REGION I, EPA-NEW ENGLAND

     IMMUNOASSAY GUIDELINES
                 FOR
PLANNING ENVIRONMENTAL PROJECTS
         U.S. EPA-NEW ENGLAND
                Region I
         Quality Assurance Unit Staff
Office of Environmental Measurement and Evaluation
              October 1996

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                                               PREFACE

Region I, EPA-New England promotes the development and use of innovative technologies to improve environmental
monitoring and remediation activities, while reducing both the time and expense involved.  One such technology
gaining increasing momentum in the field of environmental testing is immunoassay (LA).  IA can provide real-time
field analysis of a wide variety of environmental parameters at a fraction of the cost and time for conventional full
protocol laboratory analyses.  LA techniques can be used effectively in the hazardous waste remediation process to
delineate the extent of contamination and to ascertain that cleanup activities have been successfully completed for a
particular project.

However, for LA or any other field measurement technology to gain regulatory acceptance, the technology must be
properly employed and must produce valid data that are usable for their intended purpose in project decision making.
Use of this document, in conjunction with proper project planning, strict adherence to vendor procedural requirements,
and good quality assurance/quality control practices; will result in the effective application of environmental IA
techniques.

IA is an innovative technology that is in a fluid state of development.  The project planner should be cognizant of
product changes in the environmental LA market, and should rely heavily on vendor consultation  and training to ensure
proper LA kit selection and operation.
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                 REGION I, EPA-NEW ENGLAND IMMUNOASSAY GUIDELINES
                        FOR PLANNING ENVIRONMENTAL PROJECTS

                                   TABLE OF CONTENTS
Preface
1.     Introduction	IA GUIDELINES -1
2.     Environmental Immunoassay	IA GUIDELINES - 3
3.     Environmental IA Kits 	IA GUIDELINES - 8
4.     Kit Sensitivity	LA GUIDELINES -14
5.     Kit Selectivity	IA GUIDELINES - 15
6     Kit Operational Concerns	IA GUIDELINES -18
7.     Practical Planning for Projects Using Immunoassay	LA GUIDELINES - 26
8.     Data Comparability and Usability 	LA GUIDELINES - 36
9     References	LA GUIDELINES - 40

Appendices

Appendix A    Glossary
Appendix B    Technical Immunoassay Description
Appendix C    Immunoassay Tables
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                  REGION I, EPA-NEW ENGLAND IMMUNOASSAY GUIDELINES
                         FOR PLANNING ENVIRONMENTAL PROJECTS
PREFACE
                                                                                         i
1.0    INTRODUCTION	IA GUIDELINES-1
      1.1    Information Sources 	LA GUIDELINES-1

2.0    ENVIRONMENTAL IMMUNOASSAY	LA GUIDELINES-3
      2.1    Glossary of Terms	IA GUIDELINES-3
      2.2    What is Immunoassay?	LA GUIDELINES-3
      2.3    Formats for Environmental LA Kits 	LA GUIDELINES-4
              2.3.1    Basic Format	LA GUIDELINES-5
              2.3.2    Antibody Attachment to Solid Phase	IA GUIDELINES-5
              2.3.3    Reagent and Sample Additions  	LA GUIDELINES-7
              2.3.4    Colorimetric Determination	LA GUIDELINES-7
      2.4    Immunoassay and Environmental Chemicals 	LA GUIDELINES-7

3.0    ENVIRONMENTAL LA KITS 	LA GUIDELINES-8
      3.1    Currently Available Environmental LA Kits  	LA GUIDELINES-8
      3.2    Field Kits Versus Laboratory Based Kits	LA GUIDELINES-9
      3.3    Quantitative, Semi-Quantitative, and Qualitative LA Kits	LA GUIDELINES-9
              3.3.1    Quantitative LA Results	LA GUIDELINES-10
              3.3.2    Semi-Quantitative LA Results	LA GUIDELINES-10
              3.3.3    Kit Standardization and Quality Control	LA GUIDELINES-11
                        3.3.3.1   Calibration	LA GUIDELINES-12
                        3.3.3.2   Blanks	LA GUIDELINES-12
                        3.3.3.3   Duplicates	LA GUIDELINES-13
                        3.3.3.4   Performance Evaluation Samples 	IA GUIDELINES-13
      3.4    Kit Detection Ranges	IA GUIDELINES-13

4.0    KIT SENSITIVITY	LA GUIDELINES-14

5.0    KIT SELECTIVITY 	LA GUIDELINES-15
      5.1    KitReactivity	LA GUIDELINES-16
              5.1.1    Compounds with Little Sensitivity  	LA GUIDELINES-17
      5.2    Cross-Reactivity 	LA GUIDELINES-17

6.0    KIT OPERATIONAL CONCERNS	LA GUIDELINES-18
      6.1    Temperature Range Considerations and Shelf Life	LA GUIDELINES-19
              6.1.1    Storage Conditions	IA GUIDELINES-19
              6.1.2    Operating Temperature Range	LA GUIDELINES-19
              6.1.3    Shelf Life	LA GUIDELINES-20
      6.2    Water Characteristics	LA GUIDELINES-20
      6 3    Soil Characteristics 	LA GUIDELINES-20
      6.4    Extraction Solvents	LA GUIDELINES-21

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      6.5    Soil Moisture	IA GUIDELINES-22
      6.6    Operational Consistency	IA GUIDELINES-23
      6.7    Cost and Time Considerations  	IA GUIDELINES-23
              6.7.1     Time Savings	IA GUIDELINES-25
              6.7.2     Cost Considerations 	IA GUIDELINES-25
      6.8    Disposal of LA-Related Waste	LA GUIDELINES-25

7.0    PRACTICAL PLANNING FOR PROJECTS USING IMMUNOASSAY	IA GUIDELINES-26
      7.1    Gathering LA Information	IA GUIDELINES-26
      7.2    Determining Project Data Quality Objectives (DQOs)	LA GUIDELINES-28
      7.3    Ascertaining LA Needs	LA GUIDELINES-28
              7.3.1     LA Kit Selection Factors	LA GUIDELINES-30
      7.4    Obtaining Vendor Training	LA GUIDELINES-35
      7.5    Ordering LA Kits and Ancillary Equipment	LA GUIDELINES-35
      7.6    Finalizing the QAPjP/SAP 	LA GUIDELINES-35

8.0    DATA COMPARABILITY AND USABILITY 	IA GUIDELINES-36
      8.1    Data Comparability	LA GUIDELINES-36
      8.2    Data Usability 	LA GUIDELINES-38

9.0    REFERENCES  	LA GUIDELINES-40

APPENDIX A

      GLOSSARY	GLOSSARY-1

APPENDIX B

      TECHNICAL IMMUNOASSAY DESCRIPTION 	 APPENDIX-B-1

APPENDIX C

      IMMUNOASSAY TABLES  	APPENDIX-C
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                         REGION I, EPA-NE IMMUNOASSAY GUIDELINES
                          FOR PLANNING ENVIRONMENTAL PROJECTS
1.0    INTRODUCTION

These guidelines provide the most current and comprehensive information available concerning environmental
applications of immunochemical analysis and immunoassay (IA) techniques. Their purpose is to assist IA users in
planning environmental sampling and analysis projects. Integration of immunoassay techniques into the project plan
can cut costs and decrease time spent in the field. Using the information provided in these guidelines, the IA user
should be able to successfully choose an LA kit to meet the project's Data Quality Objectives (DQOs), perform the IA
analyses correctly, and produce usable LA data that meet the project objectives.

Proper planning, including establishing Data Quality Objectives (DQOs) is critical to project success.  If project DQOs
require fast turnaround field analyses at low costs to perform site activities such as hot spot screening, plume
delineation, removal/remedial site clean-up, etc., then LA techniques may be applicable.

IA techniques are not applicable to sites with unknown site conditions and contaminants, or to sites that do not have
established clean-up criteria. Sites with a single contaminant, or only one type or chemical class of contamination,
are the most ideal sites for LA use. LA test kits may not be applicable to sites contaminated with complex mixtures of
chemicals due to interferences arising from the contaminant sources.

This document  is divided  into nine sections.  Section 1.0 provides  an overview of the  document's  purpose,
organization, and information sources. Section 2.0 contains a discussion of the basic principles of immunochemistry
and the format of environmental LA kits.  Section 3.0 provides information on characteristics and availability of
environmental LA kits, and Sections 4.0 through 6.0 specify the physical and chemical constraints to LA use that must
be considered for project success. Section 7.0 builds upon the information furnished in Sections 1.0 through 6.0 to
walk the project planner through the process of selecting LA kits to meet project DQOs.  The comparability and
usability of LA data for making site decisions are discussed in Section 8.0. Finally, pertinent references are provided
in Section 9.0.

These guidelines are not intended to endorse one product or vendor over another for any analytical parameter. They
will be revised as needed to incorporate products that are currently in development and testing stages or that are
otherwise unavailable at this time.

1.1    Information Sources

The information utilized to prepare this guidance document was obtained from the following sources:
Organization Name
Address
D-Tech Strategic Diagnostics
128 Sandy Drive
Newark, DE 197 13
ENSYS
PO Box 14063
Research Triangle Park, NC 27709
Contact Name(s)
Joe Dautlick
Dr. Kevin Carter
Yli Vallejo
Karen McKenzie
Telephone Numbers
(302) 456-6789
(919)941-5509
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Organization Name
Address
Ohmicron
375 Pheasant Run
Newtown, PA 18940
Quantix/Idetek
1245 Ream wood Avenue
Sunnyvale, CA 94089
BioNebraska, Inc.
3820 NW 46th Street
Lincoln, NB 68524
Hach Company
PO Box 389
Loveland, CO 80539-0389
California EPA
DTSC/HML
2151 Berkley Way
Berkley, CA 94704
U.S. Army Corp. Of Engineers
Sylvanus Environmental
Contact Name(s)
Mary Hayes
Tim Lawruk
Richard Lankow
Craig Schweitzer
Brett Poor
Shirley Holmes
Robert Hass
Dr. G. Wolfgang Furs
Kira-Pratt Lynch
Dr. Stephan B Friedman
Telephone Numbers
(800)544-8881
(408) 745-0544
(402) 470-2345
(970) 669-3050
(510)540-2803
(510)540-3076
(206) 764-6918
(919)545-0552
Additional IA information can also be found in the following Publications and Methods from Test Methods for
Evaluating Solid Waste. Physical/Chemical Methods, SW-846,3rd Edition:
Publication
Method 4000
Method 4010A
Method 40 15
Method 4020
Method 4030
Method 4035
Method 4040
Method 4041
Recommended Format and Content for Documentation Supporting New Submittals
Immunoassay
Screening for Pentachlorophenol by Immunoassay
Soil Screening for 2,4 Dichlorophenoxy Acetic Acid by Immunoassay
Soil Screening for Polychlorinated Biphenyls by Immunoassay
Soil Screening for Petroleum Hydrocarbons by Immunoassay
Soil Screening for Polynuclear Aromatic Hydrocarbons by Immunoassay
Soil Screening for Toxaphene By Immunoassay
Soil Screening for Chlordane by Immunoassay
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Method 4042
Method 4050
Method 4051
Soil Screening for DDT by Immunoassay
TNT Explosives in Water and Soils by Immunoassay
Hexahydro-l,3,5-Trinitro-l,3,5-Triazine (RDX) ir. Soil and Water by
Immunoassay
Section 9.0 of this guidance document contains other pertinent IA references.

2.0     ENVIRONMENTAL IMMUNOASSAY
2.1
       Glossary of Terms
IA users must understand  the scientific  and non-scientific terms related to immunochemical processes  and
immunoassay techniques prior to considering its use for a project, LA users include personnel such as project planners,
project chemists, and field samplers. The term "IA user" is employed in Sections 2.0 - 6.0 to denote a range of
personnel that might encounter and/or utilize IA technology, while the term "project planner" is used in Sections 7.0
and 8.0 to denote actual project planning responsibilities.

Appendix A contains a comprehensive glossary which defines the terms used throughout these guidelines and the
vendor literature.  Basic IA principles are described in Section  22, while a more detailed technical discussion ot"
immunoassay, which is suitable for more advanced IA users, is provided in Appendix B.

2.2     What is Immunoassay'.'

Clinical chemists have utilized immunoassay techniques to detect and quantify- proteins, hormones, and drugs for
decades  Currently, lAs are used in home pregnancy tests as well as in commercial laboratories to detect the presence
of the HIV virus and the use of illicit drugs

Immunoassay for environmental chemicals began in the 1970s  with the analysis of selected pesticides.  The most
common version of environmental IA analysis is called ELISA (Enzyme Linked Immunosorbent Assay). ELISA is
an immunoassay method that uses antibodies and enzyme conjugates  to detect and quantify target compounds,
otherwise known as compounds of interest (COIs), in field samples.

                                 Antibodies are proteins produced by the
                                 mammalian  immune  system  that  can
                                 specifically bind with COIs.  An antibody
                                 and  its target  chemical  compound  fit
                                 together like a lock and key as depicted in
                                 Figure 1.  An  enzyme conjugate is  an
                                 enzyme to which  a COI is bound  as
                                 depicted in Figure 2  The  COI portion of
                    Antibody  the enzyme conjugate can bind with the
                                 antibody as  can the  COI  present in the
                                 samples (shown in Figure 3).  ELISA tests
                                 are considered to be "competitive" assays
                                 because the sample-derived COI competes
                                 with the enzyme conjugate COI (which is
Figure 1
Antibody / COI
Lock and Key Mechanism
Figure 2

Enzyme Conjugate COI
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                Solid
                Phase
             Solution
                                     Figure 3
Antibody Binding
kept at a constant concentration) to bind to the finite number of antibody sites. The greater the concentration of
sample-derived COI relative to the enzyme conjugate COI, the larger the proportion of antibody sites that are occupied
by the sample-derived COI.

The enzyme portion of the enzyme conjugate serves as a catalyst to change a colorless compound to a measurable
colored product that can be detected instrumentally.  The amount of color produced is inversely proportional to the
amount of sample-derived COI. More color equals less sample-derived COI. Less color equals more sample-derived
COI because all the antibody sites are bound to sample-derived COI and there is less enzyme conjugate present to
catalyze the color reaction.

2.3    Formats for Environmental IA Kits

Each vendor has developed an IA format that they believe to be the most applicable to environmental samples and the
most easily used by project personnel.  Almost all environmental IA kits presently available use the basic format that
is described below. The predominant enzyme used in the enzyme conjugate is horseradish pcroxidase

BioNebraska's kit for the detection of mercury does not use the enzyme conjugate COI system.  Instead, their kit uses
a metal sensitive protein that is fixed to the wall of a test tube. The metal sensitive protein binds to sample-derived
mercury.  The protein/mercury complex then binds to an antibody/enzyme that catalyzes the release of a color agent.
Therefore, the color developed is proportional to the mercury present, not inversely proportional as the other kits are
designed.
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Certain IA kits may have advantages over kits produced by other vendors for dealing with particular sample matrix
or interference problems. However, each vendor's procedures have been engineered and validated for their speciOc
kits.  One vendor's procedures, therefore, cannot be used for another vendor's kit.

       2.3.1   Basic Format

       The basic format for an environmental IA kit is depicted in Figure 4 on the following page and can be
       described as follows:

           1.  The stationary antibody is engineered to react with the COL

          2.  The sample-derived COI and the enzyme conjugate COI compete for sites on the fixed antibody.  This
              is the incubation step that must be precisely controlled.

          3.  The antibody/COI complexes are fixed by design or fixed on a membrane by filtration or by magnetic
              forces.  The complexes are washed to remove the sample matrices, excess enzyme conjugate, and
              reagents.

          4.  The enzyme portion of the enzyme conjugate COI remains complexed to certain antibody sites and
              catalyzes the reaction of the color agents to form a color that is inversely proportional to the amount
              of sample-derived COI.

          5.  The color reaction is stopped chemically.

          6.  The colored product is measured using UV/Visible spectrophotometry.

       Each vendor has a variation of the general procedure described above.  Detailed descriptions of each vendor's
       procedures can be found in their kit's literature. The formats are essentially the same except for how and where
       the engineered antibody is attached to the solid phase, how the sample and reagents are mixed and in what
       order, and how the analytical measurement (colorimetric determination) is performed (as discussed below).
       Regardless of a vendor's IA kit format, the solid phase, antibody, enzyme conjugate and color agents are
       manufactured in a manner that is highly reproducible.

       2.3.2   Antibody Attachment to Solid Phase

       The following types of fixed antibody formats are utilized hi environmental IA kits:

              Antibody attached to the walls of a styrene polymer tube
              Antibody attached to the surface of a polymeric well
              Antibody attached to polymeric latex particles
              Antibody coated onto para-magnetic particles
              Antibody attached to a porous membrane
              BioNebraska uses a metal binding protein fixed to the wall of a test tube and the antibody is then
              added to bind to the immobilized mercury
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                       Figure 4   Immunoassay Reaction Scheme  - ELISA
                       Reagent   Standard
Reagent     Blank
Reagent    Sample
    Step 1

   Reagent
     &
   Sample
   Addition
    Step 2

  Incubation
    Step 3

    Wash
    Step 4

   Addition
   of Color
   Reagents
   StepS

 Colorimetric
Determination
                         Tube 1

                        Standard
                               TubeS

                           Positive Sample
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       2.3.3   Reagent and Sample Additions

       The sequence and timing of reagent and sample additions is critical to the proper use of each IA kit. The
       timing of sample-derived COI and enzyme conjugate COI addition and exposure to the antibody greatly
       influences IA results.  Each vendor requires that the IA user follow the exact sequence and timing for all
       samples, blanks, QC samples and standards. Any deviation from the prescribed procedure can affect IA results
       within and between batches.

       The same warning applies to vendor washing procedures.  The vendors using particles that get trapped on a
       membrane or particles attached to the reaction cell wall via magnetism rely on a very different separatory
       procedure than the vendors whose stationary antibody phase is attached to a tube wall.

       2.3.4   Colorimetric Determination

       The process that produces the analytical signal, or so called reporter system, is unique to each vendor and for
       each IA kit. Vendors use different color agents that produce different colored reactions. The different colors
       require specific wavelengths of UV/Visible light to measure the absorbance of the final solution.  Other
       vendors use a visible light reflectance measurement of the trapped and plated enzyme conjugate/color agent
       complex.  Later in this document, the practical pros and cons of these techniques will be discussed relative
       to sample matrix interferences.

