905R95101
U.S. EPA Immunoassay
Technology Workshop
March 28-29,1995
Chicago, Illinois
Sponsored by:
With support from:
U.S. EPA Region 5
The Great Lakes Toxic Reduction Effort
Great Lakes National Program Office
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AGENDA
U.S. Environmental Protection Agency - Region 5
IMMUNOASSAY TECHNOLOGY WORKSHOP
March 28 -29,1995
U. S. EPA REGION 5
CHICAGO, IL
DAY1
Morning session
8:30 - 9:00 a.m.
Matthew Williams
Chair, U.S. EPA Region 5 Immunoassay Workgroup
Welcome and Introductions
Goals and Objectives
9:00-9:15 a.m.
David A. Ullrich
Deputy Administrator, U.S. EPA Region 5
Opening Remarks
9:15-10:30 a.m.
Immunoassay testing at
U.S. EPA
0 history
0 methods update
0 validation requirements
0 data needs/project opportunities
0 future
Barry Lesnik
Manager, U.S. EPA Headquarters Immunoassay Program
* Break *
10:30-10:45 a.m.
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Day 1 Continued...
10:45-11:45 a.m.
Part 1: Introduction to
Immunoassays
Part 2: Analytical Issues & Case
Studies
Jeffre Johnson
U.S. EPA Exposure Research Program,
EMSL - Las Vegas
LUNCH
11:45-1:00 p.m.
1:00-1:30 p.m.
Afternoon Session
Discussion Session on
Morning Presentations
1:30-2:30 p.m.
Parti:
* Break *
2:30 - 2:45 p.m.
Expect the Unexpected
in the Field
2:45-3:45 p.m.
Diane Anderson
Contractor, U.S. EPA Immunoassay Program
Part 2: Different Types of Kits
3:45-4:15 p.m.
Group Discussion on Immunoassay
Training and Certification
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DAY 2
Morning Session
8:30-9:15 a.m.
Field Study of Immunoassay Screening Methods for BTEX, PAHs and PCBs
at a Former Coal Gasification Facility
Robert M. Schulte, Delaware Department of Natural Resources and Environmental Control,
Superfund Branch.
9:15-10:15 a.m.
Immunoassay testing for PCBs in the Kalamazoo River, Michigan
Terese Van Donsel, U.S. EPA Region 5 Office of Superfund
* Break *
10:15-10:30 a.m.
10:30-11:30 a.m.
Immunoassay Screening for Sediment in the Ottawa River, Ohio
Jeff Wander, OEPA Division of Environmental & Remedial Response
Lunch
11:30-1:00 p.m.
1:00-1:30 p.m.
Immunoassay Applications by the Illinois Environmental Protection Agency
Ralph Foster, IEPA Emergency Response Division
1:30-2:00 p.m.
Evaluation of An Immunoassay for Mercury in Soil
Larry C. Waters Ph.D., Oak Ridge National Laboratory, Chemical and Analytical Sciences
Division
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Day 2 Continued...
2:00 - 2:30 p.m.
Evaluation of EnSys PAH-RISc Test Kit
R. Paul Swift, Ph.D., ICF Kaiser, ESAT Contractor for U.S. EPA Region 10
2:30-3:15 p.m.
Using Immunoassays to Sample for PAHs at Pine Street Superfund Site,
Burlington Vermont
Ross Gilleland, U. S. EPA Region 1, Office of Superfund
* Break +
3:15-3:30
3:30 - 4:00 p.m.
Comparison of Immunoassay versus laboratory and FASP Technique for
PCBs in Sediment
Dennis Wesotowski, U.S. EPA Region 5, Environmental Sciences Division, Chief of Contract
Analytical Services Section.
4:30 - 5:30 p.m.
Group Discussion & Wrap up
• Potential Sites to Apply New or Current Immunoassays
• Evaluating Costs/Benefits
• Funding Sources for Projects
• National and Regional Direction for lAs
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Table of Contents
• Immunoassay Techniques: Goals and Objectives
• U.S. EPA Developers Guide
Immunoassay Methods in SW-846: Recommended Format and Content for
Documentation Supporting New Submittals
• U.S. EPA Validated Method 4010 PCP
• U.S. EPA Validated Method 4015 2,4D
• U.S. EPA Validated Method 4020 PCB
• U.S. EPA Validated Method 4030 TPH
• U.S. EPA Validated Method 4035 PAH
• U.S. EPA Validated Method 4040 toxaphene
• U.S. EPA Validated Method 4041 chlordane
• U.S. EPA Validated Method 4042 DDT
• U.S. EPA Validated Method 4050 TNT
• U.S. EPA Validated Method 4051 RDX
• U.S. EPA Validated Kits Matrix
• A User's Guide to Environmental Immunochemical Analysis
• A Technical Discussion About Immunoassays
A Technical Discussion About Immunoassays: An EMSL-Las Vegas
Perspective
• U.S. EPA Region 5 Vendor Information Matrix
• U.S. EPA Headquarters, Expect the Unexpected in the Field
• MDNR - Region 5, Kalamazoo River Study
Evaluation of Three PCB Field-Screening Technologies: Results of a
Demonstration Conducted at the Allied Paper, Inc./Portage Creek/Kalamazoo
River Super-fund Site, Kalamazoo, Michigan.
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• OEPA - Region 5, Ottawa River Study
• Delaware DNR - Superfund Study
Field Study of Immunoassay Screening Methods for BTEX, PAH's, and
PCB's at a Former Coal Gasification Facility.
• U.S. EPA Region 10, Evaluation of a PAH Test Kit
Evaluation of the ENSYS PAH-RISc® Test Kit
• Oak Ridge National Laboratory, Evaluation of a Mercury Test Kit
Evaluation of an Immunoassay for Mercury in Soil
• U.S. EPA Region 1, Pine Street Study for PAHs
Case Study in the Use of Immunoassay Field Screening Method for Polycyclic
Aromatic Hydrocarbon (PAHs), Pine Street Superfund Site, Burlington, VT.
• Illinois EPA, Cross-Program Summary of Immunoassay Applications
• U.S. EPA Region 5, Comparison of Immunoassays versus Laboratory
• Notes/Handouts
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I !
n
Immunoassay Workgroup
Background
During the past several years the interest in and use of immunoassay testing for sediment,
soil, water and other media has increased in Region 5 and GLNPO. Associated with this
interest are questions regarding when, how, and to what degree immunoassays should be
applied, as well as the appropriate use and limitations of sampling results for making
contaminant assessment and remediation decisions. To begin answering these questions,
members of the In Place Pollutant Task Force (IPPTF), a multi-program group charged with
addressing contaminated sediment issues in the Region and GLNPO, and non-IPPTF
interested participants from other programs, formed the Immunoassay Workgroup, as a
part of the IPPTF.
Goal
The goal of the work group is to provide current and practical information about
immunoassays to EPA Region 5, GLNPO and state programs in order to advance the
knowledge and appropriate use of immunoassays to help make better decisions about the
environment while providing savings in project cost and time.
Objectives
To support this goal, the workgroup is focusing on achieving the following objectives for
the use of immunoassays:
• identify key technical advantages/disadvantages of immunoassays versus other
techniques;
• pursue consensus to resolve key technical issues; and
• assist the Region, GLNPO and states with identifying future sites to apply and
evaluate the technique.
Common Questions for the Workshop
The Immunoassay Workshop will pursue answers to common questions about
immunoassays such as:
o what are they and for which analytes can they be used?
o when should they be used at a site?
o what are the appropriate procedures for using them?
o where have they been used and what were the results?
o what are the limitations/errors associated with results?
o what is the cost/benefit of using them?
Immunoassay Technology Workshop
March 28 - 29, 1995
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IMMUNOASSAY WORKGROUP MEMBERS & PARTICIPANTS
(USEPA Region 5 and GLNPO)
Name
Alwan, Al
Eleder, Bonnie
Evangelista, Erlinda
Fenner, Ken
Fox, Rick
Harris, Willie
Hoist, Linda
Johnson, Steve
Kelley, John
Khanna, Kaushalya
Kleiman, Judy
Lubin, Arthur
Schupp, George
Wesolowski, Dennis
"Williams, Matthew
Zar, Howard
Division/Office
ESD
OSF
i Chem Lab
Water
GLNPO
ESD
Water
ESD
ESD
OSF
RCRA
ESD
ESD
s ESD
/ Water
Water
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
(312)
Phone
353-2004
886-4885
353-4331
886-6777
353-7979
353-2306
886-6758
886-1330
353-3808
353-2663
886-1482
886-6226
886-6221
886-1970
353-4934
886-1491
Fax
3-4342
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886-2591
6-7804
6-2018
3-4342
6-7804
353-4342
3-4342
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353-4788
353-4342
353-4342
353-4342
886-7804
6-7804
Mailcode
SQQ-14J
HSRW-6J
SLO-10C
WQ-16J
GR-9J
SQ-14J
WS-16J
SPB-14J
S-14
HSRL-5J
HRP-8J
SQ-14J
SQQ-14J
SQC-14J
WS-16J
WS-16J
* Chair
ESD
GLNPO
OSF
RCRA
Water
- Environmental Sciences Division
- Great Lakes National Program Office
- Office of Superfund
- Office of Resource Conservation and Recovery Act
- Water Division
Immunoassay Technology Workshop
March 28-29, 1995
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-------
IMMUNOASSAY METHODS IN SW-846
RECOMMENDED FORMAT AND CONTENT FOR DOCUMENTATION
SUPPORTING NEW SUBMITTALS
-------
1. Background
The Methods Section of the Office of Solid Waste is
responsible for the promulgation of rugged and reliable analytical
techniques in support of the Resource Conservation and Recovery Act
(RCRA) program. The methods published in Test Methods for
Evaluating Solid Waste, SW-846, are used to measure the
concentration of specific pollutants or to establish whether a
waste stream demonstrates a hazardous characteristic (e.g.
ignitability, corrosivity, reactivity or toxicity).
SW-846 currently provides reliable and sensitive laboratory
methods for the analysis of Appendix VIII analytes. However, some
of these methods may be too costly or require too much analysis
time for some applications. The Methods Section also recognizes
the savings that could be achieved by sending only contaminated
samples to analytical laboratories for quantitative analysis.
Therefore, the Methods Section has recognized the need for more
rapid, less expensive screening procedures that can be used either
in the field or in the laboratory.
The number of field or laboratory screening procedures with
potential application to the RCRA program is increasing rapidly.
The increasing number of supporting documentation packages to be
reviewed, and the time required to interpret each manufacturer's
data format, has significantly increased the amount of time that it
takes for a method to become "approved". As more and more methods
are developed, this situation will become worse. Therefore, the
Agency has prepared this guidance document to provide developers
with a list of specific information which must be included in the
documentation package submitted as demonstration of the efficacy of
the test procedure. The format and information requested in this
guidance document are based on the requirements of the Food and
Drug Administration's 501(k) Premarket Notification. This guidance
document does not supersede or replace the more rigorous
requirements described in Test Method Equivalency Petitions,
EPA/530-SW-87-008, OSWER Policy Directive No. 9433.00-2 (2/87).
That document provides the requirements for a method equivalency
petition which may be used to promulgate a method outside of the
Work Group process.
2. The Evaluation Process
Specifically, the Agency plans to review three documents
during the evaluation of a new ixnmunoassay test product; 1) the
package insert, 2) a documentation package that provides the
information specified in this guidance document, and 3) a document
(to be treated as confidential business information or CBI)
delineating the manufacturer's internal quality control criteria
for insuring lot-to-lot consistency of performance and stability
claims in product manufacture. The claims made in the package
insert will be reviewed to determine if the test product is
applicable to the RCRA program. If the test product is determined
to be applicable, the information presented in the documentation
Rev 3; February, 1995
-------
package will be reviewed to verify that it fully and clearly
supports each of the claims made in the package insert. If,
however, the documentation package does not support the claims made
in the package insert, the test product will not be accepted by the
Agency. If the test product is accepted by the Agency, only the
documents listed in items 1) and 2) will be included in the public
docket.
All documentation reviewed by the Agency in support of new
immunoassay procedures will be reviewed with the expectation that
all of the requested information is present. If this information
is not readily apparent to the reviewer, the documentation will be
returned to the submitter for revision. Therefore, submittal in
the recommended format is strongly advised. Those methods with
complete, acceptable submittals will be routed to the appropriate
SW-846 Methods Workgroup for consideration for inclusion in SW-846.
Completeness of a submittal will be evaluated using the elements
defined in Section 4 of this document as criteria. Acceptability
of a submittal will be evaluated using the following criteria:
2.1 Target Analvtes
The analytes and sample matrices targeted by the method are
regulated under RCRA, or are of interest to one or more of the
RCRA program areas.
Cross-reactivity claims must indicate the recognition of other
cross-reacting compounds relative to the target analyte(s)
specified by the method. The cross-reactivity data provided
must demonstrate that the test product is most sensitive to
those compounds identified as target analytes. Section 3.2
provides guidance on the generation of cross-reactivity data.
2 .2 Detection Limit
The detection limits targeted by the test product must have
some utility in RCRA testing, e.g., some regulatory action
limit, or concentrations designed to be used as go/no go
action indicators. The basis for the detection limit(s)
selected must be clearly stated.
2.3 False Negative/False Positive Rate
False negatives are defined as a negative response for a
sample that contains the •target analyte(s) at the stated
action level. Ideally, a candidate procedure should produce
no false negatives. The maximum permissible 'false negative
rate is 5% at the action level specified.
False positives are defined as a positive response for a
sample that contains analytes below the claimed action level.
The rate of false positives at the claimed action level will
not be specified by the OSW, but must be represented by the
developer in the package insert and supporting documentation
Rev 3; February, 1995
-------
package.
The SW-846 Methods Workgroups will be provided with the method
in SW-846 format, a summary of the performance data (detection
limit, false negative/positive rates, cross-reactivity data), and
any comments from the reviewer. Any information submitted as part
of the documentation package may be transmitted to the Workgroup,
and will become part of the public record, unless the person/group
submitting the package asserts a claim of confidentiality. Such
claims must be made on specific parts of the package rather than
the entire submittal. For example, a manufacturer's internal QA
data are likely to be confidential, and as such, will be
distributed on a limited basis and withheld from the public record.
On the other hand, there is no reason for the information included
in the package insert and/or field data to be considered
confidential, and such an assertion will result in the package
being returned to the submitter.
3. The Performance Data
3.1 False Negative/False Positives
While screening procedures need not be fully quantitative,
they should measure the presence or absence of target analytes .at
or below regulatory action levels. Therefore, initial
demonstration of method performance requires measurement of the
percentage of false negatives and false positives generated using
the procedure for the claimed sample matrices. These data must be
generated by analyzing split samples using both the method under
evaluation and a reference method. The reference method should be
an approved SW-846 quantitative method, including both preparation
and determinative steps. In addition to comparing determinative
data points, this comparison should demonstrate that the extraction
efficiency of the test being evaluated-correlates with (but does
not have to be equivalent to) that of the standard method. For
semivolatile analytes, Soxhlet extraction, either simple or
automated, is considered to be. the reference extraction procedure.
The percentage of false negatives and false positives should
be measured using 20-50 samples of the claimed matrix(ces), spiked
at the claimed action level, by determining the incidence of false
negative results. A sufficient volume of each spiked sample should
be prepared so that each test can be cotnpleted with one lot of
material. In addition, a sufficient number of aliquots of each
spiked matrix should be analyzed and the results compared to those
of the SW-846 reference method in order to demonstrate correlation
of results," and to characterize the sample in terms of target
analytes and potential interferences. This demonstration must be
made for each matrix for which the method is claimed to be
applicable.
3.2 Non-Target Interferences and Cross-Reactivity
A minimum of 20-50 negative samples, confirmed by an SW-846
Rev 3; February, 1995
-------
reference method, must be analyzed to demonstrate that the method
is not susceptible to matrix interferences. A separate study
should be conducted to establish the effect of non-target
interferences. For example, the immunoassay may produce a positive
response to non-target analytes similar to the targeted analytes,
or to chemically dissimilar co-contaminants. At a minimum, this
study must evaluate the response of the test product to all other
RCRA analytes (Table 1) in that compound class, as well as for the
selected lists of compounds shown in Tables 2-5. This testing
scheme is designed to ensure that all other similar RCRA analytes
and likely co-contaminants are evaluated during cross-reactivity
testing. If you choose not to test for these compounds, it becomes
more likely that the reviewer or Workgroup will return the package
for additional testing.
The package insert must present the concentration of a cross -
reactant that will give a false positive response.
3.3 Matrix Applicability
In some cases, a single test product may be applicable to more
than one matrix, utilizing extraction protocols to isolate the
target analytes into a medium suitable for testing by immunoassay.
In order to demonstrate applicability to the claimed matrices, test
data should be submitted for three different types of samples :of
each matrix (e.g., clay, sand and loamy soil). These samples
should either be characterized reference materials or spiked
matrices containing known amounts of target analytes. In either
case, bulk samples should be carefully homogenized to reduce sub-
sampling errors. The sample matrices should be selected to
represent what is regulated under RCRA (e.g., soil, oily waste or
waste waters). OSW reserves the right to reject methods based upon
non-representative analysis of matrices and analytes. Negative
controls must be analyzed with each set of samples.
Matrix-specific performance data in support of claims,
including detection limits, should be gathered by analyzing ten
replicate aliquots of three different sample matrix types spiked at
the claimed detection concentrations. The results of testing the
low concentration samples should be reported as positive or
negative response. The results of testing the high concentration
samples should be reported as either semi-quantitative results
(above, below or within a numerical range) or as positive/negative
response.
3 .4 Field Trials
Data from at least one field trial (two to three are
preferred) in support of product performance claims for a
particular matrix are required. Test products from at least three
separate manufacturing lots should be used. These data should
demonstrate that the method is applicable to analysis of the target
analyte(s) and claimed matrices in at least 30 (preferably many
more) real-world (un-spiked) samples 1) at or near the action
levels specified in the test product, 2) well above the action
Rev 3; February, 1995
-------
levels, and 3) well below the action levels. Field data generated
using an excessive proportion of samples that are not contaminated
with the target analytes, or are only contaminated with high
concentrations of the target analytes, are not useful. The field
trials must provide a comparison between the test product results
and results generated using a reference method. As with the
generation of false negative/positive data, the reference method
should be an approved SW-846 quantitative method, including both
preparation and determinative steps. Furthermore, the results of
these field trials should support the false negative/positive rate
claims presented by the develo'per for the method.
Field trials are not to be performed by the developer or
personnel employed by or involved in the development of the method.
Field trials studies are to be performed by a credible group that
is not affiliated with the developer. The field trial plan,
including the study objectives, methods, sample description,
reference method employed, data and conclusions should be provided
in the documentation supplied to OSW. Individuals performing field
evaluations should be willing to discuss the study with EPA
reviewers upon request, and such individuals should be identified
in the documentation.
4. The Documentation Package
The documentation package should provide substantive
documentation to support the claims being made about the test
product, including each of the 24 elements described in this
section.
4.1 General Information - The following information is required as
background material. This will become part of the public docket
that supports all method proposals and promulgations:
4.1.1 Name and Address of Manufacturer
4.1.2 Proprietary and Common Names of the test product(a)
4.1.3 Intended Use of the test product(s) - This section
should address both target analytes and applicable matrices,
as well as detection limits.
4.1.4 Summary of the Test - This section should briefly
explain the principle of the immunoassay and the quantitation
system used in the test product. For example,
In general, the method is performed using a water sample
or an extract of a water sample. The sample/extract and
an enzyme conjugate reagent are added to immobilized
antibody-. The enzyme conjugate "competes" with the
target analyte present in the sample for binding to
immobilized antibody. The test is interpreted by
comparing the colorimetric (yellow) response produced by
testing a sample to the colorimetric (yellow) response
Rev 3; February, 1995
-------
produced by simultaneous testing of standard(s).
4.2. The Test Product
4.2.1 Reagents - Provide a list of all reagents included in
the test product, and a separate list of all reagents that are
necessary for performance of the test, but are noc included in
the test product. Specify reagent, number of containers
provided, and the volume and concentration provided or
necessary.
4.2.2 Instrumentation - Provide a list of all instrumentation
included with the test product, and a separate list of all
equipment/instrumentation that are necessary for performance
of the test, but are not included in the test product.
4.2.3 Storage Conditions - Provide the recommended range of
storage temperatures, as well as any other storage
recommendations (i.e., humidity specifications, protection
from light). Also provide the design of storage stability
testing (i.e., actual vs. accelerated) and summary tables of
the results of this testing.
4.2.4 Physical, Biological or Chemical Indications of
Instability or Deterioration - This section should provide
specific indicators that may be used as evidence that one or
more parts of the test product are unstable and should not be
used.
4.3. The Test
4.3.1 Warning or Precautions for Users - This section should
address any specific safety concerns presented by the test
product material or performance of the test. General safety
precautions (i.e., wear eye protection) need not be addressed.
4.3.2 Limitations of the Procedure - Describe any limitations
of the test when used as described in Section 4.3.4. For
example, it may be critical that some test steps be performed
exactly as written (i.e., number or volume of washings, timing
between steps). Describe and substantiate the following:
o The acceptable temperature range across which the test
product will exhibit the claims being made,
o The storage stability of the test product,
o" The number of tests that may be performed
simultaneously
4.3.3 Specimen Collection and Preparation - Provide any
specific instructions for sample collection (i.e., minimum
sample volume or size) or preparation (i.e., removal of
particulate matter) that are necessary for successful
Rev 3; February, 1995
-------
performance of the test.
4.3.4 Assay Procedure
4.3.4.1 Instructions for Preparation of Reagent and
Substrate - If the substrate or any of the reagents
cannot be used as received in the test product, provide
instructions for their preparation.
4.3.4.2 Assay Procedure - Provide step-by-step
instructions for performance of the assay.
4.3.5 Stability of the Final Reaction - Provide data that
describe the length of time that the final reaction (i.e.,
color change) is stable. This information is critical in
evaluating how many tests may be run simultaneously, as well
as determining when tests must be repeated if the analyst does
not read test results promptly.
4.3.6 Manufacturer's Internal Quality Control - Provide
sufficient quality control data to support the assertion that
intra- and inter-lot test product variability are controlled.
Also discuss measures to ensure long-term production of the
test product (i.e., quantity of stored antibody, provisions
for production of additional antibody). Internal QA data are
confidential, and as such, will be distributed on a limited
basis, with the consent of the developer, and withheld from
the public record.
4.4. The Data
4.4.1 The Dose-Response Curve - A dose-response curve must be
provided which provides a graphical representation of the
signal generated by the test product vs. the concentration of
target analyte required to generate that response.
4.4.2 Performance Data
4.4.2.1 Reproducibility
4.4.2.1.1 Intra-Assay - Provide data demonstrating
the reproducibility of the test product when
samples are analyzed repeatedly using test products
from one manufacturing lot.
4.4.2.1.2 Inter-Assay - Provide data demonstrating
the reproducibility of the test product when
samples are analyzed repeatedly using test products
from different manufacturing lots.
4.4.2.2 Bias
4.4.2.2.1 Dilution Study - Provide data
demonstrating the bias introduced by serial
dilution of samples (i.e., is the recovery of
Rev 3; February, 1995
-------
target analyte a function of concentration?).
4.4.2.2.2 Recovery Study - Provide data
demonstrating that the test being evaluated
exhibits consistent recovery during any extraction
step(s). Table 6 provides an example format for
presentation of data from the recovery study.
4.4.2.2.3 Correlation Study - Provide data
correlating immunoassay product test results with
the results generated using an SW-846 reference
method. These data must be used to calculate false
negative/positive rates at or near the action
level. Table 7 provides an example format for
presentation of false negative/positive data.
Example tables for presentation of the study data
are provided in Table 8 (Table 8a is for test
products configured at a single action level, and
Table 8b is for test products configured with
multiple action levels).
4.4.2.3 Cross Reactivity - Provide data that illustrate
the cross-reactivity of the test product for non-targeted
analytes relative to the claimed target analyte(s) . The
data provided must include all other RCRA analytes in
that compound class (Table 1) , as well as the analytes in
the selected lists of compounds shown in Tables 2-5.
This testing scheme is designed to ensure that all other
similar RCRA analytes and likely co-contaminants are
evaluated during cross-reactivity testing. The cross-
reactivity data provided must demonstrate that the test
. product is most sensitive to those compounds identified
as target analytes. All data are to be normalized to the
response of the target analyte. Table 9 provides an
example format for presentation of the cross-reactivity
data.
4.5. Bibliography - Provide copies of published material relevant
to the test product being evaluated.
4.6. Formatted Method -' One copy of the method prepared in SW-846
format (See Attachment 1, Method 4000)
Rev 3; February, 1995
-------
Table 1 - RCRA Analytes
Table la - (TABLE 2-1 from Chapter Two of SW-846)
DETERMINATIVE ANALYTICAL METHODS FOR ORGANIC COMPOUNDS
Compound
Applicable Methcd(s)
Acenaphthene
Acenaphthylene
Acetaldehyde
Acetone
Acetomtnle
Acetophenone
2-Acetylaminof1uorene
l-Acetyl-2-thlourea
Acifluorfen
Acrolein (Propenal)
Acrylamide
Acrylonltrlle
Alachlor
Aldicarb (Temik)
Aldlcarb Sulfone
Aldnn
Allyl alcohol
Ally! chloride
2-Aminoanthraquinone
Aminoazobenzene
4-Aminobiphenyl
2-Amino-4,6-dinitrotoluene (2-Am-DNT)
4-Amino-2.6-dinitrotoluene (4-Am-DNT)
3-Amino-9-ethylcarbazole
Anilazine
Aniline
o-Anisidine
Anthracene
Aramite
Aroclor-1016 (PCB-1016)
Aroclor-1221 (PCB-1221)
Aroclor-1232 (PCB-1232)
Aroclor-1242 (PCB-1242)
Aroclor-1248 (PCB-1248)
Aroclor-1254-(PCB-1254)
Aroclor-1260 (PCB-1260)
Aspon
As ill am
Atrazine
Azinphos-ethyl
Azinphos-methyl
Barban
Bentazon
Benzal chloride
Benzaldehyde
8100. 8250/8270. 8310. 8410
8100. 8250/8270. 8310, 8410
8315
8240/8260, 8315
8240/82&0
8250/8270
8270
8270
8151
8030/8031, 8240/8260, 8315,
8316
8032, 8316
8030/8031, 8240/8260, 8316
8081
8318
8318
8080/8081, 8250/8270, 8275 .
8240/8260
8010, 8240/8260
8270
8270
8250/8270
8330
8330
8270
8270
8250/8270
8270
8100. 8250/8270, 8310, 8410
8270
8080/8081, 8250/8270
8080/8081, 8250/8270
8080/8081. 8250/8270
8080/8081, 8250/8270
8080/8081, 8250/8270
8080/8081, 8250/8270
8080/8081. 8250/8270
8141
8321
8141
8141
8140/8141, 8270
8270
8151
8121
8315
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
Benz(a)anthracene
Benzene
Benzidine
Benzo(b)fluoranthene
Benzo(j)fluoranthene
Benzo(k)fluoranthene
Benzole acid
Benzo(g,h,1)perylene
Benzo(a)pyrene
p-Benzoquinone
Benzotn chloride
Benzyl alcohol
Benzyl benzoate
Benzyl chloride
BHC (Hexachlorocyclohexane)
a-BHC (alpha-Hexachlorocyclohexane)
/3-BHC (beta-Hexachlorocyclohexane)
5-BHC (delta-Hexachlorocyclohexane)
7-BHC (Llndane, gamma-Hexachlorocyclohexane)
Bis(2-Chloroethoxy)methane
Bis(2-Chloroethyl)ether
Bis(2-Chloroethyl)sulf1de
B1s(2-Chloroisopropyl) ether
B1s(2-Ethylhexyl) phthalate
Bolstar (Sulprofos)
Bromoacetone
Bromobenzene
Bromochloromethane
Bromodi chloromethane
4-Bromof1uorobenzene
Bromoform
Bromomethane
4-Bromophenyl phenyl ether
Bromoxynil
Butanal
n-Butanol
2-Butanone (Methyl ethyl ketone, MEK)
n-Butyl benzene
sec-Butyl benzene
tert-Butylbenzene
Butyl benzyl phthalate
2-sec-Butyl-4,6-din1trophenol (DNBP, Dinoseb)
Captafol
Captan
Carbaryl (Sevin)
8100, 8250/8270, 8310, 8410
8020, 8021, 8240/8260
8250/8270
8100, 8250/8270. 8310
8100
8100, 8250/8270. 8275, 8310
8250/8270, 8410
8100, 8250/8270, 8310
8100, 8250/8270, 8275. 8310.
8410
8270
8121
8250/8270
8061
8010. 8121, 8240/8260
8120
8080/8081, 8121, 8250/8270
8080/8081. 8121. 8250/8270 -
8080/8081, 8121, 8250/8270
8080/8081. 8121. 8250/8270
8010. 8110. 8250/8270, 8410
8110. 8250/8270. 8410
8240/8260
8010. 8110, 8250/8270, 8410
8060/8061, 8250/8270, 8410
8140/8141
8010. 8240/8260
8010. 8021. 8260
8021, 8240/8260
8010, 8021, 8240/8260
8240/8260
8010, 8021, 8240/8260
8010, 8021, 8240/8260
8110, 8250/8270, 8410
8270
8315
8260
8015, 8240/8260
8021, 8260
8021, 8260
8021, 8260
8060/8061, 8250/8270, 8410
8040, 8150/8151, 8270, 8321
8081. 8270
8081, 8270
8270, 8318
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
Carbazole
Carbofuran (Furaden)
Carbon disulfide
Carbon tetrachloride
Carbophenothion (Carbofenthion)
Chloral hydrate
Chloramben
Chlordane (technical)
a-Chlordane
7-Chlordane
Chlorfenvinphos
Chloroacetonitrlle
4-Chloroanlline
Chlorobenzene
Chiorobenzllate
2-Chloro-1.3-butadiene
1-Chlorobutane
Chiorodibromomethane (Di bromochloromethane)
Chloroethane
2-Chloroethanol
2-Chloroethyl vinyl ether
Chloroform
1-Chlorohexane
Chioromethane
5-Chloro-2-methyl aniline
Chloromethyl methyl ether
4-Chloro-3-methyl phenol
Chloroneb
3-(Chloromethyl)pyrid1ne hydrochlorlde
1-Chloronaphthalene
2-Chloronaphthalene
2-Chlorophenol
4-Chlorophenol
4-Chloro-1.2-phenylenedlamine
4-Chloro-1.3-phenylenedlami ne
4-Chlorophenyl phenyl ether
Chloroprene
3-Chloropropene
3-Chloropropionitrile
Chloropropylate
Chlorothalonll-
2-Chlorotoluene
4-Chlorotoluene
Chlorpyrifos
Chlorpyrlfos methyl
Chrysene
8275
8270. 8318
8240/8260
8010, 8021. 8240/8260
8141, 8270
8240/8260
8151
8080, 8250/8270
8081
8081
8141, 8270
8260
8250/8270, 8410
8010, 8020, 8021, 8240/8260
8081, 8270
8260
8260
8010, 8021, 8240/8260
8010, 8021, 8240/8260
8010, 8240/8260
8010, 8240/8260
8010, 8021, 8240/8260
8010, 8260
8010, 8021, 8240/8260
8270
8010
8040, 8250/8270, 8275, 8410
8081
8270
8250/8270, 8275
8120/8121. 8250/8270, 8410
8040, 825(M«27Gi'62?$,'841G
8410 ' '"
8270
8270
8110. 8250/8270, 8410
8010. 8240/8260
8260
8240/8260
8081
8081
8021, 8260
8010, 8021. 8260
8140/8141
8141
8100, 8250/8270, 8310. 8410
Rev 3; February, 1995
-------
TABLE 2-1
(Continued)
Compound
Applicable Method(s)
Coumaphos
Coumarin Dyes
p-Cresidine
o-Cresol (2-Methylphenol)
m-Cresol (3-Methylphenol)
p-Cresol (4-Methylphenol)
Cresols (Methylphenols, Cresylic acids)
Crotonaldehyde
Crotoxyphos
Cyclohexanone
2-Cyclohexyl-4,6-dini trophenol
2.4-D
Dalapon
2,4-DB
DBCP
2.4-D. butoxyethanol ester
DCPA
DCPA dlacid
4,4'-DDD
4.4'-DDE
4,4'-DDT
Decanal
Demeton-0, and -S
2.4-D.ethylhexyl ester
Diallate
2.4-Dlamlnotoluene
Dlazlnon
D1benz(a,h)acr1d1ne
D1benz(a.j)acr1d1ne
D1benz(a.h)anthracene
7H-Dibenzo(c,g)carbazole
Dlbenzofuran
D1benzo(a,e)pyrene
D1benzo(a,h)pyrene
D1benzo(a,1)pyrene
Dlbenzothlophene
Dlbromochloromethane (Chlorodibromomethane)
1.2-D1bromo-3-chloropropane
1,2-Dibromoethane (Ethylene dibromide)
D1 bromof1uoromethane
Dlbromomethane
Dl-n-butyl phthalate
Dlcamba
Dichlone
1,2-Oichlorobenzene
8140/8141, 8270
8321
8270
8250/8270. 8410
8270
8250/8270, 8275. 8410
8040
8260. 8315
8141. 8270
8315 '
8040. 8270
8150/8151, 8321
8150/8151, 8321
8150/8151, 8321
8081
8321
8081
8151
8080/8081. 8250/8270
8080/8081. 8270
8080/8081, 8250/8270
8315
8140/8141, 8270
8321
8081, 8270
8270
8140/8141
8100
8100, 8250/8270
8100. 8250/8270, 8310
8100
8250/8270. 8410
8100. 8270
8100
8100
8275
8010. 8021. 8240/8260
8010. 8011. 8240/8260, 8270
8010. 8011, 8021. 8240/8260
8260
8010. 8021, 8240/8260
8060/8061. 8250/8270, 8410
8150/8151, 8321
8081. 8270
8010. 8020. 8021. 8120/8121,
8250/8270. 8260. 8410
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
1.3-Dichlorobenzene
1,4-Dichlorobenzene
3,3'-Dichlorobenzidine
3,5-Dlchlorobenzoic acid
l,4-D1chloro-2-butene
cis-l,4-D1chloro-2-butene
trans-1.4-D1chloro-2-butene
D1chlorodif1uoromethane
1,1-Dlchloroethane
1.2-Dichloroethane
1,1-Dlchloroethene (Vinylldene chloride)
c1s-l,2-D1chloroethene
trans-l,2-Dichloroethene
Dlchlorofenthlon
Dichloromethane (Methylene chloride)
2,4-Dlchlorophenol
2,6-Dlchlorophenol
Dlchlorprop
1,2-Dlchloropropane
1,3-Dlchloropropane
2,2-Dichloropropane
l,3-01chloro-2-propanol
1,1-Dlchloropropene
cis-1.3-Dichloropropene
trans-1.3-Dichloropropene
Dichlorvos (Dlchlorovos)
Dichrotophos
Dlcofol
Dleldrln
1,2,3,4-Diepoxybutane
Diethyl ether
01 ethyl phthalate
DIethylstllbestrol
DIethyl sulfate
1.4-Difluorobenzene
Dlhydrosaffrole
Dlmethoate
3,3'-DImethoxybenzi di ne
DImethyl aminoazobenzene
2,5-Dlmethylbenzaldehyde
7.12-Dimethylbenz(a)anthracene
3,3'-D1methylbenzid1ne
a,a-DImethylphenethylami ne
2,4-DImethyl phenol
8010, 8020. 8021, 8120/8121.
8250/8270, 8260. 8410
8010. 8020. 8021, 8120/8121,
8250/8270. 8260, 8410
8250/8270
8151 « •
8010, 8240
8260
8260
8010, 8021, 8240/8260
8010, 8021, 8240/8260
8010, 8021, 8240/8260
8010, 8021. 8240/8260
8021, 8260
8010, 8021. 8240/8260
8141
8010. 8021. 8240/8260
8040. 8250/8270. 8275. 841Q
8040. 8250/8270
8150/8151. 8321
8010. 8021, 8240/8260
8021. 8260
8021. 8260
8010. 8240/8260
8021. 8260
8010, 8021, 8240/8260
8010, 8021. 8240/8260
8140/8141. 827Q. 8321
8141. 8270
8081
8080/8081. 8250/8270
8240/8260
8015. 8260
8060/8061. 8250/8270, 8410
8270
8270
8240/8260
8270
8141, 8270, 8321
8270
8250/8270
8315
8250/8270
8270
8250/8270
8040, 8250/8270
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
Dimethyl phthalate
Di nitrobenzene
1.2-Di nitrobenzene
1.3-D1 nitrobenzene (1.3-DNB)
1,4-DInitrobenzene
4.6-D1nitro-2-methylphenol
2,4-Dinltrophenol
2.4-Dinitrotoluene (2,4-DNT)
2.6-D1nitrotoluene (2.6-DNT)
Dinocap
Dlnoseb (2-sec-Butyl-4,6-dlnltrophenol, DNBP)
Oi-n-octyl phthalate
Dl-n-propyl phthalate
Dioxacarb
1,4-DIoxane
Dioxathion
Diphenylamine
5,5-Diphenylhydantoi n
1,2-D1phenylhydrazi ne
Disperse Blue 3
Disperse Blue 14
Disperse Brown 1
Disperse Orange 3
Disperse Orange 30
Disperse Red 1
Disperse Red 5
Disperse Red 13
Disperse Red 60
Disperse Yellow 5
Disulfoton
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Endrin aldehyde
Endrin ketone
Epichlorohydrin
EPN
Ethanol (Ethyl alcohol)
Ethion
Ethoprop
Ethyl acetate
Ethyl benzene
Ethyl carbamate
Ethylene dibromide
Ethylene oxide
8060/8061, 8250/8270. 8410
8090
8270
8270. 8330
8270
8250/8270. 8410
8040, 8250/8270, 8410
8090, 8250/8270. 8275, 8330.
8410
8090, 8250/8270, 8330. 8410
8270
8040, 8150/8151, 8270, 8321
8060/8061, 8250/8270, 8410
8410
8318
8240/8260
8141, 8270
8250/8270, 8275
8270
8250/8270
8321
8321
8321
8321
8321
8321
8321
8321
8321
8321'
8140/8141, 8270, 8321
8080/8081, 8250/8270
8080/8081, 8250/8270
8080/8081, 8250/8270
8080/8081. 8250/8270
8080/8081, 8250/8270
8081, 8250/8270
8010. 8240/8260
8141, 8270
8015. 8240/8260
8141, 8270
8140/8141
8260
8020, 8021. 8240/8260
8270
8010, 8011, 8021, 8240/8260
8240/8260
Rev 3; February, 1995
-------
TABLE 2-1
(Continued)
Compound
Applicable Method(s)
Ethyl methacrylate
Ethyl methanesulfonate
Ethyl parathion
Etridiazole
Famphur
Fenitrothion
Fensulfothlon
Fenthion
Fluchloralin
Fluoranthene
Fluorene
Fluorescent Brightener 61
Fluorescent Brightener 236
Fluorobenzene
2-Fluoroblphenyl
2-Fluorophenol
Fonophos
Formaldehyde
Halowax-1000
Halowa^-1001
Halowax-1013
Halowax-1014
Halowax-1051
Halowax-1099
Heptachlor
Heptachlor epoxlde
Heptanal
Hexachlorobenzene
Hexachlorobutadlene (1,3-Hexachlorobutadi ene)
Hexachlorocyclohexane
o!-Hexachlorocyclohexane (a-BHC)
/3-Hexachl orocycl ohexane (/3-BHC)
5-Hexachlorocyclohexane (6-BHC)
7-Hexachlorocyclohexane (-y-BHC)
Hexachlorocyclopentadi ene
Hexachloroethane
Hexachlorophene
8240/8260
8250/8270
8270
8081
8141, 8270, 8321
8141
8140/8141. 8270. 8321
8140/8141. 8270
8270
8100, 8250/8270, 8310, 8410
8100, 8250/8270, 8275, 8310,
8410
8321
8321
8260
8250/8270
8250/8270
8141
8315
8081
8081
8081
8081
8081
8081
8080/8081, 8250/8270
8080/8081, 8250/8270
8315
8081. 8120/8121, 8250/8270.
8275. 8410
8021, 8120/8121, 8250/8270.
8260. 8410
8120
8080/8081. 8120/8121. 8250.
8270
8080/8081. 8120/8121, 8250,
8270
8080/8081, 8120/8121, 8250,
8270
8080/8081, 8120/8121, 8250.
8270
8081, 8120/8121. 8250/8270.
8410
8120/8121, 8250/8270. 8260,
8410
8270
Rev 3; February, 1995
-------
TABLE 2-1
(Continued)
Compound
Applicable Method(s)
Hexachloropropene
Hexahydro-1.3,5-tnmtro-1.3.5-tnazine (RDX)
Hexamethylphosphorarmde (HMPA)
Hexanal
2-Hexanone
HMX
1,2,3.4,6,7.8-HpCDD
1,2,3,4,6.7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
1,2,3,4.7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3.7.8,9-HxCDD
1,2,3,4,7.8-HxCDF
1,2,3,6,7.8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7.8-HxCDF
Hydroqulnone
3-Hydroxycarbofuran
5-Hydroxydicamba
2-Hydroxypropionitrile
IndenoC1,2,3-cd)pyrene
lodomethane
Isobutyl alcohol (2-Methyl-l-propanol)
Isodrln
Isophorone
Isopropylbenzene
p-Isopropyltoluene
Isosafrole
Isovaleraldehyde
Kepone
Leptophos
Malathion
Mai elc anhydride
Malononltrlle
MCPA
MCPP
Merphos
Mestranol
Methacrylonltrlle
Methanol
Methapyrllene.
Methlocarb (Mesurol)
Methomyl (Lannate)
Methoxychlor (4,4'-Methoxychlor)
Methyl acrylate
Methyl-t-butyl ether
3-Methylcholanthrene
8270
8330
8141. 8270
8315
8240/8260
8330 •
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8280/8290
8270
8318
8151
8240/8260
8100, 8250/8270, 8310
8240/8260
8240/8260
8081, 8270
8090, 8250/8270, 8410
8021. 8260
8021. 8260
8270
8315
8081, 8270
8141, 8270
8141, 8270
8270
8240/8260
8150/8151, 8321
8150/8151. 8321
8140/8141. 8321
8270
8240/8260
8260
8270
8318
8318. 8321
8080/8081, 8250/8270
8260
8260
8100, 8250/8270
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
2-Methyl-4 6-dinitrophenol
4,4' -MethyleneDis(2-chloroamline)
4,4'-Methylenebis(N,N-dimethylam line)
Methyl ethyl ketone (MEK. 2-Butanone)
Methylene chloride (Dichloromethane)
Methyl iodide
Methyl isobutyl ketone (4-Methyl-2-pentanone)
Methyl methacrylate
Methyl methanesulfonate
2-Methylnaphthalene
2-Methyl-5-nitroaniline
Methyl parathlon
4-Methyl-2-pentanone (Methyl isobutyl ketone)
2-Methylphenol (o-Cresol)
3-Methylphenol (m-Cresol)
4-Methylphenol (p-Cresol)
2-Methylpyrldine
Methyl-2.4.6-tnnitrophenylnitramine (Tetry1)
Mevinphos
Mexacarbate
Ml rex
Monochrotophos
Naled
Naphthalene
Naphthoquinone
1,4-Naphthcqmnone
1-Naphthylamine
2-Naphthylamine
Nicotine
5-N1troacenaphthene
2-N1troan111ne
3-N1troamline
4-Nitroaniline
5-N1tro-o-amsid1ne
Nitrobenzene (NB)
4-Nitrobiphenyl
Nitrofen
2-N1trophenol
4-N1trophenol -
2-Nitropropane
Nitroqulnol1ne-1-oxide
N-Nitrosodibutyl amine
N-Ni trosodi ethyl ami ne
N-Ni trosodimethyl ami ne
N-Ni trosodi phenylami ne
8040
8270
8270
8015. 8240/8260
8010, 8021. 8240/8260
8010. 8240/8260
8015. 8240/8260
8240/8260
8250/8270
8250/8270. 8410
8270
8270. 8321
.8015, 8240/8260
8250/8270, 8410
8270
8250/8270, 8275, 8410
8270
8330
8140/8141. 8270
8270
8081, 8270
8141. 8270, 8321
8140/8141. 8270. 8321
8021, 8100. 8250/8270. 8260,
8275. 8310. 8410
8090
8270
8250/8270
8250/8270
8270
8270
8250/8270. 8410
8250/8270, 8410
8250/8270, 8410
8270
8090, 8250/8270, 8260, 8330,
8410
8270
8081, 8270
8040. 8250/8270. 8410
,8040, 8151, 8250/8270, 8410
8260
8270
8250/8270
8270
8070, 8250/8270, 8410
8070, 8250/8270, 8410
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound Applicable Method(s)
N-Nitrosodi-n-propylamine 8070. 8250/8270. 8410
N-Nitrosomethylethylamine 8270
N-Nitrosomorpholine 8270
N-Nltrosopipendine 8250/8270
N-Nitrosopyrrolidine 8270
o-Nitrotoluene (2-NT) 8330
m-Nitrotoluene (3-NT) 8330
p-Nltrotoluene (4-NT) 8330
5-Nitro-o-toluidine 8270
trans-Nonachlor 8081
Nonanal 8315
OCDD 8280/8290
OCDF 8280/8290
Octahydro-1.3.5,7-tetranitro-1.3.5.7-tetrazocine
(HMX) 8330
Octamethyl pyrophosphoramlde 8270
Octanal 8315
4,4'-Oxydianil1ne 8270
Parathion 8270
Parathlon. ethyl 8141
Parathion, methyl 8140/8141
PCB-1016 (Aroclor-1016) 8080/8081. 8250/8270
PCB-1221 (Aroclor-1221) 8080/8081, 8250/8270
PCB-1232 (Aroclor-1232) 8080/8081, 8250/8270
PCB-1242 (Aroclor-1242) 8080/8081, 8250/8270
PCB-1248 (Aroclor-1248) 8080/8081, 8250/8270
PCB-1254 (Aroclor-1254) 8080/8081, 8250/8270
PCB-1260 (Aroclor-1260) - 8080/8081, 8250/8270
PCNB 8081
1,2,3,4,7-PeCDD 8280/8290
1,2,3,7,8-PeCDD 8280/8290
1.2,3,7,8-PeCDF 8280/8290
2,3,4,7,8-PeCDF 8280/8290
Pentachlorobenzene 8121, 8250/8270
Pentachloroethane 8240/8260
Pentachlorohexane 8120
Pentachloroni trobenzene 8250/8270
Pentachlorophenol 8040, 8151, 8250/8270, 8410
Pentafluorobenzene 8260, 4010
Pentanal 8315
trans-PermethFln ' 8081
Perthane 8081
Phenacetln 8250/8270
Phenanthrene 8100. 8250/8270, 8275, 8310.
8410
Phenobarbltal 8270
Phenol 8040, 8250/8270, 8410
Rev 3; February, 1995
-------
TABLE 2-1
(Continued)
Compound
Applicable Method(s)
1,4-Phenylenediamine
Phorate
Phosalone
Phosmet
Phosphamidion
Phthallc anhydride
Picloram
2-Picoline
Piperonyl sulfoxlde
Promecarb
Pronannde
Propachlor
Propanal
Propargyl alcohol
B-Proplolactone
Propionitrile
Propoxur (Baygon)
n-Propylamine
n-Propylbenzene
Propylthiouracil
Pyrene
Pyrldlne
RDX
Resorcinol
Ronnel
Safrole
Simazlne
Solvent Red 3
Solvent Red 23
Stirophos (Tetrachlorvlnphos)
Strobane
Strychnine
Styrene
Sulfall ate
Sulfotepp
2,4,5-T
2,4,5-T, butoxyethanol ester
2,4,5-T, butyl ester
1,2,3,4-TCDD
1,2,7,8-TCDD .
1,2.8,9-TCDD
1,3,6,8-TCDD
1,3,7.8-TCDD
1,3,7,9-TCDD
2,3.7,8-TCDD
1,2,7,8-TCDF
8270
8140/8141, 8270, 8321
8270
8141. 8270
8141, 8270
8270
8151
8240/8260, 8250/8270
8270
8318
8250/8270
8081
8315
8240/8260
8240/8260
8240/8260
8318
8240/8260
8021, 8260
8270
8100, 8250/8270. 8275, 8310,
8410
8240/8260, 8270
8330
8270
8140/8141
8270
8141
8321
8321
8140/8141/ 8270
8081
8270, 8321
8021, 8240/8260
8270
8141
8150/8151. 8321
8321
8321
8280
8280
8280
8280
8280
8280
8280/8290
8280
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound
Applicable Method(s)
2,3.7.8-TCDF
TEPP
Terbuphos (Terbufos)
Terphenyl
1,2.3.4-Tetrachlorobenzene
1.2.3.5-Tetrachlorobenzene
1.2.4.5-Tetrachlorobenzene
Tetrachlorobenzenes
1,1,1,2-Tetrachloroethane
1.1.2,2-Tetrachloroethane
Tetrachloroethene
2,3,4,6-Tetrachlorophenol
Tetrachl orophenols
Tetrachlorvinphos (Stirophos)
Tetraethyl dlthlopyrophosphate
Tetraethyl pyrophosphate
Tetrazene
Thiofanox
Thlonazlne
Thlophenol (Benzenethiol)
TOCP (Tri-o-cresylphosphate)
Tokuthlon (Prothiofos)
m-Tolualdehyde
o-Tolualdehyde
p-Tolualdehyde
Toluene
Toluene dllsocyanate
o-Toluidine
Toxaphene
2,4,5-TP (Silvex)
2,4.6-Tribromophenol
1,2.3-Trichlorobenzene
1,2,4-Trichlorobenzene
1,3,5-Trichlorobenzene
1.1,1-Trichloroethane
1,1.2-Trichloroethane
Tn chl oroethene
Trichlorof1uoromethane
Trichlorfon
Trichloronate.
2,4,5-Trichlorophenol
2,4.6-Trichlorophenol
Tn chl orophenols
1,2,3-Trichloropropane
0.0,0-Triethyl phosphorothioate
Tnfluralin
8280/8290
8141
8141. 8270
8250/8270
8121
8121 . .
8121, 8250/8270
8120
8010, 8021, 8240/8260
8010. 8021, 8240/8260
8010. 8021. 8240/8260
8250/8270
8040
8140/8141. 8270
8270
8270
8331
8321
8141. 8270
8270
8141
8140/8141
8315
8315
8315
8020, 8021. 8240/8260
8270
8270
8080/8081, 8250/8270
8150/8151. 8321
8250/8270
8021. 8121. 8260
8021, 8120/8121. 8250/8270,
8260. 8410
8121
8010. 8021, 8240/8260
8010. 8021. 8240/8260
8010. 8021. 8240/8260
8010. 8021, 8240/8260
8141, 8321
8140/8141
8250/8270, 8410
8040, 8250/8270, 8410
8040
8010, 8021. 8240/8260
8270
8081, 8270
Rev 3; February, 1995
-------
TABLE 2-1.
(Continued)
Compound Applicable Method(s)
2.4.5-Tnmethylamline 8270
1.2,4-Tnmethyl benzene 8021. 8260
1.3.5-Trimethyl benzene 8021. 8260
Tn methyl phosphate 8270
1.3,5-Trinitrobenzene (1.3.5-TNB) 8270, 8330
2.4.6-Trinitrotoluene (2.4,6-TNT) 8330
Tri-o-cresyl phosphate (TOCP) 8141
Tri-p-tolyl phosphate 8270
Tris(2.3-Dibromopropyl) phosphate (Tns-BP) 8270. 8321
Vinyl acetate 8240/8260
Vinyl chloride 8010, 8021. 8240/8260
o-Xylene 8021, 8260
m-Xylene 8021, 8260
p-Xylene 8021, 8260
Xylene (Total) 8020, 8240
Rev 3; February, 1995
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Table Ib - (TABLE 2-32 from Chapter Two of SW-846:
ANALYSIS METHODS FOR INORGANIC COMPOUNDS
Compound
Aluminum
Antimony
Arsenic
Barium
Beryllium
Bromide
Cadmium
Calcium
Chloride
Chromium
Chromium, hexavalent
Cobalt
Copper
Cyanide
Fluoride
Iron
Lead
Lithium
Magnesium
Manganese
Mercury
Molybdenum
Nickel
Nitrate
Nitrite
Osmium
Phosphate
Phosphorus
Potassium
Selenium
Silver
Sodi urn
Strontium
Sulfate
Sulfide
Thallium
Tin
Vanadium
Zinc
Applicable Method(s)
6010.
6010,
6010,
6010.
6010,
9056
6010,
6010,
9056.
6010,
7195,
6010,
6010,
9010.
9056
6010.
6010.
6010,
6010,
6010,
7470!
6010.
6010.
9056.
9056
7550
9056
6010
6010.
6Q10,
6010.
6010,
6010.
9035.
9030.
6010.
7870
6010.
6010.
6020.
6020.
6020.
6020.
6020.
6020,
7140
9250,
6020.
7196.
6020,
6020,
9012,
7380.
6020,
7430
7450
6020.
7471
7480,
6020.
9200
7610
7740.
6020,
7770
7780
9036,
9031
6020.
7910,
6020,
7020
7040
7060
7080
7090
7130
9251
7190
7197
7200
7210
9013
7381
7420
7460
7481
7520
7741
7760
9038
7840
7911
7950
, 7041. 7062
, 7061, 7062
: 7081
. 7091
,7131
, 9252, 9253
. 7191
. 7198
. 7201
, 7211
. 7421
, 7461
, 7742
, 7761
. 9056
. 7841
, 795-1
Rev 3; February, 1995
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Table 2
REPRESENTATIVE ORGANOCHLORINE PESTICIDES
The following chlorinated pesticides from the Best
Demonstrated Available Technology (BOAT) list are representative
of analytes for Method 8081. All should be sufficiently resolved
to ensure good quantitation using two columns such as the DB-608
or DB-1701 (or equivalents). Suggested low and high
concentrations should be appropriate for relatively
uncontaminated solid matrices such as soil. Higher
concentrations may be required for highly contaminated samples.
COMPOUND
Aldrin
/3-BHC
5-BHC
•y-BHC (Lindane)
a-Chlordane
y-Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endrin
Endrin aldehyde
Heptachlor
Heptachlor epoxide
LOW (ua/ka)
5
5
5
5
5
5
10
5
5
5
5
5
10
5
5
5
HIGH (ua/ka)
250
250
250
250
250
250
500
250
250
250
250
250
500
250
250
250
Rev 3; February, 1995
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Table 3 - REPRESENTATIVE SEMIVOLATILE COMPOUNDS
The following compounds are representative of analytes for
Method 8270. Although many of these compounds are considered
difficult, all can be extracted from waste matrices using
conventional techniques. Suggested low and high concentrations
should be appropriate for relatively uncontaminated solid
matrices such as soil. Higher concentrations may be required for
highly contaminated samples. Phthalate esters are not spiked but
should be reported as a measure of contamination.
COMPOUND LOW (ua/kcr) HIGH (uq/kcr)
Phenol 250 12,500
o-Cresol 250 12,500
2-Methyl phenol 250 12,500
2,4,6-Trichlorophenol 250 12,500
Pentachlorophenol 250 12,500
1,2-Dichlorobenzene 250 12,500
Naphthalene 250 12,500
2-Chloronaphthalene 250 12,500
Anthracene 250 12,500
Chrysene 250 12,500
Benzo(a)anthracene 250 12,500
Benzo(a)pyrene 250 12,500
Fluoranthene 250 12,500
Indenod, 2,3-cd)pyrene 250 12,500
Benzo(g,h,i)perylene 250 12,500
o-Toluidine 250 12,500
p-Nitrotoluene 250 12,500
2,6-Dinitrotoluene 250 12,500
2-Nitroaniline 250 12,500
Di-n-propylnitrosamine 250 12,500
4-Bromophenyl phenyl ether 250 12,500
3,3'-Dichlorobenzidine 250 12,500
In addition, surrogates should be added such that 50 /xg or 100
would be present in final extracts, assuming 100% recovery.
Nitrobenzene-ds 50
2-Fluorobiphenyl 50
p-Terphenyl-d14 50
Phenol-ds 100
2-Fluorophenol 100
2,4,6-Tribromophenol 100
Rev 3; February, 1995
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Table 4
REPRESENTATIVE VOLATILE COMPOUNDS
The following compounds are representative of analytes for
Method 8260; many are TCLP analytes. All can be purged from
waste matrices using Method 5030. Suggested low and high
concentrations should be appropriate for relatively
uncontaminated solid matrices such as soil. Higher
concentrations may be required for highly contaminated samples.
COMPOUND LOW (gg/kg) HIGH (ug/kg)
Vinyl chloride 5 • 250
Dibromochloromethane 5 250
1,1-dichloroethane 5 250
Chloroform 5 250
Carbon tetrachloride 5 250
Trichloroethylene 5 250
1,1,1-Trichloroethane 5 250
Benzene 5 250
Toluene 5 250
Ethylbenzene 5 250
Chlorobenzene 5 250
Nitrobenzene 5 250
Methyl ethyl ketone 5 250
Carbon disulfide 5 250
The following compounds are representative of non-purgeable
volatiles which currently require azeotropic distillation.
Suggested low and high concentrations should be appropriate for
relatively uncontaminated solid matrices such as soil. Higher
concentrations may be required for highly contaminated samples.
COMPOUND LOW (ua/ka) HIGH (ug/kq)
1,4-Dioxane 10 500
n-Butanol 10 500
iso-butanol 10 500
Ethyl acetate 10 500
Pyridine 10 500
Rev 3; February, 1995
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Table 5
REPRESENTATIVE METALS
The following metals include the TCLP analytes. Suggested
low and high concentrations are based on analysis by ICP, except
for mercury, which is generally analyzed by cold vapor AA. The
suggested concentrations should be appropriate for relatively
uncontaminated solid matrices such as soil. Higher
concentrations may be required for highly contaminated samples.
ELEMENT LOW (ucr/kcr) HIGH (ua/ka)
Arsenic 2500 • 75,000
Barium 100 5,000
Cadmium 200 10,000
Chromium 350 17,500
Copper 300 15,000
Lead 2000 100,000
Silver 350 17,500
Zinc 100 5,000
Mercury 100 5,000
Rev 3; February, 1995
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Table 6
Example Format for Data from the Recovery Study
Compound
Blank
Blank
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Benzo (a) anthracene
Benzo (a) anthracene
Benzo (a) anthracene
Benzo (a) anthracene
Benzo (a) pyrene
Benzo (a) pyrene
Benzo (a) pyrene
Spike
(ppm)
0
0
1
1
1
10
10
10
1.6
1.6
16
16
8.3
8.3
83
Soil
Wake
PAH-116
Wake
PAH-116
PAH-141
Wake
PAH-116
PAH-141
Wake
PAH-116
Wake
PAH-116
Wake
PAH-116
PAH-116
PAH RISC™
Results
<1
<1
1-10
1-10
1-10
>10
>10
>10
1-10
1-10
>10
>10
1-10
1-10
>10
Rev 3; February, 1995
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Table 7
Example Data Format for False Negative/False Positive Data
Probability of False Negative and False Positive Results
for PAHs at a 1 ppm Action Level
Spike Concentration
Phenanthrene (ppm)
0
0.4
0.8
1.0
Probability of
False Positive
(Mean ± SD)
0% ± 0%
23% ± 17%
94% ± 13%
N/A
Probability of
False Negative
(Mean + SD)
N/A
N/A
N/A
0% ± 0%
Results were obtained from spiking four different validation
lots, using 3 operators, 12 matrices for a total of 201
determinations at each concentration of phenanthrene.
N/A = No false positive possible above action limit.
No false negative possible below action limit.
Rev 3; February, 1995
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Table 8a
Example Data Format for Results of Correlation Study
SAMPLE SCREENING
NUMBER RESULT (units)
001 >10
002 >10
003 <10
004 >10
005 >10
006 >10
007 >10
008 >10
009 >10
010 >10
Oil >10
012 <10
013 <1Q
014 <10
015 >10
015D >10
016 >10
017 >10
018 >10
019 >10
020 <10
021 <10
022 <10
022D <10
023 >10
024 <10
a - Y Acceptable agreement
FP = False Positive
FN = False Negative
REFERENCE '
METHOD RESULT (units)
5.98
1.27
0.11
6 .71
1.37
0 .68
0.55
2 .00
1.30
0.17
1.15
ND (>0.05)
1.13
0.18
9.13
9.84
2110
2.55
45.4
6.70
0.07
0.06
0.54
0.72
20.8
0.06
AGREEMENT3
Y, FN, FP
FP
FP
Y
FP
FP
FP
FP
FP
FP
FP
FP
Y
Y
Y
FP
FP
Y
FP .
Y
FP
Y
Y
Y
Y
Y
Y
Rev 3; February, 1995
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Table 8b
Total PAH Content of Field Samples Using PAH RISc™ test product
Sample
ID
PAH-1
PAH- 2
PAH- 3
PAH-4
PAH- 5
PAH- 6
PAH- 7
PAH- 8
PAH- 9
PAH-10
PAH- 11
PAH- 12
PAH-
12Dup
PAH-13
PAH-14
PAH- 15
PAH-16
PAH-17
PAH-18
PAH- 19
PAH- 20
PAH- 21
1 ppm
Test
<1
*
*
*
*
*
*
>1
*
*
*
*
*
*
*
*
10 ppm
Test
<10
*
*
*
*
*
>10
*
*
*
*
*
*
*
*
*
*
*
*
*
GC/MS Lab
Result (ppm)1
0 .2
12.2
16.0
0.0
0.5
8.7
148
182
4.4
0.2
0.0
85.4
85.4
28.5
0.3
0.6
0.0
1.8
3.4
6. 7
0. 9
43 .2
False +/-
Eva!
@
1 ppm
+
+
+
Eval
@
10
ppm
+
+
+
+
+
"Sum of all" PAHs detected.
Rev 3; February, 1995
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Table 9
Example Format, Cross-Reactivity Data
Compound
Aroclor 1248
Bif enox
1-Chloronaphthalene
2 , 5 -Dichloroaniline
2,4-Dichlorophenyl-benzenesulfonate
Dichlorof enthion
2 , 4-Dichloro-l-naphthol
Diesel fuel
Gasoline
Hexachlorobenzene
Pentachlorobenzene
Tetradifon
1,2, 4 -Trichlorobenzene
-Soil Equivalent
Concentration (ppm)
Required to Yield a
Positive Result
1
500
•• 10,000
>10, 000
irooo
10, 000
>10, 000
>10,000
>10,000
>10,000
>10,000
125
10,000
Rev 3; February, 1995
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METHOD 4000
IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Invnunoassay is an analytical technique useful for the separation,
detection and quantitation of both organic and inorganic analytes in diverse
environmental and waste matrices. Immunoassay methods are used to produce two
types of quantitative results: 1) range-finding or screening results indicative
of compliance with an action level, and 2) assay values.
1.2 Commercially-available testing products present immunoassay protocols
that are rapid, simple and portable. These products can be used effectively in
both laboratory and field settings, and require limited training. These test
products substantially increase the number of data points that can be generated
within a given time period, and permit an operator to analyze a number of
samples simultaneously, within a relatively short period of time. Results are
available immediately upon completion of the test, and can assist in the on-site
management of personnel and equipment, as well as the data management activities
of the laboratory.
1.3 Section 11.0 provides a glossary of basic immunoassay terms.
1.3.1 The glossary is not intended to be comprehensive, but to
provide basic definitions that will assist in understanding product
inserts and publications relating to immunoassay technology.
1.3.2 The performance of test products will vary from manufacturer
to manufacturer. The performance claims and limitations of each test
product will be provided in the package insert. The package insert of
each test product purchased should be read to determine if the performance
is acceptable for a given application.
2.0 SUMMARY OF METHOD
2.1 The immunoassay test products available will often vary in both format
and chemistry. The characteristics of a. specific product are described in the
package insert provided by the manufacturer. This summary is, therefore, general
in scope, and is intended to provide a general description of the more common
elements of these methods.
Immunoassay test products use an antibody molecule to detect and quantitate
a substance in a test sample. These testing products combine the specific
binding characteristics of an antibody molecule with a detection chemistry that
produces a detectable response used for interpretation. In general, antibody
molecules specific for the method's intended target are provided at a predefined
concentration. A reporter (i.e., signal generating) reagent, composed of the
target compound conjugated to a signal producing compound or molecule (e.g.,
enzymes, chromophores, fluorophores, luminescent compounds, etc.), is also
4000 - 1 Revision 0
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provided. The concentration, affinity, and specificity of the products's
antibody influences performance, as does the chemistry of the reporter reagent.
The reporter reagent and antibody molecules of a given product are binding
partners, and complex in solution. The addition of a positive sample containing
the target substance to this solution results in a competitive binding reaction
for the antibody sites. The antibody concentration, and therefore binding
capacity, is limited to prevent the simultaneous binding of both the reporter and
sample molecules. The concentration of reporter reagent that can bind to the
antibody is inversely proportional to the concentration of substance in the test
sample. Immunoassay methods may be heterogeneous (i.e., requiring a wash or
separation step), or homogeneous (i.e., not requiring a separation step). In
commonly available heterogeneous testing products, the antibody is immobilized
to a solid support such as a disposable test tube, and the bound reporter reagent
will be retained after removing the unbound contents of the tube by washing.
Therefore, a negative sample results in the retention of more reporter molecules
than a positive sample. The analysis of a standard containing a known
concentration results in the immobilization of a proportional concentration of
reporter reagent. A positive sample (i.e., containing a higher concentration
than the standard) results in the immobilization of fewer reporter molecules than
the standard, and a negative sample (i.e., containing less that the standard)
will immobilize more.
2.2 A chemistry of the detection of the immobilized reporter is used for
interpretation of results. The reporter molecule may be a conjugate of the
target molecule and a directly detectable chromophore, fluorophore, or other
specie, or conjugated to an enzyme that will act upon a substrate to produce the
detectable response. Immunoassay testing products have a quantitative basis, and
will produce a signal that is dependant on the concentration of analyte present
in the sample. For environmental immunoassay methods, the signal produced is
exponentially related to the concentration of the compounds present. Many
immunoassay methods use enzymes to develop chromogenic response, and are termed
enzyme immunoassays. Assays that generate a chromogenic response are analyzed
photometrically, and use the principles of Beer's Law (Absorbance = Extinction
Coefficient x Concentration x Path Length) to determine the concentration of
analyte in a sample.
Immunoassay methods can provide quantitative data when configured with a series
of reference standards that are analyzed and used to construct a standard curve.
The signal generated from the analysis of a test sample is used to determine
concentration by interpolation from the standard curve. Alternatively, these
testing products can be configured to determine if a sample is positive or
negative relative to a single standard.
Individual immunoassay testing products are reviewed and accepted by the EPA-OSW
for the detection of sample analytes in specified matrices. A variety of testing
products, produced by several different developers, may be available for the same
compound(s) and matrices. Each of theses methods have been formulated using
independently developed reagents that may result in significantly different
performance characteristics and limitations.
The performance c+" the immunoassay testing products ultimately relates to the
characteristics of the antibody, reporter molecule, and sample processing
4000 - 2 Revision 0
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chemistry. The dose-response characteristics of a method, the position of the
standard relative to the claimed action level, and the stated cross-reactivity
characteristics of the selected test product, provide relevant information
regarding the performance and recognition profile of the selected test product.
The precision, and ultimately the sensitivity of an immunoassay method, is a
function of the signal-to-noise characteristics of its dose-response curve, and
its operational consistency. Methods having a high slope and low non-specific
signal generation produce the most sensitive and precise methods. Signal
imprecision applied to a dose-response curve having a shallow slope exhibits
proportionally greater imprecision in the calculated concentration than would a
method having a steeper slope. In an action level testing product, this would
cause the reference standard to be positioned further from the action level,
increasing the incidence of false positive results. Similarly, a method having
less non-specific signal generation (higher signal-to-noise ratio) will be more
sensitive and precise when other characteristics (i.e., dose-response slope) are
held constant.
Immunoassay methods are used to detect contamination at a specific concentration
below the claimed detection level for the test product. For example, an
immunoassay used to detect PCB contamination in soil at 1 ppm will include a
reference preparation containing less than 1 ppm. The reference preparation
concentration is positioned to minimize the incidence of false negative results
at the claimed detection level. For remediation and monitoring applications,
where action levels of interest are defined, immunoassay methods should exhibit
a negligible incidence of false negative results, and minimal false positives.
For a single point action level test, the concentration of analyte relative to
the action level is selected by the developer, and is influenced by the precision
(i.e., intra-assay, inter-person, inter-lot, inter-day, etc.), sample matrix
interferences and other performance characteristics and limitations of the basic
method. The concentration of analyte in the reference materials should be less
than, but close to, the claimed action level. The concentration selected for the
standard defines the concentration that will produce a 50% incidence of false
positive results by the test product. While this issue is one representing
limited liability to the operator, it is a practical issue that often requires
attention. An immunoassay method for the detection of 1 ppm of PCB using a
standard containing 0.8 ppm of PCB will experience a 50% false positive incidence
in samples containing 0.8 ppm of PCB, and some incidence of false positive
results in a sample containing between 0.8 and 1 ppm. A similar immunoassay that
uses a standard containing 0.4 ppm will experience a 50% false positive incidence
in samples containing 0.4 ppm of PCB, and some incidence of false positive
results in a sample containing between 0.4 and 1 ppm. The closer the standard
concentration is to the action level, the better the overall performance.
2.3 Cross reactivity characteristics illustrate the specificity of the
underlying immunochemistry. The antibody molecules used by a test product bind
to a target compound and then participate in the process of generating the signal
used for interpretation. Antibody molecules bind by conformational
complimentarity. These molecules can be exquisitely specific, and can
differentiate subtle differences in the structure of a compound. The binding
characteristics of reagents in different test products can vary, and influence
the recognition profile and incidence of false results obtained by the method.
4000 - 3 Revision 0
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Immunoassay methods should detect the target analytes claimed by the test product
and exhibit limited recognition for compounds and substances not specified.
3.0 INTERFERENCES
3.1 Non-target analytes may bind with the antibody present, producing a
false-positive result. These non-target analytes may be similar to the target
analytes, or they may be chemically dissimilar co-contaminants. During
evaluation of each test product for RCRA testing applications, studies were
conducted to determine these "cross-reactive" constituents. At a minimum, these
studies evaluated the response of the test product to all other similar RCRA
analytes in that analyte class, as well as for selected lists of non-RCRA
analytes. This testing scheme is designed to ensure that all other similar RCRA
analytes and likely co-contaminants are evaluated during cross-reactivity
testing. The results of these studies are presented in each method in tabular
form, providing separate data sets for each test product evaluated.
3.2 Interference in the binding of an antibody to its target compound,
or reporter molecule reagent, may occur when testing sample matrices with
confounding contaminants or circumstances (e.g., oil, pH, temperature, some
solvents). Immunoassay products contain sample processing technology that has
been developed and validated for use with specified matrices. Interferences
incurred from the testing of incompatible matrices may prevent the testing
product from meeting its performance claims, and increase the number of false
positive or false negative results. Individual immunoassay products designate
the intended sample matrices.
3.3 Immunoassay products differ in shelf-life and storage requirements.
Test products that are operated outside of the shelf-life and storage temperature
recommendations may not provide the claimed performance.
3.4 Some test products have designated temperature ranges for operation.
When these products are used, all tests must be performed within the specified
operating temperature limits, or else false negative/positive results may exceed
performance claims.
4.0 APPARATUS AND MATERIALS
4.1 Each test product will specify the apparatus and materials provided,
as well as any additional apparatus and materials necessary for performance of
the test.
5.0 REAGENTS
5.1 The two basic reagents used in immunoassay analysis are the antibody
(e.g., anti-PCP) and reporter conjugate reagent (e.g., PCP molecules bound to an
enzyme).
5.1.1 The formation of antibodies to haptenic molecules (i.e., most
environmental contaminants) is induced by the derivatization and coupling
4000 - 4 Revision 0
January 1995
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of molecules of the target analytes to large carrier molecules such as
albumin, hemocyanin or thyroglobulin. The increased size and complexity
of the immunogen (antigen) conjugate, once injected, is sufficient to
stimulate the immune system to produce an antibody response. The
effectiveness of the immunogen in producing antibodies having the
prerequisite binding characteristics and recognition profile is influenced
by the surface density of the chemical groups on the carrier molecule, the
nature of the bridge chemistry used, the point of attachment, the
immunization protocol, immunogen concentration, adjuvants (i.e., immune
response stimulants), and the species of the host animal.
5.1.2 An enzyme-reporter conjugate reagent is synthesized by
coupling a target analyte or derivative of a target analyte to an enzyme,
such as horseradish peroxidase. Enzymes enhance the sensitivity of the
method by action on a substrate and the production and catalytic
amplification of the detection signal. A single enzyme molecule used in
immunoassay methods will convert approximately 106 molecules of a target
analyte into a detectable product within one minute at ambient
temperature.
5.2 Each test product will specify the reagents provided, as well as any
additional reagents necessary for performance of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 Testing of solid waste by immunoassay requires production of a
reproducible, particulate free leachate. It is critical that this leachate be
produced using a solvent that allows the reproducible extraction and recovery of
the target analytes, and is compatible with the antibody/enzyme conjugate of the
immunoassay system used. Buffers, detergents, and solvents, used together or in
combination, have been used effectively for extraction. Filtration of
particulate matter may be integrated into the immunoassay test, or accomplished
as a separate step within the protocol.
6.2 The immunoassay test products included in SW-846 methods will provide
explicit waste- or medium-specific directions for handling samples and extraction
of target analytes.
6.3 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
7.0 PROCEDURE
7.1 The specific procedure for each immunoassay test product is supplied
by the manufacturer in the package insert.
7.2 The recognition characteristics, sensitivity, detection ranges(s),
effective operating temperature, interferences and cross-reactivity of the test
will depend on the product being used.
4000 - 5 Revision 0.
January 1995
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7.3 Immunoassay methods include both a sample processing and immunoassay
component. It should be noted that the immunochemical reagents and sample
processing components supplied with each product is specific to each
manufacturer. Methods available from different manufacturers for the same
compound and application may have significantly different performance
characteristics.
8.0 QUALITY CONTROL
8.1 The performance of the tests cited in the immunoassay methods in this
manual has been reviewed, and found to be consistent with the claims that are
made in the manufacturer's literature. In order to meet this performance
expectation, the analyst must:
o Follow the manufacturer's instructions for the test product being
used,
o Use test products before the specified expiration date,
o Use reagents only with the test products for which they are
designated,
o Use the test products within their specified storage temperature and
operating temperature limits.
8.2 It is important to evaluate the performance claims and limitation
provided with each testing product to determine its application to a specific
matrix and testing program.
