduced in the  calibration standards and in the
      internal  standard spiking  solution (Subsection F.13.).  One of the
      calibration  standard solutions should be prepared to contain TCDD
      representing a  concsntration near, but  above, the method detection limit.
      The other TCDD  concentrations should cover tne range of concentrations
      expected  in  the samples to be analyzed.

 2.    Using injections  of  2 to 5 yl, tabulate m/e area responses against the
      concentration of  TCDD and  internal standard in each calibration standard,
      and calculate response factors (RF) for TCDD using Equation 1.

                                    (As) (C1s)
                            ^F  =   	                        Eq. 1
                                     III-337

-------
     where

             As  =  SIM response for TCDO m/e 320
A-jS  =  SIM response for internal standard
        ("C1?-TCDD at m/e 332 or
         37CTj-TCDD at m/e 328)
                    (3
            Cis  =  concentration of the internal  standard,  yg/1

             Cs  =  concentration of TCDD, yg/1.


     If the RF value over the working range is a  constant (<10% RSD),  the RF
     can be assumed to be invariant and the average RF can be used for
     calculations.  Alternatively, the results can be used to plot a
     calibration curve of response factors, As/AiS vs. Cs.

I.   DAILY PERFORMANCE TESTS

     The working calibration curve or RF must be  verified on each working day
by the measurement of one or more TCDD calibration standards.  If the  response
for TCDD varies from the predicted response by more than 10  percent, the test
must be reoeated using a fresh calibration standard.  Alternatively, a new
         on curve .r.ust oa preparad.
     oefore using any cleanup procedure, the inalyst must process a series of
calibration standards through the procedure to validate elution patterns and
the absence of interferences from the reagents.


        TABLE 1.  CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMIT
                                (Water Samples)8
===================================== 3=^============ ===========================
                                    Retention Time            Detection Limit
   Method                               (min)                     (yg/D


   Glass Capillary Column               34.5                       0.002
= = = ss ==z = s = = ===3: = ==3==:5=== = ==== ==== = === = = = === === = = = = === = = = = = = = = = = = = = = = = = = = = = = =
Column conditions:  SILAR-10C coated on a 50 m x 0,25 mm I.D. glass column
vor equivalent), with helium carrier gas at 30 cm/sec l-'near velocity, split-
less injector.  Column temperature programmed:  isothermal, 100C for 3
minutes, then 20C/min to 180C, and 2C/min to 250C.
                                    111-338

-------
J.   ANALYTICAL PROCEDURES
     1.1  Analysis of Hazardous Waste Samples  for Dioxin.  Reserved.
                                    111-339

-------
2.1  Analysis of Water Samples for Dioxin
          Analytical  Procedure:   available
          Sample Preparation:   available

     2.1.1  Reference

            U.S. Environmental Protection Agency,  "2,3,7,8-Tetrachloro-
            dibenzp-p-dioxinMethod 613,"  Methods  for  Organic  Chemical
            Analysis  of Municipal  and Industrial Wastewater.   U.S.  EPA,
            EPA-600/4-82-057,  1982.

     2.1.2  Method Summary

            A 1-liter sample of water is spiked with an  internal  standard
            of labeled TCDD.  The  spiked sample is then  extracted with
            methylene chloride using separatory funnel techniques.  The
            extract is concentrated,  solvent-exchanged into hexane  and
            concentrated to a  volume of 1.0  ml  or  less.

            Capillary column GC/MS conditions  are  described that allow
            for the separation and measurement  of TCDD in  the  extract.

     2.1.3  Applicability

            This is a gas cnromatograpmc/mass  spectrometnc 3C/MSJ
            method applicable  to. the determination of TCDD *n  municipal
            and industrial discharges.   The  range  of.the procedure  Can 5e
            modified"by adjusting  the sample size  and/or the degree of
            extract concentration.

            This metnod shoulc be  restricted co use  only by or under  the
            supervision of analysts  experienced in the use of  gas
            chromatograph/mass spectrometers and skilled in the  interpreta-
            tion of mass spectra.   Each analyst should demonstrate  the ability
            to generate acceptable results with this method using the quality
            assurance procedures in  Subsection  G.

     2.1.4  Precision and Accuracy

            The method detection limit (MDL) for TCDD using this procedure
            is 0.002  ug/1 (MDL is  the minimum  concentration that can  be
            measured  and reported  witn 99% confidence tnat the value  is
            greater than zero).  The reported MDL was determined on a
            secondarily-treated  sewage effluent.  This value may vary
            with sample matrix.

            Single-operator recoveries from  reagent  water  fortified at 0.005
            ug/1, industrial wastewater fortified  at 0.005 ug/1, and munic-
            ipal wastewater fortified at 0.025  ug/1  were 95.4    10.2%, 85.8
             6.6%, and 92.4  18.7%, respectively (R o).8
                                    III-340

-------
2.1.5  Sample Extraction

       CAUTION:  It is recommended that all  of the following
       operations be performed in a limited  access laboratory with
       the analyst wearing full  protective covering for all  exposed
       skin surfaces.

       Depending on the sample bottle, either mark the liquid level
       on the bottle for later volume determination or pour  nearly
       1 liter of sample into a 1-liter graduated cylinder,  recording
       the sample volume.   Pour the sample into a 2-liter separatory
       funnel.  Check the pH of the sample with wide-range pH paper and
       adjust to within the range of 5 to 9  with sodium hydroxide or
       sulfuric acid.   Spike each sample with 20 nanograms of internal
       standard.

       Rinse the sample bottle or graduated  cylinder with 60 ml
       methylene chloride and add the solvent to the sample  in the
       separatory funnel.   Extract the sample by shaking the funnel
       for 2 minutes with periodic venting to release vapor  pressure.
       Allow the organic layer to separate from the water phase for a
       minimum of 10 minutes.  If the emulsion interface between layers
       is more than one-third the size of the solvent layer, the analyst
       nust employ mechanical techniaues *,o  romolete the phase sepa-
       ration.  The optimum tecnnique depends upon cne sample, uut may
       include stirring, filtration of the emulsion through  -jluss wool,
       or centrifugation.   Collect the metnylene cnioride extract in a
  '  ;  250-ml Erlenmeyer flask.

       Ado a seccna 60-uii  volume if ,-neth'y era irv.or-.ca :s cne
       separatory funnel and complete che extraction procedure a
       second time, combining the extracts in the 250-ml flask.

       Perform a third extraction in the .same manner.

       Discard the extracted aqueous portion of the sample,
       retaining the separatory funnel for washing steps.  Pour the
       combined extracts through a glass funnel containing anhydrous
       sodium sulfate, and collect it in a 500-ml Kuderna-Danish
       (K-D) flask equipped with a 10-ml concentrator tube.   Rinse
       the Erlenmeyer flask and funnel with  20 to 30 ml methylene
       chloride to complete the quantitative transfer.

       Add 1 or 2 clean boiling chips to the flask and attach a
       three-ball Snyder column.  Prewet the Snyder column by adding
       about 2 ml methylene chloride to the  top.  Place the  K-D
       apparatus on a hot water bath (60 to 65C) so that the concen-
       trator tube is partially immersed in  the hot water, and the
       entire lower rounded surface of the flask is bathed in vapor.
       Adjust the vertical position of the apparatus and the water
       temperture as required co complete ;ne concentration  in 15 :c 20
                               III-341

-------
minutes.  At the proper rate of distillation, the balls of the
column will  actively chatter but the chambers will  not flood.
When the apparent volume of liquid reaches 1 ml, remove the
K-D apparatus and allow it to drain for at least 10 minutes
while cooling.

Momentarily remove the Snyder column, add 50 ml hexane and a
new boiling chip to the K-D flask, and replace the Snyder
column.  Increase the temperature of the water bath to
85 to 90C.   Prewet the Snyder column by adding about 1 ml
hexane to the top.  Evaporate the solvent to a volume of
approximately 1 ml, remove the K-D apparatus from the water
bath and allow it to drain and cool for at least 1C) minutes.

Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml hexane.

Rinse the 2-liter separatory funnel used in the initial
extraction twice with reagent water and discard the wash.
Pour the hexane extract from the concentrator tube into the
separatory funnel.  Rinse the concentrator tube four times
with 10-ml  aliquots of hexane.  Combine all of the rinses in
the separatory funnel.

Add 50 ml of 10 N sodium nydroxiae solution co cne funnel and
shake for 30 to 60 seconds.  Discard the aqueous phase.

Wash the organic layer with 50 ml reagent water.  Discard the
aaueous ohase.
                       

Wash the hexane layer with ar least two 30-ml aliquots of
concentrated sulfuric acid.  Continue washing the hexane
layer with 50-ml aliquots of concentrated sulfuric acid until
the acid layer remains colorless. - Discard all acid fractions.

Wash the hexane layer with two 50-ml aliquots of reagent
water.  Discard the aqueous phases.

Prepare a glass funnel packed with sodium sulfate.   Pour the
hexane extract through the funnel and collect in the K-D
flask used in the first concentration.  Rinse the separatory
funnel with several hexane washes.  Pour each wash through
the sodium sulfate column and collect in the K-D flask to
effect a quantitative transfer.

Add 1 or 2 clean boiling chips and concentrate the extract to
approximately 1 ml.  Rinse the K-D reservoir with a few ml of
hexane and collect the solvent in the concentrator tube.

Concentrate the extract to 1 ml by blowing down under a stream
of clean nitrogen.  Stopper ;rie .concentrator tube  vith 2 ^l
                        III-342

-------
       stopper and store refrigerated and protected from light if
       GC/MS analysis or cleanup will not be performed immediately.

       If the original  sample volume was not measured in a graduated
       cylinder, refill the sample bottle to the marked level  and
       measure the volume taken.

2.1.6  Sample Cleanup

       Cleanup procedures may not be required for relatively clean
       sample matrices.  The cleanup procedures recommended in this
       method have been used for the analysis of various clean water
       and industrial effluent samples The single-operator precision
       and accuracy data presented were obtained using the recommended
       cleanup procedures.

       The alumina column is frequently used to overcome interferences
       and the silica gel column has been used to overcome background
       problems.  Other cleanup procedures have been described to
       overcome special interference problems.3-6

       Alumina Column Cleanup

       Fill  a 300-mm x iO-mm-I.D. chromatograpny column ?ntn actv/ataa
       alumina to the 150-mm level, tapping the column gently  to
       settle the alumina.  Add 10 mm anhydrous sodium sulfate on top
       of the alumina.

       Preeiuie :he column with 30 nl '-.exane at ; -ate :f 1 nl/min.
       Discard the eluate and, just prior to exposure of the sodium
       sulfate layer to the air, transfer the 1.0-ml  sample extract
       onto the column.  Use two 2-ml washes of the concentrator
       tube with hexane to complete a quantitative transfer.

       Just prior to exposure of the sodium sulfate layer to the  air
       during elution,  add 50 ml of 3% methylene chloride/97%  hexane
       (v/v) and continue the elution of the column.   Discard  the
       eluate.

       Elute the column with 50 ml  of 20% methylene chloride/80%
       hexane (v/v) into a 500-mi K-D flask equipped with a 10-ml
       concentrator tube.  Concentrate the sample to 1 ml  using
       standard K-D techniques.  Analyze by GC/MS (see paragraph  2.1.7)

       Silica Gel  Cleanup

       Fill  a 400-mrn x  11-mm-I.D. chromatography column with silica  gel
       to the 300-mm level, tapping the column gently to settle the
       silica gel.

       Add 10 mm anhydrous sodiunTsulfate to the top of the silica
       gel.

                               TII-343

-------
       Preelute the column with 50 ml  of 20% benzene/80% hexane (v/v)
       at a rate of 1 ml/min.   Discard the eluate and,  just prior  to
       exposure of the sodium  sulfate  layer to the air,  transfer the
       extract to the column.   Rinse the extract container with
       two 2-ml washes of 20%  benzene/80% hexane and transfer the
       washes to the column.

       Just prior to exposure  of the sodium sulfate layer to air
       during elution, add 40  ml of 20% benzene/80% (v/v)  hexane to the
       column.  Collect the eluate in  a clean 500-ml  K-D flask  equipped
       with a 10-ml concentrator tube.

       Concentrate the eluate  to 1.0 ml using standard  K-D techniques.
       Analyze by GC/MS (see paragraph 2.1.7).

2.1.7  GC/MS Analysis

       Calibrate the GC/MS system daily as described 1n Subsection H.
       The volume of calibration standard must be measured or be
       the same as all sample  injection volumes.   A volume of 2 to 5
       ul is suggested.

       Analyze standards and samples with the mass spectrometer
       operating in the selected ion monitoring (SIM) mode,  using  a
       dwell time to give at least seven points per peak,   "or low
       resolution-mass spectrometry (LRMS), use ions at m/e 320, 322,
       arid 257 for 2,-3,7,8-TCDD and either the ion at m/e  328 for
       37Cl4-2,3,7,8-TCDD or m/e 332 for 13C12-2,3,7,8-TCDD.   For
       high-resolution mass spectrometry (HRPIS),  use ions  at m/e
       319.3965 ana 321.2936 ror :,3,?,3-TC3D and sither the  on
       at m/e 327.8847 for -37C!4-2,3,7,8-TCDD or the ion at m/e
       331.9367 for 13C12-2,3,7,8-TCDD.  Total and Extracted Ion
       Current Profiles for TCDD are presented in Figure 2.   Electron
       capture GC (GC/ECD) may be used to screen samples and to elimi-
       nate samples from GC/MS analysis if the results  from GC/ECD
       analysis are below the  GC/MS detection limit.  An example of a
       GC/ECD chromatogram for two TCDD isomers 1s shown 1n Figure 3.
       GC/ECD should not be used for quantisation of samples above
       the GC/MS detection limit.

       The method performance  data reported in Subsection  G were gath-
       ered using a final extract volume of i.O ml.  'If lower detection
       limits are required, the extract may be concentrated further
       by carefully evaporating the extract to dryness  under a  gentle
       stream of nitrogen with the concentrator tube in a  water bath
       at 40C.  Redissolve the extract 1n the final  desired  volume
       of hexane or tetradecane.

       The following criteria  must be  met to make a qualitative
       Identification of TCDD:
                               III-344

-------
           Normalized Instrument Response
U'J
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rt ii>  i ro 01 3 ro fire i re   oi o ro
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rere TDrt- reo> 03 rt-o *< 3
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-------
   Column:  6' x 2 mm I.D. glass,  3% OV-210 on 80/100 Supelcoport.
   Carrier:  5% methane/argon
   Flow  rate:  27 ml/min.
   Column temperature:  180C isothermal
   Injector temperature:  235C
   Detector temperature:  300*C
                     Retention Time - Minutes

Figure 3.   Electron  capture detector cnromatogram of 45 ng/ml 1,2,3.4-TCDD
           i5.26  minutes) and SO ,ig/ml :,3,7,3-TCDD  ;3.78 rninuces).-
                                 II1-346

-------
co-eluting impurity may be suspected.   In this  case,  another
set of ions characteristic of the TCDD  molecule should  be  ana-
lyzed.  A good second choice of ions is m/e  257 and m/e 259
for TCDD and the related ions for the  internal  standard.
These ions are in a cluster indicating  the loss of one  chlorine
and one carbonyl group from TCDD.  Suspected impurities such
as DDE, ODD, or PCB residues can be confirmed by checking  for
their major fragments, but may require  another  injection using
different SIM ions or full repetitive mass scans.  If the
response for 37ClA-TCDD Is too high, PCB contamination  at  m/e
328 can be checked by using the PCB ion at m/e  326.   These con-
taminants can be removed by alumina column cleanup.

If broad background interference restricts the  sensitivity of
the GC/MS analysis, the analyst should  employ additional
cleanup procedures and reanalyze by GC/MS.  In  those  circum-
stances where cleanup procedures do not yield a definitive
conclusion, the use of high-resolution  mass  spectrometry is
suggested.*
                        111-347

-------
2.2  Analysis of Water Samples for TCDD
          Analytical  Procedure:   evaluated
          Sample Preparation:   evaluated

     2.2.1  Reference

            Harless,  R. L., E. 0. Oswald,  W.  K.  Wilkinson,  A.  E.  Dupuy,
            D. D. McDaniel, and H. Tai, "Sample  Preparation and Gas
            Chromatography - Mass Spectrometry Determination of
            2,3,7,8-Tetrachlorod1benzo-p-dioxin."   Anal.  Chem. 52:
            1239-1245 (1980).*                                ~

     2.2.2  Method Summary

            One liter of water is fortified with isotopically-labeled
            TCDD and  extracted with multiple  portions  of  methylene
            chloride.  The residue 1s solvent-exchanged into hexane
            and washed with KOH, sulfuric  acid,  and reagent water.
            Following neutralization and drying, the extract is concen-
            trated and cleaned up on an alumina  column.   The extract is
            passed through a second alumina column, solvent-exchanged
            into benzene, and analyzed by  GC/MS.

     2.2.2  \pplicability

            This procedure is applicaole to the  determination  of  TCDD in
            aqueous samples.  The 1 imte of detection is affected  by
            sample size, percent recovery  during extraction, sample
                   effects, ?nd electronic noise during analysis.
     2.2.4  Precision and Accuracy

            The described method produced a mean recovery of 80% for
            2.5 to 10 ng 37ClA-TCDD (internal  standard)  and a mean accuracy
            of 23% for 1 to 1250 pg TCDD in quality assurance samples.   The
            precision of the GC/high resolution mass spectrometry (GC/HRMS)
            technique was determined to be 20% relative to 2 to 10 pg
            TCDD quantification standards during daily operations.

     2.2.5  Sample Preparation

            Thoroughly mix the original water sample and transfer 1 liter
               a 2000-ml separatory funnel.  Fortify the sample with 2.5 ng
                       surrogate standard and mix.
            Extract the sample with three 100-ml  portions of methyl ene
            chloride.  Combine the extracts In a  500-ml  flask.   Add a
            Snyder column and evaporate the extract to dryness  on a steam
            bath.  Dissolve the residue in 100 ml  hexane and transfer
            nuantitativelv into a seoaratorv funnel.
                                    II1-348

-------
2.2.6  Extract Cleanup

       Wash the hexane extract with 25 ml  IN KOH.  Discard the
       aqueous phase.

       Wash the sample with four 50-ml portions of concentrated
       sulfuric acid.  Discard the acid wash.

       Wash the sample with 25 ml  of reagent water.  Neutralize the
       sample mixture by the addition of powdered N32C03.   Separate
       the phases and discard the  aqueous  layer.

       Transfer the extract to a drying column consisting  of a 1-cm
       I.D. x 50 cm glass column containing 10 cm of anhydrous
       powdered NaC03.  Collect the hexane in a  Kuderna-Danish
       evaporative concentrator.  Concentrate the sample to a volume
       of 3 ml.

       Prepare two alumina chromatographic columns by packing 4.5 cm
       of neutral alumina into a clean and dry 15-cm x 0.5-cm
       disposable Pasteur pipet.  Top the  alumina with 0.5 cm
       anhydrous granular NapSO,;.   Wash the column with 4  ml  of
       methylene chloride and force residual  solvent from  the column
       using a stream of ary n'troaen.  'Store  the jolumns  'n  an oven,
       at 225C for a minimum of 24 hours.  Prior to use,  equilibrate
       the columns to room temperature by  olacement in a aesiccator
       containing Drierite.

       Prewet one of the columns with 1 ml of hexane-   Transfer the
       sample concentrate to tne coiumn.   tlute tne coiumn *itn
       6 ml carbon tetrachionde ana discara tne  eiuate.  lute
       the column with 4 ml methylene chloride and collect the
       eiuate in a 12-ml distillation receiver.

       Add a carborundum boiling chip to the receiver and  cap it with
       a micro-Snyder column.  Place the apparatus in a hot water bath
       and evaporate the methylene chloride just  to dryness.

       Add 2 ml hexane to the residue and  evaporate the sample just
       to dryness.  Repeat the process with a  second 2 ml  portion
       of hexane.

       Dissolve the residue in 3 ml  hexane and transfer the sample
       to the second alumina column that has  been prewet with
       1 ml hexane.

       Elute the column with 6 ml  carbon tetrachloride and discard
       the eiuate.  Elute the column with  4 ml  methylene chloride
       and collect the eiuate in a clean 12-ml  distillation receiver.
                               III-349

-------
Using a hot water bath, evaporate the methylene chloride just
to dryness.  Add 2 ml  benzene to the receiver and concentrate the
extract to a volume of 100 pi.

Quantitatively transfer the sample to a 2-ml  graduated Chroma-
flex sample tube (Kontes Glass Company, K-422560).   Utilizing
a slow stream of dry nitrogen, carefully concentrate the
benzene solution to a final volume of 60 yl.

If the analyses are not to be completed immediately, seal  the
extract in glass tubing (3 mm x 7 cm) and store at  subzero
temperatures.
                         III-350

-------
3.1-  Analysis of Methylene Chloride Extracts of  Sediment/Soil  Samples  for
     2,3,7,8-TCOO
          Analytical  Procedure:   evaluated
          Sample Preparation:   evaluated

     3.1.1  Reference

            Harless,  R. L., E.  0. Oswald,  M. K. Wilkinson,  A.  E.  Dupuy,
            D.  D. McDanlel, and H. Tai ,  "Sample Preparation and Gas
            Chromatography - Mass Spectrometry Determination of
            2,3,7,8-Tetrachlorodibenzo-p-dioxin."  Anal.  Chem.  52:
            1239-1245 (1980). 4

     3.1.2  Method Summary

            Ten to twenty g of  well-mixed  soil/sediment is  fortified with
            Isotopically labeled TCDD and  placed in a  100-ml  boiling
            flask.  The sample  is then refluxed for 2.5 hours  with alcoholic
            KOH and extracted with multiple portions of methylene chloride.
            The residue is solvent-exchanged into hexane and washed with
            KOH, sulfuric acid,  and reagent water.   Following  neutraliza-
            tion and drying, the extract is concentrated and cleaned up on
            a series  of alumina columns.  The residue  is  solvent-exchanged
            into benzene, concentrated,  and analyzed by C/MS.

     3.1.3  Applicability

            This procedure is applicable to the determinations of TCDD  in
            solid-chase samoles such as  soil or sediment.   The limit of
            detection is affected oy sample *e:gnt ana j-;se,  percent
            recovery during extraction,  sample matrix  affects,  and instru-
            ment noise during analysis.

     3.1.4  Precision and Accuracy

            The described method produced  a mean recovery of 80%  for
            2.5 to 10 ng 37C1^-TCDD (internal  standard) and a  mean accuracy
            of 23% for 1 to 1250 pg TCDD  1n quality assurance samples.
            The precision of the GC-HRMS technique was determined to be 20%
            relative to 2 to 10 pg TCDD  quantification standards  during daily
            operations.

     3.1.5  Sample Preparation

            Remove extraneous objects from the sample  and mix  thoroughly.
            Weigh out a 10 to 20 g portion of the homogenized  sample and
            transfer to a 100-ml boiling flask.
            Add 5 to 10 ng of ^'Cla-TCDD surrogate  standard,  20 ml  ethyl
            alcohol, and 40 ml  of 45% ootassium hydroxide  solution  to  the
            sample.   Heat the sample and rerlux anile  stirring  r'or  J.5  rsours.
                                    III-351

-------
       After cooling, transfer the mixture to a separatory funnel.
       Rinse the flask with 20 ml  ethyl  alcohol  and add to the
       separatory funnel.  Rinse the flask with 20 ml  hexane and add
       the wash to the sample.

       Extract the sample with 25 ml hexane.   Transfer the hexane
       layer to a clean separatory funnel.  Repeat the sample
       extraction with three additional  25-ml portions of hexane.
       Combine the hexane extracts.

3.1.6  Extract Cleanup

       Wash the combined hexane extracts with 25 ml IN KOH.  Discard the
       aqueous phase.

       Wash the sample with four 50-ml  portions of concentrated
       sulfuric acid.  Discard the acid  wash.

       Wash the sample with 25 ml  reagent water.  Neutralize the sample
       mixture by the addition of powdered N32C03.  Separate the phases
       and discard the aqueous layer.

       Pass the hexane layer through a  drying column consisting of
       a 1-om-I.D. x 50-cm glass column  containing 10  crn anhydrous
       powdered Na2C03.  Collect the hexane in a Kuderna-Oamsn
       avaporative concentrator.  Concentrate the sample to a volume
       of 3 ml.
             
-------
Dissolve the residue in 3 ml  hexane and transfer the sample
to the second alumina column  that has been prewet with 1 ml
hexane.  Elute with 6 ml  carbon tetrachloride and discard
the eluate.  Elute the column with 4 ml methylene chloride
and collect the eluate in a 12-ml distillation receiver.

Using a hot water bath, evaporate the methylene chloride just
to dryness.  Add 2 ml benzene to the receiver and concen-
trate the extract to a volume of 100 yl.  Quantitatively
transfer the sample to a 2-ml graduated Chromaflex sample tube
(Kontes Glass Company, K-422560).  Utilizing a slow stream of
dry nitrogen, carefully concentrate the benzene solution to a
final volume of 60 yl.

If the analyses are not to be completed immediately, seal the
extract in glass tubing (3 mm I.D. x 7 cm) and store at subzero
temperatures.

-------
3.2  Determination of 2,3,7,8-TCDD in Methanol  Extracts of Soil  and
     Sediment
          Analytical  Procedure:   evaluated
          Sample Preparation:   available

     3.2.1  Reference

            U.S. Environmental  Protection Agency,  "Determination of
            2,3,7,8-TCDD in Soil  and Sediment." U.S.  EPA,  Region VII
            Laboratory, Kansas  City, Kansas.   37 p.   February 1983.H

     3.2.2  Method Summary

            A 10-gram soil sample is spiked with an  internal  standard
            of isotopically-labeled 2,3,7,8-TCDD.  The wet sample is mixed
            with 20 grams anhydrous sodium sulfate prior to extraction
            with hexane/methanol  using a jar  extraction technique.  Optional
            cleanup procedures  to aid in the  elimination of interferences
            that may be encountered are provided.  The extract is concen-
            trated to a volume  of 0.10 ml. Capillary column GC/MS condi-
            tions are described which allow for the  separation and measure-
            ment of 2,3,7,8-TCDD in the extract.

     3.2.3  Apolicabillty

            This method is. intended for use In  the determination of
            2,3,7,8-TCDD in soil  and sediment at levels of 1 part per
            biTlion and higher.   The method is'specific for the 2,3,7,8-
            TCDD isomer, since  it employs capillary  columns which separate
            that isomer from :he ;tner 21 "CD  ''corners.  ~ots1 T.DD :zn
            also be estimated by this method.  Determination of other
            specific TCDD isomers depends on  the availability of the
            specific Isomer and the separation  from  other interfering
            Isomers.  The final  measurement process  utiMras low resolution
            mass spectrometry.   Because of the  increased possibility for
            Interferences at levels below 1 part per billion, the user  is
            cautioned in extending the method range  below that concen-
            tration.

            This method should  be restricted  to use  only by or under the
            supervision of analysts experienced 1n the use of gas
            chromatography/mass spectrometry  and  skil'ed in the inter-
            pretation of mass spectra.

     3.2.4  Precision and Accuracy

            The nominal detection limit for this method is 1.0 part per
            billion.  However,  for certain samples this detection limit
            may not be achievable because of  interferences.  On other
            relatively clean samples, the estimated  detection limit may be
            iower.


                                    II1-354

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       The following method recovery values for isotopically labeled
       2,3,7,8-TCDD from fortified soil-samples have been reported in
       three different laboratories (mean recovery  one standard devia-
       tion):   Lab A, 70  12%, 50 data points; Lab B,  59  23%,  85 data
       points; Lab C, 72  16%, 11 data points.