2.4    Immunoassay and Environmental Chemicals

Certain compounds within a chemical class are more toxic than  others and those are the compounds most tested
because of their adverse effects on human health and the environment. Environmental IA kits have been engineered
to detect a single target compound or one or more structurally similar target compounds within a chemical class,
depending upon the compounds present in the chemical class, the molecular size of the target compound(s), and the
specificity of the engineered antibody.

Individual  contaminant compounds,  such  as   pentachlorophenol  (PCP),  trinitrotoluene  (TNT),  2,3,7,8-
tetrachlorodibenzodioxin (2,3,7,8-TCDD), and most of the pesticides, insecticides, and herbicides are analyzed as
single target compounds in individual test kits.

However, IA  is not as specific for some chemical classes of common site contaminants which originate from  a
commercial product or waste by-product. Some examples of these products and by-products are:

       •  Polychlorinated Biphenvls (PCBsV a chemical class comprised of 209 different individual congeners.
          PCBs were used in dielectric fluids for capacitors and transformers, hydraulic fluids, plasticizers, and
          carbonless papers.

       •  Polynuclear Aromatic Hydrocarbons (PAHs): a class of chemicals comprised of multi-cyclic unsaturated
          6 ring compounds mat may or may not be substituted with aliphatic (straight chain) branches.  This class
          has been characterized traditionally by the analysis of 16 or 18 major compounds, but it contains many
          more compounds not normally identified and quantitated.  PAH compounds are traditionally found in
          asphalts, creosotes, coal tars, coal gas residues and heavy grade fuel oils or crude oils.

       •  Total Petroleum^ Hydrocarbons (TPHV a class of chemicals that originate from man-made fuels and
          contain both aromatic and aliphatic compounds. There are many fuels being formulated today for various

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           energy producing purposes.  The most commonly used are gasoline, diesel, and fuel oil #2.  For this
           reason, vendors developed IA kits to:  1) analyze BTEX (benzene, toluene, ethyl benzene, and xylenes),
           which are compounds found in these fuels, or 2) analyze for the fuel mixtures (by using the fuel as a
           standard). Other fuels that are used in aircraft engines and jet turbines, such as aviation gas, JP-4, and JP-
           5, are also of interest in site remediation projects and are IA sensitive.

All three chemical classes can be found at many sites individually or together in mixtures and they can be analyzed
as individual target compounds, product mixtures, or the sum of individual compounds within a particular class.

Vendors engineer IA kits to respond to one target compound, and that target compound may be chosen to represent
a particular chemical class (such as PCBs) or a particular product (such as fuel oils).

IA kit engineers must decide what compound to target from each chemical class during the initial design phase. For
instance, if all of the most prevalent PCB Aroclors found as site contaminants contain one of a specific penta, hexa,
or hepta congener, then that congener would be chosen as the target compound for development of the antibody and
enzyme conjugate used in the IA kit Each Aroclor mixture containing the target compound would then have a certain
level of sensitivity to the resultant LA kit.

Similarly, LA  kit designers must determine what target compound is present in all petroleum hydrocarbon mixtures
that are commonly spilled in the environment. Obviously, some custom manufactured hydrocarbon mixtures would
be excluded  from LA analysis if they did not contain that target compound. Products such as fuel oils, jet fuels,
gasolines, and range oils contain similar compounds at measurable concentrations.

Environmental weathering of petroleum hydrocarbons must also be taken into account when engineering an LA kit.
The petroleum hydrocarbon chosen as the target compound should not be susceptible to weathering, otherwise the kit
will be less sensitive to the weathered fuels. If the LA kit designer chooses a target compound, such as m-xylene, that
is present at measurable concentrations in manmade fuels and is less susceptible to environmental weathering, then
the resultant  LA kit would be sensitive to all fuel oil mixtures at some relative sensitivity.  Petroleum hydrocarbon
mixtures which do not contain m-xylene, such as lubricating oils, would thus not be sensitive to the kit.

The effectiveness of each LA kit for sample analysis will depend upon: 1) the various product mixtures present in the
sample, 2) the kit's sensitivity to the target compound and structurally similar compounds, and 3) the presence of
interferences in the sample. The availability and characteristics of environmental LA kits produced by various vendors
will be discussed in Section 3.0.

3.0     ENVIRONMENTAL IA KITS

3.1     Currently Available Environmental LA Kits

Tables  la and Ib, referred to as the vendor's matrix, contain a compilation of environmental LA kits produced by
particular vendors as of July 1996. The information listed in these tables does not constitute an endorsement by EPA
of any particular vendor or any specific LA kit.  Information is provided solely for reference in identifying potential
IA kit sources. One vendor that produces environmental LA kits failed to respond to EPA Region I's request for
information. Those who did respond have complete information included in this document.  Several vendor mergers
were on-going during preparation of this document. The information presented here is as comprehensive as  possible
under the circumstances.
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3.2    Field Kits Versus Laboratory Based Kits

Most vendors have designed their environmental IA kits for use in both field and laboratory settings.  Laboratory kits
are usually designed for better automation and sample through-put. All of the available field kits can be used by a
fixed or field laboratory as a screening tool prior to sample preparation and/or instrumental analysis.

The one characteristic that differentiates field kits from laboratory based kits is the number of samples analyzed per
batch.  The sequence of standard, blank, samples and QC samples, then the standard and blank set again, constitute
a batch sequence in both settings.  Only the finite number of samples between the standard and blank sets changes.
Field kits recommend performing less samples (4-6) between standard and blank sets, whereas laboratories will set
up banks made up of several batches or sequences of up to ten samples each (possibly 40 samples at one time). The
ratio of QC samples to field samples should be the  same for both settings (approximately 10% duplicates and 5%
spikes or PE samples). The exact number of QC samples will depend on the minimum number of samples run in a
batch.  Quality control requirements for environmental IA kits will be discussed in Section 3.3.3.

The 2,3,7,8-TCDD kit produced by ENSYS is designed only for fixed laboratory use due to the toxicity of the reagents,
their ban from on-site use, the requirement for a solvent exchange step, and the disposal requirements for the derived
waste.

3.3    Quantitative, Semi-Quantitative, and Qualitative IA Kits

Most vendors have designed their IA kits to be used in one or more of the following modes depending, in part, on the
number of standards analyzed by the IA user:

       •   Kits that produce results that are analytically quantitative from a specified lower detection limit to a linear
           upper limit.

       •   Kits that produce semi-quantitative results one of two ways:  1) above or below  a specified detection limit
           (Action Level or so called Go/No-Go test) or 2) between an upper and lower  concentration range.

       •   Kits that produce qualitative results.  These kits are designed to detect the presence or absence of a
           specific COI. The detection limit for this type of kit is usually conservatively set (based on field trials)
           by the manufacturer but may be uncertain due to the composition of the sample matrix and the presence
           of interferences.

Most environmental IA kits are utilized in the either the quantitative or semi-quantitative mode.  Therefore, the use
of LA kits that produce qualitative results will not be addressed in this document.

In general, environmental LA kits produced by D-Tech, Ohmicron, and Quantix/Idetek are designed to provide
quantitative or semi-quantitative results while those produced by ENSYS and BioNebraska are designed to provide
semi-quantitative results only.

Regardless of whether LA kits are used to produce quantitative or semi-quantitative results, there are two conditions
which must be met for LA data to be considered usable.

The first condition is that quality control (QC) procedures must be performed at the correct frequency and must meet
the criteria specified in the pre-approved project  Quality  Assurance Project Plan/Sampling and Analysis Plan
(QAPjP/SAP). These key QC elements are discussed in Section 3.3.3.

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The second condition is that IA results for a representative number of samples (10% minimum) must be confirmed
through the use of split samples.  Split samples should be collected throughout the entire sampling and analysis
episode and should be prepared and analyzed using conventional full protocol analytical methods performed in a fixed
laboratory or field laboratory (mobile or transportable) setting.  The split sampling results obtained using both
analytical methods must not deviate from the criteria specified in the pre-approved project QAPjP/SAP.  Data
comparability and usability will be discussed in Section 8.0.

       3.3.1    Quantitative IA Results

       Quantitative results can be obtained with IA technology as proven by the work performed daily in the medical
       diagnostic field. Environmental IA kits can be used to produce quantitative results when the two conditions
       listed above have been met along with the following requirements:

           1.   A multi-level linear calibration curve is generated which brackets the detection limit and the highest
               sample concentration. The calibration curve must meet the requirements specified in the pre-approved
               QAPjP/SAP. Deviations from the calibration curve at the upper and lower ends of the curve will yield
               inaccurate IA results.  Also, calibration stability and accuracy must be periodically checked through
               the analysis of continuing calibration check samples (at a rate specified by the vendor) that meet the
               criteria specified in the pre-approved QAPjP/SAP.

           2.   A project-specific split sampling field study is performed prior to full scale implementation of LA
               techniques at a site, and the LA results correlate with the conventional full protocol fixed or field
               laboratory results.  The split sampling results obtained using both analytical methods must not deviate
               from the criteria specified in the pre-approved project QAPjP/SAP.

       Quantitative LA  is enhanced if the source of the COI (i.e., product) is known and is used as a calibration
       standard for LA analysis.

       3.3.2   Semi-Quantitative LA Results

       The SW-846 methods that have been promulgated to date are performed in one of the semi-quantitative modes
       and are applicable for screening soil and water samples. LA kits for those methods are designed to estimate
       the concentration of a COI above or below a specified detection limit with only one standard as a reference
       point.  The concentration of that standard should be less than, but  in the same order of magnitude as, the
       project required Action Level. The concentration of the COI relative to the LA kit detection limit is engineered
       into the kit by the vendor, and is influenced by analysis precision, sample matrix interferences and other
       performance characteristics and limitations of the basic method.

       Vendors also produce semi-quantitative LA kits that are designed to estimate the concentration of a COI above
       a lower limit (for example, >1 ppm) and below an upper limit (for example, <10 ppm). These kits require the
       analysis of two standards as reference points and are, therefore, generally more accurate than the single
       standard semi-quantitative kits discussed above. Future formats will follow this general scheme.

       LA vendors have intentionally designed the possibility of false positives into their semi-quantitative kits so
       that LA users will make conservative site removal/remedial clean-up decisions.  False positives are generally
       defined as a positive response for a sample that contains the COI below the claimed detection limit.
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It follows, therefore, that semi-quantitative IA kits should yield virtually no false negatives. False negatives
are defined as a negative response for a sample containing the COI at or above the stated detection limit.  In
a situation where immunoassay will be used to detect PCB contamination in soil at 1 ppm, the IA kit has been
designed so that the 1 ppm PCB reference standard actually contains less than 1 ppm PCB. This reference
standard is positioned to minimize the incidence of false negative results at the claimed detection limit.

The vendor measures the false negative rate of a semi-quantitative IA kit by analyzing split samples using the
IA kit and a separate full protocol analytical method in a Gxed laboratory. SW-846 allows semi-quantitative
IA kits to produce a maximum of 5% false negatives at the specified detection limit.  In general, SW-846
methods are designed for LA use in a Geld setting to delineate contamination or to confirm clean-up during
site removal/remedial  activities.  False negatives cannot be tolerated when site clean-up and closure is the
sampling objective.

Each vendor that produces a semi-quantitative LA kit generates method performance data for the percentage
of false positives and false negatives. LA users should obtain and review vendor literature to determine the
false positive and false negative rates for individual LA kits.

When quantitative IA results are produced in accordance with the requirements in Section 3.3.1, false positives
and false negatives are not an issue because the detection limit  and highest sample concentrations are
bracketed by a linear calibration curve. (It should be noted that the concentration of a particular kit's detection
limit must be chosen conservatively so that it is above the kit's finite limit of detection. Otherwise, false
negatives may become an issue with samples mat have COI concentrations at or near the kit's chosen detection
limit.)

3.3.3   Kit Standardization and Quality Control

Every analytical process that determines the identification and/or concentration of target compounds in some
media must have associated quality control (QC) elements.  Key QC elements must be performed for all LA
kits that are utilized in a field or laboratory setting, and the criteria specified in the pre-approved project
QAPjP/SAP must be achieved for each key QC element The key QC elements for LA analyses include process
calibration, the analysis of continuing calibration checks, blanks,  duplicates, and performance evaluation
samples.  Note, in developing the required QC elements for a particular project, the LA user should consult
each vendor's LA kit instructions, which contain recommended QC requirements that have been tested and
validated by the vendor.

Documentation that all key QC elements were performed and met project requirements is essential, regardless
of intended data use. The preparation and analysis of each batch of samples, including related standards, QC
samples and blanks, should be recorded in a field or laboratory notebook, run logs, and/or tabulated forms.
Note, sample preparation should include kit lot numbers and expiration dates, and the ambient temperature
at which the tests were performed.

Samples should not be analyzed until project QC criteria have been met. When pre-approved project QC
criteria have not been met, appropriate and effective corrective actions must be immediately implemented and
documented. Samples analyzed after the last in control QC sample  should be re-prepared and/or reanalyzed
after effective corrective action has been implemented.  The project QAPjP/SAP should document the
corrective actions that will be taken when each key QC element does not meet the project QC criteria. The
content of QAPjPs/SAPs will be discussed in Section 7.6.
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3.3.3.1     Calibration

IA calibration using standards of known concentrations is performed to determine the sensitivity and
detection/calibration range for the IA kit.

Calibration of a semi-quantitative IA kit performed in the Action Level (Go/No-Go test) mode, uses
one calibrator comprised of a standard that contains the target compound at the detection limit.  A
reagent blank should also be included to provide the reference color generated  when no target
compound is present Calibration of semi-quantitative IA kits can also be performed in the detection
range mode.  Here, two calibrators are analyzed to delineate a detection range (i.e., a 1 ppm standard
and a 10 ppm standard). Regardless of the semi-quantitative mode employed, the concentration of
the single standard or lowest of two standards must be below the project Action Level.

Calibration of quantitative IA kits must be performed using multiple calibrators and a calibration
(dose/response) curve must be developed. Usually, one of the calibrators is a zero point. In some
situations, the product mixture specific to the site can be procured, prepared and diluted properly, and
used as the calibrator for the IA analysis. For this to work effectively, the LA user must develop their
own dose/response curve for that product mixture. That calibration curve must have a set correlation
coefficient ("r" or goodness of fit) to be acceptable. The normal "r" would be 0.995, but an acceptable
correlation coefficient may be as low as 0.990, depending upon the specific LA kit. The LA user must
consult the vendor to obtain the proper "r" specification.

Continuing calibration checks must be performed to evaluate calibration accuracy and stability for
each batch analyzed using semi-quantitative and quantitative  LA kits.  Multiple standard initial
calibrations should be performed at the beginning of each batch of samples. If samples are from
different areas of the site, or temperature or weather conditions change, then full calibrations should
be performed before and after each batch.  In the field setting, every 4 to 6 samples should  be
bracketed by a continuing calibration standard.  The absorbance of the continuing calibration standard
should not vary more than 20% from the absorbance of that standard in the initial calibration.  Vendor
LA kit instructions usually define how many samples can be successfully analyzed between standards.
If the continuing calibration standard is not within 20%, then a full calibration should be performed
and all samples run prior to the non-compliant standard should be rerun..

3.3.3.2     Blanks

Analysis of blanks must be performed to evaluate the presence of contaminants originating from
sampling and analysis activities. Equipment blanks should be collected and analyzed to evaluate the
effectiveness of equipment decontamination procedures performed in the field.  Reagent blanks
should be prepared and analyzed with every batch, or chosen sequence of samples, to evaluate the
purity and reactivity of reagents used in the LA kits and assist the LA user in ascertaining the kit's
response when no target contaminants are present.  Blanks represent the highest absorbance of color
and indicate the lack of the COL  They are also used to compare the color generated by the blank with
the color generated at the detection limit concentration.  Blanks should be run with each batch of
samples and should not show contamination above the kit's detection limit. If contamination is found
in the reagents or the equipment rinsates, then  steps must be taken to determine the cause and
eliminate the contamination. Samples should not be analyzed until the blanks meet the vendor's
recommended acceptance criteria.
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               3.3.3.3     Duplicates

               Duplicates must be analyzed to evaluate sampling and analysis precision. Field duplicates measure
               the precision of the IA test as well as the sample homogeneity. Field duplicate precision should fall
               within 30% D for waters and 50% D for soils. Duplicate precision should be tighter for IA kits that
               are utilized in a laboratory setting.  Duplicates should be prepared and analyzed at a frequency of 1
               per 10 samples, or 1 per batch of samples prepared, whichever is greater.  Duplicates should be
               performed at a greater frequency where samples are known to be less homogeneous.

               3.3.3.4     Performance Evaluation Samples

               Performance evaluation (PE) samples (or some form of independent control sample) must be analyzed
               to evaluate qualitative and quantitative accuracy for each IA kit batch following the requirements
               contained in the EPA Region I Performance Evaluation Program Guidance, dated July 1996 or most
               recent revision.  PE samples should contain the target compound at or near the project Action Level.
               Depending on the batch size of the individual analysis episode, a PE sample should be run at least
               once in 20 samples or once per day, whichever is greater.  The PE sample must be analyzed under the
               identical conditions that the calibrations, blanks, field samples and PE samples are analyzed. If many
               sets or batches are analyzed under the same conditions during one  day, then one PE per day is
               recommended. If there are changes in field conditions (temperature and  relative humidity) during the
               sampling episode, then a PE sample should be analyzed more frequently.  Poor PE sample score
               results may indicate incomplete sample extraction, operation  of the LA kit outside its required
               operating temperature ranges, or inconsistent timing of reagent additions and performance of batch
               processes.

3.4    Kit Detection Ranges

Each LA kit is designed to function within a particular detection and/or calibration range, depending on whether the
kit produces quantitative, semi-quantitative, or qualitative data. The LA user must ensure that LA kit detection limits
are lower than project Action Levels.

 LA kits are usually more sensitive than is needed for most environmental studies.  This  sensitivity generally requires
the LA user to dilute soil sample extracts to bring the COI concentrations into the LA kit's detection/calibration range.
Therefore, the LA user must determine the COI concentrations expected in the soil samples prior to kit selection.
Historical site information and previously generated site data are the best sources for obtaining those details. The LA
user then must ascertain sample homogeneity, sample weight/volume to be extracted, and required dilution factors to
be utilized on project samples.