8.3 Refer to Chapter One for standard quality control procedures.
9.0 METHOD PERFORMANCE
9.1 A false negative is defined as a negative response for a sample
containing the target analytes at or above the stated action level. False
negative rate is measured by analyzing split samples using both the test product
and a separate reference method. False negative data are provided in each method
for each test product evaluated.
.9.2 A false positive is defined as a positive response for a sample that
contains analytes below the specified action level. Like false negatives, false
positive rates are measured by analyzing split samples with both the test product
and a separate reference method. False positive data are provided in each method
for each test product evaluated.
9.3 Cross-reactivity and recognition profile data are provided at the end
of each method in tabular form, providing separate data sets for each test
product evaluated. Using these data, the analyst can evaluate if contaminants
are present which are likely to produce a false negative response, and the
magnitude of that response.
9.4 For single-point tests, sensitivity data are provided demonstrating
the concentration of target analyte(s) that can be detected with greater than 95%
confidence.
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9.5 Data are provided demonstrating the bias of the testing products
accepted. These data may be from:
o serial dilution of samples (i.e., is the recovery of target analyte
a function of concentration?),
o sample recovery studies, and
o studies correlating the results of the testing product with a
reference method.
9.6 Data are provided demonstrating that the extraction efficiency of the
test being evaluated correlates with that of the referenced method.
10.0 REFERENCES
1. S.B. Friedman; "Doing Immunoassays in the Field", Chemtech, December 1992,
pp 732-737.
2. Roitt, L., Brosstoff, J., Male, M., (eds.), Immunology, J.B. Lippincott
Co., Philadelphia, Pennsylvania, 1989
3. Stites, Daniel P., Terr, Abba I., (eds.), Basic and Clinical Immunology,
Appleton and Lange, Norwalk, Conneticut, 1991
4. Odell, W.D. and Daughaday, W.H., Principles of Competitive Protein-Binding
Assays, J.B. Lippincott Co., Philadelphia, Pennsylvania, 1971
5. Ishikawa, E., Kawai, T., Miyai, K. (eds.), Enzyme Immunoassay, Igaku-Shoin,
Tokyo, Japan, 1981
6. Tijssen, P. (ed.), Practice and Theory of Enzyme Immunoassays, Volume 15,
Elsevier, NY, NY, 1985
7. Butler, John E. (ed.)> Immunochemistry of Solid-Phase Immunoassay, CRC
Press, Boca Raton, Florida, 1991
8. Ngo, T.T., Lenhoff, H.M., Enzyme-Mediated Immunoassay, Plenum Press, New
York, 1985
9. 510K of the Federal Food, Drug and Cosmetics Act, Section 21, CFR 807.87
11.0 GLOSSARY OF TERMS
Antigen A molecule that induces the formation of an
antibody.
Antibody A binding protein which is produced in response
to an antigen, and which has the ability to bond
with the antigen that stimulated its production.
B Lymphocyte A type of lymphocyte that, upon stimulation,
(B Cell)
4000 - 7 Revision 0
January 1995
-------
differentiates
cell.
into an antibody-secreting plasma
Carrier
C o m p e t i t i
Immunoassay
v e
Cross-Reactivity
Dose-Response
Curve
ELISA
Enzyme Conjugate
Enzyme Immunoassay
An immunogenic substance that, when coupled to a
hapten, renders the hapten Immunogenic.
An immunoassay method involving an in-vitro
competitive binding reaction.
The relative concentration of an untargeted
substance that would produce a response
equivalent to a specified concentration of the
targeted compound. In a semi-quantitative
immunoassay, it provides an indication of the
concentration of cross-reactant that would
produce a positive response. 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%.
Representation of the signal generated by an
immunoassay (y axis) plotted against the
concentration of the target compound (x axis) in
a series of standards of known concentration.
When plotting a competitive immunoassay in a
rectilinear format, the dose-response will have a
hyperbolic character. When the Iog10 of
concentration is used, the plot assumes a
sigmoidal shape, and when the log of signal is
plotted against the logit transformation of
concentration, a straight line plot is produced.
Enzyme Linked Immunosorbent Assay is an enzyme
immunoassay method that uses an immobilized
reagent (e.g.,antibody adsorbed to a plastic
tube), to facilitate the separation of targeted
analytes (antibody-bound
target substances (free
using a washing step, and
generate the signal used
of results.
components) from non-
reaction components)
an enzyme conjugate to
for the interpretation
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.
An immunoassay method that uses an enzyme
conjugate reagent to generate the signal used for
interpretation of results. The enzyme mediated
response may take the form of a chromogenic,
4000 - 8
Revision 0
January 1995
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False Negatives
False Positives
Hapten
Hapten-Carrier
Conjugate
Heterogeneous
Immunoassay
Methods
Homogeneous
Immunoassay
Methods
Immunoassay
Immuhogen
Ligand
Lymphocytes
fluorogenic, chemiluminescent or potentiometric
reaction, (see Immunoassay and EL1SA)
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.
A positive interpretation for a sample is defined
as a positive response for a sample that contains
analytes below the action level.
A substance that cannot directly induce an immune
response (e.g., antibody production), but can
bind to the products of an immune response (e.g.,
antibody) when that response is induced by an
alternate mechanism. Chemical contaminants of
the environment are haptens.
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.
Immunoassay methods that include steps for the
separation of substances that become bound to the
antibody from those that remain free in solution.
Immunoassay methods that do not require the
separation of bound and free substances, but that
utilize antibody molecules that can bind and
directly modulate the signal produced by the
reporter molecule (e.g., enzyme conjugate).
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)
A substance having a minimum size and complexity,
and that is sufficiently foreign to a genetically
competent host to stimulate an immune response.
The molecule, ion or group that forms a complex
with another molecule.
One of the five classes of white blood cells
found in the^circulatory system of vertebrates.
4000 - 9
Revision 0
January 1995
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A mononuclear cell 7-12 /im in diameter containing
a nucleus with densely packed chromatin and a
small rim of cytoplasm.
Monoclonal Identical copies of antibody molecules that have
Antibodies a common set of binding characteristics.
Polyclonal A group of antibody molecules that differ in
Antibodies amino acid composition and sequence, and that
exhibit binding characteristics. Polyclonal
antibodies are produced from a simulation of
multiple clones of lymphocytes.
4000 - 10 Revision 0
January 1995
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METHOD 4010A
SCREENING FOR PENTACHLOROPHENOL BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4010 is a procedure for screening solids such as soils,
sludges, and aqueous media such as waste water and leachates for
pentachlorophenol (PCP) (CAS Registry 87-86-5).
1.2 Method 4010 is recommended for screening samples to determine whether
PCP is likely to be present at defined concentrations (i.e., kits are available
which give positive results at 0.005 mg/L for aqueous samples, and at 0.5, 10 or
100 mg/kg in solid samples). Method 4010 provides an estimate for the
concentration of PCP by comparison with a standard.
1.3 Using the test kits from which this method was developed, 95% of
aqueous samples containing 2 ppb or less of PCP will produce a negative result
in the 5 ppb configuration. Also, 95% of soil samples containing 125 ppb or less
of PCP will produce a negative result in the 5000 ppb test configuration.
1.4 In cases where the exact concentration of PCP is required, additional
techniques (i.e., gas chromatography (Method 8040) or gas chromatography/mass
spectrometry (Method 8270)) should be used.
1.5 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using a water sample or an extract
of a water sample. The sample/extract and an enzyme conjugate reagent are added
to immobilized antibody. The enzyme conjugate "competes" with PCP present in the
sample for binding to immobilized .anti-PCP antibody. The test is interpreted by
comparing the response produced by testing a sample to the response produced by
testing standard(s) simultaneously.
3.0 INTERFERENCES
3.1 Compounds that are chemically similar may cause a positive test (false
positive) for PCP. The test kits used in preparation of this method were
evaluated for interferences. Tables 1A and IB provide the concentration of
compounds which will give a false positive test at the indicated concentration.
3.2 Other compounds have been tested for cross reactivity for PCP and have
been demonstrated not to interfere with the specific kits tested. Consult the
information provided by the manufacturer of the kit used for additional
4010A-1 Revision 1
January 1995
-------
information regarding cross reactivity with other compounds.
3.3 Storage and use temperatures may modify the method performance. Follow
the manufacturer's directions for storage and use.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: PENTA RISc™ (EnSys, Inc.), EnviroGard™ PCP in
Soil (Millipore, Inc.), or equivalent. Each commercially available test kit will
supply or specify the apparatus and materials necessary for successful completion
of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-10.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used for
quality control procedures specific to the test kit used. Additionally, guidance
provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
4010A-2 Revision 1
January 1995
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8.6 Method 4010 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 This method has been applied to a series of groundwater, process
water, and wastewater samples from industries which use PCP, and the results
compared with GC/MS determination of PCP (Method 8270). These results are
provided in Table 2. These results represent determinations by two. laboratories
using the PENTA RISc™ test kit.
9.2 This method has been applied to a series of soils from industries
which use PCP and the results compared with GC/MS determination of PCP via method
8270. These results are provided in Table 3. These results represent
determinations by two laboratories using the PENTA RISc™ test kit.
9.3 Sensitivity of the EnviroGard PCP in Soil Test Kit was determined by
establishing the "noise" level expected from matrix effects encountered in
negative soil samples and determining the corresponding TPH concentration by
comparison to the analyte-specific response curve. Eight different soils which
did not contain PCP were assayed. Each of these soils was extracted in
triplicate and each extract was analyzed in three different assays. The mean and
the standard deviation of the resulting %Bo's (%Bo = [(OD8limp|e/ODnegiitlvecontro,)xlOO])
were calculated and the sensitivity was estimated at two standard deviations
below the mean. The sensitivity for Method 4010 was determined to be 80% Bo at
a 95% confidence interval. Based on the average assay response to PCP, this
corresponds to 2 ppm PCP. These data are shown in Table 4.
9.4 The effect of water content of the soil samples on the EnviroGard™ PCP
in Soil test kit was determined by assaying three different soil samples which
had been dried and subsequently had water added to 30% (w/w). Aliquots of these
samples were then fortified with PCP. Each soil sample was assayed three times,
with and without added water, and with and without home heating oil (HHO)
fortification. It was determined that water in soil up to 30% had no detectable
effect on the method. These data are shown in Table 5.
9,5 The effect of the pH of the soil extract on the EnviroGard™ PCP in
Soil test kit was determined by adjusting the soil pH of three soil samples.
Soil samples were adjusted to pH 2 - 4 using 6N HC1 and pH 10 - 12 using 6N NaOH.
Aliquots of the pH adjusted soil samples were fortified with PCP and the
unfortified and fortified samples were extracted. These extracts were assayed
three times. It was determined that soil samples with pH ranging from 3 to 11
had no detectable effect on the performance of the method. These data are shown
in Table 6.
9.6 The bias of the EnviroGard™ PCP in Soil test kit was estimated by
fortifying three different soil samples at two different concentrations (10 and
100 ppm PCP). Each fortified sample was extracted three times and each extract
was assayed three times. Recovery for individual determinations ranged from 60%
to 125%. Average recovery for each individual extract ranged from 72% to 101%.
Overall average recovery for all samples was 86%. These data are summarized in
4010A-3 Revision 1
January 1995
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Table 7.
9.7 The effect of co-contamination of soil samples with oil on the
EnviroGard™ PCP in Soil test kit was investigated. Three soil samples were
adulterated with diesel oil and aliquots were fortified with PCP. The samples
were extracted and the extracts each assayed three times. It was determined that
no interference was detected in samples with up to 10% oil contamination. The
data from samples adulterated at 10% are shown in Table 8.
9.8 A field trial was conducted at a contaminated site using the
EnviroGard™ PCP in Soil test kit. Method 4010 was used to identify soil which
had been contaminated with PCP from wood treatment operations. A total of 33
samples were analyzed including 5 field duplicates. For the field duplicates,
the reference method demonstrated an average coefficient of variation of 16%.
For Method 4010 average coefficient of variation was 31%. Since Method 4010 is
not quantitative, quantitative values were estimated. These data are shown in
Table 9. At the 10 ppm cutoff, there were 0/33 (0%) false negatives and 0/33
(0%) false positives. At the 100 ppm cutoff, there was 1/33 (3%) false negatives
and 1/33 (3%) false positives. These data are shown in Table 10.
10.0 REFERENCES
1. J.P. Mapes, K.D. McKenzie, L.R. McClelland, S. Movassaghi, R.A. Reddy, R.L.
Allen, and S.B. Friedman, "Rapid, On-Site Screening Test for
Pentachlorophenol in Soil and Water - PENTA-RISc™", Ensys Inc., Research
Triangle Park, NC 27709
2. J.P. Mapes, K.D. McKenzie, L.R. McClelland, S. Movassaghi, R.A. Reddy, R.L.
Allen, and S.B. Friedman, "PENTA-RISc™ - An On-Site Immunoassay for
Pentachlorophenol in Soil", Ensys Inc., Research Triangle Park, NC 27709
3. PENTA-RISc™ Instructions for Use, Ensys Inc.
4. EnviroGard™ PCP in Soil Test Kit Guide, Millipore, Inc.
4010A-4 Revision 1
January 1995
-------
Table 1A - Cross Reactivity for PCP
PENTA RISc™ Test Kit
Compound"
2,6-Dichlorophenol
2,3,4-Trichlorophenol
2,4,6-Trichlorophenol
2,4,5-Trichlorophenol
2,3,5, 6-TetrachTorophenol
Tetrachlorohydroquinone
Concentration (mg/Kg)
in Soil to Cause a
False Positive for
PCP at 0.5 mg/Kg
700
400
16
100
1.2
500
Concentration (M9/L)
in Water to Cause a
False Positive for
PCP at 5 jxg/L
600
600
100
500
7
>1500
" Compounds assayed at 3.75 /iM (molar equivalent of PCP at 1000 /ag/L), except
where noted.
Table IB - Cross Reactivity for PCP
EnviroGard1" PCP in Soil Test Kit
Compound
Pentachlorophenol
2,5-Dichlorophenol
2,6-Dichlorophenol
2,3,4-Trichlorophenol
2,3,5-Trichlorophenol
2,3,6-Trichlorophenol
2,4,5-Trichlorophenol
2,4,6-Trichlorophenol
Lower Limit of
Detection (mg/kg)
10
1000
1000
1000
500
500
500
500
The following compounds were tested and found to yield
negative results at 1,000 ppm:
2,3,5,6-Tetrachloronitrobenzene PCB (Aroclor
3,5-Dichlorophenol TNT
2,4-Dichlorophenol DDT
2,3-Dichlorophenol PAHs
4-Chlorophenol Chlordane
1248)
4010A-5
Revision 1
January 1995
-------
Sample
Type
Groundwater
Proceaa water
Waatewater
Run-off
Table 2 - Comparison of PENTA RISc™ Teat Kit with GC/MS - Aqueoua Matrix
Screening Reiuhl (ppm)
0.005
>
>
>
>
>
>
>
>
>
0.05
<
>
>
>
>
>
>
>
<
>
>
>
>
>
<
>
0.1
>
<
<
>
>
<
>
<
>
0.5
<
<
<
<
<
<
1
>
>
>
<
>
<
<
<
<
<
>
<
>
>
>
5
>
>
<
<
<
>
.
<
>
>
50
<
<
<
Concentration
meatured by
GC/MS (ppm)
3.5
0.35
<0.1
8.2
2.8
2.9
0.21
0.17
0.12
0.6
1.4
<0.1
0.17
<0.1
0.034
0.098
0.084
0.086
2.1
0.073
0.026
0.006
0.169
0.239
0.190
0.114
0.346
1.1
19
4.3
AGREEMENT*
Y, FP, FN
FP
y
Y
Y
Y
Y
FN
Y
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
FP
Y
FP
Y
FP
FP i'
Y
Y
Y
Y
4010A-6
Revision 1
January 1995
-------
CoqWMM «
OOMSIB-)
1100
a
0.31
0.71
311
IJ
t4
t
1.9
46
<1
11
3.3
4
11
If
33
it
a
74
IS
1.1
14.3
<1
<1
>
<
<
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
<
<
<
>
<
>
>
<
>
>
>
>
>
>
>
<
<
>
in*, s
"T«ll»«i
•tl^bd
5
>
>
<
<
>
<
>
>
<
>
<
>
<
>
>
>
>
>
>
>
>
<
>
<
<
<
<
<
<
>
<
>
>
>
>
<
>
>
<
<
<
aoc/us t
*->
»
>
<
<
<
>
<
<
<
<
>
<
<
<
<
<
<
<
>
>
>
>
<
<
<
<
<
<
<
<
>
<
>
<
>
>
<
<
>
<
<
<
1 11 lini!
AOIBBMBHT
t.rt.m
y
m
Y
PN
r
y
Y
Y
y
re
i
Y
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
rr
Y
Y
m
Y
Y
Y
FP
Y
Y
Y
Y
4010A-7
Revision 1
January 1995
-------
TABLE 4
EnviroGard1" PCP in Soil Test Kit' Sensitivity
Part 1
Average Response with Negative Soils
Average %Bo Standard
Soil* Soil Type (n = 9) Deviation
SI
S2
S3
S4
S5
S6
S7
S8
LOAM
CLAY
SAND
LOAM
SAND
CLAY
LOAM/SAND
SAND/LOAM
97.6
100.1
101.4
99.4
100.2
97.4
102.6
97.5
3.0
1.4
2.8
4.9
3.1
2.7
0.3
3.6
AVERAGE 99.5 5.2
NOTE: (%Bo = [(ODsample/ODnegative control)xlOO])
Part 2
Average Response with Pentachlorophenol Calibrators
PCP Average Average
Cone, (ppm) Absorbance %Bo
0
5
20
50
200
1.142
0.828
0.556
0.382
0.162
N/A
72.6
48.7
33.4
14.1
NOTE: (%Bo * [(ODsample/ODnegative controlJxlOO])
Part 3
Method Sensitivity
Based on Part 1 and Part 2 Above:
Average %Bo - 2 SD = 89.2 which is equivalent to 1.6 ppm PCP
Average %Bo - 3 SD = 84.0 which is equivalent to Z. 3 ppm PCP
NOTE: (%Bo - [(ODsample/ODnegative control )xlOO])
4010A-8 Revision 1
January 1995
-------
TABLE 5
EFFECT OF WATER CONTENT IN SOIL SAMPLES"
Soil % Water Fortified? Rep. 1 Rep. 2
Mean Std. Dev. ± 2 SD Range
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
0
30
0
30
0
30
0
30
0
30
0
30
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
104.5*
101.9
38.9
49.2
97.8
105.1
40.2
48.8
98.3
111.5
43.3
46.5
106.5
106.3
47.2
51.1
105.7
109.7
47.5
47.2
107.1
103.1
47.2
49.8
99.7
95.2
40.2
48.2
96.7
93.9
42.7
44.8
99.7
95.1
43.2
48.0
103.6
101.1
42.1
49.5
100.1
102.9
43.5
46.9
101.7
103.2
44.6
48.1
3.5
5.6
4.4
1.5
4.9
8.1
3.7
2.0
4.7
8.2
2.3
1.7
96.6
89.9
33.3
46.5
90.3
86.7
36.1
42.9
92.3
86.8
40.0
44.7
- Ill
- 112
- 50.9
- 52.5
- 110
- 119
- 50.9
- 50.9
- Ill
- 120
- 49.2
- 51.5
* All values shown are %Bo [- (OD.^./ODneoativecontro))xlOO]
' EnviroGard™ PCP in Soil (Mi Hi pore, Inc.)
4010A-9
Revision 1
January 1995
-------
TABLE 6
EFFECT OF pH OF SOIL SAMPLES'
oil
SI
SI
SI
SI
SI
SI
S2
S2
S2
S2
S2
S2
S3
S3
S3
S3
S3
S3
oh Adj.
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
Fortified?
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
Reo. 1*
103.1
88.7
85.2
52.7
57.1
44.6
105.6
104.4
93.4
47.8
51.4
43.3
92.3
96.6
87.7
55.2
55.3
44.3
Reo. 2
98.6
96.9
90.9
44.8
44.6
41.6
93.9
91.3
87.7
45.1
44.4
40.7
101.8
91.9
99.8
49.5
48.3
39.3
Reo. 3
98.6
100.2
98.0
45.8
45.2
45.9
102.5
105.8
105.8
44.3
54.1
44.0
100.4
98.5
96.3
55.9
42.0
48.0
Mean
100.1
95.3
91.3
47.8
48.9
44.0
100.7
100.5
95.6
45.7
50.0
42.7
98.2
95.7
94.6
53.6
48.5
43.9
Std.
2
5
6
4
7
2
6
8
9
1
5
1
5
3
6
3
6
4
Dev.
.6
.9
.4
.3
.0
.2
.1
.0
.3
.8
.0
.8
.2
.4
.2
.5
.7
.4
± 2
94.
83.
78.
39.
34.
39.
88.
84.
77.
42.
40.
39.
87.
88.
82.
46.
35.
35.
SD
9 -
5 -
5 -
2 -
9 -
6 -
5 -
5 -
0 -
1 -
0 -
1 -
8 -
9 -
2 -
6 -
1 -
1 -
Range
105
107
104
56.4
62.9
48.4
113
117
114
49.3
60.0
46.3
109
103
107
60.6
61.9
52.7
* All values shown are %Bo [= (00.^/00^^.^JxlOO]
• EnviroGard™ PCP in Soil (Mi Hi pore, Inc.)
4010A-10 Revision 1
January 1995
-------
TABLE 7
Test Kit8 Bias
Soi1# Fortification(ppm) Extraction! Recovered(ppm)* % Recovery
SI 10 19 91
SI 10 29 86
SI 10 39 88
SI 100 1 84 84
SI 100 2 78 78
SI 100 3 76 76
Average »»»»»»»»»»»»»»»»»»»»»»»»>»» 84
S2 10 1 10 100
S2 10 28 76
S2 10 . 3 8 76
S2 100 1 101 101
S2 100 2 98 98
S2 100 3 88 88
Average >»»»»»»»»»»»»»»»»»»»»»»»»»» 90
S3 10 17 72
S3 10 28 76
S3 10 38 81
S3 100 1 95 95
S3 100 2 90 90
S3 100 3 87 87
Average »»»»»»»»»»»»»»»»»»»»»»»>»»» 84
Overall Average %Recovery = 86
EnviroGard™ PCP in Soil (Millipore, Inc.)
4010A-12 Revision 1
January 1995
-------
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
TABLE 8
Effect of Co-contamination with Diesel Oil3
Soil# Adulterated Fortified Rep.#l Rep.#2 Rep.#3
NO
YES
NO
YES
NO
YES
NO
YES
NO
YES
NO
YES
* Figures are %Bo =[(OD.ample/ODneoativecontrol)*100]
a EnviroGard™ PCP in Soil (Millipore, Inc.)
Mean
NO
NO
YES
YES
NO '
NO
YES
YES
NO
NO
YES
YES
103.2*
93.4
52.7
50.9
103.1
85.4
47.8
44.6
98.9
103.8
55.2
50.4
92.5
99.4
44.8
49.7
98.3
95.1
45.1
50.8
95.4
99.7
49.5
50.6
99.8
106.2
45.8
44.6
102.3
99.9
44.3
49.0
108.1
101.4
55.9
56.7
98.5
99.7
47.8
48.4
101.2
93.5
45.7
48.1
100.8
101.6
53.6
52.6
4010A-13
Revision 1
January 1995
-------
Method 8270
Determination #1
Determination #2
Average
Standard Deviation
% Coefficient of Variation
Immunoassay*
Determination #1
Determination #2
Average
Standard Deviation
% Coefficient of Variation
TABLE 9
Field Duplicates3
059
9600
10300
9950
495
5.0
4480
3370
3920
785
20
Sample ID
086
6.59
6.88
6.74
0.20
3.0
073
74.8
78.2
76.5
2.4
3.1
79.5
122
101
30.0
30
074
836
1520
1178
484
41
604
421
512
129
25
2.4
5.0
3.7
1.8
50
087
34.0
51.8
42.9
12.6
29
36.0
24.0
30.0
8.5
28
* For the purpose of this comparison, quantitative values were calculated for
the immunoassay.
EnviroGard1" PCP in Soil (Millipore, Inc.)
4010A-14
Revision 1
January 1995
-------
TABLE 10
Immunoassay8 Compared to Method 8270
Test Interpretation at 10 ppm PCP
Sample ID
059
0590
060
061
063
064
065
066
067
068
069
070
071
072
073
073D
074
074D
075
076
077
078
079
080
081
082
083
084
085
086
086D
087
087D
Method 8270
9600
10300
1010
2740
1610
1980
1580
57.8
110
47.7
798
2890
289
326
74.8
78.2
836
1520
3690
4590
2040
1720
792
2550
125
2400
270
1140
57.7
6.59
6.88
34.0
51.8
Immunoassay
Concurrence?
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
EnviroGard™ PCP in Soil (Millipore, Inc.)
4010A-15
Revision 1
January 1995
-------
TABLE 10 (continued)
Immunoassay3 Compared to Method 8270
Test Interpretation at 100 ppm PCP
Sample ID
059
059D
060
061
063
064
065
066
067
068
069
070
071
072
073
073D
074
074D
075
076
077
078
079
080
081
082
083
084
085
086
086D
087
087D
Method 8270
9600
10300
1010
2740
1610
1980
1580
57.8
110
47.7
798
2890
289
326
74.8
78.2
836
1520
3690
4590
2040
1720
792
2550
125
2400
270
1140
57.7
.59
.88
34.0
51.8
Immunoassay
>100
>100
>100
>100
>100
>100
>100
<100
>100
<100
>100
>100
>100
>100
<100
>100
>100
>100
>100
>100
>100
>100
>100
>100
<100
>100
>100
>100
<100
<100
<100
<100
<100
Concurrence?
YES
YES
YES
YES
YES
YES
•YES
YES
YES
YES
YES
YES
YES
' YES
YES
False Positive
YES
YES
YES
YES
YES
YES
YES
YES
False Negative
YES
YES
YES
YES
YES
YES
YES
YES
4010A-16
Revision 1
January 1995
-------
TABLE 10 (continued)
Immunoassay8 Compared to Method 8270
Test Interpretation at 100 ppm PCP
Sample ID
059
059D
060
061
063
064
065
066
067
068
069
070
071
072
073
073D
074
074D
075
076
077
078
079
080
081
082
083
084
085
086
086D
087
087D
Method 8270
9600
10300
1010
2740
1610
1980
1580
57.8
110
47.7
798
2890
289
326
74.8
78.2
836
1520
3690
4590
2040
1720
792
2550
125
2400
270
1140
57.7
6.59
6.88
34.0
51.8
Immunoassay
>100
>100
>100
>100
>100
>100
>100
<100
>100
<100
>100
>100
>100
>100
<100
>100
>100
>100
>100
>100
>100
>100
>100
>100
<100
>100
>100
>100
<100
<100
<100
<100
<100
Concurrence?
YES
YES
YES
YES
YES
YES
YES
YES
. YES
YES
YES
YES
YES
YES
YES
False Positive
YES
YES
YES
YES
YES
YES
YES
YES
False Negative
YES
YES
YES
YES
YES
YES
YES
YES
4010A-16
Revision 1
January 1995
-------
METHOD 4015
SCREENING FOR 2.4 DICHLORORPHENOXY ACETIC ACID BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4015 is a procedure for screening soils and aqueous matrices
to determine whether 2,4 dichlorophenoxy acetic acid (2,4-D) (CAS Registry 94-75-
7) is likely to be present at concentrations above 0.1, 0.5, 1.0 or 5,0 mg/kg in
soil, and in aqueous matrices above 10 mg/L (the toxicity characteristic
regulatory action level) and 10 ng/L (ground water monitoring). Method 4015
provides an estimate for the concentration of 2,4-D by comparison against
standards.
1.2 Using the test kit from which this method was developed, >95% of
aqueous samples confirmed to have concentrations of 2,4-D below detection limits
will produce a negative result in the 10 ppm test configuration.
1.3 In cases where the exact concentration of 2,4-D is required,
additional techniques (i.e., gas chromatography Method 8151) should be used.
1.4 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using an extract of a soil
sample, or directly on an aqueous sample. Filtered extracts may be stored cold,
in the dark. An aliquot of the aqueous sample or extract and an enzyme-2,4-D
conjugate reagent are added to immobilized''2,4-D antibody. The enzyme-2,4-D
conjugate "competes" with 2,4-D present in the sample for binding to 2,4-D
antibody. The enzyme-2,4-D conjugate bound to the 2,4-D antibody then catalyzes
a colorless substrate to a colored product. The test is interpreted by comparing
the color produced by a sample to the response produced by a reference reaction.
3.0 INTERFERENCES
3.1 Compounds that are chemically similar may cause a positive test
(false positive) for 2,4-D. The data for the lower limit of detection of these
compounds are provided in Tables 1A and 1C. Consult the information provided by
the manufacturer of the kit used for additional information regarding cross
reactivity with other compounds.
4015-1 Revision 0
January 1995
-------
3.1.1 Solutions of Silvex alone, and Silvex/2,4-D mixtures, were
prepared in TCLP buffer to demonstrate the potential effect of a
structurally similar, environmentally significant cross-reactant on the
immunoassay screening results. At one-half of the action level for 2,4-D
(5ppm), 200 ppm of Silvex are required to be present to generate a false
positive response. These results are summarized in Table IB.
3.2 Storage and use temperatures may modify the method performance.
Follow the manufacturer's directions for storage and use.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: 2,4-D RaPIDw Assay kit (Ohmicron), EnviroGardm
2,4-D in Soil (Millipore, Inc.), or equivalent. Each commercially available test
kit will supply or specify the apparatus and materials necessary for successful
completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-9.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
4015-2 Revision 0
January 1995
-------
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4015 is intended for field or laboratory use. The appropriate
level of quality assurance .should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 Sensitivity of the EnviroGard* 2,4-D in Soil Test Kit was determined
by establishing the "noise" level expected from matrix effects encountered in
negative soil samples and determining the corresponding 2,4-D concentration by
comparison to the analyte-specific response curve. Eight different soils which
did not contain 2,4-D were assayed. Each of these soils was extracted in
triplicate and each extract was assayed in three different assays. The mean and
the standard deviation of the resulting %Bo's (%Bo = [(ODsample/ODneflativecontrol)xlOO])
were calculated and the sensitivity was estimated at two standard deviations
below the mean. The sensitivity for Method 4015 was determined to be 80% Bo at
a 95% confidence interval. Based on the average assay response to 2,4-D, this
corresponds to 0.16 ppm 2,4-D. These data are shown in Table 2.
9.2 The effect of water content of the soil samples was determined by
assaying three different soil samples which had been dried and subsequently had
water added to 30% (w/w). Aliquots of these samples were then fortified with
2,4-D. Each soil sample was assayed three times, with and without added water,
and with and without 2,4-D fortification. It was determined that water in soil
up to 30% had no detectable effect on the method. These data are shown in Table
3.
9.3 The effect of the pH of the soil extract was determined by adjusting
the soil pH of three soil samples. Soil samples were adjusted to pH 2 - 4 using
6N HC1 and pH 10 - 12 using 6N NaOH, Aliquots of the pH adjusted soil samples
were fortified with 2,4-D. Each soil sample was assayed unadjusted and with pH
adjusted to 2-4 and 10-12, both unfortified and fortified. It was determined
that soil samples with pH ranging from 3 to 11 had no detectable effect on the
performance of the method. These data are shown in Table 4.
9.4 The method bias was estimated by fortifying three different soil
samples at two different concentrations (0.3 and 2 ppm 2,4-D). Each fortified
sample was extracted three times and each extract was assayed three times.
Recovery for individual determinations ranged from 27% to 151%. Average recovery
for each individual extract ranged from 70% to 120%. Overall average recovery
for all samples was 99.7%. These data are summarized in Table 5.
9.5 The probabilities of generating false negative and false positive
4015-3 Revision 0
January 1995
-------
results at a 10 ppm action level are shown in Table 6. M
9.6 The results obtained from spiking 2,4-D into TCLP leachates and other
aqueous samples are reported in Table 7. Each matrix was diluted 1:1000 and
tested by immunoassay 5 times. The results are reported as positive (+) or
negative (-). Municipal water results are based on a 52 ppb cutoff to determine
positive from negative, and were diluted 1:7.
9.7 Comparison of the results from immunoassay and GC (Method 8150)
testing of aqueous samples are presented in Table 8.
9.8 A field trial was undertaken to evaluate the ability of the
EnviroGardw 2,4-D in Soil Test Kit to identify 2,4-D contaminated soil at a
remediation site. A total of 30 soil samples were evaluated by both the
immunoassay and Method 8151. Interpretation of the results at 200 ^g/kg
resulted in 0/32 (0%) false negatives and 1/32 (3%) false positives. This
corresponds to specificity 95% and sensitivity of 100%. These data are shown in
Table 9.
10.0 REFERENCES
1. 2,4-D RaPIDw Assay kit Users Guide, Ohmicron.
2. EnviroGard,,, 2,4-D in Soil Test Kit Guide, Millipore, Inc.
3. Lawruk, T.S., Hottenstein, C.S., Fleeker, J.R., Hall, J.C., Herzog, D.P.,
Rubio, P.M., "Quantitation of 2,4-D and Related Chlorophenoxy -.erbicides by
A Magnetic Particle-Based ELISA"' 1993, (manuscript suomitted for
publication).
4. Hayes, M.C., Jourdan, S.W., Lawruk, T.S., and Herzog, D.P., "Screening of
TCLP Extracts of Soil and Wastewater for 2,4-D by Immunoassay", USEPA Ninth
Annual Waste Testing and Quality Assurance Symposium, 1993.
4015-4 Revision 0
January 1995
-------
TABLE 1A
Cross-Reactivity8 of Chi orophenoxy Compounds and
Structurally Unrelated Pesticides
Compound
2,4-D
2,4-D propylene glycol ester
2,4-D ethyl ester
2,4-D isopropyl
2,4-D methyl ester
2,4-D sec-butyl ester
2,4-D butyl ester
2,4-D butoxyethyl ester
2,4,5-T methyl ester
2,4-D iso-octyl ester
2,4-D butoxy-propylene ester
2,4-DB
MCPA
2,4,5-T
Si 1 vex methyl ester
4-Chlorophenoxyacetic acid
MCPB
Silvex (2,4,5-TP)
Dichlorophenol
Dichloroprop
Triclopyr
MCPP
Mecoprop
. Pentachlorophenol
Picloram
Concentration Giving
a Positive Result
(ppm TCLP Leachate)
10
0.52
0.54
0.96
1.09
1.40
1.60
2.00
12.0
20.0
20.6
95
110
130
665
815
980
1375
2380
5000
>10,000
>10,000
>10,000
>10,000
>10,000
Percent Cross -
Reactivity
100
1900
1850
1040
917
714
625
500
86
50
49
11
9
8
1.5
1.2
1.0
0.7
0.4
0.2
<0.1
<0.1
<0.1
<0.1
<0.1
4015-5
Revision 0
January 1995
-------
TABLE 1A
Cross-Reactivity8 of Chlorophenoxy Compounds and
Structurally Unrelated Pesticides
Compound
Alachlor
Aldicarb
Aldicarb sulfate
Aldicarb sulfoxide
Atrazine
Benomyl
Butyl ate
Captan
Captofol
Carbaryl
Carbofuran
Dicamba
1 ,3-Dichloropropene
Dinoseb
Metolachlor
Metribuzin
Simazine
Terbufos
Thiabendazol
Concentration Giving
a Positive Result
(ppm TCLP Leachate)
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>1 0,000
>10,000
>10,000
>10,000
>10,000
Percent Cross -
Reactivity
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0 . 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
i
a 2,4-D RaPID,. Assay kit
TABLE IB
Cross -Reactivity8 of 2,4-D witn Si 1 vex
Si 1 vex Concentration 2,4-D Concentration
(ppm) (ppm)
0
0.5
1.0
2.0
100
200
0
0.5
1.0
2.0
100
200
0
0
0
0
0
0
5.0
5.0
5.0
5.0
5.0
5.0
Screening Result
-
+
a 2,4-D RaPIDw Assay kit
4015-6
Revision 0
January 1995
-------
TABLE 1C
CROSS REACTIVITY'
Compound
2,4-D Acid
2,4-D butyl ester
2,4-D Dichlorophenol
2,4-D isobutyl ester
2,4-D isopropyl ester
2,4-D methyl ester
2,4-DB
2,4-DB butyl ester
Dichloroprop
Diclofop
MCPA
2,4,5-T acid
Concentration Required for
Positive Interpretation (ppm)
0.2
0.025
1.5
0.2
0.2
0.1
0.2
0.9
6.0
42.5
0.8
7.0
" EnviroGardw 2,4-D in Soil Test Kit (Millipore Corporation)
4015-7
Revision 0
January 1995
-------
TABLE 2
Sensitivity of the EnviroGard,, 2,4-D in Soil Test Kit
Part 1 - Average Response with Negative Soils
Soil*
SI
S2
S3
S4
S5
S6
S7
Average
Soil Type
LOAM
LOAM
SAND/ LOAM
CLAY
CLAY
LOAM/SAND
SAND
LOAM
Average %Bo
(n=9)
90.0
89.6
89.3
86.3
90.0
86.9
88.8
86.9
88.5
Standard
Deviation
1.7
2.3
2.1
1.9
2.3
2.6
2.8
2.9
6.5
Part 2 - Average Response with 2,4-D Calibrators
2,4-D Calibrator
Concentration (ppm)
0
0.1
0..5
1.0
5.0
Average
Absorbance
1.442
1.186
0.776
0.600
0.301
Average %Bo
N/A
82.2
53.8
41.7
20.9
Part 3 - Method Sensitivity
Based on Part 1 and Part 2 Above:
Average %Bo - 2 SD = 75.6 which is equivalent to 0.16 ppm 2,4-D
Average %Bo - 3 SD = 69.1 which is equivalent to 0.23 ppm 2,4-D
(XBo = [(OD8ampiyODneortivecontrol)xlOO])
4015-8
Revision 0
January 1995
-------
TABLE 3
Effect of Water Content of Soil Sampleson the EnviroGardw 2,4-D in Soil Test Kit
Soil % Water Fortified? Rep. 1 Rep. 2 Rep. 3 Mean Std. Dev. ± 2 SD Range
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
0
30
0
30
0
30
0
30
0
30
0
30
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
98
96
61
63
98
96
47
37
98
97
41
43
.7*
.0
.4
.1
.5
.0
.6
.6
.7
.3
.0
.1
99.9
95.4
62.0
59.9
90.7
95.4
47.0
37.7
94.1
97.2
39.3
40.4
102.9
93.7
73.1
69.4
97.8
96.8
46.0
40.0
105.2
95.9
48.8
47.4
100.5
95.0
65.5
64.1
95.7
96.1
46.9
38.4
99.4
96.8
43.1
43.6
2.2
1.2
6.6
4.8
4.3
0.7
0.8
1.3
5.6
0.8
5.1
3.5
96
92
52
54
87
94
45
35
88
95
32
36
.1 -
.6 -
.3 -
.5 -
.1 -
.7 -
.3 -
.8 -
.2 -
.2 -
.9 -
.6 -
105
97.4
78.7
73.7
104
97.5
48.5
41.0
111
98.4
53.3
50.6
All values shown are %Bo [= (OD8amp|e/OD
negative control
)xlOO]
4015-9
Revision 0
January 1995
-------
TABLE 4
Effect of pH of Soil Samples on the ErwiroGard,,, 2,4-D in Soil Test Kit
Soil pH Adj. Fortified? Rep. 1* Rep. 2 Rep. 3 Mean Std. Dev. ± 2 SD Range
SI
SI
SI
SI
SI
SI
S2
S2
S2
S2
S2
S2
S3
S3
S3
S3
S3
S3
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
95.5
102
96.8
46.0
50
43.0
94.3
91.7
89.7
50.5
56.3
46.9
82.2
95.0
86.1
52.2
55.2
59.4
92.5
105
98.3
47.5
51.9
52.4
90.6
95.8
94.2
52.6
58.1
54.2
92.0
85.1
84.4
63.6
59.5
54.3
88.7
93.1
79.4
48.6
43.7
39.1
90.8
85.9
81.0
50.2
44.3
46.4
85.4
86.9
103 '
49.4
66.6
54.9
92.2
100
91.5
47.4
48.5
44.8
91.9
91.1
88.3
51.1
52.9
49.1
86.5
89.0
91.2
55.1
60.4
56.2
3.4
6.2
10.5
1.3
4.3
6.8
2.1
5.0
6.7
1.3
7.5
4.4
5.0
5.3
10.4
7.5
5.8
2.8
85.4
87.6
70.5
44.8
39.9
31.2
87.7
81.1
74.9
48.5
37.9
40.3
76.5
78.4
70.4
40.1
48.8
50.6
- 99.0
- 112
- 113
- 50.0
- 57.1
- 58.4
- 96.1
- 101
- 102
- 53.7
- 67.9
-57.9
- 96.5
- 99.6
- 112
- 70.1
- 72.0
- 61.8
* All values shown are %Bo [- (OD88mple/ODneoat(V,,rontro))xlOO]
4015-10
Revision 0
January 1995
-------
TABLE 5
Bias of the EnviroGardw 2,4-D in Soil Test Kit
Soilfl Fortification (pom) Extraction^ Recovered (ppm)* % Recovery
SI
SI
SI
SI
SI
SI
Average »>
S2
S2
S2
S2
S2
S2
Average >»
S3
S3
S3
S3
S3
S3
0.3
0.3
0.3
2
2
2
»»»»»»»:
0.3
0.3
0.3
2
2
2
»»»»»»»:
0.3
0.3
0.3
2
2
2
1
2
3
1
2
3
>»>»»»»»»:
1
2
3
1
2
3
>»»»»»>»»;
1.
2
3
1
2
3
0.21
0.24
0.23
1.87
2.12
2.40
»»»»»»»»:
0.29
0.29
0.30
2.05
1.89
2.22
>>»»»»»»»:
0.31
0.31
0.31
2.28
2.30
2.24
70.0
80.0
76.6
93.5
1Q6
120
»» 91.0
96.7
96.7
100
102
94.5
111
»» 100
103
103
103
114
115
112
Average »»»»»»»»»»»»»»»»»»»»»»>»»»» 108
Overall Average %Recovery =99.7
4015-11
Revision 0
January 1995
-------
Table 6
Probability of False Negative and False Positive Results for 2,4-D RaPID,
Assay kit at a 10 ppm Action Level In TCLP Extract from Organic Soil
Spike Concentration
2,4-D (PPM)
5
7.5
10
15
Probability of False
Positive (%)
0
70
N/A
N/A
Probability of False
Negative (%)
N/A
N/A
0
0
Results were based on ten replicate spiked samples. Cutoff
levels were established using 30 replicates of each solution
tested in 3 immunoassay batch runs.
N/A =
limit.
No false positives possible above/below the action
4015-12
Revision 0
January 1995
-------
Table 7
2,4-D Spiking Results on Aqueous Environmental Matrices8
ID f
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Matrix/Spike
TCLP Buffer
TCLP Buffer + 15 ppm
TCLP Buffer + 10 ppm
TCLP Buffer + 5 ppm
Sandy Extract"
Sandy Extract +15 ppm
Sandy Extract + 10 ppm
Sandy Extract + 5 ppm
Organic Extract0
Organic Extract + 15
Organic Extract + 10
Organic Extract + 5
Effluent #1
Effluent #1 + 15 ppm
Effluent #1 + 10 ppm
Effluent #1+5 ppm
Effluent #2
Effluent #2 + 15 ppm
Effluent n + 10 ppm
Effluent #2+5 ppm
Runoff
Runoff + 15 ppm
Runoff + 10 ppm
Runoff + 5 ppm
2,4-0 Test Results
Rl
_
+
+
-
-
+
+
-
- .
+
+
-
_
+
+
-
.
+
+
-
.
+
+
-
R2
_
+
+
-
-
+
+
-
-
+
+
-
.
+
+
-
.
+
+
-
-
+
+
-
R3
.
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
R4
_
+
+
-
-
+
+
-
-
+
+
-
_
+
+
-
-
+
+
-
-
+
+
-
R5
..
+
+
-
-
+
• +
-
-
+
+
-
_
+
+
-
.
+
+
-
-
+
+
-
%POS
_
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
-
+
+
-
%NEG
_
+
+
-
-
+
+
-
-
+
+
-
_
+
+
-
-
+
+
-
-
+
+
-
4015-13
Revision 0
January 1995
-------
1 Table 7
2,4-D Spiking Results on Aqueous Environmental Matrices9
25
26
27
28
Municipal Water
Municipal Water + 140 ppb
Municipal Water + 70 ppb
Municipal Water + 35 ppb
2,4-D Test Results
„
+
+
-
_
+
+
-
_
+
+
-
_
.
_
-
_
_
_
-
.
-
_
-
-
-
.
-
a 2,4-D RaPID, Assay kit
b Sandy Soil TCLP Extract
c Organic Soil TCLP Extract
4015-14
Revision 0
January 1995
-------
Table 8
2,4-D Splicing Results
2,4-D RaPID. Assay kit vs. Method 8151
ID*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1.5
16
17
18
19
20
21
22
23
Matrix/Spike
TCLP Buffer
TCLP Buffer + 15 ppm
TCLP Buffer + 10 ppm
TCLP Buffer + 5 ppm
Sandy Extract"
Sandy Extract +15 ppm
Sandy Extract + 10 ppm
Sandy Extract + 5 ppm
Organic Extract6
Organic Extract + 15 ppm
Organic Extract + 10 ppm
Organic Extract + 5 ppm
Effluent #1
Effluent #1 + 15 ppm
Effluent #1. + 10 ppm
Effluent #1+5 ppm
Effluent #2
Effluent #2+15 ppm
Effluent #2 + 10 ppm
Effluent #2+5 ppm
Runoff
Runoff +15 ppm
Runoff + 10 ppm
Inrounoassay
Results
5/5 Negative
5/5 Positive
5/5 Positive
5/5 Negative
5/5 Negative
5/5 Positive
5/5 Positive
5/5 Negative
5/5 Negative
5/5 Positive
5/5 Positive
5/5 Negative
5/5 Negative
5/5 Positive
5/5 Positive
5/5 Negative
5/5 Negative
5/5 Positive
5/5 Positive
5/5 Negative
5/5 Negative
5/5 Positive
5/5 Positive
Method 8151
2,4-D (ppm)
nd
13.0
11.0
5.6
nd
*
5.9, 5.2
*
nd
*
10.0, 9.5
*
*
*
11.0, 7.8
3.6
*
11.0
8.8, 9.5
*
nd
*
9.7, 8.6
Correlation
IA vs. GC
Yes
Yes
Yes
Yes
Yes
*
No
*
Yes
*
Yes
*
*
*
Yes
Yes
*
Yes
Yes
*
Yes
*
Yes
4015-15
Revision 0
January 1995
-------
METHOD 4020
SCREENING FOR POLYCHLORINATED BIPHENYLS BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4020 is a procedure for screening soils and non-aqueous waste
liquids to determine when total polychlorinated biphenyls (PCBs) are present at
concentrations above 5, 10 or 50 mg/kg. Method 4020 provides an estimate for the
concentration of PCBs by comparison with a standard.
1.2 Using the test kit from which this method was developed, 95% of soil
samples containing 0.625 ppm or less of PCBs will produce a negative result in
the 5 ppm test configuration. Using another commercially available test kit, 97%
of soil samples containing 0.25 ppm or less of PCBs will produce a negative
result in the assay and greater than 99% of the samples containing 1.0 ppm or
more will produce a positive result. Tables 2-5, 7, 10, and 11 present false
positive and false negative data generated from commercially available test kits.
Using a test kit commercially available for screening non-aqueous waste liquids,
>95% of samples containing 0.2-0.5 ppm or less of PCB will produce a negative
result.
1.3 In cases where the exact concentrations of PCBs are required,
quantitative techniques (i.e., Method 8082) should be used.
1.4 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed. In general, the method is
performed using a sample extract. Sample and an enzyme conjugate reagent are
added to immobilized antibody. The enzyme conjugate "competes" with PCB present
in the sample for binding to immobilized anti-PCB antibody. The test is
interpreted by comparing the response produced by testing a sample to the
response produced by testing standard(s) simultaneously.
3.0 INTERFERENCES
3.1 Chemically similar compounds and compounds which might be expected to
be found in conjunction with PCB contamination were tested to determine the
concentration required to produce a positive test result. These data are shown
in Tables 1A, IB, 1C, and ID.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: PCB RISc™ (EnSys, Inc.), EnviroGard™ PCB in
Soil (Millipore, Inc.), D TECH™ PCB test (Strategic Diagnostics Inc.), PCB
4020-1 Revision 0
January 1995
-------
RISc™ Liquid Waste Test System (EnSys, Inc.), or equivalent. Each commercially
available test kit will supply or specify the apparatus and materials necessary
for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1. Also refer to Reference 9 for the collection and handling of non-
aqueous waste liquids.
6.2 Samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-11.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used for
quality control procedures specific to the test kit used. Additionally, guidance
provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4020 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 A study was conducted with the PCB RISc™ test kit using fourteen
4020-2 Revision 0
January 1995
-------
standard soils and three soil samples whose PCB concentration had been
established by Method 8082. Replicates were performed on seven of the standard
soils and on one of the soil samples for a total of 25 separate analyses. Each
of two different analysts ran the 25 analyses. Results indicated that "<"
assignments are accurate with almost 99% certainty at the 50 ppm level while ">"
assignments can be up to about 96% inaccurate as the sample concentration
approaches that of the testing level. Corresponding certainties at the 5 ppm
level are 92% and 82% respectively. Tables 2 and 3 summarize these results.
9.2 Table 4 presents method precision data generated using the PCB RISc™
test kit, comparing immunoassay test results with results obtained using Method
8082.
9.3 Method precision was determined with the EnviroGard PCB in Soil test
kit by assaying 4 different soils (previously determined to contain 5.04, 9.78,
11.8, and 25.1 mg/kg by Method 8082), at three different sites, using three
different lots of assay kits, three times a day for 9 days. A total of 81
analyses were performed for each soil. Error attributable to site, lot, date,
and operator were determined. Separately, the relative reactivity of Aroclors
1242, 1248, 1254, and 1260 were determined. Based on Aroclor heterogeneity, and
method imprecision, concentrations of Aroclor 1248 were selected that would
result in greater than 99% confidence for negative interpretation. A study was
conducted (Superfund SITE demonstration) on 114 field samples whose PCB
concentration were also determined by Method 8082. 32 of the field samples were
collected in duplicate (as coded field duplicates) and assayed by standard and
immunoassay methods. The results for all 146 samples are summarized in Tables
5 and 6.
9.4 Grab samples were obtained from sites in Pennsylvania, Iowa and
Illinois using a stainless steel trowel. Each sample was homogenized by placing
approximately six cubic inches in a stainless steel bucket and mixing with the
trowel for approximately two minutes. The soils was aliquotted into 2 six ounce
glass bottles. The samples were tested on site using the D TECH PCB test kit,
and sent to an analytical laboratory for analysis by Method 8082. These data are
compared in Table 7.
9.5 Tables 8 and 9 present data on the inter- and intra-assay precision
of the PCB RISc™ Liquid Waste Test System. The data were generated using 11
samples, each spiked at 0, 0.2 and 5 ppm, and assayed 4 times.
9.6 Tables 10 and 11 provide data from application of the PCB RISc™
Liquid Waste Test System to a series of liquid waste samples whose PCB
concentration had been established by Method 8082.
10.0 REFERENCES
1. J.P. Mapes, T.N. Stewart, K.D. McKenzie, L.R. McClelland, R.L. Mudd, W.B.
Manning, W.B. Studabaker, and S.B. Friedman, "PCB-RISc™ - An On-Site
Immunoassay for Detecting PCB in Soil", Bull. Environ. Contam. Toxicol.
(1993) 50:219-225.
2. PCB RISc™ Users Guide, Ensys Inc.
4020-3 Revision 0
January 1995
-------
3. R.W. Counts, R.R. Smith, J.H. Stewart, and R.A. Jenkins, "Evaluation of PCB
Rapid Immunoassay Screen Test System", Oak Ridge National Laboratory, Oak
Ridge, TN 37831, April.1992, unpublished
4. EnviroGard PCB in Soil Package Insert, Millipore Corp. 2/93.
5. Technical Evaluation Report on the Demonstration of PCB Field Screening
Technologies, SITE Program. EPA Contract Number 68-CO-0047. 2/93.
6. D TECH™ PCB Users Guide , SDI/Em Sciences
7. Melby, J.M., B.S. Finlin, A.B. McQuillin, H.G. Rovira, J.W. Stave, "PCB
Analysis by Enzyme Immunoassay", Strategic Diagnostics Incorporated,
Newark, Delaware, 1993
8. Melby, J.M., B.S. Finlin, A.B. McQuillin, H.G. Rovira, "Competitive
Enzyme Immunoassay (EIA) Field Screening System for the Detection of
PCB", 1993 PCB Seminar, EPRI, September 1993
9. T.A. Bellar and J.J Lichtenberg. The Analysis of Polychloringated
Biphenyls in Transformer Fluid and Waste Oils. U.S. EPA Research and
Development, EPA/EMSL-ORD, Cincinnati, Ohio (June 24, 1980). Revised
June 1981, EPA 600/4-81-045.
10. PCB RISc™ Liquid Waste Test System, User's Guide, EnSys Environmental
Products, Inc.
4020-4 Revision 0
January 1995
-------
TABLE 1A
CROSS REACTIVITY OF DIFFERENT COMPOUNDS"
Compound
1 -Chi oronaphthal ene
1,2,4-Trichlorobenzene
2 , 4-Di chl orophenyl -benzenesul f onate
2,4-Dichloro-l-naphthol ''
Bifenox
Diesel fuel
Pentachl orobenzene
2,5-Dichloroaniline
Hexachl orobenzene
Gasoline
Dichlorofenthion
Tetrad if on
Soil Equivalent Concentration (ppm)
Required to Yield a Positive Result
10,000
10,000
1,000
>10,000
500
>10,000
>10,000
>10,000
>10,000
>10,000
10,000
125
(a) PCB RISc'M test kit, Ensys, Inc. publication
4020-5
Revision .0
January 1995
-------
TABLE IB
CROSS REACTIVITY OF DIFFERENT COMPOUNDS'
Compound
Aroclor 1248
Aroclor 1242
Aroclor 1254
Aroclor 1260
1,2-, 1,3-, & 1,4-Dichlorobenzene
1 , 2 , 4-Tri chl orobenzene
biphenyl
2,4-dichlorophenol
2,5-dichlorophenol
2 , 4 , 5- tri chl orophenol
2,4,6-trichlorophenol
Pentachl orophenol
% Cross Reactivity
100
50
90
50
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
' EnviroGard PCB Test Kits (Millipore Corporation)
4020-6
Revision 0
January 1995
-------
TABLE 1C 1
CROSS REACTIVITY OF DIFFERENT COMPOUNDS' |
Compound
Aroclor 1016
Aroclor 1221
Aroclor 1232
Aroclor 1242
Aroclor 1248
Aroclor 1254
Aroclor 1260
Aroclor 1262
Aroclor 1268
MDLb
(ppm)
5.7
25.5
9.0
1.5
0.8
0.5
0.75
0.5
3.8
1C 50C
(ppm)
83
300
105
31
24
10
10
10
40
% CROSS REACTIVITY6
12
3
10
32
42
100
100
100
25
METHOD: The compounds listed were assayed at various concentrations and
compared against an inhibition curve generated using Aroclor
1254. The concentration of the compound required to elicit a
positive response at the MDL as well as the concentration
required to yield 50% inhibition compared to the standard curve
were determined.
D TECH'M PCB test kit
The IC50 is defined as the concentration of compound required to produce a
test response equivalent to 50% of the maximum response.
c The Minimum Detection Limit (MDL) is defined as the lowest concentration of
compound that yields a positive test result.
d % Crossreactivity is determined by dividing the equivalent Aroclor 1254
concentration by the actual compound concentration at IC50
4020-7
Revision 0
January 1995
-------
TABLE ID
CROSS REACTIVITY OF DIFFERENT COMPOUNDS'
Compound
1 -Chi oronaphthal ene
1 , 2 , 4-Tr i chl orobenzene
2 ,4-Di chl oro- 1 -naphthol
Bifenox
Pentachl orobenzene
2,5-Dichloroaniline
Hexachl orobenzene
Dichlorofenthion
Tetrad if on
% Cross-Reactivity
0.05%
0.05%
<0.20%
<0.10%
<0.05%
<0.05%
<0.05%
0.05%
<0.10%
Soil Equivalent
Concentration (ppm) Required
to Yield a Positive Result
10,000
10,000
>10,000
500
>10,000
>10,000
>10,000
10,000
125
.TM
(a) PCB RISc M Liquid Waste Test System, Ensys, Inc.
4020-8
Revision 0
January 1995
-------
CM
CD
<:
PPM DILUTION3
in
O
LL.
00
LU
O
a:
LU
s
H-
LU
O
CM
O
CM
CO
r-.
VO
in
CO
CM
o
a.
Q.
0)
(O
(U
<~
h-
•
•
•
•
*
co
CM
00
CM
in
10
CM
co
CM
CO
CO
a>
c? S
3) r~T Q)
(O 'r~
O
LU O.
O
V
CO
o
in
o
o
o
CM
^
in
00
•
•
a>
«2 Q,^
-o ^ 01
ro '^
to a>
LU Z
co
CO
"e^
a.
O
LU Q-
o
CM
O
co
o
o
-
t
•
•
•
•
•
•
O>
S. S-
E Lt_ "+•*
UJ Z
o in
en
c CM
o <-<
I/i >,
> rt»
O) 3
a: c
03
O
CM
O
0
oo
(•Ml
cc
aa
a.
-------
Table 4
Comparison of PCB RISc Test Kit with GC
Sample ID
101
284
292
199
264
257
259
265
200
170
198
172
169
171
202
163
165
168
166
164
204
253
203
258
106
161
167
Screening Test
Results
<5 ppm
<5 ppm
<5 ppm-
<5 ppm
<5 ppm
<5 ppm
<5 ppm
<5 ppm
<5 ppm
5-50
<5 ppm
5-50
5-50
5-50
<5 ppm, 5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
1 AGREEMENT*
Y, FP, FN
<0.5 ppm
<0.5 ppm
<0.5 ppm
0.5 ppm
1 ppm
1.8 ppm
4 ppm
4.5 ppm
5 ppm
5.8 ppm
2.2-5.8 ppm
6.2 ppm
7.2 ppm
7.2 ppm
1.3-7.2 ppm
8.7 ppm
9 ppm
9 ppm
9.3 ppm
11.9 ppm
12.8 ppm
13 ppm
13.5 ppm
15 ppm
15-19 ppm
15.3 ppm
16.2 ppm
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
4020-10
Revision 0
January 1995
-------
Table 4
Comparison of PCB RISc Test Kit with GC
Sample 10
247
148
205
162
175
176
197
243
252
178
201
254
238
248
250
242
256
249
245 .
241
246
261
240
267
239
104
108
Screening Test
Results
5-50
>50
5-50 .
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50
5-50, >50
>50
5-50
>50
5-50
>50
>50
>50
5-50
>50
>50
>50
>50
>50
>50
>50
GC Results
18 ppm
18-34 ppm
. 20 ppm
20.4 ppm
21.2 ppm
21.6 ppm
32 ppm
32 ppm
32 ppm
43.7 ppm
43 ppm
56 ppm
46-60 ppm
44-60 ppm
68 ppm
30-69 ppm
73 ppm
96 ppm
102 ppm
154 ppm
154 ppm
204 ppm
251 ppm
339 ppm
460 ppm
200-3772 ppm
531-1450 ppm
AGREEMENT'
Y, FP, FN
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
Y
Y
Y
Y=Yes, FN=False Negative, FP=False Positive
4020-11
Revision 0.
January 1995
-------
Table 5
Comparison of EnviroGard PCB Kit with GC
SAMPLE
NUMBER
001
002
003
004
005
006
007
008
009
010
on
012
013
014
015
015D
016
017
018
019
020
021
022
022D
023
024
SCREENING GC RESULT0
RESULT0'" [8082]
>10 5.98
>10 1.27
<10 0.11
>10 6.71
>10 1.37
>10 0.68
>10 0.55
>10 2.00
>10 1.30
>10 0.17
>10 1..15
<10 NDf
<10 1.13
<10 0.18
>10 9.13
>10 9.84
>10 2110
>10 .2.55
>10 45.4
>10 6.70
<10 0.07
<10 0.06
<10 0.54
<10 0.72
>10 20.8
<10 0.06
AGREEMENT"
Y, FN, FP
FP°
FP
Y
FP°
FP
FP
FP
FP
FP
FP
FP
Y
Y
Y
FP°
FP°
Y
FP
Y
FP°
Y
Y
Y
Y
Y
Y
4020-12
Revision 0
January 1995
-------
Table 5 (continued)
SAMPLE
NUMBER
024D
025
026
027
028
028D
029
030
031
032
033
034
035
035D
036
037
037D
038
039
040
041
042
042D
043
043D
044
SCREENING GC RESULT0
RESULTc'd [8082]
<10 0.05
>10 11.7
<10 1.96
<10 0.06
<10 0.22
<10 0.22
<10 0.23
<10 1.15
<10 0.26
>10 47.6
>10 6.00
>10 34.0
<10 NDf
<10 NDf
>10 816
<10 0.06
<10 0.04
>10 1030
<10 0,68
>10 4.25
<10 ND1
>10 0.52
>10 0.47
>10 1.69
>10 1.74
<10 0.59
AGREEMENT'
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP8
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
FP
FP
FP
FP
Y
4020-13
Revision 0
January 1995
-------
Table 5 (continued)
SAMPLE
NUMBER
045
046
046D
047
047D
048
049
050
050D
051
052
053
054
055
056
057
058
059
060
060D
061
062
063
063D
064
065
SCREENING GC RESULT0
RESULT0"*1 [8082]
<10 NDf
<10 NDf
<10 NDf
<10 0.09
<10 0.10
<10 NDd
<10 NDd
>10 3.60
>10 4.41
<10 NDf
>10 4.21
<10 0.96
<10 0.52
<10 2.40
<10 0.51
<10 NDf
<10 0.69
>10 7.86
>10 0.62
<10 0.58
>10 580
>10 2.35
<10 0.09
<10 0.15
>10 19.0
>10 3.08
AGREEMENT6
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
FP
FP
Y
FP
Y
Y
Y
Y
Y
Y
ppo
FP
Y
Y
FP
Y
Y
Y
FP
4020-14
Revision 0
January 1995
-------
Table 5 (continued)
SAMPLE
NUMBER
066
067
068
069
069D
070
071
071D
072
073
074
075
076
077
078
079
080
081
081D
082
0820
083
0830
084
0840
085
SCREENING GC RESULT
RESULT^ [8082]
<10 1.98
<10 0.08
<10 0.50
<10 ND(
<10 NDf
<10 NDf
<10 0.05
<10 NDf
<10 0.04
>10 15.8
>10 13.3
>10 23.0
>10 46.7
<10 NDf
>10 2.27
>10 42.8
<10 3.77
<10 0.69
<10 0.45 .
<10 NO'
<10 0.24
<10 0.48
<10 0.41
>10 1.16
>10 1.08
>10 428
AGREEMENT6
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
FP
FP
Y
4020-15
Revision 0
January 1995
-------
Table 5 (continued)
SAMPLE
NUMBER
085D
086
086D
087
087D
088
088D
089
090
090D
091
091D
092
092D
093
094
095
095D
096
097
097D
098
098D
099
100
100D
SCREENING GC RESULT6
RESULTc'd [8082]
>10 465
<10 1.42
<10 1.25
<10 0.08
<10 NDf
>10 2.70
>10 1.77
>10 45.0
<10 1.01
<10 1.40
>10 1630
>10 1704
<10 1.21
<10 NDf
<10 0.30
<10 0.36
>10 17.5
>10 31.2
<10 0.06
<10 1.23
<10 0.29
>10 1.17
>10 0.83
<10 NDf
>10 177
>10 167
AGREEMENT'
Y, FN, FP
Y
Y
Y .
Y
Y
FP
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
FP
Y
Y
Y
4020-16
Revision 0
January 1995
-------
Table 5 (continued)
SAMPLE
NUMBER
101
102
102D
103
104
105
106
107
108
109
109D
110
111
112
113
114
SCREENING GC RESULT0
RESULT0'" [8082]
>10 1.21
>10 293
>10 177
>10 40.3
>10 7.66
<10 0.21
<10 2.50
>10 14.1
>10 3.84
<10 NDf
<10 NDf
<10 NDf
<10 NDf
>10 315
>10 14.9
>10 66.3
AGREEMENT'
Y, FN, FP
FP
Y
Y
Y
FP°
Y
Y
Y
FP .
Y
Y
Y
Y
Y
Y
Y
c rag/kg (ppm)
d Screening Calibrator is 5 mg/kg Aroclor 1248
e Y=Yes, FN=False Negative, FP=False Positive
f ND = Not Detectable
8 Expected Result Based on Calibrator Concentration
4020-17
Revision -0.
January 1995
-------
Specificity:
Note 1:
Table 6
EnviroGard PCB Kit Field Performance Summary
[l-(Reported Positives/True Negatives)] = [l-(37/109)] = 66%
8 of the 37 reported positive samples had PCB contamination
levels between 5 and 10 mg/kg. Soils in this range should test
"positive" because the assay calibrator is 5 mg/kg"Aroclor 1248.
A positive assay bias is necessary to prevent false negative
results.
Eliminating these
Specificity of:
samples from the calculations produces a
[1-(Reported Positives/True Negatives)] = [1-(29/101)] = 71%
Note 2:
The distribution of false positives is not random (p < 0.05),
with a clustering at the beginning of the sample set. This
observation was included in Developers Comments which were added
to the final draft of the Technical Evaluation Report2. One
explanation for the higher frequency of false positive results
at the beginning is inexperience of the operator with the
method. If the first 20 samples are eliminated from the
Specificity analysis, the following result is obtained:
[1-(Reported Positives/True Negatives)] = [1-(20/86)] = 77%
In the SITE demonstration, the PCB Immunoassay had a 77%
positive predictive value.
Sensitivity: [l-(Reported Negatives/True Positives)] = [1-(0/31)] = 100%
In the SITE demonstration, the PCB Immunoassay had a 100%
negative predictive value.
4020-18
Revision 0
January 1995
-------
TABLE 7
COMPARISON OF D TECH PCB test kit WITH GC - TRIAL #1
ft
SAMPLE
01
02
J3
J5
J6
J7
J8
J9
J10
Jll
J12
013
014
015
016
017
018
019
020
021
022
023
024
D TECH
(ppm)
4.0-15
>50
15-50
15-50
>50 .
4.0-15
4.0-15
>50
>50
>50
15-50
>50
>50
15-50
15-50
>50
>50
>50
>50
>50
1.0
1.0
<0.5
GC
(ppm)
5.0
147
54
160
1200
12
28
463
1760
28
17
1300
186
31
36
31
130
1310
2620
11100
0.01
0.60
0.10
AGREEMENT
Y, FN, FP
Y
Y
Y
FN
Y
Y
FN
Y
Y
FP
Y
Y
Y
Y
Y
FP
Y
Y
Y
Y
FP
Y
Y
SAMPLE
025
026
028
028
029
030
031
032
033
034
035
036
037
038
039
040
041
042
043
044
045
046
047
D TECH
(ppm)
0.5
<0.5
1.0
<0.5
0.5
>50
4.0-15
0.5
0.5
1.0
1.0
>50
<0.5
0.5
0.5
<0.5
<0.5
1.0
1.0
15-50
15-50
<0.5
<0.5
GC
(ppm)
0.12
0.01
1.8
0.18
0.54
21
13
0.72
0.32
0.36
0.26
70
0.12
0.81
0.33
0.19
0.01
0.43
0.31
503.4
5.6
0.02
0.22
AGREEMENT
Y, FN, FP
FP
Y
Y
Y
Y
FP
Y
Y
Y
FP
FP
Y
Y
Y
Y
Y
Y
FP
FP
FN
FP
Y
Y
Y=Yes, FN=False Negative, FP=False Positive
4020-19
Revision 0
Oanuary 1995
-------
TABLE 7(cont)
COMPARISON OF D TECH PCB test kit WITH GC - Trial #2
SAMPLE
Gl
G2
G3
G4
G5
G6
G7
G8
G9
G10
Gil
612
G13
G14
G15
G16
G17
G18
. G19
G20
D TECH
(ppm)
15-50
4.0-15
1.0-4.0
15-50
<0.5
1.0-4.0
1.0-4.0
15-50
4.0-15
15-50
4.0-15
4.0-15
4.0-15
0.5-1.0
<0.5
1.0-4.0
4.0-15
4.0-15
1.0-4.0
>50
GC
(ppm)
18
L 11
3.4
6.5
0.01
1.4
0.30
7.5
33
8
11
24
4.3
1.3
0.01
3.2
18
4.6
'2.3
37
AGREEMENT
Y, FN, FP
Y
Y
Y
FP
Y
Y
FP
FP
FN
FP
Y
FN
Y
Y
Y
Y
Y
Y
Y
FP
I
4020-20
Revision 0
January 1995
-------
TABLE 7(cont)
COMPARISON OF D TECH PCB test kit WITH GC - Trial #3
SAMPLE
W1A
W2A
W3A
W4A
W5A
W6A
W7A
W8A
W9A
W10A
W11A
W12A
W13A
W14A
W15A
W16A
W17A
W18A
W19A
W20A
W21A
W22A
W23A
D TECH
(ppm)
4.0-15
4.0-15
1.0-4.0
4.0-15
>50
>50
>50
4.0-15
1.0-4.0
0.5-1.0
15-50
15-50
15-50
4.0-15
1.0-4.0
1.0-4.0
4.0-15
1.0-4.0
4.0-15
>50
>50
1.0-4.0
>50
GC
(ppm)
9.1
11
2.8
13
29
1200
57
18
1.3
0.44
120
48
19
2.7
1.3
0.3
1.4
2.2
8.2
9.3
110
L 0.6
46
AGREEMENT
Y, FN, FP
Y
Y
Y
Y
FP
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
FP
FP
Y
Y
FP
Y
Y
Y
4020-21
Revision 0
January 1995
-------
Table 8
Intraassay Precision of the PCB RISc™ Liquid Waste Test System
PCB 1248 Spike
Concentration
(ppm)
0
0.2
5
Signal %RSD
(OD450nJ N=44
(11 data sets)
6.4%
5.9%
7.9%
Statistical Percentage of
False Results Compared to
Standards
<0.02%
4.1%
1.4%
Table 9
Interassay Precision of the PCB RISc™ Liquid
Waste Test System
PCB 1248 Spike
Concentration (ppm)
0
0.2
5
Signal %RSD
(OD450nm) N=44
(11 data sets)
6.4%
8.3%
8.5%
4020-22
Revision .0
January 1995
-------
Table 10
Comparison of PCB RISc1" Liquid Waste Test with Method 8082
Sample
ID
302
303
304
306
307
308
310
311
331
380
381
382
383
384
385
387
388
389
390
391
394
395
396
398
399
400
401
402
403
404
Sample Matrix
Condensate '
Condensate
Condensate
Condensate
Condensate
Condensate
Condensate
Condensate
Transformer Oil
Transformer Oil
Transformer Oil
Transformer Oil
Transformer Oil
Transformer Oil
Transformer Oil
Coolant
2,4-D Rinse Water
Waste Solvent
Herbicide
Paint/Solvent
Waste Solvent
Waste Solvent
Waste Oil
Chlor. Solvent
Paint
Pump Oil
Waste Solvent
Herbicide
Paint/Solvent
Printing Solvent
GC Results
Aroclor
NDb
NO
1242
1242
1242
1242
1254
1242
1260
PCBC
PCB
PCB
PCB
PCB
PCB
PCB
1254
1242
ND
1254
1242/1260
1242/1260
1260
NO
NO
NO
ND
ND
ND
ND
Cone, ppm
ND
ND
25
5
<10
58
25
200
183
20
38
163
176
336
6400
10
<10
29
<2
9
11/17
III
323
<5
<50
<50
<35
<50
<5
<5
IA Results
Test
Results
<5
<5
>5
>5
<5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
<5
>5
<5
>5
>5
<5
>5
<5
<5
<5
<5
<5
<5
<5
Corr.
with GC
Results
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
4020-23
Revision 0.
January 1995
-------
Table 10 (continued)
Sampl e
ID
405
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
Sample Matrix
Waste Solvent
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
GC Results
Aroclor
ND
ND
ND
ND
ND
ND
ND
ND
Waste Oil ! ND
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
Waste Oil
ND
PCB
ND
ND
ND
ND
ND
ND
ND
ND
ND
Cone, ppm
IA Results
Test
Results
<50 1 <5
ND
ND
ND
ND
ND
ND
ND
ND
ND
50
ND
ND
ND
ND
ND
ND
ND
ND
ND
Number of False Positive Results
Rate
Number of False Negative Results
Rate
>5
<5
<5
<5
<5
<5
<5
<5
<5
>5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Corr.
with GC
Results
yes
Fpd
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
1/32
3.1%
0/18
0.0%
" Trial 1 data
b ND = Not Detectable
c PCB = Aroclor was not determined
d FP = False positive
4020-24
Revision 0
January 1995
-------
Table 11
Correlation of PCB RISc™ Liquid Waste Test and Method 8082 Results
Using Spiked and Unspiked Liquid Waste Field Samples
ID
001
002
003
004
005
006
007
008
009
010
on
012
013
014
015
016
017
018
019
020
021
022
023
024
025
026
027
Matrix
Aromatic solvent
Aviation gas
Chiller oil
Compressor oil
Coolant + water
Coolant oil
Coolant oil
Cutting oil
Cutting oil
Degreaser still
bottom
Dope oil
Draw Lube oil
Fleet crankcase
oil
Floor sealer
Fuel oil
Hi-BTU oil
Honing oil
Hydraulic oil
Hydraulic oil
Hydraulic oil
Machine oil
Mineral oil
Mineral spirits
Mineral spirits +
ink
Mixed flammables
Mixed solvents
Naphtha
GC
Results
Unspiked
opm
<5
<5
<5
<5
<5
NRb
NR
<5
<5
.<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
NR
<5
<5
<5
<5
<5
<5
Immunoassay Result
Unspiked
ppm
<5
<5
<5
<5
<5
NR
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
>5
<5
<5
<5
Spiked (5
ppm 1248).
>B
>5
>5
>5 .
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
25
NR
>5
>5
>5
>5
>5
>5
Interp.