       In Table 2, data are presented indicating the method precision
       based on duplicate analyses.  The data are presented for the same
       lab running the same sample (intralab precision), different labs
       running samples taken at the same place at the same time
       (interlab precision), and different labs running samples taken
       at the same place but on different days (total precision).

3.2.5  Phase Separation

      .CAUTION:  When using this method to analyze for 2,3,7,8-TCDD,
       all of the following operations should be performed in a
       containment laboratory with the analyst wearing full protective
       covering for all exposed skin surfaces.10

       An initial centrifugation step is provided for the phase
       separation of very wet soil or sediment samples.  If used,
       analyze the separated water phase using procedures specified
       for water V3ubs2cf;sn J -.) and continue processing the solid
       phase.   If phase separation is not used, proceed to the sample
       extraction step.

       Place a 30-g aliquot in a suitable centrifuge bottle.   Place
       ;he sampie*ana i jsuntsr-balanes ** i "rsntr^yae.  Centrifuge
       the samp!a for 20 ninutss ?t 2000 rom.  Remove the sample  ana
       mark the phase interface on the bottle to estimate the relative
       volume of each phase.  Using disposable pipettes, transfer the
       liquid layer into a clean bottle for analysis as a water sample.

3.2.6  Sample Extraction

       Two procedures are provided for extracting dioxins from the
       soil or sediment matrix.  Option A is a relatively simple  jar
       extraction with methanol and hexane.  Option B is a more
       rigorous Soxhlet extraction with toluene.

       Option A

       Transfer a 10-g aliquot of the solid sample directly into  an
       extraction jar (250 ml  capacity, or larger).

       Add 1 ml of a 2.5rng/ml solution of isotopically-labeled 2,3,7,8-
       TCDD directly to the soil.  The isotopically-labeled TCDD
       should be added at several sites over the surface of the soil.

       Add 20 q of solvent-extracted anhydrous sodium suifate ana mix
       tnorougniy using a -jtainlass :tee!  :pcon or icatula.  ^llow

                               III-355

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              TABLE 2.   SUMMARY OF DUPLICATE  2,3,7,8-TCDD  RESULTS
                             (In parts  per  billion)

Paired Results                  Mean  Result          Percent Relative Difference

<1
1.4;
0.9,
1.5,
29.3,
1.8,
0.5,
(Intralab)
<1
0.5
1.2
1.5
20.0
1.1
0.4


0.95
1.05
1.5
24.65
1.45
0.45

0
95
29
0
38
' 48
22
                (Interlab)

   118, 110                     114                              7
    <1, <1                                                      0
   104, 65                       d4.o                           46
   175, 170                     172                              3
   1.5, 4.4                       2.95                          98
   0.4, 1.1                       0.75                          93
   9.2, 8.5                       8.85                        '   8
    24, 27.3  *                  25.3                           \:
  20.i, 15.4                     17.75                          26
  14.5, 13                       13.75                          11

                 (Total)

   7.0, 9.0                       8.0                           25
   7.0, 3.9                       5.45                          57
   9.0, 3.9                       6.45                          79
  24.7, 1.5                      13.1                          177
   118, 270                     194                             78
   110, 270                     190                             84
    50, 52                       51                              4
   140, 240                     190                             53
   2.6, 0.9                       1.75                          97
    <1, 0.4                       -                              0
    <1, <1                                                      0
    <1, <1                                                      0
    <1, <1                        -                              0
    <1, <1                                                      0
                                    II1-356

-------
the mixture to stand under ambient conditions.  Mix again after
2 hours and allow to stand for at least 6 hours.  Should the
soil/sodium sulfate mixture form lumps which cannot be easily
broken, it will be necessary to grind the mixture in a glass mortar
with a glass pestle.

Mix the soil/sodium sulfate mixture just before adding solvent.
Add 20 ml methanol, stir, and then add 150 ml hexane.

Seal the sample and place the jar on a wrist-action shaker,
platform shaker, magnetic stirrer, or equivalent device.
Extract the sample vigorously for a minimum of 3 hours.

Prepare a glass funnel with solvent-rinsed filter paper (Whatman
No. 4 or equivalent).  Filter the sample extract.  Thoroughly
rinse the extraction jar, its contents, and the filter residue
with hexane.  Combine the filtered rinses with the sample extract.

Concentrate the hexane/methanol extract to 1 ml using Kuderna-
Danish or rotary evaporator techniques.  When using rotary
evaporator concentration techniques, care must be taken to
carefully rinse the apparatus between samples to prevent cross-
contamination of samples.  The extract is now ready for cleanup.

Option B

Thoroughly clean the Soxhlet apparatus oy operating ror 2 flours
with toluene prior to use.  '

Transfer a 10-g aliquot of sampie airectly  . nto j jir.^b: e
glass container sucn as" a Deaer or flask.  Add 1 .nl of a
25-ng/ml solution of isotopically labeled 2,3,7,8-TCDO directly
to the sample.  The isotope should be added at several sites
over the surface of the sample.

Add 20 g of solvent-extracted anhydrous sodium sulfate and mix
thoroughly using a stainless steel spoon or spatula.  Allow the
mixture to stand under ambient conditions.  Mix again after 2 hours
and allow to stand for at least an additional 6 hours.  Mix again
just before transferring the sample to an extraction thimble.

Add 10 g of anhydrous sodium sulfate to the extraction thimoie.
Transfer the sample (soil plus sodium sulfate) to the thimble
and cover with a layer of clean glass wool.  Rinse the container
with toluene and transfer the washing to the extraction apparatus.

Put 250 ml of pesticide-grade toluene into the extractor.
Operate the apparatus for a minimum of 25 cycles.

Transfer the toluene extract to a 500-ml round-bottom flask.
Rinse the extraction apparatus with iwo Q-ini /oiumes of
toluene and add the cashes to the extract.  Concentrate the

                        III-357

-------
       extract in a rotary evaporator (Biichi/Brinkman,  or equivalent)
       at 60 to 70*C under a vacuum of 23 to 27  inches  of mercury.   When
       the toluene volume has been reduced to 2  to 3 ml,  stop the  evapor-
       ation.   Transfer the extract to an 8-ml glass culture  tube  for
       further concentration.  This is accomplished on  an N-Evap
       Analytical  Evaporator (Organomation Associates,  Inc..)  at 50*C
       with a gentle stream of filtered nitrogen.

       Rinse the round-bottom flask with at least  three 3-rnl  volumes
       of methylene chloride.  Transfer each rinse to the culture
       tube for concentration with the extract.  When the volume of
       the extract has been reduced to 200 to 300  pi, add 1 ml  hexane
       to the culture tube.  Continue concentrating until  the volume is
       reduced to 200 to 300 ul.   Add 1 ml hexane  and reduce  the extract
       volume to 200 to 300 ul.   The concentrated  hexane  extract is now
       ready for cleanup.

3.2.7  Sample Extract Cleanup

       Cleanup procedures may not be necessary for a relatively clean
       sample matrix (i.e., sandy soils).  However, most  sample types
       will require some cleanup.  Extract cleanup must be performed
       if any of the following conditions are observed:

       1.  The samole extract cannot be concentrated to 1 ml.

       2.  Interferences prevent  observation or  measurement of the
           isotopically-labeled 2,3,7,8-TCDO..

       3.  Interferences are present in the retention time winaow  dt
           mass 320, 322, or 257.

       4.  The required detection limit of 1 ppb cannot be achieved.

       5.  The sample extract is  extremely dark  colored and  viscous.

       The following cleanup options are recommended.  Other cleanup
       procedures may be used if the isotopically-labeled 2,3,7,8-
       TCDD recovery is consistently greater than 50%.

       Option A

       Pack a 1-cm I.D. x 10-cm chromatography column with 1  g
       silica gel and 4 g of 40% w/w sulfuric acid/modified silica gel.
       Pack a second chromatography column (1 cm I.D. x 30 cm) with
       6 g alumina and top with a 1-cm layer of  sodium  sulfate.
       Add hexane to the columns until they are  free of channels or
       air bubbles.  This can be readily achieved using a small
       positive pressure (5 psi)  of clean nitrogen.

       Drain the hexane to just aoove the cop or the silica get ana pi ace
       the hexane extract  on *OD of +he silica qel.  Rinse the extract

                               III-358

-------
container with two 0.5-ml volumes of hexane and add to the
column.  Elute the extract from the silica gel column directly
onto the alumina column with 45 ml hexane.  Discard the
silica gel.

Place 20 ml hexane on the alumina column and elute until the
liquid has dropped below the sodium sulfate layer.  Discard
the eluted hexane.

Elute the column with 20 ml of 20% (v/v) methyl ene chloride/
hexane and collect in a 125-ml Erlenmeyer flask.

Reduce the volume of TCDD-containing eluent using a gentle
stream of filtered nitrogen.  When the volume has been
reduced to 1 to 2 ml , transfer aliquots, one at a time, to a
2-ml conical mini-vial for further concentration until the
entire fraction has been transferred.  Rinse the Erlenmeyer
flask with 1 ml hexane and transfer the rinse to the mini-
vial.  Repeat the rinsing procedure.  The contents of the
mini-vial  must not be allowed to go to dryness during the
extract concentration process.  Finally, rinse the walls of
the mini-vial with 500 ul hexane.  Store the extract in a
freezer until analysis.  Just prior to analysis, reduce the
hexane volume almost to dryness, and add toluene to achieve a
final volume of 100 ul.

Option B
        a glass macro-column (2 cm O.D. x 23 cm, and tapered 1
cm).  ^acK tne jcuain *rcn i ;;i;g ;f r:l irn'ied ^lass wool.
followed successively oy 1 g silica, 2 g ""h'ca containing
33% (w/w) 1 M NaOH, 1 g silica, 4 g silica containing 44%
(w/w) concentrated HgSO^, and 2 g silica.  Add hexane co the
column until it is free of channels or air bubbles.  Quan-
titatively transfer the concentrated sample extract to the
column and elute with 45 ml hexane.  Collect the entire eluate
and concentrate to a volume of less than 1 ml in a centrifuge
tube.

Construct a chromatography column by packing a 5-ml disposable
pipet (cut off at the 2-ml mark) with a plug of silanized
glass wool and add 1 g activated Woeim oas-ic alumina 'activated
at 600*C for 24 hours) to the tube.

Quantitatively transfer the concentrated extract to the top of
the column using 2 ml  hexane.

Elute the column with 5 ml 3% methyl ene chloride in hexane
(v/v) and retain the entire column eluate for analysis.

Elute both columns vmh 0 :ni 30% methylene chloride fv/y)
'r\ s,exane ^nd retain the eluates for analysis.

                        III-359

-------
       Combine  the eluates  and  concentrate to  a  volume  of  less  than
       1  ml.  Quantitatively  transfer  the  extract  to  a  2-ml  conical
       mini-vial.   Concentrate  the  extract to  near dryness and  store
       at 5C.

       Prior  to GC/MS analysis  reconstitute the  extract by adding
       toluene  and adjusting  the  final  volume  to 100  yl.

       Option C

       Certain  very dirty samples may  require  preliminary  cleanup
       prior  to column chromatography.   For those  situations, the
       following procedure  is suggested.

       Wash the organic extract with 30 ml  20-percent aqueous
       potassium hydroxide  by shaking  for  10 minutes.  Remove and
       discard  the aqueous  layer.

       Wash the organic extract with 25 ml  doubly-distilled water'
       by shaking for 2 minutes.  Remove and discard  the aqueous layer.

       CAUTIOUSLY add 50 ml concentrated sulfuric  acid  to  the organic
       extract  and shake for  10 minutes.   Allow  the mixture to  stand
       antil  the aaueous and  orqanic layers seoarate  (approximately
       10 minutes).  Remove ana discard cne aqueous .ayer.   Repeat
       cne acid washing procsdure until ,10-color is visible in  the
       acid layer.

       Add 25 ml doubly-distilled water-to the organic  extract  and
       snaice  r'or 2. nnnut3.   '.emcve ana alscara  *he -iquaous "-.yer.
       Add 10 g anhydrous sodium  sulfate :o the  extract to dry  the
       organic  layer.

       Transfer the organic extract to a centrifuge tube.   Concentrate
       to near  dryness by placing the  tube in  a  water bath at 55C
       while  passing a gentle stream of filtered,  prepurified nitrogen
       over the surface of  the  extract. Reconstitute in hexane
       before proceeding with the column chromatography (either
       Option A or Option B).

3.2.8  GC/MS  Analysis

       Table  3  summarizes typical gas  chromatographic capillary
       columns  and operating  conditions.  Other  columns and/or
       conditions may be used as  long  as isomer  specificity is
       demonstrated by the  introduction of a mixture  containing all
       22 TCDD  isomers. -Thereafter, a calibration mixture containing
       fewer  isomers should be  analyzed on a daily basis in order  to
       verify the performance of  the system.
                               III-360

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                 TABLE 3.  RECOMMENDED GC CAPILLARY  CONDITIONS
=======================
Column
2,3,7,8-TCDD R.T.
Helium Linear Velocity
Initial Temperature
Initial Time
Split! ess Time
Program Rate
Final Temperature
Final Hold Time
.Split Flow
Septum Purge Flow
Capillary Head. Pressure
A (Silar IOC)
34.5 min
30 cm/sec
100'C
3 min

20C/min
180C**
15 min



====================
B (SP2340)
22 min
0.7 ml /min
at 608C
60'C
3 min
1 min
25C/min
250'C
15 min
30 ml /min
j .til /min
30 psl
====================
C (DB-5)*
13 min
1 ml /min
80C
1/min
1 min
15'C/min
300C
15 min
30 ml /min
." -n /mi n
15 psi
         not'soecific for the 2,3,7,8-TCDD  isomer.
"then 2"/min to 250UC.5

            Calibrate the analytical  system daily  as  described  in  Sub-
            section H.   The volume  of calibration  standard  injected must
            be measured, or be the  same for all  injections.

            Immediately before analysis,  adjust  the sample  extract volume
            to 100 ul.

            At the option of the analyst, a standard  amount of  isotopically
            labeled 2,3,7,8-TCDD (different from the  one  used as a surrogate)
            may be added to the extract tojponitor variations in instrument
            sensitivity.  For example,  if ""u^-TCDD  :s used as the
            surrogate,  then ^Cjo-TCDD (about  25 ng)  can  be added  to the
            extract just before GC/MS analysis.  The  response from this
            standard can be used to calculate  percent recovery of  the
            Isotopically-labeled surrogate.  It  must  not  be used to compute
            the concentration .of native TCDD.

            Analyze standards and samples with the mass spectrometer
            loeratino in the selected ion monitoring  (SIM)  mode using a
            dwell  time  co ^tve at ^aast ieven  points  per  oeak.  r">r LRMS.
            -jse Ions st ij/e 320, 322, and 257  for  2,3,7,8-TCDD and either

                                   III-361

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the 1on at m/e 328 for 37r/]-TCDD or m/e 332 for 13r,-TCDD.  For
HRMS, use Ions at m/e 319.8965 and 321.8936 for 2,3,7,8-TCDD
and either the ion at m/e 327.8847 for 37ci-TCDD or m/e 331.9367
for 13C-TCDD.

In order to achieve the stated detection limit, the instrumen-
tation must be sensitive enough to provide a signal for all
three ions in at least a 2.5-to 1 signal-to-noise ratio for an
injection of 100 picograms.

If a lower detection limit is required, the extract may be
carefully evaporated to dryness under a gentle stream of
nitrogen with the concentrator tube in a water bath at about
40C.  This must be done immediately before GC/MS analysis.
Redissolve the extract in the desired final volume of solvent.

Inject a l-to-5 til aliquot of the sample extract.

The presence of 2,3,7,8-TCDD is qualitatively confirmed if
the following criteria are met:

1.  Isomer specificity is demonstrated and verified.

2.  The 320/322 ratio i< within -.he -2nge of 0.57 to 0.87.
3.  Ions 320, 322, and 257 must all  be present and produce maxi-
    mum instrument response simultaneously.   The signal  to noise
    ratio must be 2.5-to-l or better for all  3 ions.

4.  The elusion time or native ,., ,7,6-7CDD  ,nust equal  iwi^nin 3
    seconds) the elution time r'or che isotopicaily labeled
    2,3,7,8-TCDD.

5.  Five percent of the positive samples should be confirmed
    by HRMS.  Alternately, 5 percent of the  positives can be
    confirmed by obtaining partial scan spectra from mass 150 to
    mass 350.
For quantitation, measure the response of the m/e 32
2,3,7,8-TCDD and the rn^e 332 peak for 13C12-2,3,7,8-
the m/e 328 peak for 37C1d-2,3,7,8-TCDD.   Calculate
                                                  320 peak for
                                                   -TCDD or
                                                    the
concentration of native 2,3,7,8-TCDD using the response factor
(RF) and the following equation:


         Concentration, ng/g  =  {As)(Is)/(Ais)(RF)(VO

where:

        AS  =  SIM resoonse for 2,3,7,8-TCDD ion at m/e 320



                        III-362

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       A^s  =  SIM response for the internal standard ion
               m/e 328 or 332
        Is  =  Amount of internal standard added to each
               sample (ng)

         W  =  Weight of soil in grams

Co-el uting Impurities are suspected if all criteria except the
isotope ratio criteria are achieved.  In this case, another
SIM analysis can be performed.  The ions at m/e 257 and m/e
259 are indicative of the loss of one chlorine and one carbonyl
group from 2,3,7,8-TCDD.  If the ions m/e 257 and m/e 259 give
a chlorine Isotope ratio that agrees to within 10% of the
same cluster in the calibration standards, then the presence
of TCDD can be confirmed.  Co-eluting ODD, DDE, and PCB residues
can be confirmed, but will require another injection using the
appropriate SIM ions or full  repetitive mass scans.  If the
response for 37C!-2,3,7,8-TCDD at m/e 328 is too large, PCB
contamination is suspected and can be confirmed by examining
the response at both m/e 326 and m/e 328.  The 37C1 -2,3,7,8-
TCDD internal standard gives negligible response at m/e 326.
These pesticide residues can be removed using the alumina
column cleanup.

If broad background interference restricts the sensitivity of
the GC/MS analysis, the analyst should employ additional
cleanup procedures and reanalyze by GC/MS.
In those ffrrjTr.s tineas whe^e these orocedures do not yield a
definitive conclusion, then the use of mgn- resolution ,nass
spectrometry is suggested.
                        III-363

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4.1  Analysis of Hexane Extracts .of Biological  Tissue for TCDD
          Analytical  Procedure:   evaluated
          Sample Preparation:   evaluated

     4.1.1  Reference

            Harless,  R. L., E. 0. Oswald, M.  K.  Wilkinson, A.  E.  Oupuy,
            D. D. McDaniel, and  H. Tai,  "Sample  Preparation and
            Gas Chromatography/Mass Spectrometry Determination of
            2,3,7,8-Tetrachlorodibenzo-p-dioxin." Anal.  Chem.  52:
            1239-1245 (1980).4                                ~

     4.1.2  Method Summary

            Fish tissue is digested in  alcoholic KOH for 2.5 hours.   The
            sample is then subjected to  multiple extractions with hexane.
            The combined hexane  extracts are  washed with KOH,  sulfuric
            acid, and water.   Following  neutralization and drying, the
            extract is concentrated and  cleaned  up on an alumina  column.
            The extract is passed through a second alumina column,
            exchanged into benzene, and  analyzed by GC/MS.

     4.1.3  Applicability

            This procedure is  applicable to the  determination  of  TCDD in
            fish and  other lean  tissue.   The  limit of detection is
            affected  by sample weight and size,  percent  recovery  during
            extraction, sample matrix effects, and electronic  noise
            during analysis.

     4.1.4  Precision and Accuracy

            The methodology produced a  mean recovery of 80% for 2.5  to 10  ng
            37Cl4-TCDD (internal standard) and a mean accuracy of 23% for
            1 to 1250 pg TCDD  in quality assurance samples.  The  precision of
            the GC-HRMS technique was determined to be 20% at TCDD  levels of
            2 to 10 pg during  daily operations.

     4.1.5  Sample Preparation

            Blend the fish tissue in a  blender to homogenize the  sample.
            Weigh out a 10- to 20-g portion of  cne blended tissue and
            transfer to a 100-ml boiling flask.

            Add 5 to 10 ng of  3?C1-TCDD surrogate standard, 20 ml ethyl
            alcohol,  and 40 ml 45% potassium  hydroxide solution.   Heat the
            sample and reflux, while stirring  for 2.5 hours.

            After cooling, transfer the mixture  to a separatory funnel.  Rinse
            the flask with 10  ml ethyl  alcohol and add the rinse  to  the sample
                                    III-364

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       mixture.  Rinse the flask with 20 ml hexane and add the wash to
       the sample mixture.

       Extract the sample with 25 ml hexane.  Transfer the hexane layer
       to a clean separatory funnel.  Repeat the sample extraction with
       three additional 25-ml portions of hexane.  Combine the hexane
       extracts.

4.1.6  Extract Cleanup

       Wash the combined hexane extracts with 25 ml IN KOH.  Discard the
       aqueous phase.

       Wash the sample with four 50 ml-portions of concentrated sulfuric
       acid.  Discard the acid wash.

       Wash the sample with 25 ml reagent water.  Neutralize the sample
       mixture by the addition of powdered N32C03.  Separate the phases
       and discard the aqueous layer.

       Pass the hexane layer through a drying column consisting of a
       1-cm-I.D. x 50-cm glass column containing 10 cm anhydrous powdered
       Na^COs.  Collect the hexane in a Kuderna-Danish evaporative concen-
       trator.  Concentrate tne sample tc -,  'oiume of 3 7,1.

       Prepare two alumina chromatographic columns by packing i,5 cm
       neutral alumina into a clean and dry 15-cm x 0.5-cm disposable
       Pasteur pipet.  Top the alumina with 0.5 cm anhydrous granular
       ,-ia2^<\'  ^asn ;r.e coi'jrr.n  
-------
            El lite the column with 6 ml  carbon tetrachloride and discard the
            eluate.   Elute the column with 4 ml  methylene chloride and  collect
            the eluate in a 12-ml  distillation receiver.

            Using a  hot water bath, evaporate the methylene chloride just  to
            dryness.  Add 2 ml benzene to the receiver and concentrate  the
            extract  to a volume of 100 ul  Quantitatively transfer the sample
            to a 2 ml-graduated Chromaflex sample tube (Kontes Glass Company,
            K-422560).  Utilizing a slow stream of dry nitrogen, carefully
            concentrate the benzene solution to a final  volume of 60 yl.

            If the analyses are not to be completed immediately, seal the
            extract  in glass tubing (3 mm I.D. x 7 cm)  and store at subzero
            temperatures.

K.   CALCULATIONS

     Calculate the concentration of TCDD in the sample using the response
factor (RF) determined in Subsection H and Equation 2.


                                                    (AS)(IS)
                 Concentration (pg/1 or ug/g)  =	 Eq.  2
                                                  (Ais)(RF)(V0)
where:

         As  =  SIM response for TCDD ion at m/e 322

        A-JS  -  SIM -esponse *or the 'nternal  standard "on at m/e 322

         Is  =  Amount of internal  standard added to each extract (g)

         V0  =  Sample volume (liters)  or mass (grams).

     For each sample, calculate the percent recovery of the internal  standard
by comparing the area of the ion peak measured in the sample to the area of
the same peak in the calibration standard.

     Report results in micrograms per liter or micrograms per gram.  When
duplicate and spiked samples are analyzed, report all data obtained with the
sample results.

     For samples processed as part of a set for which the spiked sample
recovery falls outside the control  limits established in Subsection G,  or for
which the internal standard recovery is below 50%, the data for TCDD must be
labeled as suspect.
                                    111-366

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L.   WIPE TEST PROCEDURE

     Use the bottom of a 1-quart paint can to determine the size of the area
to be wipe-tested.  The diameter of the paint can corresponds to an area of
about 85 cm2.

     Moisten an ashless 12.5-cm diameter piece of filter paper (Whatman 41  or
equivalent) with 1 ml  of hexane and wipe the desired area using a circular
motion.

     Fold the paper and place in a glass funnel  in the manner the paper would
normally be used for filtering.  Elute the paper with two 5-ml  portions of
hexane, collecting the hexane ^n a 2-oz. bottle.

     Concentrate the hexane to approximately 1 ml by blow-down.

     Wash the hexane by shaking with 1 ml  concentrated sulfuric acid.

     Transfer the hexane to a 1-ml  autosampler vial  for analysis by electron
capture GC.  The detection limit of the test is on the order of 1 ug of
2,3,7,8-TCDD per wipe.  Less than 1 ug per sample indicates acceptable clean-
liness'; anything higher warrants further cleanup.8  More than 10 ug on  a wipe
sample indicates an acute hazard which requires  prompt remedial  attention.8


                                   REFERENCES
1.   u.j. cnv'ironmeniai  Protection Agency.   <"!-?seri"at-!on ind Maximum
     Time for the Priority ollutants."  U.S.  PA.  environmental  Monitoring
     and Support Laboratory, Cincinnati, Ohio.   (In preparation).

2.   American Society for Testing Materials.   "Standard Practice  for  Prepara-
     tion of Sample Containers and Preservation."   ASTM Annual  Book of Stand
     ards, Part 31, D 3694.  ASTM, Philadelphia, Pennsylvania,  p. 679
     (1980).

3.   U.S. Environmental  Protection Agency.   "Analytically Determined  Method
     Detection Limits for Priority Pollutants.   Methodology  as  Method
     Performance Criteria."  U.S. EPA,  Environmental  Monitoring and Support
     Laboratory, Cincinnati, Ohio.  ,'In preparation).

4.   Harless, R. L., E.  0. Oswald, M. K. Wilkinson, A.  E.  Dupuy,  D. D.
     McDaniel, and H. Tai .  "Sample Preparation and Gas Chromatography/Mass
     Spectrometry Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin."
     Anal. Chem. 52:  1239-1245 (1980).
5.   Lamparski,  L.  L.  and Nestrick,  T.  J.   "Determination  of Tetra-,  Hepta-,
     and
     per
                                                                   ,
     and Octachlorodibenzo-p-dioxin Isomers  in Paniculate  Samples  at  Parts
     per Trillion Bevels."   Anal.  Chem.  52:   2045-2054  '1980).
                                    III-367

-------
6.   Longhorst, M. L. and L. A.  Shadoff.   "Determination  of  Parts-per-Trillion
     Concentrations of Tetra-,  Hexa-,  and Octachlorodibenzo-p-dioxins  in Human
     Milk."  Anal. Chem. 5:  2037-2044 (1980).

7.   U.S. Environmental Protection Agency.   "Handbook  of  Analytical  Quality
     Control in Water and Wastewater Laboratories."  U.S.  EPA,  Environmental
     Monitoring and Support Laboratory, Cincinnati,  Ohio.  EPA-600/4-079-019
     (March, 1979).

8.   U.S. Environmental Protection Agency.   "2,3,7,8-Tetrachlorodibenzo-p-
     dioxinMethod 613", Methods for Organic  Chemical  Analysis  of Municipal
     and Industrial Wastewater,  Longbottom,  J.  E., and J.  L.  Lichtenburg, Eds.,
     U.S. EPA, EPA-600/4-82-057, 1982.

9.   U.S. Environmental Protection Agency.   "Determination of 2,3,7,8-
     Tetrachlorodibenzo-p-Dioxin (2,3,7,8-TCDD)  in Water,"  U.S.  EPA,  National
     Enforcement Investigation  Center, Denver,  Colorado,  p.  19   (No  date).

