United States
Environmental Protection
Agency
&EPA
Environmental Monitoring
Systems Laboratory
P.O. Box 93478
Las Vegas NV 89193-3478
EPA/600/4-91-028
September 1991
Preissuecopy
Research and Development
Superfund Innovative
Technology Evaluation
Program Demonstration
Plan for Westinghouse
Bio-Analytic Systems
Pentachlorophenol
Immunoassays
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EPA/600/4-91/220
September 1991
SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM
DEMONSTRATION PLAN FOR
WESTINGHOUSE BIO-ANALYTIC SYSTEMS
PENTACHLOROPHENOL IMMUNOASSAYS
by
M. E. Silverstein, R. J. White, and R. W. Gerlach
Lockheed Engineering & Sciences Company
Las Vegas, Nevada 89119
and
J. M. Van Emon
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, Nevada 89193-3478
EPA Contract Numbers 68-03-3249 and 68-CO-0049
Work Assignment Manager
E. N. Koglin
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193-3478
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89193-3478
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NOTICE
The information in this document has been funded wholly or in part by the U.S. Environmental
Protection Agency. Work was initiated under Contract No. 68-03-3249 and completed under
Contract No. 68-CO-0049 to Lockheed Engineering & Sciences Company. It has been subjected to
the Agency's peer and adminstrative review, and it has been approved for publication as on EPA
document. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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ABSTRACT
This plan provides a detailed design and description of the demonstration and evaluation program
for the Westinghouse Bio-Analytic Systems immunoassay technologies specific for the analysis
of pentachlorophenol. The immunoassays measure parts per billion concentrations of
pentachlorophenol in water. This demonstration is being conducted under the Superfund
Innovative Technology Evaluation (SITE) Program.
The main focus of this demonstration is to evaluate on site a semiquantitative immunoassay field
analysis kit for its utility as a rapid field screening tool. The results obtained from the field kit
analyses will be compared to those obtained from a quantitative high-sample-capacity plate
immunoassay also deveJoped by Westinghouse Bio-Analytic Systems. In addition, both
immunoassay techniques will be compared to the standard gas chromatography/mass
spectrometry procedure for pentachlorophenol determination. The demonstration will be
performed at the MacGillis & Gibbs Superfund Site in New Brighton, Minnesota, a National
Priorities List site known to have ground water contaminated with pentachlorophenol. The
immunoassay demonstration will be performed in tandem with a SITE demonstration of a
bioremediation technology (a bioreactor developed by BioTrol, Inc.), that is designed to
biodegrade pentachlorophenol in water.
The demonstration plan provides the sampling plan and design specific to the immunoassay
technologies demonstration, including testing duration, test site description, logistical and
equipment considerations, communications between analysis and management locations, sample
collection and handling protocols, sample identification and tracking systems, and chain-of-
custody and sample shipping procedures. The quality assurance plan for this demonstration is
provided in Appendix A. The demonstration will enable data users and reviewers to assess the
performance of the technology in terms of its usefulness and limitations for the Superfund
Program.
This plan is submitted in partial fulfillment of contract number 68-03-3249 and number 68-CO-0049
by Lockheed Engineering & Sciences Company under the sponsorship of the U.S. Environmental
Protection Agency. The development of this plan began in May 1989. The plan was implemented
from July through September of 1989 for the field and laboratory operations.
in
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CONTENTS
Notice ii
Abstract iii
Figures vii
Tables viii
Abbreviations and Acronyms ix
Acknowledgements x
Executive Summary xi
1. Introduction 1
1.1 Overview of the SITE Program 1
1.2 Immunoassay Program Description 2
1.3 Overview of the SITE Demonstration for the WBAS
Pentachlorophenol Immunoassays -3
1.4 Purpose of this Demonstration 4
1.5 Parties Involved and Responsibilities 5
2. Description of Immunoassay Technology 9
2.1 Introduction 9
2.2 Background 9
2.3 PCP Antibodies and Immunoassays 10
2.3.1 Field Analysis Kit Immunoassay 10
2.3.2 Plate Immunoassay 15
2.4 EPA Evaluation of the WBAS Monoclonal-Based (Plate)
Immunoassay for Pentachlorophenol 15
3. Testing Program Schedule and Duration 19
4. Sampling and Analysis Designs 21
4.1 Test Site Location and Background 21
4.2 Logistics 21
4.3 Equipment 22
4.4 Communications 22
4.5 Photographs 22
4.6 Sample Handling 22
4.7 Sample Collection Procedures 28
4.8 Chain of Custody 31
4.9 Sample Packing and Shipping 31
4.10 Shipment of Performance Standard Ampules 31
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CONTENTS Continued
4.11 Sample Identification 31
4.12 Sample Coding 33
4.13 Field Data Forms 33
4.14 Analysis Design 37
4.15 Predemonstration Plan Design and Strategy 38
4.16 Predemonstration On-Site Analysis 41
5. Data Analysis and Management 43
5.1 Data Handling 43
5.2 Overview of Data Base Design and Data Base Management 43
5.3 Overview of Data Analysis 43
6. Health and Safety 47
References 49
Appendices
A. Quality Assurance Project Plan for the SITE Demonstration of the
Westinghouse Bio-Analytic Systems Immunoassays for
Pentachlorophenol 51
B. EPA Fact Sheet: SITE Demonstrations of Two Technologies at the
MacGillis & Gibbs Site; Map of the MacGillis & Gibbs Site
Location 83
C. Standard Analytical Procedures and Methods References
for the BioTrol SITE Demonstration 91
D. Sample Splitting and Shipping Instructions for the Immunoassay
SITE Demonstration 95
E. List of Addresses for Shipping Bioreactor and QA/QC Samples
for the Immunoassay SITE Demonstration 97
F. Instructions for Completing the Field Data Form for the Field Kit
Analyses 101
G. Descriptions and Schematics for Daily Sample Analysis 107
H. Instructions for Handling and Analyzing QA and QC Samples 119
I. Material Safety Data Sheets (MSDS) for Pentachlorophenol and
Methanol 125
J. Calculation of Matrix Spike Percent Recovery 145
Glossary of Immunoassay Terms 147
VI
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FIGURES
Number Page
1.1 Organizational structure for the SITE Program demonstration
of the WBAS pentachlorophenol immunoassays 7
2.1 WBAS immunoassay field analysis kit for pentachlorophenol 12
2.2 Portable microwell reader spectrophotometer 13
2.3 WBAS immunoassay field analysis kit procedural steps 14
2.4 WBAS plate immunoassay procedural steps 16
4.1 BioTrol, Inc., Biological Aqueous Treatment System and the sample
collection points for the WBAS immunoassay demonstration 27
4.2 Daily sample flow (demonstration phase) 29
4.3 Sample bottle label 30
4.4 Chain-of-custody form 32
4.5 Field data form 35
4.6 Comparison scheme of pentachlorophenol analysis methods employed
in the WBAS immunoassay field kit demonstration 40
5.1 Data flow (demonstration phase) 44
VII
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TABLES
Number Page
2.1 Specificity of Anti-pentachlorophenol Antibodies
Determined by Cross-reactivity Testing 11
4.1 Resources and Supplies Necessary to Conduct the
WBAS Immunoassay Field Kit Analyses for the SITE Demonstration 23
4.2 Equipment Necessary to Conduct the WBAS Plate
Immunoassay Analyses for the SITE Demonstration 25
4.3 WBAS Immunoassay SITE Demonstration-Sample Codes
and Definitions 34
4.4 Known or Anticipated Performances for PCP Analysis 39
viii
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ABBREVIATIONS AND ACRONYMS
AFMMP Advanced Field Monitoring Methods Program
ANOVA analysis of variance
cc cubic centimeter
CLP Contract Laboratory Program
CFR Code of Federal Regulations
DCP-THY dichlorophenol conjugated with thyroglobulin
DOT U.S. Department of Transportation
EPA U.S. Environmental Protection Agency
EMSL-LV Environmental Monitoring Systems Laboratory - Las Vegas, Nevada
GC gas chromatography
GC/MS gas chromatography/mass spectrometry
id identification
L liter
LESC Lockheed Engineering & Sciences Company
M molar
mL milliliter
mM milliMolar
mm millimeter
MSDS Materials Safety Data Sheet
N Normal
nm nanometer
NPL National Priorities List
PAH polyaromatic hydrocarbons
POP pentachlorophenol
POTW publicly owned treatment works
ppb parts per billion
ppm parts per million
QA quality assurance
QAPjP Quality Assurance Project Plan
QC quality control
RREL Risk Reduction Engineering Laboratory
RSD relative standard deviation
SAIC Science Applications International Corporation
SARA Superfund Amendments and Reauthorization Act of 1986
SAS Statistical Analysis System
SITE Superfund Innovative Technology Evaluation
SOP standard operating procedure
WBAS Westinghouse Bio-Analytic Systems
lA. microliter
IX
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ACKNOWLEDGEMENTS
A number of employees of Lockheed Engineering & Sciences Company assisted in the design and
preparation of this demonstration plan. The authors greatly appreciate the contributions of Anne
C. Neate, Pamela S. O'Bremski, William D. Munslow, Thomas C. Chiang, Lynn A. Gurzinski, Brian
C. Cordova, Donna W. Sutton, and Vicki A. Ecker.
Individuals with other organizations also provided assistance and advice: William Hahn, Herbert
Skovronek, Gary Vaccaro, Rita Schmon-Stasik, Steve Stavrou, Diana Baumwoll, and Nick Rottunda
(Science Applications International Corporation, Paramus, New Jersey; McLean, Virginia; and San
Diego, California); Thomas Chresand (BioTrol, Inc., Chaska, Minnesota); and Stephen Soileau
(Westinghouse Bio-Analytic Systems, Rockville, Maryland).
Finally, guidance and support in developing and preparing this plan within the framework of the
Superfund Innovative Technology Evaluation Program was provided by the following U.S.
Environmental Protection Agency staff: Mary K. Stinson, Project Manager (Risk Reduction
Engineering Laboratory, Cincinnati, Ohio); David G. Easterly, Quality Assurance Officer, George T.
Flatman, Statistician, and Eric N. Koglin, Matrix Manager (Environmental Monitoring Systems
Laboratory, Las Vegas, Nevada).
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EXECUTIVE SUMMARY
This demonstration plan has been prepared under the Superfund Innovative Technology
Evaluation (SITE) Program and provides a detailed design and description of the SITE
demonstration of the Westinghouse Bio-Analytic Systems (WBAS) immunoassay technologies
specific for the analysis of pentachlorophenol. The immunoassays measure parts per billion
concentrations of pentachlorophenol in environmental water samples.
The primary objective of this demonstration is to evaluate on site a semiquantitative
immunoassay field kit for its utility as a rapid field screening tool. This demonstration plan
provides the design and protocols required to obtain the information needed for this evaluation.
The field kit will be compared to a quantitative high-sample-capacity plate immunoassay
developed by WBAS that was previously evaluated at the U.S. Environmental Protection Agency
(EPA), Environmental Monitoring Systems Laboratory, Las Vegas. Nevada (EMSL-LV). Both of
these immunoassay techniques will be compared to standard EPA methods used for the
analyses of pentachlorophenol in water by gas chromatography/mass spectrometry. The
demonstration will be conducted at the MacGillis & Gibbs Superfund Site in New Brighton,
Minnesota. This is a National Priorities List site known to have ground water contaminated with
pentachlorophenol. The immunoassay demonstration will be performed in tandem with a SITE
demonstration of a bioremediation technology (a bioreactor developed by BioTrol, Inc.) that is
designed to biodegrade pentachlorophenol in water.
Immunoassays are based on receptor molecules called antibodies which are developed in
response to a particular target analyte. Quantification of the extent of contamination in an
environmental sample is based on the ability of a specific antibody to bind to its target analyte.
Immunoassays are normally based on competition for antibody binding between a known amount
of analyte labeled with an indicator, such as an enzyme, and an unknown amount of analyte from
a sample. The indicator produces a colored product that is used for quantitation. Color intensity
is determined by the amount of analyte present. Immunoassays can be configured for
quantitative, semiquantitative, or qualitative analyses.
The immunoassay field kit methodology requires about 30 minutes to perform and has a
detection limit of about 3 parts per billion (ppb). It has a linear dynamic range from about
3 to 40 ppb and uses no more than 2 milliliters (mL) of sample to obtain analytical results. The
methodology employs a portable spectrophotometer for standard curve generation and
quantification of pentachlorophenol concentrations and it requires a clean, sheltered work area
(e.g., out of the wind and direct sunlight).
XI
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The quantitative (i.e., plate) immunoassay is based on a 96-well microtiter plate format. The
minimum detectable level of pentachlorophenol is approximately 30 ppb; however, when the
required sample dilutions are considered, the minimum detection level is 90 ppb, based on an
18-mL sample. The method has a linear dynamic range of 30 to 400 ppb. Several 96-well
microtiter plates can be processed in tandem. Thus, hundreds of samples can be analyzed
during the 2.5-hour analysis time required for this method. The plate immunoassay also can be
performed under field conditions similar to those required for the field kit. However, for this
demonstration, only the field kit will be evaluated under field conditions because this methodology
is particularly suited for use by field personnel who may have limited analytical chemistry
experience.
This demonstration plan provides the specific sampling plan and design for the immunoassay
technologies demonstration, including testing duration, test site descriptions, logistical and
equipment considerations, communications between analysis and management locations, sample
collection and handling protocols, sample identification and tracking systems, and chain-of-
custody and sample shipping procedures. A detailed quality assurance plan for this
demonstration is provided in Appendix A.
The sample collection, sample analysis, and data analysis strategies presented in this plan are
designed to address the critical issues related to assessing the general and practical
applications of immunoassay technology in the measurement and monitoring aspects of the
Superfund Program. The results obtained from samples analyzed on site during the
demonstration of the field analysis kit immunoassay technique will be compared to results
obtained by analyzing splits of the same samples. For this comparison, standard EPA sample
preparation and analysis methods for determining pentachlorophenol in water by gas
chromatography/mass spectrometry will be used. Split field samples will also be analyzed off
site by both immunoassay techniques (field kit and plate) at EMSL-LV and WBAS laboratories.
The demonstration plan includes detailed standard operating procedures for sample analysis and
data management. The quality assurance plan is designed to ensure that important data quality
and methodological performance criteria are examined. A series of performance evaluation
(audit) samples, as well as blank and replicate samples, are incorporated in the analytical
scheme to assess the within-method performance parameters of the immunoassay and to
perform between-method comparisons. The data management system is tailored to the sample
analysis and quality assurance programs to provide a timely means of performing the data
analysis. Data management will also provide a mechanism for documenting and tracking the
data generated from the different analysis sites and by the various methods.
As pentachlorophenol is considered a toxic substance and is a suspected carcinogen, the plan
addresses health and safety aspects associated with handling and disposing of materials
contaminated with pentachlorophenol.
It is anticipated that a fair performance evaluation of the pentachlorophenol immunoassay
technologies can be determined by following the specifications in this demonstration plan. Thus,
upon completion of the demonstration, data users and reviewers will be able to assess the
performance of the particular pentachlorophenol immunoassays in terms of their utility and
limitations in the Superfund Program.
XII
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1. INTRODUCTION
This demonstration plan provides the specific sampling and analytical design for the evalution of
a semi-quantitiative immunoassay technique used for the rapid detection of pentachlorophenol
(PCP) in environmental water samples. The demonstration of this field screening technology,
developed by Westinghouse Bio-Analytic Systems (WBAS), will be conducted under the Superfund
Innovative Technology Evaluation (SITE) Program.
1.1 OVERVIEW OF THE SITE PROGRAM
The Superfund Amendments and Reauthorization Act of 1986 (SARA) charged the U.S.
Environmental Protection Agency (EPA) with effecting more timely and cost-effective remedies at
the Nation's Superfund sites. The costs incurred for site characterization are a direct result of
sampling, analysis, and the associated quality assurance activities. If field screening methods
can yield immediate or short-turnaround environmental data, the use of these methods will result
in major cost savings. The cost-effectiveness of clean-up efforts will be improved dramatically.
More cost-effective and timely remediation will decrease the human and ecological risks around
Superfund sites and enhance the ability to manage such risks.
The EPA SITE Program was established to satisfy the mandate in Section 311(b) of SARA, which
requires the EPA to establish 'a program of research, evaluation, testing, development and
demonstration of alternative or innovative treatment technologies .. . which may be utilized in
response actions to achieve more permanent protection of human health and welfare and the
environment." The two categories of technologies included in the SITE Program are (1) treatment
technologies that may serve as alternatives to land disposal of hazardous wastes and
(2) monitoring and measurement technologies for contaminants occurring at hazardous waste
sites. The Monitoring and Measurement Technologies Program is that component of SITE
established to address the second category.
The SITE Program provides the Agency with a good mechanism to identify and demonstrate
innovative or alternative site characterization technologies, existing within and outside the Federal
government, which may provide cost-effective, better, and faster means to detect and monitor
contaminants at uncontrolled hazardous waste sites. This Program also provides developers
with the means to rigorously evaluate the performance of their technologies and have the results
and recommendations widely distributed, thereby enhancing the market for those technologies.
Products from the various research, development, and demonstration activities conducted under
this Program will enhance the Agency's ability to perform statistically valid sampling and field
analytical programs that yield effective site characterization coupled with immediate or quick-
turnaround environmental data acquisition.
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The Monitoring and Measurement Technologies Program portion of SITE is also the core of the
Advanced Field Monitoring Methods Program which was implemented in fiscal year 1988 to
provide a mechanism to identify, test, evaluate, and accelerate the use of innovative and
alternative field monitoring and measurement technologies, primarily in support of the Regional
Superfund staffs. The Advanced Field Monitoring Methods Program enhances the SITE Program
by adding an in-house methods research element and additional technology transfer through the
preparation, testing, and promulgation of standard methods and the development of protocols for
the successful use of technologies by field personnel.
The process of selecting and accepting measurement and monitoring technologies into the SITE
demonstration program begins by compiling a list of candidate technologies identified through a
variety of sources. The technologies are then screened and assessed for anticipated
performance and application to Superfund site characterizations and for their ability to meet one
or more of the following criteria:
• can be used on-site (outdoors or in a mobile laboratory);
• is widely applicable to a variety of sites;
offers a high potential for solving critical problems for which current approaches are
less than satisfactory;
• costs significantly less than current methods;
• has significantly better performance than current methods (e.g., better data quality,
faster sample preparation or analysis time); and
• uses techniques that are easier and safer to perform than current methods.
Once the technology has been accepted into the SITE Program, the developer and the EPA will
enter into a series of negotiations and planning discussions intended to culminate in a rigorous
and comprehensive field demonstration to evaluate the utility of the technology. Activities
required to prepare and execute of a demonstration are described in this plan.
After on-site demonstration activities are completed, a detailed and thorough evaluation of the
demonstration results and comparisons will be fully documented in an evaluation report. This
report will be prepared by the EPA and reviewed by Agency and non-Agency experts for
independent assessment and peer review of the results and conclusions presented.
1.2 IMMUNOASSAY PROGRAM DESCRIPTION
Immunoassays are increasingly being recognized as cost-effective alternatives to
chromatographic and spectroscopic procedures for use in rapid, large-scale environmental
monitoring studies. Immunoassay techniques have been applied to the analysis of many
hazardous substances and possess several attributes that make them suitable for field screening
methods. In general, immunoassays have proven to be sensitive, selective, precise, rapid, cost-
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effective, and applicable to a wide range of contaminants. Several different immunoassay
formats for environmental chemistry are possible.
Identifications of immunoassays of Agency interest are sought from private industry and
academia through solicitations in scientific journals and the Commerce Business Daily. These
assays are evaluated and, if appropriate, adapted for Agency use. Immunoassays have been
applied to many compounds and compound classes which are of interest to the EPA.
Although specific immunoassays have been developed for hazardous compounds, many of these
systems have not been configured to analyze real-world environmental samples. Agency restraint
in utilizing immunoassay technology is partially due to the lack of fully developed methods for
environmental matrices. Demonstration that an immunoassay is properly developed will lead to
implementation of the technology into environmental monitoring and exposure assessment
studies. Thus, the Agency would be supplied with rapid, low-cost monitoring capability for
compounds difficult to analyze by conventional methods.
Specific immunoassay methods that have been developed for a particular environmental
application are submitted to EMSL-LV for characterization and evaluation. Such testing ensures
that the intended environmental application of the method is appropriate. According to Agency
guidelines for methods evaluations, this process requires the determination of such performance
parameters as precision, within- and among-laboratory bias, between-method bias, method
detection limits, matrix effects, interferences, limit of reliable measurement, and ruggedness of
the method. Real-world samples are used when available, particularly when the immunoassay
program can coordinate analytical support with other concurrent Agency studies. Analytical
results generated from the immunoassay technique can then be compared to those results
obtained from existing (e.g., Contract Laboratory Program [CLP]) analytical methods.
If a particular immunoassay is of the appropriate design and quality, based upon a preliminary
laboratory evaluation of the method, a SITE demonstration may be conducted. Specific
descriptions of the immunoassay techniques used in this SITE demonstration can be found in
Section 2 and in the Quality Assurance Project Plan (QAPjP) (Appendix A).
1.3 OVERVIEW OF THE SITE DEMONSTRATION FOR THE WBAS PENTACHLOROPHENOL
IMMUNOASSAYS
The technologies for which this demonstration plan has been prepared are a semiquantitative
immunoassay field analysis kit and a quantitative 96-well microtiter plate immunoassay for the
rapid screening of PCP in aqueous samples. The technologies were developed by Westinghouse
Bio-Analytic Systems (WBAS) of Rockville, Maryland. The methods are designed to provide a
quick and inexpensive means of detecting PCP in water under field or mobile laboratory
conditions. The major focus of the SITE study is to demonstrate and evaluate the field analysis
kit. The quantitative (plate) assay has already undergone an extensive laboratory evaluation at
EMSL-LV (Section 2.4). The quantitative assay can be easily performed under conditions similar
to those required for the field analysis kit (i.e., field laboratory).
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An opportunity is available to coordinate the WBAS immunoassay demonstration with the field
and analytical operations of another planned SITE demonstration that will evaluate a technology
for use in Superfund site remediations. Specifically, BioTrol, Inc., of Chaska, Minnesota, has
developed a biological reactor designed to degrade POP (as well as polyaromatic hydrocarbons)
into carbon dioxide, water, and inorganic chloride.
The performance of the BioTrol bioreactor will be demonstrated at the MacGillis & Gibbs site
located in New Brighton, Minnesota. The MacGillis & Gibbs site, which is on the EPA National
Priorities List (NPL), contains ground water contaminated with PCP as the result of a wood
preservative treatment operation. The PCP-contaminated ground water will be pumped into the
bioreactor and discharged as treated effluent (Appendix B). Preliminary data, as described in
Appendix B, show that the ground water from the well supplying the bioreactor influent contains
about 50 ppm PCP and the treated effluent contains about 1 ppm. Composite samples will be
collected once every 24 hours for a period of 6 weeks at various critical points in the bioreactor's
system, including the conditioned (i.e., pH-adjusted and nutrient-added) ground-water influent and
the fully treated effluent. These samples will be processed and analyzed for a variety of organic,
inorganic, and physical characteristics, including the use of gas chromatography/mass
spectrometry (GC/MS) analysis for PCP (EPA Method 8270 after Method 3510 extraction). The
complete description of the BioTrol bioreactor technology and its demonstration under the SITE
Program can be found in the bioreactor demonstration plan (SAIC, 1989).
This bioreactor demonstration presents an excellent opportunity to simultaneously test the
effectiveness of the remediation technology and the measurement and monitoring technology,
while minimizing logistical and analytical costs. The QAPjP for the immunoassay demonstration
(Appendix A) has been designed to ensure the collection of enough data to make the necessary
methodological and statistical comparisons between the WBAS immunoassays and an
established GC/MS method and to assess the quality of the data acquired using the
immunoassays, especially the field kit.
Although the primary goal of this demonstration is to compare the field immunoassay PCP data
to those obtained by GC/MS, both immunoassays will be compared to each other and the plate
immunoassay results will also be compared to GC/MS results obtained from analysis of split
samples. In previous studies, data from the plate immunoassay have been compared to data
from GC analyses. Section 2.3.2 describes the plate immunoassay technology and Section 2.4
presents a discussion of the results of those previous studies. This demonstration is expected to
complement and supplement the previous studies. The comparisons of these three methods
(field kit, plate, GC/MS) will maximize performance information on measurement and monitoring
technologies with a minimum of time, cost, and resources.
NOTE: Issues relevant to the performance and the evaluation of the bioreactor demonstration
are not within the scope of this document, nor within the scope of the WBAS field kit
demonstration, and will be documented elsewhere.
1.4 PURPOSE OF THIS DEMONSTRATION
The main purpose of this demonstration is to evaluate a rapid, on-site immunoassay technique
for the detection of PCP in environmental water samples. Preliminary performance data from
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testing in a controlled laboratory environment have been generated by using specified
concentrations of POP spiked into laboratory-grade water. In addition, these tests included
analyses of various natural and bioreactor matrix water samples (more detail is provided in
Section 4). This field demonstration provides the opportunity to assess the ruggedness of the
field kit immunoassay technology under field conditions using environmental samples known to
be contaminated with POP. The same water samples used to test the WBAS field kit will be
analyzed for a suite of other chemical, physical, and biological parameters (see Appendix C for
these analytes and methods). Therefore, these analyses present an opportunity to assess site-
specific interferences. Comparisons will be made among the data generated from the field kit
immunoassay performed on- and off-site, the laboratory immunoassay, and the GC/MS results.