FP
4020-25
Revision 0
January 1995
-------
Table 11 (continued)
ID
028
029
030
031
032
033
034
035
036
037
038
039
040
041
042
043
044
045
046
047
048
049
050
051
052
053
054
055
Matrix
Oil
Oil
Oil
Oil
Oil
Oil
Oil + 1,1,1-
trichloroethane
Oil sludge
Oil + freon
Oil + mineral
spirits
Oil + scum
solution
Oily water
Paint thinner
Paint thinner
Paint thinner
Paint waste
Paint waste +
thinner
Perce + oil
Petroleum
distillates
Petroleum naphtha
Pumping oil
RAC-1 SKOS
Sk oil
Sk oil
Smog Hog
Toluene + hexane
Toluene + stain
1,1,1-
Trichloroethane
GC
Results
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
NR
<5
<5
<5
<5
<5
Immunoassay Result
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
>5
<5
<5
<5
<5
<5
*i
<5
<5
<5
<5
• >5
<5
<5
<5
<5
<5
<5
<5
<5
>5
Spiked (5
ppm 1248)
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
Interp.
FP
FP
FP
4020-26
Revision 0
January 1995
-------
Table 11 (continued)
ID
056
057
058
059
060
061
062
063
064
065
066
067
068
069
070
071
072
073
074
075
076
077
078
079
080
081
082
Matrix
1,1,1-
Trichloroethane
1,1,1-
Trichloroethane
1,1,1-
Trichloroethane
1,1,1-TCE +
methanol
Trichloroethylene
Trichloroethylene
Tr i chl oroethyl ene
Turpentine
Used n-
butyl acetate
Used oil + freon
Used oil + freon
Used oils
Used petroleum
Used petroleum
Used synthetic oil
Varnish + stain
Varsol
Waste coolant +
oil
Waste ink +
solvent
Waste naphtha
Waste oil
Waste oil
Waste oil
Waste oil
Waste oil
Waste oil
Waste oil
GC
Results
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Immunoassay Result
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
• <5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Spiked (5
ppm 1248)
>5
25 •
>5
>5
>5 '
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
Interp.
4020-27
Revision 0
January 1995
-------
Table 11 (continued)
ID
083
084
085
086
087
088
089
090
091
092
093
094
095
096
097
098
099
100
Matrix
Waste oil
Waste oil
Waste oil +
kerosene
Waste oil + gas
Waste paint
Waste paint
Waste paint
Waste paint
Waste paint
Waste paint
Waste SC-49
solvent
Waste solvent
Waste stoddard
Waste toner
Waste tramp oil
Waste transmission
fluid
Xylene
Not Recorded
No. of False Positive
Results
Rate
No. of False Negative
Results
Rate
GC
Results
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Immunoassay Result
Unspiked
ppm
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
>5
<5
6/99
6.1%
Spiked (5
ppm 1248)
25
>5
>5
>5
>5
>5
25
>5
>5
>5
>5
>5
>5
^5
>5
>5
>5
NR
Interp.
FP
FP
0.98
0.0%
a Trial 2 data
b NR = not run
4020-28
Revision 0
January 1995
-------
METHOD 4030
SOIL SCREENING FOR PETROLEUM HYDROCARBONS BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4030 is a procedure for screening soils to determine whether
total petroleum hydrocarbons (TPH) are likely to be present. Depending on the
testing product selected, samples may be used to locate samples with low (<40-100
ppm), medium, and high (>1000 ppm) concentrations of contaminates, or the
determineif TPH is present at concentrations above 5, 25, 100, or 500 mg/kg.
Method 4030 provides an estimate for the concentration of TPH by comparison
against standards, and can be used to produce multiple results within an hour of
sampling.
1.2 Using the test kit from which this method was developed, 95 % of
samples containing 25 ppm or less of TPH will produce a negative result in the
100 ppm test configuration.
1.3 The sensitivity of any immunoassay test depends on the binding of the
target analyte to the antibodies used in the kit. The testing product used to
develop this method is most sensitive to the small aromatic compounds (e.g.,
ethylbenzene, xylenes, and naphthalene) found in fuels. Refer to the package
insert of the testing product selected for specific information about
sensitivity.
1.4 The sensitivity of the test is influenced by the nature of the
hydrocarbon contamination and any degradation processes operating at a site.
Although the action level of the test may vary from site to site, the test should
produce internally consistent results at a particular site.
1.5 In cases where a more exact measurement of TPH concentration is
required, additional techniques (i.e., gas chromatography Method 8015 or infra-
red spectroscopy Method 8440) should be used.
1.6 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using an extract of a soil
sample. Filtered extracts may be stored cold, in the dark. An aliquot of the
extract and an enzyme-TPH conjugate reagent are added to immobilized TPH
antibody. The enzyme-TPH conjugate "competes" with hydrocarbons present in the
4030-1 Revision 0
January 1995
-------
sample for binding to immobilized anti-TPH antibody. The test is interpreted by
comparing the respone produced by a sample to the response produced by a
reference reaction.
3.0 INTERFERENCES
3.1 Compounds that are chemically similar to petroleum hydrocarbons may
cause a positive test (false positive) for TPH. The data for the lower limit of
detection of these compounds are provided in Tables 1A and IB. Consult the
information provided by the manufacturer of the kit used for additional
information regarding cross reactivity with other compounds.
3.2 Storage and use temperatures may modify the method performance.
Follow the manufacturer's directions for storage and use.
3.3 Appropriate standards must be used (i.e. , diesel standards for diesel
analysis, JP-4 for analysis of JP-4, etc.), or excessive false negative or false
positive rates may result.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: PETRO RISc Soil Test (EnSys, Inc.), EnviroGard™
Petroleum Fuels in Soil, (Mi Hi pore, Inc.), or equivalent. Each commercially
available test kit will supply or specify the apparatus and materials necessary
for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for auccessful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-12.
4030-2 Revision 0
January 1995
-------
7.2 Appropriate standards must be used to prevent excessive rates of
false negative or false positive results.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4030 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 A single laboratory study was conducted with the PETRO RISc Soil Test,
EnSys, Inc., using five contaminated soil samples. The samples were contaminated
with oxygenated gasoline, oxygenated gasoline 24 hours after contamination, low
aromatic diesel (purchased in California), normal diesel (purchased in Northern
Virginia), and JP-4 jet fuel. Five replicate determinations were made using the
kits, and the data compared with values obtained using GC/FID (Method 8015) and
IR (Method 8440). Several different analysts ran the immunoassay analyses.
Samples two- to five-fold below the action level generally gave readings less
than the action level. Samples two fold above the action level gave readings
greater than the action level. Samples at or near the action level give mixed
results (e.g., both less than and greater than the action level). Tables 2-6
summarize these results.
9.2 Sensitivity of the EnviroGard Petroleum Fuels in Soil Test Kit was
determined by establishing the "noise" level expected from matrix effects
encountered in negative soil samples and determining the corresponding TPH
concentration by comparison to the analyte-specific response curve. 8 different
soils which did not contain TPH were assayed. Each of these soils was extracted
in triplicate and each extract was assayed in three different assays. The mean
and the standard deviation of the resulting %Bo's (%Bo = [(ODsample/ODneoative
control)xlOO]) were calculated and the sensitivity was estimated at two standard
deviations below the mean. The sensitivity for Method 4030 was determined to be
4030-3 Revision .0
January 1995
-------
80% Bo at a 95% confidence inteval. Based on the average assay response to home
heating oil (HHO), this corresponds to 5.8 ppm. These data are shown in Table
7.
9.3 The effect of water content of the soil samples was determined by
assaying three different soil samples which had been dried and subsequently had
water added to 30% (w/w). Aliquots of these samples were then fortified with
HHO. Each soil sample was assayed three times, with and without added water, and
with and without HHO fortification. It was determined that water in soil up to
30% had no detectable effect on the method. These data are shown in Table 8.
9.4 The effect of the pH of the soil extract was determined by adjusting
the soil pH of three soil samples. Soil samples were adjusted to pH 2 - 4 using
6N HC1 and pH 10 - 12 using 6N NaOH. Aliquots of the pH adjusted soil samples
were fortified with home heating oil. Each soil sample was assayed unadjusted
and with pH adjusted to 2-4 and 10-12, both unfortified and fortified. These
extracts were assayed three times. It was determined that soil samples with pH
ranging from 2 to 12 had no detectable effect on the performance of the method.
These data are shown in Table 9.
9.5 Two field studies were conducted at contaminated sites using the PETRO
RISc Soil Test, EnSys, Inc.. In Field Trial 1, the method was used to locate
soil contamination resulting from a leaking above ground gasoline tank. In Field
Trial 2, the method was used to evaluate diesel fuel contamination in a railroad
contaminated soil, sludge, and wastewater impound. Overall, a high degree of
correlation was observed between the standard method and the immunoassay method.
The application of the immunoassay method to 23 samples (46 analyses) resulted
in eight conclusive false positive results (17%) and three conclusive false
negative results (7%). Tables 10 and 11 summarize these results. There was
agreement for 76% of the samples tested in the two trials for which data are
presented.
9.6 Two field trials were undertaken to investigate the ability of the
EnviroGard Petroleum Fuels in Soil Test Kit to identify soil samples which were
contaminated with TPH. In trial 1 the method was used to identify soil which was
contaminated with gasoline from, leaking underground storage tanks. The
immunoassay was compared to Method 8015. Twenty samples were analyzed by both
methods. Interpreting the results at a cutoff of 100 ppm resulted in 1/20 (5%)
false negatives and 0/20 (0%) false positives. In trial 2, the method was used
to identify soil which was contaminated with JP-4 jet fuel from leaking
semi-submerged storage tanks. The immunoassay was compared to Method 8015. Ten
samples were analyzed by both methods. Interpreting the results at 1,000 ppm
resulted in 0/10 (0%) false negatives and 1/10 (10%) false positives. Overall,
for both field trials, there were 1/30 (3.3%) false negatives and 1/30 (3.3%)
false positives. These data are summarized in Table 12.
10.0 REFERENCES
1. PETRO RISc™ Users Guide, Ensys Inc.
4030-4 Revision 0
January 1995
-------
Marsden, P.J., S-F Tsang, and N. Chau, "Evaluation of the PETRO RISc™ kit
Immimoassay Screen Test System", Science Applications International
Corporation under contract to EnSys Inc., June 1992, unpublished
EnviroGard™ Petroleum Fuels in Soil Test Kit Guide, Millipore, Inc.
4030-5 Revision 0.
January 1995
-------
Table 1A
CROSS REACTIVITY"
Compound
Gasoline
Diesel fuel, regular #2
Jet A fuel
Kerosene
Fuel oil #2
Mineral Spirits
Light lubricating oil
Lithium grease
Brake fluid
Chain lubricant
Toluene
o-Xylene
m-Xylene
p-Xylene
Ethyl benzene
Hexachl orobenzene
Trichloroethylene
Acenaphthene
Naphthalene
Creosote
2-Methylpentane
Hexanes, mixed
Heptane
iso-Octane
Undecane
Soil Equivalent Concentration (ppm)
Required to Yield a Positive Result
100
75 .
75
100
100
<30 .
7,000
10,000
>10,000
>10,000
200
50
100
300
50
<30
1,000
<30
<30
<30
150
250
300
30
>10,000
a PETRO RISc Soil Test, EnSys, Inc.
4030-6
Revision 0
January 1995
-------
TABLE IB
CROSS REACTIVITY'
Compound
1,2,4 - Trimethyl benzene
m - Xylene
Acenaphthylene
Acenapthene
p - Xylene
Naphthalene
1,3,5 - Trimethyl benzene
Fluorene
Phenanthrene
o - Xylene
Ethyl benzene
Toluene
Propyl benzene
Chlordane
Benzene
Toxaphene
Concentration Required
Positive Interpretation
for
(ppm)
0.1
0.3
0.3
0.4
0.5
0.7
2
2
2
3
5
7
11
45
70
70
The following compounds were tested and found to yield negative results
for concentrations up to 1000 ppm:
PCB (Aroclor 1248) TNT
Pentachlorophenol DDT
EnviroGard1" Petroleum Fuels in Soil, Millipore, Inc.
4030-7
Revision 0
January 1995
-------
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Soil*
Part 1
Soil Type
TABLE 7
METHOD SENSITIVITY
Average Response with Negative Soils
Average %Bo (n = 9)Standard Deviation
SAND
S2
S3
S4
S5
S6
S7
S8
91.4
LOAM
CLAY
LOAM
CLAY
LOAM/SAND
SAND/ LOAM
LOAM
4.1
83.1
84.4
80.9
89.7
91.2
89.0
90.0
3.2
3.1
1.3
1.7
0.2
0.3
1.4
AVERAGE
87.5
4.0
Part 2 - Average Response with Calibrators
Average %Bo
Calibrator
Concentration (ppm) Average Absorbance
0
5
15
50
125
1.339
1.097
0.825
0.427
0.219
N/A
81.9
61.7
31.9
16.3
Part 3 - Method Sensitivity
Based on Part 1 and Part 2 Above:
Average %Bo - 2 SD = 79.6 which is equivalent to 5.8 ppm
Average %Bo - 3 SD = 75.6 which is equivalent to 7.0 ppm
(Wo = [(OD8ample/ODneortivecontrJxlOO])
4030-13
Revision 0
January 1995
-------
TABLE 8
EFFECT OF WATER CONTENT IN SOIL SAMPLES
Soil % Water Fortified? Rep. 1* Rep. 2 Rep. 3 Mean Std. Dev. ± 2 SD Range
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
0
30
0
30
0
30
0
30
0
30
0
30
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
101
100
59
60
57
74
40
44
70
81
41
61
.3
.5
.2
.9
.5
.3
.5
.1
.5
.1
.3
99.1
115.5
65.8
74.7
53.9
91.8
40.9
67.8
85.6
109.4
46.6
76.7
111.8
109.1
69.6
83.1
72.3
85.2
45.6
68.4
76.7
103.4
60.7
63.1
104.1
108.4
64.9
72.3
61.4
83.8
42.3
60.2
77.5
98.1
49.5
67.0
6.8
7.5
5.3
11.7
9.7
8.7
2.9
13.6
7.8
14.7
10.1
8.4
90
93
49
49
42
66
36
33
61
68
29
50
.4 -
.4 -
.9 -
.2 -
.0 -
.4 -
.5 -
.0 -
.9 -
.7 -
.3 -
.2 -
117.7
123.4
75.5
96.0
80.8
101.2
48.1
87.4
93.1
127.5
69.7
83.8
* All values shown are %Bo [= (OD88mpiyODneQ8tivecontrol)xlOO]
4030-14
Revision 0
January 1995
-------
TABLE 9
EFFECT OF pH ON SOIL SAMPLES
oil
SI
SI
SI
SI
SI
SI
S2
S2
S2
S2
S2
S2
S3
S3
S3
S3
S3
S3
DH Adj.
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
• Basic
Fortified?
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
Reo.l*
88.9
106.9
101.2
64.3
52.9
69.3
76.2
101.2
89.9
59.4
68.1
47.8
83.4
89.3
80.6
60.2
58.8
53.4
Rep. 2
93.2
66.0
90.3
55.7
41.1
61.7
86.4
82.4
72.1
• 60.3
62.3
51.7
88.4
84.9
84.2
53.6
58.5
41.8
Rep. 3
92.8
88.1
90.6
58.0
49.4
57.5
83.1
99.5
77.7
53.7
59.3
39.4
85.3
91.0
90.3
58.8
62.0
59.9
Mean
91.6
87.7
94.0
59.3
47.8
62.8
81.9
94.4
79.9
57.8
63.2
46.3
85.7
88.4
85.0
57.5
59.8
.51.7
Std.
2
21
6
4
6
6
5
10
9
3
4
6
2
3
4
3
1
9
Dev.
.4
.5
.2
.5
.1
.0
.2
.4
.1
.6
.5
.3
.5
.1
.9
.5
.9
.2
± 2
86.
.44.
81.
50.
35.
50.
71.
73.
61.
50.
54.
33.
80.
82.
75.
47.
56.
33.
SD
8 -
7-
6 -
3 -
6 -
8 -
5 -
6 -
7 -
6 -
2 -
7 -
7 -
2 -
2 -
7 -
0 -
3 -
Range
96.4
109.2
106.4
68.3
60.0
74.8
92.3
115.2
98.1
65.0
72.2
58.9
90.7
94.6
94.8
64.5
63.6
70.1
* All values shown are %Bo [= (OD8amplyODneoirtivecontrol)xlOO]
4030-15
Revision 0
January 1995
-------
Table 10
PETRO RISC Soil Test
Field "Trial 1
Sample ID
AST-01
AST-02
AST -03
AST-04
AST-05
AST-06
AST -07
AST-08
AST-09
IR Method (ppm)
<20
520
1700
130
20
40
400
640
1600
100 ppm Test
Result
< 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
AGREEMENT'
Y, FP, FN
Y
Y
Y
Y
FP
FP
Y
Y
Y
1000 ppm Test
Result
< 1000
> 1000
> 1000
< 1000
< 1000
< 1000
< 1000
< 1000
> 1000
AGREEMENT"
Y, FP, FN
Y
FP
Y
Y
Y
FN
FN
FN
Y
Y Immunoassay and GC or IR results agree
FP False Positive
FN False Negative
4030-16
Revision 0
January 1995
-------
Table 11
PETRO RISC Soil Test
Field Trial 2
Sample
ID
1-B
2 -A
2-B
2-C
3-B
3-C
4-A
4-B
5-A
5-B
5-C
6-B
8
9
GC
Extractables
(ppm)
5720
610
370
2270
4870
760
66
303
20400
26300
267
550
59300
26500 .
TRPH
(ppm)
20800
14700
6800
1950
18600
1180
4100
2100.
29600
28600
330
22700
64400
12900
75 ppm Test
Result
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
> 75
AGREEMENT"
Y, FP, FN
TRPH
Y
Y
Y
Y
Y
Y
FP"
Y
Y
Y
Y
Y
Y
Y
GC
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
750 ppm Test
Result
> 750
> 750
> 750
> 750
> 750
< 750
< 750
< 750
> 750
> 750
> 750
> 750
*> 750
> 750
AGREEMENT8
Y, FP, FN
TRPH
Y
FP
FP
Y
Y
FN"
Y
Y
Y
Y
FP
FP
Y
Y
GC
Y
Y
Y
Y
Y
FN
FN
FN
Y
Y
FP
Y
Y
Y
Y Immunoassay and GC or IR results agree
FN False Negative
FP False Positive
FN" False Negative, but within 25% of GC or IR results
FP" False Positive, but within 25% of GC or IR results
4030-17
Revision 0.
January 1995
-------
TABLE 12
IMMUNOASSAY COMPARED TO METHOD 8015
Field Trial 1: Gasoline (Interpretation at 100 ppm)
Sample ID
MW-18-1
MW-18-2
MW-18-3
MW-18-A1
MW-18-A1 Duplicate
MW-18-A2
DBS
MW-12-3
MW-13-1
MW-13-3
MW-13-4
MW-17-3
MW-17-4
MW-17-5
MW-16-2
MW-16-2 Duplicate
MW-19-2
MW-19-3
MW-14-1
MW-17-A1
Method 8015 (ppml
270
15
15
20
15
1500
300
250
40
50
20
250
180
180
11,500
11,500
10
70
280
560
Field Trial 2: JP-4 Jet Fuel
Sample ID
TB1 6.5-7.0
TB2 6.5-7.0
TB1 5.0-5.5
TB2 5.0-5.5
TBS 5.0-5.5
TBS 6.5-7.0
TBS 5.0-5.5
TB5 6.5-7.0
TB4 6.5-7.0
TB4 5.5-6.0
Method 8015 (ppm)
15,900
16,800
900
100
ND(<5)
29,500
5,000
2,000
19,000
5,900
Immunoassav
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Positive
(Interpretation at
Immunoassav
Positive
Positive
Negative
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Concurrence?
False Negative
YES
YES
' YES
YES
YES
YES
YES
YES
. YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
1,000 ppm)
Concurrence?
YES
YES
YES
False Positive
YES
YES
YES
YES
YES
YES
4030-18
Revision 0
January 1995
-------
METHOD 4040
SOIL SCREENING FOR TOXAPHENE BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4040 is a procedure for screening soils to determine whether
toxaphene (CAS Registry 8001-35-2) is present at concentrations above 0.5 /tg/g.
Method 4040 provides an estimate for the concentration of toxaphene by comparison
against standards.
1.2 In cases where the exact concentration of toxaphene is required,
additional techniques (i.e., gas chromatography (Method 8081) or gas
chromatography/mass spectrometry (Method 8270)) should be used.
1.3 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using an extract of a soil
sample. Filtered extracts may be stored cold, in the dark. An aliquot of the
extract and an enzyme-toxaphene conjugate reagent are added to immobilized
toxaphene antibody. The enzyme-toxaphene conjugate "competes" with toxaphene
present in the sample for binding to immobilized toxaphene antibody. The
enzyme-toxaphene conjugate bound to the toxaphene antibody then catalyzes a
colorless substrate to a colored product. The test is interpreted by comparing
the color produced by a sample to the response produced by a reference reaction.
3.0 INTERFERENCES
3.1 Compounds that are chemically similar may cause a positive test
(false positive) for toxaphene. The test kit used to develop this method was
evaluated for interferences, and found to be relatively insensitive to other
organochlorine pesticides (e.g., Lindane, DDT and DDE). The data for the lower
limit of detection of these compounds are provided in Table 1. Consult the
information provided by the manufacturer of the kit used . for additional
information regarding cross reactivity with other compounds.
3.2 Storage and use temperatures may modify the method performance.
Follow the manufacturer's directions for storage and use.
4040-1 Revision 0
January 1995
-------
4.0 APPARATUS AND MATERIALS 4
4.1 Immunoassay test kit: EnviroGard™ Toxaphene in Soil (Millipore,
Inc.), or equivalent. Each commercially available test kit will supply or
specify the apparatus and materials necessary for successful completion of the
test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soils samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-5.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4040 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
4040-2 Revision 0
January 1995
-------
9.0 METHOD PERFORMANCE
9.1 A single laboratory study was conducted using five spiked soil
samples and 30 real-world soils contaminated with Toxaphene. Results using the
immunoassay kit were compared with results obtained using Method 3540/8081.
Table 2 presents the data from this study. These data show a positive bias in
the immunoassay of almost 18%, compared to a 14% negative bias in Method
3540/8081.
9.2 Table 3 provides the results of single determinations in soil from
New Mexico.
9.3 Tables 8 and 9 provide data on the precision of Method 4040.
10.0 REFERENCES
1. EnviroGard™ Toxaphene Soil Test Kit Guide, Millipore, Inc.
2. Marsden, P.O., S-F Tsang, V. Frank, N. Chau, and M. Roby "Comparison of the
Millipore Immunoassay for Toxaphene with Soxhlet Extraction and Method 8081",
Science Applications International Corporation, under contract to Millipore
Inc., May 1994, unpublished.
4040-3 Revision 0
January 1995
-------
TABLE 1. POSSIBLE SOIL INTERFERENCES (a)
Compound
Diesel fuel
Endrin
Endosulfan I
Endosulfan II
Dieldrin
Heptachlor
Aldrin
technical Chlordane
gamma -6HC (Lindane)
alpha-BHC
delta-BHC
Soil Equivalent Concentration j*g/kg
. (ppb) Required to Yield a Positive
Result
45000
6
6
6
6
6
20
14
300
1000
1000
The following compounds were found to yield.a negative result for concentrations
up to 200,000 fig/kg:
Gasoline
Pentachlorophenol
DDT
PCB (Aroclor 1248)
Trinitrotoluene
(a) Millipore, Inc. product literature
Table 2
Comparison of Method 4040 and Method 3540/8081
Spike
Concentration
Ug/g)
0.25
0.50
1.0
2.5
5.0
EnviroGard™ Toxaphene in Soil
Results
(^g/g)
0.27
0.66
1.02
2.8
6.7
Average
% Recovery
108
132
102
112
134
117.6
Method 3540/Method 8081
Results
Ug/g)
0.19
0.33
0.83
2.9
5.5
% Recovery
75
66
84
116
110
85.6
4040-4
Revision Q
January 1995
-------
Table 3.
Results for New Mexico Soil Samples
Sample ID
Lab 1
Ml
M2
M3
M4
M5*
M6*
M7
LMS12
M8*
M9
M10
Mil*
Ml 2*
M13*
M14
M15*
M16
M16-MS
M16-MSD
M17
Lab 2
28,89
70,104
54,89
103,50
10,30
45,33
Nazilini
soil #12
0,33
23,104
78,33
64,5
53,75
33,75
17,75
65,33
82,75
82,75
82,75
19,50
Toxaphene
Method 3540/8081
(Lab 1)
0.09 J
0.04 J
0.04 J
0.01 J
40
19.3
<0.50
<0.50
0.26 J
1.0
0.14 J
0.27 J
27.2
0.14 J
0.48 J
0.21 J
6.0
6.0
4.8
M9/9)
Method 4040
(Lab 2)
0.3
0.9
0.8
0.2
58
54.8
0.2
1.7
1.1
2.6
2.1
11
42
0.9
2.8
1.8
NA
NA
6
AGREEMENT"
Y, FN, FP
FP
FP
FP
FP
Y
FP
Y
FP
FP
FP
FP
FP
Y
FP
FP
FP
-
-
Y
4040-5
Revision 0
January 1995
-------
Table 3. (continued)
Sample ID
Lab 1
M18
M19
M20
M21
M22
M23*
M24*
M24-MS
M24-MSD
M25*
M26
M27
M28
M29
M30*
M31
Lab 2
97,104
48,104
0,50
102,75
84,50
25,33
0,75
0,75
0,75
12,40^it
0,89
0,104
98,89
104,33
76,89
40,50
Toxaphene (jig/g)
Method 3540/8081
(Lab 1)
0.049 J
0.054 J
1.3
0.15 J
0.058 J
89.6
0.5
3.7
3.6
35.6
0.16 J
0.88
0.41 J
0.30 J
0.10 J
323
Method 4040
(Lab 2)
0.6
1.1
2.3
0.3
0.9
130
1.9
NA
NA
45.5
6.9
2.1
3.4
0.7
5.8
460
AGREEMENT"
Y, FN, FP
FP
FP
Y
Y
FP
Y
FP
-
-
Y
FP
FP
FP
FP
FP
Y
NA = not analyzed
J = an estimate value. This is used to indicate the result is less than the
sample quantitation limit but greater than zero.
* DDE identified using GC/MS analyses
4040-6
Revision 0
January 1995
-------
Table 4
Optical Measurement Precision for Spiked Samples
Spike Level
(ppm)
0.40
4.0
0.40
4.0
0.40
4.0
Mean O.D.
(450 nm)
0.798
0.450
0.753
0.397
0.713
0.385
Percent RSD
5.6
9.7
9.0
4.7
6.6
7.8
n
8
8
8
8
8
8
O.D.
(2 ppm spike)
0.540
0.540
0.501
0.501
0.541
0.541
Table 5
Recovery and Precision of Three Types of Spiked Soils
Spike Cone, (ppm)
1.0
10.0
1.0
10.0
1.0
10.0
Mean percent
recovery
91.1
96.9
84.1
89.4
122.4
101.7
Percent RSD
20.0
10.4
14.6
4.2
8.8
2.0
n
3
3
3
•3
3
3
overall percent recovery (n = 9), 1 ppm = 99 + 16%
overall percent recovery (n = 9), 10 ppm= 96 + 5%
4040-7
Revision 0
January 1995
-------
METHOD 4041
SOIL SCREENING FOR CHLORDANE BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4041 is a procedure for screening soils to determine whether
chlordane (CAS Registry 57-74-9) is present at concentrations above 20, 100 or
600 /zg/kg. Method 4041 provides an estimate for the concentration of chlordane
by comparison against standards.
1.2 In cases where the exact concentration of chlordane is required,
additional techniques [i.e., gas chromatography (Method 8081) or gas
chromatography/mass spectrometry (Method 8270)] should be used.
1.3 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using an extract of a soil
sample. Filtered extracts may be stored cold, in the dark. An aliquot of the
extract and an enzyme-chlordane conjugate reagent are added to immobilized
chlordane antibody. The enzyme-chlordane conjugate "competes" with chlordane
present in the sample for binding to chlordane antibody. The enzyme-chlordane
conjugate bound to the chlordane antibody then catalyzes a colorless substrate
to a colored product. The test is interpreted by comparing the color produced
by a sample to the response produced by a reference reaction.
3..0 INTERFERENCES
3.1 Compounds that are chemically similar may cause a positive test
(false positive) for chlordane. The test kit used to develop this method was
evaluated for interferences. The data for the lower limit of detection of these
compounds are provided in Table 1. Consult the information provided by the
manufacturer of the kit used for additional information regarding cross
reactivity with other compounds.
3.2 Storage and use temperatures may modify the meth.od performance.
Follow the manufacturer's directions for storage and use.
4041-1 Revision 0
January 1995
-------
4.0 APPARATUS AND MATERIALS A
4.1 Immunoassay test kit: EnviroGard1" Chlordane in Soil (Millipore,
Inc.), or equivalent. Each commercially available test kit will supply or
specify the apparatus and materials necessary for successful completion of the
test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 2-5.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4041 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
4041-2 Revision 0
January 1995
-------
9,0 METHOD PERFORMANCE
9.1 Method sensitivity was determined by establishing the "noise" level
expected from matrix effects encountered in negative soil samples and determining
the corresponding Chlordane concentration by comparison to the analyte-specific
response curve. 8 different soils which did not contain Chlordane were assayed.
Each of these soils was extracted in triplicate and each extract was assayed in
three different assays. The mean and the standard deviation of the resulting
%Bo's (%Bo = [(OD.ampiyODne0(rtivecontr?()xlOO]) was calculated and the sensitivity was
estimated at two standard deviations below the mean. The sensitivity for Method
4041 was determined to be 80% Bo at a 95% confidence interval. Based on the
average assay response to Chlordane, this corresponds to 6.4 peg/kg Chlordane.
These data are shown in Table 2.
9.2 The effect of water content of the soil samples was determined by
assaying three different soil samples which had been dried and subsequently had
water added to 30% (w/w). Aliquots of these samples were then fortified with
Chlordane (100 /xg/kg). Each soil sample was assayed three times, with and
without added water, and with and without Chlordane fortification. It was
determined that water in soil up to 30% had no detectable effect on the method.
These data are shown in Table 3.
9.3 The effect of the pH of the soil extract was determined by adjusting
the soil pH of three soil samples. Soil samples were adjusted to pH 2 - 4 using
6N HC1 and pH 10 - 12 using 6N NaOH. Aliquots of the pH adjusted soil samples
were fortified with Chlordane (100 /*g/kg). Each soil sample was assayed
unadjusted and with pH adjusted to 2-4 and 10-12, both unfortified and fortified.
It was determined that soil samples with pH ranging from 3 to 11 had no
detectable effect on the performance of the method. These data are shown in
Table 4.
9.4 A field trial was undertaken to evaluate to ability of the
EnviroGard™ Chlordane in Soil Test Kit to identify chlordane contaminated soil
at a remediation site. A total of 32 soil samples were evaluated by both Method
4041 and Method 8080. Interpretation of the results at a 1 mg/kg cutoff resulted
in 2/32 (6.3%) false negatives and 0/32 (0%) false positives. Interpretation of
the results at a cutoff of 10 mg/kg resulted in 0/32 (0%) false negatives and
2/32 (6.3%) false positives. These data are shown in Table 5.
10.0 REFERENCES
1. EnviroGard™ Chlordane in Soil Test Kit Guide, Millipore, Inc.
4041-3 Revision 0
January 1995
-------
TABLE 1. CROSS REACTIVITY (a)
Compound
Chlordane
Endrin
Endosulfan I
Endosulfan II
Dieldrin
Heptachlor
Aldrin
Toxaphene
gamma -BHC (Llndane)
alpha-BHC
delta-BHC
Concentration Required for
Positive Interpretation (/*g/kg)
5
3
3
3
3
3
10
100
300
1000
1000
The following compounds were found to yield a negative result for
concentrations up to 200,000 /xg/kg:
Gasoline PCB (Aroclor 1248)
Pentachl orophenol Tri ni trotol uene
4041-4
Revision 0
January 1995
-------
TABLE 2
METHOD SENSITIVITY
Soil#
SI
S2
S3
S4
S5
S6
S7
S8
Part 1 -
Soil Type
LOAM/SAND
LOAM
CLAY
CLAY
CLAY
LOAM/SAND
SAND
SAND
Average Response with Negative Soils
Average %Bo (n = 8) Standard Deviation
92.8
86.2
85.5
95.4
83.9
88.5
81.4
95.8
2.0
1.0
8.8
1.1
2.6
1.8
2.7
0.8
AVERAGE
88.7
4.5
Part 2 - Average Response with Chlordane Calibrators
Chlordane
Concentration (/zg/kg)
Average Absorbance Average %Bo
0
5
25
125
500
1.043
0.882
0.598
0.322
0.159
N/A
84.4
57.2
30.8
15.2
Part 3 - Method Sensitivity
Based on Part 1 and Part 2 Above:
Average %Bo - 2 SO = 79.7 which is equivalent to 6.4 jt9/kg Chlordane
Average %Bo - 3 SD = 75.2 which is equivalent to 8.6 /*g/kg Chlordane
4041-5
Revision 0
January 1995
-------
TABLE 3
EFFECT OF WATER CONTENT IN SOIL SAMPLES
Soil %_
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
0
30
0
30
0
30
0
30
0
30
0
30
•tifi€
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
sd? Rep. 1
95.2
96.0
40.5
42.2
85.8
78.7
37.7
39.8
76.6
87.4
40.0
40.8
Reo. 2
101
99.2
38.5
43.0
87.1
84.9
39.5
38.8
76.6
88.7
40.2
37.1
Reo. 3
94.5
96.0
35.9
43.0
85.5
79.8
40.6
37.0
73.0
85.7
35.7
38.7
Mean
97.0
97.1
38.3
42.8
86.1
81.1
39.3
38.5
75.4
87.3
38.7
38.9
Std. Dev.
3.7
1.8
2.3
0.5
0.9
3.3
1.5
1.4
2.1
1.5
2.5
1.9
± 2 SD
89.6 -
93.5 -
33.7 -
41.8 -
84.3 -
74.5 -
36.3 -
35.7 -
71.2 -
84.3 -
33.7 -
35.1 -
Range
104
101
42.9
43.8
87.9
87.8
42.3
41.3
79.6
90.3
43.7
42.7
4041-6
Revision 0
January 1995
-------
I
TABLE 4
EFFECT OF pH OF SOIL SAMPLES
oil oH Adj. Fortified? Reo
SI
SI
SI
SI
SI
SI
S2
S2
S2
S2
S2
S2
S3
S3
S3
S3
S3
S3
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
97
97
107
43
43
44
87
94
89
43
44
42
72
85
89
44
40
40
. 1* Reo. 2
.5
.4
.6
.6
.8
.8
.2
.3
.9
.6
.3
.3
.3
.4
.5
.5
.6
87.8
114
114
47.5
51.8
50.8
86.3
108
99.3
48.9
55.9
59.2
74.5
105
83.8
49.5
. 52.1
37.1 .
Reo. 3
94.8
94.7
93.8
38.5
34.1
32.0
87.6
80.5
77.9
33.9
41.5
36.5
79.3
75.7
85.9
32.6
34.7
43.9
Mean
93.4
102
105
43.2
43.2
42.5
87.3
94.1
88.8
42.2
47.4
46.0
75.4
88.8
86.4
42.2
42.4
40.5
Std. Dev.
5.0
10.7
10.1
4.5
8.8
9.6
0.8
13.5
10.7
7.7
7.6
11.8
3.6
15.1
2.8
8.7
8.9
3.4
± 2 SD
83
80
84.
34
25
23
85
67
67
26
32
22
68
58
80
24
24
33
.4 -
.8 -
.7 -
.2 -
.6 -
.3 -
.7 -
.1 -
.4 -
.8 -
.2 -
.4 -
.2 -
.6 -
.8 -
.8 -
.6 -
-.7 -
Range
103
124
125
52.2
60.8
61.7
88.9
121
110
57.6
62.6
69.6
82.6
119
92.0
59.6
60.2
47.3
All values shown are %Bo [= (ODsampl(/ODn
iegative conti
rol)xlOO]
4041-7
Revision 0
January 1995
-------
TABLE 5
Correlation to Method 8081
Test Interpretation at 1 mg/kg
Sample ID Method 8081 (mg/kg)
Immunoassay (mg/kg) Results Agree?
co-ss-2
co-ss-3
co-ss-4
co-ss-5
co-ss-6
co-ss-7
co-ss-8
co-ss-9
co-ss-10
co-ss-13
co-ss-14
co-ss-15
co-ss-17
co-ss-20
co-ss-21
co-ss-22
co-ss-23
co-ss-24
co-ss-25
co-ss-26
co-ss-27
co-ss-28
co-ss-28-170
co-ss-29
co-ss-30
co-ss-31
co-ss-32
co-ss-33
co-ss-34
co-ss-35
co-ss-36
co-ss-41
45
4.9
25
1.4
2.7
2.5
<1.0
7.9
6.0
5.2
2.9
2.1
<1.0
2.8
51
1.4
9.6
2.6
14
1,8
2.9
4.2
5.9
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
NEGATIVE
YES
YES
YES
FALSE NEGATIVE
YES
YES
YES
YES
YES
YES
YES
YES
YES
FALSE NEGATIVE
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
4041-8
Revision 0
January 1995
-------
TABLE 5 (continued)
Correlation to Method 8081
Test Interpretation at 10 rag/kg
Sample ID Method 8081 (mg/kg)
Immunoassay (mg/kg) Results Agree?
co-ss-2
co-ss-3
co-ss-4
co-ss-5
co-ss-6
co-ss-7
co-ss-8
co-ss-9
co-ss-10
co-ss-13
co-ss-14
co-ss-15
co-ss-17
co-ss-20
co-ss-21
co-ss-22
co-ss-23
co-ss-24
co-ss-25
co-ss-26
co-ss-27
co-ss-28
co-ss-28-170
co-ss-29
co-ss-30
co-ss-31
co-ss-32
co-ss-33
co-ss-34
co-ss-35
co-ss-36
co-ss-41
45
4.9
25
1.4
2.7
2.5
<1.0
7.9
6.0
5.2
2.9
2.1
<1.0
2.8
51
1.4
9.6
2.6
14
1.8
2.9
4.2
5.9
POSITIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
YES
YES
YES
YES
YES
YES
YES
FALSE POSITIVE
FALSE POSITIVE
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
4041-9
Revision 0
January 1995
-------
METHOD 4042
SOIL SCREENING FOR DDT BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4042 is a procedure for screening soils to determine whether
l,l,l-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) (CAS Registry 50-29-3) and
its breakdown products (ODD) DDE, and DDA) are present at concentrations above
0.2, 1.0 or 10 mg/kg. Method 4042 provides an estimate for the sum of
concentrations of DDT and daughter compounds by comparison against standards.
1.2 In cases where the exact concentration of DDT is required, additional
techniques [i.e., gas chromatography (Method 8081) or gas chromatography/mass
spectrometry (Method 8270)] should be used.
1.3 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using an extract of a soil
sample. Filtered extracts may be stored cold, in the dark. An aliquot of the
extract and an enzyme-DDT conjugate reagent are added to immobilized DDT
antibody. The enzyme-DDT conjugate "competes" with DDT present in the sample for
binding to DDT antibody. The enzyme-DDT conjugate bound to the DDT antibody then
catalyzes a colorless substrate to a colored product. The test is interpreted
by comparing the color produced by a sample to the response produced by a
reference reaction.
3.0 INTERFERENCES
3.1 Compounds that are chemically similar may cause a positive test
(false positive) for DDT. The test kit used to develop this method was evaluated
for interferences. The data for the lower limit of detection of these compounds
are provided in Table 1. Consult the information provided by the manufacturer
of the kit used for additional information regarding cross reactivity with other
compounds.
3.2 Storage and use temperatures may modify the method performance.
Follow the manufacturer's directions for storage and use.
4042-1 Revision 0.
January 1995
-------
4.0 APPARATUS AND MATERIALS |
4.1 Immunoassay test kit: EnviroGard™ DDT in Soil (Millipore, Inc;), or
equivalent. Each commercially available test kit will supply or specify the
apparatus and materials necessary for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated -
in Tables 2-5. •
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Method 4000 and Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action .level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
8.6 Method 4042 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
4042-2 Revision 0
January 1995
-------
fe 9.0 METHOD PERFORMANCE
9.1 Method sensitivity was determined by establishing the "noise" level
expected from matrix effects encountered in negative soil samples and determining
the corresponding DDT concentration by comparison to the analyte-specific
response curve. 8 different soils which did not contain DDT were assayed. Each
of these soils was extracted in triplicate and each extract was assayed in three
different assays. The mean and the standard deviation of the resulting %Bo's
(%Bo = [(OD.amp(yODn^lltiv. ..^JxlOO]) were calculated and the sensitivity was
estimated at two standard deviations below the mean. The sensitivity for Method
4042 was determined to be 81.4% Bo at a 95% confidence inteval. Based on the
average assay response to DDT, this corresponds to 0.044 ppm DDT. These data are
shown in Table 2.
9.2 The effect of water content of the soil samples was determined by
assaying three different soil samples which had been dried and subsequently had
water added to 30% (w/w). Aliquots of these samples were then fortified with DDT
(1.0 mg/kg). Each soil sample was assayed three times, with and without added
water, and with and without DDT fortification. It was determined that water in
soil up to 30% had no detectable effect on the method. These"data are shown in
Table 3.
9.3 The effect of the pH of the soil extract was determined by adjusting
the soil pH of three soil samples. Soil samples were adjusted to pH 2 - 4 using
6N HC1 and pH 10 - 12 using 6N NaOH. Aliquots of the pH adjusted soil samples
•were fortified with DDT (1.0 mg/kg). Each soil sample was assayed unadjusted and
with pH adjusted to 2-4 and 10-12, both unfortified and fortified. It was
determined that soil samples with pH ranging from 3 to 11 had no detectable
effect on the performance of the method. These data are shown in Table 4.
9.4 A field study was conducted at a contaminated site using a
commercially available test kit (EnviroGard™ DDT in Soil Test Kit, Millipore
Corp.). The immunoassay was used to identify soil which had been contaminated
with DDT. The standard method (Method 8080) was performed at a certified
laboratory and the results were compared to the immunoassay. When interpreting
the results at a 0.2 ppm cutoff, the immunoassay yielded 0/32 (0%) false
negatives and 2/32 (6.3%) false positives. When interpreting the results at a
1.0 ppm cutoff, the immunoassay yielded 1/32 (3.1%) false negatives and 2/32
(6.3%) false positives. These data are shown in Table 5.
10.0 REFERENCES
1, EnviroGard™ DDT in Soil Test Kit Guide, Millipore, Inc.
4042-3 Revision 0
January 1995
-------
TABLE 1. CROSS REACTIVITY (a)
Compound
p,p'-DDT
p,p'-DDD
P,P'-DDE
ojj'-DDT
o,p'-DDD
o,p'-DDE
DDA
Chloropropylate
Chi orobenzi late
Dicofol
Chloroxuron
Monolinuron
Thiobencarb
Tebuconazole
Neburon
Tetradifon
Diclofop
PCB (Aroclor 1248)
Concentration Required for
Positive Interpretation (ppm)
0.04
0.01
0.18
4.0
0.4
3.0
0.002
0.007
0.03
0.14
24
25
5
7
17
1.2
70
90
The following analytes are not detected at or above 100 ppm:
2,4-D 4-chlorophenoxyacetic acid Chlordane
Pentachlorophenol Chlorbromuron Chlortoluron
Dicamba Diflubenzuron Diuron
Lindane Linuron MCPA acid
MCPB Mecoprop Gasoline
Diesel Fuel 2,4,6-Trinitrotoluene Toxaphene
4
4042-4
Revision -Q
January 1995
-------
TABLE 2
Method Sensitivity
Soil#
SI
S2
S3
S4
S5
S6
S7
S8
Part 1
Soil Type
LOAM
CLAY
SAND
LOAM
LOAM/SAND
CLAY
LOAM/SAND
SAND/LOAM
- Average Response with
Average %Bo (n = 9)
. 87.0
93.2
97.2
87.7
88.1
100.8
103.6
89.6
Negative Soils
Standard Deviation
7.5
2.3
2.6
1.2
2.3
2.1
0.3
4.5
AVERAGE
93.4
6.0
Part 2 - Average Response with DDT Calibrators
DDT
Concentration (ppm)
Average Absorbance
Average %Bo
0
0.1
1.0
10.0
50.0
1.133
0.897
0.569
0.362
0.259
N/A
79.4
50.3
32.0
22.9
Part 3 - Method Sensitivity
Based on Part 1 and Part 2 Above:
Average %Bo - 2 SD = 81.4 which is equivalent to 0.044 ppm DDT
Average %Bo - 3 SD = 75.4 which is equivalent to 0.097 ppm DDT
4042-5
Revision .0
January 1995
-------
TABLE 3
EFFECT OF MATER CONTENT IN SOIL SAMPLES
Soil 1
SI
SI
SI
SI
S2
S2
S2
S2
S3
S3
S3
S3
K Water
0
30
0
30
0
30
0
30
0
30
0
30
Fortified?
No
No
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Reo. 1
79.7*
89.1
49.8
55.8
85.2
94.8
54.4
56.3
96.2
95.6
54.8
59.4
Rep
79
84
62
59
96
94
47
53
91
90
52
55
. 2
.3
.0
.1
.9
.2
.3
.0
.8
.3
.5
.9
.0
Reo. 3
83.7
85.9
46.3
58.0
97.9
95.0
56.1
60.2
100.0
96.4
54.8
54.5
Mean
80.9
86.4
52.8
57.9
93.1
94.7
52.5
56.8
95.8
94.2
54.2
56.3
Std.
2
2
8
2
6
0
4
3
4
3
1
2
Dev.
.4
.6
.3
.1
.9
.3
.8
.2
.3
.2
.1
.7
± 2 SD
76.1 -
81.2 -
36.2 -
53.7 -
79.3 -
94.1 -
42.9 -
50.4 -
87.2 -
87.8 -
52.0 -
50.9 -
Range
85.7
91.6
69.4
62.1
106.9
95.3
62.1
63.2
104.4
100.6
56.4
61.7
* All values shown are %Bo [= (OD8ample/ODneoativecoMrol)xlOO]
4042-6
Revision 0
January 1995
-------
TABLE 4
EFFECT OF pH OF SOIL SAMPLES
Soil oH Ad.i. Fortified? Rep. 1* Rej
3 Mean Std. Dev. ± 2 SD Range
SI
SI
SI
SI
SI
SI
S2
S2
S2
S2
S2
S2
S3
S3
S3
S3'
S3
S3
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
None
Acidic
Basic
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
91.4
79.7
80.5
57.5
54.2
52.8
94.7
87.8
87.9
51.7
52.2
52.0
99.1
86.4
94.9
56.2
54.5
54.6
91.3
87.0
84.5
60.3
60.6
60.2
90.6
100.1
81.6
56.9
61.0
53.5
94.2
84> 3
100.3
. 54.3
53.5
57.2
78.3
86.8
78.5
55.1
55.2
53.3
94.5
100.9
98.3
48.3
55.2
48.9
98.2
85.5
92.9
52.8
53.9
62.9
87.0
84.5
81.2
57.6
56.7
55.5
93.2
96.3
89.3
52.3
56.1
51.5
97.2
85.4
96.1
54.4
54.0
58.2
7.5
4.1
3.0
2.6
3.4
4.1
2.3
7.3
8.5
4.3
4.5
2.3
2.6
1.1
3.8
1.7
0.5
4.2
72.0
76.3
75.2
52.4
49.9
47.3
88.6
81.7
72.3
43.7
47.1
46.9
92.0
83.2
88.5
51.0
53.0
49.8
- 102
- 92.7
- 87.2
- 62.8
- 63.5
- 63.7
- 97.8
- Ill
- 106
- 60.9
- 65.1
- 56.1
- 102
- 87.6
- 104
- 57.8
- 55.0
- 66.6
* All values shown are %Bo [= (ODsarnpl(/ODn
negative contn
4042-7
Revision 0
January 1995
-------
TABLE 5
Comparison to Method 8081
Test Interpretation at 0.2 mg/kg
Sample ID
co-ss-2
co-ss-3
co-ss-4
co-ss-5
co-ss-6
co-ss-7
co-ss-8
co-ss-9
co-ss-10
co-ss-13
co-ss-14
co-ss-15
co-ss-17
co-ss-20
co-ss-21
co-ss-22
co-ss-23
co-ss-24
co-ss-25
co-ss-26
co-ss-27
co-ss-28
co-ss-28-170
co-ss-29
co-ss-30
co-ss-31
co-ss-32
co-ss-33
co-ss-34
co-ss-35
co-ss-36
co-ss-41
Method 8081 (mo/kg)
3.6
0.55
2.3
<0.05
0.15
0.3
0.1
0.8
0.23
0.79
0.58
0.35
<0.05
0.18
0.06
<0.05
<0.05
1.2
0.12
<0.05
<0.05
0.16
,18
.69
0.
0.
0.73
0.68
<0.05
0.32
0.23
0.52
1.0
<0.05
Immunoassay (mq/kq)
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
POSITIVE
POSITIVE
POSITIVE
POSITIVE
NEGATIVE
Results Agree?
• YES
YES
YES
YES
FALSE POSITIVE
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
FALSE POSITIVE
YES
YES
YES
YES
YES
YES
YES
YES
YES
4042-8
Revision 0
January 1995
-------
TABLE 5 (continued)
Comparison to Method 8081
Test Interpretation at 1.0 rag/kg
Sample ID
co-ss-2
co-ss-3
co-ss-4
co-ss-5
co-ss-6
co-ss-7
co-ss-8
co-ss-9
co-ss-10
co-ss-13
co-ss-14
co-ss-15
co-ss-17
co-ss-20
co-ss-21
co-ss-22
co-ss-23
co-ss-24
co-ss-25
co-ss-26
co-ss-27
co-ss-28
co-ss-28-170
co-ss-29
co-ss-30
co-ss-31
co-ss-32
co-ss-33
co-ss-34
co-ss-35
co-ss-36
co-ss-41
Method 8081 (mg/kg)
3.6
0.55
2.3
<0.05
0.15
0.3
0.1
0.8
0.23
0.79
0.58
0.35
<0.05
0.18
0.06
<0.05
<0.05
1.2
0.12
<0.05
<0.05
0.16
.18
.69
0.
0.
0.
73
0.68
<0.05
0.32
0.23
0.52
1.0
<0.05
Immunoassav (mq/kq)
POSITIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
POSITIVE
POSITIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
Results Agree?
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
FALSE POSITIVE
FALSE POSITIVE
YES
YES
YES
YES
FALSE NEGATIVE
YES
4042-9
Revision 0
January 1995
-------
O
>
CQ
Q
ti
H
-------
&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/540/R-94/5G9
March 1994
A User's Guide to
Environmental
Immunochemical
Analysis
-------
/Margins for all text pages-
. same as originals.
I
EPA/540/R-94/509
March 1994
A USER'S GUIDE TO ENVIRONMENTAL IMMUNOCHEMICAL ANALYSIS
Shirley J. Gee
Bruce D. Hammock
Department of Entomology
University of California
Davis, CA 95616
and
Jeanette M. Van Emon
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
EPA Cooperative Research Grant 891047
Project Officer
Jeanette M. Van Emon
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY-LAS VEGAS
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
Printed on Recycled Paper
-------
NOTICE
The information in this document has been funded in part by the United States
Environmental Protection Agency through its Office of Research and Development under
assistance agreement #CR819047-01 to the Department of Entomology, University of
California at Davis. It has been subject to the Agency's peer and administrative review, and it
has been approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
-------
ABSTRACT
Immunochemical methods for the analysis of environmental contaminants are relatively
new on the analytical chemistry scene. These methods are based on the use of a specific
antibody as a detector for the analyte of interest. Immunoassays are rapid, sensitive, and
selective, and are generally cost effective for large sample loads. They have been applied to
diverse chemical structures (i.e. triazines, sulfonylureas, organophosphates, polychlorinated
biphenyls, cyclodienes) and are adaptable to field use. These characteristics make
immunochemical analysis a valuable tool for use by the environmental analytical chemist.
This document is designed to facilitate the transfer of this valuable technology to the
environmental analytical chemistry laboratory. Field personnel who may need to employ a
measurement technology at a monitoring site may also find this manual helpful.
This document is a tutorial designed to instruct the reader in the use and application of
immunochemical methods of analysis for environmental contaminants. A brief introduction
describes basic principles and the advantages and disadvantages of the technology, and
gives a listing of references which supply more detail. Preparation of the laboratory for use of
this technology and the general scientific considerations prior to using the technology are
discussed. Detailed step-wise procedures are given for analysis of selected analytes, triazine
herbicides, carbaryl, paraquat, and p-nitrophenols in environmental samples as well as triazine
mercapturates in urine samples. In addition to the specific immunoassay methods, a series of
support techniques necessary to perform immunochemical methods are described. These
support techniques include pipetting, sample preparations, testing for matrix effects, optimizing
reagent concentrations, data analysis, recordkeeping, and equipment maintenance. A general
troubleshooting guide is provided to aid both the novice and experienced analyst.
This document provides specific instruction for certain analytes, but also serves to
familiarize the novice reader with many generic concepts needed to successfully utilize
immunochemistry technology including: applications, sampling, sample preparation,
extraction, cleanup, quality assurance, methods development and optimization, data handling
and troubleshooting. It is not necessary for the reader to actually perform the immunoassays
given in this User's Guide to obtain familiarity with these concepts. The Guide is written so
that the information presented can be applied to other immunoassays not given here. Thus,
the strength of the Guide is its universal applicability to immunoassay methods.
in
-------
TABLE OF CONTENTS
Section Page "
Notice ii
Abstract iii
List of Figures vi
List of Tables vi
Abbreviations and Acronyms vii
Acknowledgments viii
Executive Summary ix
1. Introduction
1.1 Brief History 1
1.2 Advantages/Disadvantages 1
1.3 Principles 2
1.4 Applications 4
1.5 Bibliography 6
2. Preparing the Laboratory
2.1 Laboratory Resources 7
2.2 General Laboratory Supplies 7
2.3 Chemicals 7
2.4 Immunochemical Reagents 7
2.5 Immunochemical Supplies 7
2.6 Instrumentation 8
3. Laboratory Considerations
3.1 Assay Optimization 9
3.2 Protocol Design 9
3.3 Sample Preparation 10
3.4 Matrix Considerations 10
3.5 Data Handling 10
3.6 Quality Control 11
3.7 Pipetting Techniques 12
3.8 Troubleshooting 12
3.9 Safety Considerations 12
3.10 Waste Disposal 13
3.11 References 13
4. Immunoassay Tutorials for Selected Environmental Analytes 14
4.1. Analysis of Triazines in Environmental Samples
Utilizing a Double Antibody-Coated Microtiter
Plate ELISA Method 15
4.2. Analysis of Triazines in Environmental Samples
Using a Single Antibody-Coated Microtiter
Plate ELISA Method 22
IV
-------
TABLE OF CONTENTS (con't)
Section Page
4.3 ELISA Method for Analysis of Carbaryl in Environmental and
Biological Samples 29
4,4 ELISA Method for the Analysis of Paraquat in Environmental
Samples 35
4.5 ELISA Method for the Analysis of 4-Nitrophenols in Environmental
Samples 41
4.6 ELISA Method for the Analysis of Triazine Mercapturates
in Urine 48
5. Tutorials for Support Techniques 54
5.1 Pipetting Techniques 55
5.2 Considerations in Sampling and Sample Preparation
for Immunoassay Analysis 57
5.2.1 Solid Phase Extraction (SPE) of s-Triazine
Herbicides from Water 60
5.2.2 Solid Phase Extraction (SPE) of Atrazine
Mercapturate from Urine 61
5.3 Approaches to Testing for Matrix Effects 62
5.4 Data Analysis Guidelines 64
5.5 Optimization of Reagent Concentration by
Checkerboard Titration 67
5.6 Record Keeping 73
5.7 Preparation of Buffers for Use in the ELISA 74
5.8 Preparation of Calibration Standards and
Samples Using the 8X12 Array 76
5.9 Outline for a Quality Assurance Document for
Using Immunoassay Methods 78
5.10 Guidelines for the Efficient Use of 96-Well
Microtiter Plates 79
5.11 General Troubleshooting Guidelines to Optimize the
Enzyme Immunoassay Method Performance 80
5.12 Maintenance and Performance Validation of a 96-Well
Microplate Reader 83
5.13 Performance Checks, Calibration and Maintenance
of Air Displacement Pipettors 85
6. Glossary of Commonly Used Terms in Immunoassay 86
Appendices
Appendix I. Performance Assurance for Air Displacement Pipettes 1
Appendix II. Performance Log and Performance Test Worksheets for
Air Displacement Pipettors 1
-------
LIST OF FIGURES
Figure Page
1. Schematic of the procedure for conducting an immobilized
antigen ELISA 3
2. Schematic of an immobilized antibody ELISA 4
3. Typical ELISA template for placement of standards and samples 10
4. Model 4-parameter calibration curve 11
5. Schematic of double antibody-coated microtiter plate ELISA 15
6. Schematic of single antibody-coated microtiter plate ELISA 22
7. Schematic of the antigen-coated plate ELISA format 29
8. Schematic of the antigen-coated plate ELISA format for paraquat 35
9. Schematic of the antigen-coated plate ELISA format 41
10. Schematic of the double antibody-coated ELISA 48
11. Representation of the forward and repetitive pipetting techniques 55
12, Comparison of diluted sample and diluted standard for the assessment of
matrix effects 63
13. Model 4-parameter calibration curve 65
14. Example protocol for titer determination: coating antigen or anti-analyte
antibody 67
15. Example protocol for titer determination: anti-analyte antibody
or enzyme-labeLled hapten 68
16. Plot of checkerboard titration data 70
17. Plot of checkerboard titration data 70
18. Schematic of the 8x12 array for sample preparation 77 i
19. Typical ELISA template 79
Appendix:
1. Normal distribution 2
2. Measured values vs. specification 4
LIST OF TABLES
Table Page
1. Troubleshooting guidelines to optimize the enzyme immunoassay
method performance 80
2. Troubleshooting guidelines for the use of air displacement pipettors 85
Appendix:
1. Weight class standards for balance calibration 5
2. Z-factors 8
VI
-------
I
ABBREVIATIONS AND ACRONYMS
Ab
Ag
AP
ATR-N(C5)-HRP
BLK
BSA
BTX
CONA
DMSO
EIA
ELISA
EMSL
EPA
HPLC
HRP
ICso
IgG
KLH
MSDS
OVA
PBS
PBS-Tween
PBS-Tween/Azide
PCB
PQ
SIM-N(C2)-AP
SFE
SITE
SPE
TMB
UC
Antibody
Antigen
Alkaline phosphatase
Triazine hapten-labeled horseradish peroxidase conjugate
Blank
Bovine serum albumin
Benzene, toluene, xylene
Conalbumin
Dimethylsulfoxide
Enzyme immunoassay
Enzyme linked immunosorbent assay
Environmental Monitoring Systems Laboratory
Environmental Protection Agency
High performance liquid chromatography
Horseradish peroxidase
Concentration producing 50% inhibition
Immunoglobuiin
Association constant
Dissociation constant
Keyhole limpet hemocyanin
Materials Safety Data Sheets
Ovalbumin
Phosphate buffered saline
Phosphate buffered saline containing Tween 20
Phosphate buffered saline containing
Tween 20 and sodium azide
Polychlorinated biphenyls
Paraquat
Triazine hapten-labeled alkaline phosphatase conjugate
Supercritical fluid extraction
Superfund Innovative Technology Evaluation
Solid phase extraction
3,3'5,5' -Tetramethylbenzidine
University of California
VII
-------
ACKNOWLEDGMENTS
The authors thank Don Gurka and Llewellyn Williams of the Environmental Protection *
Agency, Environmental Monitoring Systems Laboratory, Las Vegas, Nevada, Rosie Wong of
American Cyanamid, Princeton, New Jersey, and George Fong, Florida Department of Agricul-
ture, Tallahasee, Florida for helpful review and comments. The authors also thank Al Reed
and Virginia Kelliher, Senior Environmental Employment (SEE) Program enrollees assisting
the Environmental Protection Agency under a Cooperative Agreement with the National
Association for Hispanic Elderly for their helpful review and comments. The authors especially
thank Al Reed for his expert editorial assistance.
VIII
-------
EXECUTIVE SUMMARY
Immunochemistry has broad applications for a wide variety of environmental
contaminants. However, the potential for applying immunochemical methods to environmental
measurements is just beginning to be realized. Immunochemical methods are based on
specific antibodies combining with their target analyte(s). Many specific antibodies have been
produced for targets of environmental and human health concern. Such antibodies can be
configured into various analytical methods. The most popular immunochemical technique in
environmental analyses today is immunoassay. Immunoassays have been shown to detect
and quantify many compounds of environmental interest such as pesticides, industrial
chemicals, and products of xenobiotic metabolism. Among the most important advantages of
immunoassays are their speed, sensitivity, selectivity and cost-effectiveness.
Immunoassays can be designed as rapid field-portable, semi-quantitative methods or
as standard quantitative laboratory procedures. They are well suited for the analysis of large
numbers of samples and often obviate lengthy sample preparations. Immunoassays can be
used as screening methods to identify samples needing further analysis by classical analytical
methods. Immunoassays are especially applicable in situations where analysis by
conventional methods is either not possible or is prohibitively expensive.
Environmental immunoassays have broad applications for monitoring studies. The
EPA has used immunoassay methods for monitoring groundwater and cleanup activities at
hazardous waste sites. Immunoassays can also be used as field screening tools to confirm
the absence and or presence of particular contaminants or classes of contaminants for special
surveys. Other federal and state agencies are employing immunoassay technology where
appropriate such as for extensive monitoring studies that generate a large sample load.
In addition to detection methods, other immunochemical procedures can be used for
environmental analysis. Immunoaffinity techniques now used extensively in pharmaceutical
and biotechnology applications can be adapted to extract, and cleanup environmental
samples. Selective and sensitive sample collection systems such as air and personal
exposure monitors can be designed based on the principal of immunoaffinity. Although
immunoaffinity procedures are not addressed in this tutorial, they are mentioned here to
illustrate to the reader that immunochemicai methods can be adapted to a wide variety of
monitoring situations.
The U.S. EPA Environmental Monitoring Systems Laboratory at Las Vegas, Nevada
(EMSL-LV) has a program to develop and evaluate immunochemical methods for
environmental analysis. The EMSL-LV immunochemistry program consists of the following
major components: identification of need for an immunochemical method, identification of
existing technologies, development of new technologies, adaptations of existing technologies,
evaluations of existing technologies, field demonstration of portable technologies, and finally
technology transfer. Overall program goals, as well as prioritization of compounds for
methods development, are based upon input from client EPA Program Offices as well as the
EPA Regions. Analytical needs are defined as to target analytes, matrices, detection limits
and application of the method.
Methods and immunologic reagents have been developed for the polychlorinated
biphenyls (PCBs), BTX (benzene, toluene, xylene) and various pesticides and nitroaromatic
IX
-------
compounds through the EMSL-LV immunochernistry program. Additional methods are under
development for pyrethroid and organophosphorus pesticides.
The EMSL-LV immunochernistry program conducts laboratory and field evaluations to
assess method performance. The evaluation, characterization and testing of a particular
analytical method is necessary to ensure the intended use of the method is met. Evaluations
are conducted according to EPA guidelines requiring the determination of precision, within and
among laboratories bias, method detection limit, matrix effects, interferences, limit of reliable
measurement and ruggedness of the method. Demonstrations under the Superfund
Innovative Technology Evaluation (SITE) program have been used to document method
performance under real-world environmental conditions. SITE demonstrations of
immunoassay methods for the PCBs, pentachlorophenol, and BTX have been completed,
other demonstrations are being planned. After a SITE demonstration the methods can be
submitted to the Superfund Field Screening Methods compendium for inclusion and
distribution.
Technology transfer activities include providing guidance and training to EPA regional,
EPA headquarters, and state personnel on the use of immunoassays. A computer animated
graphics program has been developed to provide instruction on the theory and applications of
immunoassays. This graphics program may be a useful training aid to the tutorials contained
in this document. Other instructional activities planned include the development of training
videos for performing immunoassays. A "hands on" workshop at the EMSL-LV is also being
considered. Individual training for EPA personnel has been conducted and will remain an
option for interested individuals.
Another vehicle to facilitate the implementation of immunochemical methods are
annual meetings of researchers, developers and end-users of immunochemical methods. The
EMSL-LV has sponsored two meetings to discuss the direction of immunochemical methods
research, development, application, and acceptance within the regulating and regulated
communities. The last Immunochernistry Summit Meeting was held in September 1993 and
included representatives from EPA and other federal and state agencies, large chemical
companies, biotechnology companies, and research institutes. It is anticipated that this type
of meeting will continue to be an annual forum for concerns and issues regarding
environmental immunochemical methods.
Considering the advantages and versatility of immunochemical methods, it is surprising
that the technology has not been more widely accepted by environmental analytical chemists.
Although many immunoassay methods have been reported in the literature, their potential has
not been practically realized. Part of the problem is misunderstanding and perhaps skepticism
on the part of analytical chemists. A thorough understanding of the advantages and
limitations of immunoassay methods is essential to applying the technology in situations where
they offer the most promise. It is the intent of this document to dispel the mystery in
understanding and performing an environmental immunoassay.
This document presents six specific immunoassay methods. The methods are based
on the same working principle but illustrate different applications of the technology for various
analytical situations. Two methods are presented to describe immunoassays for lipophilic
analytes using the triazine herbicides as examples. Although either method can be used for
environmental samples, both are presented to illustrate different formats for the same analyte.
The third method is for the insecticide carbaryl which is applicable for both environmental and
-------
I
biological samples. Methods for p-nitrophenol, paraquat and for various triazine mer-
capturates are examples of water soluble analytes. The triazine mercapturates method
illustrates the application of immunoassay for urine samples and hence exposure assessment
studies. Accompanying solid phase extraction procedures to extract triazines from water and
atrazine mercapturate from urine are also provided. All of the methods described are
intended to serve as examples of the utility of immunoassay technology.
In addition to the six specific immunoassay methods, this document also describes
analytical laboratory techniques necessary to perform immunoassays. Suggestions for
general laboratory considerations such as protocol design, sample preparation, data handling
and analysis, and safety precautions are also given. Examples of troubleshooting and quality
control practices are included which can be applied to assays not contained in this tutorial.
Protocols for preparing buffers, determining reagent integrity and for optimizing assay
conditions are also useful for immunoassays in general. An appendix of commonly used
terms in immunoassay should facilitate understanding of the technology.
Although immunoassays are now being employed for environmental analysis, there
may still be a need for training non-analysts in the use of immunoassay or updating the
experienced analytical chemist on an unfamiliar analytical format. It is hoped that the
methods and procedures found in this users guide will be beneficial and help to standardize
the immunochemical analysis of small molecules. Comments and written requests for
additional information may be directed to Jeanette M. Van Emon, U.S. Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, P. O. Box 93478,
Las Vegas, Nevada 89193-3478.
XI
-------
SECTION 1
INTRODUCTION
This manual is designed to introduce the analytical chemist to the general concepts
and use of immunoassays for the analysis of pesticides and other small molecules. By writing
this manual we hope to encourage the analytical chemist to consider this technology among
the repertoire of methods available for solving analytical problems. As with any other
analytical technique, it will be just as important for the analyst to be able to identify when
immunochemical technology is appropriate, as it is to learn how to conduct an
immunochemical analysis. Field personnel who may need to employ a measurement
technology in the field may also find this manual helpful.
The manual is organized first to provide some general information on the technology,
second to provide tutorials consisting of some specific examples of immunoassays and thirdly
to provide guidelines and information on those procedures specific to immunochemical tech-
niques which may not currently be in use in the typical analytical chemistry laboratory. All of
these procedures were developed in an academic laboratory and may need to be adjusted to
meet regulatory requirements of the various agencies within the government as to method
performance and their procedural guidelines. This caveat also can apply to a Contract Laboratory.
1.1 Brief history
The development of chromatographic instrumentation by pesticide analytical chemists
was closely paralleled by the development of immunoassay techniques by clinical analytical
chemists. Immunoassays are routinely used in clinical situations for the analysis of proteins,
hormones and drugs. The success that these immunochemical procedures has achieved in
the clinical area is being transferred to the area of pesticide analysis.
The first antibodies developed against pesticides were reported in the 1970's. Recently
this technology has been refined for use in the pesticide analytical chemist's laboratory to the
point that commercial test kits are now available. With the availability of kits, it is imperative
that the analyst understand the underlying principles of the methodology in order to evaluate
the strengths and limitations of any one "kit" for their specific application. Since many of these
easy-to-use test kits are designed for the non-analyst, it is important that these users also
have the same fundamental understanding.
1.2 Advantages/Disadvantages
Immunoassays are a useful complement to the analytical chemist's repertoire of
methods for the detection of pesticides and other environmental chemicals. Immunoassays
are rapid, sensitive and selective for the analyte of interest and generally cost effective for
large sample loads. Immunoassays have been applied to diverse chemical structures and are
adaptable to field use. As with any technology there are disadvantages. Antibodies may bind
to structural analogs of the analyte of interest (termed cross-reactivity). This technology is not
easily adapted to a multianalyte method, since each antibody binds primarily to a single
analyte or class of analytes. Reagent stability is often cited as a problem, but can be
overcome based on knowledge gained from the clinical field. This technology also requires a
large sample load to justify development of a new assay for an analyte of interest, due to the
expense of producing antibodies and establishing the procedure. For intermittent analysis, it
might be more cost effective to use existing commercially prepared test kits.
-------
1.3 Principles
Immunoassays rely on the reaction of an analyte or antigen (Ag) with a selective
antibody (Ab) to give a product (Ab Ag) that can be measured. This reaction is characterized
by the Law of Mass Action (shown below), thus immunoassays are physical assays.
Ab + Ag -« Ab-Ag
In its most generic form, immunoassay is an analytical method dependent on the
specific binding of an antibody with its target analyte. This specific interaction can provide
quantitation of the target analyte. Many types of labels have been used for quantitating
immunoassays including radioactivity, enzymes, fluorescence, phosphorescence,
chemiluminescence, and bioluminescence. Each of these labels has its own particular
advantage. However, the use of enzymes and colorimetric substrates is probably the most
common for environmental analysis. Several different types of enzyme immunoassays (ElAs)
have been developed, the two broadest categories being heterogeneous and homogeneous
enzyme immunoassays. Heterogeneous immunoassays require a separation of bound and
free reagents throughout the assay. This is easily accomplished by simply washing the solid
phase (e.g. test tube, microtiter plate wells, cuvettes, etc) with buffer and surfactant.
Homogeneous immunoassays do not require separation or washing steps. However, in these
immunoassays the enzyme label is required to function in the sample matrix which often
poses difficulties. Due to this restriction, homogeneous immunoassays are popular in the
clinical field, while heterogeneous assays are used predominately for environmental matrices.
A common heterogeneous immunoassay is the enzyme-linked immunosorbent assay
(ELISA). The methods in this guide are based on the ELISA format schematically shown in
Figure 1 (adapted from Wie & Hammock, 1982). The following is a generic description for
preparing the microtiter plates and reagents for an ELISA. Preparation of microtiter plates: A
constant amount of the coating antigen is bound to the surface of polystyrene microtiter plate
wells by passive adsorption. After a pre-determined incubation period the wells are washed to
remove unbound coating antigen.
Preparation of ELISA reactants: 1) A constant amount of anti-analyte antibody (first
antibody) is incubated with increasing amounts of analyte in separate test tubes (tubes B, C,
and D, Figure 1). This incubation period enables the formation of analyte-antibody
complexes. The number of analyte-antibody complexes formed and the remaining amount of
free reactants is dependent upon the amount of analyte present in the samples or standards.
2) The incubation mixture is added to the prepared microtiter plate wells. The coating
antigen competes with remaining free analyte for available antibody. A washing step removes
all materials not bound to the microtiter well. 3) A second antibody covalently coupled to an
enzyme (the enzyme label) is next added which binds to the first antibody now bound to the
coating antigen. If the first antibody was developed in a rabbit, the appropriate second
antibody would be goat anti-rabbit IgG covalently coupled to alkaline phosphatase (or another
enzyme label). Excess second antibody is then washed out. 4) Finally substrate is added to
produce a color change. This ELISA is typically called an inhibition assay since a high
concentration of analyte in the samples or standards inhibits the first antibody from binding to
the coating antigen on the microtiter plate well. This is due to the number of analyte-antibody
complexes formed in the initial incubation (tubes A-D Figure 1). The amount of enzyme
-------
Log Concentcation
- L fr'1 Polystyrene
*: Coating Antigen
E Enzyme coupled to
fl Second Antibody
^ First antibody
1 Hapten/Analyte
•y Substrate to
Figure 1. Schematic of the procedure for conducting an immobilized
antigen ELISA.
product formed is directly
proportional to the amount of first
antibody bound to the plate and
is inversely proportional to the
amount of analyte in the samples
and standards (tubes A-D, Figure
1). In this example, the
maximum color intensity is
observed in the wells containing
the contents from tube A (Figure
1) where all the available first
antibody is bound to the coating
antigen. As increasing amounts
of analyte are added, the color
intensity decreases leading to a
sigmoidal analyte dose response
curve similar to that shown in
Figure 1.
There are several
variations in this "format". For
example, the first antibody and
analyte may be added directly to
the coated microtiter plate well.
A second, commonly used,
format is the direct competition
assay. In this immunoasssay format, the antibody is immobilized on the solid phase. Analyte
in the sample competes with a known amount of enzyme-labelled hapten for binding sites on
the immoblized antibody (Figure 2). In step 1, the anti-analyte antibody is adsorbed to the
microtiter plate well. In the competition step, the analyte and a hapten-labelled enzyme are
added to the microtiter plate well. All unbound reagents are washed out. The final step is the
addition of substrate. As in the first format described above, the production of color is
inversely related to the concentration of analyte. This particular format is commonly employed
in the commercial immunoassay test kits.
Formats will vary and it would be useful to know the format being tested as it may be
important to the performance of the assay. For example in the second ELISA schematic, the
sample is in contact with the enzyme labelled hapten. If the enzyme is sensitive to a matrix
effect, then you will get inhibition of the enzyme activity which may then lead to a false
positive result. If this is the case, the format of choice, would be the first format in which, the
sample does not come in contact with the enzyme.
Another important thing to remember with regard to formatting, is that the same
immunoassay reagents can be formatted for a highly quantitative laboratory test, for a semi-
quantitative test, or for rapid yes/no field tests. In general, assays that are simple and very
rapid, tend to be less sensitive. Assays designed for laboratory use may perform less repro-
ducibly in the field. As with any analytical method, immunoassays are designed to perform to
certain specifications under the conditions given. An assay designed to measure an analyte
in a groundwater sample in the laboratory may not perform the same when analyzing ground-
water at a field workstation, or even analyzing surface, instead of groundwater, in the
-------
3).