10.  National Research Council.   Committee on  Hazardous Substances in  the
     Laboratory.  "Prudent Practices for Handling Hazardous  Chemicals  in
     Laboratories," National Academy Press,  Washington, D. C.,  1981.

11.  U.S. Environmental protection Agency.   "Determination of 2,3,7,8-TCDD
     1-n Soi* and Sediment."  ;j.3. ~?A, Region  VII Laboratory, Kansas City
     Kansas.  37 p.  February 1983.
                                    III-368

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                                    SECTION  9

       METHODS  FOR  THE  DETERMINATION  OF  POLYCYCLIC  AROMATIC  HYDROCARBONS


 A.    SCOPE

      Polycyclic aromatic  hydrocarbons (PAHs)  are  also  referred  to  as  poly-
 nuclear  aromatic hydrocarbons  or  polycyclic aromatic compounds.  To avoid
 undertainty,  the nomenclature  suggested  by  Bartle et al.*  for naming  PAH
 compounds is  used throughout this section.  These compounds  are  usually deter-
 mined by a  chromatographic  technique.  Due  to the sensitivity and  potential
 selectivity of  the  ultraviolet absorbance/fluorescence detection in analysis  of
 solutions of  these  compounds2, a  high-performance liquid chromatographic  (HPLC)
 method- is presented here.   Several  PAH compounds  may also  be determined by
 base/neutral  extraction-gas chromatography  mass/spectrometry (GC/MS)  (Sec-
_tion  3).  However,  packed-column  gas  chromatograohy does not adeauately resolve
 the following four  pairs  of compounds: antr.racsne ana  phenar.thrsne, :hrysene  and
 benzo[a]anthracsne, benzcCb^ucranthene and  benzoC'O^luoranthene > or 'libenzo-
 [ahjantnracene  and  indenoti ,,3-cajpyrene.  fusee siiica capillary column GC/MS
 can separate  these  four pairs  of  compounds, but anthracene and  phenanthrene,
 chrysene and  benzo[a]anthracene,  and  benzo[b]fluoranthene  and b'enzo[k]fluor-
 used  cc  analyze  unfamiliar  samples,  compound  1 dent If '"cations  should  be  sup-
 ported by  at  least  one  additional  qualitative technique.   Thin  layer chroma-
 tography with fluorescence  detection is  appropriate for this  use.  Fluorescence
 spectra  of collected  fractions  can also  be  '.still zed ^or confirmation of
 identification.^

 B.    SAMPLE HANDLING  AND  STORAGE

      Due to the  variable  nature of the sample matrix,  both hazardous waste
 sampling methods and  sample handling, and storage  procedures  for  polycyclic
 aromatic hydrocarbon  determinations  are  quite matrix-dependent.   Because  of the
 potential  photoreactive nature  of  the compounds, ail samples  should^be  pro-
 tected from intense light. 5

      Grab  samples for water analysis must be  collected in  glass containers. 6
 Conventional  sampling practices? should  be  followed, except that  the bottles
 should not be prerinsed with sample  before  collection. 6  Composite water
 samples  should be collected in  glass containers and samples should be kept
 refrigerated  during the compositing  period. 6   Automated equipment should  be as
 free  as  oossible of potential sources of contamination.  Samples  should be
 refrigerated  at  -^C r'rom  ihe i'rne  jf ;oilecfion ':nti"!  processing.  Csmoles,
 extracts.  ?nd standards should  be  stored in amber  or foil -wrapped bottles to


                                     III-369

-------
minimize photolytic decomposition.  If residual  chlorine is suspected of
being present in an aqueous sample, sodium thiosulfate in excess (10%) of
that needed to neutralize the chlorine should be added.  If necessary, a
field test kit for measurement of chlorine may be used on a separate sample
aliquot to determine the amount of sodium thiosulfate required.  Addition of
250 mg/liter of sodium thiosulfate will neutralize 5 mg/liter chlorine.  All
samples should be extracted within 7 days of collection and analysis must be
completed within 40 days of sampling. 6

     Samples of soil or sediment should be stored by freezing. 8  Sediment
samples may be acidified with hydrochloric acid  to prevent bacterial decom-
position of the PAHS. 9  Mercuric chloride can be used for the same purpose. 10
Samples can be freeze-driedll12,13 or air-dried at ambient or elevated
temperatures. 9 .14 ,15, 16

     Tissue samples (both shellfish and vegetable) should be stored in opaque
or foil-wrapped containers to prevent  photolytic decomposition.  Shellfish
can be externally cleaned, shucked and drained of excess fluid.  A repre-
sentative 4- to 5- gram portion can be dried at  80C for 48 hours for deter-
mination of moisture content. 14,15, 16   All work  with tissue samples should
be performed under subdued yellow tungsten light. 17, 18

     Particulate samples to be analyzed for oolycyclic aromatic hydrocarbons
are usually collectad using glass fjbar "v.tars  -ind hign-v'olume air samplers. 5
Approximately *Q mg of participates ^hould be ".oil acted.  The samples should
be protected from exposure to light.  Samples  dra fairly staole for up to 1
year if stored in the dark and refrigerated. 5
C.   INTERFERENCES

1.   Method Interferences

     Method interferences caused by contaminants in solvents, reagents,
     glassware, and other hardware used in sample processing may lead to
     discrete artifacts and/or elevated baselines in the chromatograms.  All
     of these materials must be routinely demonstrated to be free from inter-
     ferences under the conditions of the analysis by running laboratory
     reagent blanks.  Glassware must be scrupulously cleaned. 20  clean all
     glasswar3, as soon as possible after jse by rinsing *ith the last solvent
     used in it.  This should be followed by detergent-washing with hot
     water, and rinses with tap water and distilled water.  Glassware, except
     volumetric glassware should then be drained dry and heated in a muffle
     furnace at 400C for 15 to 30 minutes.  Some thermally stable materials,
     such as PCBs, may not be eliminated by this treatment.  Solvent rinses
     with acetone and pesticide-quality hexane may be substituted for the
     muffle furnace heating.  Glassware can also be cleaned with chromic
     acid cleaning solution, followed by rinsing with distilled water, oven-
     iry'ng it !ROC, and -o1vent---insinq.'-  After "leaning, ilassware ^ho'jld
     be sealed and stored in a clean environment to prevent any accumulation


                                    III-370

-------
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                                            1    
                      in-371

-------
     of dust or other contaminants.   Store glassware  Inverted  or capped with
     aluminum foil.

2.   Matrix Interferences

     Matrix Interferences may be caused by contaminants  that are coextracted
     from the sample.  The extent of matrix Interferences will vary consider-
     ably from sample to sample.  Cleanup procedures  can be used to overcome
     many of these Interferences, but unique samples  may require additional
     cleanup approaches to achieve the Method Detection  Limit  (MOD.  The MDL
     values for various PAH compounds are given  1n Table 1.  For organisms,
     or for sediment samples containing small  levels  of  PAH contamination,
     the dimethylsulfoxide (DMSO) partitioning sample cleanup  (Subsection
     J.3.5) should be performed after the column chromatographlc cleanup.
     Use of the DMSO cleanup as a first step for such samples  will result 1n
     large volumes of solvents and formation of  emulsions.22

D.   SAFETY

     The carcinogenic and mutagenlc  properties of many polynuclear aromatic
hydrocarbons (PAH) are well established.21  Benzo[a]anthracene, benzo[a]-
pyrene, and d1benzo[ah]anthracene have been tentatively  classified as known
or suspected human/mammalian carcinogens.6  Consequently, care must be
taken *o 3vo1d spilling solutions of PAH material on  hands or  other areas
of the skin.  Manipulations Involving neat PAH compounds or concentrated
solutions should be performed In a fume hood and/or  primary containment
area.5f23  Personnel performing these procedures must be familiar with
current Occupational Safety and Health Administration regulations regarding
af<9 handling of he .Hs.  VH nersonnel  Involved 1n the analysis must have
access to a reference file of material aata handling  sneers pertaining  co
these chemicals.  Additional references dealing  with  iaboratory .safety
should be consulted to ascertain that the laboratory  has an effective safety
program.23,24,25,26

I.   APPARATUS

1.   Sampling equipment, for discrete or composite water sampling (either of
     the two options listed below 1s acceptable).

     1.1  Grab sample bottle - Amber glass, 1-Hter  or 1-quart volume,
          fitted with screw caps lined with Teflon.   Foil may  be substi-
          tuted for Teflon 1f the sample Is not  corrosive.  If amber bottles
          are not available, protect the samples from light.   The container
          must be washed, rinsed with acetone or methylene chloride, and
          dried before use.

     1.2  Automatic sampler - Must Incorporate glass  sample containers  for the
          collection of a minimum of 250 ml.  Sample  containers must be kept
          refrigerated at 4*C and protected from light during  compositing.  If
          the sampler uses a oerlstaltic pumo, a minimum length of compressible
          sHlcone ruooer tuoing may oe usea.  ^erore jse,  ;ne ."ompressible


                                    III-372

-------
 TABLE  1.   METHOD  DETECTION  LIMITS FOR SOME POLYNUCLEAR AROMATIC HYDROCARBONS
5=BSSS=S3SSS=SS=SSS=S=BSSBS===SB==S====== SS = = = = = = 5SB=S = S = = SSSBSSB = = B = = = = = = = = = = =

     Compound                                                       MDL
                        Hazardous Waste Samples*27  (oil)
     Benzo[a]pyrene
     Naphthalene*
     Acenaphthylene*
     Acenaphthene*
     Fluorene*
     Phenanthrene
     Anthracene
     Fluoranthene
     Pyrene
     Benzo[a]anthracene
     Chrysene
     BenzoCbjf1uoranthene
     Benzo[k]fluoranthene
     Benzo[a]pyrene
     Dibenzo[ah]anthracene
     Benzo[ghi]pery1ene
    Not available
    Benzo[a]pyrene
                                Water Samples16
                             Soil/Sediment Samples



                                 Tissue Samples*21



                                  A1rSamples28
                                                                 0.02 ug/g
                                                                 1.8*
                                                                 2.3*
                                                                 1.8*
                                                                 0.21*
                                                                 0.64
                                                                 0.66
                                                                 0.21
                                                                 0.27
                                                                 0.013
                                                                 0,15
                                                                 j.JIS
                                                                 0.017
                                                                 j.323
                                                                 0.030
                                                                 0.076
0.1 ng/g
                                                               10 pg/m3
     Benzo[a]pyrene
BSBBSSSBSSSSSBBSSBBSSSSSBSSSSSBSSBSSSSSSSSSSSSBSSSSSSSSSBSBS
 *UV detection
  Isocratic elution for 5 min with  acetonitrile  +  water  (4+6),  linear  grad
  ient elution to 100 percent acetonitrile  in  25 min; linear velocity =  2
  mm/sec.
 ^Fluorescence detection, unless  otherwise  Indicated.
 +Barley malt matrix

                                   III-373

-------
          tubing should be thoroughly rinsed with methanol,  followed by
          repeated rinsings with distilled water to minimize potential  for
          contamination of the sample.   An integrating flow  meter is required
          to collect flow proportional  composites.

2.   High-volume air sampler (Haskin Scientific Ltd., Montreal,  Canada, or
     equivalent), with glass fiber filters, 20 x 25 cm (Gelman type A,  or
     equivalent) and recorder paper and ink.

3.   Blender.

4.   Ultrasonic vibrator (Branson DHA-1000, or equivalent).

5.   Clinical centrifuge (International  Model  CL, or equivalent).

6.   Wasserman clinical centrifuge tubes.

7.   Rotary evaporator.

8.   Chromatographic columns, 10 mm x 200 mm and 14.5 mm x 250 mm.

9.   Analytical concentrator.

     9.1  (N-Evap, or equivalent) or

     9.2  Kuderna-Danish evaporative concentrator (K-D).

          9.2.1  Micro-Snyder column for K-D.

10.  '-Mgh-Per^crsanc.? L-i^u*c ^hromatogriDh (Perkin-Elmer Series  2/2,  or eauiv-
     alent), with columns 0.25 x 25 cm and preparative-scale oonaed polar
     aminocyano column (for oil  samples only)  (Perkin-.Elmer  ODS-HC  SIL-X-1,
     or equivalent), ultraviolet absorbance detector (Perkin-Elmer  LC-55, or
     equivalent), and filter fluorescence detector (Varian Fluorichrom, or
     equivalent) or fluorescence spectrophotometer equipped  with a  flow cell
     (Perkin-Elmer, 204A or equivalent), and 25-pl  injection syringes (or
     autosampler), chart recorders, and accessories.

11.  Tri-carb Scintillation Counter Model C-2425, with Automatic External
     Standard Option (Packard Institute Company, Downers Grove,  Illinois)
     or equivalent.

12.  Circular metal punch, 46 mm diameter.

13.  Analytical balance, with sensitivity of 0.1 mg.

14.  Soxhlet extractor, with extraction thimbles.

15.  Hotplate/stlrrer, combination.

15.  r!ash 3vaoorator 'Siichner,  or e-aulvalent).


                                    111-374

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17.  Separatory funnels, 125 ml, 250 ml,  500 ml,  and 2,000  ml,  with  Teflon
     stopcocks.
18.  Amber vials, 10 to 15 ml capacity,  with Teflon-lined caps  or septa.
19.  Flasks, pear-shaped, 300 and 500 ml.
20.  Buchner funnel, with coarse-porosity fritted disc (Kontes  K-955000,  or
     equivalent).
21.  Water bath - Heated, capable of (2C)  temperature control.
F.   REAGENTS
1.   Acetonitrile, HPLC grade.
2.   Alumina - Aluminum oxide 90; active neutral  (activity  Stage  I);  particle
     size 0.063-0.200 mm (70-230 mesh ASTM).
3.   Benzo[a]pvrene (B[a]P), radio-labeled:   3H-B[a]P ca. 0.1 ng  (25,000  dpm) per
     sample; l^C-B[a]P (for high levels)  ca.  3  ng (1,000 dpm) per sample.
4.   Cyclohexane, distilled  in .jlass [Burdic!: ind .lackson Snectrograde, or
     equivalent).
5.   Cyclohexanone, pesticide grade.
o.   ueiomzeu water, intsrfar^ncs-'-^e,
7.   Dimethyl sulfoxide, spectrophotometric  grade.
8.   Ethanol.
9.   F1or1s1l, 60/100 mesh (Matheson, Colemen,  and Bell, or equivalent).
10.  Isooctane, distilled in glass.
11.  Methanol, pesticide grade.
12.  Methylene chloride, pesticide grade.
13.  Permafluor V scintillation  cocktail  (Packard Inst. Co., Downers Grove
     Illinois), or equivalent.
14.  Potassium hydroxide pellets.
15.  Sephadex LH-20.
15.  311ica yet 50 - Particle sfiG ^.363  *o  1.200 urn '70/230 mesh ASTM).
17.  Sodium suifate, annyarous.
                                    iii-J/3

-------
18.  Sodium thiosulfate, granular (ACS).

19.  Stock standard solutions -  Stock  standards  can  be  prepared from  pure
     standard materials [phenanthrene  (Phn),  fluoranthene  (F), pyrene (Py),
     benzo[a]anthracene (B[a]A), benzo[b]fluoranthene  (Bbf), benzo[e]pyrene
     (B[e]Py), benz[a]pyrene (B[a]Py), dibenz[ah]anthracene  (dB[ah]A),
     benzo[b]chrysene (B[b]Ch),  indeno[l,2,3-cd]pyrene  (I[cd]Py), benzo[ghi]
     perylene (B[ghi]P), dibenzo[ai]pyrene (dB[ai]Py),  and corenone (Cor),
     available from Div. of Chem. and  Phys.,  FDA,  Washington, D.C.  Some PAH
     compounds are also available from Chemical  Repository,  Illinois  Insti-
     tute of Technology Research Institute (IITRI; Chicago,  Illinois)].  Store
     in Teflon-sealed bottles, protected  from light, at 4C.  If compound
     purity as used is greater than 96 percent,  the  mass may be used  without
     correction in calculation of the  concentration.  Stock  standard  solutions
     should be replaced after 6  months, or sooner  if comparisons with check
     standards indicate a problem.

G.   QUALITY CONTROL

1.   Any laboratory using these methods should operate  a formal quality control
     program.  The minimum requirements of such  a  program  consist of  an initial
     demonstration of laboratory capability and  the  analysis of spiked samples
     as a continuing check on performance.  The  laboratory should maintain
     performance records to define the quality of  data  that  are generated.
     Ongoing performance checks  should be :onparsd with established performance
     criteria to determine *f the results of analyses  are  within accuracy and
     precision limits expected of ihe  method-

2.   Before performing any analyses, the  analyst shoald demonstrate the ability
     to janenta -ssultc ;f xccsptnbla ace-racy  ind  precision.  Tlr's  ^ay be
     accomplished by analyses of four  or  more samnJes  fortified, at a repre-
     sentative concentration, with the compound(s) of  interest.5

     2.1  One or more unfortified samole(s) should be  processed, to determine
          background levels, and the level of fortification  should be twice the
          background level.

     2.2  The average percent recovery (R) and standard deviation  (s) of the
          percent recovery should be calculated.  These should then be compared
          to the values for average recovery (X) and standard deviation (p)
          expected for each parameter.  If s > 2p  or |X -  R| > 2p, the analyst
          should review potential sources of error in  the  procedure,  and the
          test should be repeated.  The analysis should not  be performed until
          these parameters are satisfactory.

     2.3  The results of the determination of the  average  recovery (R) and the
          standard deviation (s) of the average  recovery should be used to
          calculate the Upper and Lower Control  Limits:*9

               Upper Control Limit (UCL)     R + 3s
               Vower Control Limit 'LCD   *  * - 3s


                                    III-376

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     2.4  The UCL and LCL can be used-to construct control  charts29 to assess
          trends in analytical performance.   Ten percent of the samples
          processed should be collected in duplicate and analyzed as fortified
          and unfortified samples in order to calculate-R and s.

     2.5  The laboratory should ascertain that all glassware and reagent inter-
          ferences are under control, by analyzing a reagent blank each time  a
          sample or set of samples is analyzed.

     2.6  It is recommended that the laboratory  adopt additional  quality assur-
          ance practices for use with this method.  The specific practices that
          are most productive depend upon the needs of the  laboratory and the
          nature of the samples.  Field duplicates may be analyzed to monitor
          the precision of the sampling technique.  When doubt exists over the
          identification of a peak on the chromatogram, confirmatory techniques
          such as thin-layer chromatography (TLC) or chromatography with a
          dissimilar column or detector must be  used (see Subsection K).  This
          may include the use of a mass spectrometer.  Whenever possible, the
          laboratory should perform analyses of  standard reference materials
          and participate in relevant performance evaluation studies.

     2.7  In the analytical procedures for soil/sediment (Subsection J.3) and
          for animal tissue (Subsection J.4.2) samples, use is made of a radio-
          iabeied benzo[a]pyrene "ntarnal  standard to "indicate method recov-
          ery. 22  use of this internal  standard  in calculation of results
          totally corrects for losses of benzoCa^pyrene during purification and
          analysis.22  The application of the same recovery figure to other
          PAHs in this method corrects for mechanical losses, dilution changes,
          or otfier factors ^hich affect the  samol"? as a whole, but does not
          correct for any differential  losses wmcn occur co a varying degree
          in the compound(s) of interest.22   if  substantial  amounts of PAHs
          compounds are present in the sample, l^C-labeled  benzo[a]pyrene
          is used, while for lower levels  -^-labeled internal  standard is more
          appropriate.22

     2.8  When using the vegetable tissue method (Subsection J.4.2), the
         .recovery from the silica/alumina column chromatographic cleanup is
          measured before use.  Recoveries for each PAH should be greater than
          or equal to 90 percent.21  For extract concentration, it is recom-
          mended that extracts from samples  containing the  more volatile PAHs
          (e.g. anthracene and biphenyl)  be  concentrated by use of a Kuderna-
          Danish evaporative concentrator, to prevent losses.20

H.   CALIBRATION

1.   Liquid chromatograph operating parameters which produce separations as good
     as those indicated in Table 1 and Figures 2 and 3 should be established.
     Due to rapid advances being made in chromatography, the analyst may modify
     operating parameters, as long as the  modifications are not detrimental to
     R, s (as defined -n Subsection G). or the separation.
                                    III-377

-------
 Column: HC-ODS SIL-X
 Mobile phase: 40% to 100% Acetonitrile
                 in water
 Detector: Fluorescence
 1. Phenanthrene
 2. Anthracene
 3. Fluoranthene
 4. Pyrene
 5. Benzo [a] anthracene
 6. Chrysene
 7. Benzo [b] fluoranthene
 8. Benzo [k] floranthene
 9. Benzo [a] pyrene
"0. Oibenzo fa.h] anthracene
11. Benzo fg.h.i] peryiene
12. Indeno [1,2.3-cd] pyrene
1 1
4
i
8
i
12
I
16
i
20
i
24
i
28
I
32

36
                      Retention Time, minutes
      Figure 2.   Chromatogram of PAH standards (from reference 6).

                              III-378

-------
        1. Naphthalene
        2. Acenaphthalene
        3. Acenaphthene
        4. Fluorene
        5. Phenanthrene
        6. Anthracene
        7. Fluoranthene
        8. Pyrene
        9. Benzo [a] anthracene
       10. Chrysene
       11. Benzo [b] fluoranthene
       12. Benzo [k] fluoranthene
       13. Benzo [a] pyrene
       14. Dibenzo [a,h] anthracene
       15. Benzo [g.h.i] perylene
       16. Indeno [1,2.3-cd] pyrene
5 6
10
                                                                   16
i       I       i       I      I        I       I       I
0      4       8      12     16     20     24     28
                     Retention Time, minutes
                 32     36
          Figure 3.   Chromatogram of PAH compounds with JV detection.
                             ' from ""^fersnc2 ^)
                                 III-379

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2.   Liquid Chromatograph External  Standard Calibration

     2.1  Prepare calibration standards at three or more concentrations  for
          each compound of interest by adding volumes of one or more stock
          standard solutions to a volumetric flask and diluting to  volume with
          acetonitrile.

     2.2  Inject 2.0 to 5.0 ul  of each calibration standard and tabulate peak
          height response or area response as a function of mass injected for
          each compound for both ultraviolet absorbance and for fluorescence.

     2.3  These results can be used to construct calibration curves.   If the
          response is linear (<10 percent relative standard deviation, RSD),
          passage through the origin can be assumed and the average ratio of
          peak height or area per mass can be used 1n place of a calibration
          curve.  The ratio of fluorescence to ultraviolet.absorbance  response
          factors should not change with time.

3.   Liquid Chromatograph Internal  Standard Calibration

     Calculate the response factor as follows:


                  Response Factor (RF)  =  {AS)(C^S)/(A1-S)(CS)             Eq. 1

     where:

           Ais  =  Response for the internal standard

           As    -  .vasponsa ror ;n3 -amp]a

           C^s  =  Concentration of the internal standard

           Cs   =  Concentration of the sample.

4.   The working calibration curve or the response factor should be verified  on
     each working day by analysis of calibration standards.   If the responses
     vary from the expected response by more than either 10 percent (absolute
     basis) or more than twice the RSD, the test should be repeated,  using a
     new calibration standard.  Alternatively, a new calibration curve or
     calibration factor should be prepared for the compound.
                                    III-380

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I.   ANALYTICAL PROCEDURES

1.1  Analysis of Hazardous Wastes for Polycyclic Aromatic Hydrocarbons
          Analytical  Procedure:   available
          Sample Preparation:   available

     1.1.1  Reference

            Hertz, H. S., J. M.  Brown, S. N.  Chesler,  F.  R.  Guenther, L.  R.
            Hilpert,  W.  . May,  R. M. Parris, and S. A. Wise,  "Determination
            of Individual Organic Compounds in Shale Oil."  Analytical
            Chemistry, 52:1650-1657.   1980.

     1.1.2  Method Summary

            This method is a chromatographic  method which uses preparative
            liquid chromatography followed by analytical-scale liquid chro-
            matography.   An oil  sample is diluted and  fractionated according
            to the number of aromatic rings in the molecule.   The  concentrated
            fractions are analyzed by reversed-phase high-performance liquid
            chromatography with  ultraviolet and fluorescence detection.

     1.1.3  Applicability

            This method has been demonstrated to De applicable *c  ^ale-oil
            samoies ana oetroleuro-contaminated marine  sediments.26,27

     1.1.4  Precision and Accuracy

            Precision information /or ;ne prccsuure ': presented *n T2b1e ?,
            The method detection limits are 1'stsd -'n  Table  1.
                 TABLE 2.  PRECISION FOR HPLC/HPtC ANALYSIS OF SHALE OIL
            assssaszssssssssaaasasa333asssssa33=3=33ass3333333333:: 33=333=
            Compound                  Concentration (ppm)     Standard Deviation
            Acridine                          6                     1.2
            3enzo[a]pyrene                   21                     1.5
            Fluoranthene                     53                     3
            Pyrene                          108                     8
     1.1.5  Sample Preparation

            1.1.5.1  Dilution/Extraction
                     Dilute oil samples to a level  of ca.  0.1 g/ml  in methyl ene
                     cnlonae.  extract ^.000 9 jr "less oily :ediment xith
                                    111-381

-------
                ultrasonic agitation for ca.  2  hours  with  ether,  con-
                centrate by evaporation,  solvent-exchange  the  extract  into
                hexane,  and concentrate to approximately 1 ml.

       1.1.5.2  HPLC Fractionation

                Inject an aliquot containing  approximately 15  mg  of  sample
                onto a preparative-scale aminosilane  column.   Elute  the
                sample with pentane^?  or 2 percent  methylene chloride  in
                hexane^O at a  flow of  ca.  5 ml/min.   These conditions
                result in a separation by the number  of aromatic  rings in
                the PAH  compound.   Table 3 lists  some PAH  compounds  and
                their retention characteristics on  the aminosilane
                column.31  Use previous injections  of standards of the
                compounds of Interest  to determine  specific retention
                volumes  for the PAH compounds of  interest.  Collect  frac-
                tions of column effluent 1n centrifuge tubes,  add 50 ul
                acetonitrile,  and reduce the  volume to 50  ul.  Figure 4
                shows a  typical fractionation.