The following elements will be addressed and implemented in the demonstration:
• site and logistical considerations and support (coordinating with another
demonstration to minimize necessary costs, efforts, and resources);
• sampling and analysis plan;
• quality assurance project plan;
• data handling and analysis plan; and
• health and safety plan.
These elements are discussed in greater detail in the following sections.
1.5 PARTIES INVOLVED AND RESPONSIBILITIES
The responsibilities of the EPA Environmental Monitoring Systems Laboratory in Las Vegas,
Nevada (EMSL-LV), with assistance from its prime contractor, Lockheed Engineering & Sciences
Company (LESC), for the demonstration of the WBAS immunoassays include:
/
• designing, overseeing, and implementing the elements of this demonstration plan;
• acquiring the necessary confirmatory data; and
• evaluating and reporting on the performance of the technology.
WBAS, the developer of the immunoassays being demonstrated, is responsible for:
• providing technical assistance to the personnel using the field kit on-site,
• performing immunoassay analyses of split samples, and
• supplying a sufficient number of field kits and laboratory reagents (for the plate
immunoassay) to fully satisfy the requirements set forth in this demonstration plan.
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The main function of the Risk Reduction Environmental Laboratory (RREL), through its prime
contractor, Science Applications International Corporation (SAIC), Paramus, New Jersey, is to
perform the BioTrol bioreactor demonstration. Aspects of the immunoassay demonstration for
which RREL and SAIC are responsible include:
• performing the field immunoassay on the prescribed water samples on site;
• providing the logistical support designated in this plan; and
• analyzing the influent and effluent samples by GC/MS and reporting the results.
For the immunoassay demonstration, BioTrol, Inc., the developer of the bioreactor being
demonstrated as a remediation technology, is responsible for:
• providing predemonstration test samples and
• providing technical assistance.
Figure 1.1 shows the organizational structure and key personnel. Specific task responsibilities for
all of these parties are detailed in the QAPjP (Appendix A).
6
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SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION
DEMONSTRATION PROGRAM
MacGILLIS & GIBBS SUPERFUNO SITE
JOINT DEMONSTRATION
MONITORING AND MEASUREMENTS
TECHNOLOGIES PROGRAM
• Environmental Monitoring Systems
Laboratory - Las Vegas
Manager - Eric Koglin
• Lockheed Engineering & Sciences
Company
Program Lead - Mark Silverstein
WBAS IMMUNOASSAY DEMONSTRATION
• Environmental Monitoring Systems
Laboratory - Las Vegas
Project Manager - Jeanette Van Emon
• Lockheed Engineering & Sciences
Company
Technical Lead - Richard White
Westinghouse Bio-Analytic Systems
Technical Lead - Stephen Soileau
TREATMENT TECHNOLOGIES PROGRAM
Risk Reduction Engineering
Laboratory - Cincinnati
Director - Robert Olexsey
Branch Chief - Steve James
Section Chief - John Martin
BIOTROL BIOREACTOR DEMONSTRATION
Risk Reduction Engineering
Laboratory - Cincinnati
Project Manager - Mary Stinson
Science Applications International
Corporation
Project Manager - William Hahn
BioTrol, Inc.
Technical Lead - Tom Chresand
Figure 1.1. Organizational structure for the SITE Program demonstration of the WBAS
pentachtorophenol immunoassays.
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2. DESCRIPTION OF IMMUNOASSAY TECHNOLOGY
2.1 INTRODUCTION
Immunoassays are analytical techniques based on receptor molecules called antibodies.
Quantification of the extent of contamination in an environmental sample is based on the ability
of a specific antibody to bind to its target analyte. Specific antibodies can be developed to detect
either a single analyte or small groups of related compounds. Laboratory-based immunoassays
can accommodate an extremely high sample capacity, while portable immunoassays enable on-
site analysis. Thus immunoassay techniques are quite versatile and can be applied to many
analytical situations.
2.2 BACKGROUND
Immunoassay development is composed of several distinct steps: hapten design and synthesis;
conjugation of hapten to carrier; immunization of animals; antibody production (either polyclonal
or monoclonal); and choice of immunoassay format.
Most organic environmental contaminants are small molecules that are not large enough to
stimulate the immune system to produce specific antibodies. However, when conjugated to
carriers such as proteins, small molecules may elicit the production of antibodies. In these
instances the small molecule is termed a hapten. Immunoassays are based on the phenomenon
that the antibodies produced in response to the hapten-carrier conjugate will be able to bind to
the free hapten (or target analyte) without the carrier being attached.
Antibodies are secreted by plasma cells in response to the hapten-carrier conjugates. Various
immunization protocols can be used to stimulate these cells for specific antibody production.
Several factors (e.g., the state of the animal's immune system, characteristics of the hapten-
carrier conjugate, species response, and genetic factors) contribute to a successful immunization
regime.
Polyclonal antibodies are obtained from blood serum. Within the polyclonal antiserum are
antibodies of varying selectivity and sensitivity. It is only those antibodies which combine with
the free hapten (or target analyte) that are useful for immunoassay development. Some of the
other antibodies present may pose difficulties in configuring an immunoassay. With the aid of
hybridoma technology, it is possible to select a single parent cell that produces an antibody with
the desired specificity and clone it to produce millions of identical cells. These preparations are
termed monoclonal, as they are a homogeneous reagent of only one antibody type. Monoclonal
antibodies can be produced in large scale to provide a continuous supply of a specific antibody.
-------
Immunoassays can be successfully developed using either polyclonal or monoclonal antibodies.
Polyclonal antibodies are easier to produce and may even be superior to monoclonals in some
immunoassay formats. Immunoassays based on monoclonals tend to produce steeper
quantitation curves and may eliminate problems of multispecificity. Although many assay
configurations are possible, each is based on the binding of a specific antibody to its target
analyte(s). Assays are normally based on competition for antibody binding between a known
amount of analyte and an unknown amount of analyte in a sample. An enzyme or radiolabel is
bound to either the specific antibody or the analyte standards to detect and quantify the amount
of unknown present. A common detection method is to determine the amount of color generated
by the enzyme label and a chromogenic substrate.
Because many immunoassay formats can be used in the field, the technology can be used to
provide on-site monitoring, enabling the collection of data in real time. Immunoassays are
excellent methods for screening large numbers of samples. Such screening procedures would
minimize the number of negative samples analyzed by expensive spectroscopic and
chromatographic methods.
2.3 POP ANTIBODIES AND IMMUNOASSAYS
Westinghouse Bio-Analytic Systems (WBAS) has developed rabbit polyclonal antiserum and rat
monoclonal antibodies that are selective for POP. Both the polyclonal antiserum and monoclonal
antibody display similar selectivities towards POP and related compounds (Table 2.1). However
the polyclonal antiserum is somewhat more selective in its reactivity.
2.3.1 Field Analysis Kit Immunoassav
A polyclonal-based immunoassay field analysis kit (Figure 2.1) for detecting PCP in water has
been developed by WBAS. The detection limit of this assay is approximately 3 ppb with a total
analysis time of about 30 minutes. The linear dynamic range of the method is from 3 to 40 ppb.
The method employs a portable spectrophotometer (Figure 2.2) for standard curve generation.
The field kit provides a semiquantitative analysis of PCP in water samples. Qualitative results
can also be obtained by setting the appropriate threshold values. This polyclonal immunoassay
format (the field kit) will be tested under both field and laboratory conditions for the purpose of
the SITE demonstration.
The field analysis kit employs an 8-well microtiter strip. Each well has a maximum capacity of
200 ml and is precoated with PCP-specific antibody by WBAS (proprietary method). Water
samples are buffered and placed in the microtiter wells. A constant, known amount of enzyme-
labelled PCP is then added to the well with the buffered water sample. Thus, competition
between the sample and labeled PCP occurs for the antibody immobilized in each well on the
strip (Figure 2.3). After a 5-minute incubation, all unbound labeled and unlabeled PCP is removed
by washing. An enzyme substrate and chromogen are added, producing a colored solution. The
reaction between the substrate and chromogen is stopped by charging the pH and the final
colored end product is read spectrophotometrically. Figure 2.3 shows the field kit analysis steps
described above. Quantification of PCP is based on the competition between PCP in the sample
and the known amount of labeled PCP. The greater the amount of PCP in the water sample the
lighter the color produced at the end of the assay. Conversely, if the water sample has only a
10
-------
TABLE 2.1. SPECIFICITY OF ANTI-PENTACHLOROPHENOL ANTIBODIES DETERMINED BY
CROSS-REACTIVITY TESTING
Percent Cross-Reactivity with Pentachlorophenol"
Compound
Pentachlorophenol
2,3,5,6-Tetrachlorophenol
2.4.6-Trichlorophenol
2.3,6-Trichlorophenol
2,6-Dichlorophenol
Tetrachlorohydroquinone
2.3,4-Trichlorophenol
2,3,5- Trichlorophenol
2,4-Dichlorophenol
2,5-Dichlorophenol
3,5-Dichlorophenol
3,4-Dichlorophenol
2,3-Dichlorophenol
4-Chlorophenol
Phenol
Pentachloroaniline
Pentachlorobenzene
2,3-Dinitrotoluene
2.4-Dinitrotoluene
2.4,5-Trichloronitrobenzene
Molar ICjo"
2.2 (± 0.3) x 10*
5.3 ( ± 0.6) x 10 6
1.8 ( ± 0.3) x 10'5
2.5 (± 0.1) x105
1.2 (± 0.1) x104
2.8 (± 0.1) x10'4
4.5 ( ± 0.3) x 10 4
4.3 ( ± 0.3) x 10"*
NIC
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Rat Monoclonal
—
42.0
12.0
8.8
1.8
0.8
0.5
0.5
0
0
0
0
0
0
0
0
0
0
0
0
Rabbit Polyclonal
—
19.0
7.0
7.0
0.4
0.7
11.0
2.5
N/A
0.1
0.1
0.1
0.1
0.2
0.1
0.1
N/A
0.1
0.1
0.1
• Molar concentration of compound that inhibits 50 percent antibody binding in immunoassays.
b [ICjo PCP/ICjo compound] x 100
c NI = Not Inhibitory; 1.0 x 10 3 M
Source: Courtesy of WBAS
-------
to
PhNTACHLOKOPHKNOL
Hl-I.DANALYSIS KIT
Figure 2.1. WBAS immunoassay field analysis kit for pentachlorophenol.
-------
Figure 2.2. Portable microwell reader spectrophotometer.
13
-------
V -4
YYYY YYYY
PCP-spedTc enMbodr Is passively td-
sorbed in the wets ol a polystyrene mt-
ctotiur strip.
£nzyme-Ubeled PCP and PCP from an »n-
ytnmntnul i*mp* compel* tor birring
Sim* on th» tnttxxtf.
A buHarrinst removes IB unbound maten-
•b. BoOi the enzyme-labeled POP tnd PCf
Irom tho sample are no* bound to the
antibody.
O O O O
OOOOOOO
A chr
ogenlc substrate Is added to react
• O * • •
• O • O o»
0*O*
wHf> the bound eniyme.
The reaction between the enzyme and
substrate produces a colored end-product
The aosoroance a measured by a speclro-
pnotometer 10 quantity the unknown amount
ol POP in lha sample.
Y « PCP -Specific Antibody
_ .PCP
v (SUndard or trom Sample)
F • Entyrne- Labeled PCP
i (PCP • PeroxMase)
• » Colored End -Product
^ s Ught Pint
t^J^XJa . Polystyrene Ulcratlter Strip
Figure 2.3 WBAS immunoassay field analysis kit procedural steps.
14
-------
small amount (or no) POP present, a dark color is produced because more of the enzyme-labeled
PCP is able to bind to the immobilized antibody.
2.3.2 Plate Immunoassav
The monoclonal antibody was used to develop a quantitative immunoassay based on a 96-well
microtiter plate format (see Figure 2.4 for schematics of the assay procedures). The
immunoassay can detect PCP in drinking and environmental water samples. Although the
immunoassay involves an overnight incubation step, it requires only 2-1/2 to 3 hours of hands-on
time to complete the analysis. However, many 96-well plates can be processed at one time thus
enabling a high sample capacity. Day one of the analysis is as follows: microtiter plates of
polystyrene are sensitized by passive adsorption with 100 /A. of a solution of the coating antigen
DCP-THY (i.e., dichlorophenol conjugated with thyroglobulin) in a carbonate buffer (pH 9.6). The
microtiter plates are covered with acetate sealers and stored overnight at 4 °C. Day two
analysis activities include: buffering water samples with 70 mM phosphate buffer (pH 7.4)
containing 1.4 M NaCI and adjusted to pH 7.4. The sample preparation is completed by diluting
an aliquot of buffered sample into 25 percent by volume 2-propanol. The 25 percent 2-propanol
extracts are diluted in dilution buffer containing 25 percent 2-propanol for determination of
concentration range and final analysis. The dilutions typically used for range findings are
undiluted, 1:10, and 1:100. Dilutions are made in 12- by 75-mm test tubes using a total volume of
1 mL and performing the liquid measurements with a Pipetteman P-1000 (Rainin Corporation).
Fifty fjL of prepared sample or standard is added into triplicate wells of the sensitized microtiter
plate prepared on day one. Fifty ^L of the anti-PCP antibody is added next, and the plate is
incubated for one hour. The free PCP in the sample binds to the anti-PCP antibodies and inhibits
the binding of the antibody to the solid-phase adsorbed DCP-THY conjugate. After the incubation,
the anti-PCP antibodies not bound to the adsorbed PCP-THY conjugate are removed by rinsing the
plate with phosphate buffer. The amount of antibody bound is indirectly determined by adding an
enzyme-labeled second antibody which binds to the anti-PCP antibody. After an incubation and a
final washing, the substrate of the enzyme is added, and the amount of bound enzyme is
monitored by the development of a colored product. The intensity of the color is directly
proportional to the amount of anti-PCP antibody bound to the solid-phase and inversely
proportional to the concentration of PCP in the sample. Quantification is achieved by comparison
with a standard curve.
The minimum detectable level of PCP in the plate immunoassay is estimated to be 30 ppb. When
the required dilutions are taken into account, the minimum detection level of PCP in a water
sample is 90 ppb. If an optional reverse-phase extraction step is used, the detection limit can be
lowered to 25 ppb. If EPA Method 604 is used to extract the water samples, the minimum
detectable level is below 1 ppb.
2.4 EPA EVALUATION OF THE WBAS MONOCLONAL-BASED (PLATE) IMMUNOASSAY FOR
PENTACHLOROPHENOL
The monoclonal-based immunoassay (referred to in this plan as the plate immunoassay) for PCP
was submitted to the EMSL-LV by WBAS for evaluation. The study (Van Emon and Gerlach, 1990)
consisted of comparing the following five methods of analysis for PCP: (1) immunoassay using
no extraction (i.e., direct immunoassay); (2) immunoassay detection following solid-phase
15
-------
The PCP-protein coating antigen is pas-
sively adsorbed in the ore»s ol a polystyrene
microtiler plate. POP from an unknown
sample and PCP-specilic antibody added.
The POP In the sample and the immobilized
PCP-protein coaling antigen compete lot
binding sites on the antibody.
A butler rinse removes aU unbound materi-
als. Only me aruitxxty bound 10 the immobi-
lized PCP-protein coaling antigen remains.
o oo
O O Q Q Q
An enzyme-libeled conjugate Is added and
binds only to the PCP-specilic antibody.
After a bulter rinse removes al unbound
eruyme-labeled conjugate, a chromogenic
substrate is added.
•~ \* ^ w
• Q • O 0+0
The reaction between the enzyme and
chromogenic substrate produces a colored
end-product.
The aosorbanee fe measured oy I spearo-
photometer to quantify the unknown amount
olPCP in the sample.
\/
PCP-SoeoUc Antibody
PCf ProtetnCoriuome
= Enzym* LatMtod
S^ondAnUbody
(IgO UtMne
Ptxwpiuw**)
T «PCP
(Standard or from Sample)
O = Chromogenic Substrate
• « Colored End - Product
/P = Ughl Path
3 Polystyrene MleroDler PUIe
Figure 2.4. WBAS plate immunoassay procedural steps.
16
-------
extraction; (3) immunoassay detection following EPA Method 604 extraction (EPA, 1979); (4) gas
chromatographic (GC) detection following solid-phase extraction; and (5) GC detection (EPA
Method 604 - Phenols). Samples analyzed consisted of spiked ground water, surface water, and
drinking water. Each water type was collected from different geographic locations. Samples
were split and spiked at two different levels of PCP. In addition, several samples were spiked
with a mixture of PCP and related phenolic compounds. These additional compounds were
chosen based upon the target analytes of Method 604. The inclusion of these compounds was
essential to more fully evaluate the possibility of interference in the immunoassay, particularly as
the immunoassay data were to be compared with those of the Agency's GC method.
Based on the conditions of this evaluation (Van Emon and Gerlach, 1990), no practical difference
was observed between: (1) the plate immunoassay and GC detection of Method 604 extracts;
(2) the plate immunoassay and GC detection of solid-phase extracts; (3) laboratories for PCP
detection by plate immunoassay using the WBAS solid-phase or EPA Method 604 extraction
protocols; and (4) precision of direct immunoassay obtained by two laboratories. As a stand-
alone method the immunoassay has important applications for screening and environmental
monitoring. This monoclonal-based plate immunoassay will also be evaluated in the SITE
demonstration and compared with the polyclonal-based immunoassay field kit.
17
-------
3. TESTING PROGRAM SCHEDULE AND DURATION
The testing program schedule and duration for the WBAS immunoassays have been designed to
conform to the scheduling of the SITE demonstration of the BioTrol bioreactor.
From late May through mid-July 1989, with guidance from EMSL-LV. WBAS will conduct a variety
of performance checks of the field kit in a laboratory environment. These checks will assess
such operating and data quality parameters as the linear dynamic range of the calibration curve,
within- and between-strip precision and accuracy, and matrix effects and interferences. A few
days prior to the technology field demonstration, SAIC field personnel will conduct on-site "pilot"
testing of the WBAS field kit to determine the soundness of the logistical and analytical
procedures and to perform PCP concentration range checks on bioreactor system samples.
These tests are detailed in the discussion of predemonstration activities (sections 4.15 and 4.16).
The WBAS field kit demonstration is planned to take place in three one-week (six-day) intervals,
alternating one week on and one week off, over a period of six weeks. On or about July 24, 1989,
the first set of demonstration samples is expected to be collected, analyzed on site with the
WBAS field kit, and split and shipped to the off-site laboratories for confirmatory analyses. The
last samples are expected to be analyzed on site on or about August 26.
19
-------
4. SAMPLING AND ANALYSIS DESIGNS
4.1 TEST SITE LOCATION AND BACKGROUND
The test site for the PCP field kit immunoassay technology is the MacGillis & Gibbs Superfund
Site in New Brighton, Minnesota. This is an NPL site that is well characterized with respect to the
concentrations of PCP in the ground water. The EPA Fact Sheet (Appendix B) describes the site
and the bioreactor demonstration in more detail and also contains a map showing the general
location of the MacGillis & Gibbs site.
4.2 LOGISTICS
It is the responsibility of EMSL-LV to coordinate all logistical considerations, to ensure that all the
necessary equipment and other resources are available on site, and to ensure that the proper
lines of communication and the principal contacts at each on- and off-site location have been
identified.
The MacGillis & Gibbs demonstration site selection and the sampling and sample shipment
procedures were already in place for the BioTrol bioreactor demonstration at the onset of the
planning for the field activities of the WBAS field analysis kit demonstration (Section 1.3). The
bioreactor demonstration design was prepared by RREL with technical assistance provided by
BioTrol, Inc.
Because RREL is responsible for implementing the sampling and analytical plans of the bioreactor
SITE demonstration (SAIC, 1989), it was determined that it would be most efficient for RREL
personnel to perform all the field-related activities for the WBAS field analysis kit demonstration.
These activities include:
• providing sampling and shipping containers;
• collecting, splitting, and shipping samples;
• performing immunoassay field kit analysis for PCP (NOTE: WBAS will ensure that
RREL personnel are adequately trained to perform the field test before field work
starts);
• providing adequate space for sample analyses and equipment storage (e.g.,
refrigerator, mobile laboratory or other clean, safe, and sheltered work station);
• providing a health and safety plan and a plan for disposal of the solid and liquid
wastes generated on site from the field kit testing; and
21
-------
• providing timely access to the GO/MS analytical results for POP determinations, as
well as analytical results of the other chemical and physical measurements required
for the BioTrol bioreactor demonstration.
4.3 EQUIPMENT
To ensure that all equipment is available on site in order to perform all the analyses designed into
this plan, types and quantities of equipment must be anticipated, procured, and delivered to the
site before field activities begin. Table 4.1 lists the equipment required for the field kit
immunoassay demonstration. Table 4.2 lists the equipment required to perform the plate
immunoassay.
4.4 COMMUNICATIONS
EMSL-LV will coordinate the communications network and ensure that the primary points of
contact have been identified, including the names, telephone numbers, and addresses at each
critical on- and off-site location. This network will ensure that sample and data flow are proper;
all logistical and technical issues are quickly and properly addressed; any necessary corrective
actions are addressed and approved by management; and corrective actions are documented and
communicated to the principal party responsible for overseeing each operation.
4.5 PHOTOGRAPHS
A photographic log will be kept for all photographs taken to document on-site procedures and
operation. This log will include date, time, subject, frame, roll number, and photographer. The
photographer should review all photographs or slides for agreement with the log.
4.6 SAMPLE HANDLING
RREL personnel will collect samples to be analyzed for the WBAS field immunoassay
demonstration daily (see Section 3 for scheduling) using a composite sampling apparatus
(Section 4.7). These samples will be collected from two specified points in the BioTrol bioreactor
(Figure 4.1): the ground-water influent (after conditioning; pH adjustment with NaOH and nutrient
addition of nitrogen and phosphorus) and the treated effluent. Field equipment rinse blank water
samples will also be collected from each sampling device at each sampling point (influent and
effluent) on alternating days. Thus, three samples will be collected per day: one influent, one
effluent, and one associated equipment wash blank from one of the two sampling points. In
addition, once each week a grab sample (Section 4.7) will be collected from the influent ground
water before it enters the conditioning system.
For each of the three daily samples described above, RREL field personnel will prepare split
samples in amber glass, Teflon-lined, screw-cap bottles as follows:
• one (1) split bottle @ 30 mL and
• two (2) split bottles @ 250 mL
22
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TABLE 4.1. RESOURCES AND SUPPLIES NECESSARY TO CONDUCT THE
WBAS IMMUNOASSAY FIELD KIT ANALYSES FOR THE SITE DEMONSTRATION
1. Refrigerator shelf space or ice chest cooler large enough for several kits, QA standards,
and samples.
2. Work space to perform test (3 ft by 3 ft) out of wind, rain, and sunlight.
3. For the shipment of samples
a. Shipping containers, envelopes
b. Blue ice or ice
c. Shipping popcorn
d. Small shipping boxes
e. Sample bottles, amber glass, 250 ml_ with Teflon-lined screw caps
f. Bottle labels showing:
- Sample #
- Collection date
- Sample type
- Holding time
- Name
g. Shipping labels
4. For sample preparation and dilution
a. Stock EDTA solution (1M)
b. 10 mL disposable glass pipets
c. Pipet bulbs
d. Pipetman™ P-1000, P-200
e. Disposable tips for P-1000 and P-200
f. Disposable glass test tubes (13 mm x 100 mm) or sample vials with Teflon-lined caps
for making dilutions
g. Filters (0.2 ^m), disposable syringes, 50 cc, if necessary
h. Parafilm™
i. Beakers and other glassware for collecting, measuring volume, and diluting samples,
if necessary
j. Test tube racks for disposable glass test tubes
(continued)
23
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TABLE 4.1. (Continued)
5. Miscellaneous
a. Copies of field forms or carbonless copy
b. Pens
c. Markers for labels
d. Clip board
e. Ruler
f. Paper towels
g. Thermometers, high and low range
h. pH paper, hardness testing paper
i. Hand calculator, preferably with log and statistical functions
6. Waste disposal
a. Black cans or lined boxes for solid waste disposal
b. Hazardous waste labels
c. 17-C cans for liquid waste disposal, if necessary
d. Laboratory dishwashing detergent, dishpan
7. Health and safety
a. Disposable vinyl gloves
b. Safety glasses
c. Laboratory coat
8. For immunoassay analysis
a. 6 WBAS PCP Immunoassay Field Kits per week shipped from WBAS
b. Performance standard ampules, 25 ppm, prepared by EMSL-LV and shipped to
the site
c, A Dynatech Micro-Well Reader with power adapter and battery, Model 1 (Cat.