2).
Enzyme
substrate
Colored
product
Antl-
hapUn
antibody
Y Y Y
Figure 2. Sch&matic of an immobilized antibody EL1SA.
laboratory. That is not to say the
assay is not performing correctly.
It is only to say that it will perform
differently, although the difference
should be consistent under given
conditions.
As a final note to for-
matting and optimization; the
tutorial methods given in this
user's manual use the reading of
absorbances after a fixed period
of time or until the zero control
sample attains a given absorb-
ance value as an endpoint. An
alternative to the endpoint mode
is to use a kinetic read mode. In
this case, the absorbances in the
wells of the microtiter plate are
read at fixed intervals (several
seconds) immediately after addi-
tion of the substrate. In this way,
the rate of the enzyme reaction is
monitored. A major benefit of
kinetics reading is the minimi-
zation of well-to-well variation for replicate analyses due to the time difference in the addition
of substrate. Other advantages to this method include a decrease in analysis time and a
decrease in the amount of reagent needed to obtain a useful signal. In addition, one can
check the linearity of the enzyme reaction, thus further verifying integrity of the assay.
1.4 Applications
An easy way to introduce immunoassay into the analytical laboratory is to use specific
immunoassays as screening methods to determine dilution levels for routine instrumental
analysis. Used in this manner, immunoassays can minimize instrument down time by
protecting sensitive components such as electron capture detectors. The U.S. Environmental
Protection Agency has used immunoassay methods for monitoring cleanup activities at
hazardous waste sites. Many EPA Regions have expressed satisfaction in utilizing
immunoassay methods for these types of field monitoring activities. In some Regions
immunoassay methods are used for groundwater monitoring as a screening tool. For these
monitoring situations, a sample yielding a positive result, is confirmed by an alternative
method. The EPA, in conjunction with the state of Idaho, is currently evaluating the use of
immunoassay to monitor water in the vadose zone for pesticides, as a way of determining the
efficiency of irrigation management practices to prevent leaching. Additional studies are being
conducted by the EPA such as those through the Superfund Innovative Technology Evaluation
program (Gerlach et al., 1993). Other state and federal agencies (e.g. the U.S. Army Corps of
Engineers, the U.S. Geological Survey, the U.S. Department of Agriculture, the National
Institute of Occupational Safety and Health, and the U.S. Food and Drug Administration) are
implementing or evaluating the use of immunochemical methods for their respective
monitoring programs.
€
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The California Department of Food and Agriculture Chemistry Laboratory is an
example of a State regulatory laboratory introducing immunoassay for part of their normal
operation. Their goal is to replace, in a cost effective manner, instrumental analysis for
specific compounds in their routine compliance monitoring program with immunoassay. This
includes considerations of protocol design, dealing with outliers, curve fitting techniques,
consideration for generating "defensible" data in terms of processing and analysis and having
rapid, real time access to quality assurance data.
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1.5 Bibliography
References by Voller and Tij'ssen provide information on general principles of enzyme *
immunoassays. The other references listed are reviews prepared in this laboratory giving
overviews on the development of immunoassays.
Gerlach, R. W., White, R. J., O'Leary, N. F. D. and Van Emon, J. M. 1993. Superfund
Innovative Technology Evaluation (SITE) Program Evaluation Report for Antox BTX
Water Screen (BTX Immunoassay). EPA/540/R-93/518, U.S. Environmental Protection
Agency, Las Vegas, Nevada. 91 pp.
Hammock, B. D. and R. O. Mumma. 1980. Potential of immunochemical technology for
pesticide analysis. In Advances In Pesticide Analytical Methodology, pp. 321-352, (J.
Harvey Jr. and G. Zweig, eds.) American Chemical Society Symposium Series, ACS
Publications, Washington, D.C.
Hammock, B. D., S. J. Gee, R. O. Harrison, F. Jung, M. H. Goodrow, Q. X. Li, A. D. Lucas, A.
Szekacs, and K. M. .S. Sundaram. 1990. Immunochemical technology in
environmental analysis: addressing critical problems. In: Immunochemical Methods
for Environmental Analysis, pp. 112-139 (J.M. Van Emon and R.O. Mumma, eds.),
ACS Symposium Series 442.
Jung, F., S. J. Gee, R. O. Harrison, M. H. Goodrow, A. E. Karu, A. L Braun, Q. X. Li and B.D.
Hammock. 1989. Use of immunochemical techniques for the analysis of pesticides.
Pest. Sci. 26:303-317.
Tijssen, P. 1985. Practice and Theory of Enzyme Immunoassays. Elsevier, New York, ™
549 pp.
Van Emon, J. M., J. N. Seiber and B. D. Hammock. 1985. Applications of immunoassay to
paraquat and other pesticides. In: Bioregulators for Pest Control, pp. 307-316 (P.A.
Hedin, ed.), American Chemical Society Symposium Series 276, Washington D.C.
Van Emon, J. M., J. N. Seiber, and B. D. Hammock. 1989. Chapter 17: Immunoassay
techniques for pesticide analysis. In: Analytical Methods for Pesticide and Plant
Growth Regulators: Advanced Analytical Techniques, Vol. XVII, (J. Sherma, ed.),
Academic Press. New York. pp. 217-263.
Voller, A., A. Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31 :507-520.
Wie, S. I. and B. D. Hammock. 1982. Development of enzyme-linked immunosorbent assays
for residue analysis of Diflubenzuron and BAY SIR 8514. J. Agric. Food Chem.
30:949-957.
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SECTION 2
fe PREPARING THE LABORATORY
The materials listed below may be purchased from a number of commercial sources.
Examples of some items are given for the convenience of the reader.
2.1 Laboratory Resources
Very little additional space or resources are necessary to be able to analyze samples
using immunochemical techniques. The majority of space would be necessary for the
preparation of samples, similar to that already used for other analytical techniques. Electrical
outlets and a vacuum line or pump are the only services required.
2.2 General Laboratory Supplies
Magnetic stirrers/Magnetic stir bars
Vortex mixers
Weigh boats/paper/spatulas
Paper towels
Laboratory wipes (i.e Kimwipes)
Laboratory plastic film (i.e. Parafilm)
Assorted glassware (beakers, erlenmeyers, graduated cylinders)
Felt tipped markers
Benchtop absorbent paper
Assorted borosilicate glass test tubes
Assorted disposable gloves
Label tape
Soap, brushes, wash tubs
2.3 Chemicals
1) Assorted acids/bases and solvents (HCI, NaOH, methanol, acetonitrile, dimethyl
sulfoxide, dimethyl formamide, 2-propanol, etc.)
2) Buffer salts (sodium chloride; sodium phosphate, mono and dibasic; potassium
phosphate, mono and dibasic; sodium carbonate; sodium bicarbonate; diethanolamine;
sodium citrate; potassium chloride, etc.)
2.4 Immunochemical Reagents
The reagents described in the following procedures are examples. To facilitate the
transfer of technology to an environmental monitoring laboratory for routine monitoring studies,
similar reagents are available through commercial sources. The American Association for the
Advancement of Science publishes a yearly "Guide to Biotechnology Products and
Instruments" which includes sources of antibodies for environmental compounds. Similarly,
the American Chemical Society publishes a "Biotech Buyers' Guide."
2.5 Immunochemical Supplies
This supply list is designed to supplement the list of supplies that already exists in the
typical analytical laboratory. It is divided into two areas. In the first area, it assumes the
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analyst is evaluating a test kit which contains all the necessary tubes or plates and reagents. -
In the second area, it assumes the analyst is evaluating component reagents and will have to •
already have some standard reagents or chemicals. These supplies will vary depending on
the format of the assay. Either kit assays or component reagents should always come
supplied with a complete protocol, listing any unusual reagents that might be needed.
1) Pipet tips for multichannel pipettor
2) Pipet tips for single channel pipettors.
3) Multichannel pipet reservoirs.
4) Vacuum tubing and one 1 L and one 4 L vacuum flask for hand held plate washer
5) Several 8 L carboys for wash buffers, and stock buffers
6) Assorted test tubes or other tubes for diluting samples. Individual test tubes may be used
to make dilutions. Test tubes (2 ml) are available in a 8X12 array for use with the
multichannel pipettors. For very small volumes, dilutions may be made in 96 well
microtiter plates that are NOT designed for high binding or use in the ELISA (i.e. Item #2
listed below).
These are some of the supplies and reagents that may be necessary when evaluating
component reagent assays:
1) High binding flat bottomed 96 well microtiter plates - "ELISA plates" (i.e. Nunc
Immunoplate II or equivalent)
2) Flat bottomed 96 well tissue culture plates - used only for diluting samples (i.e. Dynatech
plates or equivalent)
3) Acetate plate sealers
4) Sodium azide (used as a preservative in buffers)
5) Goat anti-rabbit IgG antibody conjugated to alkaline phosphatase or horseradish
peroxidase (second antibody, - depends on format and animal source of primary antibody)
6) Substrates (p-nitrophenylphosphate or 3,3',5,5'-tetramethylbenzidine) - depends on
enzyme label used.
7) Tween 20 detergent. Surfactant to prevent nonspecific binding.
8) Bovine serum albumin (Fraction V). Sometimes used as a "blocking" agent, i.e. to cover
up potential sites for nonspecific binding.
2.6 Instrumentation
1) Variable volume 12 or 8-channel pipettors (approximately $700)
2) Single channel pipettors (various volume ranges, approximately $200 each)
3) Plate washer (may be a simple as a wash bottle, or as complex as an automated plate
washer, approximately $3000).
4) For microplate-based assays a spectrophotometer designed as a 96-well microplate
reader or a strip reader will be necessary for quantitation. A plate reader may cost as
much as $20,000. There are also smaller hand held or benchtop spectrophotometers
which will read an 8-well strip. These are often used for laboratory-based assays that can
be conducted in the field. Most tube-based assays are semi-quantitative in that you might
determine the concentration by eye compared to a standard. These are also adaptable
though, to reading in a spectrophotometer for more quantitative data.
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SECTION 3
LABORATORY CONSIDERATIONS
3.1 Assay Optimization
When evaluating immunoassays, it is important to keep in mind that these are
governed by the Law of Mass Action. The reagents are thus in an equilibrium condition. The
assay then is subject to fluctuations due to temperature (of the reagents and of the laboratory
in which the assays are conducted) and length of incubation time. Since reactions are
occurring at the surface of the microtiter plate, shaking the plate to mix the contents of the
wells may affect the local concentration of reactants. Each of these factors should then be
controlled in order to improve the precision of the measurements. Typically assays are
conducted with reagents which have been equilibrated to room temperature. If room
temperature is not constant (within 3 - 5 degrees of variation), than assays should be
conducted using a forced-air incubator. Shaking the plates periodically during incubation
periods may also improve precision. For immunoassays utilizing 30 minutes or longer
incubation periods, the reactants have likely come nearly to equilibrium and thus conducting
assays with precise timing is unnecessary. However, for immunoassays utilizing shorter
incubation periods, precise timing will improve precision.
3.2 Protocol Design
The methods used most commonly in the analytical laboratory are based on the
96-well microtiter plate format. There are numerous permutations and combinations of ways
that samples and standards can be placed on a 96-well microtiter plate. The number of
calibration wells and the known concentrations used for the calibration curve affect the
precision of the determinations of the unknowns, as does the choice of the number of
replicates of each unknown. Within the framework of a 96-well microtiter plate, how does one
maximize the number of samples analyzed while maintaining the best possible accuracy and
precision. A statistically based method for determining the weight of these factors has been
presented by Rocke et al. (1990). In broad terms there is a tradeoff between efficiency in the
number of samples that can be run per plate vs the additional precision obtained by running
more replicates of each sample or standard. Samples are generally analyzed at several
dilutions. For example, 1:2, 1:4 and 1:8. Values obtained for at least one of the dilutions
should fall near to the center of the calibration curve. This approach is taken in the event that
a positive response is due to a matrix effect. If multiple dilutions are analyzed then
discrepancies among the calculated values may indicate an effect of matrix. If a single
dilution is analyzed then a matrix effect may not be revealed until the sample is confirmed by
an independent method. However, if the matrix is known to not interfere in the analysis, a
single dilution may be analyzed. If the result is too high, then further dilutions can be made.
Last, efficiency of analysis may dictate splitting replicates of unknowns between microtiter
plates. This allows the achievement of desired accuracy at the lowest cost. A typical layout
for a 96-well microtiter plate is shown in Figure 3.
A typical plate format should have a calibration curve with enough replicates as shown
in Figure 3. In fact, it is recommended that a calibration curve be run on every plate because
the reactants are governed by the Law of Mass Action, they are in a dynamic equilibrium. If a
given plate is subject to differences in manipulation time, temperature of incubation or other
factors which may effect the equilibrium, the samples on that plate can be compared to a
calibration curve subjected to those same variables. See also tutorial 5.10. Guidelines for the
Efficient Use of 96-Well Microtiter Plates.
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1 2 3 4 5 S 7 3 9 10 11 12
A
B
C
0
e
F
G
H
CO
2
-------
Analysis and Quality Control of Assays: A Practical Primer", by R. P. Chenning Rogers in
Practical Immunoassay, editor Wifrid R. Butt; published by Marcel Dekker, Inc., N.Y. 1984. If
the choice of standards provides a complete definition of the shape of the curve, (i.e., the
curve has at least 2 to 3 points each defining the upper and lower asymptote and at least 4
points defining the linear region), the 4-parameter fit of Rodbard (1981) is the method of
choice for data analysis in the authors' laboratories. It is important that enough standard
concentrations are used to ensure that the curve is well defined and constant for these
concentrations. Without this information, the computer could force an improper fit (Gerlach et
al., 1993). The equation for the 4-parameter fit is:
y = (A-D)/(1 + (x/C)AB) + D
where y is the absorbance, x is the concentration of analyte, A and D are the upper and lower
asymptotes respectively, B is the slope and C is the central point of the linear portion of the
curve, also known as the IC50 (Figure 4).
cr
w
o
o>
o
n>
A
B
Log Concentration
Figure 4. Model 4-parameter calibration curve.
The best quantitation of unknowns is carried out when unknown absorbances fall in the
central portion of the linear region of the calibration curve. The use of the 4-parameter fit
extends the usefulness of the upper and lower concentrations of the calibration curve.
However, the values calculated from these upper and lower concentrations have greater error
associated with them. To save on reagents, and to keep the error on the estimation of
concentrations of unknowns to a minimum, concentrations for standard curves should be
performed in the linear range after the complete standard curve has been defined with upper
and lower asymptotes. A semi-log curve fit should then be used to fit the data to this
truncated calibration curve and the absorbance values for unknowns should fall in the central
portion of the linear region of this calibration curve. If a kit is being used, the package insert
should indicate the standard curve analysis method to use based on the range of standard
concentrations used for the calibration curve. See also tutorial 5.4. Data Analysis Guidelines.
3.6 Quality Control
There are several approaches to quality control for immunoassays. The first is to
monitor the parameters of the standard curve to ensure that these remain within the desired
coefficient of variation range. Second, it is important to establish relevant quality control
11
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standards (i.e. positive and negative controls). These too, should be monitored on a regular
basis for variations around a determined mean. This may be evaluated for example, by
construction of Shewhart charts (Wernimont & Spendley, 1985). See also tutorial 5.9. Issues
in Quality Control and Quality Assurance.
3.7 Pipetting Techniques
Pipetting is an integral part of this immunochemical technology. Assuming the error
derived from the specific assay design are fixed, the next largest source of error in analytical
data derived from immunoassay is from pipetting errors (Li et al., 1989). Another important
aspect of pipetting error is related to the light path in the microplate reader. For some 96-well
microplate readers, the light path is through the bottom of the microtiter plate well, thus the
path length is directly related to the height of the solution in the microtiter plate well. See the
tutorial 5.1. Pipetting Techniques for full details.
3.8 Troubleshooting
Troubleshooting is probably the most useful skill that any analytical chemist can
develop. The most common problems in immunoassays are poor precision among microtiter
well replicates, spurious color development and no or low color development. Poor plate
washing and pipetting technique are the largest contributors to spurious color development.
No or low color development is most likely due to a reagent failure. The type of 96-well
microtiter plate used is also an important factor. Some plates will bind antigens differently,
and some have greater variability in binding capacity from well to well which would contribute
to variability. Generally selecting a manufacturer whose plate gives reproducible assay
performance parameters for a given assay is the best solution. Another significant factor is
temperature. The reactions that are occurring on the plate are based on the Law of Mass
Action. They are therefore equilibrium reactions and are sensitive to temperature. Reagents
should be used at room temperature, and during analysis, plates should be protected from
wide fluctuations in temperature (i.e. if the laboratory ambient temperature varies more than
3-5 degrees during the day or under field conditions). With the 96 well microtiter plates, the
tendency is for the outer wells to reach temperature sooner than the inner wells, which then
has an effect on the equilibrium reactions. Variations in final absorbances are generally
manifested in what is called an "edge effect." Conducting incubations in a forced-air incubator
may eliminate problems due to temperature fluctuations. Temperature-related effects on
equilibrium are more likely to be seen in assays whose incubation times are very short.
Problems specific to a given assay (for example, stability of standards) are addressed in the
individual tutorials. A comprehensive troubleshooting guide is currently beyond the scope of
this manual. When evaluating test kits or component assays, it is best to keep open lines of
communication with the supplier in order to obtain answers to questions and obtain assistance
in troubleshooting. See tutorial 5.11. General Guidelines for Troubleshooting.
3.9 Safety Considerations
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Specific safety considerations for the target
analytes and organic solvents that may be used in sample preparation are given with each
tutorial method where appropriate.
12
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3.10 Waste disposal
Disposal of hazardous wastes is given in each tutorial method. This technique utilizes
a number of disposable items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips,
sample diluent vessels). In general, the only hazard would be due to the presence of target
analyte in any of these items. Proper disposal may depend on the analyte and the regulations
in effect at your work site. Recycling is encouraged where appropriate.
3.11 References
Gerlach, R. W., White, R. J., Deming, S. N., Palasota, J. A. and Van Emon, J. M. 1993. An
evaluation of five commercial immunoassay data analysis software systems. Anal.
Biochem. 212:185-193.
Li, Q. X., Gee, S. J., McChesney, M. M., Hammock, B. D. and Seiber, J. N. 1989.
Comparison of an enzyme-linked immunosorbent assay and a gas chromatographic
procedure for the determination of molinate residues. Anal. Chem. 61:819-823.
Li, Q. X., Zhao, M. S., Gee, S. J., Kurth, M. J., Seiber, J. N. and Hammock, B. D. 1991.
Development of enzyme-linked immunosorbent assays for 4-nitrophenol and
substituted 4-nitrophenols. J. Agric. Food Chem. 39: 1685-1692.
Miller, J. C. and Miller, J. N. 1984. Statistics for Analytical Chemistry, Ellis Horwood, Ltd.,
Chichester, England, pp. 100-102.
Rocke, D., Bunch, D. and Harrison, R. O. 1990. Statistical design of ELISA protocols. J.
Immunol. Meth. 132:247-254.
Rodbard, D. 1981. Mathematics and statistics of ligand assays. An illustrated guide. In
Ligand Assay, Langan, J., Clapp, J. J., eds.; Masson: New York; pp. 45-99.
Wernimont, G. T. and Spendley, W. 1985. Use of Statistics to Develop and Evaluate
Analytical Methods, Association of Official Analytical Chemists, Arlington, VA.
13
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SECTION 4
IMMUNOASSAY TUTORIALS FOR SELECTED ENVIRONMENTAL ANALYTES M
Six specific immunoassay tutorials are given here (tutorials 4.1 - 4.6). The assay
principles are identical for all six, differing only in the analyte detected and the format of the
reagents. In general, the specific assays that are presented may be used for additional
matrices. However, other matrices may require optimization of the assay. The most
important consideration is the interference that may be a result of that matrix. The level of
interference will determine the amount of sample preparation required prior to analysis. For
example, with water soluble analytes, very little or no sample preparation is usually required.
For lipophilic analytes, it may be necessary to introduce water miscible co-solvents into the
assay. Further hints on preparing samples for analysis by ELISA appear in tutorial 5.2. The
first two protocols describe immunoassays for triazine herbicides as examples of lipophilic
analytes. The third protocol is for the insecticide carbaryl. The last three protocols are for
p-nitrophenol, paraquat, and triazine mercapturate and are used as examples of water soluble
analytes.
For further information regarding these tutorial methods contact Shirley J. Gee,
Department of Entomology, University of California, Davis, CA 95616, telephone
916-752-8465, telefax 916-752-1537, E-mail address: SJGEE@UCDAVIS.EDU.
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4.1 ANALYSIS OF TRIAZINES IN ENVIRONMENTAL SAMPLES UTILIZING A
DOUBLE ANTIBODY-COATED MICROTITER PLATE ELISA METHOD
Introduction:
The general assay design is shown in Figure 5. This assay is a competitive enzyme
immunoassay which utilizes a capture or trapping antibody for the first coating which binds the
triazine-specific antibody in a second coating step. A hapten-enzyme conjugate is used as
the label. This assay has been optimized for detection of atrazine. Due to the structural
similarity of triazines as a class, some cross reactivity with other triazines occurs. See Note 1.
This assay utilizes a horseradish peroxidase (HRP) enzyme label. Do not use sodium azide
in any of the buffers or wash solutions, as it inhibits HRP enzyme activity. All directions
for the preparations of buffers and other solutions used in this tutorial method are given in
tutorial 5.7.
Assay Protocol:
Coating the Microtiter Plate with Trapping
Antibody.
1 .
Coat the microtiter plate with the
trapping antibody. Make a solution
of goat anti-mouse IgG antibody that
is diluted 1/2000 in pH 9.6 carbonate
buffer (coating buffer). Add 100^1
to each well of a high binding ELISA
microtiter plate. See Note 2.
Cover the microtiter plate with a plate
sealer and incubate at 4°C overnight.
See Note 3.
Wash the microtiter plate 5X with
PBS-Tween and tap dry. The wash
procedure involves flooding each well
with buffer repeatedly to remove
unbound reagents.
11).
7.8).
1).
Enzyme
substrate
Colored
product
Figure 5. Schematic of double antibody-coated
microtiter plate ELISA. Line indicates a wash step.
Numbers correspond to the steps in the assay
protocol.
Perform the second coating step.
Make a solution of anti-triazine
antibody (AM7B2.1) that is diluted
1/3200 in pH 9.6 carbonate buffer (coating buffer). Add 100 nL to each well of the
microtiter plate which has previously been coatsd with goat anti-mouse IgG antibody.
Cover the microtiter plate with a plate sealer and incubate at 4°C overnight.
5. Wash the double antibody-coated microtiter plate 5X with PBS-Tween and tap dry.
15
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Then freeze or use immediately in the ELISA step 7 below. This microtiter plate is
termed the "coated" plate. See Note 4. 4
Competitive Inhibition Steps.
6. Prepare standards, samples, and quality control samples (See Note 5.) in PBS-Tween.
This step can utilize the wells of a microtiter plate (of the type used for dilution only,
termed "mixing" plate, see Materials section). Using this technique several standard
curves can be prepared simultaneously (one per row) using a multichannel pipettor.
Multiple dilutions of samples may also be prepared in this manner. Samples can then be
transferred to the coated microtiter plate using the multichannel pipettor. See tutorial 5.8
for an example of this 8x12 array.
7. Add 50 jiL of standard or sample from the mixing plate to each well of the coated plate.
8. Add 50 M.L of ATR-N(C5)-HRP (hapten-labeled enzyme conjugate) that has been diluted
1/3000 to 1/6000 in PBS-Tween to each well of the coated plate, except those wells that
serve as blanks. In the wells that serve as blanks, replace the hapten-labeled enzyme
conjugate with buffer. See Note 6.
9. Cover the coated plate containing standards, samples and enzyme label with a plate
sealer and incubate 15 minutes at room temperature.
10. Wash the coated plate 5X with PBS-Tween and tap dry.
11. Add 100 |iL of substrate solution to each well of the coated plate and cover the plate ^
with a plate sealer. Incubate at room temperature for 15 minutes. (See tutorial 5.7 for
preparation of the substrate.)
12. Add 50 H.L of 4N sulfuric acid to each well of the coated plate to stop the enzyme
reaction.
13. Read at 450-650 nm. See Note 7. The maximum absorbance obtained is about 0.6-0.8
in the wells containing antibody, but no atrazine (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is about 1.0 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-1000 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine and dissolving in 2
ml of dimethylsulfoxide (DMSO). DMSO was chosen because it is water miscible, does not
interfere in the assay at the concentrations used and is not volatile. The primary stock is
diluted 1/100 in DMSO to make a working stock. The working stock is diluted 1/100 in PBS-
Tween to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of DMSO in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water.
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Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the sample indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect, than a simple cleanup step may be used (see Note 5). For example, tutorial
5.2.1 shows the sample preparation method for water in the analysis of triazines. Since
triazines are lipophilic and relatively nonvolatile, they are easily extracted from water using
solid phase extraction. The compounds are eluted from the column in ethyl acetate. Since
ethyl acetate is not a suitable solvent for immunoassay analysis, it is evaporated to dryness
and the residue taken up in PBS-Tween. If concentrations of triazines in the sample are very
high, some cosolvent may be necessary to solubilize the residue (i.e. methanol). Use as little
cosolvent as possible. If the cosolvent is found to interfere with the assay, running the
standard curve in the equivalent concentration of cosolvent can normalize for the interference.
This may compromise the parameters of the calibration curve compared to running the
calibration curve in buffer, but the change is reproducible. Another approach to avoiding
interference is to take advantage of the assay sensitivity. Many interferences can simply be
"diluted away." See tutorial 5.3 for approaches to evaluating matrix effects.
Notes:
1. Percent Cross Reactivity of AM7B2.1 (Atrazine = 100%)
Simazine 32 Hydroxyatrazine 2
Prometon 3 Hydroxysimazine 0
Terbutryn 19
(See Schneider et al., 1993 Table III, for a more complete list of compounds tested.)
2. The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
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3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these double antibody-coated microtiter plates can be stored
frozen for more than one month with no change in assay characteristics (i.e. IC50, slope
or maximum absorbance). We have also found that coating for more than overnight
before use or freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the no analyte control in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage. If strong
matrix effects occur, the protocol describe in tutorial 4.2 may be worth trying. There will
be about a 10 fold decrease in assay sensitivity, however in our hands this format
appeared more resistant to matrix and modifiers (such as cosolvents) in the sample
(Lucas et al., 1991).
6. Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in a
checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (ATR-N(CS)-HRP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. ATR-N(C5)-HRP has the following structure and is
conjugated to horseradish peroxidase. It should be stored in the freezer. Periodically
tests of the enzyme activity should be run to assure no loss of activity on storage of the
stock solution. Working dilutions should be made up immediately before use and the
excess discarded. A change in the assay performance parameters will be an indication
of possible degradation of the hapten-enzyme conjugate. Provided by Dr. Bruce
Hammock, Department of Entomology, University of California, Davis, CA 95616.
Revision 0
18 March 22, 1993
-------
Cl
(CH3)2-
H
0
N(CH2)5CN-HRP
H H
6-{{4-Chloro-6-[(1 -methylethyi)amino)-
1,3,5-triazirv2-yOamino}hexanoic acid
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
O
II
SCH2CH2CN-Protein
H
NCH2CH3
H
3-{{4-Ethylamino)-6-[(1 -methylethyl)aminol-
1,3,5-triazin-2-yl}thio}propanoic acid
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
This antibody was provided by Dr. Alex Karu, Hybridoma Center, University of California at
Berkeley, 1050 San Pablo Avenue, Albany, CA 94706. Other triazine antibodies are
commercially available.
19
Revision 0
March 22, 1993
-------
Purchased Reagents:
The following materials are listed for the convenience of the reader. Similar products ^
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
2) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
5) 3,3'5,5'-Tetramethylbenzidine (Sigma Catalog No. T-2885 or equivalent. Use only the
highest purity.)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any 4|
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing ™
vapors or skin contact. 3,3'5,5'-Tetramethylbenzidine is an irritant; avoid breathing vapors.
This compound is used in dimethylsulfoxide, which may promote dermal absorption. Avoid
skin contact. It is assumed the analyst will have in place procedures for the safe handling of
organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Revision 0
20 March 22, 1993
-------
Karu, A. E., R. 0. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow,
»A. Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M.( L. H. Stanker, B. E. Watkins, and
D. W. Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
Lucas, A. D., P. Schneider, R. O. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and
D. E. Rolston. 1991. Determination of atrazine and simazine in water and soil using
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
Schneider, P. and B. D. Hammock. 1992. Influence of the ELISA format and the hapten-
enzyme conjugate on the sensitivity of an immunoassay for s-triazine herbicides using
monoclonal antibodies. J. Agric. Food Chem. 40:525-530.
Revision 0
21 March 22, 1993
-------
4.2 ANALYSIS OF TRIAZINES IN ENVIRONMENTAL SAMPLES
USING A SINGLE ANTIBODY-COATED MICROTITER PLATE ELISA METHOD
Introduction:
The general assay design is shown in Figure 6. This assay is a competitive enzyme
immunoassay which utilizes a capture or trapping antibody for the coating. The principle is
the same as shown for tutorial 4.1, except that the triazine-specific antibody is not
precaptured. Instead the triazine-specific antibody is reacted in free solution with the analyte
and is trapped by the adsorbed capture antibody. This assay is about 10X less sensitive than
the method described in tutuorial 4.1, but .is more resistant to matrix and modifiers (such as
cosolvents) in the sample (Lucas et al., 1991). This assay has been optimized for detection of
atrazine. Due to the structural similarity of triazines as a class, some cross reactivity with
other triazines occurs. See Note 1. All directions for the preparations of buffers and other
solutions used in this tutorial method are given in tutorial 5.7.
12).
1).
Assay Protocol:
Coating the Microtiter Plate with Trapping
Antibody.
1. Coat the microtiter plate with the trapping
antibody. Make a solution of goat anti-
mouse IgG antibody that is diluted 1/2000 in
pH 9.6 carbonate buffer (coating buffer).
Add 100 viL to each well of a high binding
ELISA microtiter plate. See Note 2.
2. Cover the microtiter plate with a plate sealer
and incubate overnight at 4°C. See Note 3.
3. Wash the single antibody-coated microtiter
plate 5X with PBS-Tween/Azide and tap dry.
The wash procedure involves flooding each
well with buffer repeatedly to remove
unbound reagents. This plate is termed the
"coated" plate. Then freeze or use
immediately in ELISA step 9 below. See Note 4.
Competitive Inhibition Steps.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing plate"; see Materials section) for the preparation of dilutions
and for premixing reagents prior to their addition to the coated plate. Using this
technique several standard curves can be prepared simultaneously (one per row) using
a multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
i
Figure 6. Schematic of single antibody-coated
microtiter plate ELISA. Lines indicate a wash
step. Numbers correspond to the steps in the
assay protocol.
22
Revision 0
September 28, 1989
-------
5. Add 40 M-L of standard or sample to each well of the mixing plate.
6. Add 100 M.L of SIM-N(C2)-AP (hapten-labeled enzyme conjugate) that has been diluted
1/3000 to 1/6000 in PBS-Tween/Azide to each well of the mixing plate, except those
wells that serve as no-enzyme-conjugate blanks. In the microtiter plate wells that serve
as blanks, replace the enzyme conjugate with buffer. See Note 6.
7. Add 100 JO.L of anti-triazine antibody (AM7B2.1 medium) diluted 1/200 to 1/600 in PBS-
Tween/Azide to each well of the mixing plate,
8. Cover the mixing plate with a plate sealer and incubate 60 minutes at room temperature.
9. Transfer 50 nL from each well of the mixing plate using a 1 2-channel pipettor to the
respective wells of the coated microtiter plate.
10. Cover the coated plate with a plate sealer and incubate 60 minutes at room temperature.
11. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
12. Add 100 (iL of 1 mg/mL substrate solution (freshly made, one 5 mg tablet per 5 ml_ 10%
diethanolamine substrate buffer) to each well of the coated plate and cover with a plate
sealer. Incubate at room temperature for about 60 minutes (see Note 7).
13. Read at 405-650 nm. See Note 8. The maximum absorbance obtained is about 0.5-0.6
in wells containing antibody, but no atrazine (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is 20 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-2000 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine and dissolving in 2
ml of dimethylsulfoxide (DMSO). DMSO was chosen because it is water miscible, does not
interfere in the assay at the concentrations used and is not volatile. The primary stock is
diluted 1/50 in DMSO to make a working stock. The working stock is diluted 1/100 in PBS-
Tween/Azide to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of DMSO in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the curve fit
model. For example, if a semi-log curve fit is utilized, then the calibration curve would only
utilize those standard concentrations which would yield a straight line.
23 Revision 0
September 28, 1989
-------
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the sample indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5). For example, tutorial
5.2.1 shows the sample preparation method for water in the analysis of triazines. Since
triazines are lipophilic and relatively nonvolatile, they are easily extracted from water using
solid phase extraction. The compounds are eluted from the column in ethyl acetate. Since
ethyl acetate is not a suitable solvent for immunoassay analysis, it is evaporated to dryness
and the residue taken up in PBS-Tween. If concentrations of triazines in the sample are very
high, some cosolvent may be necessary to solubilize the residue (i.e. methanoi). Use as little
cosolvent as possible. If the cosolvent is found to interfere with the assay, running the
standard curve in the equivalent concentration of cosolvent can normalize for the interference.
This may compromise the parameters of the calibration curve compared to running the
calibration curve in buffer, but the change is reproducible. Another approach to avoiding
interference is to take advantage of the assay sensitivity. Many interferences can simply be
"diluted away." See tutorial 5.3 for approaches to evaluating matrix effects.
Notes:
1. Percent Cross Reactivity of AM7B2.1 (Atrazine = 100%)
Simazine 40 Hydroxysimazine 3
Prometon 6 Both mono-N-dealkylated 1
Hydroxyatrazine 5 N,N'-di-dealkylated 0.1
2. The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these single antibody-coated plates can be stored frozen for more
than one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
24 Revision 0
September 28, 1989
-------
quality controls, assuming no degradation or loss of analyte on storage. If strong
» matrix effects do not occur, the protocol given in tutorial 4.1 may be worth trying. The
advantage to method 4.1 is that the sensitivity is about 10X better than method 4.2, but
method 4.1 is more susceptible to matrix and modifiers (such as cosolvents) in the
sample (Lucas et al., 1991).
6. Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in
a checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (SIM-N(C2)-AP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
7. In order to facilitate the manual handling of several i.e., (10-25) coated plates in an
experiment, the length of incubation with the substrate has been optimized for 60
minutes. By adjusting reagent concentrations according to results obtained in the
checkerboard titration format, the assay may be optimized for shorter incubation times.
8. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. SIM-N(C2)-AP has the following structure and is
conjugated to alkaline phosphatase. It should be stored in the refrigerator.
Periodically tests of enzyme activity should be run to assure no loss of activity on
storage of the stock solution. Working dilutions should be made up immediately before
use and the extra discarded. DO NOT FREEZE - each freeze-thaw cycle will kill a
significant part of the conjugate-enzyme activity. A change in the assay performance
parameters will be an indication of possible degradation of the hapten-enzyme
conjugate. Provided by Dr. Bruce Hammock, Department of Entomology, University of
California, Davis, CA 95616.
25 Revision 0
September 28, 1989
-------
Cl
I
H
H
N-[4-Chloro-6-(ethylamino)-
1,3,5-triazin-2-yq-p-alanine
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
This antibody was provided by Dr. Alex Karu, Hybridoma Center, University of California at
Berkeley, 1050 San Pablo Avenue, Albany, CA 94706. Other triazine antibodies are
commercially available.
0
II
SCH2CH2CN—Protein
H
N
-------
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
*J 2) 96 Well microtiter plates
a) High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b) Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; Sigma Catalog No. P-1379, or
equivalent)
5) p-Nitrophenyl phosphate substrate tablets (5 mg tablets, Sigma Catalog No. 104-105
or equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Karu, A. E., R. O. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow, A.
Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M., L. H. Stanker, B. E. Watkins, and D. W.
Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
27 Revision 0
September 28, 1989
-------
Lucas, A. D., P. Schneider, R. 0. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and D. -
E. Rolston. 1991. Determination of atrazine and simazine in water and soil using fl
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
Schneider, P. and B. D. Hammock. 1992. Influence of the ELISA format and the hapten-
enzyme conjugate on the sensitivity of an immunoassay for s-triazine herbicides using
monoclonal antibodies. J. Agric. Food Chem. 40:525-530.
28 Revision 0
September 28, 1989
-------
4.3 ELISA METHOD FOR ANALYSIS OF CARBARYL
IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES
Introduction:
The general assay design is shown in Figure 7. This assay is a competitive enzyme
immunoassay which utilizes a carbaryl structural mimic covalently bound to a protein (termed
coating antigen) adsorbed to the microtiter plate surface. The sample containing carbaryl
competes with the carbaryl mimic on the coating antigen for a fixed amount of the anti-
carbaryl antibody. The amount of antibody bound is detected using a goat anti-rabbit IgG
antibody bound to alkaline phosphatase (termed second antibody). The analyst is referred to
Voller et al. (1976) for more details on this format. This assay has been optimized for the
detection of carbaryl. There is no cross reactivity with the major degradation product, 1 -
naphthol or naphthalene. Cross reactivity with other carbamate compounds is <5%. All
directions for the preparations of buffers and other solutions used in this tutorial method are
given in tutorial 5.7.
/Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1.
Coat the microtiter plate with the carbaryl
hapten conjugated to conalbumin (5-
CONA). (See Note 1.) Make a solution of
5-CONA that is 0.5 ^g/rnL in pH 9.6
carbonate buffer (coating buffer). Add 100
lit to each well of a high binding ELISA
microtiter plate. See Notes 1 and 2.
Cover the microtiter plate with a plate
sealer and incubate overnight at 4°C. See
Note 3.
11.
5,8.
Enzyme
substrate
Colored
F
lev
•L a
^^.
product
Enzyme-labelled
anti-rabbit
antibody
H Competing
free
hapten
Well of polystyrene 96-well plate
3.
Figure 7. Schematic of the antigen-coated plate
ELISA format. The lines represent wash steps.
The numbers correspond to the numbered steps
in the protocol.
Wash the coated microtiter plate 5X with
PBS-Tween/Azide and tap dry. The wash
procedure involves flooding each well with
buffer repeatedly to remove unbound
reagents. Then freeze or use immediately in ELISA step 5 below. This plate is termed
the "coated" plate. See Note 4.
Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
29
Revision 0
March 17, 1993
-------
multichannel pipettor. Multiple dilutions of samples may also be prepared in this M
manner. Samples can then be transferred to the coated microtiter plate using the ^j
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 50 M,L of standard or sample from the mixing plate to each well of the coated
plate.
6. Add 50 |iL of Antibody #2114 diluted 1/40,000 in PBS-Tween/Azide to each well of the
coated plate, except those wells that serve as no-antibody blanks. (The final
concentration in the well is 1/80,000.) In the wells that serve as blanks, replace the
antibody with buffer. See Note 6.
7. Cover the coated plate with a plate sealer and incubate 1 hour at room temperature.
8. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
9. Prepare a solution of goat anti-rabbit IgG-alkaline phosphatase that is diluted 1/5000 in
PBS-Tween/Azide, Add 100 nL of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
11. Add 100 JJ.L of substrate solution (1 mg/mL p-nitrophenylphosphate in 10%
diethanolamine buffer) to each well of the coated plate and cover with a plate sealer.
Incubate for 30 minutes at room temperature. 4M
12. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is about 0.7-
0.8 in the wells containing antibody, but no carbaryl (zero analyte standard). The IC50
(or midpoint of the calibration curve) for this assay is 2-5 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-100 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade carbaryl and dissolving in 2
ml of methanol. Methanol was chosen because it is water miscible, does not interfere in the
assay at the concentrations used and is not very volatile. The primary stock is diluted 1 /100
in methanol to make a working stock. The working stock is diluted 1/1000 in PBS-
Tween/Azide to make the highest concentration to be tested. This assures that a reasonable
amount of analytical standard is weighed and ultimately, the concentration of methanol in the
assay is quite low. If other solvents are used to make the stock solution, care should be
taken to ensure that this solvent does not interfere in the assay and that the solvent is
miscible with water. For this protocol we prepare the stock solutions in methanol and stored
them at-20°C.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
30 Revision 0
Mtrch 17, 1993
-------
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
• response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the samples indicate that there is no matrix effect,
then the sample is buffered then placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5).
A simple strategy to avoid interference is to take advantage of the assay sensitivity.
Many interferences can simply be "diluted away". Water, honey, milk and urine have been
tested for interferences following analysis of simple dilutions of these matrices. Water had no
effect on the assay performance. Honey had to be diluted 1/1000, milk 1/50,000 and urine
1/50 to eliminate influences on the standard curve. Soil was extracted with chloroform/
methanol. The solvent was evaporated and the residue taken up in methanol, then diluted
1/10 with PBS-Tween/Azide. This extract only needed to be diluted 1/25 to eliminate matrix
interferences. This assay can be used when samples contain up to 10% methanol. If other
solvents are used in sample preparation, their effects need to be tested (Marco et al., 1993).
Interference by a known media, such as 25% methanol can be corrected by running the
standard curve in the equivalent media. This may compromise the parameters of the
standard curve compared to running the standard curve in buffer, but the change is repro-
ducible. See tutorial 5.3 for approaches to evaluating matrix effects.
Notes:
1. The optimal amount of 5-CONA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (5-CONA) and
anti-carbaryl antibody (Rabbit #2114) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
2. We have tested other formats with these antibodies. Coating the plate with the
antibody (similar to tutorial 4.2) improved the sensitivity, however the maximum signal
to noise ratio was not as favorable as with the antigen coated plate format. In addition,
10X more antibody was required for the antibody-coated plate format.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. ICSO, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
31 Revision 0
March 17, 1993
-------
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage. This antibody
can be used when samples contain up to 10% methanol. If other solvents are used in
sample preparation, their effects need to be tested. Matrix effects have been
demonstrated for soil, urine, honey and milk. Soil and urine have the least effect on
the assay performance, whereas milk has a significant effect even at dilutions of
1/25000.
6. The amount of anti-carbaryl antibody should be optimized in a checkerboard titration
where varying amounts of anti-carbaryl antibody and enzyme-labeled hapten are used
in the ELISA to optimize assay performance. Changes in assay performance may be
compensated for by reoptimizing reagents. See tutorial 5.5 for the checkerboard
titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Coating antigen. 5-CONA has the following structure and is conjugated to conalbumin.
A small aliquot of the stock may be stored in the refrigerator if used regularly. The
remainder should be stored frozen in small aliquots. Working dilutions should be made
up immediately before use and the extra discarded. Too many freeze-thaw cycles may
affect the integrity of the coating antigen. A change in the assay performance
parameters will be an indication of possible degradation of the coating antigen.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California,
Davis, CA 95616.
32 Revision 0
March 17, 1993
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C-N—(CH2)5COOH
N-(2-Naphthoy1)6-aminohexanoic acid
2) Hapten specific antibody. Rabbit polyclonal antibody #2114 - final bleed directed
against the following hapten conjugated to keyhole limpet hemocyanin:
H 0 H
i i i
N-C -N—(CHjJsCOOH
1 -(S-CaitooxypentyO-3-< 1 -naphthy<)urea
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA 95616.
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
33
Revision 0
March 17, 1993
-------
1 ) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
4) p-Nitrophenylphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all immunoreagents may be treated with bleach before disposal.
References:
Marco, M. P., Gee, S. J., Cheng, H. M., Liang, Z. Y. and Hammock, B. D. 1993.
Development of an Enzyme Linked Immunosorbent Assay for Carbaryl. J. Agric. Food
Chem. 41:423-430.
Schneider, P. and Hammock, B. D. Influence of the ELISA Format and the Hapten-Enzyme
Conjugate on the Sensitivity of an Immunoassay for s-Triazine Herbicides Using
Monoclonal Antibodies. J. Agric. Food Chem. 40:525-530 (1992).
Voller, A., A. Bartlett, and D. E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
34 Revision 0
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4.4 ELISA METHOD FOR THE ANALYSIS OF PARAQUAT
IN ENVIRONMENTAL SAMPLES
Introduction:
The general assay design is shown in Figure 8. This assay is a competitive enzyme
immunoassay which utilizes a paraquat structural mimic covalently bound to a protein (termed
coating antigen) adsorbed to the microtiter plate surface. The sample containing paraquat
competes with the paraquat mimic on the coating antigen for a fixed amount of the anti-
paraquat antibody. Quantitation of paraquat is determined by detecting the amount of anti-
paraquat antibody bound to the coating antigen. The bound antibody is detected using an
anti-rabbit IgG antibody labeled with alkaline phosphatase (termed second antibody). The
analyst is referred to Voller et al. (1976) for more details on this format. This assay has been
optimized for detection of paraquat. There is no cross reactivity with the major degradation
products, or other bipyridinium compounds such as diquat. All directions for the preparations
of buffers and other solutions used in this tutorial method are given in tutorial 5.7.
SPECIAL NOTE: Paraquat is known to bind to glass surfaces. Recovery studies indicate
that it does not bind to polystyrene (Van Emon et al., 1986). Avoid handling standards or
samples in glass containers. Polystyrene or polypropylene containers are highly
recommended.
Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1. Coat the microtiter plate with the
paraquat hapten PQ-C2 conjugated
to bovine serum albumin (PQ-C2-
BSA). (See Note 1.) Make a
solution of PQ-C2-BSA that is 1.25
|ig/ml_ in pH 9.6 carbonate buffer
(coating buffer). Add 100 M.L to each
well of a high binding ELISA micro-
titer plate. See Note 1.
2. Cover the microtiter plate with a plate
sealer and incubate overnight at 4°C.
See Note 2.
11.
5. e.
Enzyme
tubs (rate
Enzyme-labelled
anti-rabbit
antibody
H Competing
frea
hapten
Well of polystyrene 96-well plate
Figure 8. Schematic of the antigen-coated plate
ELISA format for paraquat. The lines represent wash
steps. The numbers correspond to the numbered
steps in the protocol.
Wash the coated microtiter plate 5X
with PBS-Tween/Azide and tap dry.
The wash procedure involves flood-
ing each well with buffer repeatedly
to remove unbound reagents. Then freeze or use immediately in ELISA step 5 below.
This is termed the "coated" plate. See Note 3.
35
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March 17, 1993
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Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 4) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 50 H.L of standard or sample from the mixing plate to each well of the coated
plate.
6. Add 50 |il_ of Antibody #21 diluted 1/5,000 in PBS-Tween/Azide to each well of the
coated plate, except those wells that serve as no-antibody blanks. (The final
concentration in the well is 1/10,000.) In the wells that serve as blanks, replace the
antibody with buffer. See Note 5.
7. Cover the coated plate with a plate sealer and incubate 30 minutes at room
temperature.
8. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
9. Prepare a solution of goat anti-rabbit IgG-alkaline phosphatase that is diluted 1/5000 in
PBS-Tween/Azide. Add 50 y.L of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
11. Add 100 nL of substrate solution (1 mg/mL p-nitrophenyphosphate in 10%
diethanolamine buffer) to each well of the coated plate and cover with plate sealer.
Incubate for 30 minutes at room temperature.
12. Read at 405-650 nm. See Note 7. The absorbance obtained is between 0.4-0.5 in the
wells containing antibody, but no paraquat (zero analyte standard). The IC50 (or
midpoint of the calibration curve) for this assay is 1 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-4 ng/mL. The primary stock
solution is prepared by weighing 20 mg of analytical grade paraquat and dissolving in 2 ml of
double distilled water in a polystyrene or polypropylene container and storing at room
temperature. Appropriate dilutions are made in double distilled water in order to prepare the
highest concentration to be tested.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
36 Revision 0
March 17, 1993
-------
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
As noted above, paraquat binds to glass. Do not use glass in sample preparation.
The amount of sample preparation needed will depend on the matrix. The first approach
would be to attempt to analyze the sample with little or no sample preparation. For example,
water samples may be buffered, then analyzed directly in the assay. For matrices which
require extraction, a simple sonication with 6N HCI has proven useful for food matrices (Van
Emon et al.,1987). The HCI is then evaporated to dryness. The residue is resuspended in
PBS-Tween/Azide, then analyzed. For lipid matrices, such as oils, extraction with water may
provide suitable recovery. The aqueous phase may then be analyzed directly in the
immunoassay. This assay has been used successfully to measure paraquat in serum and
lymph without any sample preparation. These antibodies have also been used to "extract"
paraquat from air filter samples (see Van Emon et al., 1986).
Notes:
1. The optimal amount of PQ-C2-BSA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (PQ-C2-BSA)
and anti-paraquat antibody (Rabbit #21) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
2. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
3. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
4. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metais, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
5. The amount of anti-paraquat antibody should be optimized in a checkerboard titration
where varying amounts of anti-paraquat antibody and coating antigen are used in the
ELISA to optimize assay performance. Changes in assay performance may be
compensated for by reoptimizing reagents. See tutorial 5.5 for the checkerboard
titration format.
37 Revision 0
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-------
6. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Coating antigen. PQ-C2-BSA has the following structure and is conjugated to bovine
serum albumin. A small aliquot of the stock may be stored in the refrigerator if used
regularly. The remainder should be stored frozen in small aliquots. Working dilutions
should be made up immediately before use and the extra discarded. Too many freeze
-thaw cycles may affect the integrity of the coating antigen. A change in the assay
performance parameters will be an indication of possible degradation of the coating
antigen. Provided by Dr. Bruce Hammock, Department of Entomology, University of
California, Davis, CA 95616.
2) Hapten specific antibody. Rabbit polyclonal antibody #21 - final bleed directed against
the following hapten conjugated to conalbumin:
N-(4-Cart>oxy«lh- 1-yO-W-methylNpyiidHhjm
1) Coaling antigun.
HjC—
-(CHj)4COOH
N-<4-Cart>oxybuM -yW-mettryfcipyfidllum
2) Hapten specific antibody.
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA 95616.
38
Revision 0
March 17, 1993
-------
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
1) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
4) p-Nitrophenyphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
References:
Van Emon, J. M., J. N. Seiber and B. D. Hammock. 1985. Applications of immunoassay to
paraquat and other pesticides. In: Bioregulators for Pest Control, pp. 307-316 (P.A.
Hedin, ed.), American Chemical Society Symposium Series 276, Washington D. C.
Van Emon, J., B. Hammock, and J. N. Seiber. 1986. Enzyme-linked immunosorbent assay
for paraquat and its application to exposure analysis. Anal. Chem. 58:1866-1873.
39 Revision 0
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Van Emon, J., J. Seiber, and B. Hammock. 1987. Application of an enzyme-linked
immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and
potatoes. Bull. Environ. Contam. Toxicol. 39:490-497.
Voller, A., A. Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
40 Revision 0
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4.5 ELISA METHOD FOR THE ANALYSIS OF 4-NITROPHENOLS
IN ENVIRONMENTAL SAMPLES
Introduction:
The general assay design is shown in Figure 9. This assay is a competitive enzyme
immunoassay which utilizes a 4-nitrophenol structural mimic covalently bound to a protein
(termed coating antigen) adsorbed to the microtiter plate surface. The sample containing 4-
nitrophenol competes with the 4-nitrophenol mimic on the coating antigen for a fixed amount
of the anti-4-nitrophenol antibody. Quantitation of 4-nitrophenol is determined by detecting the
amount of anti-4-nitrophenol antibody bound to the coating antigen. The bound antibody is
detected using a goat anti-rabbit IgG antibody labeled to alkaline phosphatase (termed second
antibody). The analyst is referred to Voller et al. (1976) for more details on this format. This
assay has been optimized for detection of 4-nitrophenol and certain monosubstituted 4-
nitrophenols. There is very little cross reactivity (<2%) to substituted nitrobenzenes, 2- or 3-
nitrophenol, other substituted phenols and 4-nitropyridine-N-oxide. See Note 1 for an
indication of relative cross reactivity of nitrophenols. All directions for the preparations of
buffers and other solutions used in this tutorial method are given in tutorial 5.7.
SPECIAL NOTE: This assay is sensitive to changes in pH, as might be expected for
4-nitrophenol which may be ionized. Control of pH in antibody binding steps is critical to
precise assay performance.
Assay Protocol:
Coating the Microtiter Plate with Coating
Antigen
1. Coat the microtiter plate with the
4-nitrophenol hapten conjugated
to ovalbumin (C-OVA). (See
Note 2.) Make a solution of C-
OVA that is 2.0 u.g/mL in pH 9.6
carbonate buffer (coating buffer).
Add 100 (xL to each well of a
high binding ELISA microtiter
plate.
2. Cover the microtiter plate with a
plate sealer and incubate
overnight at 4°C. See Note 3.
3. Wash the coated microtiter plate
5X with PBS-Tween/Azide and
tap dry. The wash procedure
involves flooding each well with
11.
9.
5,6.
1.
Enzyme
substrate
Colored
product
Rabbit
anti-H
antibody
Hapten-
protein |_j
conjugate
Enzyme-labelled
anti-rabbit
antibody
H Competing
free
hapten
Well of polystyrene 96-well plate
Figure 9. Schematic of the anitgen-coated plate ELISA
format. The lines represent wash steps. The numbers
correspond to the numbered steps in the protocol.
41
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March 17, 1993
-------
buffer repeatedly to remove unbound reagents. Then freeze or use immediately in
ELISA step 8 below. This plate is termed the "coated" plate. See Note 4. M
Competitive Inhibition Step.
4. Prepare standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
5. Add 120 |iL of standard or sample to the wells of a mixing plate.
6. Add 120 M.L of Antibody #1812 diluted 1/1000 in PBS-Tween/Azide to each well of the
mixing plate, except those wells that serve as no-antibody blanks. (The final
concentration in the well is 1/2000.) In the wells that serve as blanks, replace the
antibody with buffer. See Note 6.
7. Cover the mixing plate with a plate sealer and incubate overnight at room temperature.
8. Add 50 |j,L from each well of the mixing plate to the respective wells of the coated
plate. Cover the coated plate with a plate sealer and incubate at room temperature for
3 hours.
9. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
10. Prepare a solution of goat anti-rabbit IgG-alkaline phosphatase that is diluted 1/2500 in
PBS-Tween/Azide. Add 50 jol of this solution to each well of the coated plate. Cover
the coated plate with a plate sealer and incubate for 1 hour at room temperature.
11. Wash the coated plate 5X with PBS-Tween/Azide. Tap dry.
12. Add 100 ^tL of substrate solution (1 mg/mL p-nitrophenylphosphate in 10%
diethanolamine buffer) to each well of the coated plate and cover with plate sealer.
Incubate for 30 minutes at room temperature.
13. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is between
0.4-0.5 in the wells containing antibody, but no 4-nitrophenol (zero analyte standard).
The IC50 (or midpoint of the calibration curve) of this assay is 8-10 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-2000 ng/mL. 4-Nitrophenol is
highly water soluble. Standard solutions can be prepared in distilled water and stored at room
temperature. Methanol is an alternative solvent. Solutions may be kept in a sealed container
and stored in a refrigerator. The primary stock solution is prepared by weighing 20 mg of
42 Revision 0
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analytical grade 4-nitrophenol and dissolving in 2 ml of double distilled water. This primary
stock is diluted appropriately in double distilled water to give the highest concentration to be
tested. Stocks that have been alternatively prepared in methanol, should also be diluted in
double distilled water. This assures that the concentration of methanol in the assay is quite
low. If other solvents are used to make the stock solution, care should be taken to insure that
this solvent does not interfere in the assay and that the solvent is miscible with water.
Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation. For
example if the matrix is water and tests of the samples indicate that there is no matrix effect,
then the sample is buffered, and placed directly into the assay. If the sample does manifest a
matrix effect than a simple cleanup step may be used (see Note 5). This assay has also
been used to analyze soil containing parathion. Parathion, extracted from soil using
supercritical fluid extraction was analyzed as 4-nitrophenol by ELISA after oxidation to
paraoxon using dimethyldioxirane followed by hydrolysis (Wong et al., 1991). This is a good
example of extraction and derivatization techniques which demonstrate the general principle in
immunoassay of using volatile extraction solvents and derivatizing agents to minimize
interferences with the subsequent immunoassay. This assay will tolerate up to 25% methanol
or 5% ethyl acetate. Several other solvents were tested at 5% (ethanol, acetonitrile, and
dimethyl formamide) and showed no effect on the calibration curve. Thus samples can be
prepared and analyzed in these solvents, if the concentration remains below 5%. Another
simple strategy to avoid interference is to take advantage of the assay sensitivity. Many
interferences can simply be "diluted away".
Notes:
1. Relative Cross Reactivity for Rabbit #1812
Compound Percent Cross-Reactivity
4-Nitrophenol 100
2-Chloro-4-nitrophenol 190
2-Amino-4-nitrophenol 104
3-Methyl-4-nitrophenol 92
2,4-Dinitrophenol 48
43 Revision 0
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2. The optimal amount of C-OVA needed for the assay should be determined in a
checkerboard titration format where varying amounts of coating antigen (C-OVA) and
anti-4-nitrophenol antibody (Rabbit #1812) are used in the ELISA to optimize assay
performance. This is particularly important when any new reagent is utilized or when
assay performance parameters begin to change.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these antigen-coated plates can be stored frozen for more than
one month with no change in assay characteristics (i.e. IC50, slope or maximum
absorbance). We have also found that coating for more than overnight before use or
freezing results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in water samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
6. The amount of anti-4-nitrophenol antibody should be optimized in a checkerboard
titration where varying amounts of anti-4-nitrophenol antibody and coating antigen are
used in the ELISA to optimize assay performance. Changes in assay performance
may be compensated for by reoptimizing reagents. See tutorial 5.5 for the
checkerboard titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading in two wavelengths can eliminate
absorbance discrepancies due to flaws in the plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Coating antigen. C-OVA has the following structure and is conjugated to ovalbumin. A
small aliquot or the stock may be stored in the refrigerator if used regularly. The
remainder should be stored frozen in small aliquots. Working dilutions should be made
up immediately before use and the extra discarded. Too many freeze-thaw cycles may
affect the integrity of the coating antigen. A change in the assay performance
44 Revision 0
March 17, 1993
-------
parameters will be an indication of possible degradation of the coating antigen.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California,
Davis, CA 95616.
2) Hapten specific antibody. Rabbit polyclonal antibody #1812 - final bleed directed
against the following haptepi conjugated to keyhole limpet hemocyanin at the 2-
position:
CHjCOOH
N02
4-Nilropheny* acetic acid
1) Coating antigen. C OVA
2-Hydroxy-5-nitrobenryl bromide
2) Hapten specific antibody.
Antibodies should be stored frozen in small aliquots to minimize freeze-thaw cycles.
Provided by Dr. Bruce Hammock, Department of Entomology, University of California, Davis,
CA 95616.
Purchased Reagents and Materials:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may yield satisfactory results, however the authors have
not evaluated the performance of these alternative materials.
1) 96-Well microtiter plates
a). High binding ELISA plates (i.e. Nunc Immunoplate II (Catalog No. 442404 or
equivalent) for coating.
b). Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
45
Revision 0
March 17, 1993
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2) Acetate plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
3) Tween 20 (polyoxyethylene-sorbitan monolaurate; i.e. Sigma Catalog No. P-1379 or
equivalent)
4) p-Nitrophenylphosphate tablets (i.e. Sigma Catalog No. 104-105, 5 mg tablets or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
46 Revision 0
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References:
Li, Q.X., Zhao, M.S., Gee, S.J., Kurth, M.J., Seiber, J.N. and Hammock, B.D. 1991.
Development of enzyme-linked immunosorbent assays for 4-nitrophenol and
substituted 4-nitrophenols. J. Agric. Food Chem. 39:1685-1692.
Voller, A., A, Bartlett, and D.E. Bidwell. 1978. Enzyme immunoassays with special reference
to ELISA techniques. J. Clin. Pathol. 31:507-520.
Wong, J.M., Li, Q.X., Hammock, B.D. and Seiber, J.N. 1991. Method for the analysis of 4-
nitrophenol and parathion in soil using supercritical fluid extraction and immunoassay.
J. Agric. Food Chem. 39:1802-1807.
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4.6 ELISA METHOD FOR THE ANALYSIS OF
TRIAZINE MERCAPTURATES IN URINE
Introduction:
The general assay design is shown in Figure 10. This assay is a competitive enzyme
immunoassay which utilizes a capture or trapping antibody for the first coating and which
binds the triazine mercapturate-specific antibody in a second coating step. A hapten-enzyme
conjugate is used as the label. This assay has been optimized for detection of atrazine
mercapturate. Due to the structural similarity of triazines as a class, some cross reactivity
with other triazines occurs. See Note 1. All directions for the preparations of buffers and
other solutions used in this tutorial method are given in tutorial 5.7.
Assay Protocol:
Coating the Microtiter Plate with
Trapping Antibody.
1.
4.
Coat the microtiter plate with
the trapping antibody. Make a
solution of goat anti-mouse
antibody that is diluted 1/2000
in pH 9.6 carbonate buffer
(coating buffer). Add 100 ^L to
each well of a high binding
ELISA microtiter plate. See
Note 2.
Cover the microtiter plate with
a plate sealer and incubate
overnight at 4°C. See Note 3.
Wash the coated microtiter
plate 5X with PBS-Tween/Azide
and tap dry. The wash
procedure involves flooding
each well with buffer repeatedly
to remove unbound reagents.
11).
7,8).
1).
Enzyme
substrate
Colored
product
Antl-
hapten
antibody
Anti-
mouse
antibody
Figure 10. Schematic of a double antibody-coated ELISA.
Line indicates a wash step. Numbers refer to steps in
protocol.
Perform the second coating step. Make a solution of anti-triazine antibody (AM7B2.1)
that is diluted 1/3200 in pH 9.6 carbonate buffer (coating buffer). Add 100 JJ.L to each
well of the microtiter plate which has previously been coated with goat anti-mouse IgG
antibody. Cover the microtiter plate with a plate sealer and incubate overnight at 4°C
overnight.
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March 22, 1993
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5. Wash the double antibody-coated microtiter plate 5X with PBS-Tween/Azide and tap
dry. Then freeze or use immediately in ELISA step 7 below. This microtiter plate is
termed the "coated" plate. See Note 4.
Competitive Inhibition Steps.
6. Prepare the standards, samples, and quality control samples (See Note 5) in PBS-
Tween/Azide. This step can utilize the wells of a microtiter plate (of the type used for
dilution only, termed "mixing" plate, see Materials section). Using this technique
several standard curves can be prepared simultaneously (one per row) using a
multichannel pipettor. Multiple dilutions of samples may also be prepared in this
manner. Samples can then be transferred to the coated microtiter plate using the
multichannel pipettor. See tutorial 5.8 for an example of this 8x12 array.
7. Add 50 |nL of standard or sample from the mixing plate to each well of the coated plate.
8. Add 50 (xl_/well of SIM-N(C2)-AP (hapten-labeled enzyme conjugate) that has been
diluted 1/10000 in PBS-Tween/Azide to each well of the coated plate, except those
wells that serve as blanks. In the wells that serve as blanks, replace the hapten-
labeled enzyme conjugate with buffer. See Note 6.
9. Cover the coated plate containing standards, samples and enzyme label with a plate
sealer and incubate 30 minutes at room temperature.
10. Wash the coated plate 5X with PBS-Tween/Azide and tap dry.
11. Add 100 ixL of 1 mg/mL substrate solution (freshly made, one 5 mg tablet per 5 ml
10% diethanolamine substrate buffer) to each well of the coated plate and cover with
plate sealer. Incubate at room temperature for 15-30 minutes.
12. Read at 405-650 nm. See Note 7. The maximum absorbance obtained is about 0.3-
0.4 in the wells containing antibody, but no atrazine mercapturate (zero analyte
standard). The IC50 (or midpoint of the calibration curve) for this assay is 1 ng/mL.
Preparation of Standards and Samples:
The concentration range to be tested in this protocol is 0-200 ng/mL. The primary
stock solution is prepared by weighing 20 mg of analytical grade atrazine mercapturate and
dissolving in 2 ml_ of dimethylsulfoxide (DMSO). DMSO was chosen because it is water
miscible, does not interfere in the assay at the concentrations used and is not volatile. The
primary stock is diluted 1/100 in DMSO to make a working stock. The working stock is diluted
1/100 in PBST to make the highest concentration to be tested. This assures that a
reasonable amount of analytical standard is weighed and ultimately, the concentration of
DMSO in the assay is quite low. If other solvents are used to make the stock solution, care
should be taken to insure that this solvent does not interfere in the assay and that the solvent
is miscible with water.
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Ten nonzero standards, a zero analyte standard and a no-enzyme-label blank
comprise the calibration curve. This provides at least two concentrations at which nearly
complete or complete inhibition occurs, and at least two concentrations at which little or no
inhibition occurs. These are particularly important if the intention is to define the full sigmoidal
response curve and utilize the 4-parameter curve fit for data analysis. (See tutorial 5.4 Data
Analysis Guidelines for a discussion of 4-parameter and other curve fitting methods.) If other
curve fitting methods are used, the concentrations should be adjusted to best fit the particular
curve fit model. For example, if a semi-log curve fit is utilized, then the calibration curve
would only utilize those standard concentrations which would yield a straight line.
The amount of sample preparation needed will depend on the matrix. The first
approach would be to attempt to analyze the sample with little or no sample preparation.
Another approach to avoid intereference would be to take advantage of the assay sensitivity.
Many interferences can simply be "diluted away." In this tutorial we found urine samples vary
widely in composition. For example some may be more dilute, more concentrated with salts
or protein, etc., more or less colored, and may depend on diet, etc. We have found for
measuring atrazine mercapturate in urine, a dilution of the urine in PBS-Tween/Azide to 25%
is adequate to remove interferences in the samples we tested. In the event that the matrix
effects cannot be diluted out, to quantitate the sample, a solid phase extraction using a phenyl
column may be used. (See tutorial 5.2.2). See tutorial 5.3 for approaches to evaluating
matrix effects.
Notes:
1. Percent Cross Reactivity of AM7B2.1 (Atrazine mercapturate = 100%)
Simazine 9 Hydroxysimazine <0.1
Atrazine 30 Both mono-N-dealkylated <0.1
Prometon 2 N,N'-di-dealkylated <0.1
Hydroxyatrazine 2 Cyanazine 32
2. The amount of trapping antibody needed should be determined in a checkerboard
titration format where varying amounts of trapping antibody (goat anti-mouse IgG) and
anti-triazine antibody (AM7B2.1) are used in the ELISA to optimize assay performance.
This is particularly important when any new reagent is utilized. See tutorial 5.5 for
details on the checkerboard titration format.
3. A tight sealing plate sealer should be used to avoid evaporation. In a quantitative test,
small changes in volume can affect the assay precision and accuracy.
4. We have found that these double antibody-coated plates can be stored frozen for more
than one month with no change in assay characteristics (i.e. IC50> slope or maximum
absorbance). We have also found that coating for more than overnight, before use or
freezing, results in an increase in well to well variability.
5. CAREFUL TESTING OF MATRIX EFFECTS PRIOR TO SAMPLE ANALYSIS IS
CRITICAL. The most commonly found contributors to matrix effects in urine samples
are variation in pH, presence of trace metals, excessive salt, or dissolved organic
50 Revision 0
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matter. Matrix effects are usually manifested as an inhibition of color in a sample
which should contain no analyte. Sometimes the effect is an increase in absorbance
above that obtained in the zero analyte standard in a sample which should contain no
analyte. Approaches to dealing with matrix effects are given in tutorial 5.3. Quality
control samples need to be chosen based on the type of sample being analyzed.
Spiked samples known to be free of analyte and/or real field samples can be used as
quality controls, assuming no degradation or loss of analyte on storage.
6. Enzyme-labeled hapten and the anti-triazine antibody dilutions should be optimized in
a checkerboard titration where varying amounts of anti-triazine antibody (AM7B2.1) and
enzyme-labeled hapten (SIM-N(C2)-AP) are used in the ELISA to optimize assay
performance. Changes in assay performance may be compensated for by reoptimizing
reagents. See tutorial 5.5 for the checkerboard titration format.
7. Absorbance variability is decreased by shaking the plate before reading to mix the
contents of the microtiter plate wells. Reading at two wavelengths can eliminate
absorbance discrepancies due to flaws in the microtiter plate.
Materials:
Specialized Reagents:
The immunochemical reagents described in this protocol were provided by the
indicated academic research laboratories. Similar reagents may be available commercially.
1) Hapten-enzyme conjugate. SIM-N(C2)-AP has the following structure and is
conjugated to alkaline phosphatase. It should be stored in the refrigerator. Run periodic tests
of enzyme activity to assure no loss
of activity on storage of the working
solution. Working dilutions should be
made up immediately before use and
the extra discarded. DO NOT
FREEZE - each freeze-thaw cycle
will kill a significant part of the
conjugate-enzyme activity. A change
in the assay performance parameters
will be an indication of possible
degradation of the coating antigen.
Provided by Dr. Bruce Hammock,
Department of Entomology,
University of California, Davis, CA
95616.
Cl
I It °
Jk A. ii
CH3CH2—N N NCH2CH2CN-AP
H
H
N-[4-C hloro-6-(ethytamino)-
1,3,5-triazirv2-y(]-p-alanine
H
2) Hapten specific antibody. Monoclonal AM7B2.1 cell culture medium containing
antibody directed against the following antigen:
51
Revision 0
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o
11
SCHjCHaCN—Protein
(CH3)2-N
H
""^'
H
3-{{4-E»hylamino)-6-l(1-methylethyl)aminol-
1,3,5-triazirv-2-yl}thio}propanoic acid
Antibodies should be stored
frozen in small aliquots to minimize
freeze-thaw cycles. This antibody
was provided by Dr. Alex Karu,
Hybridoma Center, University of
California at Berkeley, 1050 San
Pablo Avenue, Albany, CA 94706.
Purchased Reagents:
The following materials are
listed for the convenience of the
reader. Similar products are available
from other vendors and may yield
satisfactory results, however the authors have not evaluated the performance of these
alternative materials.
1) Goat anti-mouse IgG antibody (i.e. Boehringer-Mannheim #605 24 or equivalent)
2) Microtiter plates
a) High binding ELISA plates (i.e. Nunc Immunoplate II Catalog No. 442404 or
equivalent) for coating.
b) Mixing plates (i.e. Dynatech Catalog No. 001-012-9299 or equivalent) for
preparing dilutions.
3) Plate sealers (i.e. Dynatech Catalog No. 001-010-3501 or equivalent)
4) Tween 20 (polyoxyethylene-sorbitan monolaurate; Sigma Catalog No. P-1379, or
equivalent)
5) p-Nitrophenyl phosphate substrate tablets (5 mg tablets, Sigma Catalog No. 104-105 or
equivalent)
Safety Considerations:
Read the Materials Safety Data Sheet (MSDS) provided by the manufacturer for any
compound with which you are not familiar. Sodium azide is a teratogen; avoid breathing
vapors or skin contact. It is assumed the analyst will have in place procedures for the safe
handling of organic solvents and samples containing the analyte.
Waste Handling and Disposal:
The analyst should already have in place procedures for the disposal of organic
solvents and samples containing the analyte. This technique utilizes a number of disposable
items (i.e. polystyrene 96-well microtiter plates, plastic pipettor tips, sample diluent vessels).
In general, the only hazard would be due to the presence of target analyte in any of these
52
Revision 0
March 22, 1993
-------
items. Proper disposal may depend on the analyte and the regulations in effect at your work
site. Recycling is encouraged where appropriate. The antibody, antigens and enzyme labels
are biologically based reagents. These items are non-hazardous and non-infectious. As a
precaution, all reagents may be treated with bleach before disposal.
References:
Goodrow, M. H., R. O. Harrison and B. D. Hammock. 1990. Hapten synthesis, antibody
development, and competitive inhibition enzyme immunoassay for s-triazine herbicides.
J. Agric. Food Chem. 38:990-996.
Karu, A. E., R. O. Harrison, D. J. Schmidt, C. E. Clarkson, J. Grassman, M. H. Goodrow, A.
Lucas, B. D. Hammock, J. M. Van Emon, and R. J. White. 1991. Monoclonal
Immunoassay of Triazine Herbicides: Development and Implementation. In:
Immunoassays for Trace Chemical Analysis: Monitoring Toxic Chemicals in Humans,
Food, and the Environment, (Vanderlaan, M., L. H. Stanker, B. E. Watkins, and D. W.
Roberts, eds.), pp. 59-77, ACS Symposium Series 451.
Lucas, A. D., A. D. Jones, M. H. Goodrow, S. G. Saiz, C. Blewett, J. N. Seiber, and B. D.
Hammock. 1993. Determination of atrazine metabolites in human urine: Development
of a biomarker of exposure. Chem. Res. Toxicol. 6:107-116.
53 Revision 0
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SECTION 5
TUTORIALS FOR SUPPORT TECHNIQUES
The following tutorials describe techniques that the analyst will likely use while
conducting immunoassays. Some techniques will be familiar as these are common to
analytical chemistry. Other techniques will be new to the analyst.
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5.1 Pipetting Techniques
The following techniques generally apply to all positive air displacement pipettes.
General:
Never set the volume selector to volumes above or below the specified range for the
pipettor, or it will require recalibration. Always operate the plunger button slowly and smoothly
at all times. Never let the plunger button snap back. Ensure that clean pipette tips are firmly
pushed onto the tip cones of the pipette and that there are no foreign bodies inside the tips.
Wet the newly attached pipette tips with the solution being pipetted before any actual pipetting
takes place. This is done by filling and emptying the pipette tips 2-3 times. Hold the pipette
vertically during liquid intake. Pipettors should be stored in an upright position.
Pipetting Techniques:
Forward technique: (See Figure 11.)
1)
2)
3)
4)
Depress the operating button
to the first stop.
Dip the tips just under the
surface of the liquid in the
reservoir and slowly release
the operating button. This
action will fill the tips.
Withdraw the tips from the
liquid, touching them against
the edge of the reservoir to
remove excess liquid.
Deliver the liquid by gently
depressing the operating
button to the first stop. After a
delay of about a second,
continue to depress the
operating button all the way
down to the second stop. The
action will empty the tips.
Forward Technique
dispense
nil
Ready position -p -j-
Flrst stop
Second stop
Repetitive Technique
dispense refill
I I I
Figure 11. Representation of the forward and repetitive
pipetting techniques.
Release the operating button to the ready position. If necessary, change the tips and
continue with the pipetting.
55
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Repetitive Technique: (See Figure 11.)
This technique is suitable for dispensing liquids having a high viscosity or which have a
tendency to foam easily. The technique is also recommended for dispensing very small
volumes and is the method of choice for the authors' laboratories.
1) Depress the operating button all the way down to the second stop.
2) Dip the tips just under the surface of the liquid in the reservoir and slowly release the
operating button. This action will fill the tips. Withdraw the tips from the liquid,
touching them against the edge of the reservoir to remove excess liquid.