1.1.6  High-Performance  Liquid Chromatography

       Quantify PAH compounds  within each fraction  (see Table  3)  by  HPLC
       using an octadecy!silane column and a  water-acetonitrile gradient
       (50 to 100 percent acetonitrile 1n 30  mm  at i ml/nun),  tnnancea
       sensitivity ?nd selectivity can oe ootauied  oy use  of both detec-
       tors in series.  See Table 6 (page HI-393)  for ultraviolet absorb-
       ance and fluorescence characteristics  of several PAH compounds.
       Figure 5 shows a  chromatogram of a samole  with both ultraviolet
       absorbance and fluoresencs Uex -  -i/0  nm;  *m  = 400 nm) detection.
       Figure 6 snows fluorescence spectra ootainea ror individual peaks
       in the chromatograms in Figure  5 (Xex  =  270  nm).
                               III-382

-------
                 TABLE 3.  LOGARITHM OF RETENTION INDEX (I)
                           FOR SOME PAH COMPOUND$31
                       ====================================================
                                    log I
                               Octadecylsilane         Aminosilane Column
     Compound _ (sol vent: acetoni tri'1 e/water)   (sol vent :hexane)

Two-Ring Aromatics

Naphthalene                         2.00                      2.00
Biphenyl                            2.57                      2.25
Acenaphthalene                      2.73                      2.00

Three-Ring Aromatics

Fluorene                            2.78                      2.61
Anthracene                          3.02                      2.95
Phsnanthrene                        3. CO                      3.00

four-Ring Aromat-ics

Fluoranthene                        3.42                      3.39
2enro[a]*l'jorene                    3.74                      3.46
Benzo[b]f!uorene                    3.78                      3. S3
Pyrene                              3.51                      3.68
Naphthacene                                                 3.93
Benz[a]anthracene                   4.00                      4.00
Chrysene                            3.94                      4.03

Five-Ring and Larger Aromatics

Benzo[a]pyrene                      4.57                      4.30
Perylene                            4.43                      4.47
Benzo[ghi]pery1ene                  >5                        4.61
Indeno[l,2,3-cd]pyrene              >5                        4.72
Dibenzo[ac]anthracene               4.84                      4.33
D1benzo[ah]anthracene               >5                        4.93
                                  III-383

-------
                                  .Ill
                                        Tim* ~f
Figure 4.  HPLC fractional ion  of  an  oily sediment sample extract with yBonda
           pak NH2-  Numbers refer to  the fractions collected for subsequent
           analysis.   Conditions: pentane at 3 ml/min, 1.0 absorbance unit
           full-scale  (aufs),  1.0 ml  injected (from Reference 31).
                              UV JN_^
                             2S4nm
                             Fluor*tc*nc
                              x: 270
                              Mn:4OO
                                      10   20    30
                                     Retention Tim*, minut**
                                                      40
      Figure  5.   Reversed-phase analysis of PAH fraction (4 in Figure  4).
                  Conditions:   50 to 100 percent acetonitrile in water,  linear
                  gradient  in  30 min at 2 ml/min., 0.05 aufs, 200 yl  injected
                  (20  percent  of fraction).  Upper chromatogram:  UV  absorption
                  jetecv.on ut 254 -TH.   '.jwer "hromatogram: "uorsscance emission
                  detection at 400 nm with excitation at 270 nm from  Reference 27).
                                     III-384

-------
                        (Numbers Indicate Wiv*4ngth* in rim)
Figure 6.   Fluorescence  emission spectra at  270-nm excitation  of peaks
                  1-7 1n  Figure 5 (from Reference 26).
                                 111-385

-------
2.1  Analysis for Polycyclic Aromatic Hydrocarbons In Water Samples
          Analytical  Procedure:   available
          Sample Preparation:  available

     2.1.1  Reference

            U.S. Environmental  Protection Agency,  "Polynuclear Aromatic  Hydro-
            carbons - Method 610."   Methods of Chemical  Analysis  of Municipal
            and Industrial  Wastewater,  EPA-600/4-82-057,  U.S.  EPA (1982).6

     2.1.2  Method Summary

            A measured volume of water (ca. 1  liter)  is  solvent-extracted  into
            methylene chloride.   The methylene chloride  extract is  dried and
            concentrated to 10 ml or less, the solvent is exchanged to cyclo-
            hexane, and cleanup (as  necessary) is  performed.   Following  cleanup,
            the solvent is  exchanged to acetonitrile  and analysis is performed
            by HPLC,  using  ultraviolet (UV) and fluorescence  detectors.  A
            silica gel column cleanup procedure is provided to help alleviate
            matrix interference problems.

     2.1.3  Applicability

            This method is  a chromatograohic method applicable to the deter-
            mination  of the compounds listed ,n faDies 4 ana  5 in .vater  :nd
            wastewater samnles.   Table 4 gives the method detection limits.
            The actual method detection limits ootainea  may vary  with sample .
            size, the extent of extract concentration, the nature of the inter-
           ferences  present, and the sample cleanup  techniques used.

            This method should only  be used by analysts  experienced in high-
            performance liquid chromatographic techniques, or under the  super-
            vision of such  personnel.  Each analyst should demonstrate the
            ability to generate results of acceptable precision and accuracy,
            using the procedure of Subsection  G.

     2.1.4  Precision and Accuracy

            The method detection limit (MOD is the minimum concentration  of
            analyte which can be measured and  reported with 99 percent con-
            fidence that the value  is above zero.   The MDL values listed in
            Table 4 were obtained using reagent *ater, but similar results were
            obtained using  representative wastewater  samples.

            This procedure  was evaluated by a  single  laboratory,  using trip-
            licate fortified samples analyzed  on two  different days.6 The
            average recovery and standard deviation values are given in  Table 5.

            The method has  been shown to give  linearity  of recovery from forti-
            fied water samples over  the concentration range of 8  times the MDL
            to dOO times the MDL, except  :hat  benzo[nhi"]pery1ene  -ecoveries  at.
            80 times MOL and 800 times MDL were 35 and 45 percent,  respectively.0

                                    II1-386

-------
                 METHOD DETECTION  LIMITS  FOR  WASTEWATER  SAMPLES6
 =================:===========================================================-=;
                        Retention  Time     Capacity  Factor     Method  Detection
 Parameter                    (min)               (k1)             Limit (ug/1)a
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fl uoranthene
Pyrene
BenzoCalanthracene
Chrysene
Benzo[b]f 1 uoranthene
Benzo[k]fl uoranthene
Benzo[a]pyrene
Dibenzo[ah]anthracene
Benzo[
-------
    TABLE 5.  SINGLE-OPERATOR ACCURACY AND PRECISION FOR WASTEWATER SAMPLES6
B3SSS3a333333S33S3B3333:SSB3a33S33SS3aa3a3aSaS33SB3S3S3aBBS3S33S33a33SaBa33SBS3


Parameter
Average
Percent
Recovery
Standard
Deviation
(*)
Spike
Range
(ug/1)

Number of
Analyses

Matrix
Types
Acenaphthene               88
Acenaphthylene             93
Anthracene                 93
Benzo[a]anthracene         89
Benzo[a]pyrene             94
Benzo[b]fluoranthene       97
Benzo[ghi]perylene         86
Benzo[k]fl uoranthene       94
Chrysene                   88
Dibenzo[ah]anthracene      87
Fluoranthene              116
Fluorene                   90
Indeno[l,2,3-cd]pyrene     94
Naphthalene                78
Phenanthrene               98
Pyrene                     96
 5.7
 6.4
 6.3
 6.9
 7.4
12.9
 7.3
 9.5
 9.0
 5.8
 9.7
 7.9
 6.4
 8.3
 8.4
 8.5
11.6-
 250-
 7.9-
0.64-
0.21-
0.24-
0.42-
0.14-
 2.0-
 0.4-
 0.3-
 6.1-
0.96-
  20-
 3.8-
 2.3-
-25
450
11.3
0.66
0.30
0.30
3.4
6.2
6.8
1.7
2.2
23
1.4
70
5.0
6.9
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
            filtration of the emulsion through glass wool, centrifugation,
            or other physical'methods.  Collect the methylene chloride
            extract m a tSC-rd I.-'ienmeyer  .-'asjc.

            Add a second 60-ml volume of methylene chloride to the  sample
            bottle, rinse, and repeat the extraction procedure a second
            time, combining  the extracts in the Erlenmeyer flask.
            Perform a third extraction in the same manner.

     2.1.5.4  Assemble a Kuderna-Danish (K-D) concentrator by attaching  a
              10-ml concentrator tube to a  500-ml evaporative flask.
              Other concentration devices or techniques may be used in
              place of the .K-D 1f the requirements of Subsection G.2. are
              met.

     2.1.5.5  Pour the combined extracts through a drying column containing
              10 cm anhydrous sodium sulfate, and collect the extract in the
              K-D concentrator.  Rinse the  Erlenmeyer flask and column with
              20 to 30 ml methylene chloride to complete the quantitative
              transfer.

     2.1.5.6  Add one or two clean boiling  chips to the,evaporative flask
              and attach a three-ball Snyder column.  Prewet the Snyder
              column oy adding aoout i mi .netnyiene chloride "o  '.he '.op.
              1ace the K-0  aooaratus on a  hot water bath (60 to 65C) so
                                     III-388

-------
         that the concentrator tube is partially Immersed In the hot
         water, and the entire lower rounded surface of the flask is
         bathed with hot vapor.  Adjust the vertical position of the
         apparatus and the water temperature as required to complete
         the concentration in 15 to 20 minutes.  At the proper rate of
         distillation the balls of the column will  actively chatter but
         the chambers will not flood with condensed solvent.  When the
         apparent volume of liquid reaches 1 ml, remove the K-D appara-
         tus and allow it to drain and cool for at least 10 minutes.
         Remove the Snyder column and rinse the flask and its lower
         joint into the concentrator tube with 1 to 2 ml methylene
         chloride.  A 5-ml syringe is recommended for this operation.
         Stopper the concentrator tube and store refrigerated if further
         processing will not be performed immediately.  If the extracts
         will be stored longer than two days, they should be transferred
         to Teflon-sealed screw-cap bottles and protected from light.

2.1.5.7  Determine the original sample volume by refilling the sample
         bottle to the mark and transfering the water to a 1,000-ml
         graduated cylinder.  Record the sample volume to the nearest
         5 ml.

2.1.6  Sample Cleanup

       Cleanup procedures rcay not be necessary for a relatively clean  sam-
       ple matrix.  The cleanup procedures ."^commended in :his method  have
       been used for the analysis of various clean waters and industrial
       effluents..  If particular circumstances demand the use of an altern-
       ative cleanup procadure, ^he analyst, tnould determine the elution
       profile and demonstrate that the "ccvery or eacn compound of
       interest is no less than 85 percent.

2.1.6.1  Silica Gel Column Cleanup

         Before the silica gel cleanup technique can be utilized, the
         extract solvent must be exchanged to cyclohexane.  Add a 1- to
         10-ml aliquot of sample extract (in methylene chloride) and a
         boiling chip to a clean K-D concentrator tube.  Add 4 ml cyclo-
         hexane and attach a micro-Snyder column.  Prewet the micro-
         Snyder column by adding 0.5 ml methylene chloride to the top.
         Place the micro-K-0 apparatus on a boiling (100'C) water bath
         so that the concentrator tube is partially immersed in the hot
         water.  Adjust the vertical position of the apparatus and the
         water temperature as required to complete  concentration in 5
         to 10 minutes.  At the proper rate of distillation the balls
         of the column will actively chatter but the chambers will  not
         flood.  When the apparent volume of the liquid reaches 0.5 ml,
         remove the K-D apparatus and allow it to drain for at least 10
         minutes while cooling.  Remove the micro-Snyder column and
         rinse its lower joint into the concentrator tube xlth ^ mini-
         mum of cyclohexane.  Adjust the extract volume to about 2 mi.
                               III-389

-------
  Prepare a slurry of 10 g activated silica gel  in methylene
  chloride and place this in a 10-mm-I.D.  chromatography column.
  Gently tap the column to settle the silica gel  and drain the
  methylene chloride.  Add 1 to 2 cm anhydrous sodium sulfate
  to the top of the silica gel.

  Preelute the column with 40 ml  pentane.   Discard the eluate,
  and just prior to exposure of the sodium sulfate layer to the
  air, transfer the 2 ml of cyclohexane sample extract onto the
  column, using an additional 2 ml  cyclohexane to complete
  the transfer.

  Just prior to exposure of the sodium sulfate layer to the air,
  add 25 ml pentane and continue elution of the column.  Discard
  the pentane eluate.

  Elute the column with 25 ml methylene chloride/pentane (4 +
  6, Y+V) and collect the eluate in a 500-ml K-D flask equipped
  with a 10-ml concentrator tube.  Elution of the column should
  be at a rate of about 2 ml/min.

  Concentrate the collected fraction to less than 10 ml by K-D
  techniques as described in paragraph 2.6.1 using pentane to
  '"'rise the walls of the 
-------
            Column: HC-ODS SIL-X
            Mobile pha0: 40% to 100% Acetonttrite
                       in water
            Detector: Fluorescence
2
             
            3
            4
            6. Bvnzo () amhrKn*
            6. ChryMn*
            7 Bwuo (b) fluor*nthn*
            8 Bvfizo IK) flonntn^nv
            9 Bvnzo [) pyran*
            10 OitMnzo (*.h) nttrcn
            11 B*nm (g.h.ij p*rvton
            12. bvteno [1.2.3-cd] pyiww
                     I
                    8
 I
16
 I
20
12   16   20   24   28

  Retention Time, minute*
32   36
Figure 7.  Liquid  chromatogram of polycyclic  aromatic hydrocarbons
           with  Hjorsscsnca detacticn  '*;m  rfer*rc-s 6).
    can be compared to spectra of standards  recorded under the  same  con-
    ditions, and  can be useful for qualitative identification of
    peaks.4.31,32  Table 6 lists some polycyclic aromatic hydrocarbons
    and their  fluorescence and absorbance  characteristics in n-heptane
    solution.31

    Calibrate  the system daily as described  in Subsection H.   If  the
    internal standard approach is being  used,  the internal standard  must
    be added to the sample extract and mixed thoroughly immediately
    before injection into tne instrument.   Inject 5 to 25 ul of the
    sample extract, using a high-pressure  syringe or a constant-volume
    sample injection loop.  Record the volume  injected to the nearest
    0.1 yl, and the resulting peak size  in height or area units.
    Re-equilibrate the liquid chromatographic  column at the initial
    conditions for at least 10 minutes between injections.

    The width  of  the retention time window used to make identifications
    should be  based upon measurements of actual retention time  variations
    of standards  over ;ne course QT .; jsy.   """hree *imes the standard
                               III-391

-------
 1. Naphthalene
 2. Acenaphthalene
 3. Acenaphthene
   Fluorene
   Phenanthrene
   Anthracene
   Fluoranthene
   Pyrene
   Benzo [a] anthracene
   Chryiene
   Benzo [b] fluoranthene
   Banzo [k] fluoranthene
13. Benzo [a] pyrene
14. Dibenzo [a,h] anthracene
15. Benzo ffl.h.i] perylene
16. Indeno [1.2,3-cd] pyrene
            4.
            6.
            6.
            7
            8
            9
           10
           11
           12
5 6
10
                                                      1  13
                                                              15

1
0
i
4
i
8
i
12
t
16
i
20
*
24
28
32
36
                        Retention Time, minutes
Figure 8.   Liquid chromatogram of polycyclic  aromatic hydrocarbons with
            ultraviolet detection (254 nm)(from  reference 6).
    deviation of a retention  time for a compound can be used  to calculate
    a suggested window size;  however, the experience of the analyst
    should  weigh heavily  in the interpretation  of chromatograms.

    If the  peak height or area  exceeds the linear range of the  system,
    dilute  the extract with acetonitrile and  reanalyze.  If the peak area
    measurement ^s prevented  by the oresence  of interferences,  further
    cleanup 1s required.
                                111-392

-------
  TABLE 6.  FLUORESCENCE CHARACTERISTICS OF POLYCYCLIC AROMATIC  HYDROCARBONS
                             IN N-HEPTANE AT 298 K
                               (from reference ?.}

                                                                         RCRA
                                                         Emission        Waste
Compound                          Maxima* (nm)          Maxima*  (nm)     Number
 Excitation
Maxima* (nm)
Acenaphthalene**
Acenaphthene
Anthracene
Benz[c]acridine
(3,4-Benzacridine)
Benz[a]anthracene 230,
(1,2-Benzanthracene)
Benz[jk]f1uorene 264,
(1,2-Benzfluorene)
Benz[a]phenanthrene
( 1 ,2-8enzpnenanthrene)
Benz[a]pyrene
(\ 2-Benzoyrene)
Benz[d]pyrene 265,
(3,4-Benzpyrene)
Biphenyl
Carbazole
(Dibenzpyrrole)
Chrysene
Dibenz[ah]anthracene 2B8,
(l,2:5,6-Dibenzanthracene)
Dibenz[ai]pyrene 241,
(1,2:7 ,8-Di benzpyrene)
3,4:9,10-Dibenzpyrene . 241,
Oibenz[ae]pyrene 228,
( 1 ,2 : 4 ,5-Dibenzpyrene)
229,
228,
253.
355,
277,
341,
285,

177,
316,
284,
362,
215,
235,
290.
267,
297.
338,
294,
352,
294,
357,
292,
355,
292,
291.
324,
375
287,
358
303,

268,
331
296,
383.
215
248,
318,
306,
319.
373
314,
371,
314,
371,
302,
J7T
300
338,

327
315

288
346
394

254
331
318
332
329
391
329
391
340
320,
321,
377,
446
385,



388.
403.
305,
333,
362, 380,
393,
490*
432,
397,
335,
335. 347
399. 422,

407, 432
345, 363


397, 409
427, 454
482
316
347
402, 424
403, 417
447, 459
447, 459,
490
418, 444


U016
U018
U120
U050
U022



U050
U063
U064


                                                                   (continued)
                                    III-393

-------
                             TABLE 6.   (Continued)
                                                                         RCRA
                                   Excitation            Emission         Waste
Compound                          Maxima* (nm)           Maxima* (nm)      Number
Dibenz[ah]pyrene               270,  297,  310.  396        448.  477.  509
  (3,4:8,9-Dibenzpyrene)            418

7,12-Dimethylbenz[a]-               298.  363,  382        407,  424          U094
  anthracene
  (7,12-Dimethyl-l,2-
    benzanthracene)

Fluoranthene                   237,  263,  280,  287        461               U021
                               324,  342,  358

Indeno[l,2,3-cd]pyrene                                               U137

Benz[a]anthracene              230,  277,  2S7,  327        385;  407,  432    U018

rtapntnacene                    27 1,  278,  :93,  !94.        :?l,  504,  539
                                    4T*.  AAO,  469
Naphthalene                        - 225,  280             323.  335          U165

'eryiene                  '     ":* , 336,  1Q8,  425         *38,  *65,  449
Phenanthrene                   253. 275,  292             346,  364.  385

Pyrene                         240, 272,  305,  318,        372,  383,  394
                                    334,  363

aaaasaaaaaaaaaaassaaaaaaaaaaBBasaaaaaBaaBSSBaaaaaaaaasaaaaaaasaaaBaaaaaaaBaaasa
 *Most intense wavelengths underlined.   Wavelengths determined using a  3-nm
  spectral bandpass.

**F1 uorescence attributed to a photodecomposition product or impurity.
                                    111-394

-------
3.1  Analysis of Sediments for'Polycycl'ic Aromatic Hydrocarbons
          Analytical Procedure:   available
          Sample Preparation:  evaluated

     3.1.1  Reference

            Dunn, B. P.  and R. J. Armour, "Sample Extraction and Purification for
            Determination of Polycyclic Aromatic Hydrocarbons by Reversed-Phase
            Chromatography."  Analytical Chemistry, 52:13 (1980), pp.  2027-2031.22

     3.1.2  Method Summary

            Hydrocarbons are extracted from sediment samples by alkaline diges-
            tion, and interfering materials are removed by Florisil  column clean-
            up,  dimethyl sulfoxide partitioning, and Sephadex LH-20  column chro-
            matography.   A radio!abeled internal standard is used to correct for
            losses.  Twenty polycyclic aromatic hydrocarbons are determined by
            reversed-phase high-performance liquid chromatography, using simulta-
            neous ultraviolet absorbance and fluorescence detectors.

     3.1.3  Applicability

            This method  is applicable to wet or dry sediment samples.

     3.1.4  Precision and Accuracy

            The  overall  recovery of one polycyclic aromatic hydrocarbon,  benzc-
            [a]pyrene (B[a]P),  in sediment' samples 1s 50 to 70 percent.11,12,13
            Recoveries of B[a]P  from the KOH digestion are generally over 90 per-
            cent. --,-'-,--  3[dj? .-eccver*2s "-en ~* ir*-.':"*  column cleanuo are
            generally over 90 percent.17  The relative standard deviation of the
            method is 6.2 percent for B[a]P.l7

     3.1.5  Sample Preparation

     3.1.5.1  Sample Extraction

              Decant and discard the water layer above the sediment.  Weigh
              and dry a  representative sample for moisture determination.
              Carry out  all  extraction and concentration procedures  under
              subdued yellow light.

              Put 10 to  100  grams wet sediment,  100 ml  ethanol,  5 g  KOH,
              and radioactive B[a]P  (either 1,000 dpm 14C-B[a]P,  ca. 5 ng,
              or 25,000  dpm  3H-B[a]P,  ca.  0.1 ng)  into a flask.   Add a
              boiling stone  and  reflux.   Swirl  the contents  of the flask
              and pour Into  100-ml  centrifuge tubes.   Centrifuge  the sus-
              pension for 5  min  at 500 g,  decant the supernatant  and wash
              the particulate material  twice with  50 ml  ethanol  followed
              by stirring and resuspension,  centrifugation,  and  decantation.
              Combine the original  supernatant,  fc.he  cashes,  and  150  ml
              water In a 500-ml  seoaratory funnel.   Extract  three times with

                                    II1-395

-------
         200-ml portions of Isooctane.  Combine the extracts and wash
         four times with 200-nT! portions of warm (60*C) water.

3.1.5.2  Florlsil-Column Cleanup

3.1.5.2.1  Concentration

           Reduce the volume of the Isooctane from the extraction to
           approximately 10 ml.  Use a Kuderna-Danish evaporative
           concentrator if losses of the more volatile PAHs are of
           concern.  If not, a rotary evaporator 1s satisfactory for
           this step.  After volume reduction, dilute the extract to
           100 ml with toluene.

3.1.5.2.1  Column Chromatography

           Prepare column of 30 g Florisll (deactivated with 5 percent
           water) covered with 60 g anhydrous sodium sulfate in a
           glass chromatographic column (40 x 400 mm).  Add 100 ml
           toluene to the column and elute the toluene until the
           liquid level is just at the top of the sodium sulfate layer.
           Apply the sample (in toluene) and elute until the liquid
           level is at the top of the sodium sulfate layer.  Carefully
           add 100 ml toluene, elute, add another 100 ml toluene, and
           elute again.
                            <
3.1.5.3  DMSO Partitioning

         Add 5 ml dimethyl sulfoxide (DMSO) to the combined toluene
         eiuate  r'rom ;ne cc* Jinn and  rcncsntrate the solution under
         vacuum  (at 60") to remove the toluene.  Transfer the OMSO
         solution to a separatory funnel containing 10 ml Isooctane,
         rinse the evaporating flask with 5 ml DMSO, and add it to the
         separatory funnel.  Shake the mixture and drain the DMSO
         layer into a second separatory funnel containing 40 ml water
         and 20 ml Isooctane.  Add 10 ml DMSO to the first separatory
         funnel, shake the mixture, and drain the DMSO into the second
         separatory funnel.  Shake the second separatory funnel and
         transfer the DMSO/water phase into a third separatory funnel
         containing 20 ml isooctane.  Shake the third funnel, then
         discard the DMSO/water layer.  Combine the Isooctane layers
         from the second and tnird separatory  funnels and wash twics
         with 40 ml water.

         Reduce  the volume of  the Isooctane solution to approximately
         5 ml.   Add 10 ml ethanol/toluene (10/90) and evaporate the
         mixture to approximately 1 ml.  It 1s not necessary to dry the
         Isooctane prior to evaporation, as residual water  from the
         solvent is removed as an ethanol/toluene/water azeotrope
         during  evaporation.   Use a K-D concentrator 1f losses of the
         more  volatile ?AH  compounds  are of  concern.


                               II1-396

-------
3.1.5.4  Sephadex LH-20 Chromatography (Optional)

         The elution volumes of the Sephadex column should be checked,
         using a standard solution, before use and periodically
         thereafter.  The following procedure is written for elution of
         the PAHs between 18 ml and 36 ml.  Appropriate changes should
         be made if the initial elution volumes differ, or if they
         change due to settling of adsorbent.

         Transfer the toluene extract from the DMSO partitioning into
         a graduated 15-ml tube.  Rinse the flask several  times with
         toluene and add the rinses to the extract.  Reduce the volume
        .of the extract to approximately 0.5 ml under a stream of dry
         nitrogen with gentle wanning.  Add an equal  volume of ethanol
         and apply the sample to a column of Sephadex LH-20 (bed size
         10 mm x 200 mm) packed in toluene/ethanol (1/1).   Draw the
         material onto the column bed using gentle suction and elute
         the column with 18 ml toluene/ethanol (1/1).  Discard the first
         18 ml of eluate.  The PAH fraction is recovered from the
         column by elution with a second 18 ml of solvent.  (The column
         can be regenerated for use by washing with 36 ml  of solvent.)
         Reduce the volume of the PAH fraction to approximately 1 ml.

3.1.5.6  Hign-rerfurmancs u.'cjuid Chrcmatcgr.iiDhy

         Transfer tne purified sample "rom t~e ?MSO partitioning step
         or the Sephadex column Chromatography step into a 15-mf
         concentrator tube.  Add 100 ul  DMSO and evaporate the
         toiuene.  Store the purif^d and torcsntrated samples in the
         dark in cone vials with Teflon-1^nea screw caos cr Tsrion
         septa.

         Inject 2- to 10-ul aliquots of the ourified and concentrated
         extracts onto the column.  Figure 9 shows a chromatogram of
         a PAH standard and Figure 10 shows sample chromatograms.
         Table 7 gives appropriate operating conditions.  Table 6
         lists several PAHs and their fluorescence characteristics.

         Other chromatographic conditions which have been  used can be
         found 1n other Subsections under High-Performance Liquid
         Chromatography.

         Chromatographic peaks can be Identified by retention times,
         co-chromatography of reference compounds, and by  the response
         ratio of ultraviolet absorbance and fluorescence  detectors.
         If the analytical system is equipped with a stop-flow mecha-
         nism, qualitative identification of peaks can be  supplemented
         by a fluorescence spectrum.  Alternatively,  fractions of the
         HPLC effluent can be collected and analyzed by fluorescence
         ana/or ultraviolet ipectroscooy.4  Thin-layer chromatoqraphy
         (TLC) (see Subsection K) can also be used for confirmation  of
                               III-397

-------
     uv
                            10
                             Retention Time, minutes

                            Peak Identification is as Follows:
             1. Phenanthrene
             2. Anthracene
             3. Fluoranthene
             4. Pyrene
             5. Triphenyiene
             6. Benzo [a] anthracene
             7. Chrysene
             8. Benzo [e] pyrene
             9. Benzo fjl fluoranthene
            10. Perytene
11.  Benzo [b] fluoranthene
12.  Dibenz [a.c] anthracene (shoulder)
13.  Benzo [k] fluoranthene
14.  Benzo [a] pyrene
15.  Dibenz Ja.h] anthracene
16.  Benzo [gh,i] perylene
17.  Indeno [1.2.3-cd] pyrene
18.  Benzo [b] chrysene
19.  Coronene
20.  Dibenz [ai] pyrene
Figure 3.   Chromatogram  of polycycllc Aromatic hydrocarbon -?ference comoounds.
    Chromatography  conditions are described in the text (from reference 22).
                                   III-398

-------
                                                                 20
    UV
I
0
 T
10
 I
20
                        Retention Time, minutes
                        Peak Identification Is as Follows:
         1. Phenanthrene
         2. Anthracene
         3. Fluoranthene
         4. Pyrene
         5. Triphenylene
         6. Benzo [a] anthracene
         7. Chrysene
         8. Benzo [e] pyrene
         9. Benzo [jj fluoranthene
        10. Perylene
            11. Benzo [b] fluoranthene
            12. Dibenz [a.c] anthracene (shoulder)
            13. Benzo [k] fluoranthene
            14. Senzp (a] pyrene
            15. Dibenz [a,h] anthracene
            16. Benzo [gh.i] perylene
            17. Indeno I1.2,3-cd] pyrene
            18. Benzo [b] chrysene
            19. Coronene
            20. Dibenz [ai] pyrene
   c*nure TO.  Chromatoqram of sediment extract  (from  reference 22).
                                 III-399

-------
                           TABLE 7.   HPLC CONDITIONS
Mobile Phase:
                       A:  Water
                       B:  Acetonitrile
               Flow rate:  0.5 ml/min
               Gradient Program:
                                  1.  40% A 60% B for 6 min
                                  2.  60% B - 99% B in 13 min
                                  3.  99% B
Detectors:
               UV Absorbance Detector:  296 nm
                Fluorometric Detector:
                                  max .   340 . 38Q nm
                                  PX
                                         (Corning 7-54, 7-60 filters in series)
                                              nm
                                         'Corning 3-73, 4-76 filters in series;
              :=================================================================
              used for confirmation of identity, as can gas cnromatography/
              mass spectrometry (GC/MS) (see Section 3).3,16  The Sephadex
              LH-20 column chromatography cleanup should be used only if thr
              extract does not give satisfactory riPLC response after the
              Florisil and DMSO-partitioning cleanup steps.