Vendor instruction guides usually detail step-by-step procedures for performing their specific assay on soil matrices.
Several vendors have simplified this process by developing a formula to calculate the required dilution factor. Other
vendors have ready-to-use dilution kits available to simplify LA use.

Vendor consultation by the LA user to determine the proper kit and to obtain the correct operating instructions for the
project-specific sample matrix is critical to project success.
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4.0    KIT SENSITIVITY

IA kit sensitivity is expressed in a manner similar to other analytical techniques, i.e., detection limit or quantitation
limit.  Sensitivity is a function of the operational consistency and the signal-to-noise characteristics of the dose-
response curve.

Each vendor expresses their detection limits differently.  Some vendors use the %B/B0 convention. Here, B0 is the
absorbance of the zero standard (i.e., blank) and represents the most highly colored standard (because no sample-
derived COI is present and, therefore,  enzyme conjugate COI is bound to all antibody sites so maximum color is
produced  and measured). %B is the relative absorbance of samples or standards in relation to BO, the highest
absorbance.  Samples and standards must be less colored than the blank absorbance (B0).  The lowest detectable
concentration of pure analyte (sometimes referred to as dose) that can be differentiated from the highly colored zero
standard is defined as the detection limit. Some vendors set this detection limit as a numerical 90%B/B0.  90%B/B0
means that at values below 90% of the blank absorbance (B0), the analyst can begin to differentiate a change in color.
Likewise, at values between 90% of the blank absorbance and the blank absorbance value, the analyst is unable to
differentiate a difference in color with any certainty.

Other vendors define detection limit as the concentration necessary to result in a positive detect measured at some set
confidence limit. The confidence limit and the definition of the detection limit must be known if the IA user is to
compare vendor kits and, ultimately, choose the correct kit.

The following describes each vendor's approach to establishing the detection limit or limiting detectable dose (LDD)
as the measure of sensitivity for their LA kits.

Ohmicron:

Ohmicron defines limiting detectable dose as 90% B/B0. This means that a compound can be reliably detected at 90%
of the most colored zero standard.

D-Tech:

D-Tech defines the minimum detection limit as the lowest concentration of a compound that yields a positive test.
Each kit has a specific confidence limit that has been set by the vendor for the indicated lowest concentration (+/-18%
BTEX, +/-20% PCB, and +/-25% PAH).  A 96% level of confidence occurs within the vendor-specified range for each
kit. This means that 96% of the time, the analyst will get a positive test result.

Ouantix/Idetek:

The minimum detection limit is defined as the compound concentration required to produce 20% inhibition in the
immunoassay. This is essentially 80% B/B0. The confidence limits for these LA kits are unknown.

ENSYS:

ENSYS defines detection limit as the concentration necessary to result in a positive detect greater than 95% of the time
when tested at the stated concentration level. This approach is less limiting and allows for the determination of the
level at which the user can see a positive result compared to the zero result.
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BioNebraska:

BioNebraska defines their detection limit for mercury as a mean of three replicate values and the corresponding
standard deviation.  They do not define a method detection limit, only a detection limit for water and soil.

5.0    KIT SELECTIVITY

IA kits are designed to react to a single target compound or to one or more structurally similar target compounds within
a chemical class. This is termed reactivity.  The concept of reactivity was introduced in Section 2.4 and it will be
discussed in greater detail in Section 5.1.

Non-target compounds that are structurally similar to the target compounds may also react with the antibody. Those
non-target compounds are  considered to be "cross-reactive"  because they will compete for the finite number of
antibody binding sites, which will impact the color development and yield false positive sample results.  During IA
kit development, designers characterize cross-reactivity by adding increasing amounts of potential cross-reactive
substances to a sample and measuring their IA response.  Cross-reactivity will be discussed in greater detail in Section
5.2.

The engineering of the antibody/COI in tandem with the enzyme conjugate controls the selectivity of the LA kit to
particular target compounds and non-target compounds. Figure 5 depicts the relationship of selectivity and sensitivity
to both reactivity and cross-reactivity. Reactive and cross-reactive compounds have finite sensitivities to individual
LA kits.


                                     Figure 5       IA Selectivity
                                          SELECTIVITY
                        Target Compound                    Non-Target Compound
                            Reaction                              Reaction
                REACTIVITY      \                      (  CROSS-REACTIVITY
                    (Table 2)          /                      I           (Table 3)
                        Target Compound                    Non-Target Compound
                        Detection Limits                        Detection Limits
                                          SENSITIVITY
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S.I     Kit Reactivity

The reactivity and sensitivity of each vendor's kit to specific target compounds and product mixtures is detailed in the
following tables:

Table 2a:   TPH/BTEX Reactivity
Table 2b:   PAH and c-PAH Reactivity
Table 2c:   PCB Reactivity
Table 2d:   TNT/RDX Explosives Reactivity
Table 2e:   Individual Analyte Reactivity (mercury, pentachlorophenol, trichloroethylene, perchloroethylene, 2,3,7,8-
           TCDD, and benzene)
Table 2f:   Individual  Pesticide, Insecticide, and Herbicide Reactivity

These tables are designed to show the most sensitive compounds/mixtures within a chemical class that will react to
a particular vendor's IA  kit. The lower the detection limit number present in the tables, the more sensitive the IA kit
is to that compound/mixture. The sensitivities were established by each vendor after testing their LA kits with the
individual compounds or product mixtures.

TPH kits are normally designed to be sensitive to gasoline, kerosene, stoddard solvent, and the lighter fuel oils that
contain o-xylene, m-xylene,  and p-xylene. Some LA vendors use m-xylene as the indicator compound because it
naturally occurs in all these fuel mixtures. BTEX kits are also normally designed to be sensitive to one of the xylenes
and its quantitation represents that of BTEX (as per the design of the kit). The type of standards used to calibrate both
types of kits differentiate the use of the kit. Fuels or BTEX that are weathered and have lost the benzene, toluene,
ethylbenzene, and xylenes have little sensitivity to LA kits for the quantitation of TPH or BTEX.

All of the TPH and BTEX kits that are contained on Table 2a will react (to some degree) with toluene, ethyl benzene,
and other xylenes, and will cross-react wiiii uaputhalene and styrene. These kits will generally not react with benzene.
Because similar reactive compounds are found in common fuel products and because these LA kits have some relative
sensitivity to each of these reactive compounds (with the exception of benzene), then each common fuel product has
its own finite sensitivity to a particular vendor's IA kit.  For some kits, that sensitivity is directly related to the
concentration of the most reactive compound(s) contained within the fuel. Fuels that contain more m-xylene, such as
blended fuel oils, are overall more sensitive to LA kits.

Most PAH kits, on the other hand, are designed to be reactive to a  single representative PAH compound.  This
compound defines the type of ring structure (2, 3, or more rings) for which the LA kit will be sensitive. Phenanthrene
is 'die representative PAH compound most commonly used for general PAH kits so that those kits are most reactive
to 2, 3, and 4-ring PAH compounds (i.e., PAHs present in fuel oil #2, diesel, and kerosene).  Phenanthrene, however,
is not a good choice as a representative PAH compound if the 4, 5, and 6-ring PAHs are required to be measured (i.e.,
PAHs present in creosote, coal tar, and fuel oil #6).

For this reason, one vendor offers a carcinogenic-PAH kit which keys in on the 3, 4, 5, and 6-ring PAHs and can be
used to measure JP-4, mineral spirits, and mixtures of fuel oil, coal tar, and creosote.  The reactivity of PAH kits is
based more on each vendor's marketing philosophy relative to their niche  in the environmental market (chemical
products/mixtures to be measured that are sources of environmental contaminants). Later in this document there will
be a discussion of LA kit selection when the project planner is faced with a site that contains mixtures of PAHs
originating from multiple chemical products.


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       5.1.1   Compounds with Little Sensitivity

       Because IA kits are engineered to have reactivity to a limited set of compounds within a compound class, some
       compounds of that class will have no sensitivity or very limited sensitivity. An example is benzene in the
       BTEX class. Benzene, by itself, has little sensitivity to the TPH/BTEX kits because it is structurally different
       than the indicator compound, m-xylene (or o-xylene, p-xylene), to which the antibodies were engineered to
       bind.

       Benzene kits have been produced, but the kits require derivatization of the benzene molecule to a nitro-
       substituted benzene, which is then amenable to analysis by an IA kit specific to nitrobenzene.

       Benzene's insensitivity to TPH/BTEX kits would only inhibit a project where benzene is the only COI for a
       project. If gasoline is the product/mixture of concern for a project, then a TPH/BTEX kit can be utilized
       because it is sensitive to gasoline, even though it has little sensitivity to the benzene that is present in the
       gasoline mixture. If the exact concentration of benzene is needed for risk assessment purposes, then another
       analytical method must be utilized.

       Most PAH kits have little sensitivity to naphthalene. PAH kits are nominally engineered around phenanthrene
       or larger ring compounds, such as fluoranthene or pyrene, depending upon the vendor. If naphthalene is the
       prime COI for a project, then the TPH/BTEX kit should be used because most TPH/BTEX kits have excellent
       sensitivity to naphthalene.  If PAHs or c-PAHs are of concern for a project, then a TPH/BTEX kit can be used
       to measure naphthalene and a PAH or c-PAH kit can be used to measure other PAHs, depending on the project
       DQOs.

       Some vendor PAHs kits have little sensitivity to benzo(g,h,i)perylene and dibenzo(aji)anthracene. If those
       PAH compounds need to be measured, then another vendor's LA kit or a conventional full protocol analytical
       method must be utilized.

5.2     Cross-Reactivity

Cross-reactivity is defined in SW-846 as: "The relative concentration of a non-target substance that would produce
a response equivalent to a specified concentration of the targeted compound." In quantitative or semi-quantitative
immunoassay, it provides an indication of the concentration of cross-reactant that would produce a positive response.

Cross-reactants react with the antibody because they have similar three dimensional structures as compared to the COI.
To prevent cross-reactivity the LA engineer must produce  a highly selective antibody.  Many times this is not
economically possible and may be less advantageous for environmental pollutants. In many cases, chemicals or groups
of chemicals may have degradation products or biological metabolites that will also have sensitivity to the antibody.
This type of cross-reactivity may work to the advantage to the project because the test is more comprehensive.

Cross-reactivity can yield more robust analyses allowing the LA user broader selectivity when entire groups of
compounds such as PCBs, PAHs, TPHs, and Dioxins are being investigated. The down side to broader selectivity is
that the IA user cannot measure the  reactivity of individual compounds and has no measure of what substances
contributed to the LA response. This can be a problem when the LA user is determining the comparability of LA data
and data generated for split samples in the fixed laboratory using conventional full protocol methods. Vendors can
only test the antibody response to individual cross-reacting compounds.  No vendor has had the research time to
determine the additive effects of various combinations of cross-reactants.


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Cross-reactivity for individual compounds is often calculated as the ratio of target substance concentration to the cross-
reacting substance concentration at 50% inhibition of the immunoassay's maximum signal X 100%.  The 50% B/B0
tables that are available in the vendor literature are very important resources when the project planner is faced with
determining the effect of cross-reactive substances. Some vendors measure the amount of cross-reactant that gives
the lowest response (similar to determining the detection limit of the COI). This number allows the user to determine
what effect the individual cross-reactants will have on analysis of the sample-derived COIs.

The antibody response that occurs in a field sample, however, is the composite of all possible reactants and cross-
reactants.   This fact requires the LA. user to test representative site samples by the chosen LA technique and by
conventional full protocol analytical methods prior to initiating a full scale field sampling program. Full protocol
analytical methods produce instrument responses that are comprised of the target compounds quantitated individually
(and summed to get a total), whereas LA kits produce results that are composites of all reacting and cross-reacting
compounds. Cross-reactivity of unknown interferences may result in the IA user eventually developing a valid
correlation factor between LA results and results generated using full protocol analytical methods.

hi many situations, the IA kit can react with far more substances than can be measured by full protocol methods, thus
biasing the LA results on the high side. This can be the case with PAHs, c-PAHs, and PCB mixtures. IA kits will also
over-estimate the total compound concentrations if dry, non-organic soil  conditions are present on site and if the
extraction efficiency of the sample matrix is good. Later in this document, this situation will be discussed in detail.
Many of the LA kits have built in the bias factor because the kits have been standardized with complex mixtures of
fuels and other naturally occurring compound mixtures.

It is very  important that the LA user know the substances that may be encountered on  site and the effect those
substances will have on a particular LA kit's performance. The cross-reactivity and sensitivity of each vendor's kit to
specific target compounds and product mixtures is detailed in the following tables:

Table 3a:   TPH/BTEX Cross-Reactivity
Table 3b:   PAH and c-PAH Cross-Reactivity
Table 3c:   PCB Cross-Reactivity
Table 3d:   TNT/RDX Explosives Cross-Reactivity
Table 3e:   Individual Analyte Cross-Reactivity (mercury, pentachlorophenol, trichloroethylene, perchloroethylene,
           and2,3,7,8-TCDD)

These tables are designed to show the most sensitive compounds/mixtures within a chemical class that will cross-react
with a particular vendor's LA kit The lower the detection limit number present in the tables, the more sensitive the LA
kit is to that compound/mixture. Each vendor's pesticide application sheets should be reviewed to ascertain what
compounds/mixtures will cross-react with their particular LA kits.  Using Tables 3a through 3e, the vendor literature,
and the site information, the correct LA kit can be selected and tested on field samples, in conjunction with split
samples analyzed using a full protocol analytical method, prior to initiation of the full scale field sampling program,
to facilitate interpretation of sample data generated in the presence of reactive and cross-reactive substances during
the full scale field  sampling program.

6.0    KIT OPERATIONAL CONCERNS

Analysis of environmental chemicals using conventional full protocol analytical methods has many pitfalls and
nuances.  The same is true for performing LA analyses.   LA analyses are impacted by kit storage and operating
circumstances, field conditions, and sample matrix characteristics. These factors, in conjunction with cost and time
savings considerations, can influence LA kit selection and operation, and will be discussed in this section. In certain

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situations, the combination of physical, chemical, and ambient sampling conditions could make the use of IA less
feasible.

6.1     Temperature Range Considerations and Shelf Life

There are three basic storage and operating considerations that are critical to the effective use of IA kits:

       1.  Proper storage of the kits prior to use and when not in use.

       2.  The optimum operating temperature range of the kits.

       3.  Shelf life of the kits.

Each vendor, during IA kit development, investigates the proper kit storage conditions, the ideal operating temperature
range, and the shelf life for each LA kit as discussed below.  Table 4 details the storage temperature, operating
temperature, and the shelf life of the LA kits available at this time.

       6.1.1    Storage Conditions

       Most LA kits should be stored at less than ambient temperatures (2°C to 8°C) prior to use. Kits should not
       be used beyond their shelf lives and/or expiration dates.  LA kits  must always be  brought to ambient
       temperature just prior to use.

       Some vendors have studied the degradation of kits held at elevated temperatures (up to 37°C,  Ohmicron
       Quality Control of Immunoassays for Pesticide Residues, Mary Hayes et. al.) and found that the kits are
       susceptible to degradation at elevated temperatures.  The LA user is urged to adhere to the kit storage and
       operational temperature ranges recommended in Table 4 and in the vendor's product literature.

       6.1.2    Operating Temperature Range

       The operating temperature range of an LA kit is one of the most important criteria for generating precise and
       accurate data.  Use of LA kits in the field must be at temperatures that do not inhibit or advance the kit's
       recommended processing time sequences.   Each vendor has specific criteria for the sample extraction,
       incubation, washing, color development and stop procedure. These critical timing sequences must be adhered
       to as much as possible. It should be noted that  D-Tech's PCB literature contains a graph that depicts the
       time/temperature relationship for that kit That graph should not be used  to determine sample incubation tune;
       it should be used solely as a guide or estimate to complete the color development step.

       If large rises or drops in operating temperature occur during a field episode, then the LA user must ensure that
       standards, blanks, QC samples, and field samples are all analyzed at the  same relative temperature conditions.
       In order to obtain comparable data within and between batches as well as from one sampling episode to
       another, the operating temperature for all analyses must be within the required range.

       In Northern climates, where sampling may take place at temperatures below 50°F or even below 40°F, all LA
       kits will exhibit problems in reaction times for incubation and color development. The sluggishness of the
       kits at low operating temperatures will usually result in false negatives. This can be avoided if the LA user
       moves LA kit operation into a heated enclosure or field trailer.


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       Another problem arises when IA kits are utilized to analyze field samples during the day under normal ambient
       conditions (60-80°F) and later in the day when the temperature drops close to or below the lower limit of the
       operating temperature range (40°F).  The data generated at 60°F will not be comparable to the data generated
       at 40°F using the calibration curves and QC samples analyzed at 60° F. IA testing should be stopped if a full
       calibration sequence at 40 °F cannot be analyzed.

       There are few instances when the ambient temperature exceeds the upper limit of an LA kit's recommended
       operating temperature range. In those circumstances, the entire sequence of standards, blanks, QC samples,
       and field samples should also be analyzed at the same relative temperature.

       6.1.3  Shelf Life

       Since LA kit reagents are biological media, the vendor and the LA user must be concerned with the length of
       time that the reagents can produce usable results.  The vendor must identify the maximum length of time that
       the antibody, enzyme conjugate, and color reagents will last in order to determine the LA kit's shelf life and,
       therefore, the number of kits that can be kept in stock. Many of the vendors date each kit so that the LA user
       can tell when the kit will expire.

       The LA user must be attentive to LA kit shelf life to maintain a current inventory of usable kits.

6.2    Water Characteristics

Water analyses using LA have far less matrix-related problems than do soil analyses  because filtration  is the only
sample preparation step (i.e., extraction/dilution is not required).  However, the analysis of water  does warrant
precautions.

LA analyses of water will be affected by sample pH, high concentrations of metals or salts, high ionic strength, and the
presence of other soluble natural organic components. Insoluble organics that form emulsions, colloids, floating films
and DNAPL may create problems with sample extraction, antibody complexation and/or the color development step.
Each vendor has designed their kit to be used under specified conditions and most vendors have designed their reagent
systems to buffer the water to the correct pH and ionic strength.

Vendors have also tested their kits under non-ideal conditions and have developed recommendations for the user when
these conditions are encountered on site. In some situations, vendors can supply conditioning reagents  to mitigate
matrix interferences. Generally, vendors will only provide recommendations for possible sample adjustments. A
representative water sample should always be tested prior to initiation of the full scale sampling episode to determine
if a condition exists that will cause the LA kit to incorrectly quantitate the target compound(s). This is similar to pre-
testing soils prior to initiation of full scale field sampling episodes.