No. 011-010-6900), equipped with a 405-nm interference filter (Cat. No. 632-800-
5405)
d. Blind or semi-blind QA audit samples prepared by EMSL-LV and shipped to the
site
e. Carboy with 10 liters of laboratory deionized water for rinsing and making
dilutions
f. Clinical table top centrifuge
24
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TABLE 4.2. EQUIPMENT NECESSARY TO CONDUCT THE WBAS PLATE
IMMUNOASSAY ANALYSES FOR THE SITE DEMONSTRATION
1. Glassware (suggested specifications)
a. Vials: 4 to 6 mL, with Teflon-lined screw cap
b. Volumetric pipettes: 0.1 mL; 0.25 mL; 2.5 mL
c. Volumetric flasks: 1 mL; 5 mL; 10 mL
d. Graduated cylinder: 2 or 4 L
e. Hamilton syringe: 0.005 ml; 0.010 mL; 0.05 mL
f. Powder funnel
g. Pipetting bulbs and controllers
h. Sidearm flask (heavy walled), 1 L
2. Plasticware
a. Pipette tips (for single- and multichannel pipettors)
b. Microtiter plates (96-well, polystyrene, flat bottom)
c. Microtiter plate adhesive sealers
d. Test tubes (polypropylene conicals with screw caps): 15 mL; 50 mL
e. Nalgene carboy, 15 L
f. Polyethylene wash bottles: 250 mL; 1,000 mL
g. Reservoirs (for use with multichannel pipette), 30 mL
h. Bottle, 1 L plastic, with screw cap
3. Pipettors
a. Multichannel pipettor (variable volume): 5 to 50 M-
b. Multichannel pipettor (variable volume): 50 to 200 /A.
c. Single-channel pipettor (variable volume): 1 to 20 //L
d. Single-channel pipettor (variable volume): 20 to 200 /A.
e. Single-channel pipettor (variable volume): 200 to 1,000 p\-
f. Single-channel pipettor (variable volume): 1,000 to 5,000 //L
4. Microplate washer
5. Vacuum pump
6. pH meter
(continued)
25
-------
TABLE 4.2. (Continued)
7. Refrigerator
8. Vortex mixer
9. Magnetic stirring apparatus
a. Stirrer with top plate large enough to hold 15 L carboy
b. Stirring bar, 2 to 3 inches
10. Spectrophotometer: microplate reader equipped with 405 and 650 nm filters
(Bio-Tek Model 310)
11. Data system*
a. Compaq Computer with 640K RAM, 20 megabyte hard disk, mouse, RS232 port"
b. Epson Printer - LX80b
c. HP-Genechem Titercalc 2.0 software6
d. Spectrophotometer/computer connection (25 pin, straight-through cable)b
12. Reagents
a. Reagent grade water
b. 2-propanol
c. Hydrochloric acid (1 N)
d. Sodium hydroxide solution (1 N, 10 N)
e. Phosphate buffered saline
f. Phosphate buffered saline-Tween 20
g. Bovine serum albumin
h. Microtiter plate coating antigen
i. Monoclonal antibodies to PCP
j. Pentachlorophenol solution in 2-propanol
k. Developing reagent, alkaline phosphatase conjugates
I. Diethanolamine buffer
m. Substrate Tablets - SIGMA 104, Phosphatase Substrate Tablets
n. Dilution buffer
o. Sample buffer
" Even though it is possible to make the required calculations using a hand calculator and graph
paper, this demonstration will use the listed data system components.
b Or equivalent component.
26
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Weekly Grab
(Raw)
Influent Sample
Pump
I
Daily Routine Influent.
Composite Sample
Conditioning Tank
(Nutrients, NaOH Added)
Well
Lid
.Carbon To Air
f
Air Air Air
Clarifier
or Filter
To POTW
or
Daily Routine Effluent
Composite Sample
&
Glbbs
Source: Modiliea Horn SAlC. 1989
Figure 4.1. BioTrol. Inc., Biological Aqueous Treatment System and the sample collection
points for the WBAS immunoassay demonstration.
-------
Each 30-mL split (three per day) will be stored at 4 °C and delivered on-site to the field crew
member designated to perform the field immunoassay or will be placed in the designated
refrigerator for later analysis. One 250-mL split from each sample, totaling three samples per
day, will be stored at 4 °C and shipped to two locations, the EMSL in Las Vegas and the WBAS
laboratory in Maryland. After these samples have been analyzed according to the analysis plan
(Section 4.14), they will remain in storage at 4 °C at each location until written notification from
EMSL-LV specifies otherwise. On the days when the influent grab sample is collected, it will be
split, shipped, and handled in the same manner as the three samples described above.
Occasionally, 1-liter splits of the influent, effluent, and grab samples, totalling about 12 over the
course of the demonstration, will be prepared and sent to the EMSL-LV for GC/MS analysis. In
addition, EMSL-LV will prepare and ship blind and known performance (audit) samples and a
matrix spiking standard solution to the field and to the off-site laboratories (EMSL-LV, WBAS,
SA1C) as specified in the analysis and QA plans (Sections 4.14 and Appendix A, respectively).
Figure 4.2 provides a diagram of the sample flow described above, and Appendix 0 gives the
procedure for sample splitting and shipment.
4.7 SAMPLE COLLECTION PROCEDURES
The demonstration plan for the BioTrol bioreactor (SAIC, 1989) describes the process of sample
collection in detail and is summarized below.
The bioreactor influent and effluent samples to be analyzed using the WBAS PCP immunoassay
field analysis kit will be collected with an automatic 24-hour composite sampling device. An ISCO
or similar peristaltic pump sampler will be used. Each sample will be "time composited," collected
in approximately 150-mL portions every 20 minutes over a period of 24 hours, for a total (i.e., bulk)
sample volume of about 13 liters. The sample will be collected in a 4-gallon widemouthed
composite jug located at the base of the sampler.
After the bulk (13-liter) composite sample has been collected, the 30-mL, 250-mL, and 1-L sample
split bottles (specially cleaned for phenols analysis) will be prepared for on-site field analysis and
for shipment to the EMSL-LV, WBAS, and SAIC laboratories.
All sample split bottles will be labelled (Figure 4.3) before splitting procedures begin. Split
samples will be prepared in the same manner as the samples used for GC/MS analysis, except
for the volume of sample in the split bottles. Detailed sample collection and sample split
preparation procedures can be found in the bioreactor demonstration plan (SAIC, 1989).
The field equipment blank will be collected at the beginning of each composite sampling event.
Blanks for the composite sampler will consist of reagent water poured into a decontaminated
composite jug, pumped through the sampler, and collected directly into the appropriate bottles.
Only one influent or one effluent field equipment blank will be collected and split per day.
A single influent grab sample will be collected from a garden hose attached to a T-tap located
before the influent water conditioning tank. The sample will be collected directly into a bulk
sample collection container similar to those used to collect the composite samples.
28
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BIOTROL BIOREACTOR
CONDITIONED^
INFLUENT „
BULK SAMPLE
FOR GC/MS ANALYSIS
v 13-L COMPOSITE ^
FIELD BLANK
(After Equipment Wash)
EFFLUENT
BULK SAMPLE
FOR QC/MS ANALYSIS
v 13-L COMPOSITE .
FIELD BLANK
(Alter Equipment Wash)
WEEKLY RAW INFLUENT
(Before Conditioning)
1-L INFLUENT
1-L EFFLUENT
ONCE PER WEEK
EMSL-LV
ON-SITE SPLIT SAMPLE PREPARATION
INFLUENT
One @30mU
Two @ ZSOmL
EFFLUENT
One @ 30mL
Two (q) ZSOmL
INFLUENT
FIELD BLANK
One @ 30mL
Two @ 2SOmL
EFFLUENT
FIELD BLANK
OR
One
Two
30mL
250mL
QA/QC AMPULES
FROM EMSL-LV
ON-SITE
INFLUENT
One ® 250 mL
EFFLUENT
One @ 250mL
FIELD BLANK (Influent
or Effluent)
One @ 2SOmL
OA/OC SAMPLES
INFLUENT
One @ 30mL
EFFLUENT
One ® 30mL
FIELD BLANK (Influent
or Effluent)
One @ 30mL
OA/OC SAMPLES
4-L TO SAIC'S
GC/MS LAB
WBAS
• INFLUENT
One @ 2SOmL
• EFFLUENT
One @ 250 mL
• FIELD BLANK.(Influent
or Effluent)
One @ 250mL
• OA/OC SAMPLES
ARCHIVE AFTER ANALYSIS
ARCHIVE AFTER ANALYSIS
ARCHIVE AFTER ANALYSIS
Figure 4.2. Daily sample flow (demonstration phase).
29
-------
SMC
On* S*tr> Or/v*. Par«mui. Ntw Jtrny 07(25
Location:
Sample Date/Time:
Sample No.:
Analytis:
Collection Method:
Pretervative:
Comments:
Project No.:
Sample Location:
Purge Volume:
Collector'! Inllltli:
Figure 4.3. Sample bottle label.
30
-------
4.8 CHAIN OF CUSTODY
Sound sample collection and analysis protocols include the implementation of chain-of-custody
procedures. These procedures include sample inventory and documentation during collection,
shipment (or other transfer), and laboratory processing. Chain-of-custody forms will be used to
document the integrity of all samples. To maintain a record of sample collection, transfer,
shipment, and receipt, a chain-of-custody form will be filled out for each sample set at each
sampling location. Figure 4.4 shows the SAIC chain-of-custody form; details of the chain-of-
custody procedures can be found in the bioreactor demonstration plan (SAIC, 1989).
4.9 SAMPLE PACKING AND SHIPPING
Samples will be packed with custody tape wrapped around the neck and cap of each container
and then wrapped with plastic insulating material. Samples will be classified according to the
Code of Federal Regulations (CFR) (BNA, 9/28/90). Sample containers will be placed in thermally
insulated, rigid coolers, according to Department of Transportation (DOT) specifications. The
coolers will contain ice and absorbent packing to maintain a temperature of 4 °C during
shipment. The chain-of-custody form will be placed inside the shipping box The bioreactor
demonstration plan (SAIC, 1989) provides a detailed description of sample packing and shipping.
NOTE: If time permits, the field data forms (Section 4.13) for the enclosed samples will be
included in the box in a secured plastic bag for protection. Appendix E provides the WBAS and
EMSL-LV laboratory addresses to which the split samples will be sent.
4.10 SHIPMENT OF PERFORMANCE STANDARD AMPULES
Performance standards to be used for semiblind and known QA (audit) and QC reference
samples will be prepared in ampules at EMSL-LV and shipped at 4 °C via overnight courier to the
MacGillis & Gibbs site and to the WBAS and SAIC laboratories in standard ampule shipping
containers (30 ampules per package) (see Appendix A for use of these samples). Shipment will
follow standard DOT shipping regulations for performance evaluation samples under DOT (49
CFR 173.4) exemption and in accordance with LESC shipping Standard Operating Procedure
Number 20-86-40-3.
4.11 SAMPLE IDENTIFICATION
Sample tracking will be accomplished in the field by assigning each sample a unique number as it
is collected. This number will be traceable back to the sample day, time, and site of collection
and will also provide the tracking information for split samples analyzed on- and off-site. This
sample identification number (sample ID) will be recorded on all split sample bottle labels and
chain-of-custody and field data forms, as welt as in a field logbook A master log of all sample
IDs will be maintained by the on-site crew chief. The sample ID system is designed to conform
to the BioTrol bioreactor demonstration needs and consists of four codes representing (in order):
the stage of the bioreactor demonstration ("Stage 1" or "Stage 2"); the activity period ("A," "B," or
"C"); the sequential sample number collected in the activity period (01 to n + 1); and the sampling
point in the bioreactor system which is coded as follows:
31
-------
(Q
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CO
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(A
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Chain of Custody Record
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-------
01 - raw, unconditioned ground-water influent
02 • bioreactor conditioned influent
05 - bioreactor effluent
13 - field equipment blank
For example, the sample ID could be:
ST1-A-01-01,
which would indicate that the sample was collected at the influent point on the first day of the
first activity period of Stage 1. A complete description of the sample ID process can be found in
the bioreactor demonstration plan (SAIC, 1989).
4.12 SAMPLE CODING
Because of the complexity of the analysis design of the WBAS field kit demonstration, a series of
sample codes has been created for the samples analyzed by the field kit immunoassay technique.
These codes will identify the samples for the purpose of assessing data quality within the
method and within and between analysis locations. The sample codes and their definitions are
presented in Table 4.3, and a complete description of these samples can be found in Section 4.14,
Analysis Design, and in the QAPjP (Appendix A).
4.13 FIELD DATA FORMS
Field data forms have been designed to document important information related to each field
sample analyzed by the WBAS field kit method. These forms will be filled out on site by the field
personnel analyzing samples with the field kit, as well as by laboratory personnel at EMSL-LV and
WBAS who perform the field kit analysis. The field data form consists of 3-page carbonless
paper; one copy will be sent from the analysis site to EMSL-LV for review and entry into a data
base, one copy will be sent to WBAS for data review, and one copy will be filed on site for
reference.
The form is designed to facilitate data tracking and to document analytical and field condition
information. The format allows the information to be entered easily into a data base for data and
statistical analyses (Section 5). Figure 4.5 shows the field form and Appendix F provides a
Standard Operating Procedure (SOP) for filling out the form.
Information included on the field data form includes:
• sample collection information such as sampling date and time, sample ID, method of
collection, bioreactor information, and sampling personnel;
• environmental factors such as location and date of sample analysis, meteorological
conditions, sample and kit storage data, and analyst;
33
-------
TABLE 4.3. WBAS IMMUNOASSAY SITE DEMONSTRATION-SAMPLE CODES
AND DEFINITIONS
Quality Control Samples
FB field equipment blank (after daily decontamination of ISCO and before daily
compositing)
NC negative control sample (1 x kit dilution buffer solution)
QC daily quality control performance check sample at a known POP concentration
(20 ppm)
Calibration Standards
STD03 calibration kit standard at 3.0 ppb (0.0030 ppm) PCP
STD07 calibration kit standard at 7.1 ppb (0.0071 ppm) PCP
STD16 calibration kit standard at 16.9 ppb (0.0169 ppm) PCP
STD40 calibration kit standard at 40.0 ppb (0.0400 ppm) PCP
Routine Samples
RE routine daily effluent sample
RI routine daily influent sample
RG routine weekly influent grab sample
Duplicates and Splits
DE duplicate of an undiluted RE sample (i.e., split before any dilutions)
DI duplicate of an undiluted RI sample (i.e., split before any dilutions)
SRE split of an RE sample after the RE has been diluted into calibration range
SRI split of an RI sample after the RI has been diluted into calibration range
SDE split of a DE sample after the DE has been diluted into calibration range
SDI split of a DI sample after the DI has been diluted into calibration range
Matrix Spikes
REMS-x RE sample diluted to the range for matrix spiking which will be used in the percent
recovery calculation (x = sample to be matched with the same "x" for the matrix
spike effluent sample)
RIMS-x RI sample diluted to the range for matrix spiking which will be used in the percent
recovery calculation (x = sample to be matched with the same "x" for the matrix
spike influent sample)
MSE-x matrix spike sample of a REMS with corresponding V
MSI-x matrix spike sample of a RIMS with corresponding "x"
Audit Samples
QAA-xxx semi-blind QA standard, Lot A (xxx = LESC sample control number)
QAB-xxx semi-blind QA standard, Lot B (xxx = LESC sample control number)
Cross-Calibration Standards
CC-xxx cross-calibration color check standard for spectrophotometers (xxx = code for
theoretical optical density)
34
-------
FIELD DATA FOR ANALYSIS OF WBAS FIELD IUMUNOASSAY TEST KIT
Ptos 1 el _
Samda Collection Information
Sampts Data: o Sarnpta Tims: _ _:_ _ O SAIC Sampla No.: • _ •
Colsctlon Mstnod: Gtxnposits/GrBb ISCO tti: O Prssarxsilvs:
Btorsactor ID: Growidwaiai Wsi No.: Flow Hats: gprn;
BATS PCP Spies: Y/N; BATS Spfcs Cone: ppm
Cossetsd by: SpUt by:
Gonvnants:
Envjronmsnial Fsetors
Aiulyti* 0»t»: _______ O 0«y* Sine* CoBKtcd: _ _ O SIU Ol Analysis: OrvSitt/WBAS/VV O
Location of Analysis: Indoors/Outsids Location Tamp: _ _ *C
W Outskjs. Sunny/Ooudy/Mnajd O Windy/Calm O Othsr _ O
KM Lot No.: _ O KM Slorsgs: Days 0 Afnbtant T«mp:_ _ o: Days €> 4-C: . . O
Oaly Flsfrtg. Tsmp: . . *C O
Analyst:
Samots aosgfie Pr«-Analvsls Information
Sampfing Point Sovos: Intkjant^ffkisnt/In^ssctor(_)/Otnsr O
Sampls Appsaranos O: Color: O: Char/Turbid O: Prsdpitata: Y/N O: Oil: Y/N O: Other:.
Csntrlfugad: Y/N O H V». Appaaranca Changa: Y/N
H yas. Color O: Claw/Turbid O: Pradphata: Y/N a Oil: Y/N O: Other-. _
pH-Mrtsr: . _,_ _ o ptWspar _ _._ O Haronass-Papsr: o
Fisld Blank: Y/N SAICSampla No.: •_•__•_.O
Flald OupHcats: Y/N SAIC Sampki No.:3I_-"-._-".O
Fisld OA Audit: Y/N LESC Sample No.: -~_.O~
_o
.o
AMPLE ANALYSIS INFORMATION BY STRIP
5- mo
Cod*
A
B
S
to w
4
i i
«
(
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00
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0 «
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[ppm
0 «
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>
Figure 4.5. Field data form (page 1 of 2).
35
-------
Pig* _ ol _
FIELD DATA FOR ANALYSIS Of WBAS FIELD IMUUNOASSAV TEST KIT
SA1C SAMPLE NO.: •_•__•__ ANALYSIS DATE: ANALYST:
SAMPLE ANALYSIS INFORMATION BY STRIP
3*«np
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-------
• sample information before kit analysis such as sample appearance (before and after
centrifugation, if applicable), pH, and sample tracking data on field QA (blank,
duplicate, audit) samples; and
• kit sample analysis information such as sample codes, sample optical densities,
semilogarithmic graph paper for plotting absorbance units, and a method to
determine sample concentrations and matrix spike percent recoveries.
4.14 ANALYSIS DESIGN
The SITE demonstration plan for the BioTrol bioreactor (SAIC, 1989) contains detailed information
regarding sample collection, handling, storage, and the analysis of samples for PCP (and PAH) by
GC/MS and other analytical test methods (see Appendix C for a list of the other analytes to be
determined on the bioreactor water samples).
The sampling points in the bioreactor system chosen for sampling and analysis in the field kit
demonstration were selected because these are the first and the last points in the bioreactor
process that will be collected on a daily basis and analyzed for PCP by GC/MS. The state of the
water samples at these two sampling points in the bioreactor system will include: (1) ground-
water that has been conditioned by adding NaOH to adjust the pH to -7.3 and nutrients (i.e.,
nitrogen and phosphorus compounds) to sustain the microbes in the bioreactor and (2) effluent
water from the bioreactor before the removal of solids and before carbon filtration. Field
duplicate samples of the influent and effluent samples described above will be collected
periodically with a second composite sampler located beside the first. Samples of field
equipment blank water rinsate (field blank) samples will also be collected after daily
decontamination of the composite sampler. In addition, once per week an influent grab sample
will be collected from the bioreactor system at a point before the water is conditioned.
The analysis scheme and schedule for each operating day and schematics for the daily analyses
are presented in Appendix G. The specific standard operating procedure (SOP) for performing
analyses with the field kit, including sample dilution instructions and QC criteria, is found in
Appendix II of the QAPjP (Appendix A).
Split samples will be analyzed by using the immunoassay field kit on site and at the EMSL-LV and
WBAS laboratories. Splits of the same samples will also be analyzed by the quantitative plate
immunoassay method at EMSL-LV and WBAS. These analyses will be compared to the GC/MS
analyses for these splits performed at the SAIC laboratory in San Oiego, California (with a select
group of samples also to be analyzed by GC/MS at the EMSL-LV facility).
WBAS has also provided SOPs for the plate immunoassay and for the cleanup of samples on
solid phase extraction columns, if it is necessary to use that technique (based on the matrix spike
recovery criteria of 100% ± 25%). The SOP for sample cleanup contains detailed instructions for
sample preparation and column conditioning loading and elution. The average expected recovery
for the columns (-86 percent) was determined through studies with 14C-labelled PCP. The
document entitled "Quality Assurance Plan for Immunoassay Evaluation and Research Projects"
(White and Miah, 1989) contains information on protocols for instrument and plate variability
checks and other QA and QC requirements relevant to immunoassays.
37
-------
All SOPs for the field activities related to the WBAS field kit test will be provided to on-site
personnel during the set-up activities. Additional information will be supplied including
instructions for sample splitting, labelling, storage, and shipment; the schedule and instructions
for performing the POP field kit analysis; instructions for handling and analyzing the QA and QC
samples (Appendix H); instructions for documenting field data (Appendix F); and health and
safety information (Section 6).
Samples will be analyzed following EPA Method 8270 (Gas Chromatography/Mass Spectrometry
for Semi-Volatile Organics; EPA, 1986). This method contains detailed analysis instructions, QC
guidelines, and performance data for POP analysis by GC/MS. In addition, EPA Method 3510 (U.S.
EPA, 1986) will be used for sample extraction.
Table 4.4 presents a summary of the expected performances of each of the three analytical
methods for POP analysis used in this demonstration. Figure 4.6 shows the relationship of the
three methods as well as the location of the analysis of each. Section 5.3 provides an overview
of the ways in which the three methodologies will be compared.
4.15 PREDEMONSTRATION PLAN DESIGN AND STRATEGY
The following activities will be included in the preliminary evaluation phase of the WBAS PCP
immunoassay demonstration:
1. SOPs for the field activities (sample handling, analysis, and shipment) will be provided
to field personnel. In addition, a field form with detailed instructions and sample
codes and instructions for QA/QC sample analysis will be provided.
2. Drafts of the Immunoassay SITE Demonstration Plan and QAPjP will be disseminated
to the appropriate parties for concurrence.
3. EMSL-LV, WBAS, and SAIC personnel will cross-calibrate the microwell strip
spectrophotometer at the three analysis locations and with the laboratory
spectrophotometers at EMSL-LV and WBAS on test standard (N-2.4-DNP glycine)
curves.
4. WBAS will prepare and ship immunoassay reagents for the field kit PCP methods to
the field site (as facilities become available) and to EMSL-LV. The quantities will be
sufficient for one week of analysis. Subsequent reagent shipments will be made on a
weekly basis during the demonstration activities.
5. A list of supplies and equipment required at the field site will be circulated to the
parties responsible for procurement (WBAS, LESC, or SAIC). These supplies will be
shipped to the field site as soon as the personnel and facilities (e.g., refrigerator) are
in place.
38
-------
TABLE 4.4. KNOWN OR ANTICIPATED PERFORMANCES FOR PCP ANALYSIS
Performance
parameters
Detection limit (ppb)
Linear dynamic range
(ppb)
Precision
Accuracy
Time to analyze
Cost per sample
Key interferents
% Matrix spike
Field analysis kit
3
3-40
10-15% RSD
NA"
-30 minutes
5 samples per
analysis
~$30 per sample
See Table 2.1
50-150%
Lab plate
immunoassay
- 10-15
- 30-400
5-10% RSD
NA
-2.5 hours
10 samples per
analysis
-$25 per sample
See Table 2.1
75-125%
GC/MS (EPA
methods 8270. 3510
- 30-50
30-200
•
c
-1 hour
-$700
No major
14-176%d
recovery
Fieldability (on
site/mobile
laboratory)
Yes
Yes
No
* Robertson (1989) reports within-laboratory precision of 21% RSD; between laboratory precision
of 36% RSD based on approximately 50 laboratories.
" NA = not available.
c Robertson (1989) reports matrix spike/matrix spike duplicate accuracy of 15.5 percent based on
approximately 50 laboratories.
d Robertson (1989) reports matrix spike recoveries of 71 and 79 percent based on approximately
50 laboratories.
39
-------
HPLC
FIELD KIT
FIELD KIT
ON -SITE
i
FIELD KIT
LAB-BASED
oOOC3v ASSAY
LAB -BASED
ASSAY
SAIC -SAN DIEGO
EMSL-LV
Figure 4.6. Comparison scheme of pentachlorophenol analysis methods
employed in the WBAS immunoassay field kit demonstration.
40
-------
6. EMSL-LV will prepare QA and QC samples for both (field kit and plate) immunoassay
formats and for the GC/MS method. Performance materials obtained from EMSL-
Cincinnati will be diluted in methanol and amputated. EMSL-LV will ship the ampules,
along with SOPs for handling and analysis, to each location performing the analyses.
7. Biotrol, Inc., will send WBAS and EMSL-LV samples of PCP-contaminated well water
from the MacGillis & Gibbs site at various concentrations; surface water with low-level
POP contamination (>0.5 ppm); and archived and on-line bioreactor influent, effluent,
and conditioning tank water. These samples will be analyzed by WBAS and EMSL-LV
for preliminary matrix testing to determine if any sample pretreatment measures will
be necessary during the demonstration.
8. WBAS will provide EMSL-LV with data on accuracy, precision, detection, possible
interferences, linear dynamic range, and other performance characteristics of the field
PCP immunoassay method.
4.16 PREDEMONSTRATION ON-SITE ANALYSIS
The purpose of conducting an on-site preliminary test of the field kit is to (1) perform range-
finding and interference checks on influent and effluent samples and (2) evaluate the efficiency of
the analysis and logistical systems, including communications and data flow and documentation.
If possible during the predemonstration testing phase, a few influent, effluent, blank, and QA/QC
samples will be analyzed on-site. Split samples will be shipped to WBAS and EMSL-LV for
analysis by the field kit and plate immunoassay methods. Field forms will be filled out and
analytical SOPs will be followed to confirm proper understanding of the requirements.