     3.1.5.7  Determination of B[a]P Recovery

              Radio-labeled B[a]P is used to measure method recovery.22
              An aliquot of the DMSO solution injected into the HPLC is
              analyzed for SH- or l^c-iabeled B[a]P.  The percentage
              recovery for B[a]P is determined by comparing the recovered
              radioactivity to the amount originally added to the sample.

     3.1.5.7.1  Counting Procedure

                Radioactivity, is determined on a 50 nl aliquot of the
                purified sample in DMSO.  The DMSO solution is added to
                10 ml scintillation fluid, and radioactivity determined
                in a  scintillation counter, using either 3H or 14C settings
                as appropriate.


                                    111-400

-------
Sample counts are corrected for scintillation quenching
through the use of appropriate channel  ratio or external
standard techniques.
                    III-401

-------
4.   Analysis of Biological  Tissue Samples for Polycycllc Aromatic Hydrocarbons

     4.1  Analysis of F1sh and Shellfish Tissue for PAH
               Analytical  Procedure:   available
               Sample Preparation:  evaluated

     4.1.1  Reference

            Dunn, B. P. and R. J.  Armour, "Sample Extraction and Purification
            for Determination of Polycyclic Aromatic Hydrocarbons by Reversed-
            Phase Chromatography."  Analytical Chemistry, 52:13 (1980)
            pp. 2027-2031.22

     4.1.2  Method Summary

            A 20- to 100-g sample of tissue is digested in alcoholic potassium
            hydroxide, the resulting solution is extracted with isooctane,  and
            the Isooctane  extract 1s concentrated by evaporation.  The  concen-
            trated extract is cleaned up by Florisil column Chromatography  and
            dimethyl sulfoxide partitioning.  An optional Sephadex LH-20
            column chromatographic cleanup procedure is provided.  The  purified
            and concentrated extract is analyzed by high-performance liquid
            Chromatography using both UY absorbance and fluorescence detection.
            A radioiaoeied oenzoiajpyrene internal  standard it  used ;a  correct
            for Bosses incurred during sample ^reparation.

     4.1.3  Applicability

            This .iietnoa nas seen demonstrated :o :s uppi  "cadi?  ;o ;ne*'~. ?*ih
            (mussels, clams, oysters) and fish.13

     4.1.4  Precision and  Accuracy

            The overall recovery of B[a]P is 60 to 80 percent,  while recovery
            from the digestion step and subsequent extraction are essentially
            quantitative.17  Recoveries from the Florisll column cleanup are
            over 90 percent.17  The precision obtained 1s 6 percent.I7

     4.1.5  Sample Preparation

            Place 20 to 100 g of tissue in a 300-ml round-oottom rlask  and
            add 150 ml ethanol, 7 g KOH, two or three boiling chips, and an
            aliquot of radioactive benzo[a]pyrene (1,000 dpm 14C B[a]P,
            ca. 5 ng; or 25,000 dpm ^H B[a]P, ca. 0.1 ng; or 25,000 dpm of
            another radiolabeled PAH of interest).   Reflux the  mixture
            gently for 1.5 hours and add the digest, while still  hot, to
            approximately  150 ml water in a 1-Hter separatory  funnel.   The
            amount of water used should be adjusted to yield a  final mixture
            that is 50 to  55% water.  Rinse the flask with 50 ml  of ethanol
            and add :o :he separatory  "unne!.  "xtract :he nixture three
            times with 200 ml isooctane, combine the extracts and wash  them
            four times witn 200-iTii portions of *arm (60C) *aicer.

                                    TH-402

-------
4.1.6  FTorisi!-Column Cleanup

4.1.6.1  Concentration

         Reduce the volume of the isooctane from the extraction to
         approximately 10 ml.  Use a Kuderna-Danish evaporative
         concentrator if losses of the more volatile PAHs are of
         concern.  If not, a rotary evaporator is satisfactory for
         this step.  After volume reduction, dilute the extract
         to 100 ml  with toluene.

4.1.6.2  Column Chromatography

         Prepare a column of 30 g Florisil  (deactivated with 5 percent
         water) covered with 60 g anhydrous sodium sulfate in a glass
         chromatographic column {40 mm x 400 mm).  Add 100 ml toluene
         to the column and elute  the toluene until  the liquid level  is
         just at the top of the sodium sulfate layer.   Apply the sample
         (in toluene) and elute the column  until  the liquid level  is at
         the top of the sodium sulfate layer.  Carefully add 100 ml
         toluene, elute the column, add another 100 ml  toluene, and
         elute the column again.

4,1.7  DMSO Partitioning

       Add 5 ml dimethyl sulfoxide (DMSC) to the combined toluene
       eluate from the column and concentrate cne solution jnder
       vacuum (at 60) to remove  the toluene.  Transfer the OMSO
       solution to a separatory funnel  containing 10 ml  isooctane,
       rinse tne evaporating r'lasx ,un 5 .?.! 3MC,  -nd idd it to :he
       separatory funnel.  Shake  the mixture znd drain the DMSO ""ayer
       into a second separatory funnel  containing 40 ml  water and
       20 ml isooctane.  Add 10 ml DMSO to  the first separatory
       funnel, shake the .mixture, and drain the DMSO 'nto the second
       separatory funnel.  Shake  the second separatory funnel, and
       transfer the DMSO/water phase into a third separatory funnel
       containing 20 ml isooctane.  Shake the third funnel, then
       discard the DMSO/water layer.  Combine the isooctane layers
       from the second and third  separatory funnels and wash twice
       with 40 ml water.

       Reduce the volume of the isooctane ioiution  to approximately
       5 ml.  Add 10 ml ethanol/toluene (10/90) and evaporate the
       mixture to approximately 1 ml.  It is not necessary to dry the
       isooctane prior to evaporation,  as residual  water from the
       solvent is removed as an ethanol/to!uene/water azeotrope
       during evaporation.  Use a K-D concentrator  if  losses of the
       more volatile PAH compounds are  of concern.
                               111-403

-------
4.1.8  Sephadex LH-20 Chromatography (Optional)

       The elution volumes of the Sephadex column should be checked,
       using a standard solution, before use and periodically there-
       after.  The following procedure is written for elutfon of the
       PAHs between 18 ml  and 36 ml.  Appropriate changes should be
       made if the initial elution volumes differ, or if they change
       due to settling of adsorbent.

       Transfer the toluene extract from the DMSO partitioning into a
       graduated 15-ml tube.  Rinse the flask several  times with
       toluene and add the rinses to the extract.  Reduce the volume of
       the extract to approximately 0.5 ml under a stream of dry nitrogen
       with gentle warming.  Add an equal volume of ethanol and apply
       the sample to a column of Sephadex LH-20 (bed size 10 mm x 200
       mm) packed in toluene/ethanol (1/1).  Draw the material onto
       the column bed using gentle suction and elute the column (by
       gentle suction) with 18 ml toluene/ethanol (1/1).  Discard
       the first 18 ml of eluate.  The PAH fraction is recovered from
       the column by elution with a second 18 ml of solvent.  (The
       column can be regenerated for use by washing with 36 ml of
       solvent.)  Reduce the volume of the PAH fraction to approximately
       1 ml.

4.1.9  High-Performance liquid Chromatograpny

       Transfer the purified sample from the DMSO partitioning step
       or the Senhadex column chromatoqraohv step into a 15-ml
       concentrator tube.   Aaa 100 yi  DMSO ana evaporate the
       toluene.  Store the purified ana concentrated samples in the
       dark in cone vials with Teflon-lined screw caps or Teflon
       septa.

       Inject 2- to 10-yl  aliquots of the purified and concentrated
       extracts onto the column.  Figure 9, page 111-398, shows a
       chromatogram of PAH standards, Figure 11 shows sample chromato-
       grams, and Table 7 gives appropriate operating conditions.
       Table 6 lists several PAH compounds and their fluorescence
       characteristics.

       Chromatographic peaks can be identified by retention times, co-
       chromatography of reference compounds, and by the ratio of the
       responses of ultraviolet absorbance and fluorescence detectors.
       If the analytical system is so equipped, qualitative identifi-
       cation of peaks can be supplemented by a fluorescence spectrum.
       Alternatively, fractions of the HPLC effluent can be collected
       and analyzed by fluorescence spectroscopy for confirmation of
       identity.4  Thin-layer chromatography (TLC) (see Subsection K)
       can also be used for confirmation of identity.17  The Seohadex
       LH-20 column cnromatograpny cleanup snouid be used only if the
       extract  Hoes  lot jive satisfactory MPLC "SDonse if tar      '
       and DMSO cleanups.

-------
                                                         19
                                                                 20
J
0
                        1
                       10
 I
20
                        Retention Time,  minutes
                        Peak Identification is as Follows:
         1. Phenanthrene
         2. Anthracene
         3. Fiuoranthene
         4. Pyrene
         5. Triphenylene
         6. Benzo [a] anthracene
         7. Cnrysene
         8. Benzo [e] pyrene
         9. Benzo fj] fiuoranthene
        10. Perylene
                                   11. Benzo [b] fiuoranthene
                                   12. Dibenz [a,c] anthracene (shoulder)
                                   13. Benzo [k] fiuoranthene
                                   14. Benzo {aj pyrene
                                   15. Dibenz [a.h] anthracene
                                   16. Benzo [gh,i] perylene
                                   17. Indeno [1,2,3-cd] pyrene
                                   18. Benzo [b] chrysene
                                   19. Coronene
                                   20. Dibenz [ai] pyrene
figure ii.   Chromatograph of
                                 :smoounds ^n nussels (from reference  18).

-------
4.1.10  Determination of B[a]P Recovery

        Radio-labeled B[a]P is used to measure method recovery.22
        An aliquot of the DMSO solution injected into the HPLC is
        analyzed for SH- or l^C-labeled B[a]P.  The percentage
        recovery for B[a]P is determined by comparing the recovered
        radioactivity to the amount originally added to the sample.

4.1.10.1  Counting Procedure

          Radioactivity is determined in a 50 yl aliquot of the
          purified sample in DMSO.   The DMSO solution is added to
          10 ml  scintillation fluid, and the radioactivity determined
          in a scintillation counter, using either SH or i^c settings
          as appropriate.  Sample counts are corrected for scintillation
          quenching through the use of appropriate channel  ratio or
          external standard techniques.
                               111-406

-------
4.2  Analysis of Plant Tissues for PAH
          Analytical Procedure:  available
          Sample Preparation:  available

     4.2.1  Reference

            Joe, F. L., Jr., J. Salemme and T. Fazio,  "High Performance
            Liquid Chromatography with Fluorescence and Ultraviolet
            Detection of Polynuclear Aromatic Hydrocarbons in Barley Malt."
            J. Assoc. Off. Anal. Chem., 65:1395-1402.   198221.

     4.2.2  Method Summary

            A 100-gram sample of vegetable tissue is ultrasonically extrac-
            ted in cyclohexane, the extract is purified by water-deactivated
            silica/alumina Chromatography and dimethyl  sulfoxide partition-
            ing, and is concentrated and solvent-exchanged into aqueous
            acetonitrile/methanol.  The resulting solution is analyzed by
            reversed-phase high-performance liquid Chromatography.

     4.2.3  Applicability

            This method is applicable to the determination of PAH com-
            pounds  in barley malt tissues and :hou!d be amenable to 
-------
           TABLE 8.  FLUORESCENCE RECOVERY (PERCENT) OF PAH
                 COMPOUNDS FROM BARLEY MALT SAMPLES"
                            2.5 ppb Spike            5.0 ppb Spike
PAH
Fluorene
Pyrene
Benzo[a]anthracene
Benzo[b]f 1 uoranthene
Benzo[e]pyrene
Benzo[a]pyrene
D^benzo[ah]anthracene
Indeno[l,2,3,cd]pyrene
Benza[ghi]perylene
DibenzoCdi ]pyrene
Coronene
=======================
Recovery3
86
81
78
85
86
83
86
85
91
79
83
===============
RSD&
2.3
1.4
4.4
7.5
8.4
6.6
6.7
10.6
8.0
7.3
9.7
Recovery*
79
82
82
88
97
85
87
87
85
39
87
:==============:
RSDb
7.7
6.5
6.0
6.0
12.0
7.2
7.0
8.1
5.9
2.6
3.5
2 Average of cri'piicate aetsrminations; cxcvcai:on ^c -3
t> Relative standard deviation, percent
         (deactivated with 15 percsnt water), 5 g alumina (deacti-
         vated with 10 percent water), and 10 g anhydrous sodium
         sulfate, 1n that order, to a 250 mm by 14.5-mm column con-
         taining a glass wool plug.  Tap gently with each addition.
         Fit each column with a 250-ml solvent reservoir.

         Wash the column with 50 ml cyclohexane, stopping the
         flow when the liquid level just reaches the top of the
         sodium sulfate layer.  Test the columns as indicated in
         paragraph 4.2.6.2 before use.

4.2.6.2  Column Testing

         Prepare two columns as described above (one for a blank and
         one for recovery measurement).  Add 75 ml  cyclohexane to one
         reservoir and 75 ml cyclohexane solked with 6.25 x 10~2 yg
         each of phenanthrene (Phen), fluoranthene  (F), pyrene (Py),
         benzoiajanthracene  i'8[ajA), benzoCbjfluoranthene '3[b]F),
         benzo[e]pyrene (B[e]P), benzoCalpyrene (B[a]P), dibenzoCah]-
         anthracene iDBLanjA), inaenou,
-------
         and coronene (Cor) to the other reservoir.  Let the solvent
         percolate through the blank and spike columns.  Collect the
         two eluates in separate 500-ml  flasks.  Repeat the column
         elution with three more 75-ml  portions of cyclohexane,
         letting each drain just to the top of the sodium sulfate
         before adding the next.

         Evaporate the combined eluates to approximately 5 ml, using
         a flash evaporator with a 40C water bath.  Transfer the
         concentrates quantitatively to 15-ml concentrator tubes with
         disposable Pasteur pipets and  rinse each flask with three 1-
         ml portions of cyclohexane.  Evaporate the solutions to dry-
         ness at 30C under a gentle stream of purified nitrogen.
         Add 0.25 ml of a 0.25 vg/ml benzo[b]chrysene solution and
         subject to ultrasonic vibration for approximately 3 minutes.
         Analyze by HPLC.  Solvents (blank) must be free of interfer-
         ences and recovery for each PAH must be 90 percent or better.

4.2.6.3  Sample Chromatography

         After the column has been demonstrated to work properly,
         transfer the extract to the silica gel/-alumina column, rinse
         the Erlenmeyer flask twice with 5-ml portions of cyclohexane,
         etna add cr.em ;o ;he column.  !"!'.:: 3  :s "i par~qr3ch 4.2.6.2,
         collecting the eluate "'n a 500-ml *lask.  Evaoorate the com-
         bined eiuates to approximately 25 .711, jsing : ->TC water
         bath.

4.2.6.4  DMSO Partitioning

         Quantitatively transfer the concentrated eluate to a 125-ml
         separatory funnel  containing 15 ml dimethyl  sulfoxide (DMSO)
         which has been pre-equilibrated with cyclohexane.  Rinse the
         flask with 25 ml cyclohexane and add the rinse to the separa-
         tory funnel.  Shake vigorously for 2 minutes, let the layers
         separate, and drain the bottom (DMSO) layer into a 500-ml
         separatory funnel  containing 90 ml deionized water and 15 ml
         cyclohexane.  Repeat the extraction of the eluate with two
         additional 15-ml portions of DMSO and combine the DMSO
         extracts in the 500-ml separatory funnel.  Shake vigorously
         and let the layers separate.  Draw off k,he lower, aqueous
         phase into a second 500-ml separatory funnel containing 15
         ml cyclohexane.  Repeat the extraction, discard the aqueous
         phase, and add the organic phase to the organic phase remain-
         ing in the first 500-ml separatory funnel.  Rinse the second
         funnel with two 10-ml portions of cyclohexane and transfer
         each to the first funnel.  Wash the combined extracts with
         100 ml deionized water, shaking for 1 minute.   Let the
         phases separate and discard the aqueous phase.
                          111-409

-------
4.2.6.5  Extract Drying and Concentration

         Add 50 g anhydrous sodium sulfate to a 60-ml  Biichner funnel
         with a coarse fritted disc.  Rinse the apparatus with 50 ml
         cyclohexane, allowing the liquid to drain by gravity.

         Discard the rinse.  Filter the combined extracts from para-
         graph 4.2.6.4 into a 300-ml flask.  Rinse the separatory
         funnel with two 10 ml portions of cyclohexane and pour each
         through the Biichner funnel.  Concentrate the dried extracts
         and washes to 3 to 5 ml, using a 40C water bath.  Transfer
         the concentrate to a 15-ml  concentrator tube with a dispos-
         able Pasteur pipet.  Rinse the flask with three 1-ml por-
         tions of cyclohexane and transfer each rinse to the concen-
         trator tube.  Place the tube in a 30C water bath and
         evaporate to dryness under a gentle stream of purified
         nitrogen.  Add 0.25 ml of a solution of 0.25 vg/rol benzo-
         [b]chrysene (B[b]Ch) in a 80/20 mixture of acetonitrile:
         methanol (l:l)/water and subject to ultrasonic vibration for
         3 minutes.  Filter if necessary, and analyze the resulting
         solution by HPLC.

4.2.7  High-Performance Liquid Chromatography

       Inject 20 yl extract or standards onto the octadecylsilanc:
       "31 u.Tin, 'jsing the ~.CM vent troqram Tiven in Table 9.  Figure 12
       and Figure 13 show sample chromatograms obtained under these
       conditions.  Alternative chromatographic conditions can be
       found in other Subsections unde" Hiqh-Performance Liauid
       Chromatoqraohy.  Between inject".ons, fiusn tne sample ioop and
       port passages with approximately o mi acetorntrile/ methanol
       (1/1) to prevent cross-contamination.  Compare retention
       volumes of peaks observed with those of standards.  Inject
       standards after each third sample.  Calculate the concentra-
       tion of PAH compounds using the internal standard (B[b]Ch)
       procedure.
                          111-410

-------
                           TABLE 9.   HPLC CONDITIONS
333=333=333333333333=3=3333=33=33333=333333333333333333333333===3=33333=3333==3
Mobile Phase:
                       A:  Water
                       B:  Methanol/acentonitrile (1 + 1)
               Flow rate:  1 ml/min
               Gradient Program:
                                  1.    80% B  - 100%  B  in 20 min
                                  2.   100% B  for 20  min
                                  3.   100% B  - 80% B in 5 min
                                  4.    80% B  for 20  min
Detectors:
               UV Absorbance Detector:  289 nm
                Fluorometric Detector:  333

                                 xmax :  340  - 380 nm
                                         (Corning 7-54, 7-60 filters  in series)

                                 xmax :  >400 nm
                                         (Corning 3-73, 4-76 filters  in series)
83333333333333333==33S3SS=33333333a333333333=33S3=3==33SS=======a=33======3===3
                                    Ill-All

-------
                Fluonsctnci
                  UV
                     (f
                       I
                        .0
	1	
   :o
Tim*, minutvs
T~
 30
                  PeaK identJTication is as roiiows:

                     1. Phenanthrene
                     2. Fiuoranthene
                     3. Pyrene
                     4. Benzo [a] anthracene
                     5. Benzo [b] fluoranthene
                     6. Benzo [e] pyrene
                     7. Benzo [a] pyrene
                     8. Dibenz [ah] anthracene
                     9. Senzo [bj chrysene
                    10. Indeno [1,2,3-cd] pyrene
                    11. Benzo [ghi] perylene
                    12. Oibenz [ai] pyrene
                    13. Corenone
Figure 12.   HPLC  chromatogram of unfortified barley malt  sample.
           Top:   *1uorescence - 333 nm.  <-anqe - 0.1 uA.,
           Bottom:  UV - 289 nm, attenuation 0.02 AUFS.
                      (from -sfer^ncs 11}
                            III-412

-------
 1. Phenanthene
 2. Fluorene
 3. Pyrene
 4. Benzo [a] anthracene
 5. Benzo {e] fluoranthene
 6. Benzo [e] pyrene
 7. Benzo [a] pyrene
 8. Dibenzo [b] anthracene
 9. Benzo (bj chrysene
10. Indeno [1.2.3-cd] pyrene
11. Benzo [g.h.i] perylene
12. Dibenzo [a.i] pyrene
13. Coronene
2
i
     4
   JL
           78,9
                         i
                       10
        20
30
                    Retention Time, minutes
      Figure 13.  HPLC chromatogram of 5 ng each of 13 PAH compounds.
                      Top:  fluorescence - 133 *wu
               Bottom:  UV - 289 nm, attenuation 0.02 AUFS.
                               reference 1}
                                III-413

-------
5.1  Analysis of Air for Polycyclic Aromatic Hydrocarbons
          Analytical  Procedure:   available
          Sample Preparation:   available

     5.1.1  Reference

            Method 21516, "Methods Manual  for Chemical  Analysis of Atmospheric
            Pollutants."  Environment Canada, Alberta Environmental  Centre,
            Vegreville, Alberta, Canada, 1981.28

     5.1.2  Method Summary

            Airborne particulate matter is collected on a glass-fiber filter,  the
            sample is extracted with cyclohexane,  the solvent is flash-evaporated,
            and the residue is dissolved in methanol and analyzed by HPLC with
            a fluorescence detector.

     5.1.3  Applicability

            This method is applicable to the determination of polycyclic aromatic
            hydrocarbons in airborne particulate matter.

     5.1.4  Precision and Accuracy

                           SINGLE LABORATORY PRECISION AND RECOVERY"
                                        ('Senzof a jpyrene j
            B[a]P Amount (ng)      Coefficient of. Variation (4)      Recovery (%)
            ______.^_^_______-        

                    5                                                 103
                   10                        20.4
                  112                         5.51
                  142                                                  32   -
                  334                                                 100
                  338                         3.16
            ssss==ss=z==s=sss=sz=================================================

     5.1.5  Sample Preparation

     S.I.5.2  Sampling

              Condition a glass-fiber filter overnight at  room temperature
              and humidity or dry in a desiccator for 25 hours.  Weigh the
              filter and record the weight.  Install the filter in a high-
              volume sampler equipped with a chart recorder.

              Operate the high-volume sampler for 24 hours, with a flow
              chart recording the flow.  Weigh  the filter  disc and record
              the final weiqht in qrams.  Divide the filter disc into 5
              equal parts and process eacn according co  cne procedure yiven
              below.

                                    III-414

-------
     5.1.5.2  Extraction
              Extract each portion of the filter disc for 6 hours in a
              Soxhlet apparatus using 80 to 85 ml  pesticide grade cyclo-
              hexane.

     5.1.5.2  Concentration

              Cool the flask and remove the solvent in a flash evaporator
              at 40C.  Dissolve the residue in four 1-ml aliquots of
              methanol, transfering each into a centrifuge tube.   Centrifuge
              the methanolic solution and concentrate to 2 ml  by  blowing
              dry nitrogen over the sample.  Transfer the clear solution
              with a Pasteur pipet into a vial with a Teflon-lined septum
              cap and seal tightly.

     5.1.6  High-Performance Liquid Chromatography

            Analyze samples by HPLC using 10-ul injections and the instrument
            conditions given in Table 10.  A suggested order of sample pro-
            cessing for quality control purposes is as follows:

                 Blank
                 i szanaarcis
                 Blank
                 o sampies
                 Blank
                 2 standards
                           TABLE 10.  HPLC CONDITIONS33
2SSSSSBS5S-SSB=aSB=SBSSSB==BB3=SBaS==SSSS=SBBSSS===SBBBSS=3SSBBSBSSBSSBB=SS==SSB

Mobile Phase;
                       A:  Acetonitrile
                       B:  Water
               Flow rate:  1.0 ml/min

               Solvent program:  Isocratic;
                     A:B:  75:25 t'Y:V)
Detectors:
               UV Absorbance Detector:  289 nm
                Fluorometric Detector:  333 nm

                                 1max :  310 nm
                                  ex
                             '    xmax :  390 nm
                                 Aem
BSSSS8SSSSSSB8SSSSSSSSBSBSSBSSSSaSB=SSSBS8SBBSSSBBBSSSBSSSBSSSSSBBSSBSBS=SS==a=
                                    III-415

-------
            Sample chromatograms of standards and a sample are shown 1n Figures
            14 and 15.

            NOTE:  Routine quality control must be followed to ensure the pre-
            cision and accuracy of the method.   With every series of ten extrac-
            tions, one sample should be analyzed 1n duplicate with Its mate
            being spiked with 100 ng of B[a]P.   This will  allow the technician
            to detect any changes occurring within the total  analysis.  Also a
            blank, 2 standards, and then another blank should be run at a
            minimal interval  of 5 samples to ensure reproducibility of standard
            areas.  If these  practices are followed, the method should fall
            easily within the tolerances quoted for accuracy and precision.

J.   QUALITATIVE IDENTIFICATION

     1.0  Fluorescence Spectra

          If the HPLC system  is so equipped, a fluorescence scan can be made of
          a given peak.  Alternatively, fractions of the HPLC effluent can be
          collected and analyzed by fluorescence spectroscopy.4  Table 6 gives
          fluorescence data for a large number of PAH compounds in n-heptane
          solution.  Figure 4 shows emission spectra obtained of several peaks
          in an HPLC PAH analysis.  If both uv and fluorescence detectors are
          used i-n series, the ratio of <:he two resoonses at various wavelengths
          can be useful in ascertaining the identity of a pea*,  when oenzotdj-
          pyrene (B[a]P) and  benzol'k jfuorantnene . 8[k]F)  coelute., use has been
          made of excitation  at 307 nm and measurement of fluorescence inten-
          sity at 406 nm, to  give the 8[k]F concentration, so that the contri-
          bution of B[k"lF to  the fluorescence Intensity at 406 nm from excita-
          tion at 384 nm is known.;~  I; r.as seen round tnaz cne concentrations
          of compounds m air samples wnlcn interfere witn use-of this tech-
          nique are typically not high enough to interfere seriously.33

     2.0  Gas Chromatography/Mass Spectrometry

          Confirmation of the identity of PAHs may be accomplished by the use
          of gas chromatography/mass spectrometry (GC/MS).29  See Section 3,
          Subsection J.3 of this Chapter for protocol.

     3.0  Thin-Layer Chromatography

          Confirmation of the identity of individual PAH compounds may be
          performed by thin-layer Chromatography (TLC).3^  Samples are prepared
          as 1n the HPLC procedures, the extract reduced in volume to approxi-
          mately 0.1 ml, and  the concentrate applied to a cellulose-acetate
          thin-layer plate.