6.3    Soil Characteristics

The analysis of soils by LA is much more problematic than waters due to the magnitude and frequency of matrix-
dependent interferences and limitations.  The type of soil greatly influences water retention and adsorption of organic
compounds. The physical characteristics of the soil, mainly particle size and organic content, play a large role in
affecting the adsorption and retention of organic compounds, especially chlorinated organics. The smaller the particle
size of the soil, the larger the available surface area. The greater the surface area and organic content of the soil, the
more sites there will be for the target compounds to adsorb onto and remain attached during the extraction process.
Together, these characteristics may greatly inhibit the quantitative extraction of target compounds from soil samples.

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Sandy soils are easier to extract because they have less available surface area and  less organic content.  Soils
containing increasing amounts of silt, clay and organic content are much more difficult to quantitatively extract.

Other factors that may effect the extraction of soils include pH and the cation exchange capacity.  Some of the organic
target compounds, such as acid herbicides, may be in the salt form and, therefore, they will have poor extraction
efficiencies.

Soils that are highly colored, or that cause highly colored solutions upon extraction, can be problematic for IA. The
high color may adhere to the antibody/COI complex and interfere with the color development stage. For the D-Tech
kits that concentrate the antibody/COI complex  onto a membrane or a plastic well and then use a reflectance
measurement to measure the color, the highly colored soils/solutions may increase the color reading, yielding false
negatives or low measurements. IA kits produced by all the other vendors wash away the non-complexed sample and
form the color reaction in solution. Those kits, therefore, are less prone to this problem since the excess sample matrix
is removed prior to color development.

When PCB oils are encountered in soils or sediments, the presence of the oil will interfere with the reaction of the
antibody with the COL Although methanol extraction will normally dissolve the oil and put the oil in solution, when
the extract is introduced into the water-based buffer and conjugate solution, the oil may precipitate or form an emulsion
that coats the antibody and  prevents the enzyme conjugate COI, and  sample-derived COI from reacting with the
antibody. This action will yield results that are biased high. It is recommended that samples contaminated with PCB
oils be considered as pure oil samples, and an appropriate mineral oil waste kit be used to approximate the PCB
concentration.

6.4     Extraction Solvents

In most cases, methanol was chosen by LA kit designers as the extraction solvent for soil/solid and wipe matrices
because it is infinitely soluble in water, doesn't denature (decompose) the antibody or enzyme conjugate, and doesn't
inhibit reactions between the antibody and the COI.  Isopropanol (2-propanol) is used by Quantix/Idetek for the same
reasons as methanol, and has the added bonus that isopropanol is less toxic to the user. Many attempts have been made
by LA kit designers to use more efficient extraction solvents such as methylene chloride, hexane, acetone, and ethyl
acetate, but when those solvents were carried through IA analysis they caused unacceptable changes in the slope of
the LA calibration curve and its sensitivity.

IA analysis of waterbome or soluble contaminants is not a problem since the LA reagents are water-based and no
dilutions are needed. Methanol (or isopropanol) extracts of soils/solids and wipes must be diluted into water prior to
incubation with the antibody and the enzyme conjugate. The eventual dilution of the methanol also helps to preserve
the aqueous antibody/COI reaction.

Although methanol is the solvent used by most LA kit vendors for extraction of soil/solid and wipe samples, it has
limitations that must be addressed by the LA user during project planning. Methanol's extraction efficiency for soil
and solid matrices can be diminished in the presence of large quantities of water (soil/solid matrices containing greater
than 30% water).  Water dilutes the methanol and reduces (limits) methanol's solubilizing properties, especially for
higher molecular weight organic compounds. With high moisture soil/solid samples, the water is also weighed with
the soil/solid material, thus less soil/solid material is present to be extracted. These two factors limit the LA kit's ability
to extract and quantitate COIs at the recommended detection level.

The LA user must also be aware of the limited extraction efficiency of methanol and isopropanol for certain organic
substances that have no moisture, such as grease, tar, asphalts and  some forms of gummy or dried creosotes.  Methanol

                                          IAGUIDELINES-21                                     10/96

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and isopropanol will not dissolve the higher molecular weight organic compounds that are present in those substances
and use of a high efficiency extraction solvent in conjunction with a solvent exchange procedure should be considered
by the IA user. Solvent exchange involves using a high efficiency extraction solvent to put the COI in solution,
removing the high efficiency solvent using a gentle stream of nitrogen gas, and then reconstituting the sample extract
with methanol/isopropanol. Dimethyl sulfoxide (DMSO) is the final solvent used in dioxin kits to dissolve the extract
residue because, in its diluted form, it will not denature the antibody.

Solvent exchange procedures are not recommended unless the IA user is willing to determine that the solvent exchange
procedure can achieve the project detection limits and can quantitatively recover all COIs. This determination should
be performed  prior to initiation of the full scale field sampling program.   It should be noted, however, that
extraction/solvent exchange procedures may not be feasible for field use due to the need for additional equipment and
the use of toxic solvents on site.

Additionally, in situations where the COI is less soluble in methanol/isopropanol, the extraction step can be enhanced
with gentle heat, shaking over a longer period of time, or the use of sonication.  If the field samples require any of these
enhancements, then all the standards, blanks, and QC samples must also be run in an identical manner.

6.5    Soil Moisture

As discussed in Section 6.4, the moisture content of soil/solid samples can affect extraction efficiency.  Moisture
contents over 30% will impact the extraction efficiency for normal sand/loam soils and will exacerbate extraction
problems for highly organic soils. The incremental contribution of the water present in the soil/solid sample matrix
(up to a maximum of 30% water) can be adjusted by the dilution factor used with each kit's specific procedure or
through dry weight calculation.

Correcting for the loss of extraction efficiency due to the water content of soil/solid samples, however, is a more
complex task.  Soils or sediments with > 30% moisture may  require further water removal techniques, such as
decanting, filtration, air drying, or oven drying (if the soils/solids do not contain easily volatilized target compounds).
Air drying of the moisture laden samples may be the IA user's only recourse for IA analyses that are performed in a
field setting.  Soil/solid samples to be analyzed for volatile organics cannot be dried to remove water since the drying
procedure would cause volatilization of those compounds and would result in false negatives. Likewise, drying
soil/solid samples for petroleum  hydrocarbon analyses is not recommended due to loss of the more volatile low
molecular weight components. Soil/solid samples to be analyzed for metals, PCBs, and selected non-volatile pesticides
can be gently dried (<60°C) prior to sample extraction. For PAH analysis, gentle drying of the soil/solid samples will
cause loss of the more volatile PAH compounds such as naphthalene, acenaphthylene, phenanthrene, anthracene,
fluorene, acenaphthene, etc. If these PAH compounds are important to project DQOs, then other analytical methods
should be considered.

Each vendor provides suggestions on lowering moisture content in soil/solid samples.  These suggestions are compiled
in Table 5. When soil/solid samples have moisture contents greater than 70%, the use of immunoassay techniques may
not be applicable or advisable. Some matrices such as sands, sandy soils, or low organic content loams may dewater
rapidly.  In this case, the vendor-suggested use of coffee filters, paper napkins, diapers, or cheese cloth may be
applicable to dewater samples. To do this, the soil is placed in the filter or cloth, which is then folded into a tight ball
and squeezed to remove water through the porous surface.  Although these procedures have some success with sandy
materials, they do not work well with silt, organic sediments, or peat. For soil/solid samples containing greater than
90% moisture, the LA user may want to consider the sample matrix to be aqueous and proceed with an aqueous LA
analysis.  Project DQOs should drive this decision and such contingencies should be delineated in the pre-approved
project QAPjP/SAP.

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6.6    Operational Consistency

IA methods must be performed in a very consistent manner. Having the key QC elements, referenced in Section 3.3.3,
in place will not ensure the production of usable data if the IA user does not perform the assays consistently, under
the same operational conditions, and time each step precisely.

The operational conditions for performing IA must adhere to vendor requirements. As discussed in Section 6.1, IA
kits must be brought to ambient temperature prior to use and LA analyses must be performed within the vendor-
recommended operating temperature range. LA users must adhere to the shelf life and storage conditions recommended
by IA kit vendors to eliminate variability in reagent performance.

La addition, individual steps in the assay must be consistently performed and precisely timed. If they are not precisely
timed, then assay drift occurs. LA drift can be caused by inconsistent timing of the antibody/COI reaction and/or the
color development and stop reactions.  Immunoassays are commonly performed in batches where standards, blanks
QC samples and field samples are assayed simultaneously in a group. When the protocol calls for precise sequential
pipetting steps, there can be significant timing differences between when the immunochemical reaction begins with
the first standard versus the last field sample. Reagent additions between samples, etc. must be performed rapidly,
precisely, and consistently once the immunochemical reaction is started so  that each sample, etc.  will incubate with
the same reagent volume for the same time period.  The longer the period of incubation and/or reaction, the less
propensity for LA drift In designing the quality control program, each vendor has developed an understanding of the
critical timing issues and has addressed them by defining batch sizes and the placement of standards, blanks, and QC
samples.

The batch size is a key factor in producing consist LA results. For this reason, most field kits are limited to 4 to 6
samples, two calibrators and possibly a QC sample. Laboratory kits may use much larger batch sizes and even perform
multiple batches simultaneously in racks. Multiple batches can only be performed  using automatic multiple pipetters
and an automated rack washing system.

LA user training is another key factor in producing consistently accurate and precise LA results and will be discussed
in Section 7.0.

6.7    Cost and Time Considerations

The time required to collect environmental samples depends on the sampling technique, the physical conditions of the
environment, and the sample matrix. Samples of water, which are taken from the shore of a surface water body using
the sample bottle itself as a collection device, probably take the shortest time to collect with the least effort. Samples
of soil, that are collected from a deep soil boring using an auger and that require  compositing, probably take the most
time. This document will not attempt to estimate the time associated with sample collection efforts.

The  preparation and analysis time for use of LA in a laboratory setting will depend on the extent of laboratory
automation and the experience of the chemist LA vendors should be contacted  directly to obtain this  information. This
section will focus solely on the  time required to prepare and analyze a single sample using LA techniques in a field
setting

Each vendor has estimated the number of samples that can be successfully analyzed per batch, depending on the sample
matrix, and the time required for preparation and analysis of a sample batch.  This information in detailed in Table  6a
so that the LA user can compare the efficiency of each vendor's kit The time estimates contained therein assume that
the user is trained and practiced in the utilization of each LA kit.

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In general, IA sample preparation includes the following steps:

           Sample measurement by volume or by weight
           Adjustment of the pH and/or buffering of waters
           Filtration of the water
           Introduction of the extractant for soils
           Extraction of the sample for soils
           Filtration of the extract for soils
           Pipetting the water sample or soil sample extract into the IA container

Sample preparation for waters takes very little time. The tune required for preparation of soil samples will depend
upon:

       •   The physical characteristics of the soil and the need to segregate large chunks of gravel or organic wood
           chips and twigs,
       •   The amount of quartering and compositing required to get a representative homogeneous sample,
       •   The need to remove excess moisture from the sample, and
       •   The difficulty in quantitatively extracting the soil depending on the organic content of the soil matrix
           (possibly increasing the extraction times).

The IA user must factor site information into the vendor time estimates to obtain realistic times for site-speciOc LA use.
Analysis times for prepared water and soil samples do not vary by much because the same analytical steps take place
during the LA process.  Water samples, however, usually take slightly less time to analyze than soil extracts, since water
samples require fewer dilutions.  The time required to perform site-speciGc dilutions can be estimated by the vendor
when the LA user contacts the vendor to determine the correct kit and dilution factor to use for the project.

The analysis step, which includes the time required for pipetting, incubation, and color development, usually takes 25 -
45 minutes per sample batch (or per sample if only one sample is being analyzed). The exact analysis time will depend
upon the specific requirements of each vendor's kit and the COIs that are analyzed.

The timing sequences for each vendor's kit control the number of samples that can be accurately and precisely analyzed
in a single batch. Approximately 35 to 200 samples/person/day can be processed using LA kits, depending on the COI
tested and the extent of sample preparation.  That estimate assumes that the LA user performing the sample preparation
and analysis has experience with those particular LA kits.

Tables 6b and 6c contain the per sample costs for individual vendor LA kits.  The LA user can use the information in
Tables 6a - 6c to compare the time and cost required to prepare and analyze samples using LA kits versus conventional
full protocol analytical methods. LA has the advantage of immediate turn-around time for obtaining sample results,
whereas sample results obtained using conventional full protocol analytical methods performed in a fixed laboratory
can take over a month.

The choice of LA techniques versus conventional full protocol analytical methods is usually driven by two factors:
the project costs accrued while waiting for sample data and the quality of the sample data necessary to meet project
DQOs. When excavation or drilling equipment are present on site, the equipment idle time can be significantly more
expensive than the fixed laboratory analysis costs. The generation of quick reliable sample results to keep heavy
equipment moving can be the critical factor in lowering project costs as long as the comparability of LA data and split
sample confirmatory data meet project objectives.


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The costs and time required for preparation and analysis of project samples using IA kits versus field laboratory
analytical methods should be compared as well. The sample throughput of field laboratory methods may be equivalent
to IA, but the personnel costs associated with staffing a field laboratory with experienced chemists that can operate
the complex instrumentation competently may far outweigh the cost of personnel to perform IA analyses. The costs
associated with providing an external power source for field laboratories also negatively impacts their use.

       6.7.1  Time Savings

       Each vendor provides helpful hints to save time when performing LA analyses. However, the major factor in
       saving time on site is practical and thorough planning as well as proper training of the site personnel.
       Batching of the samples into like matrices and similar dilution groups can save processing time. Practice with
       the kits of choice prior to the field episode using site-specific samples is recommended. Prior knowledge of
       the sample matrix and its physical characteristics is crucial to successful LA use and will speed sample
       processing for the extraction and dilution steps.

       6.7.2  Cost Considerations

       The cost of IA kits can be separated  from the ancillary equipment cost for the weighing, pipetting and
       colorimetric measurement equipment because that equipment is now available from the vendors on a rental
       basis. IA users must check with each vendor to ascertain the availability of ancillary equipment. The purchase
       price for ancillary equipment may range up to $2000 and each manufacturer uses slightly different equipment
       depending on the format of their IA technique.  Rental of the ancillary equipment may be more cost effective
       for the occasional LA user or the LA user that travels between sites. In contrast, purchase of the ancillary
       equipment may be more cost effective if the LA user is planning to process many samples at a single  site over
       a long period of time. The power requirement for LA ancillary equipment is minimal and some  vendors'
       equipment is even powered by battery. Ancillary equipment costs, power requirements, and site accessibility
       should be considered when the LA user compares use of field laboratory instrumentation to LA kit  use for a
       particular  project.  Utilizing a gasoline powered generator on site to power analytical equipment may
       jeopardize the site investigation by introducing another source of BTEX and PAHs.

       LA kit costs are dependent on the number of samples in a batch. The more samples that can be processed at
       one time between the proper standards, the less the ultimate per sample cost.  Standards, blanks, and PE
       samples must also be included in the cost per sample. Vendors of LA kits offer discounts for volume sales and
       equipment rentals.

       Fixed laboratories also offer volume discounts for performing confirmatory split sample analyses using
       conventional full protocol analytical methods. Again, the more sample analyses that can be batched into 20
       sample lots, the less each sample will cost.

       With proper planning, personnel training, QA/QC, and sufficient comparable split sample confirmatory data,
       IA users will find that the less costly and faster generated LA data can be as usable as conventional full
       protocol analytical data in meeting project objectives and making valid site decisions.

6.8    Disposal of LA-Related Waste

The LA user must address the disposal of project-related wastes originating from all field sampling, decontamination,
and analysis processes that are performed on site. As is true for all site-derived wastes, wastes generated during the
IA analysis process must be disposed of in a responsible manner.

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IA waste disposal decisions are linked to the disposal decisions that are made for other site materials, such as drill
cuttings, purge water, and excess composite samples. All of these materials can be containerized or buried on the site
depending on the nature of the site activity and the Action Levels set by the regulatory body controlling the site.

IA analysis process wastes can be separated into two groups: the hazardous chemicals; and the non-hazardous, non-
contaminated disposables. QC materials (calibration standards and PE samples) and the field samples that contain
COI(s) must be considered to be contaminated and must be controlled. The packaging, pipette tips, and the reagents
that do not contain COI(s) may not need to be controlled. IA wastes that are considered non-toxic and have not come
in contact with the COI(s) usually can be disposed of in municipal trash. Some regulatory bodies will require that all
site-related waste, regardless of toxicity and weight/volume, be disposed of in a regulated manner just because these
items were on the site at some time.  The IA user must ascertain the waste disposal requirements for the site from the
subject regulatory body.

Disposal costs should also be calculated and added into the cost of on-site LA analyses. Disposal of LA-related wastes
must be addressed during the project planning stage.

7.0    PRACTICAL PLANNING FOR PROJECTS USING IMMUNOASSAY

Figure 6 depicts a decision tree, which outlines the process that project planners should follow to plan environmental
projects utilizing LA.  There are six  steps which must be followed when planning projects that will utilize LA
techniques:

       1.  Gather LA information

       2.  Determine project DQOs

       3.  Ascertain LA needs

       4.  Obtain vendor training

       5.  Order LA kits and ancillary equipment

       6.  Finalize the project QAPjP/SAP

These six steps are the subject of this section, which will attempt to compile the informational needs and to outline
the critical decision processes so that the project planner can properly choose an LA kit for a specific use.  The process
discussed in this section will also enable the project planner to help ensure that LA analyses will be performed correctly
on site and that the resultant LA data are  suitable for their intended use in site decision making.