3) Deliver the preset volume by gently depressing the operating button to the first stop.
Hold the operating button at the first stop. Some liquid will remain in the tip and
should not be included in the delivery. (For a multichannel pipettor, a quick visual
scan of the remaining liquid will show you whether the channels are delivering the
same volume as the menisci should all be lined up evenly.)
4) Dip the tips just under the surface of the liquid in the reservoir and slowly release the
operating button. This action will refill the tips. Continue pipetting by repeating the last
two steps. The remaining liquid is either discarded with the tips or pipetted back into
the container.
Maintenance/Calibration
Follow all maintenance and calibration procedures as outlined by the manufacturer.
(See tutorial 5.13. Performance checks, calibration and maintenance of air displacement
pipettors.)
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• 5.2 CONSIDERATIONS IN SAMPLING AND SAMPLE PREPARATION
FOR IMMUNOASSAY ANALYSIS
Sampling Considerations for Immunoassav Methods:
It is important to recognize that immunoassays require a much smaller sample size.
Advantages to this are that more samples may be taken for analysis in the field as less room
for storage is necessary and shipping costs are reduced for smaller sampling containers.
However, smaller sample sizes present unique problems for sampling. For example, it is
important to assure that the original sample is homogeneous prior to subsampling. For more
discussion on sampling and subsampling see van Ee et al. (1990) and EPA (1992).
Importance of the Analyte:
"Every type of material that is to be prepared for analysis presents its own practical
difficulties. The requirements for suitable sample preparation are dictated by the consistency
and the chemical characteristics of the analyte and the matrix, and by the distribution of the
analyte in the sample."
This statement by Garfield (1991) is applicable to any analytical technique, including
immunoassay. When forming an approach to sample preparation, the analyst in general is
familiar with the chemical properties of the analyte in question. Thus, the volatility, polarity,
relative stability to acid, base, heat, etc. are known. The general approach is to use as little
sample preparation as possible to get the analyte into a form suitable for analysis by a chosen
method. In the case of immunoassay, the analyte needs to basically be in a water miscible
media.
Begin by using the most simple approach. For example, if the matrix is aqueous, try
analyzing the sample directly. If the matrix is causing some interference, try checking the pH
and adjusting to neutrality. Next try diluting the matrix effect. If analyte signal is lost, then a
sample concentration/purification step will be necessary. Standard methods for sample
preparation of the compound are a good starting place for the design of your sample
preparation for immunoassay. The following guidelines should be considered in developing
approaches to sample preparation.
1). Immunoassays are conducted in aqueous media, thus samples should be prepared
with this in mind. Aqueous solubility of a lipophilic compound may not be a problem in
the concentration ranges being tested.
2). Whenever possible make use of the sensitivity of the assay. Often times matrix
interferences can be eliminated by diluting the sample.
3). Use water miscible organic solvents whenever possible. Methanol and acetonitrile
seem to be the most desirable.
4). Use as few steps as possible. Often times the immunoassay analysis method is
quicker simply because fewer sample preparation steps are necessary.
57
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5). Exploit the chemical properties of the compound in order to design simple partition
methods.
6). Explore the volatility of the compound. Can it first be extracted into organic solvent,
followed by solvent evaporation without loss of analyte? Can the residue be taken up
in a water miscible solvent. Even for relatively volatile compounds, a trapping solvent
may be used. For example the compound cou'd be extracted in ethyl acetate with a
small amount of propylene glycol added. After evaporation of the ethyl acetate, the
analyte now trapped in propylene glycol, is brought to a reasonable volume with water
or a water miscible solvent.
7). Use simple solid phase extraction methods. A wide variety of bonded phases are now
available in small columns (including reverse phase, normal phase and ion exchange).
Two example solid phase extraction methods are given in tutorials 5.2.1 and 5.2.2.
8). Consider supercritical fluid extraction (SFE) for solid samples. Samples are extracted
and concentrated in one step. The extraction media is easily removed and water
miscible trapping solvent can be used. (See Wong et al., 1991 for an example).
9). Most immunoassays can tolerate methanol or acetonitrile up to 10%. Many assays
can tolerate more, however, assay performance parameters may be altered. If you
find that you need to use more solvent to get adequate cleanup of the sample, the
assays can be run using this concentration of solvent in the calibration curve. This will
normalize for the effects of the solvent. In most cases this will mean a decrease in the
signal/noise ratio. There may also be a shift in IC50.
10). Consider using the immunoassay as a supplemental method. For example, as a
downstream detector for an HPLC, as described in a review by de Frutos & Regnier
(1993).
Importance of the Matrix:
Knowing as much as possible about the matrix with which you are working is always a
valuable asset when analyzing the sample. For soils, humic acid is known to effect
immunoassays. In addition there are some 2000 soil types in the United States alone, thus
optimization on each soil type analyzed would be important. In water samples, pH, the
presence of metals, ions or bacteria may have an effect on the assay. Optimization of the
assay for the water sample, or preparation of the sample by simple procedures such as
buffering, filtering, addition of chelate, etc. may help. With urine, variability in matrix effects
may occur due to diet, amount of liquid consumed, health, etc. Since every matrix may have
an effect, it is critical to include a procedure for the evaluation of potential matrix effects. (See
Tutorial 5.3. Approaches to Testing for Matrix Effects.)
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References:
de Frutos, M. and Regnier, F.E, 1993. Tandem chromatographic-immunological analysis.
Anal. Chem. 65:17A-25A.
EPA. 1992. Characterizing Heterogeneous Wastes: Methods and Recommendations. EPA
Report, EPA/600/R-92/033, February, 1992.
Garfield, F.M. 1991. Quality Assurance Principles for Analytical Laboratories. Association of
Official Analytical Chemists, Arlington, VA. pp 70.
van Ee, J.J. Blume, LJ. and Starks, T.H. 1990. A Rationale for the Assessment of Errors in
the Sampling of Soils. EPA Report, EPA/600/4-90/013.
Wong, J.M., Li. Q.X., Hammock, B.D. and Seiber, J.N. 1991. Method for the analysis of 4-
nitrophenol and parathion in soil using supercritical fluid extraction and immunoassay.
J. Agric. Food Chem. 39:1802-1807.
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5.2.1 SOLID PHASE EXTRACTION (SPE) OF
S-TRIAZINE HERBICIDES FROM WATER
Using this extraction system, recovery of atrazine from water was >98% for the
concentration range 0.1 ng/L to 1 mg/L using a 75 mL sample size. At <0.1 ng/L, the signal
was not distinguishable from background. At 10 mg/L, the recovery dropped significantly, likely
due to the lack of solubility of atrazine in the ELISA system. Although atrazine is reportedly
soluble to 33 mg/L in water, the presence of salts in the ELISA assays buffers significantly
decreased the solubility of atrazine. A white precipitate could be seen when attempting to
resolubilize the residue from the ethyl acetate evaporation in assay buffer. Adding about 10%
methanol helped the solubilization, and the effects of methanol could be diluted out, as this
concentration of atrazine is 1000 times larger than the IC50 of the assay.
Equipment and Supplies:
The following materials are listed for the convenience of the reader. Similar products
are available from other vendors and may likely yield satisfactory results, however the authors
have not evaluated the performance of these alternative materials.
vacuum manifold
C18 cartridges, 2.8 mL, 500 mg (i.e. Analytichem or equivalent)
reservoirs and adapters
pesticide grade hexane, ethyl acetate, acetone, methanol
double distilled water
Procedure:
1. Preclean all glassware and plasticware with acetone (except C18 cartridges).
2. Set manifold flow rate at 5 to 10 mL/min.
3. Place cartridge in manifold.
4. Wash the cartridge with the following, in order:
2 column volumes of hexane
2 column volumes of ethyl acetate
2 column volumes of methanol
2 column volumes of water
5. Attach adapter and add sample (1 liter maximum).
6. Wash cartridge with 1 column volume of water.
7. Air dry under vacuum for 5 to 15 minutes.
8. Elute cartridge with 2 mL ethyl acetate.
9. Evaporate eluate to dryness under nitrogen stream.
10. Reconstitute with PBS-Tween/Azide buffer, assay or store.
(See tutorial 5.7 for preparation of PBS-Tween/Azide buffer.)
Reference:
Lucas, A. D., P. Schneider, R. O. Harrison, J. N. Seiber, B. D. Hammock, J. W. Biggar, and
D. E. Rolston. 1991. Determination of atrazine and simazine in water and soil using
polyclonal and monoclonal antibodies in enzyme-linked immunosorbent assays. Food
Agric. Immunol. 3:155-167.
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5.2.2 SOLID PHASE EXTRACTION (SPE) OF
ATRAZINE MERCAPTURATE FROM URINE
Using this phenyl SPE system, 99% of atrazine, 70% of either of the mono
N-dealkylated products, 50% of hydroxyatrazine, and no measurable didealkylated atrazine
were retained using this SPE system. This immunoassay does cross react with atrazine,
however in urine, the mercapturate accounts for the majority of the immunoreactivity and
parent atrazine is found at levels 500-1000 times less. If, however, the presence of parent
atrazine is of concern, it may be eliminated by partitioning the urine with chloroform (1:1) and
then analyzing the aqueous phase (or subjecting it to the phenyl column for further cleanup)
for the mercapturate.
Equipment and Supplies:
The following materials are listed for the convenience of the reader. Similar products are
available from other vendors and may yield satisfactory results, however the authors have not
evaluated the performance of these alternative materials.
vacuum manifold
phenyl cartridges, 2.8 ml, 500 mg (i.e. Analytichem or equivalent)
reservoirs and adapters
pesticide grade acetone
double distilled water
hydrochloric acid
Procedure:
1. Preclean all glassware and plasticware with acetone (except phenyl cartridges).
2. Set manifold flow rate at 5 to 10 mL/min.
3. Place cartridge in manifold.
4. Wash the cartridge with the following, in order:
2 column volumes of acetone
2 column volumes of acidified water (pH 2.5-3)
5. Attach adapter and add sample (10 ml, acidified to pH 2.5-3).
6. Wash cartridge with 1 column volume of acidified water.
7. Air dry under vacuum for 5 to 15 minutes.
8. Elute cartridge with 2 mL acetone.
9. Evaporate eluate to dryness under nitrogen stream.
10. Reconstitute with PBS-Tween/Azide buffer, analyze or store. (See tutorial 5.7 for the
preparation of PBS-Tween/Azide buffer.)
Reference:
Lucas, A. D., Jones, A.D., Goodrow, M.H., Saiz, S.G., Blewett, C., Seiber, J.N. and Hammock,
B.D. 1993. Determination of atrazine metabolites in human urine: development of a
biomarkerof exposure. Chem. Res. Toxicol. 6:107-116.
Revision 0
61 March 23, 1992
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5.3 APPROACHES TO TESTING FOR MATRIX EFFECTS ^
There is potential for any sample or components within the sample to interfere with
proper quantitation of the analyte of interest with any analytical method. When presented with
a new matrix, the analyst can save a lot of time if a few key experiments are run first.
1). Ideally the analyst will have available a "blank" sample matrix that is identical to the
matrix within which the analyte of interest will be measured. The blank and the sample
are prepared identically. Prepare the standard calibration curve in this matrix and
determine if the parameters are significantly different from the standard curve in buffer.
If the curve is not different, then it can be assumed that all samples can be run in this
manner. If the curve is different, then try repeating the experiment with several
dilutions of the matrix to determine if the effect can be diluted out. As long as the
dilution is considered reasonable, such that the limit of detection in the sample is still
acceptable, than this dilution can be applied to all unknowns to be analyzed. If the
matrix cannot be diluted out appropriately, then a cleanup will be necessary. After
employing the cleanup method, the extract should be tested as above.
2). If an appropriate "blank" sample is not available, a sample can be used, preferably one
in which the analyte is present in very low levels. The method of standard additions is
then recommended for evaluating the effect of matrix (Miller & Miller, 1984). Briefly, in
this method the sample is split and one split is fortified with a known concentration of
analyte. If the concentration determined for the unknown in one split is subtracted
from the concentration determined in the split that was fortified, then the result should
be the level of the fortification. If it is not, then a matrix effect may be assumed, and a
cleanup may be necessary.
3). An alternative to cleanup in each of the above cases, would be to run the standard
curve in the matrix of interest, so that the influence on quantitation would be
normalized. For example, if methanol is needed at 30% in order to solubilize the
compound, the standard curve should be run in the presence of 30% methanol to
normalize for its effects.
4). Another way to verify the integrity of the data and determine if there is a matrix effect
is to analyze the sample at several dilutions. The dilutions chosen should result in
absorbances that lie on the linear portion of the standard curve. A plot of the
absorbance obtained from each dilution should be parallel to the slope of the
calibration curve in the absence of matrix effects (Figure 12).
62
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Q)
(0
•e
o
tf)
OS
0.6
0.4
0.2
Concentration of Analyte
Figure 12. Comparison of dilution curves to assess a matrix effect. Line A is the calibration curve.
Line B is a sample which has been analyzed at several dilutions. The non-parallel line indicates an
effect of the matrix. Line C is a sample whose dilution curve shows no effect of the matrix.
References:
Miller, J.C. and Miller, J.N. 1984. Statistics for Analytical Chemistry, Ellis Norwood, Ltd.,
Chichester, England, pp. 100-102.
63
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5.4 DATA ANALYSIS GUIDELINES ^
The data resulting from the protocols given in this manual are in the form of absorbance
values. Each sample is assayed at several dilutions. There are usually four well replicates of
a given dilution of a sample. Each concentration of standard is also assayed in four well
replicates. The following is one way to systematically evaluate the data.
Calibration curve and quality control samples:
1). Is the variation in absorbance among well replicates acceptable? Check the mean and
coefficient of variation, if the coefficient of variation is above an established value,
evaluate each well absorbance. Record outliers elsewhere.
2). Are the parameters of the standard curve and the shape of the curve acceptable?
3). Do the parameters of the quality control standards fall within acceptable standards?
Samples (unknowns):
1). Are the variation in absorbance among well replicates for a sample acceptable?
Check the mean and coefficient of variation, if the coefficient of variation is above an
established value, evaluate each well absorbance. Record outliers. The coefficient of
variation may be larger for samples than standards.
2). Do the dilutions of the sample show less inhibition with increasing amount of dilution? JB*
3). Are the absorbances within the linear portion of the standard curve?
4). If the absorbance values fall near the upper (lower limit of detectability) or lower
(complete or near complete inhibition) asymptotes of the 4-parameter fit, there will be
more variation in the calculated values. The closer the absorbance is to the
absorbance of the concentration at the IC50, the more reliable the resulting value.
Assay performance parameters:
The precision, accuracy and sensitivity should be described in the protocol that
accompanies the method.
Outliers are often a result of pipetting errors or poor plate performance. There are a
number of methods for assessing outliers that can be found in any statistical manual. For
example Dixon's Q test (Miller & Miller, 1984). As a first pass, rapid assessment, a typical
rule would be to identify absorbances which lie outside two standard deviations of the mean.
In dealing with a new assay or matrix, it will be important to verify a portion of positives
and negatives by a second Independent method, until the analyst has full confidence in the
method.
False positives - more likely because any perturbation of the system usually results in
decreased signal, resulting in a false positive.
64
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False negatives - rare if the characteristics of the method are well known, i.e. ICSO and
lower limit of detectability. Realize that these will change from matrix to matrix as it does with
any analytical technique.
Limit of detection - The limit of the detection for the assay may vary from matrix to
matrix. A detection range should be given in the protocol.
When analyzing a sample near the detection limit, one may find that the final
concentration value does not agree from dilution to dilution. As the dilution factor increases,
the apparent concentration of analyte increases. This is likely an affect of the dilution factor
multiplication. It is important to look at the absorbances and assure that they are in the linear •
portion of the standard curve.
Notes on the 4-parameter curve fitting model:
As with any analytical technique, the generation of a reproducible standard curve with
minimal error is critical. The standard curves generally resulting from immunoassays are
sigmoidal in shape. If the choice of standards provides a complete definition of the shape of
the curve, (i.e., the curve has at least 2 to 3 points each defining the upper and lower
asymptote and at least 4 points defining the linear region), the 4-parameter fit of Rodbard
(1981) is the method of choice for data analysis in the authors' laboratories. It is important
that enough standard concentrations are used to ensure that the curve is well defined and
constant for these concentrations. Without this information, the computer could force an
improper fit (Gerlach et al., 1993). The equation for the 4-parameter fit is:
y = (A-D)/(1 + (x/C)AB) + D
where y is the absorbance, x is the concentration of analyte, A and D are the upper and lower
asymptotes respectively, B is the slope and C is the central point of the linear portion of the
curve, also known as the IC50 (Figure 13).
O
B-
Q>
a
o
0> -
I I I I
Log Concentration
Figure 13. Model 4-parameter calibration curve.
65
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The best quantitation of unknowns is carried out when unknown absorbances fall in the
central portion of the linear region of the calibration curve. The use of the 4-parameter fit
extends the usefulness of the upper and lower concentrations of the calibration curve.
However, the values calculated from these upper and lower concentrations have greater error
associated with them. To save on reagents, and to keep the error on the estimation of
concentrations of unknowns to a minimum, concentrations for standard curves should be
performed in the linear range after the complete standard curve has been defined with upper
and lower asymptotes. A semi-log curve fit should then be used to fit the data to this
truncated calibration curve and the absorbance values for unknowns should fall in the central
portion of the linear region of this calibration curve. If a kit is being used, the package insert
should indicate the standard curve analysis method to use based on the range of standard
concentrations used for the calibration curve.
66
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5.5 OPTIMIZATION OF REAGENT CONCENTRATION
BY CHECKERBOARD TITRATION
The titer of the reagent refers to the dilution required to give a reasonable signal (i.e.
0.5-1.0 absorbance units) in your assay system. This procedure is recommended when
evaluating "component reagents" that have not specifically been designed to be delivered to
the user in a kit format. The specific reagent to titer will depend on the assay format. In
general, when you receive reagents for evaluation, a suggested dilution or concentration will
be given for each reagent. Due to the potential for degradation during shipping or storage, it
is usually recommended that a checkerboard titration experiment be performed to assure that
the reagent concentrations suggested are adequate. The checkerboard titration described
here tests two reagents simultaneously. Checkerboard titrations are also therefore called two
dimensional titrations. The steps in the checkerboard titration are performed identically to that
of the immunoassay tutorial for the specific compound, except that no inhibitor is used. For
the checkerboard titration the reagents are added at varying concentrations.
For the antigen-coated plate format such as tutorial 4.3 or 4.4, the reagents to titer
would be the coating antigen and the anti-analyte antibody. One could also titer the "second
antibody". This usually is unnecessary as the amount used is in excess. However, to save
on reagent, one could titer the "second antibody" to reduce the amount to the minimum
needed to assure proper assay performance.
For the antibody-coated plate formats such as tutorials 4.1 and 4.2, the reagents to
titer would be the anti-analyte antibody and the hapten-enzyme tracer. One could also titer
the "trapping antibody". This is not usually necessary as the amount used is in excess.
However, to save on reagent, one could titer the "trapping antibody" to reduce the amount to
the minimum needed to assure proper assay performance.
A
a
c
o
E
F
G
H
(2 34 5679 9 10 II 12
20 u
lOu
5 ug
2Si
125
062
031
Oug
MM.
4mL
nL
D/ml.
ug/mL
ug/ml
ug/mi
mL
»
»
•
•
>•
Figure 14. Example protocol for titer determination: coating antigen (antigen-coated plate format) or
anti-analyte antibody (antibody-coated plate format). Each concentration is added to the 12 wells in the
row.
67
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Procedure:
Antigen-coated plate format:
1. Coat the plate. Each well of the microtiter plate has added to it a fixed amount of
coating antigen. To test the range of the liter, the coating antigen is applied to the
plate at several different concentrations. (See Figure 14). The reagent dilutions are
made in coating buffer and added to each well of the microtiter plate as specified in the
specific assay procedure. Cover the microtiter plate with a plate sealer and place in
the refrigerator overnight.
2. Prepare the anti-analyte antibody. Anti-analyte antibody will also be added to the plate
at several concentrations (Figure 15). Make a dilution of anti-analyte antibody in PBS-
Tween/Azide. These solutions may be prepared the day of the titration or the night
before and left at room temperature while the coated plates are incubating in the
refrigerator.
A
a
c
o
e
f
G
H
1 1 3 4 5 6 7 9 » 10 11 12
e
-
—
i
Mj
8
1
i
i
I
1
-
i
a
i
i
1
F
i
i
1
5
3
8
i
r
i
.j
i
8
§"
I
§*
1
S"*
_i
Figure 15. Example protocol for titer determination: anti-analyte antibody (antigen-coated plate format)
or enzyme labelled hapten (antibody-coated plate format). Each dilution is added to the 8 wells in the
column.
3. On the following day, remove the microtiter plate from the refrigerator. Wash the
microtiter plate 3-5 times with PBS-Tween/Azide and tap dry. This is termed the
"coated" plate. The wash procedure involves flooding each well with buffer repeatedly
to remove unbound reagents.
4. Add 50 nL of the appropriate dilution of anti-analyte antibody solution to each well of
the coated microtiter plate as shown in Figure 15. Cover the microtiter plate with a
plate sealer.
5. Incubate for 1-2 hr. at room temperature. (Use the same incubation time as used in
the tutorial for the specific method.)
68
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6. At the end of the incubation, remove plate sealer and wash the coated plate 3-5 times
with PBS-Tween/Azide. Dry thoroughly by tapping on a paper towel.
7. Add 50 |il_ of goat anti-rabbit IgG conjugated to alkaline phosphatase to each well of
the coated plate. This solution is prepared by diluting goat anti-rabbit IgG conjugated
to alkaline phosphatase with PBS-Tween/Azide to 1/2500 or 1/5000 as recommended
by the supplier. (Use the same concentration recommended in the specific procedure.)
Note: If the source of the anti-analyte antibody is a mouse, use goat anti-mouse IgG
conjugated to alkaline phosphatase.
8. Cover the microtiter plate with a plate sealer and incubate for 1-2 hr at room
temperature. (Use the same incubation time as used in the tutorial for the specific
method.)
9. Remove an aliquot of the substrate buffer, 10% diethanolamine, pH 9.8 from the
refrigerator to allow equilibration to room temperature. Do not add the p-nitrophenyl
phosphate until just before use in step 11. Protect from light prior to use.
10. After the incubation, remove the plate sealer and wash the coated plate 3-5 times with
PBS-Tween/Azide. Dry thoroughly as above.
11. Add 100 (O.L of substrate solution to each well of the coated microtiter plate. Substrate
solution is 1 mg p-nitrophenyl phosphate/ml 10% diethanolamine buffer, pH 9.8 for the
enzyme label alkaline phosphatase.
12. Incubate at room temperature about 30 minutes or until the desired color is obtained.
13. Read on the plate reader (spectrophotometer) at 405-650 nm (for alkaline
phosphatase) as indicated in the specific immunoassay tutorial.
Data analysis.
1. Plot the absorbance on the Y axis and the concentration of coating antigen on the X
axis for each concentration of antibody. You will obtain a family of curves such as the
ones shown in Figure 16. The absorbances should increase linearly for any one curve
until reaching saturation.
2. Select the coating antigen concentration at which the absorbance no longer increases.
This is the concentration of coating antigen that will trap all of the antibody added to
the well. From Figure 16 this would be about 2.5 ug/mL Select the antibody dilution
for the above coating antigen concentration that gives a reasonable absorbance,
preferably between 0.5-1.0.
69
-------
Absortaance
0.8
234
Coating Antigen Concentration (ug/mL)
Figure 16. Plot of checkerboard titration data.
In Figure 16 this
would be the 1/1000
antibody dilution.
Larger absorbances
may give a larger
signal to noise ratio.
If increased
sensitivity is desired,
the coating antigen
concentration may be
decreased into the
linear area. As long
as the signal is
strong and the assay
conditions (time,
temperature) are held
constant, this often
results in more
sensitive assays.
Monitoring the enzyme
reaction over time
(kinetic readings) rather
than an endpoint mode should
be used under these conditions.
Alternatively, plot the absorbance
on the Y axis and the
concentration of antibody on the X
axis for each concentration of
coating antigen. You will obtain a
family of curves such as the ones
shown here (Figure 17). The
absorbances decline as the
antibody concentration decreases.
This is representative of what
would happen in the presence of
inhibitor. That is, if inhibitor is
present, there would be less
antibody in the well, thus the
absorbance would decrease. Ideal
data would show a steep slope.
Thus for small changes in the
amount of antibody inhibited, there
would be a significant change in
absorbance. From Figure 17 using
a coating antigen concentration of
1.25 ug/mL and an antibody dilution of 1/3000 would be ideal.
Select the coating antigen concentration that gives a strong signal and the antibody
dilution with a steep slope.
I
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
2468
Reciprocal Antibody Dilution X 1000
10
Figure 17. Plot of checkerboard titration data.
70
-------
5. These are tne concentrations that will be used for the ELISA when it is used to
measure a target compound in a competitive assay.
Antibody coated plate format:
1. Coat the plate. Each well of the microtiter plate has added to it a fixed amount of
trapping antibody (i.e. for tutorial 4.1, the trapping antibody is coated at 1/2000). Cover
the microtiter plate with a plate sealer and place in the refrigerator overnight.
2. For the single antibody-coated method, prepare the anti-analyte antibody. Anti-analyte
antibody will be added to the plate at several concentrations. Make a dilution of anti-
analyte antibody in PBS-Tween/Azide (in coating buffer if this a double antibody-coated
format). These solutions may be prepared the next day or the night before and left at
room temperature overnight while the coated plates are incubating in the refrigerator.
3. On the following day, remove the microtiter plate from the refrigerator. Wash 3-5X with
PBS Tween/Azide and tap dry.
4. Add 50 (J.L of the anti-analyte antibody solution to each well of the microtiter plate as
shown in Figure 14. Cover the microtiter plate with a plate sealer.
5. Incubate for 1-2 hr. at room temperature. (Use the same incubation time as used in
the tutorial for the specific method.) For the double antibody-coated method, solutions
are prepared in coating buffer and incubated in the refrigerator overnight.
6. At the end of the incubation, remove the plate sealer and wash the microtiter plate 3-5
times with PBS-Tween/Azide. Dry thoroughly by tapping on a paper towel.
7. Add 50 |iL of enzyme labeled hapten to each well of the microtiter plate in various
dilutions in PBS-Tween/Azide. (See Figure 15).
8. Cover the microtiter plate with a plate sealer and incubate for 1-2 hr at room
•temperature. (Use the same incubation time as used in the tutorial for the specific
method.)
9. Take the substrate buffer (10% diethanolamine, pH 9.8) out of the refrigerator to allow
equilibration to room temperature.
10. After the incubation, remove the plate sealer and wash the microtiter plate 3-5 times
with PBS-Tween/Azide. Dry thoroughly as above.
11. Add 100 |o.L of substrate solution to each well of the microtiter plate. Substrate solution
is 1 mg p-nitrophenyl phosphate/ml 10% diethanolamine buffer, pH 9.8 if the enzyme
label is alkaline phosphatase.
12. Incubate at room temperature about 30 minutes or until the desired color is obtained.
13. Read on the plate reader at 405-650 nm for alkaline phosphatase.
71
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Data analysis. ^
1. Plot the absorbance on the Y axis and the concentration of enzyme-labeled hapten on
the X axis for each concentration of antibody. You will obtain a family of curves that
looks like Figure 17.
2. Examine the antibody dilution curves with the steepest slopes.
3. Select the enzyme-labeled hapten dilution for the antibody dilution that gives a
reasonable absorbance, preferably between 0.5-1.0. One should also evaluate the
signal to noise ratio (i.e. the absorbance of the zero analyte standard/absorbance of
the blank). Since background or the absorbance of the blank is usually <0.10, larger
absorbances are desirable due to the larger signal to noise ratio.
4. The concentrations chosen from the checkerboard titrations will be used for the ELISA
when it is used to measure the target compound in a competitive assay.
72
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5.6 RECORD KEEPING
The following is a guideline for the keeping of records generated from immunochemical
analysis in an academic laboratory. Procedures may be altered to comply with regulations
pertaining to your particular laboratory.
All raw data, whether generated by hand or by a software program designed for
specific output, are recorded as hard copies, referenced to a raw data file name and placed in
the notebook. Raw data files are also kept on floppy disks which are catalogued in binders to
include the individual filenames. All processed data are recorded as hard copies and placed
in the notebook. All experiments will be of a quality acceptable for publication in a referred
journal in the appropriate field, i.e. Analytical Chemistry, Journal of Agriculture and Food
Chemistry, etc.
Typically, software programs associated with data collection and analysis for
immunoassay generate a large number of pages of output. To maximize efficient use of the
notebook the following suggestion is made for data output management:
1). Hard copy data: The hard copy should be given a unique page number and placed in
a looseleaf binder. The notebook should refer to the hard copy data by looseleaf
binder number and page number and the filename (if available). The hard copy in turn
should have a reference to the page in the notebook. Periodically the loose pages
should be permanently bound.
2). Electronic data: Spreadsheet data, microtiter plate reader software data, etc. can be
archived on diskette. The file should have a descriptor attached which identifies the
notebook page. The notebook should have the filename.
3). Reagents (i.e. antibodies, haptens, etc.): All reagents are labeled with a notebook
number and page number, providing a unique identifier.
Guidelines for Entries:
1). Make entires in permanent ink.
2). Use consecutive pages.
3). Date entries.
4). Identify subject matter.
5). Include sketches, diagrams, etc.
6). Explain sketches, etc.
7). Photos, drawings, etc., should be identified and permanently attached.
8). Avoid erasures.
9). Do not change entry; make new entry.
10). Periodically have quality assurance officer, laboratory manager or project leader look
73
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5.7 PREPARATION OF BUFFERS FOR USE IN ELISA ^
All buffers are prepared with double distilled water. These procedures are given with
sodium azide added as a preservative. If the buffers are used rapidly, or stored in the
refrigerator, sodium azide may be omitted. If utilizing horseradish peroxidase as the enzyme
label, it is best to omit the sodium azide, as it is inhibitory to the enzyme. In this case sodium
ethylrnercurithiosalicylate may be substituted (0.005%). PBS-Tween is recommended as the
wash buffer and for making all dilutions involving antibody or sample. It may be possible to
substitute plain water containing Tween 20 for the wash step. Tween 20 is mandatory in the
buffers to minimize non-specific binding. The following materials are given as examples.
Similar reagents from other vendors may be appropriate. Sources are listed here as a
convenience to the reader.
1) Substrate Buffer for Alkaline Phosphatase (STORE REFRIGERATED).
97 ml diethanolamine (Aldrich)
0.2 g NaN3 (Adrich)
0.1 g MgCI2.6H20 (Fisher), is a cofactor for the enzyme
Bring to 800 ml with distilled water, adjust pH to 9.8 with 6N HCI.
Bring to final volume of 1L. Check pH after prolonged storage.
2) 10OX Tween/Azide (STORE AT ROOM TEMPERATURE)
2%NaN3(10g)
5% Tween 20 (25 ml) (Sigma, polyoxyethylene-sorbitan monolaurate)
Bring to 500 ml with distilled water (Tween 20 is a detergent; add water slowly to
limit foaming.).
3) Coating Buffer (STORE REFRIGERATED)
0.795 g Na2CO3
1.465 g NaHCO3
0.1 g NaN3
Dilute to almost 500 ml with distilled water, adjust pH to 9.6 and bring to final volume
of 500 ml_. Check pH after prolonged storage.
4) 10X PBS (Phosphate Buffered Saline) (STORE AT ROOM TEMP)
640 g NaCI
16 g KH2PO4
91.96g Na2HPO4
(Add this slowly while stirring to prevent clumping of salts.)
16gKCI
Bring to approximately 7L with distilled water. Stir well until all salts dissolve.
Adjust pH to 7.5 and bring to final volume of 8L
5) 1X PBS-Tween/Azide (STORE AT ROOM TEMPERATURE)
800 ml_ 10X PBS (#4 above)
Bring to approximately 7L with distilled water.
80 mL 100X Tween/Azide (#2 above, add after most of water has been added to avoid
foaming due to Tween 20), adjust pH to 7.5 if necessary, then bring to final volume of
8L
74
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6) Substrate Buffer for Horseradish Peroxidase
Citrate-acetate buffer
13.61 g Sodium citrate (100 mM)
Bring to approximately 1L with distilled water.
Adjust pH to 5.5 with acetic acid.
1 % hydrogen peroxide
1 ml 30% H2O2 in 29 ml distilled water
Store in plastic container in the refrigerator.
0.6% 3,3'5,5'-Tetramethylbenzidine (1MB)
60 mg in 10 ml dimethylsulfoxide
Store at room temperature in the dark.
Just prior to use prepare the final substrate buffer by mixing:
0.4 mL 0.6% TMB in DMSO
0.1 ml 1% hydrogen peroxide
in 25 mL citrate-acetate buffer
Make sure buffer and TMB are at room temperature before mixing to avoid
precipitation.
75
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5.8 Preparation of Calibration Standards
and Samples Using the 8 X 12 Array
To facilitate the preparation of calibration standards and sample dilutions as well as
their transfer to the coated plate, the 8 X 12 microtiter plate array is a useful tool. In most
protocols, the analyst will prepare all the standards and samples prior to adding them to the
coated plate. Since adding reagents to the coated plate begins the equilibrium reactions, it is
desirable to add all the reagents as rapidly and uniformly (from plate to plate) as possible.
Using this array, the 8 or 12-multichannel pipettor can be used to prepare dilutions and rapidly
transfer these to the coated microtiter plate. A layout for a typical array is shown in Figure 18.
In this design, a single 8X12 array preparation of standards and samples is enough for four
coated plates (seen in step 2). This array is also useful in protocols which require a
preincubation step of the analyte and antibody prior to addition to the coated plate. In the
example below the 8x12 array is used not only for the transfer of reagents to the coated
microtiter plates, but also to prepare dilutions of the standards and samples. Alternatively,
standards and samples can be prepared volumetrically and transferred to the 8x12 array for
rapid transfer to the coated plates.
Step 1: Blanks (BLK), calibration standards (S01-S08) and samples (1A-20C) are added to a
sample preparation array. If serial dilutions of the sample or standards are used, these may
be done directly in the sample preparation array. For example, to prepare sample 1 in
dilutions of 2, 4 and 8 (1A, 1B, 1C, respectively), a volume of assay buffer is added to wells
A10, A11 and A12 of the sample preparation array. An equal volume of sample 1 is placed in
A10. After mixing, the same volume is transferred to well A11. This step is repeated for A12.
Using a multichannel pipettor, similar serial dilutions could be made simultaneously for other
samples in the column. This technique then can be expanded to the whole array.
Step 2: These four drawings, represent four coated microtiter plates. The standards and
samples from the sample preparation array (A1-A12) are transferred simultaneously using the
multichannel pipettor to rows A1-A12 on the Coated El A Plate 1. This step is repeated for
rows B1-D12 on the coated plate resulting in four well replicates from a single standard or
sample dilution. Similarly, Row B1-B12 is added to the bottom half of Coated EIA Plate 1.
76
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9 10 11 12
A
B
C
D
E
F
G
H
8LK
2A
BLK
7A
BLK
12A
BLK
17A
S01
23
S01
78
S01
12B
SO1
178
S02
2C
S02
7C
S02
12C
S02
17C
S03
3A
S03
8A
S03
13A
S03
18A
304
3B
304
8B
304
138
S04
18B
SOS
3C
SOS
8C
SOS
13C
SOS
18C
S06
4A
S06
9A
S06
14A
S06
19A
SO?
48
S07
9B
S07
14B
S07
198
SOS
4C
S08
9C
508
14C
308
19C
1A
5A
6A
10A
11A
1SA
16A
20A
18
SB
6B
10B
118
15B
168
20B
1C
5C
6C
10C
11C
15C
16C
20C
COMPETITIVE INHIBITION PLATE
1 2 3 4 5 3 7 t 3 10 11 12
12345979 9 10 11 12
B?mWIF!1
L12
COATED EIA PLATE 1
1 2 1 4 5 8 7 S 9 10 11 12
COATED EIA PLATE 3
12345373 S 10 11 12
ROW
D) tl-
12
DlA
COATED EIA PLATE 2
COATED EIA PLATE 4
Figure 18. Schematic of the 8X12 array for sample preparation.
77
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5.9 OUTLINE FOR A QUALITY ASSURANCE DOCUMENT
FOR USING IMMUNOASSAY METHODS
The following outline is to provide guidance to the reader in the development of a
quality assurance document once immunoassay methods have been added to the laboratory
methodology. It should be noted that many of the elements of the quality assurance
document are the same as may already exist for other analytical methods.
Contents:
Section 1. Introduction
General overview of the project
Objectives of the project
Section 2. Project Description
Project schedule chart
Section 3. Project Organization
Management structure
Responsibilities of participants
Section 4. Quality Assurance Objectives
Seven elements of data quality
Section 5. Quality Assurance and Quality Control Protocols
Quality control protocols for the immunoassay method
Quality control protocols for the confirmatory method
Quality control protocols for the extraction/cleanup procedures
Analysis of key samples
Section 6. Laboratory Operations
Laboratory quality assurance policies
Instrument maintenance and calibration
Standard operating procedures for cleanup, detection, and analysis
Sample handling
Section 7. Data Handling
Section 8. Assessment of Data Quality
Audits and review of data quality
Statistical evaluation of the data
Section 9. Quality Assurance Reports to Management
Briefings and status reports
Final project reports
78
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5.10 GUIDELINES FOR THE EFFICIENT USE OF
96-WELL MICROTITER PLATES
A
B
C
D
E
F
G
H
12345678 9 10 11 12
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5.11 GENERAL TROUBLESHOOTING GUIDELINES TO OPTIMIZE
THE ENZYME IMMUNOASSAY METHOD PERFORMANCE
Troubleshooting is probably the most useful skill that any analytical chemist can
develop. It is beyond the scope of this manual to provide a complete troubleshooting guide.
However, we can list the most common symptoms and best responses. When evaluating test
kits or component assays, it is best to keep open lines of communication with the supplier in
order to answer questions and provide assistance in troubleshooting.
Table 1. Troubleshooting Guidelines to Optimize the Enzyme Immunoassay Method
Performance.
Symptom
Poor well to well replication8
Low or no color development0
Color development too high
Cause
Poor pipetting technique
Poor binding plates"
Coating antigen or antibody is
degrading
Coated plates stored too long
Poor washing
Uneven temperature in the
wells
Sample carryover
Loss of reagent integrity
Incubation temperature too
coldd
Sample matrix effect
incubation too long or
temperature too high
Remedy
Check instrument, see tutorial
on pipetting, practice, calibrate
pipettor
Check new lot, change
manufacturers
Use new lot of coating reagent
Discard plates, coat a new set,
decrease storage time
Wash plates more, or more
carefully, remake buffer
Deliver reagents at room
temperature, avoid large
temperature fluctuations in the
room
Watch for potential carryover in
pipetting and washing steps
Systematically replace or check
reagents, including buffers
beginning with the enzyme
label
Lengthen incubation time or
increase temperature by using
a circulating air-temperature
controlled incubator (particularly
a problem if working in the
field)
Dilute matrix if possible, check
pH of matrix, increase the ionic
strength of the buffer
Decrease incubation time or
temperature
80
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Table I (con't)
Symptom
Change in calibration curve
parameters
High background
High plate to plate variation
Cause
Matrix effect
Degradation of reagents
For short assay times,
incubation too long
Incubation too long, favored
nonspecific binding
Used too high reagent
concentrations, favored
nonspecific binding
Poor uniformity of coating
Non uniform binding plates
Poor pipetting technique
Remedy
Dilute matrix or re-evaluate
matrix effects
Systematically check or replace
reagents, including buffers
Monitor incubation times
carefully
Monitor incubation times
carefully
Make sure the correct reagent
concentrations are used
Use new aliquot of coating
antigen or antibody
Choose new lot of plates
Check instrument, see SOP on
pipetting, practice
aA common problem in immunoassay is poor coefficient of variation on well replicates,
or spurious color development. Plate washing and pipetting are the largest contributors to
spurious color development.
The characteristics of the 96 well plate used is an important factor. Some plates will
bind antigens differently, some have greater variability in binding capacity from well to well
which would contribute to variability. Generally, the best solution is choosing a manufacturer
whose plate performance characteristics for that assay are constant.
°No or low color development is most likely due to a reagent failure. The most
common reagent to fail is the enzyme label. There are two potential problems; first that the
enzyme has lost activity; second that the conjugate has degraded and can no longer bind
efficiently to the antibody. The first case is easy to check. Dilute the conjugate about 2-5X
more than used for the assay. For example, if the method calls for a 1/2500 dilution of the
enzyme label, then make dilutions of 1/5,000 or 1/10,000 or greater. Add the substrate
solution directly to the enzyme dilution and incubate for the period of time indicated in the
method. The color development should be similar to that obtained in the assay. If the color
development is lower or there is any doubt, the enzyme label reagent should be replaced with
a new aliquot. The second case can only be remedied by replacing the reagent.
dAnother significant factor is temperature. The reactions that are occurring on the plate
are based on the Law of Mass Action. They are therefore equilibrium reactions, and are
sensitive to temperature. Reagents should be used at room temperature and during analysis
plates should be protected from wide fluctuations in temperature (i.e. try to perform all steps of
the assay at the same ambient temperature each time). If an incubator is used or the ambient
temperature is high, there may be a problem with uneven heating in the wells. With the 96-
well plates, the tendency is for the outer wells to reach temperature sooner than the inner
81
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wells, which then has an effect on the equilibrium reactions. Variations in final absorbances
are generally manifested in what is called an "edge effect". Use of a forced-air incubator can
reduce this problem. Temperature related effects on equilibrium are more likely to be seen in
assays whose incubation times are very short.
"Blocking refers to the process of covering active sites in the microtiter plate well that
may not have reacted with the coating protein (i.e. coating antigen or coating antibody). To
conduct a blocking step after the normal coating procedure, the plate is washed and
100|tiL per well of a protein such as bovine serum albumin (BSA) is added. The amount of
BSA to add to the well will depend on the degree of blocking needed. Several experiments
may need to be run in which the concentration of BSA is varied. The lowest concentration of
BSA at which the background remains low and constant should be used. A commonly used
concentration is 0.1% BSA.
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5.12 MAINTENANCE AND PERFORMANCE VALIDATION
OF A 96-WELL MICROPLATE READER
The analyst should be familiar with the manufacturer's recommendations for operation
and maintenance of the reader. The manufacturer will often describe procedures for
calibration and validating performance. Tests such as checking the linearity of the response
to the chromophore, and repeatability of readings are most commonly used. The "reversed
wet plate test" (Harrison et al., 1988) can also be used to test for bias in the reader design.
General Maintenance Tips
1). Instrument should be kept clean and as dust free as possible. If a spill occurs on the
exterior, wipe off immediately. If a spill occurs on the interior, refer to the maintenance
manual for cleaning procedure.
2). Keep the drawer or platform which holds the microtiter plate closed and the instrument
covered to protect from dust when it is not in use.
3). Do not move or obstruct the automatic movement of the microtiter plate holder.
4). Before a reading, make sure the bottom of the microplate is clean and dry. The
bottom of the plate must be clean and dry to prevent distortion of the reading and to
prevent contamination of the optic device.
5). Determine the direction of the light path for the reader. For some readers the light
path is from the bottom of the plate through the solution. Thus the distance of the light
path is the distance from the bottom of the well to the meniscus of the solution. In this
case, it is very important that pipettors be calibrated, as the volume of the solution is
the critical determinant of the light path.
6). Analyst should be familiar with error messages which may be encountered as a quick
response may be necessary in order to prevent loss of the data.
Performance Validation:
All laboratory instruments and other equipment used in an immunoassay analysis
should be maintained in proper working order. For the spectrophotometer used to read the
96-well microtiter plates, performance and calibration checks should be done on a semi-
annual basis. As an extra precaution, the performance of key instruments should be tested by
spot checks prior to initiating a large project. In most cases, the protocols for performance
checks and calibrations are contained in the user manual for a particular instrument. If these
are not available, performance protocols must be developed to ensure adequate instrument
performance. For example, the "reverse wet plate test" of Harrison et al. (1988). These
protocols should be kept in a 3-ring binder or folder along with the instrument manual.
Records of instrument calibration, maintenance and repair should also be kept in 3-ring
binders and stored in a secure location with the instrument manuals. If necessary a schedule
for more routine performance checks or calibrations should be developed and kept with the
instrument records.
83
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References
Harrison, R.O. and Hammock, B.D. 1988. Location dependent biases in 96 well microplate
readers. J. Assoc. Off. Anal. Chem. 71:981-987.
i
84
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5.13 PERFORMANCE CHECKS, CALIBRATION AND MAINTENANCE
OF AIR DISPLACEMENT PIPETTORS
This section refers to both the single channel and multichannel air displacement pipettors.
Calibration
Micropipettors should be checked for accuracy and precision using the gravimetric method.
Most manufacturers provide a description in the instruction manual. Briefly, the pipettor is set to
the test volume. Distilled water is pipetted into a pre-weighed beaker. The weight of the water is
recorded. This procedure is repeated at least 5 times and the readings averaged. The mean and
standard deviation should match the performance criteria given by the manufacturer. If the
performance criteria are not met, the procedure for calibration will be specified by the
manufacturer. When necessary, pipets should be returned to the manufacturer's service
department for cleaning, recalibration and replacement of worn parts. See Appendix I for details
on principles of performance assurance for air displacement pipettes and Appendix II for blank
sample performance assurance log and worksheets.
General maintenance
Pipettes should be checked daily for dust or other contamination on the outside surface of
the pipettor as well as for splits, cracks or chips in the surface. For procedures for general
maintenance or cleaning of the interior of the pipette, see the manufacturer's instruction manual.
Troubleshooting:
Table 2. Troubleshooting Guidelines for the Use of Air Displacement Pipettors
Symptom
Sample leaks from the tip
Inaccurate dispensing
Inaccurate dispensings with
certain liquids
Liquid is an organic solvent
Cause
Tips not attached correctly
Pipet tip is dirty. Interior of
pipet shaft is dirty due to
sample splash.
Incorrect operation by user
Pipet tips have not been pre-
wetted
Tips not attached correctly
Not in calibration
Interior parts of pipet dirty,
worn or damaged
Unsuitable calibration
Remedy
Attach tips firmly
Replace pipet tip, clean interior of
pipettor if instructions are given by
the manufacturer. See also tutorial
5.1.
See manufacturer's manual and
tutorial 5.1
Pre-wet tips by drawing up and
dispensing solution 2 or 3 times
Attach tips firmly
Recalibrate according to instructions
See manufacturer's instructions for
cleaning or replacement of parts
Some viscous liquids require that
pipettor be recalibrated
Change to a positive displacement
pipettor
General Note: These pipettors should not be used to deliver organic solvents. Positive
displacement pipettors should be used for this purpose.
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SECTION 6
Glossary of Commonly Used Terms In Immunoassay
A
Accuracy - the proximity of the value obtained to the 'true" value.
Adjuvant - a substance administered with the antigen to promote the immune response and
provide a carrier which depots the antigen for slow release. Examples: Freund's
adjuvant, RIBI adjuvant.
Affinity - the strength of the antibody recognition of the target molecule.
Amplification - a procedure which increases the signal of the assay so as to increase
detectability of the amount of bound antibody.
Analyte - the compound of interest for analysis.
Syn. target molecule.
Antibody (Ab) - refers to a group of immunoglobulins which will bind to an antigen.
Antigen (Ag) - a substance, usually a protein, which will elicit the production of specific
antibodies that will react with that substance.
Antigenic determinant - the smallest entity which can be recognized by an antibody.
Syn: epitope.
Antiserum - serum from an animal containing a group of immunoglobulins which will bind to an
antigen.
B
Bias - refers to data which varies in a predictable manner from the "true" value.
C
Carrier protein - the protein which has been covalently linked to the hapten. If the resulting
antigen is for immunizing, a protein foreign to the organism is useful. For example, we
use keyhole limpet hemocyanin as a protein to link to a hapten for an immunizing
antigen for mammals. For coating, we try to link to a protein which will have minimal
background binding to the antibody, smaller proteins such as bovine serum albumin
seem useful in this regard.
Checkerboard titration - an experimental design used in 96-well microtiter plates to optimize
for coating antigen and antibody concentrations. The coating antigen concentration is
varied by row and the antibody dilution is varied by column. The result is a plate which
gives increasing absorbance as you proceed diagonally across the plate, where more
absorbance is an indication of larger coating concentrations and/or smaller antibody
dilutions.
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Coating - the process of passively adsorbing the antigen, antibody, etc. to the solid phase
usually by noncovalent interactions at high pH.
Coating antigen - the antigen used in an ELISA which is bound to a solid phase such as a 96
well microtiter plate by noncovalent interactions.
Competitive inhibition - the process in which the target compound competes for specific
antibody.
Conjugation - the procedure which covalently binds the hapten to the carrier protein.
Syn: couple.
Couple - the procedure which covalently binds the hapten to the carrier protein.
Syn: conjugation.
Cross reactivity - the ability of compounds, structurally related to the target molecule, to bind
to the specific antibody.
Direct - an immunoassay method in which the primary antibody or an analyte mimic is labeled
so that the amount of label bound to the solid phase is directly measured.
Double antibody coated - assay format in which a trapping antibody is adsorbed ("coated") to
the solid phase, which subsequently binds or traps the analyte specific antibody.
Edge effects - the phenomenon of variability noted along the outer edges of a 96 well
microtiter plate. Most often due to uneven temperature during incubations or to poor
quality control of the microtiter plate manufacturer.
Enzyme immunoassay (EIA) - immunoassays which employ an enzyme as the label.
Enzyme linked immunosorbent assay (ELISA) - an immunoassay format in which the hapten
and coating antigen compete for the specific antibody and the amount of bound
antibody is detected by an enzyme labeled second antibody.
Syn: indirect EIA.
Enzyme tracer - the hapten covalently linked to the enzyme.
Epitope - the smallest entity which can be recognized by an antibody.
Syn: antigenic determinants.
First antibody - the antibody specific for the target analyte.
Syn: specific antibody, primary antibody
87
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Format - the manner in which the specific antibody, hapten, and target molecule are put
together for the resulting immunoassay.
H
Handle - the part of the antigen which binds the hapten to the carrier protein.
Syn: spacer, linker.
Hapten - a small molecule which cannot, by itself, elicit an antibody response but will
specifically react with an antibody.
Hapten density - the amount of hapten covalently bound to the carrier protein.
Syn: hapten loading.
Hapten loading - the amount of hapten covalently bound to the carrier protein.
Syn: hapten density.
Heterogeneous - refers to the immunoassay technique, where a separation step is required
between the bound and unbound antibody.
Heterologous - an immunoarray format in which one or all of the component reagents, e.g.,
hapten, linker, protein, differ between the coating antigen and immunizing antigen.
Homogeneous - refers to the immunoassay technique, where a step is not required to
separate bound and free antibody.
Homologous - refers to the immunoassay format, where the components are the same. For
example, an ELISA assay in which the hapten-linker combination is the same used for
coating and immunizing.
Hybridoma - the result of fusing an antibody producing spleen cell with an immortal myeloma.
The resulting hybridoma cells secrete specific antibody into the medium and can be
cultured in vitro due the properties conferred upon it by the myeloma.
I
IgG - immunoglobulin G, the most common subclass of immunogiobulins used, in
immunoassay.
Immunization protocol - the procedure used to inject animals with the goal of raising
antibodies.
Immunizing antigen - the antigen used when immunizing animals for the production of
antibodies.
Syn: immunogen.
Immunogen - synonym for antigen when emphasizing the ability of a compound to induce an
immune response.
88
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Indirect - an immunoassay method in which the amount of primary antibody bound is detected
by using anti-IgG antibodies which are usually labeled.
Label - is the technique used to visualize the bound antibody or to quantify the antibody-
antigen complex. May be an enzyme, radiolabel, fluorescent compound, etc.
Law of Mass Action - the physical law which governs immunoassay, i.e. that the antigen-
antibody interaction is a reversible one that comes to an equilibrium.
Linker- the part of the antigen which binds the hapten to the carrier protein.
Syn: spacer, handle.
M
Matrix - the substance which contains the target molecule for analysis.
Microtiter plate - polystyrene plates usually arranged in a 96-well format. Variability of the
assay may depend on the plate manufacturer, and even the particular lot.
Monoclonal antibodies- antibodies obtained from a specific clone of cells.
0
Optimization - systematic studies to select the most useful antibody, antigen, enzyme tracer,
etc. concentrations.
Plate reader - a spectrophotometer modified to read the absorbance in the wells of a 96-well
microtiter plate.
Polyclonal antibodies - antibodies obtained from the serum of an animal which contain
antibodies produced by many different cells.
Precision - the proximity of a value to the mean of a series of values obtained from repeated
measurement of the same sample.
Primary antibody - the antibody specific for the target molecule.
Syn: specific antibody, first antibody.
Radioimmunoassay (RIA) - an immunoassay in which the label is a radiotracer. Usually the
target molecule competes with labeled target for the antibody.
89
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Second antibody - in ELISA, it is the antibody against the IgG of the primary antibody and is
generally conjugated to an enzyme.
Serum - the fraction of blood obtained from the animal containing antibodies.
Single antibody coated - format in which a trapping antibody is adsorbed (coated) to the solid
phase.
Solid phase - Any support to which antigens or antibodies may be bound, for instance, 96 well
microtiter plates (polystyrene), polystyrene test tubes or nitrocellulose membranes.
Spacer - the part of the antigen which binds the hapten to the carrier protein.
Syn: handle, linker.
Specific antibody - the antibody which recognizes the target analyte.
Syn: first antibody, primary antibody.
Target molecule - the compound for which the immunoassay is being developed.
Syn: analyte.
liter - a description of the affinity of the antibody that can be variously defined. A good
definition for ELISA work is the greatest dilution that still gives an absorbance of about
0.3 at a defined coating antigen concentration and incubation conditions.
Tracer - the label used to detect bound materials.
Trapping antibody - an antibody directed against the IgG of the analyte specific antibody. For
example, an anti-mouse IgG would be the trapping antibody for a triazine antibody
raised in a mouse.
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APPENDIX I
PERFORMANCE ASSURANCE FOR AIR DISPLACEMENT PIPETTES
The use of the following material in no way implies endorsement of the manufacturer by
the authors or the U.S. EPA for the products mentioned. This document is excerpted here as
there are many general concepts regarding performance measurements for air displacement
pipettes that are applicable regardless of the manufacturer. The performance specifications
given for certain pipettes are used as examples of the type of performance specifications that
should be available for any air displacement pipette.
The following material has been excerpted with permission from "Performance Assur-
ance for Air Displacement Pipettes," AB-1, October 1988, prepared by Liquid Measurement
Quality Control, Rainin Instrument Co., Inc., Mack Road, Woburn, MA 01801-4628.
Performance Assurance
Performance assurance provides a means to monitor the performance of air displace-
ment pipetting instruments throughout their life, whether they are old or new. Each pipet
should be given a unique identifier, for example a serial number, to record their performance
history. A service record tells how long the instrument has been in use. A performance
logbook indicates the reliability of the instrument. Statistical data will aid in error analysis of
any test. With an accurate record of performance, the studies become more accurate and
more efficient.
Optimizing Liquid Measurement Performance
Optimum performance can be achieved by four easy steps:
1). Use pipet tips that have been designated for your specific pipette.
2). Reviewing instruction manuals and ensuring implementation of recommended operating
procedures can enhance pipette performance.
3). Regular maintenance and cleaning will aid in keeping instruments trouble-free.
Instructions for proper care and troubleshooting are found in each manual and provide
information on keeping the pipette in order.
4). Utilize manufacturer's service departments for cleaning, calibration and replacement of
worn or broken parts when appropriate.
Measuring Performance
Accuracy in liquid measurement is the closeness of a measured volume to the true volume
specified by the setting of the instrument.
The accuracy of a measurement is expressed in terms of its error, the difference between the
measured volume and the true volume. A small error indicates an accurate measurement.
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Two types of errors occur in liquid measurement:
Systematic error. This type of error is consistent. It represents the effect of instru-
ment calibration, operating characteristics, and conditions that are constant or that change
only in a consistent, predictable way during a series of liquid measurements. Systematic error
normally biases all measurements at a given volume setting toward volumes that are either
higher or lower than the true value.
Random error. This type of error is inconsistent and unpredictable, except for the
frequency with which errors of a given magnitude are likely to occur. Random error is
normally seen as scatter, a distribution of measured values around a most probable or mean
value. It represents the effect of uncontrolled short-term variables in the operation of a liquid
measurement system.
F
R
E
Q
U
E
N
C
Y
68.2%
. 95.4%
99.7%
-3o -2o -lo mean +10
+3o
Figure 1. Normal Distribution
The performance of a liquid measurement system at a given volume can be assessed
by performing a series of 10 to 30 measurements and determining the volume measured each
time by a method of much greater accuracy. The method most often used is weighing the
measured liquid on a properly calibrated electronic microgram balance with appropriate
corrections for liquid density and evaporation. If the frequency of occurrence is plotted vs.
measured volume for a large number of samples, a distribution similar to that in Figure 1
should be obtained.
-------
Two calculated statistical values are useful in summarizing and interpreting this type of
data:
Mean Volume. This is calculated from individual volume measurements as follows:
-
mean volume =V =-
i.e., "
1 . Add up the individual measurements.
2. Divide the total by the number of measurements.
Standard Deviation. This is calculated as follows:
standard deviation = a = \ —
n-1
i.e.,
1. Subtract the mean volume from each individual volume measurement and square each
result.
2. Add up the squared values.
3. Divide the total by one less than the number of measurements.
4. Take the square root of the result.
NOTE: Many pocket calculators and personal computer spreadsheet programs have
statistical functions that will perform both the mean volume and standard deviation calculations
automatically, once data values are entered. Using a calculator or a personal computer can
greatly simplify these calculations.
The statistical values have meanings, illustrated by Figure 1.
The mean volume is the high-probability point of the normal distribution of measured
volumes. In the scatter of volume measurements due to random error, volumes close to the
mean volume are much more likely to occur and will occur with much higher frequency than
volumes farther from the mean volume.
The standard deviation quantifies the magnitude of scatter due to random error. As
shown by Figure 1, the probability that an individual volume measurement is within one
standard deviation from the mean volume is 68.2%; the probability that an individual measure-
ment is within two standard deviations is 95.4%. Thus, in using the liquid measurement
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system at this volume setting, two out of three measurements are expected to fall within one
standard deviation of the mean volume, and 19 out of 20 are expected to fall within two
standard deviations of the mean volume.
Figure 2 illustrates the relationship between the normal distribution of measured
volumes and the specifications of the liquid measurement system at a given volume setting.
i
Figure 2. Measured Values vs. Specification
Mean error is the difference between the mean volume of actual measurements and
the true value of the volume set on the instrument. The accuracy specification provides an
upper limit to this mean error. Mean error is a measure of the systematic error component
of individual liquid measurements. The accuracy specification indicates that the instrument, as
delivered, is calibrated so that the mean volume will fall within the limit when the pipetting
system is used according to the instructions in the manual.
The precision specification is an upper limit on the standard deviation of measure-
ments performed at a given volume setting. It provides a limit to the amount of random error
that should be seen in individual liquid measurements as long as the pipetting system is
properly maintained and used in accordance with the recommendations of the manual.
As shown in Figure 2, the precision specification is normally much smaller than the
accuracy specification. This provides good assurance that the majority of individual measured
volumes will fall within the specified accuracy range. It also provides a high degree of
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assurance that accuracy checks based on four individual volume measurements can verify
proper instrument calibration. Without narrow limits on the scatter of individual measure-
ments, i.e., on standard deviation, many more data points would be necessary to check
performance.
Both accuracy and precision may be expressed as percent figures. When expressed
as a percent of nominal or set volume, the mean error figure is called percent error. When
expressed as a percent of the mean volume for a series of measurements, standard deviation
is called the coefficient of variation.
Methodology
In order to measure performance, a standard method is necessary. Standards
organizations such as the International Organization for Standardization (ISO) or the National
Committee for Clinical Laboratory Standards (NCCLS) provide standard guidelines to assess
the performance of a pipette. Here are some suggestions to develop your own program.
Calibrated Tips can easily be used on a daily basis to check the performance of an
instrument.
Set the instrument to one of the volumes of the calibration lines and aspirate a sample.
If the liquid level corresponds to the line on the tip, the system is operating properly. If the
liquid level fails to come close to the line, the instrument should be further tested on a
balance. The calibration marks on the tips are not accurate enough to be used for calibration
purposes, but they do provide some assurance of proper performance on a consistent basis.
The Gravimetric Method measures performance using an analytical balance with
distilled water as the standard. This primary method of analysis is recognized by ISO and
other organizations.
Balance. A regularly maintained microgram balance assures a good testing method
provided that the balance is calibrated with traceable weights. The following table gives
suggested weight classes from the National Bureau of Standards (NBS). Traceable weights
can usually be obtained through an authorized balance dealer or service representative.
Table 1. Weight Class Standards for Balance Calibration.
Test
Volume
1 (VL)
10
100
1000
Balance
Sensitivity
0.001 (mg)
0.01
0.1
0.1
Standard
Deviation
0.002 (mg)
0.02
0.1
0.2
Weight Class
NBS Certified
MorJ
S
S
SorS1
Weighing Accessories. The weighing vessel should not exceed 50 times the volume of
any sample to be measured. The vessel should be cylindrical so that liquid surface area will
stay fairly constant as it fills. A cover will be useful to minimize evaporative effects when
measuring smaller volumes. The cover should be loose-fitting for easy manipulation.
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The weighing vessel should be handled only with clean forceps or tweezers to prevent
errors due to deposition of moisture, dirt, oil and also to prevent heating. The balance should
be placed on a weighing table to control vibration.
Environment. The room in which testing occurs should be controlled for factors that
affect pipetting system performance and this method of measurement. This includes a draft-
free, dust-free environment with proper lighting (avoid direct sunlight). A constant temperature
(19 - 23°C / 66-73°F) should be maintained (± 1°C). The relative humidity should remain
between 45-75% in order to reduce evaporation and the build up of electrostatic potentials.
Testing Medium. Water is used as the standard in gravimetric analysis. The water
used should be distilled and gas free. It should be placed in the sampling reservoir at least
one hour ahead of time and allowed to come to a steady-state temperature in the test
environment. A thermometer (0.1 °C) will be needed to measure the temperature of the water.
Technique. Pipetting technique should follow the recommendations in the instrument
manual. In order to correct for evaporation during the weighing procedure, an amount of
water sufficient to fill the weighing vessel to a depth of at least 3 - 4 mm should be present at
the start of testing. Timing of all operations should be consistent between samples. Evapora-
tion blanks should be determined by performing the identical sequence of manipulations
without sample aspiration.
Evaporative cooling effects will create minor water-temperature differences depending
on depth and radial position in the sampling reservoir. A temperature difference of 0.5°C can
introduce additional variation on the order of 0.01% in sample measurements. For this
reason, in very rigorous testing to determine pipetting system precision, all samples should be
drawn at approximately the same depth and position in the sampling reservoir.
Calculations. To convert measured sample weight to volume, a density correction must
be applied. This correction depends on both the density of water and the density of the air in
the test area. (The density of air is used to adjust for the buoyancy of water in air, which acts
to decrease the apparent density of the sample.) The density correction is achieved by
multiplying the sample weight by a quantity called the "Z-factor." The Z-factor is the reciprocal
of the apparent density of water under the test conditions.
Because of evaporative cooling, the steady-state temperature of water in the sampling
reservoir will be lower than the air temperature, usually by two or three degrees. Also, the
density of air will vary slightly with barometric pressure and humidity.
These differences may be taken into consideration when calculating Z-factors.
However, the combined effects of these factors on Z-factor values are significant only at the
0.01% level, at least one order of magnitude below the level of discrimination needed to verify
accuracy of an air-displacement pipetting system. The Z-factor table provided ignores these
minor differences, assuming that the air temperature is the same as that of water in the
sampling reservoir and that barometric pressure is one atmosphere. The values in this table
are sufficiently accurate for routine performance assurance purposes.
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Correction for evaporation will also increase the accuracy of the data and is especially
important for small volume measurements. An evaporation blank is used to estimate the
amount of water lost during the test. This amount is added to the mean sample weight.
Table 2. Z- Factors
Water Temperature
(°C)
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
Z-Factor
(uL/mg)
1 .0020
1.0020
1 .0021
1 .0022
1 .0023
1 .0024
1.0025
1.0026
1 .0027
Water Temperature
(°C)
19.5
20.0
20.5
21.0
21.5
22.0
22.5
23.0
Z-Factor
(uL/mg)
1 .0028
1 .0029
1 .0030
1.0031
1 .0032
1.0033
1.0034
1 .0035
(Values from ISO Standard.)
The extent of an appropriate performance assurance program should be determined by
your needs. Organizations such as ISO, NCCLS, and ASTM all provide in-depth procedures
for volume analysis. Further documentation is available from those organizations upon
request.
Routine Testing
A routine test program is recommended for all air-displacement pipetting systems.
Frequency
The frequency of instrument testing should be based upon:
• Frequency of use
• Number of operators using each instrument
• Type of samples used
• Need for accurate error analysis
Levels of Checking
Level 1. Daily checks using calibrated tips. Use for easy verification during pipettings.
Problems can be detected visually by verifying levels during pipetting.
Level 2. Gravimetric method - four weighings. Use as a routine quick check on accuracy for
volumes commonly being pipetted.
-------
Level 3. Gravimetric method - ten weighings. This level of check is probably the most
comprehensive check necessary. Use to determine accuracy with confidence,
particularly when introducing a new pipette to the program. Minor service e.g.,
cleaning a partially clogged tip, may need to be performed by user or manufacturer.
Accuracy and precision determinations can be done using ten weighings at three
different volume levels to obtain the best confidence. Use this check level after
major repair, e.g., realignment of settings, or when performing critical work. Manu-
facturer's certificates are usually based on the Level 3 check.
Level 4. Gravimetric method - thirty weighings. This is the most comprehensive check to
determine accuracy and precision. Not cost effective for user to routinely return
pipette to manufacturer for this level of checking. Suggestive use would be for
checking operator technique.
Logs and Worksheets
Performance Log. The performance log is a record for each instrument that tells when
the last test was performed on the instrument and a summary of the results. This information
can help you easily assess the condition of the instrument you are about to use. This is
especially useful in a laboratory where instruments may have multiple users.
Performance Worksheets. Worksheets aid gravimetric analysis by providing simple
charts to check liquid measurement instruments.
Here are some hints for their use:
Z-Factor conversions are found on page 7 and can be used to convert pure water to
volume.
Whether to use a new tip for each weighing or the same pre-rinsed tip for all weighings
is up to you. This procedure should reflect your method in daily use.
A frequency schedule of performance tests is desirable to keep the information up to
date.
Example Lop Sheets
Examples of completed instrument logs and performance test worksheets are provided at
the end of this appendix. Forms for constructing performance logs and worksheets are given
in Appendix II.
8
-------
Example Performance Specifications
MODEL
TEST VOLUME
ACCURACY RANGE (jiL) % ERROR
PRECISION
%CV
P-20
P-100
P-200
P-1000
P-5000
P-10
P-2
P-10ML
2
10
20
20
50
100
50
100
200
200
500
1000
1000
2500
5000
1
5
10
0.2
1.0
8.0
1000
5000
10000
1.9-
9.9-
19.8-
19.65 -
49.6-
99.2-
49.5-
99.2-
198.4-
197-
496-
992-
988-
2485-
4970-
0.975 -
4.925 -
9.9-
0.176 -
0.973 -
1.97-
950-
4950-
9920-
2.1
10.1
20.2
20.35
50.4
100.8
50.5
100.8
201.6
203
504
1008
1012
2515
5030
1.025
5.075
10.1
0.224
1.027
2.03
1050
5050
10080
5.0
1.0
1.0
1.5
0.8
0.8
1.0
0.8
0.8
1.5
0.8
0.6
1.2
0.6
0.6
2.5
1.5
1.0
1.2
2.7
1.5
5
1
0.8
0.04
0.05
0.06
0.10
0.12
0.15
0.20
0.25
0.30
0.6
1.0
1.3
3.0
5.0
8.0
0.012
0.03
0.04
0.012
0.013
0.014
6
10
16
2.0
0.5
0.3
0.50
0.24
0.15
0.40
0.25
0.15
0.30
0.20
0.13
0.30
0.20
0.16
1.2
0.6
0.4
6
1.3
0.7
0.6
0.2
0.16
-------
a.
I 5
H o
o£
r-
sr
a-
vG
^
£
T
^c
fl-
^
^
o-
-^
^
o
to
ci
^J
>c *>
2 co
, •
H >
Vfi
-
k. Q.
eg
O.
x 3
< Q
w
-------
P-1000
PERFORMANCE LOG
p-
Instrument Model:
Serial Number: K-S5"
Purchase Date: ^/ifa
(QCO
Date nitia
(o/st
5/?7
1/9-7
"/*7
ultt
I/BZ
I/OB
6 iff
1/t*
(iu)
L.t
Inspection Level
# of Samples
4 10 30
-V
X
X
Comments
«; r«
Ok
OK
OK
O.K.
A/o
- dut
re
-TO
-------
P-200
QUICK CHECK WORKSHEET
Operal
Date
Model
or -Cf^
\O '4
: P-2C
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
Model
: P-2(
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
Model
• s&
)0
20 I
If -(A
Jf.St
1*1.51
\ 1-V2-
n.*3
19.5-20.5l
)0 1
20 1
20.11
2.0,17
1& ,<05
~20 JOl
^A5
\ 19.5-20.5l
P-200
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
Model
1 20 1
IT. 73
19. ?*
11.73
I 7.7*
1 1.?3
I 19.5-20.5l
: 1 P-200 1
Test Volume
Sample 1
2
3
4
Mean (mg)
Mean (ul)
Specs
1 20 1
10.O3
11. ?*
7.0.PP
* ?,^ i-
20.07.
1 19.5-20.5l
H20 Temp 2. ) . 5
Z Factor /.CJO3"2_
Serial Number: £T-fcV/2/ A
1 100 1 1 200
*»?.¥* /
*?1/>o /f
-------
APPENDIX II
PERFORMANCE LOG AND PERFORMANCE TEST WORKSHEETS
FOR AIR DISPLACEMENT PIPETTORS
The use of the following material in no way implies endorsement of the manufacturer by
the authors or the U.S.E.P.A. for the products mentioned. This document is excerpted here
as there are many general concepts regarding performance measurements for air displace-
ment pipettes that are applicable regardless of the manufacturer. The performance specifica-
tions given for certain pipettes are used as examples of the type of performance specifications
that should be available for any air displacement pipette.
The following material has been excerpted with permission from "Performance Assurance
for Air Displacement Pipettes," AB-1, October 1988, prepared by Liquid Measurement Quality
Control, Rainin Instrument Co., Inc., Mack Road, Woburn, MA 01801-4628.
The performance log and performance test worksheets provided on the following pages
can help in keeping good records. The worksheets are designed to allow serial numbers,
standard test volumes, and acceptable performance ranges, usually the instrument specifica-
tion, to be entered in the appropriate spaces. Photocopies can then be made for routine use
in recording data. Wide left margins are provided to a!!ow records to be kept in a looseleaf
binder, if desired.
-------
PERFORMANCE LOG
Instrument Model:
Serial Number:
Purchase Date:
Date Initial
nspection Level
# of Samples
4 1030
Comments
-------
QUICK CHECK WORKSHEET
Operator
Date
Model:
Test Volume
Sample
Mean (mg)
Mean foil)
Specs
Model:
Test Volume
Sample
Mean (mg)
Mean (jil)
Specs
Model :
Test Volume
Sample
Mean (mg)
Mean (u.1)
Specs
Model :
Test Volume
Sample
Mean (mg)
Mean (u,l)
Specs
H20 Temp
Z Factor
Serial Number:
1 1 1 1
1
2
3
4
I I I I
Serial Number:
I I I I
1
2
3
4
I I I I
Serial Number:
I I I I I
1
2
3
4
I I I I
Serial Number:
I I I I I
1
2
3
4
I I I I I
Comments
-------
Is
0 o
t-
-------
A Technical Discussion About Immunoassays:
An EMSL-Las Vegas Perspective
Outline
1. Introduction: What is an Immunoassay?
2. Principles of Immunoassay
3. Immunoassay as a quantitative technique
A. Matrix Effects
B. Extraction Effects
Example: Quantitative ELISA for PCBs
4. Case Studies
A. PCBs
B. PCP
C. GC Dilution Scheme
D. Upcoming Demonstration: BioNebraska Hg
Immunoassay
5. EMSL-LV Research
A. SFE-ELISA
B. SFE-ELISA (Ohmicron)
C. Immunoaffinity (Carbendazim)
D. SFE-ELISA for pesticides
E. Immunochemistry Summit
F. Immunoassay User's Guide
U.S. EPA Region 5 Immunoassay Technology Workshop
March 28-29,1995
-------
REGIONS Vendor Matrix
EM SCIENCE/STRATEGIC DIAGNOSTICS INCORPORATED
480 DEMOCRAT ROAD. P.O. BOX 70
GIBBSTOWN, NEW JERSEY 08027
CONTACT: WAYNE SAWYER PHONE: (609)354-09200 EXT. 469
COMPOUND
PCB, total
TNT/RDX
TNT/RDX
BTEX
BTEX
PAH, total
PAH, total
PCB total
DETECTION LIMIT (ppm)
0.50
0.50
S.Oppb
0.60
2.5
S.Oppb
0.6
lOug/WOcm2
MATRIX
so//
soil
water
water
soil
water
soil
surfaces
(wipes)
COST PER
D-TECH FIELD
KIT
$125.00
$128.00
$103.00
$103.00
$128.00
$103.00
$128.00
$125.00
it OF
SAMPLES PER
KIT
4
4
4
4
4
4
4
4
PLEASE NOTE:
Listing the above information does in no way constitute an endorsement by the
U.S. EPA of any of the manufacturers. This list is intended to provide
environmental agency decisionmakers general information about the various
types of immunoassay kits available.
SOURCES:
Individual vendors; and USEPA-EMSL, Las Vegas; USEPA Region 5's
Environmental Sciences Division and Water Division.
Costs are based on March 1995 quotes and are subject to change. Cost estimates were not available
where missing in the Tables.
Immunoassay Technology Workshop
March 28-29, 1995
-------
REGIONS Vendor Matrix
ENSYS ENVIRONMENTAL PRODUCTS, INC.