          Standard solutions  are applied next to the sample spot, and the plate
          is developed with ethanol/to!uene/water (17/4/4).34  The PAH bands are
          visualized with long-wave ultraviolet light.  Caution must be exer-
          cised to avoid unnecessary exposure or tne PAH rract'ons co ' ight,
                ^as been -eoortsd to decomoose them.5

                                    III-416

-------
               1. Benzo [e] pyrene
               2. Benzo [k] fluoranthene
               3. Benzo [a] pyrene
               4. Impurity from benzo [e] pyrene
Figure H.  Sample chromatogram of  PAH standards ^from reference

                            U 1-41.7

-------
                       1. Unknown
                       2. Benzo [k] fluoranthene
                       3. Benzo [a] pyrene
                       4. Unknown
figure 15.  Sample cnromatcgram jf an air sample I from reference

                            III-418

-------
          The adsorbent at a band of interest can be scraped off and the PAH
          desorbed with hot (65C) methanol  (4 x 4 ml).   The methanol  is added
          to 10 ml of 20-percent hexadecane  in isooctane, and the methanol  and
          fsooctane removed by rotary evaporation.
          The PAH concentration in hexadecane is measured fluorimetrically
          using the baseline technique.34 Samples and standards are excited  at
          an appropriate wavelength (see Table 6) and the emission spectrum
          recorded over an appropriate wavelength range.^
K.   CALCULATIONS
1.   Internal Standard Approach
     Calculate the response factor (RF) using Equation 1  (Subsection G.2.2),
     and calculate the concentration in the  sample from:
                   Concentration  =  (AS)(I1-S)/(A1-S)(RF)(M0)             Eq. 2
     where:
           \r   =  Response for the parameter to be measured
           ITS  =  Amount of internal standard added to each extract
           A-fS  =  Response for the internal  standard
           RF   =  Response Factor (see CuDsect:'cn 3.2.2;
           M0   -  Initial sample size (mass  or volume).
2.   External Standard Approach
     Obtain a calibration curve or calibration factor according to Subsection
     G.2.2,  and calculate the concentration  of the sample by interpolation
     (using a calibration curve) or by use of Equation 3:
                       Concentration  =  (As)(Vf)/(F)(M0)                Eq. 3
     where:
           As  =  Response of the sample
           Vf  =  Final extract volume
           F   *  Calibration factor; (response)/(concentration)
           M0  =  Initial sample size (mass  or volume).
3.   Recovery Corrections 'Subsections J.3, J.4.2)

-------
For analyses where 3H- or l^C-labeled B[a]P  are  used  to  indicate losses,
fractions of the HPLC effluent should be  collected which correspond to
the peak identified as B[a]P.   These  fractions should then be assayed for
3H or 14C, as appropriate.
Recovery of B[a]P is calculated by:
                       Recovery (R)   =   (CS)/C                       Eq. 4

where:
      C     Radioactivity initially added to  sample
      Cs  =  Radioactivity recovered in  sample  extract
Use of R can then be made to correct results  (see  above):
               Corrected Results  =   (Concentration)/R               Eq. 5
where:
      Concentration  =  Results of 1 or  2  (above)
                  R  =  Recovery (Eq. 4)
                               III-420

-------
                                   REFERENCES


 1.    Bar-tie,  K.  D.; M. L. Lee; and S. A. Wise.  "Modern Analytical Methods
      for Environmental Polycyclic Aromatic Compounds." Chem Soc. Rev.
      10:113-158.   (1981)

 2.    Jurgensen A.,  E. L.  Inman, Jr., and J. D.  Winefordner.  "Comprehensive
      Analytical  Figures of Merit for Fluorimetry of Polynuclear Aromatic
      Hydrocarbons."  Anal. Chim. Acta, 1^:187-194.  (1981)

 3.    Sauter,  A.  D., L. D. Betowski, T. R. Smith; V. A. Strickler; R. G. Beimer;
      B.  N.  Colby,  and J.  E.  Wilkinson.  "Fused Silica Capillary Column GC/MS
      for the  Analysis of  Priority Pollutants."  HRC CC, 4:366-384.  (1981)

 4.    Fox, M.  A., and S. W. Staley.  "Determination of Polycyclic Aromatic
      Hydrocarbons  in Atmospheric Particulate Matter by High Pressure Liquid
      Chromatography Coupled  with Fluorescence Techniques." Anal. Chem., 48:
      992-998.  (1976)

 5.    American Public Health  Association.  "Methods of Air Sampling and Anal-
      ysis." 2nd  ed.  M. Katz, Ed.  Washington, D. C.  984 p'.  (1977)

 6.    U.S. Environmental Protection Agency.  "Polynuclear Aromatic Hydrocarbons
      - Method 610."  Methods of Organic Chemical Analysis of Municipal and
      Industrial  Wastewater.  J. lonabottom and J. licntenburg, eds.
      EPA-600/4-82-057, (1982).

 7,    American Society for Testing and Materials.  "Standard Practice for
      Sampling Water.''  ASTM  Book of Standards, Section il, Vol. 11.01, D337C.
      Philadelphia,  Pennsylvania.  (1983)

 8.    Dunn,  B.  P.   "Handbook  of Polycyclic Aromatic Hydrocarbons in Sediments
      and Marine  Organisms."  Ch. 10, Handbook of Polyaromatic Hydrocarbons.
      A  Bjrfrsten, ed.  Marcel-Dekker, New York, p. 443. (1983)

 9.    Kawakami  and  Nishimura, J. Oceanogr. Soc. Japan, 3_2_:175 (1976).

10.    Prahl, F. G.  and R.  Carpenter.  "The Role of Zooplankton  Fecal Pellets
      in  the Sedimentation of Polycyclic Aromatic Hydrocarbons 1n Dacob Bay,
      Washington."   Geochim Cosmochim Acta, 3_: 1959-1972.  (1979)

11.    Bjorseth, A.,  J. Knutzen, and J. Skei.  "Determination of Polycyclic
      Aromatic Hydrocarbons in Sediments and Mussels from Saudafjord, W.
      Norway,  By  Glass Capillary Gas Chromatography."  Sci. Total Environ.
      1^:71-86.  (1979)

12.    Bieri, R. H.  M. K. Cuenan, C. L. Smith, and C. W. Su.  "Polynuclear
      Aromatic and  Polycyclic Aliphatic Hydrocarbons in Sediments from the
      Atlantic Outer Contental Shelf."  Int. J. Environ. Anal. Chem. 5:293-310.
      (1978)
                                     III-421

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13.  Gelger, W., and C. Schaffner.   "Determination  of  Polycyclic Aromatic
     Hydrocarbons in the environment by  Glass  Capillary Gas Chromatography."
     Anal. Chem., 50:243-249.

14.  Brown, R. A., and B. K.  Starnes.   "Hydrocarbons in the Water and Sediment
     of a Wilderness Lake.   II."  Marine Pollut. Bull., 9:162-165.   (1978.

15.  Maher, W. A., J. Bagg,  and J.  David Smith.  "Determination of Polycyclic
     Aromatic Hydrocarbons  in  Marine Sediments, Using  Solvent Extraction,
     Thin-Layer Chromatography and  Spectrofluorimetry."   Int. J. Environ.
     Anal. Chem., 7^:1-11.  (1979)

16.  John, E. D., M. Cooke,  and G.  Nickless.   "Polycyclic Aromatic Hydrocarbon
     in Sediments Taken from the Severn  Estuary Drainage  System."  Bull.
     Environ. Contam. Toxicol .  22_:653-659.  (1979)

17.  Dunn, B. P.  "Techniques  for Determination of  Benzo(a)pyrene in Marine
     Organisms and Sediments."  Env.  Sci.  and Tech.  10_: 1080- 1021.  (1976)

18.  Kellner, R.  "Analysis  of Airborne  Particulates by Physical Methods."
     H.  Malissa, ed., CRC  Press, p.  228.   (1978)

19.  Inscoe, M.  Anal. Chem.,  36:2505.   (1964)

20.  American Society for Testing and Materials.  "Standard Practice for
     Preparation of Sample  Containers and  for  Preservation," ASTM Book of
     Standards, Part 31, D3694. Philadelphia, Pennsylvania.  (1980)
21.  Joe, c,  1., ]r,;  J.  Salemne,  and T. ^zlo.   "High-Performance Liauid.
     Chroma tography with  Fluorescence and  Ultraviolet Detection of
     Polynuclear Aromatic Hydrocarbons  in  Barley  Malt." J. Assoc. Off. Anal.
     Chem., 65_: 1394-1402.  (1982)

22.  Dunn, B. P., and  R.  J.  Amour.  "Sample Extraction and Purification
     for Determination of Polycyclic Aromatic Hydrocarbons by Reversed-
     Phase Chromatography."  Anal.  Chem., 52_: 2027-2031.  (1980)

23.  U.S. Environmental Protection Agency.  "Laboratory Use of Toxic Sub-
     stances."  Occupational  Safety and Health Manual, Chap. 8. U.S. EPA
     Washington, D. C. (1979)

24.  American Chemical Society.   " Safety  in Academic Chemistry Laboratories."
     3rd ed., American Chemical  Society, Committee on Chemical Safety,
     Washington, D. C.  (1979)

25.  Occupational Safety  and Helath Administration.  "OSHA Safety and Health
     Standards,  General Industry." 29CFR-1910, OSHA 2206 (Revised
     January, 1976).  OSHA,  Washington, D.  C.  (1976).
                                    II1-422

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

27.  Tomkins, B. A., R. R. Reagan, J. E. Caton, and W. H. Griest.  "Liquid
     Chromatographic Determination of Benzo[a]pyrene in Natural, Synthetic,
     and Refined Crudes." Anal. Chem, 53:1213-1217.  (1981)

28.  Alberta Environmental Centre.  "Methods Manual for Chemical Analysis of
     Atmospheric Pollutants."  Method 21516, Benzo[a]p'yrene.  Alberta
     Environmental Centre, Vagreville, Alberta, Canada.  (1981)

29.  U.S. Environmental Protection Agency.  "Handbook of Analytical Quality
     Control in Water and Wastewater Laboratories."  U.S. EPA, Washington,
     D. C.  EPA-600/4-79-019.  (1979)

30.  Hertz, H. S., J. M. Brown, S. N. Chesler, F. R. Guenther, L. R. Hilpert,
     W. E. May, R. M. Parris, S. A. Wise.  "Determination of Individual Organic
     Compounds in Shale Oil."  Anal  Chem. 52_: 1650-1657.  (1980)

31.  Wise, S. A., S. N. Chesler, H. S. Hertz, L. R. Hilpert, W. E. May,
     'Chemically-bonded /Vrnnosilane Jtat-.cnary Phase -'sr the -Hqn-oer^omance
     Liquid Chromatographic Separation of Polynuclear Aromatic Compounds."
     Ana I.. Chem., 49,  pp. 2306-2310, 1977.

32.  Nielsen, T.  "Determination of Polycyclic Aromatic Hydrocarbons in
     Automobile Exhaust by Means o^ '-''gh-oer^onr-ance Liquid Chromatograohy
     with  Fluorescence Detection." J. Chromatogr.. 170:147-156.  (1979)

33.  Alberta Environmental Centre.  "Methods Manual for Chemical Analysis of
     Atmospheric Pollutants."  Method 21515.  Benzo[a]pyrene and Benzo[k]-
     fluoranthene.  Alberta Environmental Centre, Vagreville, Alberta, Canada.
     (1981)

34.  Kunte, H.  "Carcinogenic Substances in Water and Soil.  XVIII.  Determina-
     tion of Polycyclic Aromatic Hydrocarbons by Mixed Thin-layer Chromato-
     graphy and Fluorescence."  Arch. Hyg. Bakterial., 151:193-201.  (1967)
                                    II1-423

-------
IV.   PROCEDURES FOR INORGANIC SUBSTANCES
                  IV-1

-------
                                   SECTION 10

              ELEMENTAL ANALYSIS BY ATOMIC ABSORPTION SPECTROMETRY


A.   SCOPE

     Atomic absorption (AA) spectrometry provides the analytical  capability to
quantify more than 40 naturally occurring elements.  The analytical  procedure
consists of three steps:  sample preparation to transform each element into a
tractable chemical form, atomization to reduce ions to neutral atomic species,
and spectrometric measurement to quantify the amount of light of specific
wavelengths absorbed by the reduced atoms.

     The procedure can be used to distinguish the elemental  partitioning of
elements between environmentally important phases by modifying *he sample
preparation techniques (i.e., filtration to distinguish between 'soluble" and
"total"  -.oncsntraticns; extraction or ion exchange to distinquish  between
"ionic" ana ''complexea" rorms of the same element).

B.   SAMPLE HANDLING AND STORAGE

     Due to the variable nature of the sample matrix, hazardous waste sampling,
sample handling, :na ^amp1.a storage procedures for these :2t2rm nations ire
quite matrix dependent.  However, storage at reduced temperature 'iC\ in order
to retard chemical and biological activity, should be practiced.

     Grab samples for water analysis should be collected -'n  iorosilicate glass,
linear polyethylene, polypropylene, or Teflon containers.1  The containers
should be pre-washed with detergent and tap water, rinsed with 1:1 nitric acid,
distilled water:  hydrochloric acid, distilled water, and distilled deionized
water, in that order.  Chromic acid may be used to remove organic  matter from
glassware, but should not be used with plastics.  Glassware  must be well rinsed
to remove all traces of chromium after such treatment.  A commercial  product
containing no chromium, NOCHROMIX, is an available substitute for the cleaning
process.  If it can be demonstrated througn use of spiked samples  ana Blanks
that certain steps in the above-mentioned cleaning process can be omitted
without influencing analytical results, such steps may be eliminated.  Figure 1
summarizes sample handling requirements for water, soil/sediment,  tissue and
air samples.

     For the determination of dissolved metals in a water sample,  the sample
must be filtered through a 0.45-u pore size membrane filter  as soon as prac-
tical after collection.  The filtrate should then be acidified with 1:1 nitric
dcia to a pri jeiow L.  .f j precipitate "orns -ipon acidification,  the ^Itrate
should be digested ^s described for a total metals determination.

                                      IV-2

-------

< . Purpose
co 1
Container
Sample Treatment
Preservative

Water Sample
^
iV ^ 4
Soil/Sedimi .it Sample I 1 Tissue
|
* *
Sample
r
| Acidify | | Filter ] | Store Wet | | Dry ] | Freeie | | Sample j
I i
| Sure | | Acidify )
.. i
| Oiyest | | Store | | Oigex
, ' 1 ' V
1 Analyte 1 | Analyie | | Anelyre

T V '
r
I Slo. J 1 Store 1 1 Store 1
,. ~]

J ( Dig,,.. ] [ Oigett J [ Oigett |
1 v J
r
1 1 Anal, <> 1 | Analyie | | Analyre |


T. lal Soluble Tot*l Toi : Total
Water Water Sndinitnt Sedim it Sediment
Concentration Concentration Concentration Concenc<-,.iion Concentration
UP G. P G. i>
None Filter rVo..
HtlO, HNO, <'1,
(|JK2 pM<2
G. < G. P
Air l> ,' Freeze
No, None

Air Sample 1
' r
| Sample ]
^ f
Store J
1
Oiqell 1
, p
Analyte 1

Total
Paniculate
Concentration
Gl.ti
Fiber Membrane
Filter
None
None
Storage Time
t0d
            9Od
                                                                                                     indefinite
Oiyestion Solution    Sin, KJ Acid       None
                        Slruruj Acid     Sl.orK, -id     Strong Acid
                                                                                                    Strong Acid
Sa.nph} Amount      lOOSOOml     toOSOOml
                                                            25
                                                                         25g
                                                                                                     200Om>
  Figure  1.   Handling and sample  storag*.  informdi ion  for  samples to be  analyzed  for  metals.

-------
      For the determination  of  suspended metals,  a  representative volume of
 unpreserved  sample must  be  filtered through  a 0.45-y pore size filter as
 quickly  as possible  after sample  collection.  The  filter and  insoluble mate-
 rials are digested with  concentrated  nitric  acid or aqua regia and treated as
 other samples.

      Aqueous samples to  be  analyzed for total concentrations  of elements should
 be acidified to  a pH less than 2  for  preservation.  Nitric, acid is generally
 used for this purpose.   Samples treated in this  manner can be stored for up to
 6 months.

      Soil/sediment samples  may be stored  in  wet, frozen, or dried state if the
 total sample concentration  is  to  be determined.  When an operationally defined
 procedure such  as the EP Toxicity Test is to be  performed, the samples should
 be stored in a  field-moist  condition.  Plastic or  glass sample containers may
 be used.2

      Tissue  samples  should  be  weighed as  soon as possible, and may be stored as
 received, frozen, or dried.  However, consideration of the stability of the
 tissue itself must be taken into  account  to  avoid  excessive decomposition and
 associated handling  problems.   The problem of storage of tissue samples for
 elemental determination  is  not well studied.  However, if tissue samples are
 digested as  soon as  practical, the digest can then be stored  as a water sample.

      Particulates in air samples  to be analyzed  for metals are usually col-
 lected by filtration.  Filter  media jsed  for this  purpose should be glass-fiber
.or membrane  filters  (0.8 p  pore size).3.  Glass-fiber filter blanks should be
 investigated to establish the  extent  of zinc contamination and potential matrix
 effects.  Possible interf^rencas  due  to Gil^ca from glass-fiber f-'ltar--: can be
 removed by centrifugation.3 Untreated filter samples from air samoiina can be
 stored indefinitely  for  most metals.3

 C.   INTERFERENCES

 1.   General

      Since the absorption of incident radiation  is monitored  in atomic absorp-
      tion (AA)  spectrometric analyses, the absorption, scattering, or emission
      of radiation of the wavelength of interest  by any substance other than the
      element being quantified  will create a  positive or negative interference.
      Spectral interferences, or extraneous adsorption of  radiation of the
      wavelength being monitored,  can  occur due to  the presence of organic
      molecules in the flame, and  are  particularly  important at lower wave-
      lengths (below  240  nm).  Several types  of automatic background correction
      are available from manufacturers of  most AA spectrometers.  Narrowing the
      slit width of the spectrometer detector can also help eliminate this
      interference.   Emission by molecular and atomic species  can create a
      negative Interference. This interference can be corrected by the use of a
      pulsed  source.   Scattering of light  by  particles  in the  flame or by an
      excessively ~*ch Hame will  produce  ~ oositive  tnectral  ''nterferorce.
                                       IV-4

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2.   Flame Atomization

     Since instrumental  response in AA spectrometry depends  on  the  free-atom
     concentration in the beam of radiation,  anything  which  alters  the  extent
     of free-atom formation between calibration standards  and sample  solutions
     will  be an interference.   Interferences  in flame  spectrometry  may  be
     classified into three categories:  transport interferences,  evaporation
     interferences, and  gas-phase interferences.

     1)   Transport interferences arise from  a difference  between standards
          and samples in the rate of delivery of solution  to the  flame.  The
          rate of aspiration depends upon the viscosity of the  solution being
          aspirated and, to avoid these interferences, the viscosity  of stand-
          ards and samples should be the same.  Surface tension differences
          between standards and samples can also create transport interferences.
          Transport interferences can be minimized by  using  the method  of
          standard additions or by matching the viscosities  and surface ten-
          sions of the standards with the samples being analyzed.

     2)   Evaporation interferences affect the rate of evaporation  of solid
          aerosols in the flame.  The best-known example of  such  a  phenomenon
          is the effect  of phosphate on the signal  observed  for calcium in
          fame spectrometr'?c  analysis, which 4s due to formation of  2  -efrac-
          tory Ca-O-P compound upon desolvation.   Evaporation interferences can
          be minimized by three procedures:  the addition  of a  releasing agent
          which preferentially combines with  the interfering moiety,  the use of
        *  a higher temperature flame such as  the nitrous oxide-acetylene flame
 -        to break up refractory compounds, or measurement of absorbance higher
          up in the flame (to  allow more time for production of free  atomsj.

     3)   Gas-phase interferences can shift ionization equilibria and alter the
          degree of analyte ionization from sample to  sample and  between
          samples and standards.  This interference can be compensated  for by
          adding easily  ionized cations such  as cesium and potassium  to samples
          and standards  at a concentration of approximately  1000  mg/1 to serve
          as an ionization buffer.

3.   Flameless Atomization

     Flameless atomization usually occurs in  an inert  atmosphere, which ore-
    ' eludes any chemical effects such as oxide formation due to the presence of
     oxygen.  However, spectral interferences can be a problem.  Gases  gener-
     ated  by the furnace during atomization may have interfering  molecular
     absorption bands, and smoke-producing samples can cause scattering of the
     incident beam.  Also, samples with high  levels of organic  matter can
     produce molecular species with broad uv  absorption bands which may include
     the wavelength of interest.  These samples should be  digested  prior to
     analysis.

     Matrix effects are  much more probable in furnace  atomization than  m  flame
     atcmizatlcn.  To help verify the absence of natr-'x 'nterferencas.  3 *or*i-
     fied  sample and an  unfortified sample should both be  diluted by  at  least

                                      * * t c
                                      A v-5

-------
     1:1, the two solutions analyzed, and the results  compared to the expected
     results.  Agreement within 10 percent indicates an  absence of substantial
     matrix effects.   If matrix effects are present, samples  should be diluted
     and reanalyzed to determine if dilution can  eliminate  the effects, or
     whether chemical  matrix modification, use of hydrogen  in the purge gas,  or
     the method of standard additions should be used.

D.   SAFETY

     When using flame-atomization atomic absorption spectrometry, flashback
can occur as the oxidant is changed from air to nitrous  oxide.  Special burner
heads, with a modified slot, should be used for nitrous  oxide flames.  In order
to avoid flashback, the air-acetylene flame should be  lit first, the flame
stoichiometry adjusted to be quite fuel rich, and then change the oxidant
from air to nitrous oxide.  After using a nitrous oxide  flame, always turn the
nitrous oxide off first when shutting down the instrument.   In addition, stand-
ard precautions for the use of compressed gases should be foil owed.3.4

     Because many of the elements determined by atomic absorption spectrometry
are potentially hazardous, adequate ventilation and exhaust capabilities (fume
hoods and instrument exhaust) are mandatory.

     When perchloric acid is used, perchlorate salts can be formed, either i_n
situ or in the ductwork of conventional fume noods.  These  ccmpouncs are poten-
rTaTiy explosive.4  For this reason, solutions to which  perchloric acid has
been added must never be heated to dryness.  In addition, neating and evapora-
tion of such solutions should always occur in a fume hood specially designed
for use with perch'loric acid.  Such fume hoods have surfaces washed with water
in order to eliminate the potential buildup of sucn compcunas.  Standard safety
precautions must be taken with concentrated acids, particular'':/ squa regia, and
normal laboratory safety rules should be enforced for  all analysts who may
be exposed to samples or standards of potentially toxic  or carcinogenic
metals.34

E.   APPARATUS

1.   Air Sampling Equipment

     1.1   High-Volume Sampler  (Haskin Scientific Ltd., Montreal, Canada,
           or equivalent).

     1.2   Flowmeters, chart  paper, and ink.

     1.3   Glass-fiber filters, 20 cm x 25 cm.

      1.4   Membrane filters,  0.8  vsn pore  size,  (Millipore or equivalent).

2.   Soxhlet extraction apparatus, with  extraction thimbles.

      2.1   Thermometer adaptor,  1 24/40.

      2.2   Polypropylene tubes, graduated,  5 mi.

                                       IV-6

-------
 3.    Analytical  balance,  0.1  mg sensitivity.

 4.    Low-Temperature  Asher (Tracer!ab  LTA-600,  or  equivalent).

 5.    Perchloric  acid  fume hood.

 6.    Watch  glasses, ribbed, 75 mm  diameter.

 7.    Filter paper, Whatman No.  42, or  equivalent.

 8.    Centrifuge.

 9.    Atomic Absorption Spectrometer with appropriate  hollow cathode  lamps.

 10.   Hotplate.

 11.   Air compressor,  or compressed air cylinder.

 I?..   Steam  table.

 13.   Compressed-gas-cyclinder regulators (acetylene,  air,  nitrous  oxide).

 r.    REAGENTS

,1.    Acetylene,  commercial' grade (contains  acetone).

 2.   .Air.

 3.    Nitrous oxide, commercial  grade.

 4.    Aqua regia.  Prepare immediately  before  use by adding three volumes
      concentrated HC1  to  one  volume concentrated HN03.

 5.    Calibration standards, prepared from stock standards, over the  concentra-
      tion range  of interest.   These solutions can  be  prepared as indicated
      below  or  purchased commercially.

      Standard  metal solutions:   prepare a series of standard metal solutions
      containing  5 to  1000 yg/1  by  appropriate dilution  of  the following stock
      metal  solutions  rfith deionized, distilled  water  containing 1.5  ml concen-
      trated HN03/1.

      Aluminum:   dissolve  1.000 g aluminum metal in 20 ml cone. HC1 by  heating
      gently and  diluting  to 1000 ml, or dissolve 17.584 g  aluminum potassium
      sulfate (also called potassium alum),  KA1(S04)2*12H20, in 200 ml  deion-
      ized,  distilled  water, add 1.5 ml  cone.  HN03, and  dilute to 1000  ml with
      deionized,  distilled water; 1.00  ml  =  1.00 mg Al.

      Calcium:  to ?,4972  g calcium carbonate. CaCOo,  add 50 ml deionized water
      and add dropwise a minimum volume of cone. HC1 (about 10 mi)  to erfect
      complete  solution.  Dilute -.3 1000 -nl  it.h ieionized. distilled water-
      1.00 ml = 1.00 mg Ca.

-------
    Cadmium:  dissolve  1.000 g cadmium metal in a minimum volume of 1+1 HC1 .
    Dilute to 1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Cd.
    Chromium:  dissolve  2.828 g anhydrous potassium dichromate, I^CrgOy, in
    about  200 ml deionized, distilled water, add 1.5 ml cone. HN03, and dilute
    to  1000 ml with deionized, distilled water; 1.00 ml-= 1.00 mg Cr.

    Copper:  dissolve  1.000 g iron wire in 50 ml of 1+1 HN03 and dilute to
    1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Cu.

    Lanthanum solution:   dissolve 58.65 g lanthanum oxide, 1.3203, in 250 ml
    concentrated HC1 .  Add the acid  slowly until the material is dissolved
    and dilute to  1000 ml with deionized, distilled water.

    Iron:  dissolve 1.000 g iron wire in 50 ml of 1+1 HN03 and dilute to
    1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Fe.

    Lead:  dissolve 1.598 lead nitrate, Pb(N03)2, in about 200 ml of water,
    add 1.5 ml cone. HNOs, and dilute to 1000 ml with deionized, distilled
    water; 1.00 ml = 1.00 mg Pb.

    Magnesium:  dissolve 10.0135 g magnesium sulfate heptahydrate,
    MgS04-7H70, in 200 ml deionized, distilled water, add 1.5 ml cone. HN03,
    and make'up to iOOO  ml with aeionized, unfilled *atsr; A.JO .TI'I -
    1.00 nsg Mg..
     Manganese:   dissolve  3.076  g manganous  sulfate monohydrate,
     in about- 200 ml  deionized,  distilled water,  add  1.5 ml cone. HN03, and
     make up :o  1000  ml  witn  deionized,  distilled water; LOG ml =  l.CG r.g Mn.

     Molybdenum:   dissolve 1.840 g  ammonium  molybdate,  (NH^^Moyt^J'A^O,
     in deionized water  and dilute  to  1  liter.

     Nickel:  dissolve 4.953  g  nickelous nitrate  hexahydrate,, Ni(N02)2'6H20,
     in about 200 ml  deionized,  distilled water,  add  1.5 ml cone. HN03, and make
     up to 1000  ml with  deionized,  distilled water; 1.00 ml = 1.00  mg  Ni.