7.1    Gathering LA Information

Section 2.0 discussed the basic principles of immunochemistry so that project planners would be familiar with
environmental LA technology.  Using the information provided in Section 2.0, project planners can review the
characteristics and availability of specific LA kits from the various vendors outlined in Section 3.0.  If any additional
information (i.e., vendor literature) is needed in order to properly plan a project utilizing LA, then project planners
should contact the vendor directly to obtain that information.
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                          Figure 6     IA Project Planning Decision Tree
                                           / Am I an experienced
                                     s	/   project planner that
                                         \      has previously
                                          \    utilized IA kits?
                                                                            Do I
                                                                       have literature
                                                                     on the availability
                                                                      and capability of
                                                                       environmental
                                                                          lAkits?
                         Havel
                  determined my DQOs?
                  Have I ascertained my
                        IA needs?
   Consult guidelines,
determine project DQOs,
 and ascertain IA needs
                                                                     Request
                                                                  further vendor
                                                                   information
                                                                                         See these
                                                                                       guidelines for
                                                                                        information
   Contact vendors for
   further information,
    instructions, and
 availability of kits and
   ancillary equipment
                                    Have I had
                                vendor training on
                                    the selected
                                      kits?
                                  Obtain vendor training
                                      on selected kits
                                                                       Order selected kit(s) as
                                                                        needed for the project
                                              Rent or purchase
                                                equipment as
                                                   needed
                                                                                Do I
                                                                          have the ancillary
                                                                             equipment?
                                                                          Finalize project QAPjP/
                                                                                    SAP
 1.
 2.
 3.
 4.
 5.
 6.
Gather IA information
Determine project DQOs
Ascertain IA needs
Obtain vendor training
Order IA kits and equipment
Finalize project QAPjP/SAP
                                           IAGUIDELINES-27
                                                                                                    10/96

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7.2    Determining Project Data Quality Objectives (DQOs)

Before the project planner can integrate immunoassay techniques into the project's sampling and analysis scheme, the
project planner must establish the project DQOs. The project planner should obtain and evaluate as much historical
information as possible about the site conditions and history, including the results of any previous investigations at
the site, prior to DQO development so that the following questions can be answered during DQO development:

       •   What is the contamination on the site?

       •   Where is the contamination on the site?

       •   What is the source of contamination on the site?

       •   What is the purpose of sampling and analysis (data collection)?

       •   What sample matrices may be contaminated?

       •   What compounds or chemical classes (COIs) must be analyzed?

       •   What are the Action Level concentrations for the COIs?

       •   What non-target compounds or chemical classes may be present on the site?

       •   What is the intended use of the data?

If the project planner, with input from the project team (risk assessors, hydrogeologists, chemists, etc.) can answer all
of these questions completely, then the project DQOs have been developed properly and are understood.  If the planner
cannot answer these questions, then the process for DQO development must be undertaken.

DQO development will not be addressed in these guidelines. It is described, however, in the Agency document,
Guidance for the Data Quality Objective Process. EPA QA/G-4, which describes the informational needs and critical
decision processes that must be addressed during DQO development.

7.3    Ascertaining IA Needs

The COI(s) and Action Levels must be known prior to ascertaining IA needs because LA is not a technology that lends
itself to initial investigations of sites that have unknown contaminants at unknown concentrations. The presence and
estimated concentrations of non-target compounds or chemical classes on site must also be identified during the DQO
development stage. Once the project COI(s), Action Levels, non-target compounds, and intended data uses have been
established during DQO development, the project planner can proceed with ascertaining LA needs for the project as
depicted in Figure 7.
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                                   Figure?
             Ascertaining IA Needs
                                     Determine project COIs, Action Levels,
                                     and expected Don-target compounds from
                                             DQO development stage.
                                       Perform inital comparison of project
                                            COIs to available IA kits.
             (Vendor
            Tonsultation
   Kit
Selection
Process
                                       Determine kit(s) selectivity to project
                                     COIs (reactivity) and expected non-target
                                          compounds (cross-reactivity).
                                     Determine kit(s) sensitivity (quantitative
                                     and/or semi-quantitative detection limits
                                    and detection/calibration range) to achieve
                                    project action levels for COIs with minimal
                                    interferences from non-target compounds.
Evaluate kit(s) operational response
to existing project field conditions
     and sample matrix issues.
                                    Evaluate kit(s) batch size concurrently with
                                       sample costs and batch process times.
                                                         Pre-design
                                                          study  to
                                                         determine
                                                       split sampling
                                                        Comparability
                                                         correlation
                                                          factors.
                                  Determine availablity and adequacy of Vendor
                                            training for selected kit(s).
                                          Select final kit for project use
                                             IA GUIDELINES-29
                                                                     10/96

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Hie project planner should compare the COI(s) to the available IA kits (Tables la and Ib) and determine whether the
project DQOs can theoretically be met using IA techniques.  If the project planner decides that IA techniques may be
applicable to project DQOs, then the planner must answer the following questions in order to select an appropriate IA
kit:

       •   Which kit is best to use for analysis of project COI(s) at their required Action Level(s)?

       •   What type of IA data are required:  quantitative, semi-quantitative, or qualitative?

       •   What sample matrices need to be tested?

       •   What is the concentration level or range for which COIs need to be tested?

       •   What reference method for confirmation will be used for the project?

       •   Will the resultant IA data meet project objectives?

The process for selecting IA kits is discussed in Section 7.3.1, while data comparability and usability in meeting
project objectives is addressed in Section 8.0.

       7.3.1   IA Kit Selection Factors

       Now that the project planner has determined that IA kits are available to potentially meet project DQOs, the
       project planner must select the kit that best  meets the project objectives. Numerous  factors impact final kit
       selection, including kit selectivity (reactivity and cross-reactivity) and sensitivity, the need for quantitative
       vs. semi-quantitative results, expected field conditions and complexity of sample matrices, per sample costs
       and batch process times, and the availability and comprehensiveness of vendor training.

       Reactivity

       The planner must first evaluate which vendor kits are reactive to the project COI(s) as discussed in Section
       5.1. Tables 2a - 2f should be consulted to perform this evaluation.  A review of the relative sensitivity of each
       reactive compound/mixture is then required as discussed below.

       Sensitivity Mid Quantitative versus ScPV-Oliantitative Kits

       The planner must determine whether those selective vendor kits produce quantitative or semi-quantitative
       results that are sensitive enough to meet project Action Levels, especially since detection limits  and
       detection/calibration ranges are specific to each kit's particular quantitative or semi-quantitative mode. This
       determination is critical to project success, and the information provided in Sections 3.3,3.4, and 4.0 as well
       as Tables 2a - 2f should be used to make this determination.

       There is no easy way to equate different vendor detection limits  to one another.  The project planner should
       understand each vendor's definition of detection limit and how detection limits are utilized in each vendor's
       IA kit to establish detection/calibration ranges.

       As stated in Section 4.0, some vendors define the detection limit as the pre-detennined 90% B/B0 value while
       other vendors define the detection limit as the lowest concentration that gives a positive result at  a specific

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confidence limit (usually 95% confidence limit). Generally, quantitative kits use the 90%B/B0 detection limit
designation and semi-quantitative kits use the lowest detectable amount at 95% confidence limit. It should
be noted that vendor kits may have very different detection limits for the same compounds/mixtures. This is
due, in part, to vendor  use  of different antibodies that may be more or less sensitive to a specific
compound/mixture. Tables 2a - 2f list the sensitivities of specific compounds/mixtures to various vendor IA
kits. The lower the detection limit number presented in those tables, the greater the sensitivity of that IA kit
to a specific compound/mixture. The compound/mixture that has the lowest detection limit for a particular
kit is usually (but not always) the indicator compound/mixture that was used to engineer that kit.

To facilitate kit selection, the project planner should perform an initial comparison of vendor detection limits
as if they were all calculated in the same manner.  This comparison can narrow the number of kits that can
meet the Action Levels for the project COIs from many to  a few.

Next, the project planner must define which of those few kits is most applicable to the project by examining
how close each kit's detection limit is to the project Action Level and where that Action Level falls within each
kit's detection/calibration range. If the kit's detection limit is very close to the Action Level then detection
uncertainty increases and it will be difficult, if not impossible, to make site decisions using data generated with
that kit.  In addition, the expected concentration of project COI(s) should fall into the middle of the kit
detection/calibration range, where  the  most accurate identification and quantitation of COIs occurs.
Identification and quantitation at the extreme ends of the kit's detection/calibration range must be avoided.
This is generally not a problem for quantitative kits.  Vendors of semi-quantitative kits have designed the kits
so that the Action Level will fall into the middle of the kits' detection/calibration range. This type of design
helps to eliminate false negatives, but does promote some false positives. Finally, the chosen kit(s) range of
detection/calibration for project COI analysis should result in the least amount of kit manipulation, including
changes to sample weights and dilution volumes (as discussed in Sections 3.3 and 3.4). Vendor consultation
may be needed to help resolve potential detection range and sample dilution issues.

It should be noted that  some kits are not sensitive to individual compounds within a chemical class such as
benzene in the BTEX group, naphthalene and dibenzo[a4i]anthracene in the PAH group, and Aroclor 1221
in the PCB group.  Benzene alone can be measured using  a specially designed  kit made by ENSYS.
Naphthalene  can  be  measured  very well  using  the  cross-reactivity  of the  BTEX/TPH  kits.
Dibenzo[a,h]anthracene and Aroclor 1221 are not measured well by any LA kit.

During the kit selection process, the project planner must  also ensure that project objectives for generating
quantitative data for particular compounds/mixtures will be met using the kit that is ultimately chosen. For
example, a project's DQOs require quantitation of Aroclor 1242. From the historical site information, it was
determined that Aroclor 1242 was the only PCB mixture ever used on site. Aroclor 1242 is a moderately
chlorinated PCB mixture. LA kits for PCBs, however, are most sensitive to Aroclor 1254 (which is a more
highly chlorinated PCB mixture). For each applicable vendor PCB kit, one must assume, therefore, that the
minimum detection limit for Aroclor 1242 will probably be higher than the detection limit for Aroclor 1254
by  some factor. The exact factor can be calculated from the sensitivity values provided in Table 2c.  The
results obtained for Aroclor 1242 using a particular PCB kit could then be multiplied by the calculation factor
to obtain quantitative sample data for Aroclor 1242.

Use of a quantitation factor can not be applied to PAH and c-PAH kits. The molecular weight and number of
6 membered rings of the specific COI (PAH or c-PAH compound) should be matched to the kit most sensitive
to that range of PAH rings.  It will be difficult, if not impossible, to determine the number of rings present in
the PAH compounds on-site, unless previous site data has been generated and indicates the specific PAH

                                 LA GUIDELINES-31                                    10/96

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compounds that were detected. If the project objective is to collect data for human health risk assessment, then
the c-PAH kits are probably most applicable.

Cross-Reactivity

Next, the planner must factor the cross-reactivity of that select group of kits into the process. If non-target
compounds/mixtures may be present at the site, then their cross-reactivity must be evaluated using the
information provided in Section 5.2 and Tables 3a - 3e.

For individual pesticides, insecticides and herbicides, vendor literature must be obtained and examined to
determine the cross-reactivity of the various COI metabolites since it was too voluminous to include in this
document In general, there are few cross-reactants that will affect the quantitation of pesticides, insecticides,
and herbicides in field samples, unless the concentration of cross-reactant(s) is very high.

For sites that have known products containing mixtures of PAHs, petroleum hydrocarbons and BTEX, it is
important to check that a specific kit has no major cross-reactants to other site-related compounds/mixtures
such as PCBs or pesticides. The planner must always consider the difficulty in determining the additive effect
of the cross-reactants in relation to whether the kit will have the required specificity to the COI.

The planner may also use cross-reactivity to their advantage when project DQOs require the analysis of total
contaminant classes (total PAHs, total PCBs, or total TPH) rather than individual compounds. TPH kits that
measure fuel products are also sensitive to naphthalene and substituted single ring aromatic compounds. Kits
that measure PAHs and c-PAHs are also sensitive to many aliphatic substituted PAH analogs.

The following complex planning scenario illustrates use of cross-reactivity to measure site contaminants:

    Coal tar has been discharged and is the major contaminant source. Fuel oil #2 was spilled on site and may
    have mixed with the coal tar discharge and fuel oil #6 that was spilled in a different area of the site. The
    project planner must do the following to satisfy the project objectives:

    1)  Delineate the extent of Total PAH contamination on the site.

    2)  Determine the human health risk of the 8 carcinogenic PAHs.

    3)  Determine the volume of soil, which is contaminated with the coal tar and fuel oil related PAHs, that
       must be remediated.

    The project COIs include the range of high molecular weight to low molecular weight PAHs, and the
    carcinogenic PAHs that are components of all of the products found on site.

    If the project planner  is to use immunoassay kits, he/she must be cognizant of the specific PAH
    compounds that are potentially present in each product, and the ring structure and molecular weight of
    those PAH compounds.
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   •   The #2 fuel oil has naphthalene and a small amount of low molecular weight PAHs.

   •   The coal tar has a wide range of PAHs, including naphthalene.

   •   The #6 fuel oil has medium to high molecular weight PAHs.

   In order to ascertain the extent of contamination, the volume of soil to be remediated, and the human
   health risk from the carcinogenic PAHs, the planner may have to use more than 2 IA kits to satisfy the
   project objectives.

   First, all the contaminated areas would need to be characterized using a c-PAH kit to assess the human
   health carcinogenic risks.

   Next, the project planner would have to decide which additional kit(s) to use to satisfy objectives # 1 and
   #3 listed above. Several different approaches could be taken.

   The project planner could choose to use a PAH kit that is sensitive to a broad range of molecular weight
   PAHs to assess the extent of contamination and the soil volume to be remediated. The project planner
   must be aware, however, that naphthalene, which is present in measurable quantities in #2 fuel oil and coal
   tar, will not be detected using such a kit.

   On the other hand, the project planner could use a TPH/BTEX kit, in conjunction with the c-PAH kit, to
   assess the extent of contamination and the soil volume to be remediated.  The TPH/BTEX kit is sensitive
   to naphthalene in the #2 fuel oil and coal tar. The project planner must be aware, however, that the
   TPH/BTEX kit would overestimate the amount of naphthalene present in the samples, since that kit would
   also measure all of the single aromatic compounds (non-PAHs) that are present in the samples. The extent
   of contamination and the soil volume to be remediated for PAHs originating from the #6 fuel oil would
   then have to be determined using the results of samples analyzed using the c-PAH kit.

   Other combinations of kits that analyze low to medium molecular weight PAHs and medium to high
   molecular weight PAHs could also be used to determine the extent of contamination and the soil volume
   to be remediated.

Although this is a complex example, it illustrates the need for the planner to know what target and non-target
compounds/mixtures are present on site and what  IA kits  are available to analyze specific target
compounds/mixtures.

Field Conditions fl^d S prnpls Matrix Factors

The project planner has now considered selectivity (reactivity and cross-reactivity),  sensitivity, and
quantitative vs semi-quantitative use of the available kits. At this point, the project planner should be able
to match appropriate kit(s) to their intended site use, as long as those kit(s) can be used under the expected
field conditions and complexity of sample matrices that may be encountered on site.

Sections 6.1 - 6.5 discuss many of the field conditions and sample matrix factors that must be considered by
the planner for use of IA techniques to achieve project DQOs. The pertinent factors include characteristics
of water and  soil matrices, matrix extraction efficiencies, soil moisture content, and ambient conditions
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(temperature and sampling).  Vendor consultation may be needed to help resolve selected field conditions
and/or sample matrix issues prior to final kit selection.

It is critical to factor soil characteristics into the extraction and IA analysis of project soils.  Prior to final kit
selection, the project planner must evaluate potential moisture problems and the extraction efficiency of
methanol/isopropanol for separating the COI from the sample matrix. Contingency steps for each of these
circumstances should be detailed in the approved QAPjP/SAP and any pre-design studies must be performed
prior to initiation of the full scale field sampling episode.

The project planner must always consider the ambient temperature and sampling conditions for the site.  The
project planner must evaluate the ideal ambient temperature operating range of the prime kits in conjunction
with expected field conditions (ambient site temperature and location of IA testing). The planner must also
ensure that IA analyses will not be impacted by airborne site contaminants. For sampling and LA analysis of
target compounds (i.e., volatile organics, mercury, etc.) that can be affected by ambient site conditions (such
as temperature, oxidation, and moisture), appropriate contingency steps for sampling, preparation, and/or LA
analysis must be detailed by the project planner in the approved QAPjP/SAP.

Sample Costs and Batch  Process Times

After the project planner has evaluated the response of the prime kit(s) to expected field conditions and sample
matrix complexity, then the planner should examine sample costs and batch process times in conjunction with
batch size, ease of kit use, and disposal of LA-related wastes. Sections 6.7 and 6.8 contain a discussion of
those topics and Tables 6a - 6c contain information on batch processing times and per sample costs for specific
IA kits. Final selection of an LA kit will depend, in part, on the number of samples that can be analyzed per
batch, the per sample costs, and the batch processing times.

Availability of Vendor Training

The final factor pertaining to kit selection that must be considered by the project planner is the availability
and comprehensiveness of vendor training courses.  The training of personnel to use LA kits properly is an
important part of a project's QA program. Each vendor's product has a specified format and protocol that must
be adhered to much as possible. LA technologies require consistent process techniques as described in Section
6.6. Proficiency in sampling, weighing, pipetting, sample dilution, and colorimetric measurement is critical
to resultant project data quality.

Each vendor offers a training program which describes their immunoassay format and equipment operation.
Each training session usually culminates in a test of the proficiency of the LA user.  Before a particular
vendor's LA kit is utilized in the field for project work, the personnel performing LA should have attended that
particular vendor's training course and have practiced with that kit prior to initiation of the full scale field
sampling episode.

Final Kit Selection

Once all kit selection factors have been evaluated and any required vendor contact has been made to resolve
potential site-specific issues/problems pertaining to the use of particular LA kits, the project  planner can
proceed to select the most appropriate kit for achieving project DQOs.
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7.4    Obtaining Vendor Training

Once the final kit has been selected for project use, vendor training pertaining to the specific IA format and operation
of that vendor's equipment must be obtained.  All personnel that will perform IA on site must pass the vendor
proficiency test that occurs at the end of the training session. Training should be obtained prior to finalizing the
QAPjP/SAP so that the project planner can be notified of any project issues that may result from use of a particular
lAkit.

7.5    Ordering LA Kits and Ancillary Equipment

Project planners should contact the individual vendors at the telephone numbers listed in Section 1.1  as needed to
obtain current pricing and availability information, purchase LA kits, and purchase or rent the required ancillary
equipment.

7.6    Finalizing the QAPjP/SAP

The LA project planner must develop a QAPjP/SAP that details the project description and DQOs; project personnel
and their responsibilities; sampling protocols; sampling locations and numbers of samples to be collected per matrix
for each compound and/or analysis parameter, LA and confirmational analytical protocols with frequency requirements,
QC acceptance criteria, and corrective action measures; split sampling comparability acceptance criteria; and how the
QC and field sample data will be used to determine whether project DQOs have been met (Data Quality Assessment).