P.O. BOX 14063
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27709
CONTACT: AISLING MACRUNNELS
PHONE: (919)941-5509 OR (800) 242-7472
COMPOUND
total PAH
pentachlorophenol
total PCB
TNT
total petroleum
hydrocarbons
diox/n
benzene
atrazine
triazine
alachlor
metolachlor
benomyl
chktrothalonil
imazaquin
trifluralin
DETECTION LIMIT (PPMI
1.0
5.0 ppb M
0.5 ppmlsl
0.4 Is) Amctor 1254
3.0 HwlAroclor 1254
0.7
165 ppb M
10 ppmlsl
0.6 ppb
Sppb
0.05 ppb
0.1 6 ppb
0.06
0.07 ppb
0.11 ppb
0.5 ppb
1.0 ppb
1.0 ppb
MATRIX
soil
water
soil
soil
liquid waste
soil
water
soil
matrix extract
water
water
water
water
water
water
water
water
water
COST PER
KIT
$ 243.00
$243.00
t243.OO
$184.00
$130.OO
$4 1O.OO
$1 94.OO
$162.OO
tt, 2OO.OO
$60.00
$4.69-
93.38
$3.75-
$7.50
$3.38-
$6.75
$3.75-
$7.50
$4.63-
$9.25
$4.63-
$9.25
$4.25-
$8.50
$3.75-
$7.50
iOF
SAMPLES
PER KIT
4
4
4
4
4
20
4
4
10
2
40-80
40-80
40-80
4O-80
4O-8O
40-80
4O-80
4O-80
PLEASE NOTE:
SOURCES:
Listing the above information does in no way constitute an endorsement by the
U.S. EPA of any of the manufacturers. This list is intended to provide
environmental agency decisionmakers general information about the various
types of immunoassay kits available.
Individual vendors; and USEPA-EMSL, Las Vegas; USEPA Region 5's
Environmental Sciences Division and Water Division.
Costs are based on March 1995 quotes and are subject to change. Cost estimates were not available
where missing in the Tables.
Immunoassay Technology Workshop
March 28-29, 1995
-------
REGION 5 Vendor Matrix
i
MILLIPORE INTERTECH
397 WILLIAMS STREET
MARLBOROUGH, MASSACHUSETTS 01752-9162
CONTACT: ROY RIVETT PHONE: (800)645-5476, EXT. 6607
COMPOUND
PCB
PCB
PCP
PCP
PAH
PAH
TPH (total!
BTEX
BTEX
TNT
TNT
DDT
2,4-D
2,4-Dp
2,4-D(ql
toxaphene
chlordane
chlordanefpi
alachlorelqt)
alachlor
alachlorlp)
metolochtortqt)
metolochlorlp)
triazenelqt)
triazene
atrazeneipl
atminotHi sens»
aldicarb
aldicarblpl
DETECTION LIMIT (ppbi
.5PPMb,plato
10 ug & lOOug
10 PPM
5PPB
< 1 PPM
SEE INDIVIDUAL FUELS LIST
< 2PPM
< .1 PPM
< 13 PPM
< 2PPB
4OPPB
< ,2 PPM
.12PPB
1.6PPB
.2 PPM
< 20ppb
5 ppb
< . 1 ppb
.1 ppb
< 1 ppb
.2 ppb
<. 1 ppb
< .05 ppb
< . 1 ppb
.02 ppb
.01 ppb
2 ppb
.4 ppb
MATRIX
soil
wipe
soil
water
soil
water
soil
soil
water
soil
water
soil
soil
soil
water
soil
soil
water
water
water
water
water
COST PER KIT
#254. OO
t20O.OO
#270.00
#200.00
#27O.OO
$2OO.OO
$27O.OO
$270.00
$2OO.OO
#270.00
#355.OO
#270. OO
#254.00
#385.00
#325.00
#270.00
#270.00
#355.00
#325.00
$1 35.OO
$385.00
$326.00
$385.00
$326. OO
$195.00
#355.00
#354.0O
$1 95.0O
$395.00
it OF
SAMPLES
PER KIT
14-16
14-16
14-16
14-16
14-16
14-16
14-16
14-16
14-16
14-16
28-80
14-16
14-16
28-88
30-38
14-16
14-16
28-88
3O-38
14-16
28-88
30-38
28-88
30-38
14
28.-8S
28-88
14-16
$28-88
-------
i
MILLIPORE OVTERTECH
397 WILLIAMS STREET
MARLBOROUGH, MASSACHUSETTS 01752-9162
CONTACT: ROY RIVETT PHONE: (800)645-5476, EXT. 6607
COMPOUND
benomyl
benomyllp)
carbofurane
carbofuranelpl
cyclodieneslp)
isoproturonfpt
matalaxylfp)
paraquatelpl
bioresmethinfpl
chtorpyro. lethylilp)
chlorpyro. ImothylHpl
cyanazeneip)
diazinonlp)
triasulfuron
urea harbicidelp)
DETECTION LIMIT (ppbt
.2ppb
.1 ppb
.1 ppb
.1 ppb
5 ppb
< .05 ppb
.05 ppb
< 30ppt
.08 ppm
< .05 ppb
< .05 ppb
. 14 ppb
< 30ppt
< .05
< .05 ppb
MATRIX
water
water
water
water
water
COST PER KIT
$195.OO
$385.00
$1 95.OO
$386.00
$385.OO
$382. DO
* 355. 00
#355.00
#385.00
$385.00
$385.00
$382.00
$385.00
$385.OO
$385.0O
nop
SAMPLES
PER KIT
14-16
28-88
14-16
28-88
28-88
28-88
28-88
28-88
36
28-88
28-88
28-88
28-88
28-88
28-88
PLEASE NOTE:
SOURCES:
Listing the above information does in no way constitute an endorsement by the
U.S. EPA of any of the manufacturers. This list is intended to provide
environmental agency decisionmakers general information about the various
types of immunoassay kits available.
Individual vendors; and USEPA-EMSL, Las Vegas; USEPA Region 5's
Environmental Sciences Division and Water Division.
Costs are based on March 1995 quotes and are subject to change. Cost estimates were not available
where missing in the Tables.
Immunoassay Technology Workshop
March 28-29, 1995
-------
i
_REGION 5 Vendor Matrix
OHMICRON CORPORATION
375 PHEASANT RUN
NEWTON, PENNSYLVANIA 1894O
CONTACT: SUSAN BOWMAN PHONE: (215)860-5115, EXT. 679
COMPOUND
alachlor
aldicarb
atrazine
benomyl/
carbendazim
captan
carbaryl
carbofuran
chlorpyrifos
chlorothalonil
cyanazine
2,4-0
metolachlor
paraquat
procymidone
pentachloraphenol
total KB
total PAH
total BTEX
TNT
carcinogenic PAH
DETECT/ON LIMIT (ppb)
0.05
0.25
0.05
0.1
10
0.25
0.06
0.10
0.07
0.04
0.7
0.05
0.02
0.80
0.06
0. 2 ppb lw);0. 5 ppm Is)
0.7 ppb Iw); 70 Is)
20 ppb lw>; 0.2 ppm 1st
0.07 ppmls) .07 ppblw)
2 ppb (w> 10 ppb Is)
MATRIX
water, soil
water, soH
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water soil
water, soil
water, soil
COST PER KIT
$250.00
9250.00
$250.00
$710.00
$280.00
$260.00
$250.00
$300.00
$260.00
$260.00
$280.00
$250.0O
$280.00
$670.00
$335.00/1040
$495.00/1495
$495.00/1435
$ 395.00/1 04O
$395.00/1040
If OF
SAMPLES
PER KIT
30
3O
30
too
30
30
30
30
30
30
3O
30
30
100
20/80
20/80
2O/8O
2O/8O
2O/80
PLEASE NOTE: Listing the above information does in no way constitute an endorsement by the U.S. EPA of any of the
manufacturers. This list is intended to provide environmental agency decishnmakers general information
about the various types of immunoassay kits available.
SOURCES: Individual vendors; and USEPA-EMSL, Las Vegas; USEPA Region 5's Environmental Sciences Division and
Water Division.
Costs are based on March 1995 quotes and are subject to change. Cost estimates were not available where missing in the
Tables.
Immunoassay Technology Workshop
March 28-29, 1995
-------
REGIONS Vendor Matrix
QUANTIX
2611 BRANCH PIKE
CINNAMINSON, NEW JERSEY 08077
CONTACT: Craig McCaffrey PHONE: 1609)273-1566
COMPOUND
alachlor
atrazine
benomyl
chlorothalonil
imazaquin
metalachlor
trifluralin
triazine
total BTEX
total PAH
DETECTION LIMIT (ppbl
0.2
0.05
0.1
0.2
2.5
0.25
2.0
0.05
0.50 ppm (w); 5.0 ppm (s)
1.0 ppm
MATRIX
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
water, soil
COST PER KIT
$360.00
$396.00
$396.00
$396.00
$360.00
$400.00
it OF
SAMPLES PER
KIT
96
96
96
96
•
96
10
PLEASE NOTE:
Listing the above information does in no way constitute an endorsement by the
U.S. EPA of any of the manufacturers. This fist is intended to provide
environmental agency decisionmakers general information about the various
types of immunoassay kits available.
SOURCES:
Individual vendors; and USEPA-EMSL, Las Vegas; USEPA Region 5's
Environmental Sciences Division and Water Division.
Costs are based on March 1995 quotes and are subject to change. Cost estimates were not available
where missing in the Tables.
Immunoassay Technology Workshop
March 28-29, 1995
-------
DRAFT: DECEMBER 1994
PROJECT SUMMARY:
EVALUATION OF THREE PCB FIELD-SCREENING TECHNOLOGIES:
RESULTS OF A DEMONSTRATION CONDUCTED AT THE
ALLIED PAPER, INC/PORTAGE CREEK/KALAMAZOO RIVER SUPERFUND SITE
KALAMAZOO MICHIGAN
M. E. Silversiein, V. A. Ecker, T. A. Van Donsel and S. D. Cornelius
Introduction
A technology demonstration was conducted
to evaluate the performance of three rapid, on-site
field-screening techniques in the detection and
measurement of polychlorinated biphenyls (PCBs)
at the Allied Paper, Inc./Portage Creek/Kalamazoo
River (Allied Paper) Superfund Site in Kalamazoo,
Michigan. The demonstrated technologies included
two field-portable, immunoassay-based, soil analysis
kits - the EnSys, Inc. PCB RISc* Test Kit and the
Millipore Corporation EnviroGard* PCB Test Kit;
and a chloride ion-specific electrode (ISE) - the
Dexsil Corporation L2000* PCB/Chloride Analyzer.
The purpose of this demonstration, which took
place in October 1992, was to assess the
effectiveness of the field-screening techniques in
measuring PCBs (primarily Aroclors 1242 and
1254) present in soil, river sediment, and solid
paper waste (landfill) matrices at the Allied Paper
Superfund Site, and to evaluate their potential for
use at this and other sites with PCB contamination.
The Allied Paper Superfund Site includes
three miles of Portage Creek and 35 miles of the
Kalamazoo River which has been contaminated with
PCBs as the result of the recycling of carbonless
copy paper. The actual extent of PCB
contamination at this National Priorities List site is
currently being investigated as part of the Remedial
Investigation. The use of fixed-laboratory and field-
portable gas chromatographs (GCs) has been the
preferred approach in identifying and quantifying
PCBs at Superfund sites. However, since this site
encompasses such a large geographic area, it could
require the analysis of many thousands of samples
to characterize the extent of the PCB
contamination. Therefore, the U.S. Environmental
Protection Agency (EPA) and the Michigan
Department of Natural Resources (MDNR), in
conjunction with the site's Potentially Responsible
Parties (PRPs), conducted this demonstration to
determine if the PCB field-screening methods could
maximize coverage and minimize costs and time
associated with site characterization and remedial
investigation/feasibility study (RI/FS) processes.
Analytical Methods
The EnSys, Inc. PCB RISc* Test Kit is a
field-portable, immunoassay-based kit designed to
test any soil type for total PCBs. This
semiquantitative method uses tubes coated with an
antibody that binds specifically to PCBs. Samples
extracted in methanol are subjected stepwise to
enzyme-labeledanalyteconjugate,enzymesubstrate,
and chromogen. A colored end-product, which is
inversely proportional to the amount of PCBs in the
sample, is measured in a photometer. The entire
sample preparation and analysis procedure takes
approximately 45 minutes at a cost of approximately
S20 to S40 per sample. For this demonstration,
EnSys provided kits with four semiquantitative
ranges: <2, 2 to 10, 10 to 50, and >50 mg/Kg. The
Millipore EnviroGard* PCB Test Kit is also a
competitive enzyme immunoassay for field soil
extraction and photometric analysis of PCBs,
costing S20 to S40 per sample. For this
demonstration, the EnviroGard* kit was configured
with four semiquantitative ranges: <5, 5 to 10,10 to
50, and >50 mg/Kg total PCBs. The Dexsil
Corporation L2000* PCB/Chloride Analyzer is a
chloride ISE with a metered readout to
quantitatively measure PCBs in soil by converting
the amount of organic chloride measured by the
electrode into parts per million (ppm) of PCBs
(Aroclor 1242 or 1260) or Askarel (1260 +
trichlorobenzene). Sample preparation includes
extraction of PCBs (as organic chloride) from soil
with a sodium reagent. Instrumental analysis time
takes about 1 minute and the average cost is $8 to
S10 per sample analysis. The detection limit of the
L2000* ISE is 5 mg/Kg PCBs with an analytical
range of 5 to 2000 mg/Kg.
The field-screening data generated during
this demonstration were compared to results (as
specific PCB aroclors) from GC analyses (using
SW-846 Method 8081 after Method 3541 Automated
Soxhlet Extraction) conducted by the MDNR
Analytical Laboratory in Lansing, Michigan.
-------
Demonstration Design and Implementation
A Demonstration and Quality Assurance
Project Plan was prepared to ensure the generation
of sufficient data to adequately and fairly evaluate
l he performance of each PCB field-screening
technique. The critical PCB concentration ranges
at the Allied Paper Superfund Site, in terms of
potential cleanup levels for the soil, river sediment,
and paper waste matrices, were determined by
MDNR to be 1, 10, 20, 30, and 50 mg/Kg. Based
on these concentrations, a sample collection and
analysis protocol was designed. All aspects of
sample collection, preparation, homogenization,
handling, transfer, shipping, storage, and related
documentation procedures were defined to ensure
the consistency and integrity of the samples.
Twenty-four 1-gallon bulk samples were collected
and were used as discrete (indigenous) site samples
and as stock materials to create (formulate) 36
additional demonstration samples, thereby ensuring
that each of the three sample matrices contained the
full range of PCB concentrations of interest. For
ihe purpose of this report, all indigenous and
formulated sile samples are termed "field samples."
Each of the 60 field samples (20 samples per
matrix) were split into homogeneous replicates after
air drying, sieving, and riffle splitting.
A Category II Quality Assurance (QA)
Project Plan was designed (1) to meet the data
quality objectives (DQOs) established by the EPA
and MDNR managers and (2) to ensure that
confidence levels in the demonstration data required
to evaluate the field-screening technologies would
be achieved. Through QA oversight, the
demonstration was implemented so that the
intermethod comparisons could be made without
extraneous sources of variability confounding the
results. Accuracy (and bias) was assessed using
percent recovery (%R) data from the analyses of
synthetic reference materials (SRMs) composed of
commercially acquired soil spiked with Aroclor
1242 at 0.5, 1.5, 8.0, 25, 45, and 100 mg/Kg.
Precision was assessed using seven field duplicate
samples and replicate measurements of SRMs.
Representativeness was enhanced by the analysis of
samples characteristic of the site condition in terms
of matrices and ranges of PCB concentration.
Comparability was ensured by employing sample
homogenization techniques that would provide each
measurement system with equivalent samples.
Completeness was accomplished by ensuring that
sufficient numbers of samples were analyzed by
each method to conduct statistically valid
performance assessments.
MDNR and PRP representatives and
contractors were employed during the
demonstration to perform the field analyses using
the immunoassay and ISE technologies. Before the
actual demonstration, these field analysts were
trained by the technology developers on the proper
use of their respective products in a Vi-day training
session. During the demonstration, samples were
analyzed in the near-site demonstration field trailer.
The field samples were labeled and randomly mixed
in the sample stream so that site matrices, SRMs,
and field duplicate samples were distributed across
analysis batches and in such a manner that the field
and QA samples were double-blind to the analyst.
Field analysts completed field data forms
for the field-screening techniques which were
subsequently entered into the electronic data base.
All MDNR-generated GC data were submitted in
both hardcopy spreadsheets and electronically for
inclusion in the project data base. All field and
laboratory data were reviewed by the data base
manager before being entered into the data base.
In analyzing the data, the semiquantitative
immunoassays were assessed using probabilities of
false positive and false negative responses as
compared to GC results, while the quantitative ISE
comparison employed linear regression and upper
and lower bounds assessment criteria.
Conclusions on each technology's reliability,
and recommendations for their use at the Allied
Paper Superfund Site and at other sites
contaminated with PCBs are provided in an
overview, below. In addition, suggested
improvements or modifications to method protocol
or in-field applications are identified for each
technology, where appropriate. The technology
evaluation report provides a complete description of
the demonstration design, implementation, results,
conclusions and recommendations.
EnSys PCB RISc* Test Kit Results, Conclusion,
and Recommendations
The EnSys PCB RISc* Test immunoassay
results from the analysis of the 60 homogenized
Allied Paper Superfund Site field samples (and 7
field duplicates [n = 67]), as compared to the
MDNR GC data showed that 52 (77.6%) were
identified correctly, 7 (10.4%) were considered false
negative or biased low, and 8 (11.9%) were
identified as false positive or biased high. Only one
false negative (1.5%) and one false positive (1.5%)
result was inaccurate by more than one
semiquantitative range (Figure 1). If eight field
samples, which were analyzed outside of the
manufacturer's QC calibration criteria, are deleted
from the data assessment, 48 of the 59 remaining
-------
B«VS M8e TEST «• MONR GC
TABLE I. PRECISION AND ACCURACY RESULTS SUMMARY
FOR ENSYS RISs* TEST ICTT BASED ON SRM ANALYSES.
*-«* M-W O1040 D-XO
^
Figure 1. Comptraon of field samples analyied by the EnSyi PCB RlSe* Ten lot and
the MDNR CC (Points in boxes indicate correctly identified samples; points above
te fate negative, or tow bias; points below indicate Use positive, or high bias).
poorly calibrated batches (containing 8 samples) deleted
field samples (81.4%) would be correctly identified;
6 (10.2%) would be false negative, and 5 (8.5%)
would be false positive (Figure 1, inset).
The EnSys PCB RISc» Test analyzed a
total of 39 SRMs (Table 1), for which 28 (72%)
were correctly identified (i.e., true positive),
while no (0%) SRMs generated false negative
responses, and 11 (28%) SRMs generated false
positive responses (or exhibited a high bias). For
each instance where a false positive occurred, the
kit indicated a concentration in the next highest
range; 10 of the 11 false positives were from the
1.5, 8.0, and 45 mg/Kg SRM lots - concentrations
reasonably close to the high side of the kit's
respective concentration ranges. Two SRMs, one at
0.5 mg/Kg and one at 45 mg/Kg, were analyzed in
batches that were considered improperly calibrated
as per manufacturer's criterion; the 0.5-mg/Kg SRM
was correctly identified and the 45-mg/Kg SRM was
misidentified as false positive (i.e., >50 mg/Kg).
Based on these results, the EnSys PCB
RISc* Test Kit could be a valuable sample
screening tool for the Allied Paper Superfund Site
characterization and RI/FS activities. In the direct
comparison of the EnSys, Inc. PCB RISc* Test
immunoassay to the MDNR GC results, the
immunoassay kit correlated well in analyzing the
SRM
TvfCt
COM.
<*»««)•
0.5
U
8.0
25.
45.
100.
uibtoul
total
% total
Sanptas
Analyzed
(n)
7
7
7
7
7
4
39
39
100
^OaettaateiMlneduni
False negative
(to* DOS)
2raag»
lower
-
-
-
0
0
0
0
1 nap
loner
-
-
0
0
0
0
0
0
0
Arodor 12
MOtibnooi
42.
D curve out
Coma
Range
(true
positive)
71
4
5
6
2
4
28
28
72
Fake positive
(high bias)
1 range
higher
0
3
2
1
51
-
11
2 range
higher
0
0
0
-
-
-
0
n
28
• Not possible to be out by dm degree of range.
Note although it MS possible for three ranges tower/higher to be incorrectly
identified for woe coacentnnons, none were exhibited; thus, the column not shown in table.
Allied Paper Superfund Sile samples. In general,
there is a clear trend that when a GC sample was
determined to be in a particular concentration
range, the EnSys RISc* Test was able to correctly
characterize it semiquantitatively (i.e., identify the
correct concentration range). There is little risk of
generating false negative responses, and the false
positive responses tend to occur at levels near the
ends (boundaries) of the calibration ranges. For the
semiquantitative configuration used in this
demonstration, the chances are excellent that a
sample containing PCBs below 1 mg/Kg will be
measured at less than 2 mg/Kg. If this is an
acceptable level of detection, then the kit would be
suitable for low-level screening. It can be
concluded that the QC acceptance criterion for the
kit calibration standards specified in the
manufacturer's instructions is appropriate and, if
followed, should provide optimal results. Four of
the 39 kits (10%) used in this demonstration
produced calibration curves which failed to meet the
manufacturer's acceptance criteria. Samples
analyzed under these calibration standard sets
accounted for one-half of all the incorrectly
semiquantified results generated by the EnSys
RISc* Test. From this, it can also be concluded
that the QC limits established for this immunoassay
format were appropriate for identifying out-of-
control situations. Conversely, however, a 10%
failure rate in the kit-supplied calibration standards
should be taken into consideration when evaluating
the ruggedness of these kits and the budget/schedule
constraints at a site such as the Allied Paper
Superfund Site, where hundreds of RISc* kits could
be used in the screening of thousands of samples for
PCBs.
-------
Millipore EnviroGard* Test Kit Results,
Conclusion, and Recommendations
The Millipore EnviroGard* Test Kit
immunoassay results from the analysis of the 67
field and field duplicate samples (Figure 2), as
compared to the MDNR GC data, showed that 43
(64.2%) were identified correctly by the
immunoassay, 13 samples (19.4%) were considered
false negative responses (or biased low), and 11
samples (16.4%) were false positive responses (or
biased high). One batch of samples had a
particularly "bad" calibration, as the color
development of the standards was extremely
suppressed. Eight field samples were analyzed
under this calibration curve; six of which were false
negative (Note: half of all the false negative
responses generated by the EnviroGard* kit were
analyzed under this curve). If these eight samples
are deleted from the assessment (Figure 2, inset),
(he percentage of samples correctly identified would
increase to 67.8% (40 of 59), the false negative rate
would decrease to 11.9% (7 of 59), and the false
positive rate would increase to 20.3% (12 of 59).
Twenty-three SRMs were analyzed using
the EnviroGard* kit, 16 of which (69.6%) were
identified correctly, with 4 (17.4%) false negative
and 3 (13%) false positive (or high bias) responses
(Table 2). A false negative greater than one range
-from the nominal concentration was found in only
one SRM (a 25-mg/Kg), which was analyzed under
the calibration curve with the poor color
development discussed above. Five of the six
remaining SRMs that were misidentified were from
the 8.0-mg/Kg lot. This may be a result of issues
pertaining to the 5- and 10-mg/Kg calibration
standards discussed below. Overall, there was one
obviously false negative generated from the SRM
analyses.
For six-of the 12 calibration curves, the
optical densities (ODs) of the 5- and the 10-ppm
calibrators were either reversed, identical, or so
close in intensity (i.e., absorbance units [AU]) that
the difference was negligible. As a result, the
possibility of misquantification was high for samples
producing ODs in the 5 and 10 mg/Kg regions
(concentration ranges). However, since the field
analysts were not provided with acceptance/rejection
criteria for kit standard curves, no demonstration
samples were reanalyzed because of out-of-control
calibration curves. In addition, a wide range of
ODs were reported for the 12 negative control
samples (kit reagent blanks) analyzed in
conjunction with each calibration curve, fluctuating
from 1.550 to 0.240 AU. This illustrates the
inconsistency in color development from kit to kit
MLUPOAE ENVROGAKO KfT vs MDNR GC
FBLD SMfU PCi flESUJS
B«MKXMK> WT
CONCOTflAnOM NAME (ppm)
A-
-------
observable trend that when a GC sample result was
in a particular concentration range, the
EnviroGard* Kit was able to correctly identify it
semiquantitatively. Although the significant number
of false positive and false negative responses in this
comparison raise concerns about the confidence in
the kit results, much of this variability may be
caused by deficiencies in kit QC and may be easily
corrected with refinements to the kit instructions.
As with the Ensys kit, the Millipore immunoassay
was not equipped with a 1-ppm calibrator for this
demonstration. The lowest available kft standard
(calibrator) was 5 ppm. However, of the 23 field
samples and SRMs with concentrations from
nondetect to 5 mg/Kg PCBs, the EnvjroGard* kit
correctly analyzed 82.6% to be less than 5 mg/Kg,
and 10 of the 11 field samples and SRMs with PCB
concentrations below 1 mg/Kg were reported as less
than the EnviroGard* kit's detection limit of 5
mg/Kg. If 5 mg/Kg is an acceptable level of
detection, then the kit would be suitable for low-
level screening.
The EnviroGard* PCB immunoassay could
prove to be a useful tool at the Allied Paper Site.
Since ten or more samples can be analyzed under
one curve, the sample throughput would support
many samples being efficiently screened on-site for
PCB ranges. The issue of the method's calibration
procedure is significant. It should be noted that the
manufacturer currently provides QC guidelines
regarding the EnviroGard* kit protocol and
calibration curve QC acceptance limits for the OD
ratios of the negative control (reagent blank)
sample versus the positive calibrators (i.e., %Bj
inhibition formula). It is recommended that such
QC limits be enhanced for optimal performance
and these limits be made available to the field
technician. In addition, based upon field analyst
comments, increasing the volumes of reagents and
sample extracts may increase precision and accuracy
by minimizing the consequence of preparation error
during reagent and extract manipulations.
Dexsil L2000* PCB/Chtoride Analyzer Results,
Conclusion, and Recommendations
The Dexsil L2000* PCB/Chloride Analyzer
(L2000* ISE) data from the analysis of the 67
homogenized Allied Paper Super fund Site samples,
as compared to the MDNR GC results, showed that
below 50 mg/Kg PCBs, the L2000* ISE tends to
generate responses higher than the GC, indicating
a high bias in that concentration range; above 50
mg/Kg, this trend reverses and the L2000* ISE
lends to generate a lower response than the GC
(Figure 3a). Regardless of the concentration, there
is a great deal of scatter in the correlated data,
which indicates a lack of either consistent
agreement or consistent degree of bias between the
GC and ISE techniques. Below 50 mg/Kg (Figure
3b), there was essentially no correlation in the data.
In addition, a significant trend can be observed in
the lower concentration data: in the 13 instances
where the GC measured less than 5 mg/Kg, the
L2000* ISE gave results greater than 5 mg/Kg,
ranging from 5.3 to 35.5 mg/Kg. No false negative
responses were generated by the L2000* ISE.
OEXSIL12000 ISE w«. MOW* GC PCB CONCENTRATIONS
300.00
0 100 200 500
MDNR CC CONCENTRATION (ppm)
Figure 3a. Comparison of field sample analyzed bv the Dexsil L2000* Ion-Specific Electrode and
MDNR GC (all PCB concentrations! Solid line - best-fir line (R-square - U.634. slope - U 7:7
intercept - 11.00); dashed (45 ) line * line of perfect agreement.
0 10 20 30 40 50
UDNR GC CONCENTRATION (ppm)
Figure 3b.Comp*raon of field samples analyzed by the Dexsil L2000* Ion-Specific Electrode and the
MDNR GC (nondetect to 50 mg/Kg PCBs). Solid line - bat-fit line (R-square - 0.098. slope -
0.271. intercept - 16.18); dashed (45) line - line of perfect agreement.
Thus, to provide a comparison assessment of the
L2000* ISE and MDNR GC results on a per-
sample basis, the data were analyzed in terms of
relative percent difference (RPD) and percent
recovery using two data assessment criteria: (1) 60
to 140% (i.e., 100% ±40%) of the GC
measurement and (2) a factor of two (equivalent to
a 50% to 200% window of acceptance) from the GC
measurement (Figure 4).
-------
300.0
§
o
9
o
g
o
10 20 30 40
OEXSL/CC SAMPLE AMALYSS
SO
60
TABLE 3. DESOUrnVE STATISTICS FOR THE DEXSIL L20DO* ISE
BASED ON THE ANALYSIS OF SRM»
igurc 4. Cumulative percent recoveries of (he Dcxsil L2000* lon-Speafic Electrode relative to the
1DNR GC analysis of field samples. River sediment (triangle), soil (circle) and paper waste (square)
lamccs are depicted. The dau assessment criteria are plotted as 60 to 140 percent (small dashed line)
i a factor of two (large dashed line) Eight samples omitted above 300% for purpose of resolution.
Using the 60 to 140% criterion, 33 of the 67 field
samples (49.3%) are within the window, 13 (19.4%)
are below (biased low), and 21 (31.3%) are above
(biased high). When the more liberal 50 to 200
percent criterion is applied, 39 (58.2%) are inside
the window, while 10 (14.9%) are below and 18
(26.9%) are high.
A total of 30 SRMs were analyzed by the
L2000* ISE (Table 3). With respect to the low-
concentration SRMs, both the 0.5-mg/Kg and the
1.5-mg/Kg SRMs are below the manufacturer's
stated detection limit of 5 mg/Kg. All of the 0.5
and three of the 1.5-mg/Kg SRMs were reported as
concentrations below the 5 mg/Kg level. Thus, the
ability of the technique to measure nondetects or
values below 5 mg/Kg should be considered
adequate for precision and accuracy. However, the
two 1.5-mg/Kg SRMs above the detectable level of
the method yielded significantly higher
determinations than the target value (13.1 and 44.6
mg/Kg PCB). The 8.0-mg/Kg SRM yielded
extremely precise and accurate results for the five
analyses (100 %Rr-10 %RSD) and for each of the
three highest concentration SRMs (25, 45, and 100
mg/Kg), the results are very precise (9.7, 8.7, and
4.5 %RSD, respectively). Though the mean
recoveries (65 to 75%) show the L2000* ISE's
tendency to underestimate the high-concentration
SRMs, the performance is still well within the
accuracy DQO (60 to 140%). Although the scatter
in the field sample data observed in the ISE to GC
comparison is not seen with the SRMs, this scatter
is observed, when the L2000* ISE field duplicate
pair precision is assessed. Of the seven duplicate
pairs, four had RPDs above 60% and only one had
an RPD below 30%.
Based on the direct comparison of the
Dexsil L2000* ISE to the MDNR GC, the ISE
performed marginally well above 50 mg/Kg PCBs,
SRMCONC
(assyKfT
or
\s
8.0
25
45
100
NOHIUUU coaon
N
5
5
*
5
5
6
4
auoa ot t
MEAN
(•(/Kf)
U
111
IT*
40
IK
»j
643
RANGE
«n«Ai>
15-4.7
LS-444
U-4.9*
7 0 - 9.0
1M • 21-0
26.7- 314
S5J-72.0
STD
DEV
014
17.7
\»
OJ1
LSI
2JS
3.04
RSD
(%>
RECOVERY*
(*)
23.7 1 720
129
4i*
10.1
97
i7
4.5
920
I471
1 100
75.2
65.1
MJ
joctor Ii4i
1 CooeranwM
'RaMawubf
oftKfeieiofSRMuiUrm.
UM Dead L2000* ISE """ •' ••'* uued dtucuoa ham of t mt/K|
at 111 and 444 mt/Ki.
and poorly below that concentration. In general,
when the GC sample concentrations increased, so
did those analyzed by the L2000* ISE. However,
there is excessive scatter in the data, and fitting a
best fit line or the 95% confidence interval was not
particularly useful in a direct method comparison.
One particularly positive note for the use of the
L2000* at the Allied Paper Superfund Site relates
to its relative success in analyzing for PCBs in the
paper waste samples. Using the factor-of-two
criterion, the ISE was in agreement with the GC for
17 of the 22 (77.3%) of the paper waste samples
measured, and only generated two results (9.1%)
below the 50% recovery level. Unfortunately, since
only three samples were measured (by the GC) to
be below 50 mg/Kg PCBs, it is not clear if the
L2000* ISE performance on the paper waste matrix
is a consequence of the fact that there weren't
enough lower concentration samples to truly
represent the performance of the technique in the
low range, or if the technique simply performs well
in quantifying PCBs in paper waste samples,
regardless of concentration.
The use of the Dexsil L2000* as a field-
sample screening tool cannot be generally
recommended for the Allied Paper Superfund Site
RI/FS activities. The technology could have a
limited use, however. If PCB hot ;pots
(contamination significantly higher than 50 mg/Kg)
are identified by standard methods, the Dexsil
L2000* ISE could be useful in providing reasonable
estimates of PCB quantification, provided that the
decisionmaker is aware that underestimations of
PCB concentrations are possible. This could aid in
general site hot-spot mapping, and serve as a means
of alerting an off-site GC laboratory about
particularly high-concentration samples. The latter
application would reduce the potential for
overloading GC detectors; ultimately reducing
laboratory analytical time and costs. It is not
recommended that this technology be used to
quantitate samples with low levels of PCBs due to
the lack of confidence method results for samples
with concentrations below 5 mg/Kg.
-------
7
Assessment of Confirmatory GO Data Quality
The MDNR GC laboratory performance,
based on the analysis of the SRMs (Table 4),
indicates a high level of data quality. The %R for
each SRM lot was within 15% of the nominal
concentration except for the 100-mg/Kg SRM (at
133%) and there was no indication of a systematic
bias across the 0.5 to 100 mg/Kg range. The
precision estimates derived from the SRMs were
well within the DQO of 20 %RSD (with the
exception of the 100-mg/Kg lot, at 24.1%). Of the
eight field duplicates analyzed, five had RPDs below
13%, two sample pairs had RPDs of less than 30%,
and one sample pair had an RPD of 32.4%.
TABLE 4. PRECISION AND ACCURACY ESTIMATES FOR THE MDNR GC
BASED ON THE ANALYSIS OF SRMs
SRM CONC
(me/Kg)'
0.5
1.5
8.0
25
45
100
N
3
1
3
3
1
•;
MEAN
(me/Kg)
0.57
1.57
8.77
24.3
417
133.3
STD
DEV
007
0.15
1.12
1.55
6.43
311
RSD
(%)
1Z3
97
1Z8
6.3
15.1
24.1
RECOVERY
(%)
114
104.4
109.6
97.3
94.8
133.3
" Nominal concentration ot Aroclor 1242.
In order to document the validity of the
MDNR GC PCB data, a formal data validation was
conducted on 100% of the laboratory data.
Although some procedural inconsistencies and
minor deviations from standard EPA protocols were
identified, these matters were resolved before data
assessment and comparison to the field-screening
methods were initiated. The overall data quality
indicators, as appraised from internal and external
QA/QC sample results, demonstrated that the
MDNR GC data were acceptable for the intended
use.
Another means of assessing the success of
sample homogenization and laboratory
measurement precision is by comparing the
concentrations of the 36 formulated field samples to
the "recipe" used in their preparation. By plotting
the actual result for each formulated field sample
against its theoretical (or estimated) concentration,
based on the formulation component concentrations
in the proportions added, the overall integrity of
field and laboratory operations (with respect to
sample collection and handling in the field taken all
the way through sample analysis in the laboratory)
can be observed. This relationship, as depicted in
Figure 5, shows an excellent correlation and
indicates that not only was the GC laboratory
generating high quality data, but that the entire
sample handing and analysis system functioned well.
ESTIMATED «. MEASURED PCB CONCENTRATION
OF CONCOCTED SAMPLES ANALYZED BY GC
300
0 100 200 300
MDNR GC MEASURED CONC. (ppm)
Figure 5. Plot of esomaied PCB coocentnuom for the formulated field umples
IcakulawJ from recipe for (onmHinng sampk* ind MDNR GC measurement of
itoek. Y-an») venut aaual PCB oooeentnnons determined by MDNR GC (X-«as).
Solid hoc • bat-fit line (R-tquaie - 0.857. slope - 0.978. intercept - 11.05); dashed
(45*) line « line of perfect agreement. -
Overall Demonstration Conclusions and
Recommendations
The basic objective of the demonstration,
that of achieving an unprejudiced assessment of the
performance of the field-screening techniques at the
Allied Paper Superfund Site, were satisfied. This
assumption is based on the fact that (1) the
technologies were demonstrated concurrently under
identical field conditions, (2) the samples measured
in the demonstration reflected the broad range of
PCB concentrations in the soil, river sediment, and
paper waste matrices found on the site, (3) the
sample replicates analyzed by each method were
homogenous with respect to matrix and PCB
concentrations, (4) SRMs proved valuable external
precision and accuracy checks on all measurement
systems at the PCB concentrations of interest, and,
(5) acceptable-quality laboratory GC data were used
for comparison with the field-screening methods.
One aspect of the performance of the field-
screening technologies that cannot be fully
evaluated is the rate of false positive responses
based on the analysis of field equipmeni/rinse blank
samples, as these QA samples were not
incorporated into the sample batches. In all other
respects, the conclusion and recommendation
concerning the performance of each of the
demonstrated technologies can be stated with a high
level of confidence.
-------
It is concluded that on-site sample analysis
is a time-efficient tool for obtaining site data. All
of the approximately 100 demonstration field
samples and SRMs were analyzed by the field
methods on-site in less than four days, regardless of
the technology. The data generated from these on-
site analyses were reported by the analyst and
reviewed for initial quality by the EPA and State of
Michigan field managers on the day of analysis.
Thus, the ability of the field-screening methods to
rapidly process site samples is well documented.
Based on this experience, it is recommended that
field-analysis techniques be considered as tools to
complement any significant site characterization or
related measurement and monitoring effort because
of their ability to save time and resources by
acquiring data expediently.
The immunoassay Draft Method 4020 has
been accepted by the U.S. EPA Office of Solid
Waste (OSW) Organic Methods Work Group for
inclusion in the third update of the RCRA Methods
Manual (SW-846). Both the EnSys RISc® and
Millipore EnviroGard® PCB test kits have been
approved by the OSW for use with Method 4020.
As it is currently written (subject to comment and
modification), the generic procedure specifies
following the manufacturers' instructions, and
contains few QC guidelines and no mandated
performance acceptance limits. As shown in this
demonstration, data quality can be optimized and
documented by using calibration checks, negative
controls or reagent blanks, spiked or external-
source QC samples, and demonstrated calibration
standard curve acceptance limits (i.e., Bj inhibition
formula), in addition to the replicate analyses
recommended in the draft method.
With respect to planning and conducting
technology demonstrations similar to the one
described in this report, it is recommended that
three issues be adequately addressed and
implemented once the project objectives have been
clearly defined and understood by all key project
management personnel: (1) that the matrices,
analytes, and concentrations of concern for the site
in question be sufficiently represented in the real-
world study samples, (2) that the study samples be
thoroughly homogenized, and (3) that a well-
characterized, site-specific or external-source
performance evaluation or reference material be
included in the measurement process as a check on
all analytical systems.
NOTICE
The information in this document has been funded
wholly or in part by the U.S. Environmental
Protection Agency under Contract No. 68-CO-0049
to Lockheed Environmental Systems &
Technologies Company. This document is a
preliminary draft. It has not been formally released
by the U.S. Environmental Protection Agency and
should not at this stage be construed as Agency
policy. It is being circulated for comments on its
technical merit and policy implications.
Mention of corporation names, trade names, or
commercial products does not constitute
endorsement or recommendation for use. (Notice
to be amended after external review process
completed)
-------
M. E. Silverstein and V. A. Ecker are with
Lockheed Environmental Systems & Technologies
Company
Las Vegas, Nevada 89119
T. A. Van Donsel is with
U.S. Environmental Protection Agency
Region 5
Chicago, Illinois 60604
S. D. Cornelius is with
Michigan Department of Natural Resources
Lansing, Michigan 48933
K. W. Brown is the Work Assignment Manager with
Technology Support Center
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, Nevada 89193-3478
-------
FIELD STUDY OF IMMUNOASSAY SCREENING METHODS FOR BTEX,
PAH'S, AND PCB's AT A FORMER COAL GASIFICATION FACILITY
by: Robert M. Schulte and Kurt D. Olinger, CHMM; Delaware Department
of Natural Resources and Environmental Control, Superfund Branch.
ABSTRACT
The Delaware Department of Natural Resources and Environmental Control
(DNREC) Superfund Branch performed a comparative field study of three
immunoassay field screening methods versus standard SW-846 laboratory
analytical methods for detection and semi-quantification of environmental
contaminants. These immunoassay methods were designed to measure very low
concentrations of contaminants in water and soil matrices at
important regulatory action levels. The three immunoassay methods studied were
products to measure benzene, toluene, ethylbenzene, and xylenes (BTEX);
polynuclear aromatic hydrocarbons (PAH's); and polychlorinated biphenyls (PCB).
In each case, an appropriate cut-off level or range was selected and correlation was
reported in a yes/no format for detection. This study utilized samples from two sites
currently under active investigation. One a former coal gasification facility with
petroleum derived contaminants and PCB's, and the other, a dry cleaning
establishment with chlorinated solvent contamination.
This study demonstrated that immunoassay technology is well suited for the
semi-quantitative screening of soil for contaminants in the field. Results were
reliably predictive and correlated well with current standard laboratory
quantitative methods. The immunoassay methods achieved greater than 85%
agreement with laboratory results. With the exception of one comparison, all
disagreements involved immunoassay results higher than predicted by the
standard analytical method. This was expected and, in most of these cases, the
immunoassay correctly predicted the presence of contaminants and the standard
method did not, this due to the characteristics of the comparative technologies.
GC/MS techniques produce a concentration dependent individual compound result
and strict rules govern when a detected peak is reported. In contrast, these
immunoassays were designed to be class specific rather than individual compound
specific. When multiple analytes within a class of compounds are present in a
sample, at low levels, these immunoassays detect and measure them
simultaneously. Under these circumstances, immunoassay results should be
expected to report higher concentrations than standard analytical methods. As
well, the presence of known cross-reactive compounds has to be considered when
interpreting the data. Other disagreements were attributed to sampling problems
related to highly heterogeneous soils. The presence of chlorinated solvents in these
-------
samples had no significant impact on the assay results of the three immunoassay
methods.
Although PCB contamination was known to be present, the concentration
levels in most of the samples aquired for this study were low. Only a limited
number contained high PCB levels,. Therefore, the study results may be biased
towards high agreement due to the larger number of results below the practical
quantitation limits (PQL's). However, the positive results above PQL's correlated
well and were within experimental error.
INTRODUCTION
A demonstration and careful evaluation of three immunoassay screening
methods for soil samples were conducted by the Delaware Department of Natural
Resources and Environmental Control (DNREC) Superfund Branch in conjunction with
contamination investigations at two sites: a former coal gasification facility and an
active dry cleaning establishment. The immunoassay field screening test kits used
were developed by Ohmicron Environmental Diagnostics, Newtown, PA. Eight
Ohmicron products were used in this study:
RPA-I™ RaPID Photometric Analyzer
RaPID Prep™ Soil Collecion Kit
Total BTEX RaPID Assay® Kit
RaPID Prep™ Total BTEX Sample Extraction Kit
PAH's RaPID Assay® Kit
RaPID Prep™ PAH's Sample Extraciton Kit
PCB RaPID Assay® Kit
RaPID Prep™ PCB Sample Extraction Kit
The RaPID Assay immunochemistry system utilizes the priciples of enzyme
linked immunosorbent assay (ELISA). This technology uses selective antibodies
covalently bonded to suspended paramagnetic particles and couples an enzyme reaction
to produce a colorimetric response. Paramagnetic particles used as the solid-phase
allow for the precise addition of antibody and non-diffusion limited reaction kinetics.
The colorimetric response is affected by the presence of the selected contaminants and
the degree of response is inversely proportional to the concentration of the selected
contaminants in the sample. Measurements and direct concentration readouts are
produced using a dual wavelength spectrophotometer supplied as part of the RaPID
Assay system.
The three immunoassay methods studied were products to measure benzene,
toluene, ethylbenzene, and total xylene isomers (BTEX); polynuclear aromatic
hydrocarbons (PAH's); and polychlorinated biphenyls (PCB). The study was
-------
primarily focused on detection below or above specified concentrations. For each
test method, an appropriate cut-off level or range was selected and correlation was
reported in a yes/no format. Of primary study interest to DNREC and Oh micron were
the field screening results obtained from the former coal gasification facility. Using the
dry cleaning site as part of the field study allowed for the determination of possible
false positive or negative results caused by the presence of chlorinated organic solvents
in the soil matrices. A representative number of the collected soil samples were
submitted to an independent commercial laboratory, National Environmental Testing,
Inc. of Thorofare, N.J., a DNREC Superfund Branch approved laboratory, and the
DNREC Environmental Services Laboratory in Dover, DE. These labs produced the
quantitative results used for analytical verification of the immunoassay screening
results. The commercial laboratory was unaware of the field study and its involvement
in that study. By using two different analytical laboratories sampling efficiency or
inefficiency could be evaluated and would provide additional information for the
analytical comparisons particularly when differences occurred.
GENERAL DESCRIPTION OF SITE CONTAMINATION
The soil samples for the field study were collected from two Delaware State
Superfund sites located in Georgetown, Delaware; the aforementioned coal gasification
site and the dry cleaning establishment. These sites are in very close proximity to each
other, approximately 200 feet apart at their nearest point. The dry cleaning site is
located hydraulically upgradient of the coal gas facility with respect to groundwater
flow direction. Previous preliminary assessments and site inspections under the
Federal Superfund Program for both sites revealed elevated levels of BTEX, PCB's, and
PAH's in the soils at the coal gas site; tetrachloroethene (PCE), trichloroethene (TCE),
and cis-l,2-dichloroethene (cis-DCE) in the dry cleaning site soils; and PCE in a
groundwater pumping well located downgradient of the dry cleaning site and
approximately 80 feet from the coal gas site. The immunoassay field study took place
concurrent with the investigations of the two sites by the DNREC.
FIELD SAMPLING AND SAMPLE HANDLING
A total of thirty-three (33) soil samples were collected, twenty-six (26) from the
coal gas site and seven (7) from the dry cleaning site for the field study. Sample
identification, collection site, sample depth below ground surface and sampling
technique information are provided in Table 1. In addition, three (3) soil sample field
duplicate pairs, two (2) soil matrix field blanks, four (4) blind spike soil samples of
estimated concentration, and two (2) spike soil samples of known concentration were
included in the study to facilitate quality control and assurance, verification, and
documentation.
The samples were collected on two separate days, 10/19/94 and 10/26/94. The
samples collected on the first date were obtained using stainless steel hand augers.
-------
The samples collected on the second date were obtained during the drilling and
installation of monitoring wells using a split barrel sampler. They were collected in
accordance with ASTM Method D1586. Decontamination of the sampling equipment
was performed prior to, and immediately following, each sample collection. The
equipment decontamination procedure was performed hi accordance with the U.S. EPA
approved DNREC Quality Assurance Project Plan. The collected samples were
immediately placed into laboratory supplied I-Chem quality assured bottleware and
stored hi coolers containing ice for shipment to the appropriate laboratories. Sample
homogenization could not be adequately performed since the analytical parameters
included volatile compounds. This ensured that sample integrity would be preserved.
Of further concern, the coal gas site soils were highly non-homogeneous. Although part
of the heterogeneity was due to depositional processes, The heterogeneity was mostly
due to anthropogenic activity. There was a varied degree and presence of coal ash, coal
tar, gravel, brick fragments, and clayey, silty, and sandy fill material over extremely
short spatial distances both horizontally and vertically.
The samples were extracted and field screened using the recommended RaPID
Assay procedures within three days of the date of sampling. The confirmation samples
were sent to the laboratories and analytical results were received from the laboratories
within 10 to 60 days from the date of sampling. Copies of the analytical results are
provided in Appendix A.
METHODS OF DETECTION AND PROCEDURES
The detection mechanism associated with immunoassay technology is based on a
binding reaction between the contaminant (the antigen) and an immunoglobulin
molecule (the antibody). These two molecules combine at the antigen-binding site(s) of
the antibody. The surface of the antibody binding site is a slightly flexible, physical
reciprocal (mirror image) to the surface of its antigenic determinant. The combining
reaction, therefore, requires a physical as well as chemical matching and significantly
relies on molecular structure of the antigen. This matching provides for the specificity
in antigen-antibody binding. However, since many individual compounds within the
same chemical class have similar molecular structure, the specificity of the antibody
can be designed to be class specific rather than individual compound specific. This is
important since it allows simultaneous detection and measurement of a class of related
compounds when screening a site for contaminants.
In contrast with immunoassay, volatile and semivolatile gas
chromatography/mass spectroscopy methods, used in the laboratory, rely on individual
chemical identification and produce discrete concentration results for each tentatively
identified compound (TIC). There is virtually no overlap and no cross or cumulative
reactions. Detection, and subsequent reporting, are concentration dependent. For a
TIC to be reported as detected, it must be, as defined by the DNREC Hazardous
Substance Cleanuo Act - Standard Ooeratinsr Procedures for Analytical Programs
-------
(HSCA SOPCAP), greater than 10% of the total area of the closest internal standard.
Conversely, all compounds, no matter how numerous, that are less than 10% of the
closest standard peak area are not considered reportable. Also, in accordance with the
HSCA SOPCAP, only the 20 largest detected peaks greater than 10% are reported
regardless of how many others may be present.
This difference between these immunoassays and conventional GC/MS
technology must be taken into account. Immunoassay measurements produce a
cumulative result for all antibody reactive homologous chemicals in the sample.
Individually they may be present at concentrations below the detectable limit as
defined by the chromatographic equipment, PQL's, or standard operating procedures.
In general, when multiple analytes within a class of compounds are present in a
sample, at low volumes, immunoassay results should be expected to report higher
concentrations than standard analytical methods. As well, the presence of known
cross-reactive compounds has to be considered when interpreting the data.
Ohmicron has characterized and documented the reactivity of several low molecular
weight aromatic hydrocarbons detected by its BTEX kit. These include: naphthalene,
phenanthrene, anthracene, styrene, and acenapthene. Naphthalene, in particular, is
recovered to the extent of 110% (vs. the method calibration standard) so it was included
in the laboratory analytical parameters along with BTE}Cs. In order to correctly
interpret the immunoassay results in the comparisons, the laboratory data needed to be
examined for the presence of other PAH compounds; i.e., if certain PAH compounds
were present in a sample absent of BTEX's, the RaPID Assay" BTEX kit could produce
an apparently false-positive response because the laboratory results would indicate
that no BTEX compounds were present. In fact, under these circumstances, the
immunoassay is a more reliable predictor of contamination than the laboratory method.
Relative response data for a varied list of organic compounds was supplied to the
DNREC for this study and are found hi Appendix B.
The immunoassay methods were performed by the Laboratory Specialist of the
DNREC Superfund Branch following the recommended extraction procedure and assay
protocol as provided in the RaPID Prep and RaPID Assay documentation. For this
study, a total PAH concentration cut-off level of 1.0 parts per million (ppm) was
established. Results above 1.0 ppm were considered a positive result for the
immunoassay screen. The concentrations defining a positive detection for BTEX and
PCB's were 2.5 ppm and 0.5 ppm, respectively. It is important to note that the least
detectable dose (LLD) for both the PAH and BTEX kits are lower than the positive
detection concentrations set for this study.
All samples collected were sent to the Environmental Services Laboratory of the
DNREC to generate the confirmation data on PCB's and PAH'S. National
Environmental Testing, Inc. of Thorofare, N.J. (NET) data was used to provide the
laboratory information for the BTEX immunoassay result comparisons and also to
confirm the laboratory results for PCB's and PAH'S. The sample extraction method
-------
used for PCB's and PAH's was SW-846 method 3550. The analytical methods used |
were SW-846 method 8270 for PAH's, SW-846 method 8081 for PCB's, and SW-846 "
method 8240 for BTEX. The PQL's were 0.330 ppm for PAH's, 0.040 ppm for PCB's,
and 0.100 ppm for BTEX. A Data Verification Report for the laboratory analytical data
is provided in Appendix B as prepared by the DNREC Superfund Branch in accordance
with the U.S. EPA Region HI Modifications to National Functional guidelines or HSCA
SOPCAP, whichever was more strict.
IMMUNOASSAY AND LABORATORY RESULTS AND COMPARISONS
The overall correlation between the immunoassay field screening results and the
laboratory analytical results was considered good and to be within acceptable error for
field screening applications. Due to the relatively small data set and uncontrollable
variables, such as site soil heterogeneity, in-depth statistical evaluations of the
compared data were not considered necessary.
The samples collected from the dry cleaning site, the GC-xx sample series,
produced no false positive detection results in the presence of up to 21 ppm PCE, 0.5
ppm TCE, and 0.8 ppm cis-DCE in any of the three immunoassay methods. A copy of
the laboratory analytical report for these samples is provided in Appendix B.
PAH Comparative Results m
There was 86% agreement on PAH detection (31 agreeing detection results out of
36 samples) between the RaPID Assay immunoassay results and the DNREC
laboratory analytical results (Table 2). There was also a 22 out of 25, or 88%
agreement with NET, Inc. results. The five detection disagreements between
immunoassay and the DNREC laboratory" consisted of five false positive results for the
immunoassay field screening data. There were one false negative and two false
positive results when compared to the NET, Inc. laboratory data. These disagreements
are further discussed in the next section.
Results from the two analytical laboratories were not always in full agreement.
They compared at 92% agreement on PAH detection for the 25 split samples (23
agreeing results out of 25 samples). Quantification differences between these
laboraties were significant in some cases. These discrepancies are also discussed along
with five disagreeing detections in the next section. Table 2 summarizes the PAH
study results.
BTEX Comparative Results
Similarly, acceptable agreement of detection results was achieved for the BTEX
field data. For the data set, 25 out of the 30 samples (83%), had the same detection
result between the RaPID Assay BTEX kit and the laboratory. Five (5) false positive
-------
results were detected by the immunoassay screening methodology. The BTEX results
are summarized in Table 3.
PCB Comparative Results
A high correlation was achieved for the PCB screening results. Thirty-five (35)
of the 38 samples, or 92%, were in agreement. All three disagreements were false
negative results. It must be noted that most of the samples had very low
concentrations and some did not contain detectable quantities of PCB's. This could bias
the results towards high agreement. However, 2 blind spike and 1 known spike
samples agreed well. Table 4 summarizes the detection results for PCB.
INTERPRETATIONS
Quantitative results from highly contaminated soil samples at the coal gas site
collected from a region containing a varied degree and presence of coal tar and coal ash
and split between the two laboratories showed congruence of contaminant trends with
depth but fairly significant quantification differences (see the MW-2x and GCG-7x
series in Table 3). These differences were considered attributable to the sampling
protocol. As mentioned earlier in the FIELD SAMPLING AND SAMPLE HANDLING
section, homogenization was minimized due to concern for volatilization of
contaminants. A 2 foot interval of the heterogeneous material was needed to obtain
adequate sample quantity for analytical purposes. Without extensive homogenization,
it is not unlikely that each of the three groups of bottleware (one for screening and two
for the laboratories) could produce differences. These differences would also be
expected to show as detection discrepancies in the three comparisons where the
contamination interface occured within the 2 foot interval.
The heterogenity is apparent in the GCG-5x series. This sample series consisted
of a vertical sampling scheme from one soil boring containing three samples collected at
different depths: GCG-50 collected from 0 - 2 ft; GCG-53 collected from 3 - 5 ft; and
GCG-56 collected from 5 - 7 ft, below ground surface. Table 5 summarizes the detection
results for the immunoassay method and both laboratories for the PAH test. This
sample series was taken in an area where coal ash was present in the soil.
Table 5. Summary of PAH Positive (+) and Negative (-) Results.
Sample ID
GCG-50
GCG-53
GCG-56
Tfnmnnnggsgy
PAH Result
-
+
+
Immunoassay
BTEX Result
-
+
+
NET PAH
Results
+
+
-
NET BTEX
Results
-
+
-
DNREC
PAH
Results
-
-
-
-------
As can be seen in Table 5 there is consistency between the PAH and BTEX A
results for the immunoassay test methods. At each depth the two extracts were "
derived, essentially, from the same soil sample. Their correlation in presense is likely
due to PAH and BTEX both being petroleum derived and therefore, simultaneous
presence at contaminated coal gas sites should be expected. However, between all
three detection results there is little agreement. Sample heterogeneity is the logical
reason for these detection discrepancies.
The false positive detections for the PAH immunoassay results for samples GCG-
64 and MW-19 could not be directly explained other than by experimental error and
possible cross contamination during sample handling and preparation.
Of the five BTEX false positive results for the immunoassay screen, three,
samples GCG-11, GCG-10, and GCG-56, can be clearly explained as cumulative
reactivity of the BTEX antibodies with low molecular weight aromatic hydrocarbons
and other PAH compounds in these samples. Naphthalene was singled out for
inclusion with the BTEX evaluation, however, other compounds present contributed to
the assay results.
According to Ohmicron literature, phenanthrene is 41% cross reactive at 50% of
the least detectable dose (LLD) of 0.8 ppm for the BTEX method; i.e., 1.6 ppm of
phenathrene will be detected by the BTEX immunoassay method. Sample GCG-11 had 4
approximately 50 ppm of total PAH'S based on the NET data. Phenanthrene was ^
quantified at 2.0 ppm by NET, Inc. and at 1.6 ppm by the DNREC laboratory.
Acenaphthene and anthracene were also detected in GCG-11 at 3.5 ppm and 1.1 ppm,
respectively. Although this is below their respective cross reactive detection limits of
6.2 ppm and 28 ppm, their presence will be additive and will contribute to the
immunoassay result. In addition, a TIC such as 2-ethenyl naphthalene, which has no
supplied cross reactivity data, will most likely react and increase the immunoassay
result due to its similar structure to naphthalene but would not be reported by the
laboratory.
The apparent false positive BTEX result of sample GCG-10 can be explained
similarly to that of GCG-11 as described above. But more important is that NET, Inc.
did detect BTEX compounds present. However, quantification was below the positive
result concentration cut-off and therefore was considered as negative according to the
initial parameters of the study. Because of the known presence of BTEX, along with
greater than 2 ppm total PAH'S in the form of low molecular weight aromatic
hydrocarbons and other PAH compounds, it is clear that the positive result by the
immunoassay method correctly detected the presence of homologous contaminants.
Similarly, sample GCG-5 6* had numerous known and unknown PAH constituents that
the GC/MS individual, concentration dependent methodology did not report as
detected. As previously mentioned, multi-contaminant soil samples are more likely to
produce a correct positive response when analyzed with cumulative detection methods
-------
such as immunoassay than they are with laboratory quantitative methods, when
individual concentrations are low.
No confirmation can be supplied for the false positive immunoassay results for
samples MW-13 and MW-15. These are considered due to experimental error.
Considering the high levels of contamination in the samples, cross contamination
during preparation may be an explanation.
The BTEX immunoassay method correctly detected the presence of cross-reactive
compounds (detected by both laboratories) previously documented by Ohmicron as
producing positive results. Therefore, three of the five false positive results are
considered correct detection. Moreover, the correlation of the BTEX test data is more
accurately represented by a 92% agreement.
Interpretation of the highly complex chromatograms for soil extracts containing
PCB's above the PQL's for this study was significantly assisted by the immunoassay
results. These samples contained weathered Arochlor 1254 patterns and exhibited
significant matrix interference. The DNREC laboratory classified the chromatographic
results into four categories: Positive Detection; Highly Probable Detection;
Questionable Detection; and No Detection. Similar chromatographic activity patterns
were evident in the NET, Inc. data. All Questionable Detection results were less than
0.2 ppm. This level would not be detected by the immunoassay method and therefore,
would have minimal to no effect on the study conclusions. Detection disagreement
within the PCB study can also be explained by heterogeneity in the core samples. In
sample series GCG-7X, sample GCG-7 3' gave a false negative result. But reviewing
the vertical trend in PCB concentration it is clear to be decreasing with depth. The
sample GCG-7 3' is vertically positioned between the confirmed PCB presence above
and the non-detectable concentrations below. Considering a 2 foot sampling interval
and the small amount of sample used in the immunoassay method, it is possible, and
quite likely that the soil extracted for the immunoassay screen was taken below the
vertical extent of contamination and the immunoassay result is correct for that sample.
In contrast, the large amount of sample collected for the two laboratories contained
soils from both above and below the vertical extent of PCB contamination.
Sample MW-2 7' exhibited the same results within a vertical sample series as
sample GCG-7 3', except conversely. In the MW-2x series the concentrations were
increasing with depth. The sampling and sample handling inefficiencies also explain
this false negative detection.
No explanation is evident for the false negative result in sample GCG-11 other
than experimental error.
-------
CONCLUSIONS
Overall, the immunoassay field screening technique had good correlation with
the laboratory data. Further, the detection discrepancies were considered to be due to,
and explainable by, sample heterogeneity and differences in detection technology
methods between the immunoassay and the laboratory analytical methods.
It is important to emphasize the need to be aware of the cumulative method of
detection associated with this immunoassay field screening technology; especially when
results are to be compared with conventional SW-846 results. In addition, cross-
reactivity information and high quality control in sample collection protocols are
necessary to obtain appropriate data evaluations.
As technological improvements continue in the development of immunoassay
environmental testing, this reliable method of sample evaluation will become an even
more valuable, cost effective tool. Immunoassays may produce better results and be
more predictive of contamination than the current peak matching SW-846 method in
the determination of PCB concentrations when complex chemical matrices such as
highly contaminated and/or chemically degraded soils are being investigated.
10
-------
Sample Location
GCG-12
GC-13
GC-23
GC-43
GCG-71
GCG-72
GCG-73
GCG-77
GCG-8
GCG-50
GCG-53
GCG-56
GCG-61
GCG-64
GCG-67
GCG-10
GcG-11
Field Blank 10/19/94
MW-13
MW-15
MW-17
MW-19
MW-23
MW-25
MW-27
MW-29A
MW-29B
MW-33
MW-35
MW-37
MW-39
MW-311
FIELD BLANK 10/26/94
MW-4 (-50PPM)
MW-5(~5PPM)
GC-3 4(~5PPM)
GC-4 4(~5PPM)
ERA #0610-94-02(DNREC)
MW-3 5-3(DNREC)
MW-2 19-18(NET)
GCG-9(DNREC&NET)
Depth
0.00
0.00
0.00
0.00
0.00
1.00
2.00
6.00
0.00
0.00
3.00
5.00
0.00
3.00
6.00
0.00
0.00
0.00
3.00
5.00
7.00
9.00
3.00
5.00
7.00
9.00
9.00
3.00
5.00
7.00
9.00
11.00
lnterval(ft.)
2.00
3.00
3.00
3.00
1.00
2.00
3.00
7.00
1.00
2.00
5.00
6.00
1.00
4.00
7.00
1.00
1.00
1.00
5.00
7.00
9.00
11.00
5.00
7.00
9.00
11.00
13.00
5.00
7.00
9.00
11.00
13.00
Sampling Technique
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
Hand Auger
HAnd Auger
LSplit Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
Split Barrel
PCB UNKNOWN
PAH UNKNOWN
PCB UNKNOWN
PAH UNKNOWN SPIKE
PCB KNOWN SPIKE
DUP MW-3 3-5
DUP MW-2 9-11
DUP GCG 12
Table 1. Study sample source, identification, and sampling technique.
-------
Sample ID
GCG Fid Blnk
MW Fid Blnk
GC-13
GC-23
GC-33
GC-43
GC-44
GCG-50
GCG-53
GCG-56
GCG-61
GCG-64
GCG-67
GCG-71
GCG-72
GCG-73
GCG-77
GCG-80
GCG-90
GCG10
GCG-1 1
GCG-12
MW-13
MW-15
MW-17
MW-19
MW-23
MW-25
MW-27
MW-29A
MW-29B
MW-33A
MW-33B
MW-35
MW-37
MW-50
IA Result
neg
neg
neg
neg
neg
neg
pos
neg
pos
pos
pos
pos
neg
pos
pos
pos
pos
neg
pos
pos
pos
pos
neg
neg
neg
pos
pos
pos
pos
pos
pos
neg
neg
neg
neg
pos
LAB1
<0.3
0.8
<0.4
<0.4
<0.4
<0.4
NA
2.6+tic
7.2+tic
0.3
330+tic
NA
<0.4
3700+tic
2400+tiC
1060+tic
70+tic
0.5
220+tic
2.8+tic
55+tic
170+tic
<0.4
NA
NA
1.0 ppm) from
negative (<1.0 ppm) samples.
-------
Sample ID
GCG Fid Blnk
MW Fid Blnk
GC-13
GC-23
GC-33
GC-43
GCG-23
GCG-50
GCG-53
GCG-56
GCG-61
GCG-64
GCG-67
GCG-71
GCG-72
GCG-77
GCG-81
GCG-90
GCG-10
GCG-11
GCG-12
MW-13
MW-15
MW-17
MW-19
MW-25
MW-27
MW-29
MW-219-18
MW-311
IA result
neg
neg
neg
neg
neg
neg
pos
neg
pos
pos
pos
neg
neg
pos
pos
pos
neg
pos
pos
pos
pos
pos
pos
neg
neg
pos
pos
pos
pos
neg
Method 8240
BTEX (ppm)
<0.5
<1.0
<0.3
<1.S
<0.3
<0.4
62
0.2
<0.3
<0.4
2.2
<0.3
<0.3
1
34
1.5
<0.3
1.4
0.1
0.05
1.1
<0.4
<0.2
<0.3
<0.3
5.3
159
76.4
104
<0.4
Method 8270
Naphthalene (ppm)
<0.4
<0.1
<0.4
<0.4
<0.4
<0.4
440
<0.4
7.2
<0.4
' 3.1
<0.4
<0.4
' 30
660
>17
<0.4
• 5.1
<0.4
<3.7
1.8
<0.4
<0.4
<0.4
<0.4
80
220
236
210
0.04
Correlate
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
no
no
yes
no
no
yes
yes
yes
yes
yes
yes
yes
GC = Georgetown Cleaners Site, Georgetown, DE
GCG = Georgetown Coal Gas Site, Georgetown, DE
MW = Borings from monitoring wells (13-219@GCG, 311@GC)
Table 3. Comparison of Ohmicron's BTEX RaPID Assay® with SW-846 Method 8240
and the naphtalene concentration from Method 8270. In this study a cutoff concentration of 2.5
ppm for the immunoassay was established to discriminate positive (>2.5 ppm) from negative
(<2.5 ppm) samples. Confirmation analyses were performed at National Environmental Testing,
Inc., Thorofare, NJ. Results marked with * were taken from DNREC laboratory Method 8270
PAH analyses on the indicated samples.
-------
SAMPLE ID
Blind Spike-1
Blind Spike-2
Field Blank-1
Field Blank-2
Known Sample
GC-1 3'
GC-2 3'
GC-3 3'
GC-4 3'
GCG-5 0'
GCG-5 3'
GCG-5 6'
GCG-6 V
GCG-6 4'
GCG-6 7'
GCG-7 1'
GCG-7 2'
GCG-7 3'
GCG-7 7'
GCG-8 V
GCG-9 0'
GCG-101'
GCG-11 0'
GCG-12
MW-1 3'
MW-1 5'
MW-1 7'
MW-1 9'
MW-2 3'
MW-2 5'
MW-2 7'
MW-29'-11'
MW-29'-13'
MW-3 3'
MW-3 5'
MW-3 5-3
MW-3 7'
MW-3 9'
EPA METHOD 8081 (ppm)
GC Column 1
3.0
29
<0.033
<0.032
1.9
<0.034
0.084
<0.034
<0.038
0.13
0.37
<0.038
1.2
<0.033
<0.038
4.9
4.4
1.1
<0.037
<0.036
7.6
0.15
4.4
12
<0.036
<0.035
<0.037
<0.037
0.093
0.31
1.5
2.4
1.5
<0.035
<0.034
0.039
<0.034
<0.036
GC Column 2
-
29
<0.033
<0.032
-
<0.034
0.033
<0.034
<0.038
0.21
0.36
<0.038
0.84
<0.033
<0.038
8.7
2.2
0.54
<0.037
<0.036
6.1
0.19
4.2
8.7
<0.036
<0.035
<0.037
<0.037
0.040
0.19
0.99
2.5
1.1
<0.035
<0.034
0.044
<0.034
<0.036
E!A RESULT
(ppm)
4.3
>5
ND
ND
2.7
ND
ND
ND
ND
ND
ND
ND
2.6
ND
ND
2
2.1
0.2
ND
ND
1.9
ND
ND
4.8
ND
ND
ND
ND
ND
ND
ND
0.92
.68
ND
ND
ND
ND
ND
E1A SCREENING
RESULT (ppm)
0.5-10
>5
<0.5
<0.5
0.5-10
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
0.5-10
<0.5
<0.5
0.5-10
0.5-10
<0.5
<0.5
<0.5
0.5-10
<0.5
<0.5
0.5-10
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
0.5-10
0.5-10
<0.5
<0.5
<0.5
<0.5
<0.5
AGREEMENT WITH
EPA METHOD
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
NO
YES
YES
YES
YES
NO
YES
YES
YES
YES
YES
YES
YES
NO
YES
YES
YES
YES
YES
YES
YES
i
GC = Georgetown Cleaners site, Georgetown, DE
GCG = Georgetown Coal Gas site, Georgetown, DE
MW = Borings from monitoring wells
Table 4. Comparison of Ohmicron's PCB RaPID Assay® with SW-846 Method
8081. Confirmation analyses were performed in the Dover, DE laboratories of the Delaware
Department of Natural Resources and Environmental Contol.
-------
EVALUATION OF THE BNSYS PAH-RISc TEST KIT
R. Paul Swift, Ph.D.. Senior Chemist, John R. Leavell, Chemist, Chris W.
Brandenburg, Chemist, ESAT Region 10, ICF Kaiser Engineers, Inc., 7411
Beach Drive East, Port Orchard, Washington, 98366
ABSTRACT
A polynuclear aromatic hydrocarbon field screening method, utilizing
enzyme-linked immunosorbent assay techniques, was recently evaluated. The
validation study was conducted to evaluate the performance and assess the
utility of this method for use in field screening activities at hazardous
waste sites. Design criteria included analysis of field samples, analysis
of well-characterized reference materials, test kit response to various
soil types (including analyte-spiked soils), and a general performance
evaluation using selected soil types with varying PAH concentrations.
INTRODUCTION
The authors recently conducted a validation study for an Enzyme-Linked
Immunosorbent Assay (ELISA) field-screening test method, PAH-RISc*. This
semiquantitative chromogenic analytical method, developed by Ensys, Inc.,
Research Triangle Park, NC, was evaluated for use in the determination of
Polynuclear Aromatic Hydrocarbons (PAHs) in environmental soil and
sediment samples. The experimental design and interpretation of results
obtained are described below.
BACKGROUND
Field Screening
Field screening for contaminants has applications in a wide variety of
situations (1), from collecting real-time data relating to worker safety
to monitoring plume boundaries resulting from materials spills. Low cost,
rapid-turnaround sample analysis is beneficial to site characterization
and assessment, as well. Sampling locations of possible interest and
local "hot-spots" may be identified quickly, aiding in the selection of
samples to be collected for subsequent CLP analysis. Other considerations
such as remediation efforts and effluent compliance may also be monitored.
The need for rapid, reliable, semiquantitative or quantitative methods for
measuring environmental contaminants has resulted in the introduction and
development of new technologies. In addition, laboratory-based analytical
methods such as GC/MS and HPLC, among others, are finding increased use in
field applications (2).
One novel field screening method gaining regulatory acceptance utilizes
immunoassay-based testing (3). Immunoassay techniques have been used for
more than 15 years in medical and clinical settings. This technology has
received Agency acceptance for a few classes of analytes (4), and is
currently being applied to the detection of PCBs, petroleum hydrocarbons,
and pentachlorophenol. One of the earlier environmental applications in
which this technology was used was in the detection of pesticides and
pesticide residues (5).
Enzyme-Linked Immunosorbent. Assays
Immunochemical analysis is a well-established clinical diagnostic
technique which is gaining wider acceptance in other areas. In the field
of environmental analysis, antibodies have been or are being developed for
Presented at the Ninth Annual Waste Testing and Quality Assurance Symposium on July 13,
1993, in Arlington, VA
-------
a number of contaminants. All these antibodies are of animal origin, but •
advances in monoclonal preparation have yielded in-vitro propagation
methods.
In order to obtain antibodies of the desired specificity, an immunological
response must first be generated in the host. Typically, the target
analyte, or hapten, is derivatized to produce a "handle" for use in
attachment to a macromolecule. Historically, proteins such as bovine
albumin have been used as the carrier. This step is essential because
direct injection of the analyte, due to its small mass (on a biological
scale), would result in detoxification by the animal's liver. In order to
be recognized as an antigen, or immunogen, by the immune system, a. foreign
substance must have a mass which is large on a molecular scale (>10,000
Da) . When injected into the host animal these immunogens cause the
animal's immune system to generate antibodies in response to the foreign
substance. The hapten is then bound to an enzyme, such as horseradish
peroxidase, to form an enzyme-conjugate. The conjugated compound is used
as the chromogenic reagent in this test.
After the antibodies are extracted from the host, they are sorted based on
sensitivity and specificity to the hapten. In the polyclonal method, the
above steps are repeated until a desired quantity of antibody is obtained.
The test kit used in this study utilizes a monoclonal preparation of the
desired antibodies using hybridoma technology, in which antibody-producing
(spleen) cells are fused with myeloma cells. The resulting progeny cells
are able to produce relatively large amounts of the specified antibody.
In a typical test kit, antibodies of the desired specificity are then
immobilized on a solid substrate such as a small test tube. A predeter-
mined amount of sample, possibly containing analyte, is extracted with a
suitable solvent. An aliquot of extract and a fixed amount of enzyme-
conjugate are added to the antibody tube and allowed to incubate for a
given period of time. Competition between the analyte and enzyme-
conjugate for a limited number of antibody binding sites results in
binding of the haptens in proportion to their relative concentrations.
Unreacted haptens are washed from the tube and two color development
reagents added. These reagents react with the bound enzyme-conjugate,
producing a depth of color proportional to the amount of enzyme-conjugate
bound. The "intensity" of color, compared photometrically to a calibra-
tion solution containing analyte, is inversely proportional to the amount
of analyte present in the original sample.
This test method utilizes ten-fold serial dilutions of the solvent extract
for comparison with a 1 ppm calibration standard prepared in the same
manner as the samples. A comparative photometer measures the difference
in absorbance between the sample and the standard, and uses the difference
measurement rather than the parametric value for quantitation.
EXPERIMENTAL DESIGN
Four main design parameters were selected for this validation study,
including analyses of 30 samples for comparison with CLP-generated data,
analysis of four "worst-case" Superfund-class reference samples, false-
positive and then false-negative (1 ppm spike level) reaction to soil
types, and a general reliability/performance test using a given soil type
spiked with varying amounts of analyte. Given a fixed number of test
kits, the key design criterium was to conduct a comprehensive yet
conclusive set of experiments.