     Zinc:  dissolve  1.000 g  zinc metal  in 20 ml  1+1  HC1 and dilute to 1.000 ml
     with deionized,  distilled  water;  1.00 ml =  1.00  mg Zn.

6.   Deionized,  distilled  water.

7.   Extraction  reagents.

     7.1  Reagents for PDCA Extraction

          Pyrrol idine dithiocarbamic acid (PDCA):  prepare by adding 18 ml of
          analytical  reagent  grade  pyrrol idine to 500 ml chloroform in a  1-1
          flask.   Cool and add  15 ml of  carbon disulfide in small portions with
          swirl-ing, ana dilute  to  1 'itar *ith  chloroform,   "tcra  *n in imber
          bottle, refrigerated.


                                      IV-8

-------
          Ammonium hydroxide, 2 N:  dilute 3 ml  cone.  NfyOH to 100 ml  with
          deionized, distilled water.
          Bromphenol blue indicator solution:  dissolve 0.1 g bromphenol  blue
          in 100 ml 50-percent ethanol  or isopropanol.
          HC1, 5 percent (y/v):  dilute 2 ml redistilled HC1  to 40 ml  with
          distilled, deionized water.
     7.2  Reagents for APDC/MIBK Extraction
          a)  Ammonium pyrrolidine dithiocarbamate acid (APDC) solution:
          dissolve 1.0 g APDC in deionized, distilled  water and dilute to
          100 ml.  Filter through a 0.45 y pore  size membrane filter.   Prepare
          fresh daily.
          b)  Buffer:  dissolve 272 g  sodium acetate in 1500 ml  deionized,
          distilled water.  Add 125 ml  glacial  acetic  acid  and dilute  to  2
          liters.  Adjust pH to 3.8 with cone.  HN03.
          c)  Indicator:  dissolve 0.02 g methyl  red and 0.10 g  bromocresol
          green In 50 ,nl methanol.
          -i]  SuK'Jr'c 3c:d, 0.25 N:  mix 7,0 cone.  'H-?SOd with deionized.
          distilled water and dilute to 1.0 liter.
          e)  NfyOH (1+1):  mix equal  volumes cone.  NfyOH and deionized,
          distilled water.	
          f)  Methyl isobutyl ketone - HPLC grade.
8.   Hydrochloric acid, cone. (sp. gr.  1.18; 36.5 to 38.0 percent).
9.   Hydrochloric acid, constant-boiling (approximately 19  percent}.
10.  Nitric acid, cone. (sp. gr. 1.42;  69.0 to 71.0  percent).
11.  Perchloric acid - Nitric acid mixture.  Add 10  ml  of cone,  perchloric  acid
     to 90 ml of cone, nitric acid.
12.  Perchloric acid, 10 percent (v/v):  dilute  100  ml  cone,  acid  to  1 "Hter
     with water.
13.  Nitric acid, 40 percent.
14.  LiB02, anhydrous (G. F. Smith No.  304).
15.  LiF, powder (Alfa No. 87628).
VS.  ^O*.
17.  Indicating taSO,*.
                                      IV-9

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G.   QUALITY CONTROL

1.   Any laboratory using these methods should operate a formal  Quality
     Assurance program.5  The minimum requirements  of such a program consist
     of an initial  demonstration of laboratory capability and continued
     monitoring of laboratory performance.   Records of laboratory performance
     should be maintained and available for reference or inspection.  Ongoing
     performance checks should be compared  with established performance cri-
     teria to determine if the results of analyses  are within accuracy and
     precision limits expected.  An unknown performance evaluation sample
     should be analyzed at least once a year for all  metals of interest as a
     check on laboratory performance.5

2.   Other quality control steps should be  implemented to the extent pos-
     sible, including:  service contracts for maintenance and calibration of
     balances and atomic absorption spectrometer, use of Class S weights for
     periodic checks on balances, dating and replacement (as necessary) of
     chemicals, analysis of standard reference material(s) at least once a
     quarter, analysis of at least 10 percent duplicate samples, monitoring of
     standard deviation of all measurements, tabulation of mean  and standard
     deviation of data, and use of quality  control  charts.5.

H.   CALIBRATION

1.   Calibration Standards

     Calibration standards are prepared by  diluting the stock metal solutions
     ?,t *:h9 time of analysis.  For" best results, calibration standards should
     be prepared fresh each time an analysis is to be made and discarded after
     use.  Prepare a blank and at least four calibration standards in graduated
     amounts in the appropriate range.  The calibration standards should be
     prepared using the same type of acid or combination of acids at the same
     concentration(s) as will result in the samples following processing.
     Since filtered water samples are preserved with 1:1 redistilled HNOs (3 ml
     per liter), calibration standards for  these analyses should be similarly
     prepared with HN03.  Beginning with the blank and working toward the
     highest standard, aspirate the solutions and record the readings.  Repeat
     the operation with both the calibration standards and the samples a
     sufficient number of times to secure a reliable average reading for each
     solution.

     For routine samples, the Calibration Curve Technique can be used to convert
     sample absorbances to concentrations (paragraph 2).  Where the sample matrix
     cannot be accurately matched with standards, the method of standard additions
     (paragraph 3) should be used.

2.   Calibration Curves

     cor Instruments which do not give results in concentration units, a cali-
     bration curve is ootainea oy plotting measured aosoroance as a  function or
     concentration rir a  series of standards.  The absorbance of the samole is
     then compared directly to the curve to determine the concentration of the

                                       TV-i.O

-------
     unknown.  If sample absorbances fall  outside the range covered by the
     standards, they should be diluted and reanalyzed.

3.   Method of Standard Additions

     In the method of standard additions,  incremental amounts of the analyte
     being quantified in equal volumes are each added to separate but equal
     volumes of sample, and the absorbance of each mixture measured by atomic
     absorption spectrometry.   The amounts of analyte added to the sample
     aliquot should vary from 0 to 150 percent of the amount of analyte expec-
     ted to be present in the sample aliquot.

     The results of these measurements are plotted as a function of added
     analyte, and when the resulting curve is extrapolated to zero absorbance,
     the intersection with the abscissa gives the.concentration of analyte in
     the sample.  The method of standard additions is required when matrix
     interferences are known or suspected  of existing in the sample being
     analyzed.  Figure 2 presents an example of such a plot.

I.   DAILY PERFORMANCE TESTS

     A calibration curve consisting of at  least one reagent blank and three
standards should be obtained daily and subsequent calibration checks must  be
rfenfied with at laast one rsagent biank ana one standard.  If 20 or more
samples are analyzed in a day, the calibration curve must be verified by anal-
lysis of a standard (at or near the maximum concentration of the curve) after
each 20 samples.  Results must be within 10 percent of the expected value.
                                     IV-11

-------
                     I
I
     (8)     (6)     (4)      (2)      0      2       4
                        Added Standard (jig/ml)
                                     8
Figure 2.   Typical graph for standard addition method.   The sample contains
         5.6  uq/ml of the element.  (From C.  T. Kenner  and K. W. Busch,
                 Quantitative Analysis, ftactmilom,  rt. i.,  -979. /&

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J.   ANALYTICAL PROCEDURES

     1.1  Determination of Metals in LMB/LiF Fusion Pellets of Waste Samples
               Analytical  Procedure:  available
               Sample Preparation:  available

          1.1.1  Reference

                 U.S. Environmental  Protection Agency, "LMB/LiF Fusion of HWDS
                 Solid-Phase and Nonaqueous Samples for Total  Metals."  U.S.
                 EPA - National  Enforcement Investigation Center,  Denver,
                 Colorado, Method 200.62.   3 p. (no date).7

          1.1.2  Method Summary

                 A 100-mg sample is  fused  with lithium metaborate  (LMB)  and
                 lithium fluoride (LW) for 9 minutes at 975C in  a  graphite
                 crucible in a muffle furnace.  After cooling, the fusion pel-
                 let is transferred  to a polyethylene vial, dissolved  in 100 ml
                 of 4-percent HNOs,  and filtered through a prewashed 0.45-um
                 membrane filter to  remove any carbon from the graphite  cru-
                 cible that may  nave adhered to the pellet.  The digest  i-s then
                 analyzed as a liquid sample using atomic absorption spec-
                 trometry.

          1.1.3  Applicability

                 The method is primarily applicable to hazardous waste samples
                 and has keen successfully aoplied to high-silica  soil,  sedi-
                 ment, clay, sludge, ash,  sulfide bearing ore, and oil.   Because
                 of the relatively low temperature used, volatile  elements sucn
                 as arsenic and  lead may also be determined in the final  solu-
                 tion.

          1.1.4  Precision and Accuracy

                 When the LMB/LiF fusion sample procedure was used with  stand-
                 ard reference materials and the resulting digests analyzed by
                 ICAP, recoveries in excess of 90 percent were the general rule
                 (Table 2 in ICAP Section).  Atomic absorption performance
                 would be expected to be similar to that reported  *or analysis
                 of sediment samples by atomic absorption.

          1.1.5  Sample Preparation

                 Place the weighing  dishes in a vacuum desiccator  containing
                 P205 and CaS04.  Obtain and record a constant weight  for these
                 dishes.

                 Weigh an aliquot of -vet HWDS solid-ohase samole (aoproximately
                 1 g) into a pre-weighed dish.  Place the samples  in the desic-
                 cator ana apply a vacuum.

                                     IV-13

-------
       After  48 hours of desiccation, reweigh the samples plus dishes.
       (A constant weight is obtained when two weighings separated by
       6 hours agree to within 2 percent.)  The percent moisture of
       the  sample is calculated as:

                               (wet wt.) - (dry wt.)
                % moisture  =	x 100
                                     (wet wt.)

       Place  6 to 8 graphite crucibles in a preheated muffle furnace
       and  heat at 1,000C for 30 minutes.  Remove the crucibles;
       allow  to cool slightly.  Blow off excess graphite and chalky
       residue with compressed air.

       Heat the crucibles for an additional 30 minutes at 1,000C.
       Repeat the process of blowing off excess graphite and chalky
       residue with compressed air.

       Store  the prepared crucibles in a clean, closed container.

       Prepare the lithium metaborate flux by combining 3 parts LiF
       and  7  parts LiBO^ (by weight).  Mix the mixture thoroughly
       and  store in a tightly-sealed container as the flux is hygro-
       scopic.

       Preneat the muffle furnace to 795C.

       Tare the prepared crucible.  Weigh 1.0 g 1MB flux into the
     crucible,  neigh 0.100 g dry sample into the '"'ux.  Carefully
       stir the contents of the crucible with a platinum wire to mix
       the  sample and the flux.  Distribute the mixture evenly in
       the  crucible.

       Using  tongs, place the crucibles in the furnace.  Fuse the
       samples for 5 minutes.  Check the samples after 5 minutes
       (for splattering, etc.) and mix.  Return the samples to the
       furnace and fuse for an additional 4 minutes (total fusion
       time is 9 minutes).

       Remove the crucibles and place on a cinder plate to cool.
       After  the fusion product has cooled, transfer the pellet to an
       8-ml polyethylene vial by tapping the bottom of the crucible.

       Dissolve the pellet  in 100 ml of 4 percent v/v HN03.  After
       dissolution is complete, filter the sample through a prewashed
       0.45 ym membrane filter to remove any carbon from the graphite
       crucible that may have adhered to the pellet.

1.1.6  Sample Analysis

       Analyze the digests  using standard atomic absorption spectrom-
       etry proceaures as presented in Subsection J.2.

                           IV-14

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2.1  Analysis of Water Samples for Metals
          Analytical  Procedure:   evaluated
          Sample Preparation:   evaluated

     2.1.1  Reference

            U.S. Environmental Protection Agency,  "Methods  for Chemical  Anal-
            ysis of Water and  Wastes."  Environmental  Monitoring and  Support
            Laboratory.  U.S.  EPA, Cincinnati,  Ohio.   EPA-600/4-79-020,  1979.l

     2.1.2  Method Summary

            Water or wastewater samples are digested  with acid at elevated
            temperature, the digest is diluted  to  a predetermined volume, and
            analyzed by atomic absorption spectrometry.

     2.1.3  Applicability

            This method is applicable to water  samples and  municipal  and indus-
            trial wastewaters  for determination of the metals listed  in  Table 1.
            Table 1 also lists the optimum concentration range, the sensi-
            tivity, and the detection limit for each  metal.

     2.1.4  Precision and Accuracy

            Table 2 summarizes orecision and accuracy information obtained
            with the atomic absorption method for  the determination of 32
   ' r  '   '  elements using the flame atomization mode, for  29 elements using
            the graphite furnace mode, and for  2 elements using hydride  gen-
            eration procedures.   Accuracy data  are expressed as percent  b-ias
            and precision data are expressed as one standard deviation.

     2.1.5  Sample Preparation

            General guidance is provided to prepare samples for the determina-
            tion of dissolved  metals concentrations,  total  metals concentra-
            tions, and particulate metals concentrations.  The distinction
            between these phases is operationally  defined by the filter  pore
            size used in the filtration process.

     2.1.5.1  Dissolved Metals Samples

              For the determination of total dissolved metals concentrations,
              the sample should be filtered through a 0.45  ym pore size  mem-
              brane filter at  the time of collection, if possible, or as soon
              as practical thereafter (the solid material on the filter  can be
              discarded or retained for suspended  solids determination,  para-
              graph 2.1.5.3).  The filtrate should be acidified to a  pH  less
              than 2 with nitric acid.  Three milliters of  1:1 acid per  liter
              1s usually sufficient for this adjustment.  If a precipitate
              forms upon acicnfication, -digest  the filtrate as outlined  in
              oaraqraoh ?.1.5.2.

                                     IV-15

-------
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-------
=========
                    TABLE l.  (Continued)
================C==================================3
                      Flame Atomization
Metal
H,
Mn
Mo
Ni
Os
Pd
Pt
K
Re
Rh
Ru
Ag
Na
n
Wave-
length
(nm)
285.2
279.5
313.3
232.0
290.9
247.6
265.9
766.5
346.0
343.5
349.9
328.1
589.6
276.8
Opt i mum
Concentration
Range
(mg/1 iter)
0.02-0.5
0.1-3
1-40
0.3-5
2-100
0.5-15
5-75
0.1-
5-1000
1-30
1-50
0.1-4
0.03-1
1-20
Sensitivity
(mg/1 iter)
0.007
0.05
0.4
0.15
1
0.25
2
u . u-t
15
0.3
0.5
0.06
0.015
0.5
Detection
Limit
(mg/1 iter)
0.001
0.01
0.1
0.04
0.3
0.1
0.2
J  U 1
5
0.05
0.2
0.01
0.002
0.1
Flame
(type)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
(fuel rich)
Air-acetylene
(oxidizing)
NgO-acetylene
(fuel rich)
Air-acetylene
(oxiaizing;
Air-acetylene
(oxidizing)
lightly oxidizing)
N20-acetylene
(fuel rich)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Ai r-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Air-acetylene
                                                               (oxidizing)
                                                                  coni'.nuea
                                     IV-17

-------
 TABLE 1.   (Continued)
: = ss = s:sss3: = = s = = 3S= = = == =
   Flame Atomization
=================

Metal
Sn
Ti
V
In
Metal
Al
So
As**
3a
Be
Cd
Cr
Co
Cu
Au
Ir
Fe
Pb

Wave-
length
(nm)
286.3
365.3
318.4
213.9
Waveiengtn
(nm)
309.3
^ 1 -^ -
i/ .0
193.7
352.5
234.9
228.8
357.9
240.7
324.7
242.8
264.0
248.3
283.3

Optimum
Concentration Detection
Range Sensitivity Limit
(mg/liter) (mg/liter) (mg/liter)
10-300 4 0.8
5-100 2 0.4
2-100 0.8 0.2
0.05-1 0.02 0.005
Furnace Method***
Optimum Concentration
Range Sensitivity
(yg/Hter) (ug/liter)
20-200
:o-:oo
5-100
10-200
1-30
0.5-10
5-100
5-iOO
5-100
5-100
100-1500
5-100
5-100


Flame
(type)
N20-acetylene
(fuel rich)
N20-acetylene
(fuel rich)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Detection
.--it
'yg/liter>
3
*
1
?
0.2
0.1
1
-
1
1
30
1
1
(continued)
          IV-18

-------
                             TABLE 1.  (Continued)
                     ========================================
                                 Furnace Method***
Metal
Mn
Mo
Ni
Os
Pd
Pt
Re
Rh
rtU
Se1**
Ag
TT
Sn
Ti
V
Zn
Metal
Wavelength
(nm)
279.5
313.3
232.0
290.9
247.6
265.9
346.0
343.5
349.3
196.0
328.1
275.3
224.6
365.4
318.4
213.9
Wavelength
(nm)
Optimum Concentration
Range Sensitivity
(vg/liter) (ug/liter)
1-30
3-60
5-100
50-500
20-400
100-2000
500-5000
20-400
100-C009
5-100
1-25 	 . '_
5-100
20-300
50-500
10-200
0.2-4
Gaseous Hydride Methods
Working Range
(ug/liter)
Detection
Limit
(ug/liter)
0.2
1
1
20
5
20
200
5
?0
2
0.2
1
5
10
4
0.05
Detection Limit
dig/liters)
 As
193.7
2-20
 Se      196.0              2-20                                      2
s=r===========================================================================
  *Chelation-extfaction method
 'Gaseous hydride method also Available.
***Consult Instrument operators manual for appropriate conditions.
                                     IV-19

-------
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-------
TABLE 2.  (Continued)
  Flame Atomization
Metal
Feb



Pbb





Mg3

Mnb





Mo a


Mia


Os
Pd
Pt
Ka

Re
Rh
Ru
Concent ration(s)
(mg/liter)
0.840
0.438
0.024
0.010
0.367
0.334
0.101
0.084
0.037
0.025
2.1
8.2
0.^26
0.469
0.084
0.106
0.011
0.017
3.30
1.5
7.5
0.20
1.0
5.0



1.6
6.3


 
Standard
Deviation
0.173
0.183
0.069
0.069
0.128
0.111
0.046
0.040
0.025
0.022
0.1
0.2
0.070
3.097
0.026
0.031
0.027
0.020
0.007
0.02
0.07
0.011
0.02
0.04



0.2
0.5

--
 
Recovery
(Percent)

-_


 
--
-_
-_
--

100
100
_ 
-_
--



100
96
95
100
97
93



103
102


__
Accuracy
(Percent Bias)
1.8
-0.7
141
382
2.9
1.8
-0.2
1.1
9.6
25.7
_.

1.5
 - *_
2.1
2.1
 93
22
 
--
--
 
.-


_.

 ^

--

__
                                    [continued;
         IV-21

-------
TABLE 2.  (Continued)
===============================
Concentration(s)
Metal (mg/liter)
Age 0.050
Naa 8.2
52
Tia 0.60
3.0
15
Sna 4.0
20
60
Ti
Va 2.0
10
50
Znb 0.310
1.056
0.070 "
0.011
0.007

Concentration(s)
Metal (ug/liter)
Al
Sb
Asa 15
20
50
100
Baa 500
!QOO
Flame Atomi ration
Standard
Deviation
8.8
0.1
0.8
0.018
0.05
0.2
0.25
0.5
0.5

0.10
0.1
0.2
0.114
0.028
0.028
0.018
0.026
Furnace Methods
Standard
Deviation


0.75
0.7
1.1
1.6
2.5
2.2
==========
Recovery
(Percent)
10.6%
102
100
100
98
98
96
101
101

100
95
97
 
--
--
--
 

Recovery
(Percent)


90
105
106
101
96
102

Accuracy
(Percent Bias)
rel . error
__
--
__
__
--
 
--
--

~ 
-_
--
-0.7.
11.3
6.6
66.6
206

Accuracy
(Percent Bias)


. .
--
--

__
--
                                    (continued)

-------
:===============================
            TABLE 2.   (Continued)
                 s s s s s = s s s s s s a s s ass 5 = = = = =: = :
               Furnace Methods
Metal
Cd
Co
Cu
Au
Ir
Fe
Mn
Mo
Ni
Os
Pd
Pt
Re '
Rh
Ru
Sea
Concentration(s)
(ug/liter)
2.5
5.0
10.0
19
48
77
25
50
100
.5
Standard
Deviation
0.10
0.16
0.33
0.1
0.2
0.8
1.3
1.6
3.7
0.6
Recovery
(Percent)
96
99
98
97
101
102
88
92
97
92
Accuracy
(Percent Bias)
--
10
70
                                  0.4
                                  0.5
9R
100
                                                 'csntinuad)
                    IV-23

-------
                             TABLE 2.   (Continued)
Furnace Methods
Concentration(s) Standard Recovery
Metal (yg/liter) Deviation (Percent)
Aga 25 0.4 94
50 0.7 100
75 0.9 104
Tl
Sn
Ti
v
- Zn
Gaseous 1Jydride Methods
Concentration Standard Deviation
Metal (uq/liter) (ug/liter)
"-,s3 5 . r:
in 0.9
20 1.1
Sea 5 0.6
10 " 1.1
.15 2.9

Accuracy
(Percent Bias)
--

Recovery
(Percent)
Od
93
85
100
100
-101
=================
^Single-laboratory statistics
bInter!aboratory statistics
cRound-robin statistics
              Proceed to paragraph 2.1.6 for analysis of the samples.   Report
              the results as dissolved or filterable metal  concentrations.

     2.1.5.2  Total Metals Samples

              Transfer a 50- to 100-ml, well-mixed sample to a 150-ml  or
              larger beaker.  Add 5 ml concentrated HN03 to the sample and
              ayaoorate to near Hr^ness in 3 hot nlats.  Caution should be
              exercised during this process to ensure that the sample does not
              boil.

-------
         Cool the sample and add a second 5-ml  portion of concentrated
         HN03-  Cover the beaker with a watch glass and reflux the sample
         on a hot plate.  Additional acid should be added to the sample
         as necessary during the refluxing.   The heating should be con-
         tinued until the digestion process  is complete, as indicated by
         the presence of a light-colored residue.

         Following digestion, add 1 to 2 ml  concentrated HC1 and warm
         the beaker slightly.  Wash the watch glass and beaker walls with
         distilled water.  Filter the digestate to remove any remaining
         insoluble matter and adjust the volume of the filtrate to a con-
         venient volume with distilled water.

         Proceed to paragraph 2.1.6 for analyses of the samples.  Report
         the results as total metal concentrations.

2.1.5.3  Suspended Metal Samples*

         The following sample preparation technique is useful  for most
         elements determined by atomic absorption  spectrometry.  These
         procedures should be equally applicable to air particulate
         solids as well as aqueous particulate solids.  However, separate
         digestion techniques are provided for suspended solids samples to
         be :nalyred "or ;ola, ^r-idium, palladium, and olatinum 'oaragraph
         2.1.5.4), arsenic and selenium (paragraph 2.1.5.5) or rhenium and
         titanium 'paragraph 2.1.5.5).

         Filter a well-mixed portion of the  unpreserved sample through a
         Q.i?, urn pore s-'zs membrane filter.   Record the volume of samole
         filtered.  (The exact volume of sample necessary will vary
         inversely with the concentration and the  composition  or tne
         suspended solids.)

         Transfer the membrane filter to a 250-ml  beaker, add  3 mi  concen-
         trated HN03, cover with a watch glass, and heat gently.  Increase
         the temperature of the hot plate after the membrane dissolves.
         When the acid has nearly evaporated, allow the beaker and watch
         glass to cool, and add 3 ml concentrated  HN03.  Continue heating
         the sample until digestion is completed as indicated  by the pres-
         ence of light-colored residue or the complete absence of partic-
         ulate matter.

         Remove the watch glass and evaporate the  sample to near dryness.
         Add 5 ml  1:1 hydrochloric acid and  warm the beaker to dissolve
         the residue.  (If the sample is to  be  analyzed for silver or
         1f the graphite furnace method is to be used, 1 ml  of 1:1 nitric
         acid should be used in place of the hydrochloric acid.)

         Wash down the  watch glass and the walls of the beaker with de-
         *oni?.ed.  -11 stilled water.  Filter the  samole to remove any silicates
         or other remaining insoluble materials.  Adjust the volume or  cne
         uigest co a convenient -volume with  distilled watsr.

                                IV-25

-------
         Proceed to paragraph 2.1.6 to analyze the digest.   Report the
         results as the participate concentration.

2.1.5.4  Samples to be analyzed for gold, iridium, palladium, and platinum
         are acidified with 3 ml  concentrated HN03 and heated to near
         dryness.  Add 5 ml aqua  regia, heat on a steam bath for 30 min-
         utes.  Remove the watch  glass and allow the sample to evaporate
         to near dryness.  Transfer the digest to a convenient-size vol-
         umetric flask and dilute to volume with distilled  water.  Pro-
         ceed to paragraph 2.1.6  for analysis and report the results as
         the particulate concentration.

2.1.5.5  For samples to be analyzed for arsenic or selenium, add 2 ml
         30 percent hydrogen peroxide, 1 ml concentrated HN03, and heat for
         1 hour.  Transfer the digest to a 100-ml volumetric flask, add
         10 ml 1-percent nickel nitrate, and dilute to volume with distilled
         water.

         Proceed to paragraph 2.1.6 to analyze the digest.   Report the
         results as the particulate concentration.

2.1.5.6  For samples to be analyzed for rhenium, acidify with 1 ml con-
         centrated HN03.  Warm the sample on 3 steam bath for 15 minutes.
         Allow the sample to cool, filter to remove any resiauai soiias,
         and dilute the digest to a convenient volume *n'th  distilled
         water.

         When titanium analyses are to be run, the digestion procedure is
         modified siigntly.  Acidify che sample with i mi concentrated
         riNC3 and 1 ml concentrated H2S04.  Heat the .sample j/itil the
         evolution of $03 fumes.   Allow the sample to cool, filter to
         remove any residual solids, and dilute the digest  to a convenient
         volume with distilled water.

         Proceed to paragraph 2.1.6 to analyze the digest.   Report the
         results as the particulate concentration.

2.1.6  Sample Analysis

       Samoles to be analyzed by atomic* absorption spectrometry can be
       processed using three separate techniques.  These tecnniques are
       Direct Flame Atomization (paragraph 2.1.6.1), Graphite Furnace
       (paragraph 2.1.6.2), or Cold Vapor Analysis (mercury).  Samples
       can also be analyzed using chelation-extraction but specific
       guidance is not provided here.

2.1.6.1  Direct Flame Atomization.

         Prepare a series of working metal standards by diluting the
         appropriate stocx ioiutions *ith aeionizea, uisffilea water
         containing 1.5 ml concentrated HNO-^/l.  These solutions should
         be prepared fresh on the day of use.

                                IV-26

-------
 Install  the  appropriate  hollow  cathode  lamp  in  the  instrument.
 Align the  lamp  and  set the  source current according, to  the
 manufacturer's  instructions.  Turn  on the instrument  and  allow
 both the instrument  and  lamp to warm up.  This  process  usually
 requires 10  to  20 minutes.

 Select the slit width and wavelength as  appropriate for the
 analyte  being determined.   However, the  recommended wavelength
 should only  be  used  as a guide.  Because of  calibration differences,
 the actual wavelength should be based on maximum  sensitivity
 after the  instrument has completely warmed up.

 Turn on  appropriate  gases,  ignite flame, and adjust the flow of
 fuel and oxidant to  give maximum sensitivity for  the  metal being
 measured.  When using a  nitrous oxide flame, a  T-junction valve
 or alternate switching valve should be  employed for rapidly
 changing from nitrous oxide to  air to prevent flashbacks when the
 flame is turned on  or off.