41
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5.0 DATA ANALYSIS AND MANAGEMENT
5.1 DATA HANDLING
Data generated in the field will be collected and documented on the field data forms. One copy
of each form will be sent to the data base manager at EMSL-LV, one will be sent to WBAS, and
one will be retained on site. Data generated from the field kit method performed in the EMSL-LV
and WBAS laboratories will also be recorded on the same field data forms used on-site; a copy
will be sent to the data base manager and a copy will remain at the point of analysis. Data
generated from the plate immunoassay by EMSL-LV and WBAS will be sent to the data base
manager on hard copies and on a floppy diskette if necessary. Copies of the data generated
from the GC/MS analysis at the SAIC laboratory will be sent on data reporting and
documentation forms used by SAIC. Figure 5.1 provides a diagram of the data flow.
All locations will be contacted by EMSL-LV for preliminary data over the telephone until the data
forms or diskettes are received. All data will be reviewed by a QA officer for consistency in
reporting, reasonableness, transcription errors, other data reporting errors, and other issues.
After review the data will be entered into a data base (Section 5.2), from which a detailed
statistical review will be performed.
5.2 OVERVIEW OF DATA BASE DESIGN AND DATA BASE MANAGEMENT
The data collected from the SITE immunoassay demonstration will be entered and stored in a
Statistical Analysis System (SAS) data set. The SAS data set will be divided into separate files
(members), depending on the source of the data (RREL, EMSL-LV, SAIC, WBAS) and on the
analytical method (field kit immunoassay, plate immunoassay, GC/MS). (NOTE: BioTrol, Inc.,
has developed a quick turn-around high-pressure liquid chromatography method for analyzing
PCP that is not part of this demonstration. However, any data generated analyzing samples with
this method may be useful for additional comparisons.)
Variable names that identify samples will be complete and consistent across members to allow
for both within-method performance assessment and between-method comparisons as the need
arises. Each member will contain variables for all information pertinent to that analysis.
5.3 OVERVIEW OF DATA ANALYSIS
The data analysis will consist of two phases. In the first phase the quality of the data will be
assessed. This assessment will consist of a number of standard procedures. The QA and QC
data will be examined (via time trends and descriptive statistics) to ensure that consistent results
and adequately accurate measurements are available. This examination will require data from the
various blanks, audit samples, matrix spikes (Appendix J), and splits and duplicates. This form of
43
-------
r
~i
TO WBAS
SAMPLE COLLECTION DATA
SAMPLE DATA
(WEATHER. pH. COLOR, ETC.)
SAMPLE RESULTS
FIELD FORM CHECKED
AND SHIPPED
(COPY KEPT ON-SITE)
WBAS FIELD KIT
ON FORM;
LAB DATA
ON DISKETTE
| \ 1
TO EMSL-LV
DATA CHECK FOR
REASONABLENESS,
TYPO'S, ETC.
CONFIRMATION OR
REANALYSIS MAY BE
NEEDED (?)
SAIC
QC/MS DATA
PHONE. FORMS
TO FOLLOW
EMSL FIELD KIT
DATA ON FORM;
LAB DATA ON
DISKETTE
QC/MS DATA
ENTERED. COPIED INTO
DATA BASE (PC SAS)
STATISTICAL COMPARISONS,
COMPLETE VERIFICATION AND
VALIDATION, MAKE ANY DATA
BASE CHANGES
Figure 5.1. Data flow (demonstration phase).
44
-------
analysis will be conducted for each analysis method for each site. Also, based upon the results
of the splits and duplicate analyses between and within strips, a simple analysis of variance
(ANOVA) to determine relative sources of error may be carried out if it appears, from inspection of
the data, that a major component of error can be isolated. The second phase of analysis will be
the comparison between methods and sites. Of primary concern will be the comparison between
routine GC/MS values and the field kit results. Given positive results from the first phase of
analysis, it will be assumed that the GC/MS results are the "true" values with respect to the
accuracy expected from the immunoassay method. The comparison will be examined via
summary statistics, t-tests for differences, correlation of results, and XY plots. Similar analyses
are planned for comparing results from the other tests, though these latter results are expected
to provide less important information because the primary goal is to look at the strip
immunoassay under field conditions. However, efforts will also be made to estimate whether
laboratory bias is significant as well as whether method-to-method bias exists.
45
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6. HEALTH AND SAFETY
Most of the on-site health and safety considerations are described in the bioreactor
demonstration plan (SAIC, 1989). The following instructions relate to the analysis aspects of the
WBAS PCP immunoassays:
1. Because PCP is both a toxic and carcinogenic substance (see Appendix I, Materials
Safety Data Sheets), solutions containing the substance should be handled with care.
Personnel handling PCP-containing solutions will wear plastic or vinyl gloves,
laboratory coat, and safety glasses. Care should be taken to avoid skin exposure,
since the substance can be absorbed through the skin.
2. The QA and QC solutions contain methanol, an inflammable solvent, in addition to
parts per million concentrations of PCP. The ampules have a warning label and will be
shipped according to DOT regulations. These solutions should be handled with care,
as they are flammable and toxic.
3. For waste disposal of liquids contaminated with PCP, use empty glass solvent bottles,
17-C cans, or 55-gallon drums. Make sure the containers are labelled with hazardous
waste labels and that a record is made showing the solvent type and the approximate
volume and concentration of PCP.
4. Small pieces of PCP-contaminated solid waste, such as disposable Pasteur pipets and
pipet tips, should be placed in empty glass solvent bottles or in a plastic trash bag
inside of a metal can or thick-walled cardboard box These containers should be
labelled with hazardous waste labels stating that the contaminant is PCP.
5. Reusable glassware contaminated with PCP should be rinsed with acetone or
methanol prior to placing it with other glassware for dishwashing. The solvent rinses
should be collected and disposed of with other PCP waste.
6. The colorimetric substrate used in the WBAS field kit is carcinogenic and should be
handled with the same precautions applicable to the solutions containing PCP (see
above).
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REFERENCES
BNA (Bureau of National Affairs, Inc.). September 28, 1990. Chemical Regulation Reporter.
49 CFR 173.4. Exceptions for Small Quantities, Bureau of National Affairs, Inc.,
Washington, D.C.
EPA (U.S. Environmental Protection Agency). 1979. Method 604-Phenols. Federal Register
44:69464-69575.
EPA (U.S. Environmental Protection Agency). 1986. Test Methods for Evaluating Solid Waste:
Method 3510; Method 8270. EPA/SW-846. U.S. EPA Office of Solid Waste and Emergency
Response, Washington, O.C.
Robertson, G. L 1989. A Dynamic Method Validation Report on the Contract Laboratory Program
Routine Organic Analysis. Internal Report. Environmental Monitoring Systems Laboratory,
U.S. Environmental Protection Agency, Las Vegas, Nevada.
SAIC (Science Applications International Corporation). 1989. Draft Demonstration Test Plan for
BioTrol, Inc. Biological Treatment of Contaminated Ground water at New Brighton,
Minnesota. Draft Document. Science Applications International Corporation, 1 Sears
Drive, Paramus, New Jersey.
Twin Cities Testing Corporation. 1986. Remedial Investigation Report MacGillis and Gibbs
Company Site Prepared for Minnesota Pollution Control Agency. #120 86-414.
Van Emon, Jeanette M., and Robert W. Gerlach. 1990. EPA Evaluation of the Westinghouse Bio-
Analytic Systems Pentachlorophenol Immunoassays. EPA/600/X-90/146. Environmental
Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, Nevada.
White, R. J., and M. J. Miah. 1989. Quality Assurance Plan for Immunoassay Evaluation and
Research Projects. Internal Document. Environmental Monitoring Systems Laboratory,
U.S. Environmental Protection Agency, Las Vegas, Nevada.
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APPENDIX A
QUALITY ASSURANCE PROJECT PLAN FOR THE
SITE DEMONSTRATION OF THE
WESTINGHOUSE BIO-ANALYTIC SYSTEMS
IMMUNOASSAYS FOR PENTACHLOROPHENOL
The following Quality Assurance Project Plan (QAPjP)
was approved in July 1989 and was used as guidance
for the demonstration field activities.
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QUALITY ASSURANCE PROJECT PLAN FOR THE
SITE DEMONSTRATION OF THE WESTINGHOUSE BIO-ANALYTIC SYSTEMS
IMMUNOASSAYS FOR PENTACHLOROPHENOL
Prepared by:
Lockheed Engineering & Science Company
1050 East Flamingo Road
Las Vegas. Nevada 89117
Prepared for:
United States Environmental Protection Agency
Environmental Monitoring Systems Laboratory
Las Vegas. Nevada 89193-3478
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PROJECT QUALITY ASSURANCE PLAN APPROVAL
This plan is approved for use with the SITE Demonstration of the Westinghouse Bio-Analytic
Systems Immunoassay for Pentachlorophenol (PCP).
This project quality assurance (QA) plan was developed to assure that all environmental data
generated for the U.S. Environmental Protection Agency (EPA) are scientifically valid,
representative, comparable, complete, and of known accuracy. The signatures of key project
personnel below indicate concurrence with the procedures specified in the plan and a
commitment to disseminate the plan and the philosophy of quality to alt project personnel.
V. Eckers,
QA OFficer, LESC
Date
. Van Emon,
reject Manager, EAD
Date
RKJ. White (
Principal Scientist
E. Koglin
Manager, QAD
Date
W.D. Munslow
Manager, LESC
Date D.G. Easterly
QA Officer, EMSL-LV
•' Date
53
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CONTENTS
Page
Project Quality Assurance Plan Approval ii
Introduction 1
Starting Date 1
Expected Completion Date 1
Target Analyte 1
Description of Immunoassays 2
Standard Analytical Method 2
Key Reagents 2
Project Description 2
Project Organization 3
Quality Assurance Design and Responsibilities 5
Data Quality Objectives 7
Data Quality Objectives-Acceptance Criteria 7
Seven Elements of Data Quality 8
Key Interference 10
Specificity 10
Assessment of Data Quality 10
Corrective Actions 14
Performance and Systems Audits 14
Preliminary Evaluation Phase 15
Quality Control Protocols 15
Data Handling 15
Data Validation, Reduction, and Reporting 16
Statistical Evaluation 16
Reports for Management 17
Appendices
I. Sampling and Analysis Schedule for the Field PCP Immunoassay
Kit SITE Demonstration 1-1
II. Pentachlorophenol field Analysis Kit II-1
IIL Calculations IH-1
ii!
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QUALITY ASSURANCE PROJECT PLAN FOR THE
SITE DEMONSTRATION OF THE WESTINGHOUSE BIO-ANALYTIC SYSTEMS
IMMUNOASSAYS FOR PENTACHLOROPHENOL
INTRODUCTION:
One of the goals of the U.S. Environmental Protection Agency's Superfund Innovative Technology
Evaluation (SITE) Program is to evaluate the application of new and innovative technologies for
field measurement of environmental contaminants. In this SITE demonstration, the technology
being evaluated is a rapid field-deployable immunoassay. which is specific for pentachlorophenol
(POP). The analytical method employs an antibody which specifically binds to the target analyte
PCP. In this SITE demonstration, the immunoassay will be evaluated as a rapid, semi-
quantitative field screening method.
To ensure that the data resulting from this SITE demonstration are of sufficiently high quality to
meet the intended use, it is necessary to prepare a Category II Quality Assurance Project Plan.
The following QA Project Plan addresses the eleven key elements that are necessary for Category
II projects (U.S. Environmental Protection Agency, "Quality Assurance Program Plan.*
EPA/600/X-87/241. Environmental Protection Agency. Las Vegas, Nevada, 1987.)
STARTING DATE:
Preliminary evaluation phase: June 26 to July 21. 1989
Formal data collection: July 24 to August 26, 1989
EXPECTED COMPLETION DATE:
The field demonstration will end approximately August 26. 1989. The final project report should be
completed within six months after the field data collection is completed.
TARGET ANALYTE:
Pentachlorophenol (PCP) in ground water, in treated bioreactor influent, and in bioreactor effluent
samples.
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DESCRIPTION OF IMMUNOASSAYS:
1. the Rapid Field POP Immunoassay - This test is a direct competitive enzyme-linked
immunosorbent assay (EUSA) that consists of polystyrene microwell strips coated
with anti-pentachlorophenol primary antibody. The test involves direct competition for
antibody binding between a PCP-enzyme conjugate and PCP in the sample. The test
has a detection level of about 3 ppb and a linear dynamic range of about 3-40 ppb.
The test Is rapid (30 minutes) and easy to perform, and is designed to provide semi-
quantitative information about PCP levels. Thus, the method is particularly suitable for
field screening.
2. The standard laboratory competitive inhibition enzyme immunoassay (CIEIA) - This is
a microtiter plate competitive inhibition EUSA which employs an antigen-coated
(dichlorophenol-protein) solid phase. The competition is between analyte (free
antigen) in the sample and solid-phase antigen for binding to primary antibody in
solution. A secondary antibody conjugated to an enzyme is used as a label to
quantitate the amount of primary antibody bound to the antigen on the solid-phase.
The method has a detection limit of about 30 ppb and has a linear dynamic range
from 30 ppb to 400 ppb. The method is designed to be used for quantitative
laboratory analysis of water samples containing PCP.
STANDARD ANALYTICAL METHOD:
The comparison method is gas chromatography/mass spectrometry (GC/MS) by EPA Method
8270 following the EPA Method 3510 extraction protocol. BtoTrol. Inc.. will also be analyzing
selected bioreactor samples by high-pressure liquid chromatography (HPLC). These data will be
available for comparison with the immunoassay methods.
KEY REAGENTS:
Rabbit polydonal antiserum and a pentachlorophenol-enzyme conjugate are used for the rapid
field PCP immunoassay. A rat monoclonal antibody and a coating antigen of dichlorophehol-
thyroglobulin are used in the standard laboratory PCP immunoassay.
PROJECT DESCRIPTION:
The MacGillis & Gibbs Superfund SITE in New Brighton. Minnesota, is the location of a lumber
and pole company where ground water has become contaminated with PCP and polyaromatic
hydrocarbons (PAHs). In July 1989, BtoTrol. Inc., plans to demonstrate a biological reactor
consisting of bacteria that can degrade PCP and PAHs to carbon dioxide, water, and inorganic
chloride. In conjunction with the bioreactor demonstration, ground water and effluent samples
from within the bioreactor process cycle will be analyzed for PCP and PAHs (and other
compounds) using GC/MS, EPA Method 8270.
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Westinghouse Bio-Analytic Systems (WBAS) has developed a field portable immunoassay kit for
PGP determination. The kit is designed to provide a rapid, sensitive, and inexpensive field
screening method. The immunoassay and the bkxeactor SITE demonstrations will run
concurrently.
The objective of the immunoassay SHE demonstration is to evaluate the ruggedness and utility
of the WBAS rapid field immunoassay kit. This will be accomplished by taking grab samples and
spirts of composite field samples taken for GC/MS analysis as part of the BioTrol reactor
demonstration. These samples, along with splits of field blanks, field duplicates, and QA
samples, will be analyzed by the field Immunoassay kit at the site and by both the field
immunoassay kit and the standard laboratory plate ELJSA (also developed by WBAS) at two
different laboratories, EMSL-LV and WBAS. All field samples selected for on-site analysis (see
Appendix I) will be analyzed by the laboratory-based immunoassay, at both laboratories. During
the first and third weeks of the immunoassay demonstration, selected field samples will be
analyzed by the field Immunoassay kit In both laboratories (as well as on site). These analyses
will provide laboratory-versus-field site and laboratory-versus-laboratory comparison data. The
performance of the rapid field PCP assay will be evaluated and compared with the standard
laboratory plate Immunoassay and with GC/MS results.
PROJECT ORGANIZATION:
The following diagram summarizes the project organization. Splits of field samples to be
analyzed for PCP will be analyzed by the field assay at the site and by both the field assay and
the standard laboratory plate EUSA at two laboratories: EMSL-LV and WBAS. Results from
each of the methods will be compared. Immunoassay and GC/MS results from different
laboratories will also be compared.
Contact persons for participating agencies, contractors, and companies involved are listed below:
Or. Jeanette Van Emon EMSL-LV Project Manager
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Post Office Box 83478
Las Vegas, NV 89193-3478
(702) 798-2154
Mr. Richard White or LESC Technical Lead
Mr. Mark Silverstein LESC SITE Program Lead
Lockheed Engineering and Sciences Company
Environmental Programs Office, Suite 120
1050 East Flamingo Road
Las Vegas, NV 89119
(702) 798-2165 or
(702)734-3291
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Dr. Steve Soileau WBAS Technical Lead
Westinghouse Bio-Analytic Systems
15225 Shady Grove Road. Suite 306
Rockvilte, MD 20850
(301) 921-0031
Dr. Herb Skovronek SAIC Deputy Project Manager
Science Applications International Corporation
One Sears Drive
Paramus. NJ 07652-3518
(201) 599-0100
Mr. Thomas Chresand BtoTrol, Inc.. Technical Lead
BioTrol, Inc.
11 Peavy Road
Chaska. MN 55318
(612) 448-2515.
Ms. Mary K. Stinson RREL Project Manager
Chemical Engineer
Releases Control Branch
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Woodbridge Avenue (MS-104)
Edison, NJ 08837-3679
(201) 321-6683
Mr. Gene Easterly EMSL-LV QA Officer
U.S. Environmental Protection Agency
Environmental Monitoring Systems Laboratory
Post Office Box 93476
Las Vegas. NV 89193-3478
(702) 798-2108
Ms. Susan M. Brustman State Public Information
Public Information Officer Officer
Minnesota Pollution Control Agency
520 Lafayette Road
SL Paul. MN 55155
(612) 296-7769
The most Important responsibilities of participants in the site demonstration are listed below:
1. WBAS will provide sufficient quantities of both the field- and the laboratory-based
immunoassay kit reagents to complete the study. In addition, WBAS will provide
detailed protocols for both the rapid field and the standard laboratory immunoassays.
QA/OC guidelines and performance data are to accompany both immunoassay
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method protocols. WBAS la responsibla for performing preliminary matrix and analyte
stability studies with field samples. WBAS will provide adequate training for Science
Application International Corporation (SAIC) contractors in preparing and diluting
samples and running the field assay at the demonstration site. WBAS will analyze
field sample splits In Hs laboratory by both the field kit and laboratory-based
immunoassay methods and send results to EMSL-LV for timely comparison. WBAS
will act as a source of technical support to those performing the immunoassays at
the demonstration site and at EMSL-LV.
Z EMSL-LV is responstole for preparing a detailed site demonstration plan and quality
assurance project plan (QAPjP). This involves planning, scheduling and coordinating
activities associated with the SITE demonstration, and writing a detailed sampling
plan. In addition. EMSL-LV will prepare the data quality objectives and design a
system for data handling and statistical analysis. EMSL-LV is responsible for
analyzing splits of field samples by both Immunoassay methods. EMSL-LV will
provide confirmatory technical support for developmental work and assay
performance evaluation conducted at WBAS. EMSL-LV will write a detailed evaluation
report on the technology, which contains thorough statistical comparisons of method
data and addresses important QA/QC parameters. EMSL-LV will run a few QA
samples (6) and field samples (6) by GC/MS to provide interlaboratory comparison
data.
3. SAIC Is responsible for performing the field Immunoassay analyses. This will Involve
making splits of composite samples, preparing and diluting samples, analyzing
samples, and calculating and recording results. SAIC will label, prepare, and ship split
samples, and field assay results, to the two immunoassay laboratories, EMSL-LV and
WBAS. SAIC's responsibilities associated with the BtoTrol bioreactor demonstration
are detailed in the bioreactor SITE demonstration plan (SAIC, 1989). As part of the
bioreactor demonstration, SAIC will run GC/MS analysis of each influent and effluent
composite sample collected. SAIC will provide GC/ MS results for comparison with
the immunoassay results. SAIC will also provide other types of logistical support
on-site.
QUALITY ASSURANCE DESIGN AND RESPONSIBILITIES:
The LESC Technical Lead has the responsibility for drafting the QA Plan for the project. Field
immunoassay results will be reviewed (checked for errors, completeness, and consistency) by
laboratory personnel at both WBAS and EMSL-LV. If problems arise, laboratory personnel will do
the necessary experimentation and take appropriate corrective actions. Field personnel will be
notified of any necessary corrective actions.
A minimum number of splits from influent and effluent samples as well as two QA audit samples
will be interpreted during the preliminary evaluation phase. The data will be reviewed at both
immunoassay laboratories. This will provide an opportunity to make necessary corrective actions
prior to starting formal data collection.
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In the initial stages of the formal data collection (starting on approximately July 24). an auditor
(EPA) will perform an on-site technical systems audit. The purpose of these audit is to check
that important aspects of the analysis system are operational. These include sample handling;
sample tracking and storage; and procedures for diluting, analyzing, and shipping samples.
At EMSL-LV, the field and laboratory immunoassay results will be reviewed and compared to
GC/MS results for the six QA audit samples and ten selected field samples. A determination will
be made as to whether the immunoassay results satisfy the QC guidelines established for each
method.
After all the data have been collected, tabulated, and reviewed and the statistical analysis and
comparisons have been done, the final project evaluation report will be prepared. It will contain a
summary of all OA/QC activities associated with the project.
Required Quantities: The estimated minimum quantities of reagents necessary for the SITE
demonstration are as follows:
1. Approximately SO field kits, or enough for 15-17 kits for the field analysis, and 15-17
kits for both EMSL-LV and WBAS. Each of these kits should contain enough reagents
to run ten 8-well strips.
2. Enough standard laboratory immunoassay reagents to run 30-40 plates, or 15-20
plates for each of the two laboratories, EMSL-LV and WBAS.
The developer, WBAS, has provided assurance that the required quantities of immunoassay
supplies and regents have been made and will be available in time for the beginning of the
demonstration.
Storage and Stability: Both field kit and standard laboratory kit components are stable for at
least several weeks if stored at 4 degrees C. Standards and QA samples will be stored at 4
degrees C in methanol, in sealed ampoules or auto sampler vials with teflon-lined caps. The PCP
analyte is very stable under these conditions.
Sample Handling and Cleanup: For the rapid field test, effluent samples will be centrifuged and
adjusted in pH, if necessary. In case samples have high concentrations of metal ions, ft will be
necessary to add EDTA to the buffer used for sample dilution.
For the standard laboratory CIEIA, the standard operating procedure (SOP) supplied by WBAS
will be followed. This involves pH adjustment, sample preparation in 25 percent 2-propanol and
serial dilutions. Samples with internal spike values outside the acceptable range may undergo
cleanup and concentration by solid phase extraction. The effluent samples may require special
handling due to bfomass and microorganisms which could degrade residual PCP and interfere
with immunoassay components. It may be necessary to centrifuge the samples in a table top
centrifuge.
As described in the bioreactor SITE demonstration plan, chain of custody procedures will be used
for tracking of samples for the bioreactor demonstration. For immunoassay analysis in the two
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laboratories (EMSL-LV and WBAS), formal chain of custody procedures will not be necessary.
Samples will be carefully inventoried and archived when they are received.
Caltoratton: A Oynatech microwell strip reader (model number 4025-051) will be used at WBAS
and EMSL-LV laboratories and the field site. During the preliminary evaluation phase, these
readers will be cross-calibrated with each other and with laboratory-based HP/Genenchem(TM)
plate readers. This will be accomplished by making several dilutions of a yellow dye or 2.4-
dinitrophenol-glvcine at EMSL-LV and sending splits of the dilutions to the other locations for
measurement of absorbance at 405 nm on both the strip readers and the laboratory microtiter
plate readers. Prior to use, all micropipettors to be used in the study will be checked for accuracy
and precision by a gravimetric testing procedure using an analytical balance.
DATA QUALITY OBJECTIVES:
Central Decision or Question: The goal of this study is to evaluate the ruggedness and utility of
the WBAS Rapid Field Immunoassay for PCP. The primary objective is to determine whether the
rapid field test can provide useful qualitative or semi-quantitative screening information for
detecting pentachlorophenol in ground water and for monitoring the effectiveness of remedial
action at a Superfund site.
DATA QUALITY OBJECTIVES-ACCEPTANCE CRITERIA:
To be useful as a field screening tool, a field method must be capable of measuring the target
analyte with sufficient accuracy and precision to provide criteria for selecting which samples are
clearly above, below, or near a critical concentration range. The methods are intended to be used
as a supplement to conventional analytical methods used to measure pollutants in environmental
samples. Used as a screening tool, these methods can be used in selecting which field samples
need to be analyzed by conventional analytical methods. The end result will be a more efficient
and cost-effective system for analyzing field samples.
In this study we are limited as to the number and type of samples which can be analyzed. The
sampling and analysis scheme had to be designed to integrate into the plan that had been
written for the BtoTrol Inc. bioreactor SITE demonstration plan. Within the constraints of the
study, the goal Is to evaluate the ruggedness and utility of the field immunoassay method as a
rapid field screening tool. The other key objective is to obtain information regarding the
limitations and range of applicability of the method. The sampling design chosen for the
immunoassays is sufficient to achieve these goals.
To adequately demonstrate the utility of the field PCP immunoassay as a field screening method,
the following data quality objectives are proposed:
1. For field samples, the field immunoassay test result should not differ from
the GC/MS result by more than a factor of two.
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2. The maximum coefficient of variation (CV) for the QC performance
standards (20 pom), which are diluted and run by different operators on
different days, should not exceed 50 percent.