-------
Polynuclear aromatic hydrocarbons are a class of compounds which have one
or more aromatic rings, generally in conjugation. The majority of common
PAHs contain three, four or five rings, may have alkyl- or aryl- groups
bound to them, and the rings themselves may be heterocyclic. One
important three-ringed PAH is phenanthrene, because it has a physical
structure which resembles the skeleton of many other PAH compounds.
Table I PAH RISc" Soil Test Sensitivity to PAH Compounds
Number of Rings
2 rings
3 rings
4 rings
5 rings
6 rings
PAH Compound
Naphthalene
Acenaphthene
Acenaphthylene
Phenanthrene
Anthracene
Fluorene
Benzola [anthracene
Chrysene
Fluoranthene
Pvrene
Benzolfclfluoranthene
Benzo[Alfluoranthene
Benzolajpyrene
Dibenzo|a,/?]anthracene
Indenol /,2,3-ctflpyrene
BenzolsrA/lperylene
Concentration Necessary to Result in
Positive Test (ppm) *
200
8.1
7.5
1.0
0.81
1.5
1.6
1.2
1.4
3.5
4.6
9.4
8.3
>200
11
>200
* Samples with stated concentration will give positive result greater than 95% of the time when
tested at stated concentration level.
The antibody used by this test kit was developed to target, or key,
phenanthrene. According to the manufacturer, the kit is designed such
that concentration of phenanthrene necessary to result in a positive test
is 1 ppm (6). With the exception of anthracene, which is detectable at
0.81 ppm, the test is less sensitive to other PAHs. The concentrations
necessary to produce a positive result range from 1.2 ppm for chrysene to
200 ppm for naphthalene. A list of several PAH compounds (SW-846 Method
8310 Target List) and their respective detection limits has been provided
by the vendor (Table I). The reported sensitivities have been used as
-------
scaling factors in some sections of this report. For example, to produce •
an equivalent response using naphthalene rather than phenanthrene as the
target analyte, 200-times more naphthalene (on a mass basis) is required.
This relationship will be further investigated in a later section.
Phase I: Analysis of Field Samples
Several issues were raised during the initial design of the validation
study, the most obvious of these being the evaluation of the test kit with
respect to the manufacturer's claim:
This method correctly identifies 95% of samples that are PAH-
free and those containing 1 ppm or 10 ppm of PAHs. A sample
that develops less color than the standard is interpreted as
positive. It contains PAHs. A sample that develops more
color than the standard is interpreted as negative. It
contains less than 1 ppm or 10 ppm PAHs.
To substantiate this claim, 30 samples were selected from the Manchester
Environmental Laboratory (MEL) soils storage facility. These samples were
analyzed previously using either GC/MS (tentatively identified compounds
excluded but noted) or HPLC methodology; total PAH values (16 analytes)
ranged from zero (non-detect) to >182 ppm. Soil types ranged from
weathered sand to dark loamy humus.
Phase II: Analysis of Reference Samples
Four well-characterized reference samples were selected for use in this
study. These samples have been independently analyzed by a number of
laboratories and are representative of materials present at various
preremediated Superfund sites. The matrices range from marine sediment to
composited hazardous waste. All samples contain a variety of contami-
nants, including pesticides, PCBs, and in one case, tributyl tin.
Clearly, potential interferents are present in these samples. A scheme in
which three analysts conduct the analyses, with one analyst performing the
tests in duplicate, provides an empirical estimate of both precision and
accuracy.
Phase III: Reaction to Soil Types
Soil compositions vary greatly on a regional basis. Extractable humic
components, among many others, have the potential to negatively affect the
performance of the test kit. Analysis of soils of various compositions
would provide information necessary to estimate the effect of soil type on
test kit reliability. To that end, 11 samples collected from different
soil horizons throughout Washington state by the US Geological Service
were analyzed. The PAH content of each of these library soils has been
previously determined to be below the detection limit of the test kit.
Therefore, any positive test result(s) could be attributed to what have
been termed "relatively unremarkable" contaminants, which possess
structural features similar to those of phenanthrene (7).
PAH compounds adsorbed onto soil surfaces may resist methanol extraction
and the degree of adsorption may be dependent on the nature of the soil.
In order to investigate this effect, a subset of the library soils was
selected and the soils fortified, or spiked, with 1 ppm phenanthrene.
This quality control measure was used to qualitatively estimate the
distribution of phenanthrene between the soil matrix and the methanol
extract.
-------
Phase IVs Reliability/Performance Evaluation
A set of samples consisting of weathered, sandy soil was composited for
use in this phase of the validation study. Each of the samples were
previously confirmed to be PAH-free. Eight spiking levels were used,
ranging from 0.1 to 10.0 ppm normalized to the phenanthrene response. For
the spike, a three-component mixture containing a three, four and five
ringed PAH - phenanthrene, pyrene and benzo[&]fluoranthene - was
prepared. The relative amount of each component was scaled based on the
sensitivity of the test kit to the individual components.
The objective of this set of measurements was to provide an estimate of
error with respect to false positive results below the detection limit as
well as false negative results for samples with PAH concentrations above
the detection limit.
It was also hoped that these measurements would provide insight relating
to test kit response for mixtures of PAHs. Because the assay utilizes
competitive binding of the antibody-antigen for detection of the analyte,
we were interested in determining whether the presence of one PAH would
affect the sensitivity of the test to another PAH. Rather than having
only one PAH competing with the enzyme conjugate for antibody binding
sites, there could be two or more competitors. There is no a priori
reason to believe that the test response may be calculated using a simple
weighted average concentration of the individual PAHs and the stated test
sensitivities to each.
RESULTS AND DISCUSSION
This experiment was conducted under relatively ideal conditions. Three
experienced chemists performed several trial analyses in order to gain
familiarity with the kit. Work was performed at the laboratory benchtop
in a controlled environment (temperature, humidity, etc.). Quality
control/quality assurance, as specified through personal communications
and training by the manufacturer, included analysis of method blanks,
regular pipet delivery calibration verification, and rejection of
analytical sequences in which the relative absorbance of replicate
calibration standards varied by more than 0.2 absorbance units. The
analytical instructions supplied with the kit and QA/QC guidelines
provided by the manufacturer were followed without exception.
The laboratory results reported for the GC/MS and HPLC data were generated
in accordance with full QA/QC requirements following USEPA CLP and SW-846
protocols. All data reported have been validated. To maintain client
confidentiality, any unnecessary site-specific references have been
omitted.
One set of measurements was made to evaluate the response of the
comparative photometer. A series of five spiked-blank solutions were
prepared in duplicate, with concentrations of 0.5, 0.75, 1.0, 1.25 and 1.5
ppm phenanthrene. Within each set, the 1.0 ppm was treated as the
calibration standard. Aliquots of these solutions were withdrawn and
processed as if they were actual soil extract. The absorbance of each
solution was measured and recorded. The resulting "calibration" curve was
determined through a linear least-squares fit of absorbance vs. concentra-
tion. The results are illustrated in Figure I.
From the data, it appears as though the instrument response is linear
around the zero (difference) absorbance. However, the slope is relatively
small - therefore variations in absorbance affect the apparent sample
-------
Concentration ( ppm)
» tr i a I
O trial 2
Figure 1 Instrument Response
concentrations.
cautiously.
Instrument readings near zero must be interpreted
Phase Ii Thirty soil samples were selected for ELISA-PAH analysis. No
attempt was made to target the one or ten ppm concentration levels; this
portion of the study was designed to emulate characterization of samples
in the field. These samples were previously extracted (EPA Method 3540)
and characterized for base/neutral/acid extractable compounds (BNAs,
including PAHs; EPA Method 8270), volatile organic analytes (VOAs; EPA
Method 8240), metals (ICP-AES, EPA-CLP Method 200.8), and pest-
icides/polychlorinated biphenyls (pest/PCBs; GC/ECD, EPA Method 8080). In
addition to quantitating total PAH content and relative amounts of the
individual compounds, the supporting analyses were used to characterize
the soil matrices and identify potential interferences. The results are
shown in Tables II and III.
In Table II, the total PAH concentration for each sample is compared with
the ELISA results. False-positive and false-negative results are
indicated by "+" or "-", respectively. Results from GC/MS analysis of
sample 2361 indicated the presence of methyl-phenanthrenes at 3 ppm.
Although the associated data were qualified "NJ" to indicate "there is
evidence that the analyte is present, the associated numerical result is
an estimate", the levels apparently present would be sufficient to yield
positive identification at 1 ppm. This result slightly increases the
accuracy rates (indicated in parenthesis). The estimated accuracy for the
ELISA test, based analysis of the thirty samples, was 86.7% (90%) at 1 ppm
and 73.3% at 10 ppm. The frequency of false-positive results at 1 ppm was
13.3% (10%), with no false-negatives. The frequency of false- positive
results at 10 ppm was 20%, and false-negatives, 6.7%. One sample,
selected randomly, was analyzed in duplicate.
-------
Table II Analysis of Field Samples / Total PAH Content
Sample 10
4005
4006
4007
4010
4659
4300
4301
4302
4303
4640
2107
2358
2358D
2359
2360
2361
2362
2363
2364
2365
2366
2368
2369
2370
2371
2372
2373
2374
1 ppm Test
<1
*
*
tt
ft
*
»
*
*
>1
»
*
ft
»
ft
ft
tt
tt
*
»
10 ppm test
<10
•
*
*
*
«
»
H
*
ft
>10
•
tt
tt
*
tt
t
•
tt
ft
ft
ft
*
*
•
•
Lab Result (ppm)
0.2
12.2
16.0
0.0
0.5
8.7
147.7
182.3
4.4
0.2
0.0
85.4
85.4
28.5
0.3
0.6
0.0
1.8
3.4
6.7
0.9
43.2
72.8
1.3
0.3
0.4
27.9
0.0
False +/-
Evaluation
@ 1 ppm
+
+
+ '
+
Evaluation
10 ppm
+
+
+
4-
+
+
-
-------
Table II Analysis of Field Samples / Total PAH Content
1 —
Sample ID
2375
2376
2377
1 ppm Test
<1
ft
>1
•
10 ppm test
<10
ft
ft
ft
>10
Lab Result (ppm)
16.4
0.4
9.5
False +/-
Evaluation
@ 1 ppm
Evaluation
@ 10 ppm
-
' Tentatively identified compounds list indicates methyl-phenanthrenes present at 3 ppm
Table III Analysis of Field Samples / Normalized PAH Content
Sample ID
4005
40O6
4007
4010
4659
4300
4301
4302
4303
4640
2107
2358
23580
2359
2360
2361
2362
2363
2364
2365
1 ppm Test
<1
*
*
•
*
«
>1
ft
ft
ft
ft
ft
ft
ft
ft
1 0 ppm test
<10
«
»
•
»
>10
•
»
«
•
*
•
*
•
*
«
•
•
Normalized Lab
Result (ppm)
0.1
8.1
9.0
0.0
0.2
5.2
56.9
73.2
0.1
0.0
0.0
47.3
47.3
11.5
0.2
0.5
0.0
1.2
1.7
3.6
False +/-
Evaluation
(§> 1 ppm
+
+
+
+ '
Evaluation
@ 10 ppm
+
+
+
+
+
+
+
-------
Table III Analysis of Field Samples / Normalized PAH Content
Sample ID
2366
2368
2369
2370
2371
2372
2373
2374
2375
2376
2377
1 ppm Test
<1
«
*
*
»
>1
«
*
*
10 ppm test
<10
»
*
•
•
*
*
V
»
>10
9
ft
*
Normalized Lab
Result (ppm)
0.6
27.5
49.2
0.8
0.1
0.2
13.5
0.0
6.4
0.2
2.8
False
Evaluation
@ 1 ppm
+
+
+ /-
Evaluation
<§> 10 ppm
-f
-
' Tentatively identified compounds list indicates methyl-phenanthrenes present at 3 ppm
Table III shows the comparison of normalized PAH concentrations with the
ELISA results. As mentioned previously, the sensitivity of the test
varies with different PAH compounds. In order to account for this effect,
each PAH concentration was normalized by dividing its actual value by that
concentration required to produce a positive result at 1 ppm (phenanthrene
- 1). This was done to minimize any bias introduced into the test by
anti-PAH specificity. A critical assumption is that the manufacturer-
provided list of PAH sensitivities are valid in the presence of two or
more PAH compounds. Sensitivity to the methyl-phenanthrenes tentatively
identified in sample 2361 is not known, but was assumed to be similar to
that of phenanthrene. As in the previous data set, estimated accuracies
including any contribution from the methyl-phenanthrenes are enclosed in
parenthesis. The estimated accuracy was 80% (83.3%) at 1 ppm and 70% at
10 ppm. The frequency of false-positive results at 1 ppm was 20% (16.7%),
with no false-negatives. At 10 ppm, the frequency of false-positive
results was 26.6%, and false-negatives, 3.3%.
The results using total and normalized PAH concentration values are
similar. If samples with PAH concentrations exclusively near 1 and 10 ppm
had been used, a greater discrepancy would have been observed. The
majority of these samples contained levels of PAHs separated from either
test kit detection level. This demonstrates the need to apply the
screening test judiciously, with operator intervention/interpretation
applied to those samples near either limit.
Phase II: Four soil reference samples were analyzed by three different
analysts, with one analysis performed in duplicate ("D" in Table IV), to
illustrate the variation introduced by different analysts and also the
duplicate precision. Comparison of the data is shown in Table IV. There
was considerable variation in the results, apparently due to the presence
-------
of interferants, including compounds to which the ELISA test exhibits
cross-reactivity. The summary in Table IV lists the total and normalized
PAH concentrations.
These samples were selected because they present a real challenge to the
ELISA test. They are each representative of the types of materials found
at Superfund sites. Prior to analysis, the samples were sieved and
homogenized. Non-representative material was removed, but an effort was
made to preserve the physical character of the original sample. A brief
summary of each soil follows.
Sequim Bay: This matrix is typical of marine or lower-wetlands sediment,
with corresponding levels of marine salts. The consistency was
similar to water-saturated silt. Although the concentration of
various contaminants were each less than 1 ppm, a large number of
different compounds were present. This particular reference sample
has been independently characterized as many as 50 different times.
Some of the compounds present were:
Phenol - 0.24 ppm; 4-methyl phenol - 0.24 ppm; pentachlorophenol - 0.42
ppm; biphenyl - 0.79 ppm; methylnaphthalenes - 0.25 ppm; tributyl tin
chloride - 0.11 ppm; halophenyl phenyl ethers - 0.4 ppm; tetrachloroguicol
- 0.47 ppm
Table IV Analysis of Reference Samples
Sample ID
Sequim Bay
- marine sediment
- low levels of many
pesticides, chloro-
benzenes, phthalates
H.I. BSRM #XX
- Composite soil
- PCBs: 1 30 ppm
- Lead: 5000 ppm
- contains slag/ash
RTC Sample #XXX
- PCP: 2190 ppm
- substituted
naphthalenes:
2300 ppm
Soils Bldg 0003
- Isophorone: 4.4ppm
(dimethyl hexanone)
Trial
1
2
3
3D
1
2
3
3D
1
2
3
3D
1
2
3
3D
1 ppm test
<1
>1
«
X
*
X
X
»
ft
»
»
ft
ft
»
10 ppm test
<10
*
»
*
X
*
*
>10
*
ft
X
X
*
»
«
ft
*
*
1 00 ppm test
<100
»
»
*
•
«
*
*
>100
*
ft
*
ft
*
*
*
«
»
GC/MS Results
Lab Result
(ppm)
1.73
1.73
1.73
1.73
18.3
18.3
18,3
18.3
8611
8611
8611
8611
24.8
24.8
24.8
24.8
Normalized Lab
Result (ppm)
<0.1
<0.1
<0.1
<0.1
12.1
12.1
12.1
12.1
5764
5764
5764
5764
12.4
12.4
12.4
12.4
10
-------
As indicated in Table IV, the actual PAH concentration was less than
2 ppm. When normalized based on specificity to individual PAH
compounds, the apparent PAH concentration was less than 0.1 ppm.
Three of four ELISA analyses indicated that the PAH concentration
was greater than 10 ppm, and two tests yielded results greater than
100 ppm. Clearly, materials were present which respond to the test
in a similar manner as do PAHs, and exhibit a strong interference.
Unfortunately, no clear pattern was apparent in the spurious
results. The test did appear to yield either correct or false-
positive results at the 1 ppm level, depending on which set of lab
results were considered.
HI BSRM #XX: This bulk site reference material composite sample was
manufactured from soils collected at a Superfund site by the USEPA
in Region 10 and known to contain PAHs and PCBs. The matrix
consisted largely of marine sediments and silt, although there is a
significant amount of ash, slag and related fallout originating from
the operation of a secondary smelter for many years. Other man-made
debris and residue include cinders, brick material and sandblasting
waste from ship refinishing. Local "hot-spots" resulting from wood
and pole treating and indiscriminate dumping of waste oils and
electrical transformer/capacitor dielectric contributed PCBs and
PAHs to the BSRM.
The reference sample exhibited a fairly high degree of matrix
inhomogeneity on both the macroscopic and microscopic level. A
concerted effort was made to homogenize the sample prior to
analysis. The variability is reflected, in part, by the standard
deviations associated with the analytical results:
Lead - 5480 ± 2620 ppm; Arsenic - 37.8 ± 7.3 ppm; Total PAHs (7 carcino-
genic PAHs identified in MTCA) - 18.3 ± 4.6 ppm; Total PCBs - 127 ± 39
ppm.
A comparison of the sample results is shown in Table IV. All ELISA
analyses at the 1 ppm level correlate with the laboratory data. The
first two trials produced identical results at 10 and 100 ppm (true-
negatives), but disagree with the duplicate analyses performed by
the third analyst. The duplicate analyses were self-consistent.
Because the duplicate analyses were performed using the same soil-
extract, sample inhomogeneity rather than ELISA precision may have
contributed to the disparity.
RTC Sample #XXX: This sample was prepared under contract to the EPA as
part of a RCRA study and made available for use in this study.
Laboratory analyses were performed for both total metals and BNA
compounds. The levels of sodium, potassium, nickel and total PAHs
were relatively high. Some of the analytes present at significant
levels were:
Chromium - 16100 ppm; Phenanthrene - 2430 ppm; fluoranthene - 1840 ppm;
pentachlorophenol - 2190 ppm; methyl-naphthalenes - 200 ppm; carbazole -
81 ppm;
Of the reference samples, this was the most highly contaminated.
All ELISA results indicated PAHs present at 1 ppm. One analysis
yielded false-negative results at 10 and 100 ppm. This analysis is
clearly in error; with the levels of contaminants known to be
present, it seems unlikely that normal experimental or method
uncertainties were the cause. Since the 10 and 100 ppm results were
11
-------
generated using serial dilution of the 1 ppm extract, we believe •
their inaccuracy may be attributed to dilution error. ^
Soils Building 003: This sample was also a composite soil manufactured
from materials which had collected in a soils laboratory over a
period of time. A large number of assorted samples of varying
origin were blended, with aliquots withdrawn for waste disposal
characterization. Some of the material was dated and all had been
stored at room temperature. Microbial and oxidative decomposition
were expected to have yielded a variety of degradation products of
the PAHs and other contaminants. Subsequent analysis showed that
with the exception of 4.4 ppm isophorone (dimethyl hexanone), the
composition of contaminants was due predominately to PAHs, with
total and normalized concentrations of 24.8 and 12.4 ppm, respec-
tively. The matrix of the composite soil sample included clay,
silt, sand and loam.
All ELISA results indicated PAHs present at the 1 ppm level. Two
replicates yielded false-negative results at 10 ppm. Duplicate
analysis yielded correct results at 10 ppm, but false positive
results at 100 ppm. No simple explanation can be offered for this
disparity.
Phase III: Eleven soil samples collected from various soil horizons across
Washington state were analyzed using the ELISA method. The soils were
previously determined to contain undetectable amounts of PAH compounds.
The purpose of this phase of the experiment was to determine the effect of
soil type with respect to false-positive ELISA results.
Analysis of these soils yielded negative results in each case. On this ^L
basis, soil type did not appear to negatively impact the accuracy of the •
test with respect to false-positive results. ^
Four soils from this set were spiked with PAH to provide an estimate of
false-negative results with respect to soil type. These soils were
primarily clay/silt with a fair amount of humic or loamy material. A 10-
gram aliquot of each soil was spiked directly with phenanthrene to give a
final concentration of 1 ppm, allowed to weather at room temperature for
24 hours, extracted, and analyzed in duplicate. This method was preferred
to that in which the extract is spiked because adsorption of phenanthrene
on the soil surface is a potential physical interference. Resistance to
methanol-extraction would result in a negative bias and, therefore, false-
negative results. Phenanthrene was selected for use as the spike on the
basis of ELISA sensitivity, and to reduce any possible cross-reactivity
questions which may arise from the use of a mixed-PAH spike. The results
are shown in Table V.
Results from the spiking study show a high frequency (75%) of false-
negative results, indicative of the inability of the methanol extraction
fluid to completely liberate the phenanthrene from the matrix. Although
it is reasonable to expect that the methanol-based extraction is not 100%
efficient, these results yield an unacceptable percentage of false-
negative results. The bias built into the ELISA test did not appear to be
able to provide a large enough margin of uncertainty in this case.
Because a rigorous Soxhlet extraction and analysis was not performed, it
was not possible to estimate the actual efficiency of the extraction using
the ELISA method.
These limitations should be compared with those generally encountered
during laboratory-based analysis. The standard Region 10 Soxhlet
12
-------
extraction procedure involves refluxing the solid sample for a minimum of
18 hours in boiling solvent. Even then, extraction efficiency may only
approach 80% or so. Matrix spike analysis (non-RCRA) sometimes involves
adding the spike compound to the extraction solvent rather than to the
soil directly. Obviously, no weathering of the PAH/soil occurs when this
procedure is used. However, this does not change the fact that the kit
was unable to detect weathered PAH at 1 ppm in certain soil types.
Table V Reaction To Soil Types
Sample ID
8114
8123
8129
8136
8142
8500
8500 S
8501
8501 S
8506
8512
8512 S
8513
8513S
8107
1 ppm test
<1 ppm
•
•
*
•
•
*
Jt
ft
ft
*
ft
*
*
«
> 1 ppm
ft
10 ppm test
< 10 ppm
•
«
•
•
•
•
•
*
»
•
*
»
»
•
«
> 1 0 ppm
Spike result
phenanthrene
1 ppm
n/a
n/a
n/a
n/a
n/a
n/a
-
n/a
-
n/a
n/a
n/a
-
n/a
Soil description
yellow brn sandy loam
pale brn silty loam
brn silty loam
pale brn sand
pale brn sand
drk brn silt loam
-
drk brn gry sandy loam
-
lake sediments
It brn coarse loam sand
-
drk brn stony silt loam
"
It grey silty clay
Two papers previously published by the manufacturer described spiking
studies performed during validation of their PCS and pentachlorophenol
immunoassay methods (8-9). In each case the extracts, rather than the
native soils, were spiked. A similar study reported poor extraction
efficiency for PCBs spiked directly onto the soil (10). We felt it
important and more procedurally valid to spike and then weather the soil.
Phase IV: Another spiking study was performed to estimate reliability of
the ELISA method as a function of analyte concentration. Sandy, weathered
soil previously determined to be PAH-free was divided into 57 - 10 gram
aliquots, spiked directly, and allowed to interact for one hour.
A spiking solution was prepared using a three, four and five ring PAH
compound - phenanthrene (3), pyrene (4) and benzo[k] fluoranthene (5) -
such that their relative, normalized contributions on a mass basis to the
spike cocktail were equivalent. Using the 1 ppm spike as an example, a
solution was prepared such that 0.33 ppm of phenanthrene would be added to
13
-------
the soil. The same contributions were desired from the four and five-
ringed compounds, but the test is less sensitive to them. Since 3.5 ppm
of pyrene is required to produce the same response as 1 ppm phenanthrene,
3.5 times more pyrene was used (3.5x0.33). Similarly, the test is 9.4
times less sensitive to the benzo-compound, so 9.4x0.33 ppm benzo[fc]fluor-
anthene was used. Calculation shows that the total PAH concentration in
the spiked soil was actually 4.6 ppm. A similar scheme was used in the
preparation of the soils at the other spiking levels. With the exception
of the blank sample, which was analyzed once in duplicate, seven
replicates were analyzed in duplicate at each of 0.1, 0.5, 0.8, 1.0, 1.5,
2.0, 5.0, and 10.0 ppm (normalized) concentrations. Seven extractions
were performed at each level, and each extract analyzed in duplicate. The
results for normalized and total PAH concentrations are shown in Tables
VI(a) and VI(b), respectively; agreement of estimated error rates for the
different spike levels is coincidental.
Table Vl(a) Reliability Test - Normalized Concentrations
Normalized Value
(ppm)
Estimated Rate of
False Positives (%)
Estimated Rate of
False Negatives (%)
0.0
0.0
.
0.1
0.0
.
0.5
78.5
_
0.8
78.5
_
1.0
.
21.5
1.5
„
0.0
2.0
_
0.0
5.0
..
0.0
10.0
.
0.0
Table Vl(b) Reliability Test - Actual Concentrations
True Value (ppm)
Estimated Rate of
False Positives (%)
Estimated Rate of
False Negatives (%)
0.0
0.0
-
0.46
0.0
-
2.32
-
21.5
3.71
-
21.5
4.63
-
21.5
6.9
-
0.0
9.3
-
0.0
23.2
-
0.0
46.3
-
0.0
Three false-positive results were observed at both the 0.5 and 0.8 ppm
levels. The 0.5 ppm trial was repeated (extraction and analyses), and the
results were identical to those obtained in the first trial. Three false-
negative results were observed at the 1 ppm level. No false-negative
results were seen above 1 ppm.
The total PAH results showed a high rate of false-negatives. This data
would seem to indicate that normalizing the PAH concentration based on
sensitivity of the anti-PAH to the various PAH compounds is somewhat
valid. Also, the discrepancy between the spike recoveries in Phase V and
Phase VI is attributed to the difference in soil matrices. More PAH was
extracted from the sandy soil than from the loamy soil. This further
supports the conclusion that adsorption may introduce a low bias into the
test results.
CONCLUSION
It was the intent of this validation study to investigate and evaluate the
performance of the Ensys PAH-RISc* test kit. The goal was to conduct a
comprehensive set of experiments while working under the constraint of a
predetermined number of available test kits.
14
-------
This immunoassay test method performed favorably, although not quite as
well, as claimed by the manufacturer. It does appear to have the
potential to be used as a field screening tool, provided that the
limitations of its use be borne in mind.
The primary caveat is that this kit should be used by personnel capable of
proper interpretation of the results from a scientific standpoint. A
negative result at 1 ppm does not necessarily indicate that less than 1
ppm of PAH contamination exists. Relatively high concentrations of
particularly carcinogenic PAHs, such as benzo(a]pyrene, may be present on-
site at levels which are well above 1 ppm but below the levels necessary
to generate a positive test response. The sensitivity of the test kit to
the various compounds must be borne in mind when interpreting the results.
PAH-extractability may introduce low-bias at 1 ppm; the implications of
this must be considered when concentrations of PAHs are near the 1 ppm
level.
Also, proper soil sampling is a scientific technique. In order to obtain
accurate, representative results using this test kit, the operator should
follow appropriate soil collection methods.
Any samples which generate positive results, as well as a percentage of
those which test negative, should be submitted for subsequent confirma-
tional analysis by classical analytical methodology.
ACKNOWLEDGEMENTS
The authors would like to thank the U.S. EPA for supporting the study
under the Environmental Service Assistance Teams Zone 2 Contract 68D10135.
We would also like to thank Ensys Inc for providing the test kits used in
this study, and for the training rendered in their proper use. The USGS
soils and their characterization data were provided by Mr. Kenneth Ames,
USGS, and Mr. Dickey Huntamer, Washington State Department of Ecology.
Mr. Gerald Muth, USEPA, provided initial technical direction on the
experimental design.
DISCLAIMER
Although the research described in this manuscript has been funded wholly
or in part by the EPA Contract 68D10135 to ICF Technology Inc., it has not
been subjected to the Agency's review and therefore does not necessarily
reflect the views of the Agency; no official endorsement should be
inferred.
REFERENCES
(1) H.M. Fribush, J.F. Fisk, "Field Analytical Methods for Superfund",
Field Screening Methods for Hazardous Wastes and Toxic Chemicals,
Proceedings of the Second International Symposium, ICAIR Life
Systems, Inc., Las Vegas, NV, pp 25-29, February 12-14, 1991.
(2) Office of Emergency and Remedial Response, Hazardous Site Evaluation
Division, Field Screening Methods Catalog, Users Guide, EPA/540/2-
88/005, U.S. EPA, Washington, D.C., July, 1990.
(3) J.M. Van Emon and R.O. Mumma, Immunochemical Methods for Environmen-
tal Analysis, ACS Symposium Series 442, American Chemical Society,
Washington, D.C., 1990.
15
-------
(4) U.S. EPA Office of Solid Waste, SW-846 Draft Methods 4010, 4020,
4030, October, 1992.
(5) H.W. Newsome and P.G. Collins, Assoc. Off. Anal. Chem, 70, 1025,
1987.
(6) Personal communication, K.R. Carter, Ensys, Inc., 1993.
(7) S.B. Friedman, CHEMTECH, 732, 1992.
(8) J.P. Mapes, et.al., "Rapid, On-Site Screening Test for Pentachloro-
phenol in Soil and Water-PENTA RISc*, Superfund '91 Proceedings of
the 12th National Conference, HMCRI, Washington, D.C., December
1991.
(9) J.P. Mapes, et. al., "PCB-RISc* - An On-Site Immunoassay for
Detecting PCBs in Soil", Superfund ^91 Proceedings of the 12th
National Conference, HMCRI, Washington, D.C., December 1991.
(10) M. Chamberlik-Cooper, R.E. Carlson, R.O. Harrison, "Determination of
PCBs by Enzyme Immunoassay", Field Screening Methods for Hazardous
Wastes and Toxic Chemicals, Proceedings of the Second International
Symposium, ICAIR Life Systems, Inc., Las Vegas, NV, pp 625-628,
February 12-14, 1991.
16
-------
EVALUATION OF AN IMMUNOASSAY FOR MERCURY IN SOIL
Larry C. Waters, Richard W. Counts and Roger A. Jenkins
Environmental Monitoring Group, Chemical and Analytical Sciences Division,
Oak Ridge National Laboratory
Oak Ridge, TN 37831-6120
Telephone: (615) 574-4969; Fax: (615) 576-7956
INTRODUCTION
Sample analysis is a major component of environmental restoration and waste management
activities. Standard laboratory methods are both expensive and time-consuming. A major portion
of the field samples taken to the laboratory for analysis either are negative for the analyte being
tested, or are contaminated below the regulated level. Effective field screening methods could
eliminate much of this effort and expense.
An objective of this program is to define commercial screening methods that are capable of
measuring environmental contaminants of concern to the Department of Energy. The approach
involves selecting potentially useful technologies, experimentally evaluating the methods that utilize
the technology, and transferring the validated methodology to appropriate users. One aspect of the
latter involves submission of the protocols and performance data for inclusion in the DOE Methods
for Evaluating Environmental and Waste Management Samples.
An account of our experiences with an immunoassay (IA)-based method for the analysis of
mercury in soils will be presented. Additional information about the method can be found in the
following references:
Waters, L.C.; Jenkins, R.A.; Smith, R.; Stewart, J.; Counts, R., "Immunoassay for mercury in
soils: Method MB100", DOE Methods for Evaluating Environmental and Waste Management
Samples, DOE, pp. MB100-1 to MB100-17 (1994).
Waters, L.C.; Smith, R.R.; Counts, R.W.; Stewart, J.H.; Jenkins, R.A., "Evaluation of field test
kits including immunoassays for the detection of contaminants in soil and water", in
Proceedings of the 1993 U.S. EPAIA&WMA International Symposium on Field Screening
Methods for Hazardous Wastes and Toxic Chemicals", Air & Waste Management Association:
Pittsburgh, 1993; vol 1, pp 503-513.
EXPERIMENTAL METHODS
Materials
The kits used in our evaluation studies are the BiMelyze 96-Well Microtiter Plate Assay Kit
and the BiMelyze Mercury 16-Tube Assay Kit, with associated extraction kits. They were obtained
from BioNebraska, Inc., Lincoln, NE. [We are not aware of any other manufacturers of LA-based
test kits for mercury.] Materials required but not supplied in the microtiter plate kit include the
pipets required for making sample dilutions and adding samples and reagents to the plates, and a
plate reader. Because the tube kit utilizes dropper-topped bottles to deliver samples and reagents,
there is no requirement for pipettors. Tubes are read with a differential photometer. Both kits
require a user supplied balance to weigh the samples, and the nitric and hydrochloric acids used to
extract the soil. The microtiter plate format is well suited for the analysis of multiple samples in the
laboratory while the tube format is more appropriate for onsite field use.
-------
Sample preparation
A 1.0 g sample of soil is shaken intermittently with 3 mL of a 2:1 mixture of concentrated HC1
and concentrated HNO3 for 10 min. This procedure converts poorly soluble salts of mercury, e.g.,
HgS, elemental mercury, and to some extent methyl mercury, to soluble forms of Hg2+. The extract
is partially neutralized by the addition of 7 mL of buffer, effecting a 1 to 10 dilution of the sample.
A further 1 to 1000 dilution is made (total dilution equals 1 to 10,000) before the extract is analyzed.
Consequently, a 5 ppm sample is equivalent to 0.5 ppb in the assay. Although we have not used it,
the extraction procedure was recently modified to use 5 g of soil with a concomitant increase in test
sensitivity of 5 fold.
Immunoassay
The assay is performed as follows: 1) Aliquots of the diluted sample extracts are added to the
test tubes (or microtiter plate wells) which are been coated with -SH rich proteins. Hg2+ binds to
these proteins in proportion to the concentration of Hg in the sample. Unbound Hg is removed by
rinsing the tubes. 2) A Hg-specific antibody is added that binds to the Hg on the tube, or well,
surface. Unbound antibody is removed by rinsing the tubes. 3) A conjugate, consisting of a
peroxidase enzyme coupled to a second antibody that is specific for the Hg-specific antibody, is added.
This conjugate forms a complex with the Hg-specific antibody. The more Hg bound in step 1 the more
Hg-specific antibody is bound in step 2, and the more conjugate is bound in step 3. Unbound conjugate
is removed by rinsing the tubes. 4) A colorless substrate for the peroxidase component of the
conjugate is added. Oxidation of this substrate causes it to become colored; the more Hg in the
sample the more color is produced. [The intensity of the color is actually proportional to the log of
the Hg concentration.] The Hg immunoassay is different from most of the other lAs for
environmental contaminants in that the latter are formatted to allow the analyte(s) to compete with
the conjugate for binding, in which case more color indicates less analyte. '
In our evaluations, 1 gram samples of soil were used. With this amount of soil, a sensitivity
of about 5 ppm in soil is expected with a working range of up to about 80 to 100 ppm. Extracts of
samples containing higher levels can be further diluted to within the working range and reanalyzed.
As indicated in the previous section the use of 5 grams of soil increases the sensitivity to about 1
ppm. I, t
Hbi^Kxvc^ ,,. . -,',<.<* - ••'•- _?•• x .. "^ _ ep
RESULTS
Both the microtiter plate and tube formats were used in these studies. Most of the soil
samples used were retained samples taken from the East Fork Popular Creek Floodplain in the city
of Oak Ridge, Tennessee. This area was contaminated with mercury in the 1950s by releases from
an upstream nuclear weapons facility. These samples had been previously analyzed by X-ray
fluorescence (XRF) and/or neutron activation analysis (NAA). The predominant mercury species in
these samples is HgS, with lesser amounts of elemental mercury and methyl mercury. Other samples
were prepared by spiking blank soils with HgCl2.
Microtiter plate assay of soils
The microtiter plate format was used in a quantitative mode by using a plate reader capable
of converting the results obtained from simultaneously run standards to a standard curve. From this
curve, a plot of absorbance at 410 nm versus log of the concentrations of the standards, the mercury
concentration of the test samples was determined. A group of 29 field soil samples were analyzed
and the results compared with those previously obtained by XRF and/or NAA.
The samples were independently assayed 3 times with 2 replicates/assay (Table 1). Values
obtained with the immunoassay (IA) that were below 5 ppm, i.e., below the linear range of the
-------
absorbance versus log concentration plot, are reported as < 5 ppm. Values above the linear range,
i.e., > 80 ppm, were determined by using appropriate dilutions of those samples (indicated by the
suffix s). Results were then compared with those obtained by NAA and XRF (Table 1). Statistical
comparisons of the methods were performed with the data given in Table 1. [For this analysis, IA
and NAA "less than" values were given one-half that value and the XRF and IA values used were
the averages of the respective 2 (XRF) and 4-6 (IA) determinations. When compared with XRF an
R2 = 0.980 and a slope of 1.10 were obtained (Figure 1). When compared with NAA the results
obtained by immunoassay gave a best-fit straight line with an R2 = 0.980 and a slope of 1.21 (Figure
1). When results obtained by XRF were compared with those obtained by NAA, a straight line with
an R2 = 0.978 and a slope of 1.08 was produced (Figure 1). These results are interpreted to indicate
that soil mercury contents determined by the immunoassay method correlate well with those
determined by NAA and XRF and that the correlation of the immunoassay with either of the
chemical/physical methods is as good as those two methods are with each other.
Tube assay of soils
The tube format was used in a semiquantitative mode, i.e., the absorbance values obtained
for the test samples were compared to those obtained for the reference soil samples included with
the kit. These reference samples contained 0, 5 or 15 ppm mercury. By comparison, the test samples
could be categorized as containing 0 to 5, 5 to 15 or greater than 15 ppm.
Two independent experiments were done to evaluate this method. Experiment 1 included
10 field samples and 3 spiked samples, in addition to the 3 standard samples supplied in the kit.
Experiment 2 consisted of 8 field samples, 5 spiked samples and the 3 kit samples. Four of the field
samples were common to both experiments. Instead of measuring absorbances directly in the assay
tubes using a differential photometer, they were measured in 96-well microplates using an available
microplate reader. Aliquots of 200 uL were read in Experiment 1 and 100 \iL aliquots were read in
Experiment 2. Test samples were referenced against the kit standards. Wherever possible, analytical
data obtained previously by NAA, XRF and LA are given for comparison. Results are given in Table
2.
The tube assay gave the expected results with all of the samples tested except for one sample,
#14 in Experiment 1. It appears, on the basis of previous data obtained using the microtiter plate
assay (Table 1) and that shown in Table 2, that the mercury content of sample #6 (both experiments)
was significantly underestimated by NAA. Similarly, the mercury content of sample #8 (Experiment
1) also appears to have been underestimated by NAA. Like the microtiter plate assay, the tube assay
appears to be as accurate as either NAA or XRF for measuring mercury in soil. Results obtained
for the 4 field samples that were analyzed in both experiments showed good reproducibility of the
method. [It should be noted that data that are relevant to any particular remedial action level can
be obtained by simply changing the concentrations of the reference standards used.]
Analysis for mercury in sludge
The city of Oak Ridge waste water treatment facility has disposed of post-digestion sludge by
spreading it on nearby University of Tennessee pasture land. In some instances, subsequent
monitoring of the land has indicated an above-background level of mercury. Thus, the facility
operators were interested in identifying a rapid, low cost method to analyze the sludge before it was
spread on UT land. The dark, odorless sludge had a pH of about 8 and was about 30% by volume
particulate in nature.
The initial experiment was to separate the sludge into soluble and particulate fractions by low
speed centrifugation. The soluble fraction was spiked with mercury in the range of 0 to 200 ppb and
analyzed as if it were water or a diluted acid extract. As shown in Table 3, Hg was detected only in
the sample that was spiked at 200 ppb and the amount found was equivalent to only about 2 ppb.
-------
On the other hand quantitative recovery of Hg was observed with the spiked and acid extracted
participate fractions (remember that the participates extracts were diluted 1 to 10,000, i.e., to ppb,
prior to assay). These data show, furthermore, that the unspiked sludge tested did not contain
significant levels of mercury. At least two possibilities can explain the poor recovery of mercury from
the soluble fraction of sludge. First, this fraction contains high levels of proteolytic enzymes that
degrade the -SH rich proteins coating the tubes. Second, the soluble fraction contains proteins that,
themselves, bind the added Hg. In either case, interference with binding of the Hg to the tubes
would occur. The results obtained with the particulate fraction show that acid extraction will prevent
or reverse these actions. This is further illustrated by a second experiment in which whole sludge was
directly extracted with acid and as little as 2.5 ppm Hg was detected (Table 3).
Possible relevance of IA to bioavaflability of mercury in water
The Xenometrix Pro-Tox toxicity test contains a battery of genetically engineered bacterial
cell lines carrying different gene promoters, capable of responding to a variety of chemical or physical
insults. These promoters are placed upstream of a reporter gene, p-galactosidase. If a promoter is
induced, p-galactosidase is produced and measured colorimetrically. A waste water sample known
to contain mercury was tested in this system. Only two of the 16 promoters used, dinD and merR,
were significantly induced by the sample. dinD is induced by a variety of DNA damaging agents while
merR is inducible by heavy metals, particularly mercury. While we have no idea as to the nature of
the dinD inducer, the clear induction by mercury was obvious. It was of interest to try and relate the
response to the level of mercury in the sample. Total mercury content in the waste water sample had
been determined to be in the 200 to 400 ppb range. Analysis using the BioNebraska IA for mercury
indicated a level of about 8 ppb; this being a measure of the free, unbound ionic mercury. Although
it may be purely fortuitous, the level of induction of merR by the waste water sample was about 55
fold which falls in between the 27 and 69 fold induction observed by Xenometrix for 2 ppb and 20
ppb HgCl2, respectively, in a standard test. These results may suggest that the IA for mercury is a
good indicator of the concentration of mercury in water that is detected in the Pro-Tox test, i.e.,
bioavailable.
-------
TABLE 1. IMMUNOANALYSIS OF MERCURY IN CONTAMINATED SOIL SAMPLES:
Comparison with Neutron Activation and X-ray Fluorescence Methods
Sample
Identification
910807-023
910820-013
910820-030
910822-060
910822-070
910829-218
910909-267
910911-022
910926-124
911001-064
911010-051
911022-194
911107-014
911108-178
911112-060
911118-058
911118-061
911118-063
911118-065
911118-071
911118-074
910807-028S
910813-207S
911007-302S
911010-256S
911014-060S
911021-241S
911028-081S
911031-131S
Analytical Technique (ppm Hg)
Neutron
Activation
(1)
<7.6
8.6
19
<5
<2.1
8.3
56
<5.3
12
57
46
73
12
<1.5
18
<1.2
<1.3
<3.3
17
<7.7
11
121
166
589
116
87
206
94
121
X-ray
Fluorescence
(2)
-
13,15
-
7,30
-
23,33
63,81
22,52
19,24
31,33
56,69
56,59
12,20
-
24,24
-
-
-
15,31
-
-
118,122
202,233
601,684
90,116
150,159
235,259
80,106
131,131
Immunoassay
Average
2.5
16.4
46.5
9.0
2.5
33.8
77.8
39.0
36.5
63.2
74.7
73.2
20.0
2.5
29.0
3.5
2.5
2.5
38.5
2.5
42.2
125.3
174.0
740.0
102.5
139.7
262.7
109.8
107.7
%
RSD
-
27
31
13
-
10
37
39
50
34
30
16
52
-
21
-
-
-
40
-
40
23
42
8
29
21
31
48
46
Assay 1
Replicates
1 2
<5 <5
18.0
33.0 38.0
9.7 11.0
<5 <5
75.0 112.0
65.0 39.0
32.0 24.0
61.0 95.0
102.0 76.0
86.0 79.0
30.0
<5 <5
20.0 23.0
5.3 5.8
<5 <5
<5 <5
66.0 37.0
<5 <5
45.0 46.0
120.0 170.0
132.0
795.0 705.0
105.0 160.0
156.0 150.0
344.0 264.0
92.0 124.0
92.0 174.0
Assay 2
Replicates
1 2
<5 <5
15.0 23.0
57.0 66.0
8.3 8.0
<5
-------
Immunoassay (ppm)
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Immunoassay (ppm)
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X-ray Fluorescence (ppm)
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-------
TABLE 2. ANALYSIS OF MERCURY IN SOILS USING THE BiMelyze* TUBE ASSAY KIT
1
I) Sample # Spiked fppm)
Experiment lb
1 V
2 5C
3 15C
4
5
6
7
8
9
10
11
12
13
14 7.5d
15 12.5d
16 1004
1 Experiment 2e
r i o°
i
2 5C
3 15C
4
5
6
7
8
9
10
11
12 cr1
13 2.5"
14 54
15 7.5d
16 25d
Field fppm") bv:
NAA XRF lA"
.
.
-
116 90-116 102
<3.3 - "2.5"
11 - 42
<2.1 - "2.5"
<53 22-52 39
<1.5 - "2.5"
<7.7 - "2.5"
87 150-159 140
19 - 46.5
121 118-122 125
...
.
.
-
.
.
116 90-116 102
<3.3 - "2.5"
11 - 42
<2.1 - "2.5"
<1.5 - "2.5"
_t
J
J
.
.
.
.
.
Absorbance (8> 410 nm
0.077
0.131
0.216
0.357
0.104
0.293
0.096
0.292
0.127
0.088
0.337
0.259
0.264
0.122
0.164
0.264
0.046
0.068
0.097
0.159
0.055
0.134
0.056
0.065
0.059
0.054
0.050
0.054
0.054
0.066
0.071
0.118
Concentration fbv tube assay")
0
5
15
>15
0-5
>15
0-5
>I5
0-5
0-5
>15
>15
>15
0-5
5-15
>!5
0
5
15
>15
0-5
>15
0-5
-5
0-5
0-5
0-5
0-5
0-5
-5
5-15
>15
\a)- results presented in Method MB 100; (b)- a 200 *tL aliquot was read; (c)- standard soils provided with kit;
(d)- soils spiked in the laboratory; (e)- a 100 pL aliquot was read; (f)- stated to be "below detection limits of NAA method"
-------
TABLE 3. ANALYSIS OF MERCURY IN SLUDGE BY LA
Experiment 1
Experiment 2
SAMPLES1
Standards
1
2
3
4
Sludge2
Soluble 1
Soluble 2
Soluble 3
Soluble 4
Soluble 5
Paniculate 1
Paniculate 2
Paniculate 3
Paniculate 4
Standards
1
2
3
4
5
Sludge2
Whole 1
Whole 2
Whole 3
Whole 4
Whole 5
HG CONTENT
Oppb
0.5 ppb
2.0 ppb
8.0 ppb
Oppb
0.5 ppb
2.0 ppb
8.0 ppb
200 ppb
0 ppm
5 ppm
20 ppm
80 ppm
Oppb
0.25 ppb
0.5 ppb
2.0 ppb
8.0 ppb
0 ppm
2.5 ppm
5 ppm
20 ppm
80 ppm
A^o
0.000
0.026
0.240
0.346
0.000
0.000
0.000
0.000
0.235
0.000
0.062
0.298
0.344
0.000
0.021
0.125
0.318
0.381
0.000
0.057
0.184
0.339
0.367
'Samples were diluted (standards) or spiked (sludge) to the indicated concentrations.
^e soluble fractions were analyzed directly without dilution; the paniculate fractions and
whole sludge were extracted with acid and diluted prior to analysis.
-------
Case Study
in the Use of Immunoassay Field Screening Method
for Polycyclic Aromatic Hydrocarbon (PAHs)
Pine Street Canal Superfund Site
Burlington, Vermont
Ross L. Gilleland
Remedial Project Manager
EPA Region I
This paper focuses on the project management aspects of using an innovative field
screening analytical method - immunoassay - for detecting PAHs at a Superfund Site in
EPA Region I. The scoping, planning and goals for the investigation will be discussed
as well as the criteria used for selecting the actual immunoassay kit used: Quantix
PAH-50. The hurdles faced and lessons learned during the work plan review and
approval process will be emphasized. The primary objective of this investigation was to
identify ranges of surficial contamination across the 70-acre site in order to identify
areas for future toxicity testing. However, because the immunoassay data may be used
in making remedy decisions, the EPA site managers required a demonstration that the
field screening results would be representative and comparable to EPA-approved
methods (EPA Method 8270). As a result, two separate validation studies were
conducted prior to actual implementation of the planned work. Finally, the paper will
present the results of the immunoassay method and a comparison of confirmation
sample results analyzed by two independent laboratories.
The Pine Street Canal Superfund site (the location of a former coal gasification plant) is
located within the city limits of Burlington, Vermont and is connected to Lake
Champlain. Since 1993 when EPA withdrew from further consideration a controversial
proposed plan, EPA has been working with a community workgroup ("the Coordinating
Council"), which is comprised of local environmental groups, citizen representatives,
PRPs, Vermont DEC, U.S. FWS, and EPA Region I. The Coordinating Council's
scientific experts identified data gaps in assessing ecological risk at the site. In
fulfilling the data gaps, the scientists scoped out a sampling plan that required over 300
soil and sediment samples be collected across site. The 70-acre urban site consists of
approximately 21 acres of open-water canal, scrub-shrub and forested wetlands and is
surrounded by light industry, including several small businesses, a Department of
Defense armament contractor, a railroad yard, and a former petroleum tank farm.
EPA - Region I performed oversight of the PRPs work by using EPA's Environmental
Monitoring Systems Laboratory (EMSL-LV). EMSL-LV reviewed work plan documents,
provided field oversight, analyzed confirmation samples, and is preparing a split
sampling report which will evaluate the data from the immunoassay and 8270 methods.
U.S. EPA Region 5, Immunoassay Technology Workshop
March 28-29, 1995
-------
Illinois Environmental Protection Agency
Summary of Immuneassay Usage
The Illinois Environmental Protection Agency ("Agency") started using
Inmunoassay techniques 1n mid-1993, At that time two events, the flood of '93
and funding cuts, prompted the use of Immunoassay testing 1n two different
program areas of the Agency. The Division of Laboratories! 1n conjunction
with the Emergency Response Unit, utilized Imunoassays 1n a mobile laboratory
to screen palls, drums, and tanks that washed up as the flood waters receded.
The Division of Public Water Supplies, Groundwater Section, Initiated an
ambient groundwater monitoring network for pesticides. The samples were
tested using Iramunoassay procedures due to funding cuts within the Agency's
Division of Laboratories. Since those two early projects, the Emergency
Response Unit has effectively used Immunoassay technology In responding to
gasoline spills 1n Illinois.
Screening of El pod Borne Containers
Several analytical screening techniques, which Included Imnunoassay,
were used to quickly detect the presence or absence of many toxic materials In
flood borne containers. The flood of '93 affected mainly agricultural areas
and one concern was that the containers had a strong likelihood of being
pesticide or herbicide products. A second concern came from the potential
run-off from fields which might mobilize pesticides Into the river water.
These concerns, as well a& others, needed to be addressed prior to making any
disposal decisions. Was It river water? Could we Just pour out the water
with no 111 effects? Using Immunoassay technology, the unknown contents of
the. container* w*rt.tested for metolachlor, alachlor, atrazlne, tHazlnes (as
-------
a class), and tHfluralin, All of these are commonly used herbicides 1n
Illinois. With tht results from the Immunoassay analysis we were often able
to make a decision on pesticide contamination on scene without additional cost
or testing.
We decided to use imunoassay techniques rather than GC or GC/MS since
the testing needed to be simple and rapid, yet allot* Informed decisions to be
nade, A simple with abnormal or unexpected results, from any of the screening
tests, was then sent to a full service laboratory for Identification. The
main problem we found using Imenmoassay tests for this type of application 1s
that they are too specific. Just to screen an unknown requires using a
separate test kit for each possible contaminant, A preferred methodology
would allow screening a number of pesticides with one test; then following up
with a More specific method.
Ambient firoundwater Protection
There are 353 community water supply wells which make-up the groundweter
monitoring network developed by the firoundwater Section. Testing of
groundwater samples taken from these wells were screened for trltzlnes and
alachlor using Irwuno assay procedures. This sampling and listing constituted
the first of two rounds. Innunoassay screening was utilized out of necessity.
Funding cuts 1n the Agency's Of vision of Laboratories, reduced the staff
available to analyze samples. Although this shortage eliminated the ability
to test these samples using traditional laboratory methods, 1t allowed us to
evaluate Imunoassay as a screening tool and to determine the need for further
analysis.
A second round; of stapling is currently ongoing. Any wells which bad a
detection by immtousagr during tfef first rettnd; an imr tin being seat for
-------
6C analysis. After completing this phisa, the Agency should bi ibli to assess
thi reliability ind accuracy of Immunoassay tasting. One potential problem
that lay have developed Is false positives using the high sensitivity trlazlne
test. This particular aspect 1s being closely monitored during the current
round of sampling.
Emergency Response Use
Energency Response staff have used limunoassay kits to assist them 1n
decision taking at gasoline spill sites. The Inmunoassay product we use 1n
these Instances 1s a BETX detector cell with a hand held reader. Spill
locations have been throughout Illinois with many varied Incident
circumstances. We have typically used the Inmunoassay tests to either help
per1meter1ze a plume or to assess the extent of clean up completed.
Occasionally, 1t has been used to evaluate surface water contamination or
potential drinking water Impacts. We have had good results with Inmunoassay
techniques for these applications and Intend to expand their use as
applicable.
-------
METHOD 4051
HEXAHYDRO-1.3.5-TRINITRO-1.3,5-TRIAZINE (RDX) IN SOIL & WATER BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4051 is a procedure for screening waters and soils to
determine when hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX, CAS No. 121-82-4)
is present at concentrations above 5 pg/L in water and 0.5 mg/kg in soil.
Method 4051 provides an estimate of the concentration of RDX by comparison with
a reference.
1.2 Using the test kit from which this method was developed, 96% of water
samples containing 2.5 ppb or less of RDX will produce a negative result and 99+%
of waters containing 10 ppb or more will produce a positive result. In addition
99+% of soil samples containing 0.25 ppm or less of RDX will produce a negative
result and 99+% of soil samples containing 1.0 ppm will produce a positive
result.
1.3 In cases where the exact concentration of RDX is required,
quantitative techniques (i.e., Method 8330) should be used.
1.4 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed. In general, the method is
performed using a diluted water sample or an extract of a soil sample. Samples
and an enzyme conjugate reagent are added to immobilized RDX antibody. The
enzyme-RDX conjugate "competes" with RDX present in the sample for binding to
immobilized RDX antibody. The enzyme-RDX conjugate bound to the antibody then
catalyzes a colorless substrate to a colored product. The test is interpreted
by comparing the color produced by a sample to the response produced by a
reference reaction.
3.0 INTERFERENCES
3.1 Chemically similar compounds and compounds which might be expected
to be found in conjunction with RDX contamination were tested to determine the
concentration required to produce a positive test result.
3.1.1 Table 1 provides the concentrations of compounds tested with
the D TECH test kit that are required to elicit a positive response at the
4051-1 Revision 0
January 1995
-------
MDL, as well as the concentration required to yield 50% inhibition
compared to the standard curve.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: D TECH™ RDX (Strategic Diagnostics Inc.), or
equivalent. Each commercially available test kit will supply or specify the
apparatus and materials necessary for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HAULING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Water and soil samples may be contaminated, and should therefore be
considered hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance indicated in Tables 3-6.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
Do not mix reagents from one kit lot with a different kit lot.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
4051-2 Revision 0
January 1995
-------
8.6 Method 4051 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 Table 1 provides data on the minimum concentrations of possible
interferants and co-contaminants required to elicit a positive response in the
test kits evaluated.
9.2 Twenty six soil samples, known to not be contaminated with RDX, were
extracted and analyzed using the DTECH RDX kit to determine the extent of soil
matrix effects on the performance of the test kit. The results are provided in
Table 2A, and show that false positive results are not attributable to soil
components. Table 2B presents similar data generated from the analysis of thirty
uncontaminated water samples.
9.3 Thirty water samples and thirty soil samples, known to not be
contaminated with RDX, were each spiked with RDX at one-half and two times the
MDL (0.25 and 1.0 ppm respectively). These samples were analyzed with the DTECH
RDX test kit to determine the error rate of the assay. The results are presented
in Tables 3A and 3B.
9.4 Ten different soil types, all known to not be contaminated with RDX,
were spiked with RDX. The spiked soil samples were each analyzed six times with
the DTECH kit to determine the extraction efficiency of the method. The data are
presented in Table 4.
9.5 Table 5A presents the results of analysis of three soils spiked at
approximately 0.4, 1 and 3 ppm RDX. Each sample was analyzed using Method 8330
and in triplicate using the DTECH kit. Table 5B presents similar data generated
using water samples spiked at 10, 20 and 40 ppb of RDX.
9.6 Tables 6A through 6D present the results of four field trials.
Freshly collected (Table 6A, 6B and 6D) and archived (6C) soil samples, and
samples of water collected from monitoring wells (Table 6B), were analyzed by
commercial laboratories using Method 8330 and the DTech test kit. The Tables
provide results for both analyses, and evaluate the agreement between the two.
10.0 REFERENCES
1. D TECH™ TNT Users Guide , SDI/Em Sciences.
2. Haas, R.J., and B.P. Simmons, "Measurement of Trinitrotoluene (TNT) and
Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in Soil by Enzyme
Immunoassay and High Performance Liquid Chromatography (EPA Method 8330)",
California Environmental Protection Agency, Department of Toxic Substances
Control, Hazardous Materials Laboratory, March, 1995.
4051-3 Revision 0
January 1995
-------
TABLE 1
CROSS REACTANTS - D TECH™ RDX test kit
SAMPLE
RDX d
HMX d
TNT (trinitrotoluene)
Tetryl d
TNB (trinitrobenzene)
2-amino-4,6-dinitrotoluene
4-amino-2,6-dinitrotoluene
2,4-dinitrotoluene
2,6-dinitrotoluene
1,3-dinitrobenzene
nitrobenzene
2-nitrotoluene
3-nitrotoluene
4-nitrotoluene
nitroglycerine
pentaerythr i tol tetran i trate
MDLB
(ppb)
5
150
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
JC5ob
(ppb)
25
800
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
> 500
% CROSS
REACTIVITY0
100
3
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
< 1
The following compounds were not detected at or above 500 ppm (lOOx the method
MDL for RDX):
Atrazine Benzo(a)pyrene Benzo(b)fluoranthene Benzene
Aroclor 1254 Acenaphthene Dibenz(ah)anthracene Chrysene
Acetone Acenaphthalene Fluoranthene Fluorene
Toluene 1,2-Benzanthracene Benzo(k)fluoranthene Pyrene
Ethylbenzene Indeno(123-cd)pyrene Benzo(ghi)perylene Xylene
Naphthalene Methanol Phenanthrene
The Method Detection Limit (MDL) is defined as the lowest concentration of
compound that yields a positive test result.
The IC50 is defined as the concentration of compound required to produce a
test response equivalent to 50% of the maximum response.
% Cross Reactivity is determined by dividing the equivalent RDX
concentration by the actual compound concentration at IC50.
RDX = hexahydro-l,3,5-trinitro-l,3,5-triazine
HMX = octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine
Tetryl = methyl-2,4,6-trinitrophenylnitramine
4051-4
Revision 0
January 1995
-------
TABLE 2A
SOIL MATRIX EFFECTS
Soil ID #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
?7
Soil Type
Low OM Clay Loam
Sassafras Sandy Loam
Cecil Sandy Clay Loam
Davidson Clay Loam
Shontik-Casa Grande Clay
Trix Sandy Clay Loam
Trix-Casa Grande Clay Loam
Yolo Loam
Capay Silty Clay
Sycamore Silt Loam
Dennis Silt Loam
Luray Silty Clay Loam
Wooster Silt Loam
Vienna Loam
Opal Clay
Raulb Silt Loam
Rockfield Silt Loam
Cisne Silt Loam
Muscatine Silt Loam
Avonburg
Matapeake Silt Loam
Evesboro Low OM Sand
Selbyville High OM Sand
Casa Grande Clay Loam
Grundy Silty Clay Loam
Drummer Silty Clay
Non-Soil Control
State
DE
DE
GA
GA
AZ
AZ
AZ
CA
CA
CA
KA
OH
OH
SD
SO
IN
IN
IL
IL
DE
DE
DE
AZ
KA
IL
-
D TECH Result
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0,5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< O.B
4051-5
Revision 0
January 1995
-------
TABLE 2B
WATER MATRIX EFFECTS
Water ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Location
Adamsville, RI
Buttermilk Falls, PA
Hudson River, PA
Germantown, PA
Houston, TX (1)
Houston, TX (2)
Ontario, CA
Pacific Ocean, CA
S. Darthmouth, MA
Newark, DE (1)
U.S. Army Waterways
U.S. Army WES
U.S. Army WES
U.S. Army WES
U.S. Army WES
U.S. Army WES
U.S. Army WES
U.S. Army WES
Georgetown, DE
Newark, DE (2)
Burlington, IA
Burlington, IA
Lake St. Germain, Canada
Milliston, WI
Moorhead, MN
McKenzie Co., NO
Wolcott, IN
Newark, DE (3)
Smith Island, MD
Adrian, GA
DI Cnntrol
D TECH Result (ppb)
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
< 5.0
4051-6
Revision 0
January 1995
-------
TABLE 3A
False Negative and False Positive Rates, Soil Matrix3
Spike Concentration
0.25 ppm
1.0 ppm
False Positive Rate
0%
-
False Negative Rate
-
0%
a Thirty negative soils were spiked with RDX at one-half and two times the MDL
(0.25 and 1.0 ppm respectively). These samples were analyzed with the DTECH
RDX test kit to determine the error rate of the assay.
TABLE 3B
False Negative and False Positive Rates, Water Matrix3
Spike Concentration
0.25 ppm
1.0 ppm
False Positive Rate
3.3%
-
False Negative Rate
_
0%
a Thirty negative water samples were spiked with RDX at one-half and two times
the MDL (0.25 and 1.0 ppm respectively). These samples were analyzed in
triplicate with the DTECH RDX test kit to determine the error rate of the
assay.
4051-7
Revision 0
January 1995
-------
TABLE 4
DETERMINATION OF EXTRACTION EFFICIENCY FROM SOIL SAMPLES3
Soil ID : Spike
(ppm)
101:1
106:1
108:1
109:1
110:1
116:1
117:1
123:1
126:1
128:1
Non-Soil
Average
101:6
106:6
108:6
109:6
110:6
116:6
117:6
123:6
126:6
128:6
Non-Soil
Average
Mean RDX
Concentration
(ppm)
0.53
0.88
0.86
0.66
0.70
0.96
0.92
1.00
1.03
1.02
1.05
0.86
4.92
6.15
5.69
6.11
6.12
6.26
5.71
6.05
6.82
6.02
6.02
5.98
Standard
Deviation
0.19
0.13
0.23
0.22
0.14
0.12
0.42
0.45
0.25
0.18
0.13
0.23
0.54
0.84
1.09
0.93
0.46
1.21
0.72
0.8
0.33
0.62
0.83
0.75
Coefficient
of
Variation
(%)
35
15
26
34
19
13
46
45
24
18
12
27
11
14
19
15
8
19
13
13
5
10
14
13
Recovery
(*)
53
88
86
66
70
96
92
100
103
102
105
86
82
103
95
102
102
104
95
101
114
100
100
100
4051-8
Revision 0
January 1995
-------
TABLE 5A
RECOVERY OF RDX SPIKED INTO REAL SOILS.
Soil ID
106
116
128
Spike
Concentration
(ppm)
0.4
1.0
3.0
0.4
1.0
3.0
0.4
1.0
3.0
Method 8330
(ppm)
0.32
0.83
1.79
0.29
0.66
0.61
0.31(0.25)
0.73(0.73)
0.75(2.27)
D TECH
(ppm)
< 0.5
< 0.5
< 0.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
> 2.0
> 2.0
=> ? n
< 0.5
< 0.5
< 0.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
> 2.0
< 2.0
> ? n
< 0.5
< 0.5
< 0.5
< 0.5
0.5 - 1.5
0.5 - 1.5
> 2.0
< 2.0
<: ? 0
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
FP
FP
FP
Y
Y
Y
Y
Y
Y
FP
FP
FP
Y
Y
Y
FN
Y
Y
Y
Y
Y
4051-9
Revision 0
January 1995
-------
TABLE 5B
RECOVERY OF RDX SPIKED INTO WATERS
Sample ID
1
?
Spike
Concentration
(ppb)
10
20
40
10
10
20
40
20
Method 8330
(ppb)
11.1
18.0
35.7
9.0
9.?
19.4
36.5
17.1
D TECH
(ppb)
5 - 15
5 - 15
5 - 15
15 - 30
15 - 30
15 - 30
>45
> 45
> 45
5 - 15
5 - 15
5 - 15
5 - 15
5 - 15
5 - 15
15 - 30
15 - 30
15 - 30
> 45
> 45
> 45
15 - 30
15 - 30
15 - 30
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
FP
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
FP
FP
Y
Y
Y
4051-10
Revision 0
January 1995
-------
TABLE 5B
RECOVERY OF RDX SPIKED INTO WATERS
Sample ID
3
Spike
Concentration
(ppb)
10
20
40
40
Method 8330
(ppb)
9.7
18.2
35.8
31.8
D TECH
(ppb)
5 - 15
5 - 15
5 - 15
15 - 30
15 - 30
15 - 30
> 45
> 45
30 - 45
> 45
30 - 45
30 - 45
AGREEMENT9
Y, FN, FP
Y
Y
Y
Y
Y
Y
FP
FP
Y
FP
Y
Y
4051-11
Revision 0
January 1995
-------
TABLE 6A
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample ID
S4
S12
S14
S15
S19
S20
S21
Tl-2
T2-4
T6-1
T3-5
T12-3
T12-6
T20-3
T21-10
T22-4
T22-5
T22-6
T28-3
T28-4
T28-5
T28-6
T28-7
T28-8
T28-9
Method 8330
(ppm)
< 0.2
< 0.2
1.72
< 0.2
2.12
1.61
0.32
0.21
1.41
2.62
2.00
< 0.2
1.00
< 0.2
1.89
< 0.2
0.83
0.99
3.73
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
D TECH
(ppm)
< 0.5
< 0.5
1.5 - 2.0
< 0.5
1.5 - 3.0
1.5 - 3.0
< 0.5
< 0.5
1.5 - 2.0
> 3.0
0.5 - 1.5
< 0.5
0.5 - 1.5
< 0.5
1.5 - 2.0
< 0.5
0.5 - 1.5
0.5 - 1.5
> 3.0
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
AGREEMENT3
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
FP
FP
FN
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
4051-12
Revision 0
January 1995
-------
TABLE 6A
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample ID
T28-10
T28-11
T28-12
T28-13
T31-4
T12-5
Method 8330
(ppm)
0.28
1.51
1.3
0.6
1.22
0.26
D TECH
(ppm)
< 0.5
• 1.5 - 3.0
1.5 - 3.0
0.5 - 1.5
1.5 - 2.0
< 0.5
AGREEMENT8
Y., FN, FP
Y
Y
FP
Y
FP
Y
4051-13
Revision 0
January 1995
-------
TABLE 6B
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
ID
1
3
13
15
16
23
24
25
26
31
33
34
35
37
38
43
44
47
48
58
59
64
67
68
75
84
85
87
94
96
97
Method 8330
(ppm)
4.00
19.0
1.30
1.80
3.40
0.48
0.68
0.68
0.75
0.13
0.74
0.48
1.30
5.50
0.55
1.30
40.0
2.30
0.36
0.79
0.80
2.20
10.9
3.40
3.90
17.6
70.3
101
1.60
0.20
5.40
Replicate 1
D TECH
(ppm)
> 3.0
> 6.0
0.5 - 1.5
1.5 - 3.0
> 3.0
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
> 6.0
0.5 - 1.5
1.5 - 3.0
> 6.0
> 3.0
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
1.5 - 3.0
> 6.0
1.5 - 3.0
> 3.0
> 6.0
> 6.0
> 6.0
1.5 - 3.0
< 0.5
> 3.0
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
FP
FP
Y
FP
Y
FP
FP
Y
FP
Y
Y
FN
Y
Y
Y
Y
Y
Y
Y
Replicate 2
D TECH
(ppm)
> 3.0
> 6.0
0.5 - 1.5
1.5 - 3.0
> 3.0
0.5 - 1.5
< 0.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
> 3.0
0.5 - 1.5
1.5 - 3.0
> 6.0
> 3.0
< 0.5
0.5 - 1.5
1.5 - 3.0
1.5 - 3.0
> 6.0
1.5 - 3.0
> 3.0
> 6.0
> 6.0
> 6.0
1.5 - 3.0
< 0.5
> 3.0
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
FP
Y
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
4051-14
Revision 0
January 1995
-------
TABLE 6B
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
ID
98
99
105
111
113
115
119
Method 8330
(ppm)
< 0.05
< 0.05
130
< 1.0
< 1.0
3.00
36.0
Replicate 1
D TECH
(ppm)
< 0.5
< 0.5
> 60
> 3.0
< 5.0
< 5.0
> 30
AGREEMENT8
Y, FN, FP
Y
Y
Y
FP
Y
Y
Y
Replicate 2
D TECH
(ppm)
< 0.5
0.5 - 1.5
> 60
< 5.0
< 0.5
< 0.5
15 - 30
AGREEMENT8
Y, FN, FP
Y
FP
Y
Y
FN
FN
FN
4051-15
Revision 0
January 1995
-------
TABLE 6C
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample ID
1
2
3
4
5
6
7
8
9
10
19
20
11
12
13
14
15
16
17
18
21
22
23
24
25
METHOD 8330
(ppm)
17
34
48
160
650
41
360
840
69
85
17
19
4.3
1.9
4.9
27
1.2
1.0
0.82
0.78
0.67
0.94
< 0.4
< 0.4
< 0.4
D TECH
(ppm)
15 - 30
15 - 30
> 30
60 - 120
150 - 300
> 30
50 - 150
> 600
> 60
30 - 60
> 6.0
> 6.0
> 3.0
> 3.0
> 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
0.5 - 1.5
0.5 - 1.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
AGREEMENT8
Y, FN, FP
Y
FN
Y
FN
FN
Y
FN
Y
Y
FN
Y
Y
Y
FP
Y
Y
FP
FP
Y
Y
FN
FN
Y
Y
Y
4051-16
Revision 0
January 1995
-------
TABLE 6C
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample ID
26
27
28
29
30
METHOD 8330
(ppm)
< 0.4
< 0.4
< 0.4
< 0.4
< 0.4
D TECH
(ppm)
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
AGREEMENT"
Y, FN, FP
Y
Y
Y
Y
Y
4051-17
Revision 0
January 1995
-------
TABLE 6D
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
SDI Dilution
Factor
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
SDI
Results
<0.5
<0.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
0.5-1.5
0.5-1.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
8330
Results
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
AGREEMENT
Y, FN, FP
Y
Y
Y
Y
FP
Y
Y
FP
Y
Y
FP
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
4051-18
Revision 0
January 1995
-------
TABLE 6D
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
SDI Dilution
Factor
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
SDI
Results
0.5-1.5
<0.5
<0.5
1.5-3.5
<0.5
0.5-1.5
<0.5
0.5-1.5
0.5-1.5
<0.5
1.5-3.0
<0.5
0.5-1.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
1.5-3.0
<0.5
0.5-1.5
<0.5
0.5-1.5
<0.5
0.5-1.5
8330
Results
<0.17
<0.17
<0.17
0.17-0.99
<0.17
<0.17
<0.17
0.17-0.99
0.17-0.99
<0.17
1.2
<0.17
<0.17
<0.17
<0.17
3.8
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
0.17-0.99
AGREEMENT"
Y, FN, FP
FP
Y
Y
FP
Y
FP
Y
Y
Y
Y
FP
Y
FP
Y
Y
FN
Y
Y
Y
FP
Y
FP
Y
FP
Y
Y
4051-19
Revision 0
January 1995
-------
TABLE 6D
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
SDI Dilution
Factor
100
1
1
1
1
1
1
1
1
100
10
1
1
1
100
1
100
1
1
100
10
10
10
1
1
10
SDI
Results
50-150
3.0-4.5
3.0-4.5
<0.5
0.5-1.5
<0.5
<0.5
1.5-3.0
0.5-1.5
150-300
15-30
1.5-3.0
1.5-3.0
3.0-4.5
50-150
0.5-1.5
50-150
0.5-1.5
1.5-3.0
150-300
45-60
>60
30-45
1.5-3.0
4.5-6.0
>60
8330
Results
100
<0.17
<0.17
<0.17
<0.17
<0.17
<0.17
1.1
<0.17
290
46
4.8
0.17-0.99
12
150
2.6
140
7.8
3.2
340
55
67
63
2.4
6.4
73
AGREEMENT
Y, FN, FP
Y
FP
FP
Y
FP
Y
Y
FP
FP
Y
FN
FN
FP
FN
Y
FN
Y
FN
FN
FN
Y
Y
FN
Y
FP
Y
4051-20
Revision 0
January 1995
-------
TABLE 6D
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
SDI Dilution
Factor
10
1
1
1
10
1
100
10
100
1
1
1
1
1
1
1
1
1
1
1
1
1
SDI
Results
15-30
0.5-1.5
3.0-4.5
1.5-3.0
>60
>6
50-150
30-45
50-150
0.5-1.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
8330
Results
14
2.1
2.4
2
94
23
150
34
150
1.2
0.17-0.99
<15
<15
<2
<15
<5
<0.17
<15
<5
<0.17
<0.17
<0.17
AGREEMENT
Y, FN, FP
FP
FN
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
4051-21
Revision 0
January 1995
-------
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Pine Street Canal Superfund Site
Burlington, Vermont
EPA RPM's:
Sheila Eckman (617) 573 — 5784
Ross Gilleland (617) 573-5766
VT DEC Project Manager
Stan Corneille (8O2) 241-3888
Oversight Contractor
Metcalf & Eddy, Inc.
Martha Zirbel (617) 246-52OO
EPA EMSL-LV
Ken Brown (7O2) 798-227O
PRPs (Respondents):
Green mountain Power Corp.