EPA QA/R-5, EPA Requirements for Quality Assurance Project Plans (August 1994), details the requirements used
by Region I, EPA-NE for the development of a QAPjP/SAP. The QAPjP/SAP need not be extensive for an LA episode,
but it must sufficiently address all of the elements detailed in EPA QA/R-5.

DQO's must be developed in accordance with EPA QA/G-4, Guidance for the Data Quality Objective Process.
How IA data will be used in project decision making is critical to document in the QAPjP/SAP. Justification must also
be provided in the QAPjP/SAP for the final LA kit selected for use in the project. Expectations for LA data use must
be realistic in relation to the nature of the site contamination.

The QAPjP/SAP must describe, in explicit detail, the sampling protocols  that will be used for the project. The
sampling protocols must demonstrate that proper procedures will be used consistently and will result in sufficient
volume of representative sample to enable multiple IA analyses (in cases where duplicates are required) as well as split
sample confirmation analysis.  Samples of water must be taken where the water is in equilibrium with the environment.
Well waters must be sampled using methods that do not disturb the aquifer such as the EPA Region I low flow
procedure. Soils must be representative of the site conditions, and samples taken as composites must be well mixed
prior to LA analysis. Soil samples that are high in moisture must be decanted, centrifuged, filtered, or gently dried to
ensure that there is at least 70% solids prior to LA analysis. Samples of free product materials (pure oils or pure
chemical solids) must be diluted with the vendor-recommended solvent prior to analysis. Most of all, sampling must
be performed in a consistent manner for all samples collected during the project episode.

The QAPjP/SAP must also describe, in explicit detail, the analytical procedures that will be performed for sample
preparation and analysis by LA techniques as well as by conventional full protocol analytical methods  that are used
for split sample confirmation analyses. The QAPjP/SAP must compile all the key QC elements that were discussed
in Section 3.3.3 along with their frequency requirements, QC acceptance criteria, and corrective action measures when
QC acceptance criteria have not been met. LA analysis must be performed in strict accordance with vendor-specified


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procedures, with exact timing sequences and reagent additions. Inconsistencies in sampling and analysis procedures
will make data comparability difficult, if not impossible.

Detailed standard operating procedures (SOPs) for the sampling and analysis protocols can be referenced in the
QAPjP/SAP as long as the applicable SOPs are appended to that QAPjP/SAP. All SOPs that are pertinent to project
operations must be developed in accordance with EPA QA/G-6, Guidance for the Preparation of Standard Operating
Procedures for Quality-related Operations.

The QAPjP/SAP must also describe documentation requirements for the resultant IA and split sample confirmation
data. Documentation that all key QC elements were performed and met project requirements is essential, regardless
of intended data use.  For LA techniques, the preparation and analysis of each batch of samples, including related
standards, QC samples and blanks, should be recorded in a field or laboratory notebook, run logs, and/or tabulated
forms. Note, sample preparation should include kit lot numbers and expiration dates, and the ambient temperature at
which the tests were performed.  For conventional full protocol analytical methods, complete data packages should
be produced in accordance with the Region I, EPA-NE specifications contained in the Training Manual for Reviewing
Laboratory Data Package Completeness, dated June 1994.

The procedure and requirements for determining data comparability and usability in meeting project objectives must
also be  described in the project QAPjP/SAP.  Data comparability  and usability are discussed in  Section 8.0.
Documentation of LA use for a project, prior to field sampling initiation, is critical to data interpretation after the field
sampling has been completed.  Many project planners have used IA as a panacea to reduce project analytical costs and
speed up the remediation activities at contaminated sites. Used properly, with the appropriate QC procedures and a
definitive QA program, LA can be a very effective tool. Used without regard to QC requirements and QA process
controls, the resultant LA data may be unusable or uninterpretable.

8.0    DATA COMPARABILITY AND USABILITY

8.1    Data Comparability

The comparability of LA data generated on site and split sample confirmation data generated in a fixed or field
laboratory using conventional full protocol analytical methods is the most important factor for determining whether
IA data will meet the project objectives and be usable for project decision making.  The conventional  full protocol
analytical methods that are used to confirm the LA results must be scientifically valid and well documented protocols
that have been routinely accepted by regulators, since data comparability decisions are based upon a limited number
of samples analyzed by those conventional full protocol methods.

Figure 8 illustrates two approaches that can be used for determining data comparability.  One involves the generation
and application of pre-design correlation factors to adjust IA sample results prior to performing data comparability
calculations. The other approach does not utilize correlation factor adjustment of IA sample results prior to performing
data comparability calculations.  Both approaches require that data comparability  acceptance requirements be
developed and documented in an approved project QAPjP/SAP  prior to initiation of the full scale field sampling
program. Both approaches also require that split samples be collected and analyzed at a 10% frequency throughout
the duration of the full scale field sampling program to assess data comparability.
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                     Figure 8   Comparability Determination
IA
         Split Sampling
                 Conventional
       Develop a Statistical
         Project-Specific
       Correlation Factor
          for Each COI
          ( Mean + SD )
       Full Scale Sampling
                                                          Full Scale Sampling
                                                               Perform
                                                            Individual RPD
                                                             Calculations
                                                    Perform Overall Evaluation
                                                          of Comparability:

                                                      Determine % Splits that Meet
                                                      Project-Specific Comparability
                                                     Acceptance Criteria  (fromPre-
                                                         Approved QAPjP/SAP)
                                        Perform
                               K   Individual RPD
                                      Calculations
    Perform
Correlation Factor
 Adjustment of IA
  Sample Results
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Generally, split sampling data generated using IA techniques and conventional full protocol analytical methods should
meet comparability acceptance criteria of 30% relative percent difference (RPD) for water matrices and 50% RPD for
soil matrices (assuming a one-to-one correlation).  These recommended comparability acceptance criteria can be
modified to meet project objectives, as long as the project-specific comparability acceptance criteria are documented
in the pre-approved project QAPjP/SAP.

Since IA techniques and conventional full protocol analytical methods measure target compounds using different
principles, a one-to-one correlation between data sets cannot always be expected. IA kits measure the sum of the
reactive target compounds and cross-reactive non-target compounds present in the sample matrix. Conventional full
protocol analytical methods, on the other hand, measure the concentrations of individual target compounds present
in the sample matrix. Extraction efficiency differences between the full protocol method and the IA method may also
account for correlation factors that are not one-to-one.  The alcoholic solvent and extraction procedure used in IA kits
may decrease the extraction efficiency of IA for certain chemical mixtures/products when compared to the more
rigorous extraction solvents and procedures used in conventional full protocol analytical methods. The combination
of less effective extraction and broader range of detection/quantitation will make the exact project-specific correlation
factor hard to predict.  It is likely, however, that a near one-to-one correlation will be achieved for the individual
compounds listed in Tables 2e and 2£ since IA kits for those compounds are minimally impacted by the cross-reactants
that are common to other environmental parameters.  lA's cross-reactive properties could also potentially result in a
greater than one-to-one correlation (IA to conventional analysis) for the kits that characterize chemical classes such
as PCBs, PAHs, and TPH. During the project planning phase, the project planner must evaluate the impact that a
greater than one-to-one correlation factor may have on achievement of project objectives through review of historical
information concerning site conditions, history, and the results of any previous site investigations. That is one of the
reasons why it is  strongly recommended that a pre-design project-specific correlation factor for each  COI be
determined prior to initiation of the full scale field sampling program.

Ideally, during the project planning phase, a project-specific correlation factor for each COI is established based upon
actual testing of representative split field samples using both the selected LA kit and the full protocol analytical method
that will be used during the full scale field sampling program.  The project planner should utilize previously generated
LA case studies, vendor literature, and past experience to focus these pre-design studies. Once a valid correlation factor
has been established, it can be used to calculate adjusted LA sample results prior to performing comparability
calculations.  Note, however, that all LA sample results must be reported without correlation factor adjustment, and the
applicable correlation factor must be reported separately. Comparability calculations performed with LA sample results
for which correlation factors have not been generated and/or applied may cause split sampling results to exceed the
data comparability acceptance criteria contained in the approved project QAPjP/SAP (due to the increased impact of
sampling and analysis variability) and, therefore, may potentially render entire LA data sets unusable for project
decision making.

For large remediation projects requiring confirmation of site cleanup, generation and application of an accurate and
stable correlation factor for each COI in conjunction with on-going 10% split sampling comparability checks will
provide the remediator and the regulator with a comfort zone and some assurance that site cleanup is proceeding to
the required action level.

8.2     Data Usability

The usability of LA data is directly related to proper project planning (realistic project expectations and explicitly
delineated decision  objectives), implementation (proper sample collection, preparation and analysis procedures) and
assessment (data validation and split sampling data comparability evaluation) activities.  Semi-quantitative LA  analysis
is suggested by SW-846.  Quantitative LA analysis can be performed successfully when the project planner uses all

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the available site information to design the sampling and analysis protocols.  How well IA data compares to the split
sample data generated using conventional full protocol analytical methods in a flxed or field laboratory is the key to
determining data usability.  Evaluation of PE sample score results should also be incorporated into the usability
determination.  If the conventional full protocol analytical data are valid and usable, then IA data which meet
comparability acceptance criteria can also be considered valid and usable to make project decisions.

Validation of the split sampling data generated using conventional full protocol analytical methods is required, since
those conventional full protocol analytical data will be the benchmark to which the  IA data will be compared.
Validation of the conventional full protocol analytical data must be performed and documented in accordance with
the procedures and requirements contained in the Region I. EPA-New England Data Validation Functional Guidelines
for Evaluating Environmental Analyses, dated July 1996 or most recent revision. Validation of IA data should also
be performed by reviewing the results of calibration, continuing calibration checks, blanks, duplicates and PE samples.
IA data validation should be performed and documented in accordance with the requirements contained in the pre-
approved project QAPjP/SAP.

IA data that are produced for a project will be usable for their intended purpose in site decision making if the QC and
confirmation data indicate that there were no false negatives and few false positives for samples with concentrations
around the project Action Level. As stated previously, the IA kit's detection limit should be below the project Action
Level to ensure that there  are no false negatives.  Likewise, the Action  Level should fall in the middle of the
detection/calibration range of the IA kit to facilitate accurate quantitation.

If IA data are used strictly for semi-quantitative screening purposes (to delineate the extent of contamination or to
confirm site cleanup), then LA data can be considered usable when 90% of the split samples achieve the pre-established
comparability acceptance criteria.  Quantitative LA analyses should follow similarly stringent criteria.  The project
planner should identify the limiting factor for obtaining quantitatively accurate and precise project-specific LA sample
results and develop a project-specific correlation factor for each COI prior to initiation of the full scale field sampling
episode.

Using the information that has been provided in this document, the project planner should now be able to successfully
choose an IA kit to meet the project's DQOs, successfully perform the LA analyses, and produce usable LA data that
can be employed in project decision making.
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9.0    REFERENCES

Quality Control of Immunoassays for Pesticide Residues. Mary C. Hayes, Joseph X Dautlick, and David Herzog,
Ohmicron Corporation, 1992.

Immunoassay for Environmental Contaminants (Pesticides) in Food and Water. David Herzog, Ohmicron Corporation
1993.

Pesticides in Soil and Analysis by Immunoassay T Scott W. Jourdan, Ohmicron Corporation,  1992.

Petroleum Hydrocarbon Detection Using Rapid Assays ®. Ohmicron Corporation, Rev 4/96, Part No. A00255.

Rapid Assay Environmental User's Guide, Ohmicron Environmental Diagnostics, Rev 12/94b, Part No. A00155.

A User's Guide to Environmental Immunochemical Analysis. EPA/540/R-94/509, March 1994.

Field  Analytical Measurement  Technologies. Applications  and  Selection. California Military Environmental
Coordination Committee, Chemical Data Quality/Cost Reduction Process Action Team, April 1996.

Scientific Swapping. Kimberly A. Roy, Hazmat World, December 1991.

Environmental Immunochemical Methods 8"d Their Associated OA/OC. Llewellyn R. Williams, Senior Science
Advisor, U.S. EPA, Las Vegas, Nevada
Region If EPA-New Eflgtend Data Validation Functional Guidelines for Evaluating Environmental Analyses. July
1996.

EPA Region I Performance Evaluation ProgT3"> Guidance. July 1996.

Training Manual for Reviewing Laboratory Data Package Completeness. June 1994.

Guidance for the Data Quality Objective Process, QA/G-4, EPA/600/Rr96/055, September 1994.

EPA Requirements for Quality Assurance Project Plans, QA/R-5, August 1994.

Guidance for the Preparation of Standard Operating Procedures for Quality-related Operations. QA/G-6, EPA/600/R-
96/027, November 1995.

Test Methods for Evaluating Solid Waste. Physical/Chemical Methods, SW-846, 3rd Edition.
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                                           APPENDIX A

                                            GLOSSARY

Accuracy - degree to which the value obtained in an assay corresponds to the "true" value.

Adjuvant - a substance, usually injected with an antigen, which improves the immune response to the antigen.

Affinity - The strength of the binding between binding reagent and a ligand.

Analog  -  a member of a family of molecules whose major structural characteristics are identical, with only minor
structural differences.

Analyte - a compound or family of compounds in a sample to be analyzed in an assay.

Antibody (Ab) - an immunoglobulin produced in an animal in response to an antigen (or hapten combined with its
macromolecular carrier) which can react specifically to form an antigen-antibody complex.

Antibody-Binding Site  - Sites on the antibody that react with the determinant sites on antigens.

Antigen (Ag)  -  a substance which can elicit the formation of antibodies and react specifically with the antibodies
formed.

Antisera - antibody containing sera.

Avidity  - the strength of the bond between a binding reagent (antibody) and a ligand (antigen or hapten).

Bound  -  antigen which is present as a complex, attached to antibody; that fraction of the reaction mixture of an
immunoassay which contains the antigen-antibody complex.

Carrier - An immunogenic substance that, when coupled to a hapten, renders the hapten immunogenic.

Chromogen Substrate -  a substrate which produces a color when it reacts with its specific enzyme, the intensity of
the color produced being directly proportional to the amount of enzyme available to react with the chromogen
substrate.

Competitive Assay - an immunoassay based on the principle of competition between the test (unknown) antigen and
antigen labeled with an isotope or an enzyme for a limited number of antibody binding sites.

Competitive Immunoassay - an immunoassay method involving an in-vitro competitive binding reaction.

Conjugates -  enzymes linked to antigens or antibodies in such a manner that each retains the maximum amount of
their reactivity.

Control - a sample-like preparation containing a known amount of analyte or devoid of analyte that is treated in the
assay as an unknown sample.

Cross-reaction  - reaction of an antibody with more than one antigenic structure.

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Determinant - unique small three-dimensional surface sites on antigen molecules that react (combine) with antibody-
binding on antibodies.

ELISA (Enzyme-Linked Immunosorbent Assay) - a heterogeneous immunoassay utilizing enzyme-labeled antigens
or antibodies.

Enzyme - a protein capable of catalyzing a reaction of a substrate molecule to form a product. Enzymes can be highly
specific for a given substrate.

Enzyme Conjugate - a molecule produced by the coupling of an enzyme molecule to an immunoassay component
that is responsible for acting upon a substrate to produce a detectable signal.

Enzyme Immunoassay (EIA) -  an immunoassay utilizing enzyme labeled antigens, antibodies, or haptens. There
are two main types of EIA procedures; homogeneous assays and heterogenous (ELISA) assays.

False Negatives - a negative interpretation of the method containing the target analytes at or above the detection
level. Ideally, an immunoassay test product included in an SW-846 method should produce no false negatives. The
maximum permissible false negative rate is 5%, as measured by analyzing split samples using both the test product
and a reference method.

False Positives - a positive interpretation for a sample is defined as a positive response for a sample that contains
analytes below the  action level.

Free  - antigen which is not attached to antibody; that fraction of the reaction mixture in an immunoassay which
contains the free antigen.

Hapten - a small  molecule which is not antigenic in itself but when attached to a large molecule (macromolecular
earner) can stimulate the formation of antibodies. A hapten, like an antigen, can react with its specific antibodies once
they have been produced.

Hapten-Carrier Conjugate  -  The coupling of a non-immunogenic molecule (e.g., targeted analyte)  to an
immunogenic substance (e.g., bovine serum albumin, keyhole limpet hemocyanin) for the purpose of stimulating an
immune response.

Heterogeneous Immunoassay  -  a type of immunoassay that requires a separation of bound and free phases.
Generally, accompanied by incubation and washing steps (ELISA Assays).

Homogeneous Immunoassay - a type of immunoassay that does not require washing  steps i.e. it requires no physical
separation between the bound and free phases.

Immunoassay -  an analytical technique that uses an antibody molecule as a binding agent in the detection and
quantitation of substances in a sample, (see Enzyme Immunoassay and ELISA)

Immunogen  - a synonym for antigen, particularly when used to describe a substance used to elicit an immunologic
response  in an animal.

Immunity - a state, natural or acquired, in which the body is resistant to disease.


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Immunoglobulin - the class of globular proteins (gamma globulins) which are antibodies. There are Gve classes of
immunoglobulin (IgM, IgA, IgD, IgG and IgE).

Immunology -  the science that deals with study of immunity to diseases.

Ligand - a member of a binding pair, generally the smaller member. For example, in an enzyme-substrate reaction
the substrate is frequently referred to as a ligand. The antigen or hapten may be called a ligand in an antibody reaction.

Lymphocytes - One of the five classes of white blood cells found in the circulatory system of vertebrates.

Monoclonal Antibodies  - a homogeneous preparation of antibodies directed at a single antigenic determinant
produced from a single clone of an antibody producing lymphocyte hybridized with a "tumor" cell line to form a
hybridoma which continuously secretes a single antibody molecule.

Polyclonal Antibodies - a group of antibody molecules that differ in amino acid composition and sequences, and that
exhibit binding characteristics.  Polyclonal antibodies are produced from a simulation of multiple clones of
lymphocytes.

Polyclonal antiserum - an antibody containing serum which is made up of antibodies from more than one clone of
lymphocyte. Usually produced in vivo by immunising animals with an antigen.

Precision - extent to which the obtained measurements of a defined substance agree with one another, usually stated
as coefficients of variation, relative standard deviations, or confidence limits.

Quality Control - a planned system of activities whose purpose is to provide a quality product.