3. The QA audit sample field test result should be within ±50 percent of the
ejected value.
These criteria are proposed as guidelines for evaluating the quality and validity of the semi-
quantitative (or qualitative) information obtained from the test results. If the data do not satisfy
the objectives stated here, the basic objectives of the SITE demonstration may still be met.
Useful information regarding the limitations and applicability of this particular method for field
testing will have been obtained. The outcome and conclusions will be discussed in the final
evaluation report.
SEVEN ELEMENTS OF DATA QUALITY:
Critical Concentration Range: The critical concentration range of PCP analyte for this study is
approximately 0.2 ppm to 50 ppm. The ground-water samples flowing into the bioreactor will
contain approximately 45-50 ppm PCP. Effluent samples will vary in concentration. If the reactor
is working as expected, final effluent PCP values should range from 0 to about 1 ppm. Effluent
PCP levels may vary with flow rate through the reactor.
The Metropolitan Waste Control Commission (MWCC) standard value for release of PCP is 1
ppm. Bioreactor effluent, after solids removal and carbon filtration, must fall below 1 ppm before
release. The Minnesota Pollution Control Agency (MPCA) standard for PCP in water is 0.22 ppm.
Required Sensitivity: Both the rapid field and the standard laboratory immunoassays are
sensitive into the parts per billion (pob) range. Both methods are sensitive to below the MWCC
and MPCA standards.
Environmental Samples: The ground-water samples taken from one well at the MacGillis and
Gibbs site contain PCP (40-50 ppm) and polyaromatic hydrocarbons. The samples may contain
iron and other metal tons. The measured pH is about 6.8. The influent may have to be run
through an oil/water separator prior to treatment. The influent samples will be adjusted to pH 7.4
and 25 degrees C, and nutrients will be added prior to flowing into the reactor. Splits of
composite influents for GC/MS and immunoassay will be taken at this point.
Site Selection: The MacGiOis and Gibbs site is an ideal location for a demonstration of the WBAS
Rapid Field Immunoassay Kit because the ground-water samples are relatively clean (e.g., low in
sediment or turbidity) and contain high (ppm) concentrations of PCP. Since the sampling and
analysis plan was already in place for the BioTrol bioreactor demonstration, it was not necessary
or practical to design a sampling and analysis scheme from scratch. The bioreactor demon-
stration provides an opportunity to demonstrate the ability of the immunoassay method at a site
where remedial actions are under way, and where comparative Contract Laboratory Program
(CLP) data is being collected.
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Field Sampling Deaion: The sampling design for the immunoassay demonstration will coincide
with that set forth in the BioTrol bioreactor demonstration. Selected samples which are taken for
GC/MS analysis will be split and run by both the rapid field and the standard laboratory assays.
The first part of the bioreactor SITE demonstration will consist of three two-week phases, and for
each, the reactor will be run at a different flow rate. Composite samples will be collected for
GC/MS analysis six days a week. Immunoassay analysis of key samples is listed in Appendix I.
The number of splits, QA standards, duplicates and blanks selected are sufficient to provide
statistically valid conclusions from the data (Appendix I).
The following provides a brief description of each sample type and the total number that will be
analyzed. Approximately 18 influent and 12 effluent samples will be run during the three weeks
immunoassay samples are being run. An additional 1 or 2 influent and effluent samples will be
run during the preliminary evaluation phase.
Environmental Samples: Splits of selected grab samples and composite influent and effluent
samples taken for GC/MS analysis will be assayed using the rapid field immunoassay and the
standard laboratory CIEIA in the laboratories (EMSL-LV and WBAS). During the first and third
weeks of the immunoassay demonstration, selected field samples will be analyzed using the field
immunoassay kit in both laboratories. Selected samples (see Appendix I) will be run by the rapid
field immunoassay, on site. Each sample will be run at several dilutions, both for range finding
and in duplicate at optimal dilution, with and without an internal standard spike. Each day, field
blanks (instrument water rinses) will be run undiluted and diluted ten fold. In addition, on a daily
basis, a minimum of 2 to 3 negative controls (in buffer from the kit) will be run undiluted and a
single dilution of a 20 ppm performance standard will be run on one strip. This 20 ppm standard
will be prepared at EMSL-LV and will be sent to the field and to the WBAS laboratory. It will be
run as a known standard, diluted to an optimal level, and run in single wells on one strip, on
separate days.
On two days each week, field duplicate pairs (influent on one day, effluent on the other) will be
run in duplicate in both the rapid field and standard plate ELJSA. If possible, these field
duplicates will be the same ones selected by SAIC for GC/MS analysis in order to provide a direct
comparison.
On one day each week, a grab sample of raw (untreated) ground water will be taken. SAIC will
analyze a split of this sample by GC/MS. Splits of this sample will be analyzed by the rapid field
immunoassay on site, and by both immunoassay methods in the two laboratories.
QA Samples: The same QA samples (single analyte PCP and mixed phenols, with PCP) will be
analyzed by all three methods: the field immunoassay, the laboratory immunoassay and GC/MS.
These will be prepared from single lots of EMSL-Cincinnati performance evaluation samples (of
each type) by dilution in methanol. These dilute QA samples will be prepared at EMSL-LV, placed
in ampules and sent to the field site, WBAS, and to SAIC (for GC/MS analysis).
On three days each week, several dilutions of the PCP QA samples (two single analyte and one
mixed phenols) will be analyzed using the field assay on-site, using both the laboratory and the
field immunoassays, at WBAS and EMSL-LV. These samples will be run semi-blind using the field
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immunoassay on-site. That is, the recommended dilutions (but not the concentrations) will be
provided.
At least six ampules of the same diluted QA samples prepared for immunoassay analysis will be
analyzed by GC/MS at both SAIC and EMSL, during the study. At least six splits of field samples
will be analyzed by GC/MS at the EMSL-LV laboratory.
Controls and Blanks: Each day on the first strip run, an instrument rinse field blank will be run at
two different dilutions and a negative control (1X buffer from kit) will be run in duplicate. The field
blank will consist of a laboratory water rinse of the ISCO composite sample collector, after it has
been cleaned (SAIC, 1989).
Internal Standard Spikes: Each effluent field sample split will be diluted and run in duplicate with
and without a 15 ppb internal standard. This will be done after determining the proper sample
dilution by running 2-3 range-finding dilution strips. One daily influent sample per week will also
be run in duplicate, with and without a IS ppb internal standard spike.
QC Performance Standards: Each day, one 20 ppm performance standard will be am at a single
dilution on one strip in the field immunoassay, on-site. This will provide a minimum of 18
replicates of a known standard for charting and precision analysis.
KEY INTERFERENCES:
Ground-water samples may contain Iron and other metal ions, polyaromatic hydrocarbons,
mixtures of phenols, and possibly oil. The extent of interference from these sources is estimated
to be minimal, since large dilutions of samples will be done prior to analysis. The effluent
samples will contain biomass and bacteria, inorganic and organic nutrients, and a mixture of
unknown metabolites. The effects of other compounds in the matrix on the immunoassay is
unknown and will have to be studied as part of the preliminary evaluation phase.
SPECIFICITY:
The rabbit polyclonal antibody used for the field PCP immunoassay method cross-reacts with
2,3,5,6-tetrachlorophenol (19 percent), several trichlorophenols (7 percent), and tetryhydroquinone
(11 percent). Phenol and dichtorophenols do not interfere significantly (see Table A-1). The rat
monoclonal antibody used for the laboratory PCP immunoassay cross-reacts with 2,3,5,6-tetra-
chlorophenol (42 percent), 2,4.6-trtahlorophenol (12 percent), and 2,3,5,6-trichlorophenol (8.8
percent). As with the rabbit polyclonal, the monoclonal does not cross-react significantly with
phenol or dichlorophenols (see Table A-2).
ASSESSMENT OF DATA QUALITY:
Statement of Intended Use: The data obtained in this SITE demonstration is to be used by the
EPA to evaluate the ruggedness and utility of the WBAS field PCP immunoassay. The objective is
10
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TABLE A-1. PENTACHLOROPHENOL FIELD ANALYSIS KIT CROSS-REACTIVITY PROFILE
CHEMICAL PERCENT CROSS-REACTIVITY
PENTACHLOROPHENOL
2.3.5.6-TETRACHLOROPHENOL 19
2.4.6-TRICHLOROPHENOL 7
2.3.6-TRJCHLOROPHENOL 7
2.4.5-TRICHLOROPHENOL 0.4
2,6-DICHLOROPHENOL 0.7
TETRYHYDROOU1NONE 11
2,3.4-TRICHLOROPHENOL 2.5
Z4-DICHLOROPHENOL <0.1
Z5-OICHLOROPHENOL <0.1
3,5-DICHLOROPHENOL <0.1
3.4-OICHLOROPHENOL <0.1
2.3-OICHLOROPHENOL 0.2
4-CHLOROPHENOL <0.1
PHENOL <0.1
PENTACHLOROBENZENE <0.1
2.3-OINITROTOLUENE <0.1
2.4-DINITROTOLUENE <0.1
2.4.5-TRICHLORONITROBENZENE <0.1
to determine whether this immunoassay can provide qualitative or semi-quantitative field
screening information, which is useful for site characterization and monitoring remedial actions at
Superfund sites. The end result of this and possible future SITE demonstrations for this
technology will be a comprehensive report which covers specific recommendations and limitations
for use.
Accuracy: Accuracy will be assessed by comparison of the field assay results with those
expected for QA audit sample spikes and performance standards. Accuracy will also be
evaluated by comparison of the field immunoassay results with those obtained by GC/MS and the
11
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TABLE A-2. SPECIFICITY OF PCP MONOCLONAL ANTIBODIES
Compound
Pentachlorophenol
2.3,5.6-Tatrachlorophenol
2.4.6-Trichlorophenol
2.3,6-Trtentoropnenol
2,6-Dichlorophenol
Tetrachtorohydroquinone
2,3.4-Trtahlorophenol
2,3.5-Trichlorophenol
2.4-Dtenkxopnenol
2,5-Dtahlorophenol
3,5-Oichlorophenol
3.4-Otahlorophenol
2,3-Dichlorophenol
4-Chlorophenol
Phenol
Pentachloroaniline
Pentachlorobenzene
Z3-Dinitrotoloene
2.3-Oinitrotoluene
2.4-Dinrtrotoluene
2,4,5-Trichkxonitrobenzene
Molar ICM"
12 (± 0.3) xlff4
5.3 (± 0.6) x NT*
1.8 (± 0.3) x 10*
2.5 (± 0.1) xlff4
12 (± 0.1) x 10"
2.8 (± 0.1) x Iff*
4.5 (± 0.3) xlff4
4.3 (± 0.3) x Iff*
NI"
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Cross-Reactivity
wrthPCP"
__
42.0
12.0
8.8
1.8
0.8
0.5
0.5
0
0
0
0
0
0
0
0
0
0
0
0
0
* Molar concentration of compound that inhibits 50 percent antibody binding in the "normal"
competitive inhibition enzyme immunoassays.
0 PCM PCP/IC.O compound] x 100
c NI - Not inhibitory; 1.0 x Iff4 M
Source: Courtesy of WBAS
laboratory Immunoassay method. Confidence intervals for each methods test result will be
determined and the degree of overlap will be quantitated.
Precision: The precision of the field immunoassay method has been evaluated by having two
analysts run six replicates of each kit standard (five different levels). Field kit precision on-site
will be evaluated by repetitive analysis of blanks. 20 ppm performance standards (diluted), and
QA audit samples. A minimum of six single analyte PCP QA samples and three phenols mixture
(with PCP) QA audit samples will be analyzed for the entire study. A minimum of 18 replicates of
the performance standard (single dilution) and the negative control (one X sample dilution buffer)
wiU be run.
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Each week one field duplicate pair of one influent and one effluent sample will be analyzed on
duplicate strips. In addition, duplicates of each effluent tested will be run. diluted to 3-20 ppb,
and with and without internal standard spikes (15 ppb).
Internal Standard Spike Recovery: The internal standard spike recovery data for the field
immunoassay is intended to provide information on the relative levels of matrix Interference in the
field samples after dilution to the appropriate concentration range.
For the standard laboratory CIEIA, the internal standard spike recovery will be used as a QC
check on the matrix effects. Spaced samples that do not fall within ±25 percent of the expected
value may be cleaned up by solid phase extraction and reanalyzed.
Detection Limit: The detection limit for the field immunoassay test is defined as follows: any
sample with an absorbance greater than 90 percent of the absorbance range should be reported
as less than the Method Detection Limit (MOL). The approximate MDL is 1-3 ppb of POP. The
MDL for the standard laboratory CIEIA is defined the same way: any sample with an absorbance
greater than 90 percent of the absorbance range should be reported as less than the MOL The
approximate MOL is 30 ppb of PCP.
(NOTE: MOLs provided by manufacturer; no official MDL studies have been performed).
Representativeness: See the document entitled 'Quality Assurance Plan for Immunoassay
Evaluation and Research Projects* (White and Miah. 1989) for a general discussion. For this SITE
demonstration, the field samples consist of PGP-contaminated groundwater samples and
bkxeactor effluent samples. The groundwater samples may or may not be representative of
groundwater taken at other wells or sites. The effluent samples from the btoreactor are
representative of the type of matrix that would be commonly found with this type of remedial
action. The immunoassay demonstration at this site Is limited to the sampling design chosen for
the bioreactor demonstration. Although the data collected will be representative of the utility of
the test at a single site, it may be necessary to sample water from a variety of wells (and sites)
in future demonstrations to show the full capability of the method.
Completeness: See the QA document mentioned above (White and Miah, 1989) for a discussion
of this element of data quality as it refers to immunoassay projects. The completeness objective
of 90 percent (of the expected number of samples) established for immunoassay projects in
general is also applicable for this SITE demonstration.
Comparability: See the QA document mentioned above (White and Miah, 1989) for a discussion
of this element of data quality as it refers to immunoassay projects. To meet EPA requirements,
study data must be reported in the same units as other projects using the same technology. For
this SITE demonstration, comparability will be addressed by comparing the on-site field immuno-
assay results with the standard laboratory immunoassay results obtained at two laboratories
and the GC/MS results run at one or two laboratories. WBAS has written a detailed SOP
containing QC protocols and acceptance criteria for the standard laboratory Immunoassay and
the solid phase extraction procedure. WBAS has sent a protocol for the field PCP kit which
contains a QC protocol and strip acceptance criteria (see Appendix II). Copies will be sent to all
parties involved and strict adherence to the immunoassay test and QC protocols will be stressed.
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The field immunoassay kit protocol is easy to learn and WBAS will train the SAIC contractors
who will be performing the test in the field. WBAS will send kits and a detailed protocol to SAIC.
After SAIC performs the immunoassay. WBAS will discuss questions and problems with them.
An SAIC representative will travel to WBAS for more in depth training during the weeks prior to
the start of the demonstration. A representative from WBAS will be on-site to provide assistance
during the first 2-3 days of the formal demonstration.
Specificity/Interference: A phenols mixture (containing PCP) QA audit sample will be analyzed
using the field immunoassay on-site and at both laboratories by the standard laboratory immuno-
assay. At least three of these will be analyzed during the demonstration period. If possible,
depending on laboratory commitments, these standards will also be analyzed by GC/MS by both
SAIC and EMSL-LV. Analysis of this mixture will provide information on the specificity of the anti-
bodies used for the immunoassay methods (also see Tables A-1 and A-2).
All field samples analyzed by the laboratory immunoassay will be run with and without internal
spikes. A minimum number (3) of field influent and all effluent samples will be analyzed with and
without internal spikes. This will provide Information regarding the extent of sample matrix
interference. Possible matrix effects and interferences will be studied at WBAS and EMSL-LV on
representative groundwater. effluent, and surface water samples shipped from BioTrol. Inc.
during the preliminary evaluation phase of the btoreactor. The most effective sample pre-
treatment steps for influent and effluent field samples will be determined by WBAS during the
preliminary evaluation phase. The immunoassay field kit protocol and training sessions will
address all necessary sample pretreatment steps.
CORRECTIVE ACTIONS:
In the event that data from the field Immunoassay analysis of QA samples, performance
standards, or blanks fall outside the expected ranges (contained in data quality objectives and
the QC guidelines of the kit protocol) the strip analysis will be repeated. If the problem persists,
splits of field samples will continue to be made for immunoassay analysis, but field testing may
be suspended until WBAS and EMSL-LV laboratories can do sufficient testing to determine the
source of the problem. A minimum of 20-30 mL of each field sample wiU be archived at both
laboratories and on-site. Samples will be stored (at 4 degrees C) until the study is completed in
case it is necessary to reanalyze selected samples.
PERFORMANCE AND SYSTEMS AUDITS:
Performance Audit: Ongoing assessment of data quality will be accomplished through analysis
of a minimum of nine semi-blind (with only the recommended dilutions given) QA samples
(reference standard materials obtained from EMSL-Cincinnati). At least three of these will be
mixed phenols standards (EMSL-Cin reference standard C-090-02. acid extractabtes II, In
methytene chloride). Ampoules of the OA samples will be made by EMSL-LV and distributed to
the field site and to WBAS. The same nine QA samples (6 PCP only [EMSL-Cin reference
standard EV-062-03-13 in methanol] and 3 mixed phenols with PCP) will be assayed in the field
PCP immunoassay at the site and both the field and laboratory immunoassays at WBAS and
14
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EMSL-LV. An external reviewer has the responsibility for comparing the results from both the field
and laboratory methods with the expected results for the QA audit samples.
Site Systems Audit: At some time during the initial stages of the formal data collection, an on-
site systems performance audit will be conducted by an individual who is familiar with the
technology and who has been provided with a check list of critical procedural items. This list will
be prepared by EMSL-LV. The results of the audit will be summarized in the final project
evaluation report.
PRELIMINARY EVALUATION PHASE:
A minimum of two QA samples (one mixed phenols, one PCP only) and 2-3 influent and effluent
samples from the field (if available) will be analyzed by both immunoassay methods. Results
from the two methods will be compared with the expected results for standards or with HPLC
results for field samples. This will provide an opportunity to take any necessary corrective
actions, before the formal data collection starts.
QUALITY CONTROL PROTOCOLS:
QC protocols for the standard laboratory PCP immunoassay are detailed in the SOP for the
method and in the "Quality Assurance Plan for Immunoassay Evaluation and Research Projects*
(Whhe and Miah. 1989).
WBAS has provided QC protocols for the field immunoassay (see Appendix II). They have done a
solid-phase variability check on the strips and will cross-calibrate the field- and laboratory-based
strip readers, as will EMSL-LV.
QC protocols for the solid phase extraction method are contained in the SOP for the laboratory
PCP Immunoassay method.
The QC protocols contained in EPA method 0270 will be used for GC/MS analysis at both SAIC
and EMSL-LV.
DATA HANDLING:
A field form will be used for identification and tracking of samples. A draft copy is included In
Appendix II. This form contains all the necessary information regarding the details of collection,
storage, time, date, and operator; as wen as the analytical results. A copy of this form will be
sent to the WBAS and EMSL-LV laboratories. The reporting units for the immunoassay data are
parts-per-million (ppm) or ug/ml. Field and laboratory analysis data will be stored in personal
computers (PC), and backup copies will be made on floppy disks.
15
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For the field immunoassay, standard curve data points will be plotted on semi-log graphs and
fitted by hand. The same data can also be manually entered into a PC in the laboratory and fitted
by other methods, for comparison.
Laboratory immunoassay results will be handled with the Hewlett-Packard Titer-Cate* software
with 4-parameter curve fitting. Data will be stored in the computer and on backup disks.
DATA VALIDATION, REDUCTION, AND REPORTING:
On-srte. field PCP kit data will be calculated from semi-log plots of kit standards made for each
eight well strip. The field forms will then be sent daily to both WBAS and EMSL-LV laboratories.
where the field data will be initially reviewed, as received, for completeness, consistency, and
falling within expected ranges. The data are keyed into a PC using a standard spreadsheet or
statistical software. Backup copies will be kept on floppy disks. Any necessary corrective
actions (e.g., confirmation of suspect data) will be taken, and then selected samples will be
reanalyzed by the field kit in the laboratory. Samples are prepared as necessary and analyzed
using the laboratory PCP immunoassay. If samples with the internal standard spikes are outside
of specification, samples will be reanalyzed after cleanup. Data from both the laboratory and
field PCP Immunoassay analyses will be entered into the computer data base. All data entries
will be checked for accuracy by a second party. Data received from the GC/MS analyses at both
SAIC and EMSL-LV will be reviewed and entered into the computer data base for comparison with
the immunoassay data. After further detailed review by the project leader and EPA Technical
Monitor for technical validity, such as incorrect number of significant figures or inconsistency in
results for the same sample analyzed at different locations or at difference dilutions, selected
samples will be reanalyzed after necessary corrective actions are taken.
The data from GC/MS analysis of the 6-9 QA audit samples and the six selected field samples will
be reviewed. If necessary, samples will be reanalyzed by GC/MS.
AJI immunoassay and GC/MS data entered into the computer data base will be organized and
tabulated for easy comparison. The PC will be interfaced with the VAX mainframe computer and
the data will be analyzed using Statistical Analysis System (SAS) and other available statistical
computer software.
A final report containing tabulated data, interpretations, conclusions, and QA/QC summary will be
prepared. The report will be reviewed by the EPA Technical Monitor and circulated for internal
peer review. The data may also be published or presented at scientific meetings for external peer
review.
STATISTICAL EVALUATION:
The data analysis will consist of two phases. In the first phase the quality of data will be
assessed. This will consist of a number of standard procedures. QA/QC data will be examined
(via time trends and descriptive statistics) to ensure that consistent results and adequately
accurate measurements are available. This will require information from the various blanks, audit
16
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samples, splits and duplicates. This form of analysis will be carried out for each analysis method
at each she. Also, based on analysis splits and within- and between-atrip replicates, a simple
analysis of variance (ANOVA) may be performed to isolate major components of error.
The second phase of analysis will be the comparison between methods/sites. Of primary
concern will be the comparison between routine GC/MS values and the field strip test results.
Given positive results from the first phase of analysis, we will assume that the GC/MS results are
the true* values with respect to the accuracy expected from the immunoassay method. The
comparison will be looked at via summary statistics, t-tests for differences, correlation of results,
and XY plots. Similar analyses are planned for comparing the results from the laboratory-based
immunoassay method with the GC/MS results, although the primary focus will be on the field
method. We wil also try to estimate whether laboratory bias is significant and whether method
to method bias exists.
In summary, the data analysis methodology is not overly complicated. However, the number of
inter-laboratory and inter-method comparisons involved will amplify the amount of time required
to complete the analysis. See Appendix III for equations used to calculate statistics.
REPORTS FOR MANAGEMENT:
A short summary report will be written at the conclusion of the preliminary evaluation phase. The
report will contain a comparison of the data obtained by the rapid field method and the laboratory
method in the two laboratories. Results obtained by both methods on the two QA audit samples
(one PCP only, one mixed phenols, containing PCP) will be compared with the expected results for
the standards and the GC/MS results from at least one laboratory. Laboratory and field
immunoassay results will also be compared with BtoTrol HPLC results, whenever possible. The
report will contain a brief assessment of the quality of the data, as well as any significant
problems encountered. Approaches to alleviate or eliminate problems will be discussed.
The final project evaluation report will follow the guidelines written for project final reports within
the •Quality Assurance Plan for Immunoassay Evaluation and Research Projects* (White and
Mlah, 1889) as it applies to the SITE evaluation report. This report also follows the current
guidelines for SITE evaluation reports. The report will contain detailed analysis and interpretation
of the data collected and a summary of the quality assurance activities associated with the
project. A copy of the final report will be reviewed by the EPA Technical Monitor and other EPA
and LESC managers involved with the project. The report will be subject to both internal and
external peer review.
17
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APPENDIX I
SAMPLING AND ANALYSIS SCHEDULE
FOR THE FIELD PCP IMMUNOASSAY KIT SITE DEMONSTRATION
PRELIMINARY EVALUATION (bioreactor start-up) 26 days. June 26-July 21.
PART A-(bkxeactor flow rate= 1 gal/min) 6 days, July 24-29
PART B-(bk>reactor flow rate- 3 gal/min) 6 days. August 7-12
PART C-(bk>reactor flow rate- 5 gal/min) 6 days, August 21-26
Use the same sampling and analysis scheme for all three parts. Effluent samples are from
reactor 3 (final), unless otherwise stated. The samples referred to as the routine influent or
effluent is a split of the composite sample taken for GC/MS analysis, for that day.
The routine influent will be taken from the holding tank, containing treated (nutrients added, pH
adjusted to 7.2) well water. In addition, once a week, a grab sample of raw or untreated well
water will be taken for GC/MS analysis. A split of this grab sample is to be analyzed by the field
PCP immunoassay method also. If possible, do the analysis schemes in the order listed: DAY 1
first, DAY 2 second, and DAY 3 third. The short day routines are for days when the person doing
the field analysis is busy with other activities and the long day routines are for days when there
is more time available. The 4 kit standards that are run on each strip are the following: 3 ppb. 7.1
ppb, 16.9 ppb, and 40 ppb. Each day. run the OC STRIP first.
All samples should be run as soon as possible after collection. Try to run all samples within a 24
hr period after splitting. Try to complete the analysis of all the scheduled samples (see the
weekly totals list below) within each of the three 6 day periods. A minimum volume of 20 ml from
each field sample analyzed should be placed in a 30 ml amber vial, labelled with an SAIC label
(see example below) and capped tightly with a teflon Ikied screw cap. All these vials are to be
stored at 4 degrees C, in case later analysis is necessary. If time does not permit the analysis of
all the above samples during the 6 day week, unanatyzed samples are to be run on the following
week.