A.Norman Terreri (8O2) 864-5731
New England Electric Company
Joe Kwasnik (5O8) 366-9O11
Consultant:
The Johnson Company
Chris Crandell (8O2) 229-46OO
Immunoassay kit: Quantix Idetek
-------
Project Management
Aspects of Project
Planning
Implementation
Data Evaluation/Usage
Lessons Learned
-------
Planning
Site History: Previous Studies
Scoping: Data Gaps. I/A Goals
Work Plans: PRPs, Oversight
Implementation
Data Evaluation/Usage
Lessons Learned
-------
Selecting a Kit
PAH Concentrations
Compound Specificity
Detection Ranges
Precision & Accuracy
Limits for Soil Moisture
Sample Throughput
-------
Why QUANTIX was selected
Quantitative Results
Correlates with 8270 and 8310
Market Survey
Low Cross reactivity to BETEX
Positive bias to Quantix results
1:1, 1:10, 1:100, 1:1000 dilutions
from one sample extraction
Detection Range: 50ppb to
5,000ppm
Previous Experience
Idetek Technical Support Staff
-------
Planning
Implementation
Pre-Validation Sudy
Validation Study
Phase I ARI Study
Oversight Audits
Data Evaluation/Usage
Lessons Learned
-------
Pre-Validation Study
"Q" Samples
Purpose:
Idetek Site-specific
method development and
Bias testing.
10 Samples
I/A: Quantix
GC/MS (8270): MRI
Outcome:
Site-specific changes to
Quantix Method
•
-------
Validation Study
"JV" Samples
Purpose:
Test Quantix system against
GC/MS Method 8270
15 Samples
I/A: Quantix
GC/MS (8270): MRI
GC/MS (8270): IEA
GC/MS (8270): LESAT
6 samples only
Outcome:
GC/MS data not available prior
to proceeding with Phase I ARI
Study.
-------
Phase I ARI Study
"JT" and "JH" Samples
Purpose:
Identify PAH concentration
ranges on site.
Program:
300+ Samples
I/A: Quantix
10% Confirmation
GC/MS (8270): IEA
8 Referee samples
GC/MS (8270): LESAT
only
Outcome:
Data under review.
Phase I ARI Report 4/10/95
-------
Planning
implementation
Data Evaluation/Usage
Acceptability of PRP's Data
(Project Data)
Quality of I/A Data
Usability of the Data
Project Goals
Decision Making
Lessons Learned
-------
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LOCk'MEED-ESt
702 3S7 6641
TEL:: 702-397-5641
Mar 24 35
5:Q4 No.002
Comparison of Total PAH and Percent Moisture Results for Selected ARI Samples
Analyzed by Immunoassay and GC/MS
SAMPLE
ID
J.T13-52
J.T13-89
J.T15-82
JJT15-87
JJT17-53
J_T18-81
.L.T20-52
J_T21-51
TOTAL PAH CONCENTRATION
(mg/Kg)
QUANT1X™
IA
(wetwt)
80.6
2.96
1R5
142
33.2
4.70
21.3
8.71
ffiA
OOMS
(dry wt)
140.
1.02
32.5
3.56
29.8
39.5
30.2
50.8
LESAT
GC/MS
(drywt)
200.
1.57
28.9
OJ66
32,9
56.2
45.9
14.8
PERCENT
MOISTURE
IEA
72.
17.
83.
20.
46.
32.
72.
26.
LESAT
69.3
ia7
88.0
1U
51.4
35.0
66.0
33.0
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Arguments for Using
Wet Weight Quantix Results
Method developed for field ("as
is") samples.
Water displaces air in soil pores -
negligible increase in volume;
therefore, volume to mass
comparison is acceptable
Want to use results which are
most comparable to GC/MS
— wet weight
-------
Comparison of
Pre-Validation Study
SAMPLE ID
Q-l
Q-2
Q-3
Q-4
Q-5
Q-6
Q-7/8
Q-9
Q-10
Q-ll
TOTAL PAH
CONCENTRATION (mg/Kg)
GCMS
(MRI)
1.47
3.08
3.34
5.83
12.6
5.70
7.01
121.
80.1
36.5
QUANTIX™
IA
7.48
5.00
9.76
8.60
4.02
1.90
34.2
115.
182.
195.
RPD
(%)
134.
47.5
98.0
38.4
103.
95.8
132.
5.1
76.2
137.
Note: Percent moisture results unavailable at this time.
-------
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Comparison of
Validation Study
SAMPLE ID
JV-03B
JV-06
JV-05B
JV-03A
JV-02B
JV-04B
JV-02A
JV-01A
JV-04A
- JV-05A
JV-01B
JV-10
JV-09
JV-07
JV-08
TOTAL PAH CONCENTRATION
(mg/Kg)
ffiAGGMS
(dry wt)
0.56
0.72
1.24
2.29
2.75
3.14
4.24
6.51
12.6
24.7
35.5
810
94.1
331.
711.
QUANTIX™ LA
(wet wt)
1.7
156.
2.1
1.6
1.8
6.0
51.0
11.3
39.5
75.
82.
97.
595.
292.
2000.
RPD
(%)
99.1
198.
51.5
38.5
41.8
62.6
169.
53.8
103.
101.
79.0
16.8
145.
116
95.1
-------
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Comparison of
Phase I ARI Study
-------
SAMPLE ED
JH3-05
JT10-81
JT7-83
JT13-89
JH4-13
JT15-87
JH4-02
JT18-03
JT10-02
JH1-05
JH5-05
JT17-53
JT20-52
JT15-82
JT18-81
JT13-81
JT17-03
JT12-52
JT21-51
JT25-82
JT27-85
JT15-03
JT13-52
JT29-82
JT2-57
JT3-52
JT7-51
JTl-54
JT5-51
TOTAL PAH CONCENTRATION
(mg/Xg)
LEAGCMS
(dry wt)
0.11
0.45
0.54
1.02
3.19
3.56
6.86
7.93
8.28
15.0
24.4
29.8
30.2
315
39.5
42.5
43.0
48.3
50.8
662
75.3
76.6
140.
185.
212.
335.
360.
848.
2215.
QUANTEX™ IA
(wet wt)
0.23
1.18
0.13
2.96
0.53
2.42
1.10
0.23
0.28
1.30
6.3
33.2
21.3
18.5
4.70
55.1
0.49
103.
8.71
4.61
5.28
1.55
80.6
4.39
153.
158.
2700.
591
395.
RPD
(*)
70.6
88.9
122.
97.5
143.
38.3
145.
189.
187.
168.
118.
11.0
34.5
55.0
158.
25.9
1%.
72.2
141
174.
174.
192.
54.1
191.
313
71.8
153.
35.6
139.
-------
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Planning
Implementation
Data Evaluation/Usage
Lessons Learned
Planning
Workplans, Oversight
Implemetation
Data Usage
-------
Lessons Learned
Scoping/Work Plan
Clear Project Goals
Understand Data Limitations/uses
Confirmation Method
Early Oversight staff involvement
Ample Time for Review
Validation Studies
-------
Lessons Learned
Implementation
Ample Time to modify method
Ample Time to evaluate Validation
Studies to determine whether kit
works for Site
Homogenization
For More Info:
The Johnson Company
Chris Crandell
(802) 229-4600
EMSL-LV
Ken Brown
(702) 798-2270
-------
Lessons Learned
Data Evaluation/Usage
Adhere to DQO's and Data limitations
Compound Sensitivities
Moisture Content
Acceptable Project Data
Wet Weight vs. Dry Weight
-------
METHOD 4035
SCREENING FOR POLYNUCLEAR AROMATIC HYDROCARBONS IN SOIL BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4035 is a procedure for screening soils to determine when total
polynuclear aromatic hydrocarbons (PAHs) are present at concentrations above 1
mg/Kg. Method 4035 provides an estimate for the concentration of PAHs by
comparison with a PAH standard.
1.2 Using the test kit from which this method was developed, >95% of
samples confirmed to have concentrations of PAHs below detection limits will
produce a negative result in the 1 ppm test configuration.
1.3 The sensitivity of the test is influenced by the binding of the target
analyte to the antibodies used in the kit. The commercial PAH kit used for
evaluation of this method is most sensitive to the three (i.e., phenanthrene,
anthracene, fluorene) and four (i.e. benzo(a)anthracene, chrysene, fluoranthene,
pyrene) ring PAH compounds listed in Method 8310, and also recognizes most of the
five and six ring compounds listed.
1.4 The sensitivity of the test is influenced by the nature of the PAH
contamination and any degradation processes operating at a site. Although the
action level of the test may vary from site to site, the test should produce
internally consistent results at any given site.
1.5 In cases where the exact concentration of PAHs are required,
quantitative techniques (i.e., Method 8310, 8270, or 8100) should be used.
1.6 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 An accurately weighed sample is first extracted and the extract
filtered using a commercially available test kit. The sample extract and an
enzyme conjugate reagent are added to immobilized antibody. The enzyme conjugate
"competes" with the PAHs present in the sample for binding to the immobilized
anti-PAH antibody. The test is interpreted by comparing the response produced
by testing a sample to the response produced by testing standard(s)
simultaneously.
2.2 A portion of all samples in each analytical batch should be confirmed
using quantitative techniques.
3.0 INTERFERENCES
3.1 Chemically similar compounds and compounds which might be expected to
be found in conjunction with PAH contamination were tested to determine the
4035 - 1 Revision 0
January 1995
-------
concentration required to produce a positive result. These data are shown in
Tables 1 and 2.
3.2 The kit was optimized to respond to three and four ring PAHs. The
sensitivity of the test to individual PAHs is highly variable. Naphthalene,
dibenzo(a,h)anthracene, and benzo(g,h,i)perylene have 0.5 percent or less than
the reactivity of phenanthrene with the enzyme conjugate.
3.3 The alkyl-substituted PAHs, chlorinated aromatic compounds, and other
aromatic hydrocarbons, such as dibenzofuran, have been demonstrated to be cross-
reactive with the immobilized anti-PAH antibody. The presence of these compounds
in the sample may contribute to false positives.
4.0 APPARATUS AND MATERIALS
4.1 PAH RISc™ Soil Test (EnSys, Inc.), or equivalent. Each commercially
available test kit will supply or specify the apparatus and materials necessary
for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Method 4035 is intended for field or laboratory use.
7.2 Follow the manufacturer's instructions for the test being used. Those
test kits used must meet or exceed the performance indicated in Tables 3-7.
7.3 The action limit for each application must be within the operating
range of the kit used.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used for
quality control procedures specific to the test kit used. Additionally, guidance
provided in Chapter One should be followed.
4035 - 2 Revision 0
January 1995
-------
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other kits.
8.5 Use the test kits within the specified storage temperature and
operating temperature limits.
9.0 METHOD PERFORMANCE
9.1 The extraction efficiency of a commercially available test kit was
tested (PAH RISc™ Test, EnSys Inc.) by spiking phenanthrene, benzo(a)anthracene
and benzo(a)pyrene into PAH negative soil matrices (PAH-116 and PAH-141 are field
samples). The soils were spiked using detection limits established for each
compound (see Table 1), extracted and determined by immunoassay. The results for
these 3-, 4- and 5-ring PAHs (Table 4) demonstrated that they were extracted with
good recovery and yielded the correct assay interpretation.
9.2 A single laboratory study was conducted with a commercially available
test kit (PAH RISc™ Test, EnSys Inc.), using 25 contaminated soil samples. Four
replicate determinations were made on each test sample and the data compared with
values obtained using HPLC Method 8310. Several analysts performed the
immunoassay analyses. The immunoassay data agreed in all cases with the external
HPLC data obtained (Table 5).
9.3 An additional single laboratory validation study on 30 randomly
selected, PAH-contaminated field samples from multiple sites was run by the USEPA
Region X Laboratory. Results are reported in Table 6 on an as found basis, and
reported in Table 7 normalized to phenanthrene, based on cross-reactivity data
(from Table 1). The false positive rate at the 1 ppm action level was 13% for
unnormalized results and 19% for normalized results based on 31 analyses. The
false negative rate at 1 ppm was 0 in both cases. At the 10 ppm action level,
the false positive rate was 19% unnormalized and 26% normalized. False negative
rates at 10 ppm were 6% unnormalized and 3% normalized.
9.4 The probabilities of generating false positive and false negative
results at an action level of 1 ppm are listed in Table 3.
10.0 REFERENCES
1. PAH-RISc™ Users Guide, EnSys Inc.
2. P. P. McDonald, R. E. Almond, J. P. Mapes, and S. B. Friedman, "PAH-RISc™
Soil Test - A Rapid, On-Site Screening Test for Polynuclear Aromatic
Hydrocarbons in Soil", J. of AOAC International (accepted for publication
document #92263)
3. R. P. Swift, J. R. Leavell, and C. W. Brandenburg, "Evaluation of the
EnSys PAH-RISc™ Test Kit", Proceedings, USEPA Ninth Annual Waste Testing
and Quality Assurance Symposium, 1993.
4035 - 3 Revision 0
January 1995
-------
Table 1: Cross-Reactivity of Method 8310 PAHs
Compound
2 Rinqs
Naphthalene
3 Rinqs
Acenaphthene
Acenaphthylene
Phenanthrene
Anthracene
Fluorene
4 Rings
Benzo(a) anthracene
Chrysene
Fluoranthene
Pyrene
5 Rings
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo( a) pyrene
Dibenzo (a, h) anthracene
6 Rings
I ndeno ( 1 , 2 , 3 -c , d ) pyrene
Benzo (g , h , i ) peryl ene
Concentration Giving
a Positive Result
(ppm Soil Equivalent)
200
8.1
7.5
1.0
0.81
1.5
1.6
1.2
1.4
3.5
4.6
9.4
8.3
>200
11
>200
Percent
Cross -Reactivity
0.5
12
13
100
123
67
64
84
73
29
22
11
12
<0.5
9.4
<0.5
4035 - 4
Revision 0
January 1995
-------
Table 2: Cross Reactivity of Other PAHs and Related Compounds
Compound
Other PAHs
1 -Methyl naphthyl ene
2-Methyl naphthyl ene
1 -Chi oronaphthyl ene
Halowax 1013
Halowax 1051
Dibenzofuran
Other Compounds
Benzene
Toluene
CCA
Phenol
Creosote
2 , 4 , 6-Tri chl orobenzene
2,3,5,6-
Tetrachl orobenzene
Pentachl orobenzene
Pentachlorophenol
Bis(2-ethylhexyl)
phthalate
Aroclor 1254
Aroclor 1260
Concentration Giving
a Positive Result
(ppm, Soil Equivalent)
54
58
59
18
>200
14
>200
>200
>200
>200
5.4
>200
>200
>200
>200
>200
>200
>200
Percent
Cross-Reactivity
1.8
1.7
1.7
5.7
<0.5
7.2
<0.5
<0.5
<0.5
<0.5
18.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
Table 3: Probability of False Negative and False Positive Results
for PAHs at a 1 ppm Action Level
Spike Concentration
Phenanthrene (ppm)
0
0.4
0.8
1.0
Probability of False
Positive (Mean ± SD)
0% ± 0%
23% ± 17%
94% ± 13%
N/A
Probability of False
Negative (Mean ± SD)
N/A
N/A
N/A
0% ± 0%
Results were obtained from spiking four different validation lots, using
3 operators, 12 matrices for a total of 201 determinations at each
concentration of phenanthrene.
N/A = No false positive possible above action limit.
No false negative possible below action limit.
4035 - 5
Revision 0
January 1995
-------
Table 4: Spike Recovery of Phenanthrene, Benzo(a)anthracene and Benzo(a)pyrene
Compound
Blank
Blank
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Benzo (a) anthracene
Benzo (a) anthracene
Benzo ( a) anthracene
Benzo ( a) anthracene
Benzo(a)pyrene
Benzo(a)pyrene
Benzo(a)pyrene
Spike
(ppm)
0
0
1
1
1
10
10
10
1.6
1.6
16
16
8.3
8.3
83
Soil
Wake
PAH- 116
Wake
PAH- 116
PAH- 141
Wake
PAH- 116
PAH- 141
Wake
PAH- 116
Wake
PAH- 116
Wake
PAH- 116
PAH- 116
PAH RISc™
Results
<1
<1
1-10
1-10
1-10
>10
>10
>10
1-10
1-10
>10
>10
1-10
1-10
>10
4035 - 6
Revision 0
January 1995
-------
Table 5: Powerplant Field Samples (Soil) Evaluated by Immunoassay
Field Sample
Number
PAH -137
PAH- 141
PAH- 118
PAH -136
PAH- 139
PAH- 126
PAH- 127
PAH -122
PAH -138
PAH- 131
PAH -128
PAH- 132
PAH- 112
PAH- 140
PAH -130
PAH- 116
PAH- 135
PAH- 133
PAH- 119
PAH -120
PAH- 124
PAH-134
PAH- 114
PAH- 113
PAH- 115
EnSys Method
Immunoassay (ppm)
>10
<1
1-10
>10
>10
1-10, >10
>10
>10
>10
>10
>10
>10
>10
>10
>10
<1
>10
>10
>10
>10
>10
>10
>10
>10
>10
Method 8310
HPLC (ppm)
<21
<21
<26
26
<28
<32
<33
<33
33
<34
<35
<43
<48
50
54
<61
71
<91
<100
<161
<167
182
<247
<294
<343
4035 - 7
Revision 0
January 1995
-------
Table 6: Total PAH Content of Region X Field Samples Using EnSys
PAH RISc™ Immunoassay Test Kit
Sample ID
PAH-1
PAH-2
PAH-3
PAH-4
PAH-5
PAH-6
PAH- 7
PAH-8
PAH-9
PAH-10
PAH- 11
PAH-12
PAH-12Dup
PAH- 13
PAH- 14
PAH- 15
PAH- 16
PAH-17
PAH- 18
PAH- 19
PAH- 20
PAH-2 1
PAH-22
PAH- 2 3
PAH-2 4
PAH-2 5
1 ppm Test
<1
*
*
*
*
*
*
*
>1
*
*
*
*
*
*
*
*
*
*
10 ppm Xest
<10
*
*
*
*
*
*
*
>10
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
GC/MS
Lab Result
(ppm)
0.2
12.2
16.0
0.0
0.5
8.7
148
182
4.4
0.2
0.0
85.4
85. 4
28.5
0.3
0.6
0.0
1.8
3.4
6.7
0.9
43.2
72.8
1.3
0.3
0.4
False +/-
Eval 6
1 ppm
+
+
+
+
Eval @
10 ppm
+
+
+
+
+
+
of all PAHs detected.
4035 - 8
Revision 0
January 1995
-------
Table 6: Total PAH Content of Region X Field Samples Using EnSys
PAH RISc™ Immunoassay Test Kit (Contd.)
Sample ID
PAH-26
PAH-27
PAH-28
PAH-29
PAH-30
1 ppm Test
<1
*
*
>1
*
10 ppm Test
<10
*
*
*
*
*
>10
OC/MS
Lab Result
(ppm)1
27.9
0.0
16.4
0.4
9.6
False +/-
Eval @
1 ppm
Eval @
10 ppm
_
—
Table 7: Total PAH Content of Region X Field Samples Using EnSys
PAH RISc™ Immunoassay Test Kit Normalized to Cross-Reactivity
Sample ID
PAH-1
PAH-2
PAH- 3
PAH-4
PAH-5
PAH-6
PAH- 7
PAH-8
PAH-9
PAH- 10
PAH- 11
PAH- 12
PAH-12Dup
PAH- 13
1 ppm Test
<1
*
*
*
>1
*
*
*
*
10 ppm Test
<10
>10
*
it
if
*
*
*
*
*
*
*
*
OC/MS
Lab Result
(ppm)1
0.1
8.1
9.0
0.0
0.2
5.2
56.9
73.2
0.1
0.0
0.0
47.3
47.3
11.5
False +/-
Eval @
1 ppm
+
+
+
Eval @
10 ppm
+
+
+
+
+
+
of all PAHs detected.
4035 - 9
Revision 0
January 1995
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Table 7: Total PAH Content of Region X Field Samples Using EnSys
PAH RISc™ Immunoassay Test Kit Normalized to Cross-Reactivity (Contd.)
Sample ID
PAH-14
PAH-15
PAH-16
PAH-17
PAH-18
PAH-19
PAH-20
PAH-21
PAH-22
PAH-23
PAH-24
PAH-25
PAH-26
PAH-27
PAH-28
PAH-29
PAH-30
1 ppm Test
<1
*
*
*
*
*
*
>1
*
*
*
*
*
*
*
10 ppm Test
<10
*
*
it
*
*
*
*
*
*
*
*
*
>10
*
*
*
*
OC/MS
Lab Result
(PP*)1
0.2
0.5
0.0
1.2
1.7
3.6
0.6
27.5
49.2
0.8
0.1
0.2
13.5
0.0
6.4
0.2
2.8
False +/-
Eval 6
1 ppm
+
+
+
Eval 6
10 ppm
+
+
_
'Sum of all PAHs detected.
4035 - 10
Revision 0
January 1995
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METHOD 4050
TNT EXPLOSIVES IN WATER AND SOILS BY IMMUNOASSAY
1.0 SCOPE AND APPLICATION
1.1 Method 4050 is a procedure for screening waters and soils to
determine when trinitrotoluene (TNT, CAS No. 118-96-7) is present at
concentrations above 0.5 mg/kg in soil and 5 jig/L in water. Method 4050
provides an estimate for the concentration of TNT by comparison with a reference.
1.2 Using the test kit from which this method was developed, 93% of soil
samples containing 0.25 ppm or less of TNT will produce a negative result, and
99+% of soil samples containing 1.0 ppm or greater of TNT will produce a positive
result. In addition, 93% of water samples containing 2.5 ppb or less of TNT will
produce a negative result, and 99%+ of water samples containing 10 ppb or more
of TNT will produce a positive result.
1.3 In cases where the exact concentrations of TNT are required,
quantitative techniques (j.e., Method 8330) should be used.
1.4 This method is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.0 SUMMARY OF METHOD
2.1 Test kits are commercially available for this method. The
manufacturer's directions should be followed.
2.2 In general, the method is performed using a diluted water sample or
an extract of a soil sample. Samples and an enzyme-TNT conjugate reagent are
added to immobilized TNT antibody. The enzyme-TNT conjugate "competes" with TNT
present in the sample for binding to immobilized TNT antibody. The enzyme-TNT
conjugate bound to the TNT antibody then catalyzes a colorless substrate to a
colored product. The test is interpreted by comparing the color produced by a
sample to the response produced by a reference reaction.
3.0 INTERFERENCES
3.1 Chemically similar compounds and compounds that might be expected to
be found in conjunction with TNT contamination were tested to determine the
concentration required to produce a positive test result.
3.1.1 Table 1 provides the concentrations of compounds tested with
the D TECH test kit that are required to elicit a positive response at the
MDL, as well as the concentration required to yield 50% inhibition
4050-1 Revision 0
January 1995
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compared to the standard curve.
4.0 APPARATUS AND MATERIALS
4.1 Immunoassay test kit: D TECH™ TNT (Strategic Diagnostics Inc.), or
equivalent. Each commercially available test kit will supply or specify the
apparatus and materials necessary for successful completion of the test.
5.0 REAGENTS
5.1 Each commercially available test kit will supply or specify the
reagents necessary for successful completion of the test.
6.0 SAMPLE COLLECTION, PRESERVATION, AND TRANSPORTATION
6.1 See the introductory material to this chapter, Organic Analytes,
Section 4.1.
6.2 Soil samples may be contaminated, and should therefore be considered
hazardous and handled accordingly.
7.0 PROCEDURE
7.1 Follow the manufacturer's instructions for the test kit being used.
Those test kits used must meet or exceed the performance specifications indicated
in Tables 3-6.
8.0 QUALITY CONTROL
8.1 Follow the manufacturer's instructions for the test kit being used
for quality control procedures specific to the test kit used. Additionally,
guidance provided in Chapter One should be followed.
8.2 Use of replicate analyses, particularly when results indicate
concentrations near the action level, is recommended to refine information
gathered with the kit.
8.3 Do not use test kits past their expiration date.
8.4 Do not use tubes or reagents designated for use with other test kits.
Do not mix reagents from one kit lot with a different kit lot.
8.5 Use the test kits within their specified storage temperature and
operating temperature limits.
4050-2 Revision 0
January 1995
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8.6 Method 4050 is intended for field or laboratory use. The appropriate
level of quality assurance should accompany the application of this method to
document data quality.
9.0 METHOD PERFORMANCE
9.1 Table 1 provides data on the minimum concentrations of possible
interferants and co-contaminants required to elicit a positive response in the
test kits evaluated.
9.2 Twenty five soil samples, known to not be contaminated with TNT, were
extracted and analyzed using the DTECH TNT kit to determine the extent of soil
matrix effects on the performance of the test kit. The results are provided in
Table 2A, and show that false positive results are not attributable to soil
components. Table 2B presents similar data generated from the analysis of thirty
uncontaminated water samples.
9.3 Thirty water samples and thirty soil samples, known to not be
contaminated with TNT, were each spiked with TNT at one-half and two times the
MDL (0.25 and 1.0 ppm respectively). These samples were analyzed with the DTECH
TNT test kit to determine the error rate of the assay. The results are presented
in Tables 3A and 3B.
9.4 Ten different soil types, all known to not be contaminated with TNT,
were spiked with an acetone solution containing approximately 1.0 ppm TNT. This
spiking solution was later quantitated by Method 8330 and found to contain 0.77
ppm TNT. The spiked soil samples were analyzed three (3) times with the DTECH
kit to determine the extraction efficiency of the method. The data are presented
in Table 4.
9.5 Table 5 presents the results of analysis of three soils spiked at
approximately 1 and 3 ppm TNT. Each sample was analyzed once using Method 8330
and ten times using the DTECH kit.
9.6 Tables 6A and 6B present the results of two field trials. In each
trial, soil samples were obtained at a West Coast site from borings, using a
split spoon technique. The samples were homogenized by placing approximately six
cubic inches of soil into a stainless steel vessel and mixing for five minutes
with a stainless steel trowel. The soil was aliquotted into two (2) six ounce
glass bottles, tested on-site using the DTECH method and transported to
commercial laboratories (one laboratory per field trial) for analysis by Method
8330. Table 6C presents the results of a third party field trial, conducted by
the California Department of Environmental Health Services.
10.0 REFERENCES
1. D TECH™ TNT Users Guide , SDI/EM Sciences 1994
4050-3 Revision 0
January 1995
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2. Hutter,L., G. Teaney, and J.W.Stave, "A Novel Field Screening System for
TNT Using EIA", in Field Screening Methods for Hazardous Wastes and
Toxic Chemicals, Vol 1, Proceedings of the 1993 U.S. EPA/A&WMA
International Symposium, p.472, 1993.
3. Teaney, G., J.Melby, L.Hutter and J.Stave, "A Novel Field Analytical
Method for TNT", Proceedings of the American Association of Analytical
Chemists, 1993.
4. Haas, R.J., and B.P. Simmons, "Measurement of Trinitrotoluene (TNT) and
Hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) in Soil by Enzyme
Immunoassay and High Performance Liquid Chromatography (EPA Method
8330)", California Environmental Protection Agency, Department of Toxic
Substances Control, Hazardous Materials Laboratory, March, 1995.
4050-4 Revision 0
January 1995
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TABLE 1
CROSS REACTANTS
D TECH™ TNT test kit
COMPOUND
TNT (2,4,6-trinitrotoluene)
Tetryl"
1,3, 5- tri ni trobenzene
2-amino-4,6-dinitrotoluene
4-amino-2,6-dinitrotoluene
2,4-dinitrotoluene
2,6-diaminonitrotoluene
2-nitrophenol
4-nitrophenol
2,4-dinitrophenol
RDXd
HMXd
MDL"
(ppm)
0.5
3
4
13
>500
90
>500
>500
>500
>500
>500
>500
1C 50b
(ppm)
17
48
75
150
>500
390
>500
>500
>500
>500
>500
>500
% CROSS
REACTIVITY0
100
35
23
11
<1
4
<1
<1
<1
<1
<1
<1
The following compounds were not detected at or above 100 ppm:
Benzene Xylenes PCB 1254 Triazine
Ethyl benzene Toluene PCP
PAHs - an equal concentration mixture of:
Acenaphthene Acenaphthalene Anthracene
1,2-Benzanthracene Benzo(a)pyrene Benzo(b)fluoranthene
Benzo(ghi)perylene Benzo(k)fluoranthene Chrysene
Dibenz(ah)anthracene Fluoranthene Fluorene
Indeno(123-cd)pyrene Naphthalene Phenanthrene
Pyrene
The Method Detection Limit (MDL) is defined as the lowest concentration
of compound that yields a positive test result.
The IC50 is defined as the concentration of compound required to produce
a test response equivalent to 50% of the maximum response.
% Cross reactivity is determined by dividing the equivalent TNT
concentration by the actual compound concentration at IC50
Tetryl = methyl-2,4,6-trinitrophenylnitramine
RDX = hexahydro-l,3,5-trinitro-l,3,5-triazine
HMX = octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine
4050-5
Revision 0
January 1995
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TABLE 2A
SOIL MATRIX EFFECTS
Soil
133
101
100
102
106
107
109
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
127
128
130
Soil type
Avonburg Fine Sine Silt
Matapeake Silt Loam
Clay Loam
Sassafras Sand Loam
Evesboro Low Organic Sand
Pokomoke High OM Sand
Davidson Clay Loam
Shontic Casa Grande Sand
Casa Grande Clay Loam
Trix Sand Clay Loam
Trix Casa Grande Clay
Yolo Loam
Capay Silt Clay
Sycamore Silt Loam
Dennis Silt Loam
Grundy Silt Clay Loam
Luray Silt Clay Loam
Wooster Silt Loam
Vienna Loam
Opal Clay
Raub Silt Loam
Rockfield Silt Loam
Cisne
Muscatine Loam
Sandy Brae
N/A
DE
DE
DE
DE
DE
GA
AZ
AZ
AZ
AZ
CA
CA
CA
KS
KS
OH
OH
SD
SD
IN
IN
IL
IL
DE
D TECH
RANGE (ppm)
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
4050-6
Revision 0
January 1995
-------
TABLE 2B
WATER MATRIX EFFECTS
Water
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Water type
Ground Water, Burlington
Well Water, Burlington
Surface Water #1, Houston
Unknown Creek, Dartmouth
City Well Water, Ontario
Pacific Ocean, Victoria
Surface Water, Harmony Woods
Adamsville River, Adamsville
Surface Water #2, Houston
Buttermilk Falls, White Haven
Main St Pond, Germantown
Hudson River, Germantown
Atlantic Ocean
Ground Water #1, Dover
Ground Water #2, Dover
Ground Water #3, Dover
Drinking Well Water,
Ground Water, Elsmere
Ground Water, Elsmere
Ground Water, Elsmere
Lab Sample 20643
Lab Sample 20645
Lab Sample 20659
Lab Sample 20826
Lab Sample 20827
Lab Sample 20843
Lab Sample 20850
Lab Sample 20848
Ground Water, Adrian
Ground Water, Adrian
IA
IA
TX
MA
CA
CA
DE
RI
TX
PA
NY
NY
NJ
DE
DE
DE
PA
DE
DE
DE
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
GA
GA
D TECH RANGE (ppm)
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
<5.0
4050-7
Revision 0
January 1995
-------
TABLE 3A
False Negative and False Positive Rates, Soil Matrix3
Spike Concentration
0.25 ppm
1.0 ppm
False Positive Rate
7%
-
False Negative Rate
-
0%
a Thirty negative soils were spiked with TNT at one-half and two times the MDL
(0.25 and 1.0 ppm respectively). These samples were analyzed with the DTECH TNT
test kit to determine the error rate of the assay.
TABLE 3B
False Negative and False Positive Rates, Water Matrix8
Spike Concentration
0.25 ppm
1.0 ppm
False Positive Rate
7%
100%
False Negative Rate
93%
0%
a Thirty negative water samples were spiked with TNT at one-half and two times
the MDL (0.25 and 1.0 ppm respectively). These samples were analyzed with the
DTECH TNT test kit to determine the error rate of the assay.
4050-8
Revision 0
January 1995
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TABLE 4
DETERMINATION OF EXTRACTION EFFICIENCY FROM SOIL SAMPLES8
SOIL ID
101
106
108
109
110
116
117
123
126
128
SPIKING
SOLUTION
MEAN TNT CONC.
(ppm)
0.54
0.64
0.87
0.63
0.88
1.02
0.82
0.87
0.95
0.65
0.77
SD
0.04
0.06
0.18
0.08
0.15
0.15
0.15
0.23
0.26
0.11
N/A
%CV
7
9
20
13
17
17
15
26
28
16
N/A
%RECOVERY
70
84
113
82
115
115
132
113
123
84
100
aTen different TNT negative soils were spiked with an acetone solution containing
0.77 TNT. The spiked soil samples were analyzed three times with the DTECH kit
to determine the extraction efficiency of the method.
4050-9
Revision 0
January 1995
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TABLE 5
RECOVERY OF TNT SPIKED INTO REAL SOILS
Three (3) soils were spiked at approximately 1 and 3 ppm TNT.
analyzed once by Method 8330 and ten (10) times by D TECH.
Each sample was
SAMPLE ID
106-1
116-1
128-1
AMOUNT SPIKED
1.0
1.0
1.0
D TECH (ppm)
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
1.5 - 3.0
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
0.5 - 1.5
HPLC METHOD
8330
0.69
0.73
0.75
AGREEMENT"
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
FP
Y
Y
Y
Y
Y
FP
Y
Y
Y
Y
Y
FP
Y
Y
Y
Y
4050-10
Revision 0
January 1995
-------
TABLE 5 (cont)
RECOVERY OF TNT SPIKED INTO REAL SOILS
SAMPLE ID
106-3
116-3
128-3
AMOUNT SPIKED
3.0
3.0
3.0
D TECH (ppm)
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
0.5 - 1.5
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
0.5 - 1.5
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
0.5 - 1.5
1.5 - 3.0
0.5 - 1.5
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
1.5 - 3.0
HPLC METHOD
8330
1.53
2.12
2.07
AGREEMENT"
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
FN
Y
Y
Y
Y
Y
Y
Y
Y
Y
4050-11
Revision 0
January 1995
-------
TABLE 6A
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
SAMPLE ID
61-1
61-10
61-11
61-12
61-13
61-14
61-15
61-16
61-17
61-18
61-19
61-2
61-20
61-21
61-22
61-23
61-24
61-25
61-26
61-27
61-28
61-29
61-3
61-30
61-4
61-5
61-6
61-7
61-8
61-9
TET-1
TET-2
TET-3
TL-1
D TECH RANGE
(ppm)
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
< 0.2
> 1.5
< 0.2
0.5-1.0
< 0.2
< 0.2
1.0-1.5
< 0.2
< 0.2
0.2-0.5
< 0.2
< 0.2
1.0-1.5
< 0.2
> 1.5
0.5 - 1.0
> 1.5
< 0.2
0.5-1.0
0.2-0.5
0.5-1.0
< 0.2
< 0.2
0.2-0.5
METHOD 8330
TNT (ppm)
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
< 0.09
> 3.0
< 0.09
2.44
< 0.09
< 0.09
1.4
< 0.09
< 0.09
0.27
< 0.09
< 0.09
1.3
< 0.09
1.1
1.0
> 3.0
< 0.09
1.0
0.56
< 0.09
< 0.09
< 0.09
0.99
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
Y
Y
Y
FP
Y
Y
FN
4050-12
Revision 0
January 1995
-------
SAMPLE ID
TL-2
TL-3
TL-4
TL-5
TL-6
TL-7
TL-8
TL-9
D TECH RANGE
(ppm)
> 1.5
> 1.5
0.2-0.5
> 1.5
0.2-0.5
0.2-0.5
0.5-1.0
0.2-0.5
METHOD 8330
TNT (ppm)
1.2
> 3.0
0.66
> 3.0
0.66
0.71
1.46
0.92
AGREEMENT8
Y, FN, FP
FP
Y
FN
Y
FN
FN
FN
FN
4050-13
Revision 0
January 1995
-------
TABLE 6B
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Sample
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
D TECH
Range (ppm)
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
0.5 - 1.0
0.5 - 1.0
0.5 - 1.0
> 1.5
0.5 - 1.0
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
8330 TNT
(ppm)
5.75
3.32
166
2500
2.72
<2.0
<2.0
140
230
1100
23.5
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
3.23
<2.0
<2.0
4.75
<2.0
<2.0
<2.0
3.64
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
6.39
8330 TNB
(ppm)
< 1.0
< 1.0
< 1.0
18.50
< 1.0
7.02
5.12
12.2
20.2
16.9
11.5
2.95
1.30
1.89
3.94
4.54
4.57
10.5
24.3
81
1.61
2.60
2.97
6.29
< 1.0
5.05
6.62
1.94
8.53
6.77
6.75
17.6
39.2
TNT Equivalent
(ppm)
5.75-6.0
3.32-3.57
166
2504
2.72-2.97
1.76-3.76
1.28-3.28
143
235
1104
26.0
0.74-2.74
0.33-2.33
0.47-2.47
0.99-2.99
1.14-3.14
1.14-3.14
2.63-4.63
9.3
20.3
0.40-2.40
5.40
0.74-2.74
1.57-3.57
<2.25
4.90
1.66-3.66
0.49-2.49
2.13-4.13
1.69-3.69
1.69-3.69
4.40-6.41
16.2
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
4050-14
Revision 0
January 1995
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Sample
Number
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
0 TECH
Range (ppm)
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
0.5 - 1.0
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
1.0 - 1.5
> 1.5
> 1.5
> 1.5
8330 TNT
(ppm)
4.20
5.14
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
820
1200
27.6
7.43
4.98
3.32
3.42
4.32
7.57
5.12
<2.0
<2.0
33.5
2.19
7.00
2.84
<2.0
2.23
5.38
2.60
4.43
4.79
2.29
8.84
9.01
29.00
<2.0
8330 TNB
(ppm)
1.39
< 1.0
2.68
7.65
27.70
9.01
30.90
35.70
5.69
24.0
11.9
9.01
9.46
10.4
16.5
28.2
44.8
81.2
1.64
2.27
23.4
8.43
11.0
4.69
5.67
12.8
31.4
13.0
31.1
25.9
18.2
148
< 1.0
6.02
1.30
TNT Equivalent
(ppm)
4.55
5.14-5.39
0.67-2.67
1.91-3.91
6.9-8.9
2.25-4.25
7.7-9.7
8.9-10.9
821
1206
31
9.70
7.40
5.90
7.60
11.4
18.8
25.4
0.41-2.41
0.57-2.57
39.4
4.30
9.75
4.01
1.42-3.42
5.43
13.23
5.85
12.2
11.3
6.8
45.8
9.01
30.50
0.33-2.33
AGREEMENT"
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
4050-15
Revision 0
January 1995
-------
Sample
Number
78
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
95
96
97
98
99
100
101
D TECH
Range (ppm)
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
0.5 - 1.0
0.5 - 1.0
1.0 - 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
1.0 - 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
> 1.5
0.5 - 1.0
> 1.5
> 1.5
> 1.5
> 1.5
< 0.2
> 1.5
> 1.5
8330 TNT
(ppm)
<2.0
<2.0
2.49
<2.0
<2.0
<2.0
<2.0
3.98
5.67
7.05
8.04
1000
2.12
8.83
3.64
3.22
<2.0
<2.0
<2.0
<2.0
<2.0
351
116
4.29
<2.0
2.34
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
4.24
<2.0
8330 TNB
(ppm)
7.50
4.70
30.0
29.1
8.86
30.7
38.1
183
122
< 1.0
< 1.0
7.49
2.99
5.56
3.20
10.6
18.3
17.4
20.4
117
1.96
5.77
39.2
3.92
11.6
9.26
48.7
5.05
12.6
10.7
11.1
3.74
1.88
< 1.0
1.10
TNT Equivalent
(ppm)
1.88-3.88
1.18-3.18
9.99
7.28-9.28
2.22-4.22
7.68-9.68
9.59-11.6
49.7
36.2
7.05-7.3
8.04-8.29
1001
2.87
10.20
4.44
5.87
4.58-6.58
4.43-6.43
5.10-7.10
29.2-31.2
0.49-2.49
352
126
5.27
2.9-4.9
4.66
12.2-14.2
1.26-3.26
3.15-5.15
2.68-4.68
2.78-4.78
0.94-2.94
0.47-2.47
4.24-4.49
0.28-2.28
AGREEMENT8
Y, FN, FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
FN
FN
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
FN
Y
Y
Y
Y
FN
Y
Y
4050-16
Revision 0
January 1995
-------
Sample
Number
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
through
279
280
through
365
366
through
381
382
through
391
D TECH
Range (ppm)
0.5 - 1.0
1.0 - 1.5
> 1.5
> 1.5
> 1.5
0.5 - 1.0
1.0 - 1.5
0.5 - 1.0
0.5 - 1.0
0.5 - 1.0
1.0 - 1.5
> 1.5
> 1.5
> 1.5
0.2 - 0.5
0.5 - 1.0
0.2 - 0.5
0.5 - 1.0
> 1.5
> 1.5
> 1.5
0.2 - 0.5
< 0.2
< 0.2
0.2 - 0.5
< 0.2
0.2 - 0.5
0.5 - 1.0
1.0 - 1.5
8330 TNT
(ppm)
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
6.35
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
8330 TNB
(ppm)
1.28
2.70
10.5
14.1
18.4
6.35
6.66
21.8
5.29
4.49
16.3
28.7
17.7
24.1
< 1.0
2.40
4.70
11.6
56.9
45.6
67.7
2.78
1.61
4.07
3.12
<1.0
< 1.0
< 1.0
< 1.0
TNT Equivalent
(ppm)
0.32-2.32
0.68-2.68
2.63-4.63
3.53-5.53
4.6-6.6
1.59-3.59
1.67-3.67
5.45-7.45
1.32-3.32
1.12-3.12
4.08-6.08
7.18-9.18
4.43-6.43
6.03-8.03
6.35-6.6
0.60-2.6
1.18-3.18
2.9-4.9
14.2-16.2
11.4-13.4
16.9-18.9
0.7-2.7
0.4-2.4
1.02-3.02
0.78-2.78
<2.25
<2.25
<2.25
<2,25
AGREEMENT"
Y, FN, FP
Y
Y
Y
Y
Y
FN
FN
FN
FN
FN
FN
Y
Y
Y
FN
Y
FN
FN
Y
Y
Y
FN
FN
FN
FN
Y
Y
Y
Y
4050-17
Revision 0
January 1995
-------
Sample
Number
392
through
399
D TECH
Range (ppm)
> 1.5
8330 TNT
(ppm)
<2.0
8330 TNB
(ppm)
< 1.0
TNT Equivalent
(ppm)
<2.25
AGREEMENT"
Y, FN, FP
Y
4050-18
Revision 0
January 1995
-------
TABLE 6C
COMPARISON OF DTECH SOIL RESULTS WITH METHOD 8330
Third Party Field Trial
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Dilution
Factor
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
SDI
Results
<0.5
<0.5
<0.5
0.5-1.5
<0.5
0.5-1.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
<0.5
<0.5
0.5-1.5
<0.5
<0.5
8330
TNT Results
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
8330
TNT+TNB
Results
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
AGREEMENT"
Y, FN, FP
Y
Y
Y
FP
Y
FP
Y
Y
Y
Y
Y
Y
Y
Y
Y
FP
Y
Y
FP
Y
Y
Y
Y
FP
Y
Y
4050-19
Revision 0
January 1995
-------
Sample
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Dilution
Factor
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10
10
1
SDI
Results
<0.5
0.5-1.5
<0.5
0.5-1.5
<0.5
0.5-1.5
0.5-1.5
0.5-1.5
<0.5
<0.5
3.0-4.0
<0.5
<0.5
<0.5
0.5-1.5
1.5-3.0
<0.5
0.5-1.5
0.5-1.5
0.5-1.5
0.5-1.5
<0.5
0.5-1.5
<0.5
0.5-1.5
5-15
40-50
0.5-1.5
8330
TNT Results
<0.15
<0.15
<0.15
0.15-0.99
<0.15
<0.15
<0.15
0.15-0.99
<0.15
<0.15
0.15-0.99
<0.15
<0.15
<0.15
<0.15
0.15-0.99
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
<0.15
1.4
35
<0.15
8330
TNT+TNB
Results
<0.25
<0.25
<0.25
0.15-0.99
<0.25
<0.25
<0.25
0.15-0.99
<0.25
<0.25
0.25-2.0
<0.25
<0.25
<0.25
<0.25
0.15-0.99
<0.25
0.15-0.99
<0.25
<0.25
1.3
<0.25
<0.25
<0.25
<0.25
3.2
41.67
<0.15
AGREEMENT'
Y, FN, FP
Y
FP
Y
Y
Y
FP
FP
Y
Y
Y
FP
Y
Y
Y
FP
FP
Y
Y
FP
FP
Y
Y
FP
Y
FP
Y
Y
FP
4050-20
Revision 0
January 1995
-------
Sample
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Dilution
Factor
1
1
1
1
1
10
1
10
10
100
1000
10000
1000
10
100
10
10
10
10
10
10
10
10
100
10
10
10
10
SDI
Results
0.5-1.5
1.5
0.5-1.5
3.0-4.0
0.5-1.5
15-30
0.5-1.5
4-40
5-15
400-500
4000-5000
15000
15000
5-15
400-500
15-30
5-15
40
5-15
5
4-30
5-15
5-15
300-400
5-15
5-15
5-15
15-30
8330
TNT Results
0.15-0.99
0.15-0.99
<0.15
0.15-0.99
<0.15
22
-
2.1
2
360
6300
4000
530
2.8
460
4.2
1.0
5.1
1.9
1.6
2.2
1.7
2.2
180
3.1
2.8
2.5
3.2
8330
TNT+TNB
Results
0.15-0.99
0.15-0.99
<0.15
0.15-0.99
<0.15
22.48
<0.15
32
3.1
364
6327
4027
547
3.375
477
6.73
1.57
34.5
4
2.7
4.3
2
3.95
192.19
4.61
5.26
5.26
4.5
AGREEMENT'
Y, FN, FP
Y
Y
FP
FP
FP
Y
FP
Y
Y
Y
Y
FP
FP
Y
Y
FP
FP
Y
Y
Y
Y
FP
Y
Y
Y
Y
Y
FP
4050-21
Revision 0
January 1995
-------
Sample
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
Dilution
Factor
10
10
10
10
10
10
100
1
10000
10
10000
1000
1
10000
10000
10
10
100
SDI
Results
40-50
15-30
15-30
15-30
5-15
5-15
150-300
4-5
15000
40-50
15000-30000
500-1500
3.0
40000-50000
4000-5000
15-30
15-30
50-150
8330
TNT Results
1
3.8
36
3.6
2.6
3.2
78
18000
11000
36
11000
88
9.6
15000
2200
3.6
6.4
26
8330
TNT+TNB
Results
23
18.5
52.5
8.66
19.16
3.84
82
18050
11052.9
42.4
11052.9
107
10.17
15050
2220
3.9
6.7
28.76
AGREEMENT"
Y, FN, FP
Y
Y
FN
Y
Y
Y
Y
FN
Y
Y
Y
FP
FN
FP
Y
FP
FP
Y
4050-22
Revision 0
January 1995
-------
THE OHIO ENVIRONMENTAL
PROTECTION AGENCY
DIVISION OF
EMERGENCY REMEDIAL RESPONSE
Ottawa River, Lucas County, Ohio
Health Advisory Zone
Sediment Screening Survey
Step One of Stage One
March 28-29, 1995
-------
Ottawa River Site Description and Location
The Maumee Bay was once known as the most prolific fish
spawning ground in Lake Erie. This area included what was
known as the Great Black Swamp which contained a faunal
association requiring water free of clayey silts and
containing aquatic vegetation. Habitat and water quality
degradation began as far back as 1850, due to the effects of
dams, channelization, over fishing and pollution. The heavy
metal and organic chemical contamination caused by
agriculture and the heavy industry such as oil refining,
petrochemical, metal fabricating, auto parts and
manufacturing resulted in the Maumee Bay being listed as an
Area of Concern (AOC) in 1985 by the International Joint
Commission. It is one of 43 areas with pollution problems
so severe that the 14 identified beneficial uses in the
Great Lakes Water Quality Agreement are impaired. Because
the Maumee River is the largest tributary to the Great
Lakes, this pollution is readily carried into Lake Erie,
contaminating water and sediment.
In the spring of 1991, Ohio EPA produced the "Fish Tissue,
Bottom Sediment, Surface Water, Organic and Metal Chemical
Evaluation and Biological Community Evaluation" for the
Ottawa River and Tenmile Creek, a tributary in the Maumee
AOC. These data documented the pollution problems and
identified areas needing further analysis, including grossly
contaminated surface sediments. This report led to the Ohio
Department of Health issuing a fish consumption/contact
advisory for the Ottawa River from River Mile 8.8 to Lake
Erie in April 1991. This advisory was based on the
detection of extremely elevated levels of polychlorinated
biphenyls (PCBs) in sediment and fish tissues. Some
important points which are detailed in the report include:
1. Extensive PCB contamination in the lower 10 miles
of the Ottawa River. PCBs were detected in all
media sampled within the Ottawa River, with Ohio
Water Quality Standard violations noted in both
surface water and fish samples. The highest PCB
concentration (1,200 ppm) was documented in the
sediment sampled from a portion of the former
river channel which now serves as a drainage swale
for storm water discharges into the Ottawa River
upstream from the Dura landfill. The highest
total PCB levels in fish occurred at River Mile
5.2, where common carp fillets and whole body PCB
concentrations were 65 ppm and 84 ppm,
respectively.
2. A wide range of pesticides were detected in the
fish tissue and sediment samples. Most pesticides
appeared to be in low concentrations. However,
heptachlor epoxide and dieldrin were considered
-------
extremely elevated using the sediment evaluation
criteria developed by Kelly and Hite (Illinois
EPA) .
3. Five heavy metal contaminants (barium, cadmium,
chromium, lead, and selenium) were measured in
fish tissue fillet and whole body samples from the
1990 sampling sites. Eleven heavy metal
parameters were detected in the sediments from the
Ottawa River between 1986 and 1990.
4. Biological community results show non-attainment
of the Warmwater Habitat aquatic life use
designation for nearly the entire sampling area of
the Ottawa River. Based on 1986 and 1990 Ohio EPA
sampling results, the Ottawa River is in violation
of Ohio Water Quality Standards.
The Ohio EPA, Division of Surface Water (DSW), and the
Division of Emergency Remedial Response (DERR) worked
together during the various stages of this screening survey.
The DSW focused their efforts on establishing a baseline of
current water quality conditions in the AOC. Their efforts
include the collection and chemical/physical analysis of
surface water samples and the chemical analysis of sediments
at several stations throughout the AOC. The DERR began
their study efforts by initiating site inspections at
uncontrolled/unregulated known or suspected hazardous waste
disposal sites within the Ottawa River watershed. The
decision to first focus on the Ottawa River was based on the
knowledge of its severe water quality problems from past
reports and based on the fact that 21 uncontrolled hazardous
waste sites have been identified. Of these, 18 were
targeted for site inspection by either Ohio EPA or the U.S.
EPA Superfund contractors. All 18 site inspections were
completed at the end of March, 1993. The site assessment
report are on file at the Ohio EPA Northwest District
Office.
The Ottawa River health advisory zone (River Mile 8.8 to the
mouth), sediments will be evaluated by conducting sampling
and analysis on sediments in direct contact with 14
uncontrolled hazardous waste sources from RM 8.8 (Auburn
Ave. bridge) to RM 4.9 (Stickney Avenue bridge), and on
sediments in the depositional zone from RM 4.9 to the mouth
of the Ottawa River where it flows into Maumee Bay. The
upper zone from RM 8.8 to RM 4.9 is 3,9 miles of wall to
wall landfills and will require a more comprehensive survey
than the lower zone or depositional zone from RM 4.9 to the
river mouth, where more homogeneous sediment conditions are
anticipated.
-------
Maumee AOC Project
Ottawa River
The Ohio EPA DERR believes that a three stage approach would
be the most affected means to characterize the Ottawa River
sediments, with each stage implemented using the information
(results) from the previous stage. The three stages are
summarized below. During the September, 1994 approach of
the sediment screening survey, Step 1 of Stage One was the
only step implemented. The three stages will provide the
most effective and efficient means to characterize the
contaminants present in the sediments, the extent of these
contaminants, and to aid in the delineation of the source
(sources) of the contaminants. This approach will also
allow, given budgetary constraints, for the most thorough
(extensive) investigation efforts to be focused on the areas
of greatest concern to the river ecosystem (areas of
greatest contamination problems) . The information gathered
from Step 1 of the sediment survey, together with the
information gathered from other surveys and assessments
being performed as part of the RAP process will aid in
future activities to be implemented in order to achieve the
overall goal of swimmable and fishable water within the
Ottawa River Watershed.
Stage One - Sediment Screening Survey
Conduct a comprehensive screening of the shallow
sediments (<6 feet) within the 8.8 miles of the
health advisory zone based on known contaminant
problems with PCBs, PAHs, and heavy metals. The
effort of this survey will focus on the
identification of "hot spots" of contaminated
sediment areas.
Geairer.t Characterization
Conduct additional sediment investigations with
the emphasis placed on further defining the extent
of contaminated areas as identified in stage one.
Deep borings will be completed at this stage with
sediment samples analyzed for CLP TCL/TAL priority
pollutants.
Stage Three - Remedial Characterization
Conduct a sediment survey which will fill in data
gaps identified from Stage Two and will provide
information necessary to select the most effective
remedial options. Remedial options most likely to
be explored at: this stage will be no action,
removal, stabilization, biological or experimental
pilot technologies.
-------
TABLE 1 - Ottawa River Core Sediment Sampling Locations
Location on the Ottawa River
University of Toledo
Auburn Ave. Bridge
Jeep Parkway Bridge
Berdan Ave. Bridge
Detroit Ave. Bridge
Lagrange St. Bridge
Storm Sewer Outfall - 100 Yards Upstream
Storm Sewer Outfall -
(located at the mouth of the river channel
prior to the constr. of 1-75)
Storm Sewer Outfall -
(located approx. 75' east of the mouth of
the river channel mentioned above)
Railroad Trestle
Directly behind Casey's Sign & 15'- N.Bank
Stickney Ave. Bridge
Railroad Trestle
1-75 Bridge
Closed East Channel along Island
Downstream of West Bank Shore Point
Summit St. Bridge
Narrow Area mid-stream @ East Shore Point
River Mouth
River Mile
11.0
8.8
7.9
7.4
6.9
6.4
6. 1
6. 0
6.0A
5.8
5.2
4.9
4.2
3.4
2.8
2.4
1. 6
0.7
0.0
-------
3A Sample # ROSS Lab Sample # PCB Field (ppm) PCB Lab. (ppm) TOC (ppm)
56800
34000
130000
112000
59400
54700
90100
120000
81200
37700
41200
28000
42700
40900
66400
43200
74000
60400
33800
21500
135000
25600
31000
51800
96300
14400
47300
25700
5420
1/4.9/9-15
,1/4.9/15-34
1/6.0/0-6
1/6.0/30-57
1/4.2/6-23
1/4.2/23-40
1/3.4/8-19
1/3.4/19-41
1/5.8/0-6
1/6.4/0-6
:l/7.9/0-6
11/7.4/0-6
11/6.9/8-21
!1/6.9/21-31
J1/9.0/6-23
51/10.0/0-6
M/1. 6/1 2-27
{1/1.6/0-12
*1/6.1/0-16
^1/6.1/16-50
?1/6.0A/0-24
?1/5.5/0-8
11/8.8/4-17
*1/1 2.0/0-6
^1/6.0/6-30
R1/1 1.0/8-1 7
R1/1 1.0/0-8
jM/0.0/0-11
R1/0. 0/1 1-25
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
3.9
2.1
244
1045
9.6 .
8.4
1.2
5.8
11.0
2.7
0.67
0.35
2.7
1.3
6.1
4.7
3.6
1.1
1.7
<0.12
848
4.3
<0.12
1.4
72
<0.12
<0.12
1.8
<0.12
6.0
0.11
1300
2000
5.0
0.79
0.79
0.17
37
3.7
0.53
0.65
8.6
0.70
2.7
10
0.3
0.62
3.8
0.0
2500
16
0.3
3.3
190
0.22
0.099
0.66
0.29
-------
OTTAWA RIVER UNCONTROLLED WASTE SITES OVERVIEW
Ottawa River Profile
The following is a complete list of Ottawa River sites that were
investigated by either Ohio EPA DERR or U.S.EPA Superfund
Contractors in FFY 1993.
DURA AVENUE LANDFILL
Dura Avenue Landfill (DAL) is an inactive 70 acre landfill
which operated for 28 years, from December 1952 (purchased by
City) through June 1980 (termination). Municipal wastes were
received for all 28 years. Commercial and industrial wastes
were received for 16 years (1952 - 1968). Commercial disposal
stopped in July 1968 due to Ordinance No. 554-68. The site is
bordered to the north and east by industrial/commercial
property and to the south and east by Sibley Creek and the
Ottawa River, respectively. The DAL has a total estimated
fill volume of 4.65 million yd3, which may include 750,000
gallons of potentially hazardous liquid waste and 13,000 cubic
yards of potentially hazardous solid waste. Groundwater,
leachate and soil monitoring show PCBs, volatile and semi-
volatile organic compounds and heavy metals. A barrier wall,
leachate collection system, and wastewater treatment facility
have been constructed to stop the leachate flow into the
Ottawa River. A federal court mediation program is currently
underway to address the final closure of this landfill.
STICKNEY AVENUE LANDFILL
The Facility is approximately 55 acres in size and is located
across the road from the Stickney Avenue Jeep facility. The
site is covered with 0 to 3 feet of clay and is overgrown with
vegetation. The Ottawa River borders the site to the east and
north with over 2500 feet of frontage. The City of Toledo
operated the facility as a municipal waste landfill from 1958
to 1986, waste was burned for an unknown period of time.
Leachate and soil sampling indicate the presence of several
volatile and semi-volatile compounds and heavy metals.
Excessive soil erosion and leachate outbreaks exist along the
entire landfill perimeter. An Engineering Evaluation/Cost
Analysis (EE/CA) emphasizing presumptive remedies (i.e. cap,
leachate collection, methane monitoring, and bank
stabilization) has been initiated by an industrial group of
potentially responsible parties (PRP's) . This action has been
initiated by the U.S. EPA Superfund program.
TYLER STREET LANDFILL
The Facility is an inactive municipal and industrial landfill
that occupies about 77.6 acres in a primarily industrial area.
The west end of the Tyler site is currently used by Creekside
Auto Parts as an automobile junkyard which has been covered by
soil and demolition debris. The east side of the landfill is
-------
vegetated with several small ponds/marshes present due to
differential settling of the filled materials. Leachate is
discharging from the landfill into the river in several places
and the landfill is severely eroded along the riverbanks. The
City of Toledo used the site to dispose of up to 2200 cubic
yards of waste daily (between 1955 and 1968). Private parties
also reportedly disposed of about 400 cubic yards of
industrial/ commercial waste daily. Observed releases of
contaminants to the environment from leachate discharge and
soil erosion are documented. Contaminants identified to the
site include heavy metals and semi-volatile contaminants
associated with coal tar and creosote were also identified.
An Engineering Evaluation/Cost Analysis (EE/CA) emphasizing
presumptive remedies (i.e. cap, leachate collection, methane
monitoring, and bank stabilization) has been initiated by an
industrial group of potentially responsible parties (PRP's).
This action has been initiated by the U.S. EPA Superfund
program.
KING ROAD LANDFILL
The King Road Landfill is a 104 acres site operated; by Lucas
County from 1954 to the 1960s, Park Forest Development from
the 1960s to 1969, and Lucas County again from 1969 to 1976
when the landfilling ceased operation. The site is now
completely enclosed by chain link fencing and signs are posted
warning that "Hazardous Conditions May Exist." The landfill
is located in a light industrial/residential area. The
landfill was determined to have been poorly located and has
contributed to local ground water quality degradation.
Concern over the landfill currently centers around the fact
that unknown materials may have been deposited in the landfill
during its operating life. The landfill reportedly was used
for disposal of lead, cadmium, chromium, and arsenic latent
industrial waste. It was estimated that the landfill may be
discharging up to 30,000+ gallons of leachate per day. As an
interim measure The Lucas County Board of Commissioners (Lucas
County) has constructed a collection system on the north end
of the landfill to intercept leachate migrating into Ten Mile
Creek (i.e. Ottawa River).
PERSTORP POLYOLS INC. (aka DUPONT)
The facility, formerly the Dupont facility is located on 218
ares adjacent to the Ottawa River. A wastewater lagoon,
approximately 180' by 75' was located at the eastern portion
of the property near the river. Soil samples collected at the
site indicated a variety of volatile organic compounds, semi-
volatile compounds, and formaldehyde (a compound produced at
the facility and present at concentrations which may pose a
health risk) . It is thought that a portions of the Dura
Avenue Landfill extends into the boundaries of the Perstorp
Facility.
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NORTH COVE, SOUTH COVE AND WILLY'S PARK LANDFILLS
The North Cove Landfill contains two parcels of land, the
eastern portion located beneath Interstate 75, contained the
former dump area and alleged drum disposal area and the
western part of the site, known as Willys Test Track, located
at the eastern end of Hillcrest Avenue. The North Cove
Landfill was operated by AMC, Willy's Overland and Kaiser Jeep
as a landfill from 1941 to 1970. South Cove Landfill is
located northeast of Beatty Park and Jermain Park. The
property is bounded on the North by the Ottawa River, on the
south by South Cove Boulevard, on the West by Beatty Park, and
on the east by the railroad tracks. This parcel is presently
owned by the City of Toledo. Willy's Park Landfill is located
on North Cove Boulevard. This site, as well as the two
previously mentioned, are believed to have received paint
related waste. All three sites are inactive. The Ohio EPA
has initiated enforcement actions to require the PRP's to
conduct a remedial investigation/feasibility study (RI/FS) for
the site.
JOE E. BROWN PARK LANDFILL
The J.E. Brown Park Landfill is located 1/4 mile south of 1-75
and the Ottawa River in a residential area of north Toledo.
Historical information on waste materials placed into this
fill is currently unavailable. Soil samples have indicated
low level volatile and semi-volatile compounds in soil,
however, PCBs were detected in soils and in the sediments of
an adjacent ditch which connects the Ottawa River with a city
storm sewer.
Textileather (a.k.a. Gencorp)
The Facility is located along the Ottawa River and is adjacent
to the Stickney Avenue Landfill. This facility entered into
an Administrative Order on Consent (AOC) on March 18, 1992,
for initiation of an RI/FS and remedial action to remediate
onsite PCB soil contamination. Soil remediation/cleanup is
scheduled to be initiated in September, 1994.
ROYSTER CORPORATION
The facility was a fertilizer manufacturing facility that
dated back to the early 1900's with operations ceasing in the
early 1980's. The site is situated between the Dura Avenue
and Tyler Street Landfills. Organic compounds, primarily
polyaromatic hydrocarbons and pesticides, and several heavy
metals have been attributed to the site.
SHELLER GLOBE
The facility known as City Auto Stamping, located north of the
Ottawa River along Dura Avenue has two property owners, United
Technologies Automotive Systems (UT) and City Auto Stamping
(CAS). The UT portions of the site occupies 3 acres and is
-------
currently inactive. The CAS portion occupies 7 acres which is
mostly buildings and parking lot and is an active metal
stamping and electroplating facility. Various VOC's, PAH,s
and metals have been detected in the analyses of on-site soil
samples how ever off site migrations of the contaminants is
unlikely due to the lack of migration pathways.
XXKEM/INCORPORATED CRAFTS
The XXKem site is located on 13 acres, formerly occupied by
several companies which performed a series of waste solvent
recycling operations utilizing distillation as a reclamation
process. XXKem is bordered on the west by the Ottawa River,
on the north by Stickney Landfill, and on the south by
Conrail. It is possible that portions of XXKem may have been
used for dumping activities during the early days of Stickney
Avenue Landfill operations. Waste disposal activities began
as early as 1959 with sludges and waste oil. An emergency
removal action took place at this site in 1992/1993 by U.S.
EPA. Removal contractors packaged and disposed of 1000+ drums
of mixed solvent waste.
NORTHERN OHIO ASPHALT COMPANY
The facility is located on 24 ares adjacent to Ten Mile Creek
(i.e. Ottawa River). Approximately 13 of the 24 acres have
been disturbed by facility operations. An unlined settling
pond 0.16 to 0.25 in size is present onsite which was used
prior to 1977 for process water. Water was pumped from Ten
Mile Creek, used in the asphalt process, drained to the
settling pond and then returned to the creek. This practice
was discontinued in June of 1977 when a closed loop system was
installed eliminating discharge to Ten Mile Creek. The
chemical constituents of the process water discharge to Ten
Mile Creek is unknown.
HERBERT E. ORR
The facility is located north of the Ottawa River and occupies
approximately 10 acres. The plant consists of one
manufacturing building which operated as an electophoric
painting, metal stamping, and forging facility. Plant
activity began in 1985 but have since ceased, with the site
currently inactive and unoccupied. Prior to Herbert E. Orr
the site was owned and operated by the Devilbiss Company which
manufactured paint and material application equipment.
CLEVELAND METALS
The facility is located approximately 2 miles south of the
Ottawa River and occupies approximately 8 acres. A surface
impoundment approximately 1 acre in size was located on site
for the storage of waste generated from the manufacture of
metal steel abrasives. Overflow of plant wastewater
discharged directly to Fleig Ditch which flows to the Ottawa
River approximately 2 miles away.
8
-------
TOLEDO TIE TREATMENT
The Toledo Tie Treatment site is an approximately 21 acre site
located at the Arco Industrial Park. Williams Ditch runs
adjacent to the property on the west and north perimeters and
is grossly contaminated with creasote type waste products.
The site originally encompassed over 50 acres, most of which
was owned by Federal Creosoting Company from 1923 to 1959 when
the corporation was transferred to the American Creosoting
Company, site operations discontinued in 1962. Additional
investigation work is currently being scheduled by the Ohio
EPA.
OWENS-ILLINOIS TECHNICAL CENTER / OWENS-ILLINOIS HILFINGER
The Owens-Illinois Technical Center site is located at 1700
Westwood Avenue in the City of Toledo on approximately 12.5
acres. The Ottawa River is the nearest surface water body,
located approximately 0.5 miles northwest of the site. The
University of Toledo is the present owner of the site and is
in the process of developing the area.
The Owens-Illinois Technical Center facility operated from
1932 to 1986 as a Research and Development Center (RDC) for
the glass manufacturing process. The Research and Development
Center was equipped with chemistry laboratories and a large
glass furnace for pilot work. In 1981 a CERCLA notification
form was filed for the South Technical Center, indicating that
waste refractory bricks contaminated with chromium and lead
were buried in a basement of a building near the southeast
corner of the facility. The building has since been
demolished, the basement filled in, and the area paved over.
The Owens-Illinois Hilfinger facility is adjacent to the
Owens-Illinois Technical Center and slightly east of Toledo
University. Owens-Illinois Helfinger is located approximately
1000 yards south of the Ottawa River. The site occupies
approximately 10 acres and is inactive. The terrain between
the site and the Ottawa River is flat, allowing surface water
run-off to the river. The Hilfinger plant was engaged in
foundry operations, electroplating and metal finishing until
the facility was closed in 1970. Electroplating sludge and
other foundry waste was disposed of at the Hilfinger facility.
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December 21, 1994
Maumee Area of Concern Fish Tissue Analytical Results
Attached are Ohio EPA fish tissue
analytical results collected as part of the Maumee River
Remedial Action Plan (RAP) sampling activities during 1993 and 1994 in the Maumee "Area
of Concern" (AOC). Fish tissue samples were collected for chemical analyses at 21 sites in
the Toledo area including: the lowsr Maumee River, the Ottawa River, Swan Creek and
several other tributaries, as part off a two-year, $2.5 million study.
The purpose of the federally-funded study,is to provide the data and information necessary to
develop sound, science-based priorities for cleanup. The Maumee Rivei RAP is designed to
identify sources of pollutants and will likely require total cleanup costs in the hundreds of
millions of dollars. Ohio EPA's goal is to determine which sources are causing the most
damage or impact and to focus remedial efforts where they will be most effective.
Twenty (20) of 28 fish tissue san:
of various; species. The Ohio
(1) if there are contaminant
possible wildlife effects; or (2)
environment which are biologicall;
leachates, nonpoint source runoff,
not used to evaluate health concerns
Ohio sport fish tissue advisory
es analyzed (71.4% percent) were whole body composites
uses whole body fish tissue analytical data to determine:
conceiiitrations in fish which would indicate a concern about
indications of sources of pollutants released to tibe
available at concentrations of concern from sources like
or sediment sinks. Whole body .fish tissue composites are
resulting from sport fish consumption. The current
procedure uses the edible portion of the fish. .
Final conclusions about what the attached fish tissue data mean cannot be made until all
project data gersenxtee as part of tltis study arc evaluated, interpreted and assessed. Very high
total PCB fisn tissue concentrations were found in whole body fish samples collected in thc:|
lower Ottawa River. This area is impacted by PCBs from nonpoint sources, including " |
landfills, sediment sinks, contaminated soil erosion, and possibly leachates. Ohio EPA has •
been working with the City of Toledo to remediate one site, the Dura Ave. landfill, over the
past several years. It is likely that additional sites along the contaminated reach, of the i
Ottawa River will be included in f iiture actions. A fish advisory recommending that fish
from the Ottawa River not be con! aimed between 1-475 north of Wildwood Preserve (River
Mile (RM) 16.7) and Maumee Ba<
(ODH) in April, 1991.
(RM 0.0) was issued by die Ohio Department of Health
High concentrations of Total PCB;i are also present (range 1,200 to 2,700 ppb) in whole
body samples in Swan Creek. The Ohio EPA will look closely at these data, in association
with sediment and other data, to determine what follow-up action(s) will be necessary.
-------
High concentrations of Total PCS;
in the lower Maumee River are
for various species collected ia the
"ultimate" sediment PCB sink for
of the Maumee River. Fish tissue
result of all PCB sources to tins
the release of PCBs to this ecosyst
to the biota can be decreased as
tentatively scheduled for release to
Maumee Bay.
analyses. The analytical results ar
(range 1,000 to 4,700 ppb) in whole body carp collected
siijnilar to historical Ohio EPA whole body fish tissue dafa
lower Maumee River from KM 20.7 to KM 0.0. The
the Maumee River drainage basin is located in this portion
concentrations in fish in the lower Maumee River are the
rijver basin. The Ohio EPA will determine how to decrease
em and to determine if the biological availability of PCBs
of this study.
The ODH is in the process of reviewing all Ohio fillet data and will determine what advisory
recommendations, if any, are appropriate for the lower Maumee River. This information is
the public in the spring of 1995. Currently, there is a
human health advisory for Lake Bjrie carp and catfish consumption and .a consumption
advisory for catfish (do not eat) ard carp (eat no more than six meals per year) caught in j
Additional fish fillet samples were collected in Maumee Bay during 1994 for
not available at this time. "When the data become
available, the Maumee Bay fish consumption advisory will be reviewed and reevaluated to
determine if additional consumption advisory information is necessary.
i
CONTACT: Tom Balduf, Ohio EJ>A Northwest District Office, (419) 352-8461
-------
DATA SUMMARY FOR SELECT PARAMETERS IN MAOC FISH TISSUE
Location. RM
^imile Ck, 6.0
Tenmile Ck, 6.0
NBr.Tenmile Ck,
Ottawa R, 11.1
Ottawa R, 6.4
Ottawa R, 4.8
Swan Ck, 21.6
Swan Ck, 4.3
Swan Ck, 2.6
Swan Ck, 0.5
Blue Ck, 0.7
Silver Ck, 2.2
Shantee Ck, 0.7
|>uck Ck, 1.4
Grassy Ck, 0.4
Dry Ck, 4.8
Crane Ck, 13.1
Cedar Ck, 15.5
Cedar Ck, 6.5
Maumee R, 13.2
Maumee R, 13.2
Maumee R, 13.2
Maumee R, 4.9
Maumee R, 4.9
Maumee R, 4.9
4
Maumee R, 1.0
Maumee R, 1.0
Maumee R, 1.0
Species
Northern Pike
Carp
0.1 Creek Chub
Carp X Goldfish
Carp
Carp X Goldfish
Carp
Carp
Carp
Largemouth Bass
Rock Bass
Carp
Carp
Carp
White Sucker
Carp
r-_ -LChub
Carp
Creek Chub
Black Bullhead
Carp
Largemouth Bass
Carp
Channel Catfish
Largemouth Bass
Rock Bass
Channel Catfish
Carp
Tyj>e
SOFC
WBC
WBC
WBC
WBC
WBC
WBC
WBC
WBC
SOFC
WBC
WBC
WBC
WBC
WBC
WBC
- T- ^
WBC
WBC
SFFC
WBC
SOFC
WBC
SFFC
SOFC
SOFC
SFFC
WBC
T-PCB
(ppb)
ND (9.90)
563.01
91.19
575.21
29,397.79
59,378.43
1,295.89
1,948.67
2,715.82
225.50
43.19
987.23
489.92
259.28
66.84
154.73
69.4^
138.19
92.82
137.43
1,159.98
79.80
4,785.72
643.85
85.91
69.44
2,491.23
1,056.68
T-DDT
(ppb)
46.22
471.14
70.44
531.97
1,131.35
339.44
354.27
224.02
324.84
27.64
109.49
371.68
186.04
260.32
43.15
39.25
20.:-.
44.71
34.82
64.97
289.54
18.91
182.89
205.52
11.20
11.23
329.71
161.46
Dieldrin
(ppb)
8.74
112.16
187.62
45.29
97.00
151.24
208.44
56.40
79.39
6.59
33.30
11.77
4.89
3.99
4.49
8.69
ND (2.00)
12.08
ND (2.00)
10.86
36.59
5.99
44.89
72.31
3.52
3.99
54.40
18.69
T-Cd
(ppm)
<0.00609
0.0856
0.0321
0.151
0.0187
0.0444
0.111
0.107
0.159
<0.00640
<0.00574
0.0628
0.0304
<0.00642
0.0143
0.0179
O.C'-15
0.0131
0.0230
<0.00606
0.121
<0.00614
0.0453
0.0281
0.00761
0.00644
0.00724
0.0549
T-Pb
(ppm)
0.0122
0.184
0.0642
1.77
0.538
0.542
0.475
0.767
0.766
0.565
<0.0574
1.23
0.279
0.217
0.176
2.38
0.24 i
0.141
1.06
0.336
0.596
<0.00614
0.339
0.277
0.697
0.0676
<0.0579
8.32
T-Hg
(ppm)
0.228
0.0454
0.0624
0.0711
0.0227
<0.0194
0.0651
0.0357
0.0257
0.185
0.0426
0.0276
0.0140
O.0185
0.0952
0.0291
0.0394
0.0211
0.0437
0.0659
0.0655
0.132
0.421
0.0321
0.0687
0.0752
0.0701
0.0210
-------
DATA SUMMARY FOR SELECT PARAMETERS IN MAOC FISH TISSUE
f-PCB sum of all isomers detected
T-DDT sum of 4,4'-DDD, 4,4'-DDE, and 4,4'-DDT
not detected (detection limit presented in parenthesis)
iC whole body composite
SOFC skin on filet composite
SFFC skin off filet composite
WB whole body
SOF skin on filet
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