Radioimmunoassay (RIA) - an immunologic test utilizing a radiolabeled antigen, antibody, or other reactants.

Replicates - repeated but independent determinations on the same sample by the same analyst at essentially the same
time under the same conditions.

Sandwich Assay - an immunoassay technique for measuring antigen in a test sample that "sandwiches" the antigen
being measured between two antibodies.

Sensitivity - sensitivity of a laboratory procedure refers to the ability of the test to detect or respond to small changes
in concentration; the more sensitive the test, the more likely it will detect minute quantities.

Serology  - generally considered a subdivision of immunology, it is the study of blood serum reactions such as
antigen-antibody interactions and complement.

Solid Phase - separation method in which the binding reagent is immobilized by coupling to an insoluble material
(magnetic particles, coated tubes, polymers, etc.).

Specificity  - the characteristic of a laboratory test that distinguishes between true (specific) and inaccurate (non-
specific) results. Non-specific reactions may be caused by cross-reactions or interferences from matrix factors.
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Standard Curve - the dose-response curve generated in a quantitative immunoassay by running a series of reference
standards containing known concentrations of analyte.  It is used to calculate the concentration of unknown samples
from their response data.

Standard Deviation  - often the measure of precision. Mathematically defined as the square root of the sum of the
squares of the difference between the individual values of a set and the arithmetic mean of the set, divided by one less
than the number of values.

Stopping Reaction -  stopping reaction refers to the addition of a solution to prevent further increases of color from
occurring due to conversion of a chromogenic substrate. For example, the end result in ELISA assays is the intensity
of color produced over a given period of time from conversion of a substrate caused by reaction with an enzyme
conjugate. The stopping solution is used to prevent further increases in intensity of color after the given period of time.

Substrate -  substances chosen to react with enzymes that provide a sensitive method for detection of the antigen or
antibody being measured. Generally, chromogenic substrates are chosen which are colorless initially, but which form
colored products as they are converted by the enzyme reaction.

Titer - the greatest dilution of a substance used in a serologic reaction that will produce the desired result.
                                             GLOSSARY-4                                        10/96

-------
                                            APPENDIX B

                           TECHNICAL IMMUNOASSAY DESCRIPTION
Immunoassays are divided into two distinct classes that are referred to as heterogeneous and homogeneous.  The
present state-of-the-art in environmental IA analyses use a heterogeneous assay where an antigen/antibody complex,
containing the Compound of Interest, is bound to a solid substrate and a separation step is required to remove excess
free sample and reagents. Homogeneous assays do not require separation of the bound and free substances and the
antibody can directly modulate the signal produced.

IA can rely on a single antibody (monoclonal) or mixtures of antibodies (polyclonal) to trap or collect the antigen(s),
otherwise known as the Compound Of Interest (COO- Most environmental IA analyses rely on monoclonal antibodies.
Antibodies are a class of proteins known as immunoglobulins which are produced in animals in response to a foreign
substance (antigen). The small molecular weight antigen (COI) may not cause an immune response in the animal, so
it must be coupled to a "carrier" molecule which will present the small molecule (haptan) to the immune system as a
foreign substance.  The antibody is capable of reacting specifically with the  antigen to form an antigen/antibody
complex (commonly referred to as the lock and key approach).

In  immunochemical methods, the unreacted antigen  and antibody are referred to as the free phase, while the
antigen/antibody complex is referred to as the bound phase.  This highly sensitive three dimensional stereo-chemical
reaction between antigens and antibodies is the basis for immunoassay technology.

Because individual chemical compounds of low molecular weight have specific three dimensional stereo-chemical
structures (molecular geometries), there are few other compounds of exact or similar three dimensional structures that
will react with the antibody. Compounds having similar three dimensional stereo-chemical structures that react with
the antibody are said to be cross-reactive.

The development of an appropriate antibody that will bind to an antigen (COI) or a COI attached to an enzyme
(otherwise known as an "enzyme conjugate") is the most important phase of designing an immunochemical specific
test kit Monoclonal antibodies are produced by a single cell and generally have a high degree of specificity and low
cross-reactivity.  This makes them ideal for designing IA kits that are specific for one compound.  Polyclonal
antibodies contain a mixture of antibodies that detect a range of compounds having similar structures.  Because they
are less specific, polyclonal antibodies are better suited to the detection of classes of compounds and they are rarely
used in environmental analyses.

Enzyme Linked Immunosorbant Assay (ELJSA) is an enzyme immunoassay method that uses an immobilized antibody
absorbed onto a plastic well, tube, polymer particle, or magnetic particle to facilitate the separation of the targeted
analytes from the untargeted substances (free reaction components) using a washing step and an enzyme conjugate
to generate the signal. The enzyme conjugate is the COI bound to an enzyme such as horseradish peroxidase. The
signal, or reporter system,  is a colorimetric determination  of the chromogenic  (color) response used for the
interpretation or quantitation of results. Chromogenic responses are analyzed photometrically, and use the principles
of Beer's Law to determine the concentration of analyte in  a sample.

ELISA tests are competitive assays utilizing immobilized antibodies that bind to pure contaminants (COIs), enzyme
conjugates, or sometimes both in proportion to the relative concentration of the sample. The greater the concentration
of the sample-derived COI relative to the enzyme conjugate, the larger the proportion of antibody sites  that are
occupied by the COI molecules.  The antibody in most LA tests cannot bind to both at the same time. The bound

                                           APPENDK-B-1                                      10/96

-------
antibody/col/enzyme conjugate is then washed to remove sample solution and excess reagents leaving the bound COI
and enzyme conjugate antibody complex.  A chromogen (for example, hydrogen peroxide and tetramethylbenzidine
or hydrogen peroxide and orthophenylenediamine) is added to produce a color when it reacts in the presence of the
IA specific enzyme conjugate.  Specifically, the horseradish peroxidase reacts with the hydrogen peroxide to release
a proton, which in turn reduces the tetramethylbenzidine to form the colored product. The amount of response (color)
in solution is directly proportional to the amount of enzyme conjugate available to catalyze the reaction of the
chromogen and hydrogen peroxide. The presence of the enzyme conjugate bound to the antibody is proportional to
the amount of color formed. The chromogen/hydrogen peroxide (sometimes called a substrate) only reacts in the
presence of the enzyme conjugate and does not react in the presence of the COI. This process results in a color
formation in solution that is inversely proportional to the amount of chemical contaminant. More color equals less
contaminant. Less color equals more contaminant. In the presence of high concentrations of contaminants, all the
antibody sites are taken up by the chemical contaminant, thus, no color is developed because there is no enzyme
conjugate present to catalyze the chromogen color reaction.
                                           APPENDIX-B-2                                      10/96

-------
                                          APPENDIX C

                                   IMMUNOASSAY TABLES
Table values are reported from vendor literature.  For consistency, the following general guidelines were used:
values < 1.0 are reported to two decimal places, if appropriate; values * 1.0 are reported to two significant figures.
                                          APPENDIX-C

-------
Table 1a - Vendor's M
Parameter/Analyte
TPH
BTEX
Benzene
PAH
C-PAH
PCB
PCB (Wipe)
PCB (Oils)
TNT
RDX
Dioxin (2.3.7.8-TCDD)
Mercury (Hg)
Tnhalomethanes (THMs)
Pentachlorophenol (PCP)
Trichloroethylene (TCE)
atrix
D-Tech
Water

X

X




X
X



X
X
Soil

X

X

X
X

X
X



X
X

ENS
Water
X
X
. X
X





X

X
X
X

>YS
~s~r
X
X

X

X
X
X
X
X
X
X

X


Ohmicron
Water
X
X

X
X
X


X




X

Soil
X
X

X
X
X
X

X




X


Quanta
Water
X
X













(/Idetek
Soil
X
X

X











BioNel
Water











X



jraska
So//"











X



Ha
Water
X

X












ch
~So;/
X




X









APPENDIX-C
Table 1a

-------
Table 1b - Vendor's Pesti
Parameter/Analvte
2.4-D
Acetanilide
Alachlor
Aldlcarb
Atrazine
Benomvl
Bioresmethrin
Captan
Carbarvl
Carbofuran
Chlordane
Chlorothalonil
Chlorpvnfos
Chlorovrifos-methvl
Chlorsulfuron
Cvanazine
Cvclodienes
DDT
Diazinon
Endosulfan
Fenitrottmn
Hexazinone
Imazaauin
Imazapvr
Isoproturon
Undane
Metsulfuron
Metalaxvl
Methomvl
Methoprene
Metolachlor
Metribuzin
Molinate
Nicotine
Paraauat
Parathlon
Pirimiphos-methvl
Procvmldone
Silvex
Thiabendazole
Toxaohene
Triasulfuran
Triazine
TrichloroDvridinol
TricloDvr
Urea Herbicides
x = tube kit
p = plate kit
cide, Insecticide and Herbicide Matrix
ENSYS
Water
x.o
D
X. P
X. P
X. P
X. P
P


X. P


P
x(
-------
Table 2a - TPH/BTEI
Parameter /Analyte
TPH
Gasoline
Diesel
Jet A Fuel
JP-4
Kerosene
Fuel oil #2
Fuel oil #6
Mineral Spirits
BTEX
Benzene
Toluene
Ethylbenzene
o-Xylene
m-Xylene
p-Xylene
Total BTEX
Benzene (only)
{ Reactivity
D-Tech
Water
ppm*

-
-
-
-
-
-
—
—

1.2
0.6
06
0.6
1.4
1.3
0.6
NA
Soil
ppm-

-
-
-
—
-
-
-
-

5.0
2.5
2.5
2.5
5.8
5.4
2.5
NA

ENSYS
Water
ppm'

0.16
0.24
0.28
0.18
022
0.21
-
0.49

5.0
0.74
0.06
0.10
0.10
0.59
0.20
0005*
Soil
ppm*

10
15
15
15
15
15
25
40

400
40
7.0
8.5
8.0
45
10
NA

Ohmicron
Water
ppm

043
1.3
2.7
0.5
1 5
0.4
0.2
1.1

0.59
0.44
0.24
0.22
0.03
0.13
002
NA
Soil
ppm

43
13
27
20
15
13
13
11

5.9
44
2.4
22
03
1.3
02
NA
Quantix/ldetek
Water
ppm

6.0
1.0
10
1.0
50
1.0
-
NR

3.9
0.6
0.7
8.0
5.0
0.6
0.25
NA
Soil
ppm

60
25
100
25
50
25
-
NR

49
4.9
4.9
31
18
1.7
3.5
NA
* Lowest concentration that yields a positive test result
— = not tested
# = test can give presence/absence indication at two detection levels (0.005 and 0 05 ppm)
NR = Not Responsive
NA = Not Available
APPENDK-C
Table 2a

-------
Table 2b - PAH and C-PAH R
Parameter/Analyte (rings)
Acenaphthene (3)
Acenaphthylene (3)
Anthracene (3)
Benzo[a]anthracene (4)
Benzo[a]pyrene (5)
Benzo[b]fluoranthene (5)
Benzo[g.h,i]perylene (6)
Benzo[k]fluoranthene (5)
Chrysene (4)
Dibenzo[a,h]anthracene (5)
Fluoranthene (4)
Fluorene (3)
lndeno[1,2,3-cd]pyrene (6)
Naphthalene (2)
Phenanthrene (3)
Pyrene (4)
Total PAH
eactivity
D-Tech
Water
ppb'
310
310
10
42
10
53
42
-
8
1100
5
110
8
1800
420
10
8
Soil
ppm
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
06
ENSYS
Water
ppb
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Soil
ppm
81
7.5
081
1.6
83
4.6
>200
94
1.2
>200
1.4
1.5
11
200
1.0
3.5
—
Ohmicron
Water
ppb
13
10
0.54
0.77
0.50
0.91
15
0.77
0.40
26
0.32
1.6
0.78
65
070
0.20
-
Soil
ppm
1.0
1.3
0.05
0.08
0.05
0.09
1.5
0.08
0.04
2.6
0.03
0.16
0.08
6.5
0.07
0.02
-
Ohmicron
C-PAH
Water
PPb
1100
150
0.44
0.02
0.08
0.04
0.30
002
004
014
20
37
002
380
2.7
2.0
-
Soil
ppm
>100
22
0.58
0.002
0.01
0.005
0.12
0.003
0005
0015
022
3.5
0.008
36
0.43
0.09
-
Quantix/ldetek
Water
ppb
600
20
-
-
-
-
-
-
-
-
-
200
-
100
30
400
50
Soil
ppm"
6.0
0.2
>50
>50
>50
>50
>10
>50
>50
>50
0.40
1.6
>10
1.2
0.3
4.0
0.7
* Sensitivity is defined by lowest concentration of compound that yields a positive detection.
** Lower Limit of Detection (LLD)
— = not tested
APPENDK-C
Table 2b

-------
Table 2c - PCB Reactivity
Parameter/Analyte
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Aroclor 1262
Aroclor 1268
* wipe concentration = ug/IOOcm*
Surface A is used to interpret test re
Surface B is used to interpret test re
- = not tested
Table 2d - TNT/RDX Explo
Parameter/Analyte
TNT
RDX
TNT = Trinitrotoluene
RDX = Hexahydro-1.3.5-tnnitro-1.2
- = not tested
Table 2e - Individual Anal
Parameter/Analyte
Mercury
Pentachlorophenol
Trichloroethylene (TCE)
Tetrachloroethylene (PCE)
Dioxin (2,3.7.8-TCDD)
Benzene (see also BTEX)^

wii
Surf A
100
450
160
27
14
90
9.0
9.0
69
D-Tech
36"
SurfB
51
220
82
14
7.2
45
4.5
4.5
34
Soil
ppm
5.7
25
9.0
1.5
0.8
05
0.5
0.5
3.8
Wipe
ug/IOOcm*
40
500
40
20
10
5
5
—
-
ENSYS
Oil/LJq.
ppm
9
75
10
35
2.5
1
1
1
9
Soil
ppm
4
50
4
2
1
0.5
0.5
—
—
G
Wipe
ug/IOOcm1
36
—
26
12
8
5
3
6
31
'hmicro
Water
ppm
094
14
084
0.34
0.22
0.20
0.20
0.36
0.92
n
Soil
ppm
2.7
27
2.2
0.80
0.42
0.50
0.30
0.64
2.3
suits from non-porous surfaces such as smooth metal or glazed tile-like surfaces.
suits from painted surfaces, rusted metals, or concrete-like surfaces.
sives Reactivity
D-T
Water
ppb
5
5
ech
Soil
ppm
0.5
05
1.5-triazme
yte Reactivity
D-T
Water
ppm
—
(D
1.5
0.3
_
—
ech
Soil
ppm
—
(1)
5
1
_
—
ENS
Water
ppm
—
—
»YS
Soil
ppm
0.7
0.8

ENS
Water
ppb
250-
5
_
_
0.4
5
;YS
Soil
ppm
os-
os
_
_
(2)
—
Ohmi
Water
ppb
0.07
—
cron
Soil
ppm
0.25
—

Ohm
Water
ppb
—
0.06
_
_
_
—
cron
Soil
ppm
—
0.1
—
_
_
—

BioNel
Water
ppb
250
_
_
_
_
—
iraska
Soil
ppm
0.5
—
_
—
_
—
•• ENSYS Markets the BioNebraska kits
(1) Kit scheduled for release, sensitivity unknown.
(2) Laboratory analysis only MDL equal to parts per trillion (ng/L) or parts per quadrillion (pg/L) depending
on sample concentration factor.
— = not tested

APPENDIX-C
Tables 2c, 2d, 2e

-------
Table 2f - Individual Pestii
Parameter/Analyte
2.4-D @
2.4-DNT
2,4-D Butyric butyl ester
Acetanilide
Acetochlor
Alachlor @
Alachlor Sulfonic acid
Aldicarb @
Aldicarb Sulfone
Aldicarb Sulfoxide
Aldrin
Ametryn
Atrazme @
Azinphos
Benomyl @
BHC, alpha
BHC. delta
BHC, gamma (lindane)
Bioresmethrin
Captan @
Carbaryl @
Carbendazim
Carbofuran @
Chlordane
Chlorpyrifos
Chlorsulfuron
Cholorthalonil
Cyanazme @
Cyclodienes
ODD.
DOE
DDT
Diazinon
Dicamba
Dichlorprop
Dieldrin
Diquat
Dursban
cide, Insecticide, and Herbicide Reactivity
ENSYS
Water
ppb
05
—
002"
0.1
01
01
0.1"
1
5
10"
0.32"
0.01"
001
10"
0.1"
—
—
—
100
_
—
—
01"
10
0.05
0.04

0.25
5.0
_
—
_
0.03
98"
17"
0.1
30"
0.05
Soil
ppm
0.2
0.5
1.8
—
—
—
—
—
_
—
—
—
10"
—
—
2
2
1
—
_
_
_
—
0.02
_
_
—
—
_
0.01
0.18
0.2
—
—
12
0006
—
—
Ohmicron
Water
ppb
0.70
—
_
—
—
0.05
—
0.25
0.27
1.8
0.294
—
005
—
038
—
—
20*
—
001
025
0.10
0.06
—
0.10
—
007
004
_
_
—
_
—
—
_
0.55"
—
—
Soil
ppm
_
—
_
_
_
—
—
—
_
_
_
_
—
_
—
—
_
_
—
_
_
_
—
—
_
_
—
_
_
_
_
_
—
—
_
_
_
—
Quantix/ldetek
Water
ppb
_
—
_
_
_
100
—
—
_
_
_
_
_
_
50
—
_
_
—
_
_
_
—
—
_
_
500
_
_
_
_
_
—
—
_
_
_
—
Soil
ppm
...
_
_
_
_
_
_
_
—
_
—
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
—
* Determined in Cyclodiene Ohmicron kit - see vendor literature
M Lower Level of Detection (LLD) under laboratory conditions
@ = available as PE samples
- = not tested
APPENDK-C
Table 2f -1

-------
Table 2f - Individual Pesti
Parameter/Analyte
Endosulfan
Endosutfan 1
Endosulfan II
Endrin
Ethylated Atrazines
Fenitrothion
Heptachlor
Hexazinone
Hydroxy atrazine
Imazapyr
Imazaquin
Isoproturon
Metalaxyl
Methomyl
Methoprene
Metolachlor
Metribuzin
Metsulfuron
Molinate
Nicotine (in tobacco)
Paraquat
Parathion
Picloram
Pirimiphos-methyl
Procymidone @
Prometon
Prometryn
Reldan
Silvex 2,4.5-TP
Simazene
Thiabendazole
Toxaphene
Tnasulfuron
Triazine
Trichloropyridinol
Trichopyr
Trifluralin
Urea Herbicides
:ide, Insecticide, and Herbicide Reactivity
ENSYS
Water
ppb
0.08
_
—
0.15-
0.05
100
0.69-
0.1