QC STRIP
LONG 1 strip with a flsld water blank, undiluted and diluted 1 to 10, a negative control
DAY 1 (1X kit dilution buffer) and a 20 pom performance standard, diluted 1 to 1000 + 4
kit standards.
M
72
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EFFLUENT STRIPS
1-2 strips with four semi-log serial dilutions (suggested dilutions: 1 to 10, 1 to SO, 1
to 100, and 1 to 500) of the routine effluent + 4 kit standards. 1 strip with the
routine effluent diluted to about 10 ppb (3-20 ppb) run in duplicate with and
without a 15 ppb internal standard spike -f 4 kit standards.
QA SAMPLE STRIPS
2 strips with three serial two-fold dilutions of a semi-blind (see instructions) type
A QA audit sample + 4 kit standards, and one negative control
INFLUENT STRIP
1 strip with routine influent at 4 dilutions (suggested dilutions: 1 to 1000,1 to 2000,
1 to 4000 and 1 to 8000) + 4 kit standards.
LONG
DAY 2
. SAME AS LONG DAY 1.
LONG
DAY 3
• SAME AS LONG DAY 1, EXCEPT RUN TYPE B OA AUDIT SAMPLE.
OC STRIP
SHORT 1 strip with a field water blank, undiluted and 1 to 10
DAY 1 diluted, a negative control (1X kit dilution buffer) and a 20 ppm performance
standard, diluted 1 to 1000 + 4 kit standards.
INFLUENT STRIPS
2 strips with routine influent at 3 dilutions (suggested dilutions: 1 to 1000.1 to
2000, and 1 to 4000) + 4 kit standards, and one negative control.
2 strips, with duplicates of both the routine influent and its field duplicate (split).
each diluted to near the midpoint (20+/- 10 ppb) of the standard curve -f 4 kit
standards.
1-2
73
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1 strip with split of the routine influent diluted to about 10 ppb (3-20 ppb) and run
in duplicate with and without a 15 ppb internal standard spike + 4 kit standards.
SHORT
DAY 2
OC STRIP
1 strip with field water blank, undiluted and diluted 1 to 10.1 negative control
(1X kit dilution buffer) and a 20 ppm performance standard, diluted 1 to 1000.
EFFLUENT STRIPS
2 strips each with a split of the routine effluent at 4 dilutions (suggested dilutions:
1 to 10.1 to 100,1 to 50 and 1 to 500) + 4 kit standards.
2 strips, with duplicates of both the routine effluent and its field duplicate split.
each diluted to near the midpoint of the standard curve (20+/-10 ppb) + 4 kit
standards.
INFLUENT STRIP
1 strip with the routine influent at three dilutions (suggested dilutions: 1 to 1000,
1 to 2000. and 1 to 4000) + 4 kit standards and one negative control.
QC STRIP
SHORT 1 strip with a field water blank, undiluted and diluted 1 to 10. a negative control
DAY 3 (1X kit dilution buffer) and a 20 ppm performance standard diluted 1 to 1000 + 4
kit standards.
INFLUENT STRIPS
2 strips with split of the routine influent at 3 dilutions (suggested dilutions: 1 to
1000, 1 to 2000.1 to 4000) + 4 kit standards, and one negative control.
1 strip with split of the routine influent diluted to about 10 ppb (3-20 ppb) and run
in duplicate with and without a 15 ppb internal standard spike + 4 kit standards.
1-3
74
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DAY FOR INFLUENT GRAB - After running the other strips scheduled for this day. run the
following.
INFLUENT STRIPS
2 strips with split of the raw influent grab sample at 3 dilutions (suggested
dilutions: 1 to 1000, 1 to 2000,1 to 4000) + 4 kit standards, and one negatlv
control.
WEEKLY TOTALS
1) 6 routine (daily) influent samples
2) 4 routine (daily) effluent samples
3) 1 field duplicate split of a routine (daily) effluent
4) 1 field duplicate split of a routine (daily) influent
5) 1 raw (untreated) influent grab sample
6) 2 type A OA audit samples
7) 1 type B QA audit sample
8) 6 routine (daily) field water blanks (equipment rinse)
9) 6 daily replicates of the 20 ppm QC performance standard, diluted fresh each
day.
10) A minimum of 18 negative controls (1X dilution buffer from kit)
1-4
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APPENDIX II
^•^
jWestinghouse
'Bio-Analytic System
PENTACHLOROPHENOL FIELD ANALYSIS KIT
SUMMARY
The Ptntjchlorophenol Field Analysis Kit is an imnunoassay-based
)cit thac is used for the dotection of pentachlorophenol (pc_P) in
water. The detection limit of the "assay is approximately 1 parts
per billion (ppb) . However, the detection liait of the assay for
the purposes of the BioTrol field test is 3 ppb. The total tiae
to complete the test is approximately 30 min (not including sample
preparation time) .
KOTE: Please read all of *:he Instructions completely beforp
beginning vour first assay.
CONTENTS
Each PCP Field Analysis Kit should contain the following:
1) Rinse Solution (one bottle)
2) Eight-Well Strips (5)
3) PCP Standards (one bottle each)
1 part per million (ppm)
40 ppb
16.9 ppb
7.1 ppb
3 ppb
4) Negative Control (one bottle)
5) 100X Buffer (one bottle)
6) Substrate (one bottle)
7) Chromogen (one bottle)
8) Stop Solution (on* bottle) CAUTIOK? CONTAINS 0.3N
SPLFURIC ACIDt I I
9) Enzyme Conjugate (one bottle)
10) Instructions (one set)
If any reagents are missing, please contact Westinghouse Bio-
Analytic Systems Company at (301) 670-0688.
76
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Sample
aolic PCP c°n=«"«ation of the influent sample
Jill nariii SE " ?° Ppm and l Ppn' The
-------
a) Uaing a 1000 nicroliter 3ingla channel pipettor and tip,
transfer 462 aicroliters of the '1:32' sanple to tn« tube
labelled '1:100'. Mix wall, using the single channel
pipattor.
() Analyze the samples labelled '1:10', '1:32', and '1:100'
with the PCP Field Analysis Kit.
3) Quality Control Samples - The general procedure for dilution of
quality control samples is as follows. If the concentration of
the sample is known, prepare serial dilutions of the sample (be
sure no single dilution is greater than 1:10) until the sample
is diluted to 30 ppb. Label the tube with the sample number
and '30 ppb1. Prepare tvo 1:1 serial dilutions of the 30 ppb
sample in IX buffer and label the tubes '15 ppb1 and '7.3 ppb1.
If the concentration of the sample is not known, treat the
sample as you would an influent sample.
ANALYSIS KIT PROCEDURE
The time required to run tost is approximately 30 ainutes.
1. Remove strip(s) needed from the resealable bag.
MOTE: Strips are not reuseable. Remove the reagents from the
box and allow them to equilibrate at ambient temperature.
Place the strip (s) in the supplied strip holder. The square
end of the strip should be placed at the top of the holder
(The side with raised numbers). The Individual wells will be
identified by the letters that appear on the left side of the
strip holder.
2. Choose an appropriate strip format for your needs (Supplied
in the sampling protocol). Using a 200 aicrollter single
channel pipettor, add 50 microliters from the standard
bottles and appropriate sample test tubes to the appropriate
wells. Be sure and change pipet tips between each standard
and sample.
3. Using a 200 microliter single channel pipettor, add 50
microliters of the Enzyme Conjugate bottle to all the wells
being used.
4. Centlv tap the strips to mix the reactants t be careful not
to splash reagents out of the wells or into adjacent wells.
5. Walt 15 minutes. Tap the strips once or twice during this
time. During the incubation step, prepare the substrate
solution. Using a 1000 microliter single channel
pipettor, add 220 microliters of the Substrate and 220
microliters of the Chramogen to a test tube for each strip
that is being used. For example, if one is using 3 strips,
add 660 microliters of Substrate and 660 microliters of
Chromogen to a test tube. Mix well.
II-3
78
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... t
from coming loo., fro* the nold.r dJring
7. invert the atrip and dry by tapping several times on a paper
Mote: DO not wipe tha wells with a towel or heat
'
«! ",Binutes and 8t°P **• col« reaction by adding the
Stop solution. Uaing a 200 microliter .ingle channel
Si??"0?' a!d.K° tt}cro^t.r. of the Stop Solution So each
well. Touch tha pipet tip to the side of each well when
dispensing and do not allow the tip to go below the TimHrf
IE*1*!,*11!: °?Uld ?ontaBi«»te the Itlp JolSJion S^thi'bStl.
when the tip i« reintroduced into the bottle. Tap the strips
gently to effect mixing and read the strips with thrJortablJ
strip reader. Be sure the reader contain? the 405 nm mJer
Please note that the substrate solution should change from a'
blue color to a yellow color. 9
Note: caution should be used as the Stopping Reagent
contains Sulfuric Acid. *
10. The approximate PCP concentration of the samples can b.
determined by comparing the sample colors to the standard
colors. The rate of color development is Inversely
proportional to the concentration of PCP in the sample. Plot
the standard, on the sample sheets supplied by SXIC. Fit the
best straight line to the standards (by eye) and caiculat.
the sample concentrations using the best-fit line. Multiply
the concentration obtained by the dilution factor to obtain
the undiluted sample concentration.
QUALITY CONTROL
STORAGE
II-4
79
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Keep the kit refrigerated when not in use.
TO OUR CUSTOMERS:
Westinghouse Bio-Analytic Systems Co. wants to hear froa you.
Should you have any difficulties, questions, or comaents about
this product, please contact Or. Stephen 0. Soileau at (331) 670-
0688 or write to:
Weetlnghouse Bio-Analytic Systems Co.
15225 Shady Grove Rd.
Suite 306
RocJcville, Maryland 20850
II-5
80
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QUALITY CONTROL
1) The minimum acceptable O.D. for the negative control (1 X kit dilution buffer) is 0.50. If
the O.D. level falls below this level, the strip should be repeated.
2) Check that the semi-log standard curve (kit standard POP concentration vs O.D.) for
each strip is reasonably linear, by eye. An example of a reasonably linear standard
curve is shown below. Check that the O.D. values for dilutions of QA and field
samples are in the expected order. If both these criteria are not met, the strip should
be repeated.
3) If repeated strips do not meet the QC criteria, call Dr. Jeanette Van Emon at
(702) 798-2154, before proceeding with analysis.
0.7-^
0.6-
~ 0.5-3
M
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APPENDIX III
CALCULATIONS
1) Calculation of percent recovery for spikes of a solution with a standard of known
concentration.
a) Calculate the change in concentration AS expected due to the spike.
b) Measure the concentration of the solution before spiking (C0) and the
concentration of the spiked solution (Cs).
c) The percent recovery is the percent of the change in concentration relative to
the expected change.
percent recovery = 100 (C8 - C0)/AS
2) Calculation of the coefficient of variation (%CV). The coefficient of variation is a
measure of scatter or dispersion and is defined as the ratio of the standard deviation
to the mean.
%CV = 100(Sx/x)
where x = the mean concentration for a sample of size n.
Sx = the standard deviation of the mean.
3) Calculation of accuracy, or the degree of agreement of a measurement (or a limiting
mean of measurements), x, with an accepted reference or true value, t. Accuracy can
be expressed as the difference between the two values, as a percentage of the
reference or true value.
Accuracy = 100 (x-t)/t
in-1
82
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APPENDIX B
EPA FACT SHEET:
SITE DEMONSTRATIONS OF TWO TECHNOLOGIES AT THE
MACGILLIS & GIBBS SITE;
MAP OF MACGILLIS & GIBBS SITE LOCATION
83
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&EPA Fact Sheet
SITE Demonstrations of Two Technologies at the
MacGillis & Gibbs Site:
1. Soil Washing with the BioTrol Soil Treatment System
2. Biological Treatment of Contaminated Water with the
BioTrol Aqueous Treatment System
The MacGillis & Gibbs site has been proposed as a test site for the demonstration of two cleanu p
technologies under a new U.S. Environmental Protection Agency (EPA) program called the
Superfund Innovative Technology Evaluation (SITE) program. The technologies proposed for
testing at the MacGillis & Gibbs site were developed by BioTrol, Inc. of Chaska, Minnesota.
One of the technologies was designed to treat contaminated soils; the other technology treats
contaminated ground water and wastewater. If approved, the demonstrations will occur in
July or August, 1989. The purpose of this Fact Sheet is to provide information on the proposed
project and solicit public comment. EPA staff will discuss the project and ask for public
comments at the regular meeting of the City of New Brighton Environmental Quality
Commission at 7:30 PM, April 12, at New Brighton City Hall, in Council Chamber, 803 Fifth
Avenue, NW, New Brighton, Minnesota. The SITE Demonstrations will be the first topic of
discussion.
What is the Problem?
MacGillis & Gibbs, Inc. has been operating a wood treating facility on a 24-acre site in New
Brighton, Minnesota since the early 1920's. Originally, a preservative known as creosote was
used to treat wood products until the use of pentachlorophenol (penta) was initiated in the late
1940's or early 1950's. In 1970, MacGillis & Gibbs installed a chroma ted copper arsenate (CCA)
pressure treating plant and currently uses only that process. Waste management practices
associated with thecurrent wood treating operations conform to current regulations. However,
for many years MacGillis & Gibbs and the neighboring Bell Lumber & Pole facility disposed
of wastes in a low-lying area astride the properties. The wastes included treated and untreated
wood, sludge, and runoff water from the MacGillis & Gibbs treatment area. Studies show that
soil throughout the two sites and ground water under the disposal area are contaminated with
the toxic chemicals used in the wood preserving process.
The MacGillis & Gibbs and the Bell Lumber & Pole sites were nominated (as a single 68-acre
site) for inclusion on the EPA's National Priority List (NPL) in 1983. In 1984 the site was
permanently included on the NPL. Bell Lumber & Pole entered into an agreement with the
Minnesota Pollution Control Agency (MPCA) in 1985 to investigate and clean up its portion
of the site. The contamination migrating from the MacGillis & Gibbs site is being studied under
the auspices of both the EPA and the MPCA Superfund programs.
84
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What Is the SITE Program?
The Environmental Protection Agency is trying to find better solutions to hazardous waste
cleanup through its new SITE program, which was created in response to the Superfund
Amendments and Reauthorization Act of 1986. As a joint effort between EPA's Office of
Research and Development and Office of Solid Waste and Emergency Response, the SITE
program conducts carefully planned demonstration projects to test new ways to destroy,
neutralize, or otherwise detoxify hazardous wastes.
EPA will select suitable locations for SITE demonstration projects after a nationwide search to
match promising technologies with the types of wastes and conditions at selected Superfund
sites. During the first two years of the SITE program approximately twenty sites across the
country were proposed to test various technologies. MacGillis & Gibbs is one of the sites
nominated for pilot testing of two innovative treatment technologies.
Location of SITE
Demonstration at the
MacGillis & Gibbs Site
85
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Science Applications International Corporation (SAIC) is assisting the EPA in evaluating the
BioTrol technologies. For the demonstrations at the MacGillis & Gibbs site, SAIC will sample
and analyze the materials before and after treatment, and monitor operating parameters such
as temperature, flow rates, and power consumption. Finally, SAIC will help the EPA conduct
a performance and cost assessment for each demonstration to determine whether the technol-
ogy is feasi-'le for use at Superfund sites.
What Technologies Will Be Demonstrated at the MacGillis & Cibbs Site?
The EPA is evaluating the BioTrol Soil Treatment System (BSTS) and the BioTrol Aqueous
Treatment System (BATS). The BSTS is a volume reduction step for treatment of contaminated
soils. During the BSTS process, the larger particles of the soil (the sand) are separated from the
smaller soil particles (silt and clay) where the contaminants concentrate. The BATS, a
microbiological treatment process for destroying toxic organics, will be tested for cleanup of
contaminated ground water from under the MacGillis & Gibbs site. The BATS will also be used
to degrade the toxic organics in the wastewater from the BSTS test. The objective of both
treatment technologies is to produce nonhazardous materials for disposal.
Which Contaminants Will Be Treated During the SITE Demonstrations?
The BSTS will be tested on soils contaminated with wood treating chemicals including penta,
polynuclear aromatic hydrocarbons (PAHs) present in creosote, and copper, chromium, and
arsenic (from the CCA solution). It is expected that the BSTS will remove penta, PAHs, and the
CCA metals from the sand portion of the soil. The BATS will be tested on water contaminated
with penta and PAHs, both of which are expected to be removed.
Will the Proposed Demonstrations Interfere with the Studies Currently Being Conducted?
The investigations underway as a result of EPA and MPCA Superfund activities will not be
delayed or disrupted by the SITE demonstrations. In fact, the SITE data will prove useful in
evaluating treatment alternatives for the MacGillis & Gibbs site and selecting the remedial
action.
How Does the BioTrol Soils Treatment System Work?
The BSTS operates on the principle that most of the contaminants present at the site are
associated with the silt and clay particles and that removal of these particles leaves the rest of
the soil (mo.sdy sand particles) relatively clean. Thus, the BSTS is a waste volume reduction
technology. It produces a smaller, more easily treated, amount of hazardous waste. First,
excavated soils will be passed through a large screen to remove debris. Next, the soil will be
mixed with water to form a slurry. The resulting slurry will be screened again and subjected
to a series of intensive scrubbing and physical separation steps ina multi-stage washing circuit.
The slurry will be separated into a washed sand and a silt and clay slurry containing most of
the contamination.
Some of the contaminated silt and clay produced by the BSTS will be further treated
biologically by a technology jointly developed by BioTrol and. EIMCO Process Equipment
Company, the EIMCO Bio-Slurry Reactor (EBSR) will biodegrade the contaminants
concentrated in the silt and clay, producing a treated silt and clay.
The BSTS performance will be assessed with soils having two different penta concentrations:
about 200-500 ppm, and about 1500-2000 ppm. The system will be tested during continuous
24-hour operation. About 75 tons of contaminated soil will be treated during the 6-8 week SITE
86
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demonstration. Because the EBSR is built on a smaller scale than the BSTS, only part of the silt
and clay from the BSTS will be treated. Altogether, the soil treatment tests will produce about
62 tons of washed sand, 18 tons of contaminated silt and clay, 4 tons of washed and biologically
treated silt and clay, and 8 tons of wood particles. The increase in total weight of material
results from water added during the treatment. The residuals from the treatment system,
including Ur washed sand, the contaminated silt and clay, and the biologically treated silt and
clay, will be stored in drums at the site for disposal as part of the Superfund cleanup, or for
disposal offsite as a hazardous waste. The washed sand and biologically treated silt and clay
may be disposable offsite as nonhazardous waste.
All wastewater produced by the soil treatment system will be treated in the BATS reactor,
where contaminants will be broken down by naturally occurring bacteria. If the treated
wastewater meets local standards, it will be discharged to the sanitary sewer for treatment and
disposal.
During 1988. a pilot test of this technology was conducted at the MacCillis & Gibbs sito. The
mobile pilot system, treating up to 500 pounds of soil per hour, demonstrated removal of 85
to 99 percent of the pentn and PAHs from the contaminated soil. From 73 to 83 percent of the
original soil was recovered as washed sand. The favorable results of this pilot test indicate that
the proposed SITE demonstration should be successful.
Excavate
Contaminated
Soil
Screen
1
Oversize
Debris
Slurry
j
/
Multi-Stage
Washing
Circuit
c
washed
* Sand
V
/ \
Recycle
; Contami-
nated
Water
*
Water
Treatment
Systems
(BATS)
1
r ^
1 Clean
Water
V J
Contami- ^
nated
^ Silt/Clay J
\
ao-Slurry
Reactor
*
Oewaier
Treated
Silt/Clay
The BioTrol Soil Treatment System (BSTS)
87
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How Does the BioTrol Aqueous Treatment System Work?
The BATS is a microbiological system, consisting of a layer of naturally occurring microbes
growing on plastic support material in tanks, used for degrading toxic organic compounds in
water. Under the planned SITE demonstration, BioTrol will apply the BATS process to
removing the penta and PAHs from thegroundwater underlying the MacCillis & Cibbs site.
The contaminated groundwater may require pretreatment, such as oil/water separation (to
remove floating oil) or flocculation and settling (to remove suspended solids), before passing
through the BATS. The pH of the water (a measure of acidity) will be adjusted and inorganic
nutrients will be added. These additions help to optimize the performance of the microbes
used in the process. In the BATS bioreactor, BioTrol adds a specific naturally occurring
microorganism to the microbes which already exist in the groundwater. This combination of
microbes rapidly degrades the penta and PAHs into carbon dioxide, water, and inorganic
chloride, which are harmless products. A bag filter will be used to capture the excess biomass
which exits the bioreactor. This material consists of microbes, both alive and dead, which
detach from the supports and are flushed out with the water stream. The bag filter will be
replaced periodically. The small amount of residuals from the BATS, including separated oil,
flocculated and settled solids, and bag filters containing biomass, will be stored in drums at the
site for disposal as part of the Superfund cleanup, or properly disposed of of fsite as hazardous
wastes.
The SITE demonstration for the BATS will last about 60 days. A maximum of 400,000 gallons
of groundwater \vill be treated during the test. This treated water will be further treated with
carbon to remove any remaining contaminants. The water will either be recycled to MacCillis
& Gibbs for use in their treatment process or sent to the sanitary sewer for treatment and
disposal.
In a nine-month groundwater treatment test conducted at the adjacent Bell Lumber & Pole site
from September, 1986 to May, 1987, the BATS process successfully reduced 60-100 ppm levels
The BioTrol Aqueous Treatment System (BATS)
88
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of penta to less than 5 ppm in the treated water. At times, the residual penta was reduced to
less than 0.5 ppm. ['AH levels of 12 ppm were reduced to 0.5 ppm. The favorable results of
this pilot test indicate that the proposed SITE demonstration should be successful.
Have Potential Environmental Effects of the Demonstration Testing Been Evaluated?
Potential effects on air quality, water quality, wetlands and other environmentally sensitive
areas, and on threatened or endangered animals or plants have been evaluated. No adverse
effects on human health or the environment will be caused by either of the two technologies
being demonstrated.
Who Will Be at the Public Meeting?
In accordance with EPA policy, a public meeting has been scheduled for April 12,1989 at 7:30
PM. Representatives from MPC A, EPA, BioTrol, Inc., and SA1C will be present to explain the
proposed SITE demonstrations, and answer any questions that might be raised during the
meeting. The general public is invited, including the citizens of New Brighton, environmental
groups, and other interested parties.
When is the Public Comment Period?
The MPCA and EPA invite comments on the information presented in this Fact Sheet, as well
as any materials discussed during the public meeting. The public comment period will end on
May 10,1989. Written comments should be addressed to:
Minnesota Pollution Control Agency
520 Lafayette Road
St. Paul, MN 55155
Attn: Susan Brustman
Who Can I Contact with Questions about the SITE Demonstrations?
Susan M. Brustman
Public Information Officer
Minnesota Pollution Control Agency
520 Lafayette Road
St. Paul, MN 55155
(612)296-7769
Mary K. Stinson
Chemical Engineer
Releases Control Branch
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Woodbridge Avenue (MS-104)
Edison, NJ 08837-3679
(201)321-6683
Rhonda E. McBride
Remedial Project Manager
CERCI.A Enforcement Section, Region V
U.S. Environmental Protection Agency
230 South Dearborn Street
Chicago. ILftUMM
Morris J. Anderson
Vice President, Regulatory and
Governmental Affairs
BioTrol, Inc.
11 Peavey Rond
Chaslui, MN 55318
(612)448-2515
89
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WATER SURFACE FEATURES
f • n I b. rt w »* •-» i rt wt* i ^ «-t i w rt c a
MACGILLIS & GIBBS WASTE SITE ,
MPW PRtnMTDM MIMMPSnTl i M=l :
'IT HIV ..^&: V~ Vflft.- .Htt- ' - •«*.- : Si,, L
• •• "X« I -""• •••I C * i •*. t^^^^r^
^r—rr*ri"T"*'i ' *it t'V * ^
S^ffll^lEIi'^1-^1
itesiM MHrintt&
Provided by Twin City Testing Corporation
Figure B-1. MacGillis & Gibbs Site Location Map (site marked by hatched area).
90
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APPENDIX C
STANDARD ANALYTICAL PROCEDURES AND METHODS REFERENCES FOR THE
BIOTROL SITE DEMONSTRATION
Source: Draft Demonstration Test Plan for BioTrol, Inc., Biological Treatment of Contaminated
Groundwater at New Brighton, Minnesota. 1989. Draft Document, Science Applications
International Corporation (SAIC), Paramus, New Jersey, pp. 38-40.
91
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Standard Analytical Procedures and Method References
MeasurcBent
Matrix Type Method Number
Title
Method Type
Reference
<0
10
Volatile Organic* Croundwater SW 8240
Organic Extraction Croundwater SW 3510
Organic Extraction
Organic Extraction
Sealvolatile
Organic*
Oloxlna
Solid (Sorbent SH 3540
Realn)
Solid SU 3550
(Blosludge)
Solvent
Extract
SU 0270
Croundwater SU 8280
Metal* Extraction Croundwater SU 3010
(Cd.Cr.Cu.Pb.Ag.