 Atomize  deionized distilled water acidified  with  1.5  ml concentrated
 HN03/1 and check the aspiration rate for 1 minute.  If  necessary,
 adjust the aspiration rate  to 3 to  5 ml/min.  Zero  the  instrument.

 Atomize  a  standard  and adjust the burner alignment  (up, down,
 siaeways)  until  a maximum signal /asponse is obtained.

 Aspirate a series of jietal  standards chat bracket the expected
.range of sample concentrations  and  record the absorbance  of each
 standard.  Rinse the atomizer with deionized distilled  water
 containing 1.5  ,r,: ^cncsntratad  HN03/"1 ';stween ?ach  standard.

 Atomize  the  digested water  samples and  record their absorbances.
 Rinse the  atomizer  with  dilute  nitric acid between  each sample.

 Samples  scheduled for iron  and  manganese or  calcium and magnesium
 analysis should be  premixed with calcium chloride or  lanthanum
 chloride solution,  respectively.  This  is accomplished  by mixing
 four volumes of digested sample with one volume of  the  appropriate
 salt solution.

 When determining metal concentrations by atomic absorption, the
 following  sequence  of sample processing  is recommended:

 a. Analyze a set of  standards.
 b. Analyze five samples.
 c. Analyze a duplicate of the fifth sample.
 d. Analyze five.additional  samples.
 e. Analyze a duplicate of the fifth sample.
 f. Analyze a fifth  sample that  has been  spiked.
.g. Analyze a standard.
 ,1. Repeat  Jteps a
 i. Reanalyze standards.

                       IV-27

-------
         This approach will  Incorporate certain aspects of the quality
         control  program into the analytical  procedure.  Specifically,
         the suggested sequence allows for evaluation of instrument
         stability, replicate analysis, and spike recovery.

         Prepare a standard  curve by plotting the absorbance of each
         standard versus concentration for each metal.   Use the standard
         curve to convert sample absorbance to metal  concentration.
         Calculate sample concentrations as indicated in Subsection L.

2.1.6.2  Graphite Furnace Procedure

         The use of graphite furnaces or carbon rod atomizers is considered
         to be an approved test method because it is  essentially an atomic
         absorption technique.8  However, the method  is not meant for use
         with all samples.  The method should only be considered as an
         alternative to conventional flame atomic absorption spectrophotom-
         etry when one or more of the following conditions exist;!

         a. Greater analytical sensitivity is required.
         b. Sample size is limited.
         c. Samples have a high dissolved solids content and cannot be
            aspirated into a flame.

         These conditions will usually result in the  use of a graphite
         furnace with water samples oecause of che higher metal concentra-
         tions in sediments  and the relative ease of  increasing sediment
         sample size during  the digestion step.

         Once d decision has been made to utilize a graphite ^urnace,
         install  the attachment according to manufacturer's instructions.
         Align the atomizer as required and warm up the instrument.
         Optimum instrument  conditions for the graphite furnace will
         generally be identical to those used for flame atomization.
         Check individual operation manuals for differences with specific
         metals (usually arsenic and selenium).  Warm up the background
         corrector, which should always be used with  the graphite furnace.

         Prior to the analysis of a new series of samples or the use of a
         new sample cup, it  is recommended that the atomizer be decontam-
         inated.  This can oe accomplished by operating the instrument in
         the maximum temperature mode for approximately 2 seconds.

         The analysis is conducted by transferring a  known volume of stand-
         ard or sample, 5 to 20 yl, to the sample cup.   The recommended
         method of sample transfer would be through the use of an automatic
         sampler attachment.  Other methods such as the use of oxford or
         Eppendorf pipettors can be used; but an increase in analytical
         variability 1s to be expected as a result.  The sample is then
         aUDjectea to a drying cycle :o ramove solvent, in Ashing  :yc!e to
         destroy organic matter, and an atomizing cycle (Table 3).  The
         metai concentration  is quantified during che atomization cycla.

                                IV-28

-------
       TABLE 3.  GRAPHITE FURNACE OPERATING CONDITIONS FOR SELECTED METALS
==============================================================================

Conditions                AT        As        Cd        Cr        Cu        Fe
Drying Time,
sec.
Drying Temp.,
C
Ashing Time,
sec.
Ashing Temp. ,
C
Atomizing Time,
sec.
Atomizing Temp. ,
C
Purge Gas
Wavel ennth,
nm
Conditions
Drying Time,
sec.
Dryina Temo. ,
C
Ashing Time,
sec.
Ashing Temp.,
C
Atomizing Time,
sec. .
Atomizing TemD. ,
C
Purge Gas
Wavelength,
nm

30

125

30

1300

10

2700
Ar

309.3
Pb

30

125

30

500

10

2700
Ar

283.3

30

125

30

1100

10

2700
Ar

193.7
Mn

30

125

30

1000

10

2700
Ar

279.5

30

125

30

500

10

1900
Ar

228.8
Mo

30

125

30

1400

15

2800
Ar

313.3

30

125

30

1000

10

2700
Ar

357.5
Ni

30

125

30

900

10

2700
Ar

232.0

30

125

30

900

10

2700
Ar

324.7
Se

30

125

30

1200

10

2700
Ar

196.0

30

125

30

1000

10

2700
Ar

48. -j
Zn

30

1 "C

30

400

10

2500
Ar

213.9
               :============================================
                                     IV-29

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V.  SCREENING AND GENERAL SAMPLE CHARACTERIZATION PROCEDURES

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                                   SECTION 19

                   METHODS FOR THE DETERMINATION OF OXIDANTS
A.   SCOPE

     These methods are applicable .to the measurement of the oxidant content
In air^ and agueous samp1es23 as well  as to aqueous extracts of both hazardous
waste samples^ and solid phase samples such as soil or sediment.

B.   SAMPLE HANDLING AND STORAGE

     It is not possible to recommend preservation techniques or storage times
for samples that contain reactive chemical soecies.  Therefore, these proce-
dures should be completed as rapidly as possible.

C.   INTERFERENCES

1.   General

     Since the tests are not specific'for one particular chemical species,
     :ny  :uDSuanc3  at -^ac'is -jith ;atu3Vuni ;ccnde *o '
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     Peroxyacetyl  nitrate gives a response equivalent to approximately 50
     percent of that of an equlmolar concentration of ozone.   Concentrations
     In the atmosphere may range up to 0.1 ppm.

     Hydrogen sulflde and reducing dusts or droplets can act  as negative
     Interferences.

D.   APPARATUS

1.   Standard laboratory glassware.

2.   Microburet, 2 ml, 10 ml.

3.   Absorber:  all-glass midget 1mp1ngers, graduated 1n 5-ml Increments.
     (Other bubblers with nozzle or open-end Inlet tubes may  be used.   Fritted
     bubblers tend to give relatively low results.)  Implngers must be kept
     scrupulously clean and dust free.  Traces of grease can  be removed by
     alcoholic potassium hydroxide, followed by washing with  laboratory
     detergent and rinses with tap and distilled water.

4.   A1r-meter1ng device:  a glass rotameter capable of measuring a flow of
     0.5 to 3 liters per minute with a test meter to ensure an accuracy of 2
     percant *z racommended.

5.   Air pumo:  an appropriate suction dump capable jf drawing the required
     sample flow for intervals of up to 30 minutes 1s suitable.  It is
     desirable'to have a needle valve or critical orifice for flow control.6
     A trao should be Installed upstream of the pump to protect the needle
     vaive and pump against accidental flooding <;tn aosoroing reagent -no
     consequent corrosion.

6.   Spectrophotometer:  any laboratory instrument suitable for measuring the  
     aosorbance of the iriiodide Ion at 352 nm *itfi stoppered tubes or rjvettss
     (suitable for near ultraviolet use) 1s recommended.

E.   REAGENTS

     All reagents are made from analytical-grade chemicals.  They are stable
     for several months in well-stoppered bottles.

1.   Glacial acetic acid.

2.   Potassium Iodide:  crystals.

3.   Phenylarslne oxide, 0.00564 N:  dissolve approximately 0.8 g phenylarsine
     oxide powder 1n 150 ml 0.3 N NaOH solution.  After settling, decant 110 ml
     Into 800 ml distilled water and mix thoroughly.  Adjust  the pH into the
     range 6 to 7 with 6 N HC1 and dilute to 950 ml with distilled water.
     Standardize against a standardized Iodine solution prior to use.   Com-
     mercial ly-preparea pnenyiarslne oxide solutions are dvaiiaoie ,Wallace ana
     T1erman. or 
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4.   Double-distilled water,  used for all  reagents:   redistill  distilled water
     1n an all -glass still  after adding  a  crystal  each  of  potassium  permanga-
     nate and barium hydroxide.

5.   Absorbing solution (1  percent KI in 0.1  M phosphate buffer):  dissolve
     13.6 g potassium dlhydrogen phosphate (KH2P04),  14.2  g  dlsodlum hydrogen
     phosphate (Na^HPO^), or  35.8 g of the dodecahydrate salt  (^HPO^^hUO) ,
     and 10.0 g potassium Iodide 1n sequence  and dilute the  mixture  1:1 with
     double distilled water.   Keep at room temperature  for at  least  1  day
     before use.   Measure pH  and adjust to 6.8  0.2  with  NaOH or  KHoPO^  This
     solution can be stored for  several  months in a glass-stoppered  brown
     bottle at room temperature  without deterioration.  It should  not be
     exposed to direct sunlight.

6.   Stock solution 0.025M  12 (0.05N):  dissolve 16 g potassium iodide and
     3.173 g resublimed iodine successively and dilute  the mixture to exactly
     500 ml with  double-distilled water.  Keep at room  temperature at least
     1 day before use.  Standardize shortly before use  against 0.025 M Na2S?03.
     The sodium thlosulfate is standardized against primary  standard potassium
     biiodate (KH( 103)2) or potassium bichromate (I
     6.1  0.001M !2 solution:   plpet sxactly  4.00 ml  of the  0.025-M  stock
          solution into a 100-ml  low-actinic  volumetric flask  and dilute  to
          *.*i9 iiars '-ntn iosormng solution.   Dorset  "rom  strong  light.
          Discard after use.

     6.2  Calibrating iodine solution:   4.09  ml  of the 0.001-M I? solution
          (or equivalent volume for other molarlty) is diluted with  absorbing
          solution just before use to 100 ml  (final volume)  to make  the final
          concantration equivalent to 1  ui  of u3/;nl >3ee Calculations; .
          Discard this solution after use.

     Sulfur dioxide absorber:   flashfired glass  fiber paper  is impregnated with
     chromium tnoxide, as follows:^  Drop 15 mi  of aqueous  solution containing
     2.5 g chromium trioxide and 0.7 ml  cone, sulfuric acid  uniformly over
     400 cm2 of paper, dry in  an oven at 80 to 90C for 1  hour and store  1n  a
     tightly capped jar.  Half of this paper  suffices to pack  one absorber.
     Cut the paper in 6- x 12-mm strips, each folded  once  into a  V-shape, pack
     into an 85-ml U-tube or drying tube and  condition by  drawing air that has
     been dried over silica gel through  the tube overnight.  The  absorber is
     active for it ""east 1 tjonth,  When  H becomes '-Msibly vet from  samolfng
     humid air, it must be dried with dry air before  further use.
                                      V-4

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F.   ANALYTICAL PROCEDURES



     1.1  Ox'idant Capacity of Hazardous Waste Samples.  Reserved.

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2.1  Oxidant Capacity of Aqueous Samples
          Analytical Procedure:  evaluated
          Sample Preparation:  available

     2.1.1  Reference

            U.S. Environmental Protection Agency, "Oxldant Determination for
            HWDS Samples."  U.S. EPA, National  Enforcement Investigations
            Center, Denver, Colorado.  Method 330.65, 2 p. No date.

     2.1.2  Method Summary

            Potassium Iodide is added to an acidified (pH 4 or less) sample
            aliquot (aqueous phase or aqueous extract of a solid phase).  The
            sample is titrated with phenylarsine oxide, using starch as an
            indicator, to quantify the amount of liberated iodine.  The oxidant
            strength of the sample is expressed as an equivalent concentration
            of chlorine.

     2.1.3  Apolicability

            The method is suitable for both liquid and solid-phase samples.
            Mowever, *~~r solia-onase samoles, *he ^rocadur? only measures the
            extractable or exchangeable oxidant capacity as defined Dy the
            conditions ':sed to orepare the oHd-onase extract.''  The range of
            the test vrill vary with the size of the sample aliquot titrated.
            Data based on the determination or' totai resiauai chionne using
            the starch-iodine titration method is presented In Table 1.  While
            the ourpose was to determine residual chlorine rather than oxidant
            capacity, the analytical technique is tne same and the data pro-
            vides an estimate of expected procedure performance.


       TABLE 1.  PRECISION OF OXIDANT DETERMINATION FOR AQUEOUS SAMPLES2
3333BB333333B33333a33::BB3BS3333333333:S33333333333533B33B333BB33=3333B33333=33:s3
Sample
Matrix
Distilled water
Distilled water
Drinking water
River water
Domestic sewage
Raw sewage
Replicates
3
3
7
7
7
7
Chlorine
Concentration
mg/1
0.41
3.51
0.84
0.84
0.87
0.55
Std.
Qev.
mg/1
0.05
0.12
0.04
0.02
0.07
0.09
Rel.
Std. Oev.
%
12.2
3.3
4.3
2.7
7.6
16.0
                                      "-5

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2.1.5  Sample Preparation

       Obtain an aqueous aliquot of the sample to be analyzed as
       Indicated below:

       a.  Shake 2 g of solid-phase hazardous waste with 200 ml
           distilled water for 1 hour.   Decant the liquid phase  and use
           100 ml for the oxldant capacity determination.

       b.  P1pet 100 ml  of aqueous-phase hazardous waste sample  or water
           sample Into a disposable beaker.

       c.  Prepare an aqueous extract of soil /sediment samples as
           explained In paragraph (a).

       Standardize the phenylarslne oxide tltrant against a fresh Iodine
       solution.  Place 10 ml 0.0282 N  Iodine solution 1n a flask and  add
       1 g potassium Iodide.  Titrate with phenylarslne oxide to the
       starch endpoint.   Adjust to 0.00564 N.  1.00 ml = 200 g  chlorine.

       To the 100 ml aqueous extract or water sample, add 5 ml acetic
       add.  While stirring on a magnetic stirrer, add 1 g KI.   Add 1 ml
       starch indicator solution.

       After the blue color is homogeneously distributed, titrate the
       sample with 0.00564 N phenylarsine oxide (PAO) until a colorless
       condition persists 1n the sample for at least 15 seconds.  This is
       *he sndooint 
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3.1  Determination of Oxidant Capacity  in  Soil Samples.  Reserved.
                                     V-8

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4.1  Determination of Oxidant Capacity  in  Biological Tissue Samples.
     Reserved.
                                      V-9

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5.1  Determination of Oxldants 1n A1r
          Analytical  Procedure:   available
          Sample Preparation:   available

     5.1.1  Reference

            American Public Health Association,  "Methods  of A1r Sampling  and
            Analysis."  2nd ed;  APHA, Washington,  D.C.  Method  411,  pp. 556-559.
            (1977).

     5.1.2  Method Summary

            This is a method for manual  determination of  oxidants  (Including
            ozone) in air over the range of approximately 0.01  to  10 ppm  (as
            ozone).  The method is based on sampling with a midget impinger and
            determination of the iodine  liberated  from a  1-percent potassium
            iodide solution at pH 6.8   2.1*7  The Iodine is determined
            spectrophotometrically by measuring  the absorption  of  triIodide
            ion at 352 nm.  The stoich-'ometry  is approximated by the following
            equation;!


                        03 * 3 
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       upstream from the impinger.   Butt-to-butt glass connections  with
       lightly greased Tygon tubing  may  also  be  used  for  connections  with-
       out losses 1f lengths are kept minimal.   Pipet exactly  10 ml  of the
       absorbing solution Into the midget Impinger and sample  at a  flow
       rate of 0.5 to 3 liters per minute.  Note the  total  volume of the
       air sample.  If the sample air temperature and pressure deviate
       greatly from 25*C and 760 mm  Hg,  measure  and record  these values.
       Sufficient air should be sampled  so  that  the equivalent of 1  to
       10 ul  of ozone 1s absorbed.   Approximately 1 ul  of ozone can be
       obtained in the absorbing solution at  an  atmospheric concentration
       of 0.01 ppm by sampling for 30 min at  3 1/min.l Do  not expose the
       absorbing reagent to direct sunlight.   If appreciable evaporation
       has occurred, add distilled water to restore the volume to the
       10-ml  graduation mark.

5.1.6  Sample Analysis

       Assemble a train consisting of a  rotameter, U-tube with chromium
       trioxide paper (optional), midget Impinger, needle valve or  criti-
       cal orifice^ and pump.  Connections  upstream from  the Impinger
       should be ground glass or inert tubing outt lointed  witn polyvinyl
       tubing.  FluorosiUcon or fluorocarbon grease  should be used
       sparingly,  s-'oet exactly 10  ml of the absorbing loluticn Into the
       midget Impinger.  Sample at  a rate of  0.5 to 3 I/mm ror up  to 30
       min,  The T]QW rate and tne  time  of  samolinq snouia  oe  aajustsd to
       obtain a sufficiently large concentration of oxidant m the
       absorbing solution.  Approximately 1 ill of ozone can be obtained
       in the absorbing solution at  an atmosoherlc concentration of 0.01
       opm oy sampling ror 30 min 3t 3 I/mm. Measure cne  air temperature
       and pressure, and calculate  the totai  volume of cf-.e  air sampie.  Do
       not expose the absorbing reagent  to direct sunlight.

       The sample 1s absoroed and read in A0  fli  of absoroing reagent.
       Calibrating solutions are made up to 10 ml to  facilitate the
       calculations.  For greater precision these can be  made  up to
       25 ml or more.

       It is more convenient to standardize the  instrument  using iodine
       solutions than accurately-known gas  samples.  See  paragraph  5.1.2
       for postulated stoichiometr-'c relationshios.  To obtain a ranqe of
       concentration values 1n standardization,  add several amounts of
       calibrating solution from 0  to 10 ml to a series of  !0-ml volu-
       metric flasks.  Dilute to volume  with  absorbing reagent.

       Read the absorbance of each  standard at 352 nm.

       Plot the absorbances of the  standards  versus the ul  of 03 in 10 ml
       absorbing reagent.  The plot  should ^ollow Beer's  law.

       If appreciaole evaporation of the dosoroing solution nas occurrsa
       lur-'ng sampling, 5dd double-distilled  water to bring the liauid
       volume to 10 ml.

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            Within 30 to 60 minutes  alter sample  collection,  read  the  absorb-
            ance of the sample solution  1n a  cuvette  or  tube  at 352  run against
            a reference cuvette or a tube containing  double-distilled  water.

            Measure the absorbance of the unexposed reagent at 352 nm  and
            subtract the value from  the  absorbance of the  sample.

G.   CALCULATIONS

1.   Aqueous Samples

     Adjust the sample tltratlon volume  based on  the  blank tltratlon results
     (subtract the blank PAO tltratlon results or add the  net equivalent of the
     back-titratlon results).

     Calculate the oxidant content (OC)  of the solid-phase sample  as:
                                               35,450 mg   E
                 OC (ug Cl2/g)   a - x -  x -  x  1,000
                                       S          eq       G


     where:
             OC  =  oxidant concentration
             Vi    corrected volume of PAO used,  ml
             Hi    normality of "AO
              S  *  volume of sample titrated,  ml
              E  =  volume of water  extract prepared, ml
              5  -  weiqht of olid  ohase extracted, q.

     The oxidant concentration of an aqueous sample  is calculated  as:


                                   (YiHNii   35,450 mg
                  OC (mgCl2/D   - - x -
                                      S          eq


     where the terms are defined as  above with 35, 450 mg/eq  being the
     equivalent weight of chlorine.

     Air Samples

     Standard conditions are taken as 760 Torr and 25*C,  at which  the molar gas
     volume 1s 24.47 liters.

     Record the volume of the sample collected in liters. Generally  the
     correction of the sample volume to standard conditions Is  slight and  may
     be omitted.  However, for greater accuracy, corrections  may be calculated
     w Tieans of the ideal oas law.
                                      V-12

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The calibrating iodine solutions are calculated on the basis, of equiva-
lence of. 63 and 12, as indicated in Subsection E.6.2.  Hence, a 100-ml
portion of final solution equivalent to 1 yl Os/ml contains the
following amount of iodine:
                         100 x 10-6
                  U  =   	 = 4.087 x 10' moles
                  12
                                                -6
                           24.47
This is equivalent to 4.09 ml of 0.001 M (or 0.002N) \2 solution.

The total yl of 03/10 ml of reagent are read from the calibration curve.

The concentration of 03 in the gas phase in yl/1 or ppm is given by:


                               total yl ozone per 10 ml
                 03 ppm
                            volume of air sample in liters
The cone, of 03 in terms of yg/1 at 750 Torr ana 25C  is ootainea *nen
desired from the value of ul/1 by:
                                 ppm-x 48.00
                 ug 03/ liter  =  -  - 1,362
                                      .17
                                 V-13

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                                  REFERENCES
1.   American Public  Health  Association.   "Methods of A1r Sampling and Anal-
     ysis."  2nd ed., APHA,  Washington D.C.  pp. 556-559.  (1977).

2.   U.S.  Environmental  Protection Agency.   "Methods for Chemical Analysis of
     Water and Wastes."   U.S.  EPA, Environmental Monitoring and Support Labo-
     ratory, Cincinnati, Ohio.   EPA-600/4-79-020, (1979).

3.   American Public  Health  Association.   "Standard Methods for the Examination
     of Water and Wastewater."   15 Ed. Washington, D. C., (1980).

4.   U.S.  Environmental  Protection Agency.   "Oxidant Determination for HWDS
     Samples."  U.S.  EPA, National Enforcement  Investigations Center, Denver,
     Colorado.  Method 330.65,  2 p.   No date.

5.   Saltzmann, B. E., and A.  F. Wartburg, Jr.  "Absorption Tube for Removal
     of Interfering Sulfur Dioxide in Analysis  of Atmospheric: Oxidants."
     Anal. Chem. 37:779, (1965).

5.   Lodge, J. P., Jr.,  j. 3.  Pate,  3. c.  Ammons, and A. F. Swansons,
     "The Use of Hypodermic  Needles  as Critical Orifices in Mir Sampling.'1
     Air Poll. Cont.  Assoc.  J., 16:197-200,  11966).

7.   Byers, D. H., and B. E. Saltzmann.   "Determination of Ozone in Air by
     Neutral and Alkaline Iodide Procedures."   J, Am. Indust. Hyg. Assoc.,
     19:251-7, (19585.

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                                   SECTION 20

              METHOD FOR THE DETERMINATION OF REDUCTANT CAPACITY
A.   SCOPE
     This method is applicable to the measurement of sample reductant capacity
in aqueous samples and aqueous extracts of hazardous wastes, soil and sediment
samples.  The reductant capacity of each sample is expressed as an equivalent
concentration of iodine.

B.   SAMPLE HANDLING AND STORAGE

     The concentration of total reductants in the sample Is expressed as an
equivalent concentration of iodine.  However, there is no recommended
preservative or molding time  '"or 'odine.1-  As ^ rule, itany suostances that
contribute to the reductant capacity of a sample can react with oxygen.
Therefore, it ts recommended that sample container: be completely filled xnth
sample, when possible, to exclude oxygen.  Also, the samples should be stored
at 4*C and the storage period kept to a minimum.
          ~,he rsductant capacity of 'he samoie 's 
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E.    REAGENTS

1.    Potassium Iodide,  1 percent w/v:   dissolve  1 g KI  1n 100 ml of demin-
     erallzed water.

2.    Standard Iodine solution,  0.0282  N:   dissolve 25 g KI 1n a small volume of
     distilled water 1n a 1-Hter volumetric  flask.  Add the appropriate amount
     of 0.1 M stock Iodine solution  (approximately 282 ml) and dilute to
     1 liter with distilled water.   For accurate work, this solution should be
     standardized daily against an arsenite or thiosulfate solution. Commer-
     cially-prepared iodine solutions  are  available from Fisher Scientific Co.,
     Pittsburgh, Pennsylvania.

3.    Stock iodine solution, 0.1 N:   dissolve  40  g KI in 25 ml distilled water.
     Add 13 g resublimed iodine and  stir until dissolved.  Transfer to a
     1-liter flask and dilute to volume.   Standardize against a 0.1-N arsenite
     solution using a starch solution  as an endpoint indicator.  To improve
     accuracy, ensure that the  solution is saturated with COj at the end of the
     tltration by bubbling C02  through the solution or adding HCl to shift the
     carbonate equilibrium and  liberate C02-  Alternately, this solution may be
     standardized against the phenylarsine oxide solution used in the oxidarn;
     capacity determination.

4.    Starch solution:  add 1 g  starcn  to 1 liter of coiling, aistillea water.
     Stir and. let settle overnignt.  Use tne  clear suoernatant.  This solution
     may be preserved with 1.25 g salicylic acid, 4 g zinc chloride, or a
     combination of 4 g sodium  propionate  and 2  g sodium azide/1.
                                      V-16

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F.   ANALYTICAL PROCEDURES
          Analytical  Procedure:   available
          Sample Preparation:  available

     NOTE: Measurement of this parameter 1s based on the analysis of an aqueous
     phase or aqueous extract.  Therefore, all  matrices have been combined into
     a single procedure.  The reductant capacity for solid-phase samples is
     operationally defined by the extraction procedure and may not measure the
     total reductant capacity of the sample.

     1.1  Reference

          U.S. Environmental Protection Agency, "Reductant Determination for
          HWDS Samples."  U.S. EPA, National Enforcement Investigation Center,
          Denver, Colorado.  Method 331.60.  2 p.  No date.2

     1.2  Method Summary

          An aliquot of sample is added to a trliodide solution.  The
          decrease in Intensity of the yellow color, due to the presence of
          reducing agents, 1s measured spectrophotometrically at 352 nm.  The
          :otai reducing capacity jf ;he sample is expressed as an equivalent
          iodine concentration.

     1.3  Applicability

          "he .nethod  :s ;ui"abla  "or both "-'quid- ird ral'd-ohase samoles.
          However, for solid-phase samples, *he -srocsdure only Treasures tne
          extractable or exchangeable reductant concentration as defined by
          the conditions used to prepare the solid-phase extract.  A sample
          containing 10 mg/1 salflde lan be visually detected with the
          procedure and a spectrophotometer can detect 0.5 mg/1 sulflde.

     1.4  Precision and Accuracy

          Precision and accuracy Information is not available at this time.

     1.5  Procedure

          P1pet 5 ml of 1% KI solution into a series of test tubes.  Add
          0.025 ml of 0.0282 N Iodine solution to all but one of the test
          tubes.

          P1pet 0.010 ml of  sample Into one of the test tubes containing the
          Iodine solution.  The rapid bleaching of the yellow color Indicates
          the presence of a  reducing agent.  If the yellow color 1s completely
          dissipated, dilute the sample and test again until some color
          remains.
          Set the spectropnotometer wavelength at CS2 ,im.  Zero ^he s
          photometer with the 11 KI solution 1n quartz cuvettes.  Measure and
          record the absorbance of each sample and a standard iodine solution.

                                      V-17

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G.   CALCULATIONS
     The reductant content of each sample 1s calculated based on the ratio of
the sample absorbance to the absorbance of the standard Iodine solution.

                                            (X)(VI)(N)(D) x 1000
                Reductant cone, (meq/1)  a  	
                                                   (S)(V2)
where:
       X  =  absorbance reading for sample
       S  *  absorbance r