0.30
5.0
0.05
0.1
—
1000
01
—
025
0.5
10
0.02
004
1000-
_
_


0.02
0.50
0.04-
0.25
3.8
0.05
0.1
_
_
_
0.05
Soil
ppm
—
0.006-
0.006-
0.006-
—
—
0.006-
—
—
—
—
—
—
—
—
_
—
_
—
_
_
—
_
_
_
—
_
—
0.05
_
—
0.005-
—
_
_
_
_
—
Ohmicron
Water
ppb
1.2*
—
—
—
_
—
0.66
—
—
—
—
—
—
0.45
—
0.05
0.04
_
—
—
0.02
—
_
_
0.80
0.05
0.05*
—
1.4
0.03
—
2.6-
—
_
0.25
0.03
_
—
Soil
ppm
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
_
—
—
—
—
—
_
—
—
—
—
_
—
_
—
Quantix/ldetek
Water
ppb
_
_
_
_
_
—
_
—
—
_
—
_
_
_
_
100
_
_
_
_
_
_
_
_
_
—
_
—
—
_
—
_
_
50
_
—
1.0
—
Soil
ppm
_
_
_
_
_
—
—
—
—
—
—
—
_
_
_
_
_
_
—
—
_
—
_
_
—
—
_
—
—
_
—
_
—
_
—
—
_
—
* Determined in Cyclodiene Ohmicron kit - see vendor literature
— Lower Level of Detection (LLD) under laboratory conditions
@ = available as PE samples
- = not tested
APPENDEX-C
Table 2f - 2

-------
Table 3a - TPH / BTEX Crc
Parameter /Analyte
Acenaphthene
Anthracene
Benzo(a)pyrene
Benzole acid
Biphenyl
Chrysene
Creosote
1 ,2-Dichlorobenzene
2,4-Dinrtrotoluene
n-Decane
2-Ethyttoluene
4-Ethyttoluene
n-Heptane
Hexachlorobenzene
Hexane (mixed)
n-Hexane
Isooctane
MTBE
Methylcyclohexane
2-Methylpentane
Naphthalene
Nitrobenzene
2-Nrtrophenol
n-Nonane
n-Octane
o-Cresol
16PAHs
Pentachlorophenol
Phenanthrene
n-Propylbenzene
Styrene
1 ,2,4-Trimethylbenzene
1 ,3,5-Trimethylbenzene
Undecane
>ss-Reactivrty
D-Tech
Water
ppm*


6
>500

6

5
>500

5
5



>500
72

>500

11
5
>500


5
>500
>500






Soil
ppm


































ENSYS
Water
ppm*
0.01






0.02




1.6
0.1


0.11
>1200

0.45
0.008









0.07


>12
Soil
ppm*
0.5



10

1.5
2.5




130
10
65

8.5
>1000

35
0.8









7.0


>1000

Ohmicron
Water
ppm
0.17
0.06




0.10


14


2.4
0.08

63




0.03


44
34



0.08
0.27
0.07
0.04
0.14

Soil
ppm
1.7
0.6




1.0


140


24
0.8

63




03


44
34



0.8
2.7
07
0.4
14

Quantix/ldetek
Water
ppm




















008













Soil
ppm




31

1.0













2.2













* lowest concentration that yields a positive test result.
APPENDIX-C
Table 3a

-------
Table 3b - PAH and C
Parameter/Analyte
Creosote
Fuel oil #1
Fuel oil #2
Fuel oil #4
Fuel oil #6
Heating fuel
Diesel fuel
Turbine fuel
Fuel oil # 5
JP-4
JP-5
Jet A Fuel
Gasoline
Kerosene
BTEX
Benzene
Phenol
Toluene
2.4,6-Tnchlorobenzene
2.3,5,6-Tetrachloro-
benzene
Pentachlorobenzene
Pentachlorophenol
BEHP
1 -methyl-Naphthalene
2-methyt-Naphthalene
1-Chloro-Naphthalene
Halowax 1051
Dibenzofuran
Aroclor 1254
Biphenyl
•PAH Cross-Reactivity
D-Tech
Water
ppm














>100







>200






>100

Soil
ppm































ENSYS
Water
ppm































Soil
ppm
54














>200
>200
>200
>20
>200

>200
>200
>200
54
58
59
18
14


Ohmicron
Water
ppb
1 5
[280]

116]
[50]
17
36
[20]
17
[610]
[340]
>10.000
13
1700

















Soil
ppm
0.11



0.5
1.3
2.0




>1000
100
120

















Ohmicron C-PAH
Water
ppb
1.2



6.6
20
240

03


>10.000
200
2800

















Soil
ppm
0.12


2.1
1.1
32
18

13


>1000
160
830

















Quantix/ldetek
Water
ppm
0.1

4

6

1


2
10

4
6
3
















Soil
ppm
1.0

30

63

8
8

16
100

40
60
32
>500

>500






0.6
1.9




31
[ ] = LLD x 50% B/B0
APPENDK-C
Table 3b

-------
Table 3c - PCB Cross-Res
Parameter/Analyte
Biphenyl
Bifenox
Halowax 1000
Halowax 1051
Halowax 1099
2,5-Dichlorophenol
2,3,5-Trichlorophenol
Di-n-octylphthalate
Tetradifon
2,4-Dichloro-1 -naphthol
See fact sheets for others
ictivtty
D-Tech
W(\
Surf A

450
18,000

4500






K?"
SurfB

220
9000

2200






Soil
ppm

25
1000

250






ENSYS
Water
ppm











Soil
ppm

1000

1000




100
50

Ohmicron
Water
ppb
>1 0.000




>1 0.000
>1 0.000
>1 0.000



Soil
ppm











* wipe concentration = ug/IOOcm2
Surface A is used to interpret test results from non-porous surfaces, smooth metal or glazed tile-like surfaces
Surface B is used to interpret test results from painted surfaces, rusted metals, or concrete-like surfaces
APPENDIX-C
Table 3c

-------
Table 3d - TNT/RDX Explo
Parameter/Analyte
TNT
2-Amino-4,6-din'rtrotoluene
4-Amino-2.6-dmitrotoluene
2,6-Diaminonitrotoluene
2,4-Dinrtroaniline
1,2-Dinitrobenzene
1,3- Dmitrobenzene
2,4-Dinrtrophenol
2,4-Dmitrotoluene
2,6-Dinitrotoluene
HMX (3)
Picric Acid
Nitrobenzene
2-Nitrophenol
2-Nitrotoluene
3-Nitrotoluene
4-Nitrophenol
4-Nitrotoluene
RDX (2)
Tetryl (1)
1 ,3,5-Tnnitrobenzene
RDX
HMX (3)
Nitroglycerine
Nitroguanadme
PETN (4)
Tetryl (1)
TNT (5)
All others listed above
isives Cross-Reactivity
D-Tech
Water
ppb

>500
>500
>500



>500
120
>500
>500



30

>500

>500
15
20

150


>500
>500
>500
>500
Soil
ppm

>50
>50
>50



>50
12
>50
>50



3.0

>50

>50
1.5
2.0

15


>50
>50
>50
>50
ENSYS
Water
ppb





























Soil
ppm


>100





1 1
2


>100
MOO

>100

>100

0.9
10

24
8.9
10
1.0



Ohmicron
Water
ppb


0.10

.0.10
1000
2.4
80
1 0
100

025
3400
2300
025
160

1200
700
010
0.04








Soil
ppm


5.9

0.99
3300
11
240
33
360

45
>1 0.000
>1 0.000
2.4
1400

5600
2900
1.9
0.15








(1) Tetryl = Methyl-2.4,6-trinitrophenylnitramine
(2) RDX = Hexahydro-1,3,5-trinitro-1,3.5-triazine
(3) HMX = Octahydro-I.S.SJ-tetranitro-I.S.SJ-tetrazocine
(4) PETN = Pentaerythritol tetranitrate
(5) TNT = Trinitrotoluene
APPENDIX-C
Table 3d

-------
Table 3e - Individual Anal;
Parameter/Analyte
Mercury Test
Gold trichloride
Chromium nitrate
Silver nitrate
Pentachlorophenol
CCA (1)
4-Chlorophenol
Creosote
2. 3-Dichlorophenol
2,4-Dichlorophenol
2,5-Dichlorophenol
2,6-Dichlorophenol
3.5-Dichlorophenol
Diesel fuel
Hexachlorobenzene
Hexachlorocyclohexane
PCB(Aroclor1254)
Pentachlorobenzene
Phenol
2,3,4-Trichlorophenol
2.3.5-Trichlorophenol
2, 3,6-Trichlorophenol
2,4.5-Trichlorophenol
2.4,6-Trichlorophenol
Tetrachlorohydroquinone
2,3,4,6-Tetrachlorophenol
2.3.5.6-Tetrachlorophenol
TCE/PCE
Dioxin (2,3.7,8-TCDD)
yte Cross-Reactivity
D-Tech
Water
ppb




*






















*

Soil
ppm




*














.







•

ENSYS
Water
ppb





>1 0.000
>800
>1000

>1000

600

>10.000


>1000
>1400
>600
600


500
100
>1500

7

#
Soil
ppm





>1000
>1000
>1000

>1000

700

>10.000


>1000
>1000
>1000
400


100
16
500



#
Ohmicron
Water
ppb








610
880
63
270
1700

1600
5800



53
1.5
2.4
22
15
8.7
091
021


Soil
ppm








>1000
>1000
210
420
>1000

>1000
>1000



120
3.7
4.4
38
22
14
1.2



BioNebi
Water"
ppm

300
240
17



















1





•aska
Soil
ppm





























* Contact D-Tech, literature unavailable at time of publication
(1) CCA = Chromated copper arsenate
# 2,3,7.8-TCDF only analyte to show cross reactivity (approx. 20 times less sensitive than 2,3.7,8-TCDD)
APPENDIX-C
Table 3e

-------
Table 4 - Assay K
Parameter/Analyte
Storage Temp.
POP
PCB
PAH
BTEX/TPH
TNT/RDX
Pesticides
Benzene
Dioxin
Mercury
TCE/PCE
THMs
Operating Temp.
PCP
PCB
PAH
BTEX/TPH
TNT/RDX
Pesticides
Benzene
Mercury
TCE/PCE
Dioxin
THMs
Shelf Life
PCP
PCB
PAH
BTEX/TPH
TNT/RDX
Pesticides
Benzene
Dioxin
Mercury
TCE/PCE
THMs
it Storage and Operating Conditions
D-Tech
Water


Soil


40-1 DOT (4-38'C)
40-1 OO'F (4-38'C)
40-1 00"F (4-38'C)
refrigerate








40-100'F (4-38'C)






45-1 00°F (7-38'C)
45-100'F (7-38'C)
45-100'F (7-38'C)
Room temp






45-100'F (7-38'C)








expiration date
expiration date
expiration date
expiration date












ENSYS
Water

40'F (4'C)
Soil

Room tsinp
Room temp.
< 80"F (27"C)
Room temp.
Room temp
Room temp.
Room temp.
Room temp.
Room temp.

Room temp.




55-90'F (13-32-C)
40-90'F
48-90'F
60-1 00°F
40-100'F
(4-32°C)
(9-32'C)
(16-38'C)
(4-38'C)
64-81'F (18-27'C)
55-90'F (13-32-C)
50-98'F (10-37-C)


Room temp.


Room tsmp.


3 mo. room/4 mo. refrig
6mo.<80°F(27'C)
6 mo. <80°F(27'C)
12 mo
24 mo. <80*F (27'C)
6mo.<80°F(27'C)
6mo.<80'F(27°C)
6mo.<80-F(27*C)
6 mo 40°F(4°C)


6mo.<80°F(27'C)
Ohmicron
Water

Soil

3W6*F (2-8°C)
3&-46-F (2-8'C)
36M6°F (2-8*C)
36-46-F (2-8'C)
36-46'F (2-8'C)
36-46'F (2-8'C)












59-86'F (1^30'C)
5M6'F (15-30'C)
5M6'F (15-30'C)
5M6°F (15«)'C)
59^6°F (15-30'C)
5&86*F (15-30'C)












1 Year
1 Year
1 Year
1 Year
1 Year
1 Year










Quantixfldetek
Water




refrigerate











50-85'F











6 mo.







Soil



refrigerate
refrigerate










50-85'F
50-85'F










6 mo.
6 mo.







BioNebraska
Water









Soil









40'F (4'C)"




















50-98'F (10-37'C)
























6 mo. 40'F (4'C)




•All Kits are validated for 4°-37°C (39°-100°F)
** Mercury test kits stored at 4°C; extracts can be stored at room temp.
APPENDIX-C
Table 4

-------
Table 5 - Moisture Removal Guidelines

Parameter/Analyte
All parameters









ENSYS
Water Soil

Place sample on an
absorbant material.
(filter paper or coffee
filter) and ring out.
Reweigh sample.

pH should be between
3-11 See Vendor's
literature for special
cases.
Ohmicron
Water Soil

Measure sample into
a coffee filter. Wrap
filter with paper
towels and squeeze
cutwater. Remove
the soil and weigh
pH should be between
3-11 See Vendor's
literature for special
cases
BioNebraska
Water Soil

No need to remove
water, but more
sample will be needed
with high moisture
samples

Highly alkaline soils
may need more acid
to digest sample.

APPENDIX-C
Table 5

-------
Table 6a - Kit Proces
Parameter/Analyte
Total BTEX
TPH
C-PAHs
PAHs
PCB
PCB wipe
PCB oil
Pentachlorophenol
TNT
RDX
Mercury
Dioxm (2,3.7.8-TCDD)
THMs
TCE/PCE
Pesticides
Benzene
s Times (Samples per batch / Batch process time)
D-Tech
Water













4125m


Soil



4/25m
4125m
4125m


4125m
4125m






ENS
Water

10/50m

10/70m







5/70m


44/90m
5/60m
»YS
Soil

10/70m

10/70m
10/75m

4/45m
10/70m
10/40m
10740m
16790m



12/30m

Ohmi
Water
50/60m
50/60m
50/60m
50/60m
50/60m


50/60m
50/60m





60/60m

cron
~~Soil
207120m
20/1 20m
20/1 20m
20/1 20m
207120m


20/1 20m
20/1 20m





60/1 20m

Quantii
Water
3730m
5715m

5730m












i/ldetek
Soil
5730m
5730m

5730m












BioNel
Water
















iraska
Soil










16790m





m = minutes
P = Plate kit
APPENDIX-C
Table 6a

-------
Table 6b - Cost per Sa
Parameter/Analyte
Total BTEX
TPH
C-PAHs
PAHs
PCB
PCB Wipe
PCB Oil
Pentachlorophenol
TNT
RDX
Mercury
Dioxin (2.3,7,8-TCDD)
THM's
TCE/PCE
Extraction Kits
Comments:
imple
D-Tech
Water







$26
$26
$26





Soil
$26


$26
$31
$31

$26
$26
$26



$26
$25
Kits usually contain
materials for 4 tests.
Meter $299.00
Computer program
$150.00
ENSYS
Water
$25
$25

$40
$30


$40







Soil
$25
$25

$40
$30
$30
$25
$40
$21
$25

$50
$15


Sold as 12 tests per
kit. with 1.2 or
more calibrators
Ohmicron
Water
$7/$11

$157 $19
$13/$18
$13/$18


$8/$11
$7/$11






Soil
$13/$20

$20/$27
$197 $25
$19 7 $25


$14/$15
$13 7 $20






Water kits sold in
100 or 30 tube kits.
With standards and
blanks, the 30 tube
kit equates to 22
samples per kit.
Soil kits sold in 80
or 20 samples/ kit.
Includes sample
collection and
extraction kits
Prices:
Large kit /Small kit
Quantix/ldetek
Water
$32
$32

$36











Soil
$32
$32

$36











Kit includes soil
collector, sample
preparation supplies.
and extraction tubes
Meter not included
BioNebraska
Water















Soil










$21




May be purchased
as the total kit. or
just the assay kits.
or extraction kits.
APPENDK-C
Table 6b

-------
Table 6c - Individual Pest
Parameter/Analyte
2.4-D
Alachlor
Aldicarb
Atrazme
Benomyl
Bioresmethrin
Captan
Carbaryl
Carbofuran
Chlordane
Chlorpynfos
Chlorosulfuron
Chlorothalonil
Cyanazine
Cyclodienes
DDT
Diazinon
Fenitrothion
Isoproturon
Imazaquin
Imazapyr
Lindane
Metsulfuron
Metalaxyl
Methomyl
Methoprene
Metolachlor
Metnbuzin
cide, Insecticide, and Herbicide Cost per Sample
EN!
Water
$8
$8
$8

$9
$8


$8

$8
$8

$9
$8

$9
$14
$8

$9

$8
$9

$9
$14

SYS
Soil
$8








$8





$8





$14






Ohm
Water
$12
$10
$10
$10
$12

$12
$11
$10

$13

$11
$11
$12









$13

$10
$13
icron
Soil




























Quanti:
Water

$8

$8
$10







$10






$10






$8

x/ldetek
Soil




























APPENDIX-C
                               Table 6c -1

-------
Table 6c - Individual Pest
Parameter/Analyte
Nicotine Cm tobacco)
Paraquat
Procymidone
Sitvex
Toxaphene
Triasulfuron
Triazine
Trichloropyndinol
Tnclopyr
Tnfluralm
Urea Herbtctdes
Comments:
cide, Insecticide, and Herbicide Cost per Sample
EN!
Water
$9
$9
$8


$7
$8



$8
SYS
Soil




$8






Tube kits consist of 40
tubes, which equates
to 24 field samples
analyzed
Plate kits contain 96
test wells per kit.
which equates to 44
Reids samples
analyzed
Ohm
Water

$12

$13



$16
$13


icron
Soil











Available in 30 and
100 tube kits.
Prices based on
average of 20 field
samples analyzed In a
30 tube kit (remainder
needed for standards).
Quanti;
Water









$8

ic/ldetek
Soil











48 assays per kit
Microwell kits used
APPENDIX-C
Table 6c - 2

-------