Ba.Nl.Zn)
Metal* Extraction Groundwater SU 3020
(Aa.Sb.Se.Tl)
Cat Chromatography/Mass Spec-
trooecry for Volatile Organlcs
Separatory Funnel Liquid-
Liquid Extraction
Soxhlet Extraction
Sonlcatlon Extraction
Ca* Chroaatography/Maas Spec-
for Sealvolatlle Organlca:
Capillary Coluan Technique
The Analyala of Polychlorlnated
Dlbenxo-P-Dloxlna and Poly-
chlorlnated Olbenzofurana
Acid Dlgeatlon of Aqueou*
Samples and Extracta for Total
Mctala for Analyil* by Plane
Atoalc Absorption Spectroscopy
or Inductively Coupled Plasna
Spectroacopy
Acid Dlgeatlon of Aqueou*
Saaple* and Extract* for Total
Metal* for Analysis by Furnace
Atovlc Absorption Spectroacopy
Purge and trap/ SU 846
CC/MS
Solvent Llq- SU 846
Llq extraction
Soxhlet Organic SU 846
Solvent extraction
Solvent extraction SU 846
by aonlcatlon
CC/MS
CC/MS
Acid Digestion
Acid Dlgeatlon
-------
Standard Analytical Procedures and Method References
CO
CO
Measurement
Metal* Extraction
Metal* - Mercury
Metal* - Mercury
H«tal* Analysis
(Cd.Cr.Cu.Pb.Ag,
8e.Hl.2n)
Metals Analysis:
Arsenic
Ant loony
Selenlusi
Thslllusi
Chloride
Total Organic
Carbon (TOC)
Phenolic*
Oil and Crease
Matrix Type
Solid
Croundwater
Solid
Croundwater -
Dtgestate
Croundwater -
Dtgescate
Croundwater
Croundwater
Croundwater
Croundwater
Method Nuaber
SU 3050
SU 7470
SW 7471
SU 6010
SU 7060
SU 7041
SU 7740
SU 7841
SU 9232
SU 9060
SU 906 5
SU 9070
Title
Acid Digestion of Sediment,
Sludges and Soils
Mercury In Liquid Uasce (Manual
Cold Vapor Technique)
Method Type
Acid Digestion
Cold Vapor - AA
Mercury In Solid or Seolcolld
Uaate (Manual Cold Vapor Technique)
Industlvely Coupled Plasma
Atonic Emission Spectroscopy
Arsenic (AA, Furnace Technique)
Ant loony (AA. Furnace Technique)
Selenlu*) (AA, Furnace Technique)
Thalllus) (AA, Furnace Technique)
Chloride (Tit rloetr Ic . Mercuric
Nitrate)
Total Organic Carbon
Phenolic* (Spectrophotooetrlc
Manual 4AAP with Distillation)
Total Recoverable Oil and Crease
ICP
CFAA
CFAA
CFAA
CFAA
Tit rlaet rlc
Conbustlon/IR
Detector
Colorlaetrlc/
Spect rophotoaetrlc
Solvent Extraction/
Reference
SU 846
SU 846
SW 846
SU 846
SU 846
SU 846
SU 846
SU 846
SU 846
SU 846
SU 846
(Cravlnetrlc, Separating Funnel
Extraction)
Gravimetric
-------
Standard Analytical Procedures and HeChod Reference*
CO
Measurement
Residue
Alkalinity
Nutrients:
Ammonia
Nitrate
Phosphate
Field .Measurements:
PH
Dissolved Oxygen
Temperature
Matrix Type
Croundwater
Croundwater
Croundwater
Croundwater
Croundwater
Croundwater
Croundwater
Croundwater
Kethod Number
SM 209
SM 403
SM 417
SM 418
SM 424
SM 423
SM 421
SM 212
Title
Residue
Alkalinity
Nitrogen (Ammonia)
Nitrogen (Nitrate)
Phosphorous
pH Value
Oxygen (Dissolved)
Temperature
Method Type
Gravimetric
Tltrlmetrtc
Direct Measurement
Direct Measurement
Direct Measurement
Reference
SM
SM
SM
SM
SN
SM
SM
SM
*SW 846 - "Test Methods fore Evaluating Solid Waste". SU 846. Third Edition. November. 1986
SM - "Standard Methods for the Examination of Water and Wastewater" 15th Edition, 1980.
-------
APPENDIX D
SAMPLE SPLITTING AND SHIPPING INSTRUCTIONS FOR THE
IMMUNOASSAY SITE DEMONSTRATION
95
-------
SAMPLE SPLITTING AND SHIPPING INSTRUCTIONS FOR THE
IMMUNOASSAY SITE DEMONSTRATION
During each of the three 6-day spans comprising the formal demonstration, splits of every field
sample analyzed by the field immunoassay kit (all routine influents, routine effluents, field blanks,
field duplicates, raw influent grabs) are to be sent to both the EMSL-LV and WBAS laboratories.
About 500-600 ml_ is split from each well mixed field sample for immunoassay analysis. Two
large glass bottles are filled with 250-300 ml from each sample. The bottles are labelled with an
SAIC label, capped with a teflon-lined screw cap, packed in ice and shipping insulation in a
shipping cooler and shipped to the addresses below. For field analysis, 20-30 mL of each
sample* is placed in a small amber glass bottle, labelled with an SAIC label (as above). The
bottles are capped tightly and stored at 4 °C on-site. A small volume is used for one-site field kit
PCP analysis, and the remainder is archived until all the analyses for the project are completed.
Dr. Steve Soileau
Westinghouse Bio-Analytic Systems
15225 Shady Grove Road, Suite 306
Rockville. MD 20850
(301) 921-0031
Or. Jeanette Van Emon
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Post Office Box 93478
Las Vegas, NV 89123-3478
(702) 734-2154
For GC/MS Analysis at EMSL-LV on a few selected samples, the following sampling and shipping
procedure is to be used. On one day of each of the six 6-day spans (see below) an additional
1 liter of the routine (daily) influent, effluent and water blank are split off and placed in a large
(1 liter) amber glass bottle. In addition, once during the six weeks comprising the bioreactor
demonstration, a 1-liter split of the raw influent grab (selected for SAIC GC/MS analysis) sample
is to be taken and bottled. The bottles are labelled as above, capped tightly with a teflon-lined
screw cap, and carefully packed in ice and shipping insulation in a shipping cooler. The samples
are sent to the above EMSL-LV/LESC address for confirmatory GC/MS analysis.
*The small volume splits of the routine effluent samples made for field analysis and archiving are
to be centrifuged prior to bottling.
96
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APPENDIX E
LIST OF ADDRESSES FOR SHIPPING BIOREACTOR AMD QA/QC SAMPLES
FOR THE IMMUNOASSAY SITE DEMONSTRATION
97
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FIELD SITE ADDRESS (FIELD IA)
MacGillis and Gibbs
440 5th Ave. N.W.
New Brighton, Minnesota 55112
Attn: Randy Potter
ADDRESS FOR SAIC (GC/MS)
Sample Custodian
SAIC
4224 Campus Point Court
Mailstop 210
San Oiego, California 92121
ADDRESS FOR WBAS (LAB & FIELD IA)
Westinghouse Bio-Analytic Systems
15225 Shady Grove Rd.
Suite 306
Rockville, Maryland 20850
Attn: Dr. Steve Soileau
QA SAMPLE SHIPMENT
FIELD SITE
Total Analysis
No. Volume Type
16 Type A QA Amps (2 mL) each with 0.5 ml_ 8 ml_ Field IA
8 Type B QA Amps (2 mL) each with 0.5 mL 4 mL Field IA
6 20 ppm QC Performance Sample Amps (5 mL), 12 mL Field IA
each with 2 mL
6 15 ppm I.S. Spike Std. Amps (2 mL), each, 6 mL Field IA
with 1 mL
98
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WESTINGHOUSE BIO-ANALYTIC SYSTEMS
No.
14
8
14
8
6
6
Type A QA Amps (5 mL) each with 2 mL
Type B QA Amps (5 mL) each with 2 mL
Type A QA Amps (2 mL) each with 0.5 mL
Type B QA Amps (2 mL) each with 0.5 mL
20 ppm QC Performance Sample Amps (5 mL),
each with 2 mL
15 ppm I.S. Spike Std. Amps (2 mL), each,
with 1 mL
SCIENCE APPLICATIONS INTERNATIONAL CORP
Na
12
6
6
3
Type A QA Amps (5 mL) each with 5 mL
Type B QA Amps (5 mL) each with 5 mL
Type A QA Amps (2 mL) each with 2 mL
Type B QA Amps (2 mL) each with 2 mL
IA = immune-assay
Amps - Ampules
IS = internal standard
Total
Volume
28 mL
16 mL
7mL
2mL
12 mL
6mL
Total
Volume
60 mL
30 mL
12 mL
6mL
Analysis
Type
LabIA
LabIA
Field IA
Field IA
Field IA
Field IA
Analysis
Type
GC/MS
GC/MS
GC/MS
GC/MS
99
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APPENDIX F
INSTRUCTIONS FOR COMPLETING THE FIELD DATA FORM
FOR THE FIELD KIT ANALYSIS
101
-------
INSTRUCTIONS FOR FILLING OUT THE FORM ENTITLED
•FIELD DATA FOR ANALYSIS OF WBAS FIELD IMMUNOASSAY TEST KIT
GENERAL
1) A field form is to be filled out for each sample analyzed. For instance, when a daily influent (RI)
and a daily effluent (RE) sample are analyzed on the same day, a field form will be completed for each
sample.
2) The filling out of page numbers (top right corner of each form) should be done after all the analyses
for one sample are completed. 'Page 1 of _" will, by definition, be the first page (with the sample
collection, environmental factors, and sample specific information included along with the results of
the analysis of the first strip for the sample). 'Page _ of _' is designed to accommodate all
subsequent strips (i.e., range-finding, duplicate/split, matrix spike, and/or QA and QC strips) needed
to complete the analysis of a given sample on a given day.
3) The circles located after many of variables on the form are to indicate that a qualifying comment
(tag) relates to that particular piece of information. Place an 'X* or 'V or some other tag in (or legibly
over) the circle and then "footnote* it in the 'Comments' section. This should direct the data reviewers
to issues relating to that particular variable.
4) Use a ball point pen with ink that will not run. Apply sufficient pressure to ensure that all
carbonless sheets are legible. When illegible or incorrect entries are made on the form, use a single
line to cross out; then initial the line and put correct information immediately next to it if there is room
(if necessary, note correct information in the 'Comments').
5) After reviewing all the entries on the completed forms:
o send the top (WHITE) copy to Las Vegas via overnight courier along with the printout (cash
register tape) for the data from that sample
o send the middle (CANARY) copy to WBAS via overnight courier
o retain the bottom (PINK) copy on file on site
NOTE: If logistically possible, send field forms with corresponding field samples or samples from the
next shipping day; when sending forms in boxes with field samples, use plastic Zploc bags.
The specific instruction for entering field data on the forms are discussed below by "INFORMATION
BLOCK1 and by 'Variable' as they appear on the forms.
SAMPLE COLLECTION INFORMATION
(NOTE: This block contains the SAIC sample bottle label data and other information)
Sample Date: enter in the day, month, year in the following format: '23JUL89' or '04AUG89'.
Sample Time: in military time.
SAIC Sample No.: enter the sample number as described in the sampling plan (e.g., 'ST1-A-01-OV is
an 'influent sample' taken on the 'first day' of flow rate period 'A* during 'Stage V).
102
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Collection Method: circle one, usually 'Composite'.
ISCO ID: identification number noted on the sampling device.
Preservative: usually '4°C'.
Bioreactor 10: BATS identification number.
Groundwater Well No.: as applicable to MacGillis & Gibbs Site.
Flow Rate: usually either 'V or '3' or '5' gallons per minute.
BATS PCP Spike: 'yes' or 'no' (yes, for Stage 2 of the BATS demonstration).
BATS Spike Cone: concentration (ppm) of PCP spike (added to the influent in Stage 2).
Collected by: initials of field crew member who collected the sample.
Split by: initials of field crew member who prepared the splits of the composite for on-site and off-
site analysis.
Comments: any comments applicable to the information in this block, and any other information
related to the sample collection or handling.
ENVIRONMENTAL FACTORS
Analysis Date: the date the sample is analyzed, in same format as Sample Date (e.g., '23JUL89').
Days Since Collected: number of calendar days between sampling and analysis (NOTE: if 'Sample
Date' and 'Analysis Date' are the same, enter '00').
Site of Analysis: circle applicable site.
Location of Analysis: circle one, usually 'Indoors*.
Location Temp.: ambient temperature where analysis performed.
Sunny/Cloudy/Mixed/, Windy/Calm, Other: applicable to outdoor factors, if sample analyzed there.
Kit Lot No.: lot number on the WBAS kit box
Kit Storage Days: number of days kit was stored at site location (both in and out of the refrigerator),
from date of receipt to date of analysis.
Daily Refrig. Temp.: refrigerator temperature at the time the kit is removed for analysis activities.
Comments: any pertinent comments related to the information in this block.
Analyst: initials of the crew member performing the test kit analysis.
103
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SAMPLE-SPECIFIC PRE-ANALYSIS INFORMATION
Sampling Point Source: as applicable.
Sample Appearance: as applicable for color, clarity, presence of precipitate (or settleable matter) or
oil, etc.
Centrifuged: usually for effluent samples to remove biomass, etc.
Appearance Change: applicable only if there are observed differences in sample appearance after
centrifugation.
pH-Meter: result of on-site sample analysis performed by pH meter.
pH-Paper: result of on-site sample analysis performed by pH paper (NOTE: this may be optional).
Hardness-Paper: result of on-site sample analysis by hardness paper (NOTE: this may be optional).
Field Blank: indicate if a field equipment wash blank sample was collected with this sample and the
applicable SAIC Sample No. Any pertinent pre-analysis information about this blank should be noted
in the 'Comments'.
Field Duplicate: indicate if a field duplicate sample was collected with this sample and if there is an
SAIC Sample No. Any pertinent pre-analysis information about this duplicate sample should be noted
in the 'Comments'.
Field QA Audit: indicate if a QA audit sample, type A or B was analyzed with this sample (usually with
a routine effluent sample on a "long day"; see sampling schedule and protocols). Include the LESC
Sample No. (e.g., 'QAA-0221, 'QAB-004') obtained from the ampule label.
Comments: any comments related to the information in this block.
SAMPLE ANALYSIS INFORMATION BY STRIP
Strip ID: this is a strip identifier comprised of one letter and one number. The letters represent:
A = daily QC strip
B = QA strip
C - effluent range-finding strip
D = influent range-finding strip
E » effluent duplicate/split strip
F - influent duplicate/split strip
G = effluent matrix spike strip
H <• influent matrix spike strip
I = cross-calibration strip for field photometer
J to Z » as necessary, only when above letters don't apply
The number after the letter represents how many runs of a particular strip type (A-Z) were done for
that sample. Example #1: the daily QC 'Strip ID' is labelled 'A-1'; if a second QC strip must be
analyzed because of 'bad" QC data (see performance specifications), it will be labelled 'A-2'. Example
#2: If the first range-finding strip for a daily effluent ('Strip ID': 'C-1') or influent ('Strip ID': 'D-1')
sample yields inadequate dilution concentrations, a second strip with a different dilution scheme will
be analyzed (labelled 'C-2' and '0-2", respectively).
Sample Code: note the applicable sample code for each well, as they appear and are defined on the
sample code list (e.g., 'FB', 'QC. 'RE'. 'RIMS', 'SOI').
104
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Well*: these will usually be numbered 1 • 8.
OD(ABS): this is the optical density (OD) for each sample as it appears on the LCD readout of the
Dynatech photometer and is expressed in absorbance units (ABS). Report the ABS value to three
places after the decimal point in this column.
NOTE: when plotting these points on the semi-log graph below, round to two places after the decimal
point, because of the limited resolution on the graph.
Plotted Cone, (ppb): the PCP concentration of samples analyzed in the range of the calibration curve
(the curve is determined by plotting the absorbance units of the four kit standards against their
known (3.0, 7.1, 16.9, 40.0) concentrations and then fitting (estimating by eye) the best straight line
between those four points. All sample absorbance units are then plotted on that tine so that the
concentration, in ppb, can be determined.
NOTE: estimate the ppb concentration to the nearest tenth, if possible (e.g., 8.4 ppb); it is recognized
that this will be difficult for samples > 10 ppb.
to ppm: multiplying 'Plotted Cone (ppb)' by 0.001 converts ppb to ppm. NOTE: This is not necessary
for kit standards.
Dilution Factor: this is a multiplication factor through which samples diluted to fall within the linear
dynamic range of the calibration curve are converted to concentrations in their undiluted state. For
instance, if a sample is diluted 1:1000, the dilution factor is the inverse, or 1000. (NOTE: if a sample
is not diluted or is a kit standard, there is no dilution factor).
Sample Cone (ppm): the result of multiplying:
'Plotted Cone (ppb)' X '0.00V X 'Dilution factor' (if any)
The product should equal the PCP concentration (in ppm) of the sample
(NOTE: this is not applicable to the kit standards).
Amt(ppb) Spike Added: this is only used for MSE or MSI samples and is the concentration (in ppb)
of the internal standard (matrix) spike into a routine (REMS, RIMS) sample diluted into specified linear
range (~3-20 ppb) for spiking.
% Spike Rec: this is the percent spike recovery and is determined by calculating:
(sample concentration + spike) - unspiked sample concentration
concentration of spike added
specifically,
MSE - REMS MSI - RIMS
X 100 or X 100
X100
Arm Spike Added Amt Spike Added
(NOTE: numerator values are from the 'Plotted Cone (ppb)' column)
Semi-Log Graph: as presented, the X-axis is prelabelled for PCP concentrations in parts per billion
([PCP] ppb) from 2-60 ppb in log scale. The Y-axis is labelled for the optical density (OD) in
absorbance units (ABS), but no prelabelled units are given. The Y-axis is not in log scale and is left
105
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to the discretion of the analyst to label. This will depend upon the ASS readings generated from the
calibration kit standards analyzed on each strip. For instance, the ABS increments may be labelled:
0.20. 0.40, 0.60, 0.80, 1.00, 1.20;
or 0.30, 0.50, 0.70, 0.90, 1.10; 1.30;
or 0.30. 0.40. 0.50. 0.60. 0.70. 0.80;
or some other increments that 1) cover the absorbance range of the kit standards and, 2) provide
maximum resolution (using units that are 'reasonable* and easily plotted).
Comments: Any comments that are related to the plotting, diluting, calculating ppms or spike
recoveries, or other pertinent information related to sample analysis should be entered in the space
provided. The 'tag circle' provided after the 'Sample Code' can be used to denote any sample requiring
discussion here. (NOTE: It is recognized that the space provided may be awkward to write in, so
writing "sideways" is acceptable.
Page 2, and all other subsequent pages needed for the analysis of a sample, should include the 'SAIC
Sample No.' in the 'SAMPLE COLLECTION INFORMATION* block, and the 'Analysis Date1 and 'Analyst'
initials that appears in the 'ENVIRONMENTAL FACTORS' block on Page 1. All 'SAMPLE ANALYSIS
INFORMATION BY STRIP* should follow the same format as described above.
106
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APPENDIX G
DESCRIPTIONS AND SCHEMATICS FOR DAILY SAMPLE ANALYSIS
107
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SAMPLING AND ANALYSIS SCHEDULE
FOR THE FIELD PCP IMMUNOASSAY KIT SITE DEMONSTRATION
PRELIMINARY EVALUATION (bioreactor start-up) 26 days, June 26-July 21.
PART A-(bioreactor flow rate= 1 gal/min) 6 days, July 24-29
PART B-(bioreactor flow rate= 3 gal/min) 6 days, August 7-12
PART C-(bioreactor flow rate= 5 gal/min) 6 days, August 21-26
Use the same sampling and analysis scheme for all three parts. Effluent samples are from
reactor 3 (final), unless otherwise stated. The samples referred to as the routine influent or
effluent is a split of the composite sample taken for GC/MS analysis, for that day.
The routine influent will be taken from the holding tank, containing treated (nutrients added, pH
adjusted to 7.2) well water. In addition, once a week, a grab sample of raw or untreated well
water will be taken for GC/MS analysis. A split of this grab sample is to be analyzed by the field
PCP immonoassay method also. If possible, do the analysis schemes in the order listed: DAY 1
first, DAY 2 second, and DAY 3 third. The short day routines are for days when the person doing
the field analysis is busy with other activities and the long day routines are for days when there
is more time available. The 4 kit standards that are run on each strip are the following: 3 ppb, 7.1
ppb, 16.9 ppb, and 40 ppb. Each day, run the QC STRIP first.
All samples should be run as soon as possible after collection. Try to run all samples within a 24
hr period after splitting. Try to complete the analysis of all the scheduled samples (see the
weekly totals list below) within each of the three 6 day periods. A minimum volume of 20 ml from
each field sample analyzed should be placed in a 30 ml amber vial, labelled with an SAIC label
(see example below) and capped tightly with a teflon lined screw cap. All these vials are to be
stored at 4 degees C, in case later analysis is necessary. If time does not permit the analysis of
all the above samples during the 6 day week, unanalyzed samples are to be run on the following
week.
QC STRIP
LONG 1 strip with a field water blank, undiluted and DAY 1 diluted 1 to 10, a negative
DAY 1 control (1X kit dilution buffer) and a 20 ppm performance standard, diluted 1 to
1000 + 4 kit standards.
EFFLUENT STRIPS
1-2 strips with four semi-log serial dilutions (suggested dilutions: 1 to 10, 1 to 50, 1
to 100, and 1 to 500) of the routine effluent + 4 kit standards. 1 strip with the
routine effluent diluted to about 10 ppb (3-20 ppb) run in duplicate with and
without a 15ppb internal standard spike + 4 kit standards.
QA SAMPLE STRIPS
2 strips with three serial two-fold dilutions of a semi-blind (see instructions) type
A QA audit sample + 4 kit standards, and one negative control.
108
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LONG
DAY 2
INFLUENT STRIP
1 strip with routine influent at 4 dilutions (suggested dilutions: 1 to 1000, 1 to 2000,
1 to 4000 and 1 to 8000) + 4 kit standards.
- SAME AS LONG DAY 1 .
LONG
DAY 3
SHORT
DAY1
SHORT
DAY 2
SHORT
DAYS
- SAME AS LONG DAY 1, EXCEPT RUN TYPE B QA AUDIT SAMPLE.
QC STRIP
1 strip with a field water blank, undiluted and 1 to 10
diluted, a negative control (1X kit dilution buffer) and a 20 ppm performance
standard, diluted 1 to 1000 + 4 kit standards.
INFLUENT STRIPS
2 strips with routine influent at 3 dilutions (suggested dilutions: 1 to 1000, 1 to
2000, and 1 to 4000) + 4 kit standards, and one negative control.
2 strips, with duplicates of both the routine influent and its field duplicate (split),
each diluted to near the midpoint (20+/- 10 ppb) of the standard curve + 4 kit
standards.
1 strip with split of the routine influent diluted to about 10 ppb (3-20 ppb) and run
in duplicate with and without a 15 ppb internal standard spike + 4 kit standards.
QC STRIP
1 strip with field water blank, undiluted and diluted 1 to 10, 1 negative control
(1X kit dilution buffer) and a 20 ppm performance standard, diluted 1 to 1000.
EFFLUENT STRIPS
2 strips each with a split of the routine effluent at 4 dilutions (suggested dilutions:
1 to 10,1 to 100, to 50 and 1 to 500) + 4 kit standards.
2 strips, with duplicates of both the routine effluent and its field duplicate split,
each diluted to near the midpoint of the standard curve (20+/-10 ppb) + 4 kit
standards.
INFLUENT STRIP
1 strip with the routine influent at three dilutions (suggested dilutions: 1 to 1000, 1
to 2000, and 1 to 4000) + 4 kit standards and one negative control.
QC STRIP
1 strip with a field water blank, undiluted and diluted 1 to 10, a negative control
(1X kit dilution buffer) and a 20 ppm performance standard diluted 1 to 1000 + 4
kit standards.
109
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INFLUENT STRIPS
2 strips with split of the routine influent at 3 dilutions (suggested dilutions: 1 to
1000, 1 to 2000, 1 to 4000) + 4 kit standards, and one negative control.
1 strip with split of the routine influent diluted to about 10 ppb (3-20 ppb) and run
in duplicate with and without a 15 ppb internal standard spike + 4 kit standards.
DAY FOR INFLUENT GRAB - After running the other strips scheduled for this day, run the
following.
INFLUENT STRIPS
2 strips with split of the raw influent grab sample at 3 dilutions (suggested
dilutions: 1 to 1000,1 to 2000,1 to 4000) + 4 kit standards, and one negative
control.
WEEKLY TOTALS
1) 6 routine (daily) influent samples
2) 4 routine (daily) effluent samples
3) 1 field duplicate split of a routine (daily) effluent
4) 1 field duplicate split of a routine (daily) influent
5) 1 raw (untreated) influent grab sample
6) 2 type A QA audit samples
7) 1 type B QA audit sample
8) 6 routine (daily) field water blanks (equipment rinse)
9) 6 daily replicates of the 20 ppm Q C performance standard, diluted fresh each
day.
10) A minimum of 18 negative controls (1 X dilution buffer from kit)
110
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Proposed Sample Coding Scheme for WBAS Field Immunoassay Demonstration
FB - field equipment blank (after daily decontamination of ISCO and before daily compositing)
NC = negative control sample • dilution/reagent blank water (ram 1X kit dilution buffer solution
QC - daily quality control performance check sample @ 20 ppm (known concentration)
STD03 - calibration kit standard @ 3.0 ppb (0.0030 ppm) pentachlorophenol
STD07 - calibration kit standard @ 7.1 ppb (0.0071 ppm) pentachlorophenol
STD16 - calibtation kit standard @ 16.9 ppb (0.0169 ppm) pentachlorophenol
STD40 - calibration kit standard @ 40.0 ppb (0.0400 ppm) pentachlorophenol
RE - routine daily effluent sample
RI = routine daily influent sample
DE - duplicate of a raw, undiluted RE sample (i.e., split before any dilutions)
01 - duplicate of a raw, undiluted RI sample (i.e., split before any dilutions)
SRE = split of an RE sample after the RE has been diluted into calibration range
SRI - split of an RI sample after the RI has been diluted into calibration range
SDE = split of a DE sample after the DE has been diluted into calibration range
SOI =» split of a 01 sample after the 01 has been diluted into calibration range
REMS-x • RE sample diluted to the range for matrix spiking which will be used in the % recovery calculation
(x - sample to be matched with the same *x* for the MSE)
RIMS-x - RI sample diluted to the range for matrix spiking which will be used in the % recovery calculation
(x - sample to be matched with the same 'x* for the MSI)
MSE-x - matrix spike sample of a REMS with corresponding V
MSI-x » matrix spike sample of a RIMS with corresponding "x*
QAA-xxx - semi-blind QA standard, Lot A (xxx = LESC sample control #)
QAB-xxx - semi-blind QA standard, Lot B (xx - LESC sample control #)
CC-xxx - cross-calibration color check standard for photometers (xxx - code for theoretical optical density)
111
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LONG DAYS 1. 2. 3
•EFFLUENT STRIPS -
DAILY QC STOP
( ST040 )
FB
RUN FIRST
RANGE-FINDING STR*>|S)
MATRIX SPIKE
( ST003 J
• ONLV RUN IF FIRST ? COULD BE ANY
RANGE-FINDING *RE* DILUTED TO
STRIP IS 5-23 ppo RANGE
INADEQUATE
QA STRFS
f STD03 j
( STOO; )
Q*(A/B)
I 1:1000 J
QA(A/B)
1:2000
NC
NC
CAN BE RUN IN TANDEM
INFLUENT STRIP
Rl
1:2000
RUN IF
TIME PERMITS
112
-------
SHORT DAY 1
DAILY
QC STRIP
( STD03 j
RUN
FIRST
INFLUENT
RANGE-FINDING STRIP(S)
NC
SPLITS t DUPLICATES
STRIPS AT ONE DILUTION
• ONLY RUN IF
FIRST RANGE-
FINDING STRIP
IS INADEQUATE
.' COULD 8E ANY *RI* DILUTED
TO NEAR 20 ppO (CAN BE RUN
IN TANDEM) MATRIX SPIKES
MATRIX
SPIKES
» CAN BE ANY
•Rl* DILUTED
TO 5-25 PPO
RANGE
113
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SHORT DAY 2
DAILY
OC STRIP
STD03
v^y
FB
RUN FIRST
EFFLUENT
RANGE-FINDING STRIP(S)
• EFFLUENT STRIPS-
I ST007 ]
RE
1:10
SPLITS t DUPLICATES
STRIPS AT ONE DILUTION
f S7D07
( STD16 1
• ONLY RUN IF ? COULD BE ANY -RE* DILUTED TO
FIRST RANGE- NEAR 20 ppD (CAN BE RUN IN
FINDING STRIP TANDEM)
IS INADEQUATE
INFLUENT
STRIP
NC
114
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SHORT DAY 3
INFLUENT STRIPS
DAILY
QC
STRIP
( STD16 )
0
NC
RUN
FIRST
RANGE-FINDING STRIP(S)
NC
MATRIX
SPIKE
• ONLY RUN If ? COULD BE ANY
FIRST RANGE-FINDING 'BE' DILUTED TO
STRIP IS INADEQUATE S 25ppO RANGE
115
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GRAB INFLUENT SAMPLE
(Weekly Collection)
RANGE-FINDING STRIPS
NOTE «1: THESE SAMPLES AREA TO BE ANALYZED ON
DAY COLLECTED. IF POSSIBLE.
NOTE »2: IF NO OTHER DAILY QC STRIP IS RUN ON THE
DAY THIS SAMPLE IS ANALYZED.
INCLUOC A QC STRIP IN THE ANALYSIS.
NOTE *3: THIS ANALYSIS REQUIRES A SEPARATE FIELD
FORM TO BE COMPLETED.
116
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MICROWELL READER SPECTROPHOTOMETER
CROSS CALIBRATION
117
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APPENDIX H
INSTRUCTIONS FOR HANDLING AND ANALYZING
QA AND QC SAMPLES
119
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INSTRUCTIONS FOR IMMUNOASSAY ANALYSIS OF
QA AUDIT SAMPLE TYPES A AND B
1. Store all QA standard ampoules at 4 °C until use. Allow them to equilibrate
at ambient temperature before opening. Break open a new ampoule for
each analysis.
2. Break open an ampoule of Type A or B QA audit sample and, using a
rnicropipettor, make 3 serial 10-fold dilutions as described in the Field Kit
SOP, under influent sample dilution. The final dilution will be 1 mL of a 1 to
1000 dilution.
3. Label two more tubes with the sample number. Label one of the tubes with
1 to 2000 and one with 1 to 4000. Add 0.5 mL of 1X buffer to each of the
tubes.
4. Pipet 0.5 mL from the 1 to 1000 tube into the 1 to 2000 tube and mix Pipet
0.5 mL from the 1 to 2000 tube into the 1 to 4000 tube and mix
5. Assay the 1 to 1000, 1 to 2000 and 1 to 4000 dilutions of the QA samples
using the standard kit protocol on duplicate strips.
120
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INSTRUCTIONS FOR HANDLING 20 PPM QC PERFORMANCE STANDARD
1. Store the sealed ampoules at 4 °C until use.
2. Allow the ampoule to equilibrate at room temperature before breaking open.
3. At the beginning of each week, break open a new ampoule (containing 2
mL) and transfer about 1 mL from the ampoule into two 1.8 mL glass auto-
sampler vials. Label the vials with "20 ppm QC Performance Standard" and
the date. Cap the vials with a Teflon-lined septum (red side down) and
plastic screw cap. Store at 4 °C after each use.
4. Each day, pipet 100 uL out of the vial and make three log (dilute 0.1 mL to
1.0 mL) serial dilutions as described in the field kit analysis instructions.
5. Tightly recap the vial with Teflon (red) side facing the inside of the bottle
and store at 4 °C until the next day's use.
121
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INSTRUCTIONS FOR HANDLING THE 15 PPM STANDARD FOR
MAKING INTERNAL STANDARD SPIKES
1. Store the sealed ampoules at 4 °C until use.
2. Allow the ampoule to equilibrate at room temperature before breaking open.
3. At the beginning of each week, break open a new ampoule (containing 1 ml)
and transfer the contents into two 1.8 mL glass vials with a Teflon-lined (red
side) cap and label with "15 ppm Standard for Spikes" and the date.
4. Each day, after allowing the contents to reach room temperature, pipet 100
uL of the standard into a tube containing 1.8 mL of 1X dilution buffer and
mix Label as "0.75 ppm Standard for Spiking." Proceed as described in the
"Instructions for Field PCP Kit Analysis."
5. After use, tightly recap the vial with Teflon (red) side facing the inside of the
bottle and store at 4 °C until the next use.
122
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INSTRUCTIONS FOR RUNNING TYPE A AND B AUDIT SAMPLES
IN THE LABORATORY PLATE FOR ELJSA FOR POP
1. Store ampules at 4 °C. allow them to equilibrate to room temperature before opening.
2. Break open a type A or B QA sample ampule and transfer the contents into a glass
test tube.
3. Pipet 1.0 mL (volumetric) of the sample into a 10 ml_ volumetric flask and fill to the
mark with 2-propanol, and mix Transfer the remainder of the sample into a glass
auto-sampler vial with a Teflon-lined screw cap. label and store at 4 °C.
4. Pipet 2.5 mL of the 10-fold dilution in 2-propanol made in step #3 into a 10 mL
volumetric flask and bring to the mark with 25% 2-propanol in dilution buffer, and mix
5. Pipet 2.5 mL of the dilution made in step #4, into a 5.0 mL volumetric and bring to the
mark with 25% 2-propanol in dilution buffer.
6. Using the same procedure described in step #5, prepare two more serial 2-fold
dilutions in 25% 2-propanol in dilution buffer.
7. Assay the dilutions made in step #4 and the three serial 2-fold dilutions made in steps
#5 and #6 in triplicate wells in the POP plate ELISA.
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APPENDIX I
MATERIAL SAFETY DATA SHEETS (MSDS) FOR
PENTACHLOROPHENOL AND METHANOL
125
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MATERIAL SAFETY DATA SHEET OHS18150
OCCUPATIONAL HEALTH SERVICES. INC. FOR EMERGENCY SOURCE INFORMATION
11 WEST 42ND STREET. 12TH FLOOR CONTACT: 1-615-366-2000
NEW YORK, NEW YORK 10036
1-800-445-MSDS (1-800-445-6737) OR 1-212-789-35
SUBSTANCE IDENTIFICATION
GAS-NUMBER 87-86-5
RTEC-NUMBER SM6300000
SUBSTANCE: PENTACHLOROPHENOL
TRADE NAMES/SYNONYMS:
PHENOL, PENTACHLORO-: DOWICIDE 7: FUNGIFEN:
1-HYDROXYPENTACHLOROBENZENE: LAUXTOL: LIROPREM: PCP: PENCHLOROL:
PERMASAN: SANTOPHEN 20: STCC 4961380: RCRA U242: NA 2020:
C6HCL5O: OHS18150
CHEMICAL FAMILY:
HALOGEN COMPOUND, AROMATIC
MOLECULAR FORMULA CL5-C6-O-H MOLECULAR WEIGHT: 266.34
CERCLA RATINGS (SCALE 0-3): HEALTH=3 FIRE=0 REACTIVITY=0 PERSISTENCE'S
NFPA RATINGS (SCALE 0-4): HEALTHS FIRE=0 REACTIVTTY=0
COMPONENTS AND CONTAMINANTS
COMPONENT: PENTACHLOROPHENOL CAS# 87-86-5 PERCENT: 100.0
OTHER CONTAMINANTS: TECHNICAL GRADE MATERIAL MAY CONTAIN TRACES OF
CHLORINATED DIBENZODIOXINS
EXPOSURE LIMIT:
PENTACHLOROPHENOL
0.5 MG/M3 OSHA TWA (SKIN)
0.5 MG/M3 ACGIH TWA (SKIN)
10 POUNDS CERCLA SECTION 103 REPORTABLE QUANTITY
SUBJECT TO SARA SECTION 313 ANNUAL TOXIC CHEMICAL RELEASE REPORTING
SUBJECT TO CALIFORNIA PROPOSITION 65 CANCER AND/OR REPRODUCTIVE TOXICITY
WARNING AND RELEASE REQUIREMENTS- (JANUARY 1, 1990)
126
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PHYSICAL DATA
DESCRIPTION: WHITE POWDER OR CRYSTALS OR DARK-COLORED FLAKES WITH A VERY
PUNGENT ODOR WHEN HOT.
BOILING POINT: 588-590 F (309-310 C)
@ 754 MMHG (DEC) MELTING POINT: 374-376 F (190-191 C)
SPECIFIC GRAVITY: 1.978 @ 22 C SOLUBILITY IN WATER: 14 PPM @ 20 C
VAPOR DENSITY: 9.2 VAPOR PRESSURE: 0.00017 MMHG @ 20 C
OTHER SOLVENTS (SOLVENT - SOLUBILITY):
SOLUBLE IN ALCOHOL, ETHER, BENZENE, CARBITOL, XYLENE,
CELLOSOLVE, DIETHYLENE GLYCOL, PARAFFINIC PETROLEUM OILS, DILUTE ALKALI;
MODERATELY SOLUBLE IN ETHYLENE GLYCOL, CARBON TETRACHLORIDE.
OTHER PHYSICAL DATA
TECHNICAL GRADE MATERIAL MAY MELT ABOVE 338 F (170 C)
FIRE AND EXPLOSION DATA
FIRE AND EXPLOSION HAZARD
NEGLIGIBLE FIRE HAZARD WHEN EXPOSED TO HEAT OR FLAME.
FIREFIGHTING MEDIA:
DRY CHEMICAL. CARBON DIOXIDE, WATER SPRAY OR REGULAR FOAM
(1990 EMERGENCY RESPONSE GUIDEBOOK, DOT P 5800.5).
FOR LARGER FIRES. USE WATER SPRAY, FOG OR REGULAR FOAM
(1990 EMERGENCY RESPONSE GUIDEBOOK, DOT P 5800.5).
FIREFIGHTING:
MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK (1990 EMERGENCY
RESPONSE GUIDEBOOK. DOT P 5800.5, GUIDE PAGE 53).
USE AGENTS SUITABLE FOR TYPE OF FIRE. AVOID BREATHING HAZARDOUS VAPORS, KEEP
UPWIND.
127
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TRANSPORTATION
DEPARTMENT OF TRANSPORTATION HAZARD CLASSIFICATION 49 CFR 172.101:
POISON B
DEPARTMENT OF TRANSPORTATION LABELING REQUIREMENTS 49 CFR 172.101 AND
SUBPART E:
POISON
DEPARTMENT OF TRANSPORTATION PACKAGING REQUIREMENTS: 49 CFR 173.365
EXCEPTIONS: 49 CFR 173.364
TOXICITY
PENTACHLOROPHENOL:
IRRITATION DATA: 10 MG/24 HOURS OPEN SKIN-RABBIT MILD.
TOXICITY DATA: 355 MG/M3 INHALATION-RAT LC50; 225 MG/M3 INHALATION-MOUSE LC50;
40 MG/KG SKIN-RABBIT LDLO; 96 MG/KG SKIN-RAT LD50; 401 MG/KG ORAL-MAN LDLO;
27 MG/KG ORAL-RAT LD50; 117 MG/KG ORAL-MOUSE LD50; 70 MG/KG ORAL-RABBIT
LDLO; 168 MG/KG ORAL-HAMSTER LD50; 100 MG/KG SUBCUTANEOUS-RAT LD50; 70 MG/KG
SUBCUTANEOUS-RABBIT LDLO; 135 MG/KG SUBCUTANEOUS-DOG LDLO; 56 MG/KG
INTRAPERITONEAL-RAT LD50; 58 MG/KG INTRAPERITONEAL-MOUSE LD50; 135 MG/KG
INTRAPERITONEAL-RABBIT LDLO; 100 MG/KG UNREPORTED-GUINEA PIG LD50; 70 MG/KG
UNREPORTED-DOG LD50; MUTAGENIC DATA (RTECS); REPRODUCTIVE EFFECTS DATA
(RTECS); TUMORIGENIC DATA (RTECS).
CARCINOGEN STATUS: HUMAN LIMITED EVIDENCE (IARC GROUP-2B FOR CHLOROPHENOLS);
ANIMAL INADEQUATE EVIDENCE (IARC). STUDIES REVEALED A SIGNIFICANT INCREASE
IN SOFT-TISSUE SARCOMA AND LUNG, NASAL AND NASOPHARYNGEAL CANCER IN
WORKERS EXPOSED TO CHLOROPHENOLS. THERE WAS CLEAR EVIDENCE OF
CARCINOGENIC ACTIVITY IN MICE FED A TECHNICAL-GRADE AND A TECHNICAL-GRADE
FORMULATION AS SHOWN BY INCREASED INCIDENCES OF ADRENAL MEDULLARY AND
HEPATOCELLULAR NEOPLASMS AND HEMANGIOSARCOMAS (NTP TR-349).
LOCAL EFFECTS: IRRITANT- INHALATION, SKIN, EYE.
ACUTE TOXICmr LEVEL: HIGHLY TOXIC BY INHALATION, DERMAL ABSORPTION AND
INGESTION.
TAFJGET EFFECTS: POISONING MAY INCREASE THE METABOLIC RATE AND AFFECT THE
CARDIOVASCULAR AND NERVOUS SYSTEMS, LIVER, AND KIDNEYS.
128
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AT INCREASED RISK FROM EXPOSURE: PERSONS WITH RENAL OR HEPATIC DISEASES.
ADDITIONAL DATA: HOT ENVIRONMENTS MAY ENHANCE ABSORPTION AND THE TOXIC
EFFECTS.
HEALTH EFFECTS AND FIRST AID
INHALATION:
PENTACHLOROPHENOL:
IRRITANT/HIGHLY TOXIC.
150 MG/M3 IMMEDIATELY DANGEROUS TO LIFE OR HEALTH.
ACUTE EXPOSURE- MAY CAUSE IRRITATION OF THE UPPER RESPIRATORY TRACT WITH
CONCENTRATIONS GREATER THAN 1 MG/M3 PRODUCING PAIN IN THE NOSE AND THROAT,
VIOLENT SNEEZING, AND COUGH. SYMPTOMS OF SYSTEMIC POISONING MAY INCLUDE
HEADACHE, FEVER, INTENSE THIRST. EXCESSIVE PERSPIRATION, GENERALIZED
WEAKNESS, DIZZINESS. TACHYCARDIA, TACHYPNEA, DYSPNEA, CHEST PAIN,
PAIN IN THE EXTREMITIES, ANOREXIA. WEIGHT LOSS, METABOLIC ACIDOSIS.
AND GASTROINTESTINAL UPSET WITH NAUSEA, VOMITING. AND ABDOMINAL PAIN.
IN SEVERE POISONINGS, THESE EFFECTS MAY PROGRESS TO MUSCLE SPASM.
DEHYDRATION, HYPERPYREXIA, ANESTHESIA. LEUKOCYTOSIS, HYPERGLYCEMIA.
EDEMA AND HEMORRHAGE IN THE LUNGS. CEREBRAL EDEMA. STUPOR. CONVULSIONS.
AND COMA. LIVER AND KIDNEY DAMAGE MAY OCCUR. DEATH MAY BE DUE TO
VASCULAR COLLAPSE AND HEART FAILURE AND MAY OCCUR WITHIN HOURS OF THE
ONSET OF SYMPTOMS FOLLOWED RAPIDLY BY RIGOR MORTIS. IMPAIRMENT OF
AUTONOMIC FUNCTION AND CIRCULATION AND VISUAL DAMAGE WERE OBSERVED IN
SOME SERIOUS CASES OF POISONING.
CHRONIC EXPOSURE- REPEATED EXPOSURE TO LOW-LEVELS MAY CAUSE IRRITATION
OF THE NOSE, THROAT, AND LUNGS LEADING TO BRONCHITIS AND SINUSITIS.
IN ADDITION TO THE SYSTEMIC EFFECTS LISTED ABOVE. REPEATED OR PROLONGED
EXPOSURE HAS BEEN ASSOCIATED WITH THE DEVELOPMENT OF ACUTE PANCREATITIS.
LEUKOPENIA, IMMUNOLOGICAL CHANGES. APLASTIC ANEMIA, INTRAVASCULAR
HEMOLYSIS, AND POLYNEURITIS.
FIRST AID- REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY. IF BREATHING
HAS STOPPED, PERFORM ARTIFICIAL RESPIRATION. ADMINISTER OXYGEN. TREAT
SYMPTOMATICALLY AND SUPPORTIVELY. GET MEDICAL ATTENTION IMMEDIATELY.
SKIN CONTACT:
PENTACHLOROPHENOL:
IRRITANT/HIGHLY TOXIC.
ACUTE EXPOSURE- BRIEF, SINGLE EXPOSURE TO SOLUTIONS CONTAINING APPROXIMATELY
10% PENTACHLOROPHENOL MAY CAUSE IRRITATION. SOLIDS AND CONCENTRATED
SOLUTIONS MAY POSSIBLY PRODUCE SKIN BURNS. THIS MATERIAL MAY BE ABSORBED
THROUGH THE SKIN IN FATAL AMOUNTS AND PRODUCE SYSTEMIC EFFECTS AS
DESCRIBED IN ACUTE INHALATION.
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CHRONIC EXPOSURE- PROLONGED OR REPEATED EXPOSURE MAY CAUSE DERMATITIS AND
A RARE ALLERGIC SKIN RESPONSE; SOLUTIONS CONTAINING AS LITTLE AS 1% MAY
CAUSE IRRITATION. REPEATED ABSORPTION MAY RESULT IN SYSTEMIC EFFECTS AS
DESCRIBED IN INHALATION. CHLORACNE AND DISORDERS OF THE NERVOUS SYSTEM,
LIVER, AND PORPHYRIA MAY OCCUR DUE TO THE PRESENCE OF CHLORINATED
DIBENZODIOXINS.
FIRST AID- REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY. THEN REMOVE
SKIN AND HAIR CONTAMINATION BY SCRUBBING WITH SOAP AND WATER. IF BODY
TEMPERATURE IS ELEVATED, REDUCE TO 37 C BY SPONGE BATH, IMMERSION IN COOL
WATER OR BY APPLYING COOLING BLANKET. IF BODY TEMPERATURE IS ABOVE 40 C,
ICE WATER IS NECESSARY (DREISBACH, HANDBOOK OF POISONING. 12TH EDITION;
MORGAN, EPA RECOGNITION AND MANAGEMENT OF PESTICIDE POISONINGS. 3RD
EDITION). GET MEDICAL ATTENTION IMMEDIATELY.
EYE CONTACT:
PENTACHLOROPHENOL:
IRRITANT.
ACUTE EXPOSURE- EXPOSURE TO FINE DUST AND SPRAYS MAY CAUSE PAINFUL
IRRITATION, LACRIMATION, CORNEAL NUMBNESS, SLIGHT MYDRIASIS. AND
INFLAMMATION THAT MAY PROGRESS TO PERMANENT CORNEAL INJURY.
CHRONIC EXPOSURE- REPEATED OR PROLONGED EXPOSURE MAY CAUSE CONJUNCTIVITIS.
FIRST AID- WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL
SALINE, OCCASIONALLY LIFTING UPPER AND LOWER LIDS, UNTIL NO EVIDENCE OF
CHEMICAL REMAINS (APPROXIMATELY 15-20 MINUTES). GET MEDICAL ATTENTION
IMMEDIATELY.
INGESTION:
PENTACHLOROPHENOL:
HIGHLY TOXIC.
ACUTE EXPOSURE- MAY CAUSE SEVERE IRRITATION OF THE GASTROINTESTINAL TRACT
AND SYSTEMIC EFFECTS AS DESCRIBED IN INHALATION. SEVERE TOXIC EFFECTS
MAY OCCUR IN HUMANS WITH INGESTION OF 2 GRAMS.
CHRONIC EXPOSURE- MAY CAUSE EFFECTS AS DESCRIBED IN INHALATION.
ENLARGEMENT OF THE LIVER, CHANGES IN VARIOUS ENZYME ACTIVITIES, AND
OTHER HEPATIC EFFECTS WERE OBSERVED IN RATS RECEIVING PENTACHLOROPHENOL
FOR PERIODS OF THREE TO EIGHT MONTHS. FETOTOXIC EFFECTS, FETAL DEATHS AND
RESORPTIONS HAVE BEEN REPORTED IN RODENTS. INCREASED INCIDENCES OF
ADRENAL MEDULLARY AND HEPATOCELLULAR NEOPLASMS AND HEMANGIOSARCOMAS
WERE OBSERVED IN A 2-YEAR STUDY OF MICE.
FIRST AID- IF VICTIM IS ALERT AND RESPIRATION IS NOT DEPRESSED, INDUCE
EMESIS WITH SYRUP OF IPECAC. IF VICTIM IS NOT FULLY ALERT, EMPTY THE
STOMACH IMMEDIATELY BY INTUBATION, ASPIRATION, AND LAVAGE, USING ISOTONIC
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SALINE OR 5% SODIUM BICARBONATE. FOLLOW EMESIS OR LAVAGE WITH ACTIVATED
CHARCOAL GIVE SODIUM SULFATE AS A CATHARTIC. REDUCE ELEVATED BODY
TEMPERATURE TO 37 C BY SPONGE BATHS, IMMERSION IN COOL WATER OR BY APPLYING
COOLING BLANKET. (MORGAN. EPA RECOGNITION AND MANAGEMENT OF PESTICIDE
POISONINGS. THIRD EDITION). GET MEDICAL ATTENTION.
ANTIDOTE:
NO SPECIFIC ANTIDOTE. TREAT SYMPTOMATICALLY AND SUPPORTIVELY.
REACTIVITY SECTION
REACTIVITY:
STABLE UNDER NORMAL TEMPERATURES AND PRESSURES.
INCOMPATIBILITIES:
PENTACHLOROPHENOL:
OXIDIZERS (STRONG): FIRE AND EXPLOSION HAZARD.
DECOMPOSITION:
THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC AND CORROSIVE CHLORIDE
FUMES. TOXIC AND HAZARDOUS CHLORINATED PHENOLS AND OXIDES OF CARBON.
POLYMERIZATION:
HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL
TEMPERATURES AND PRESSURES.
STORAGE-DISPOSAL
OBSERVE ALL FEDERAL. STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING
OF THIS SUBSTANCE. FOR ASSISTANCE. CONTACT THE DISTRICT DIRECTOR OF THE
ENVIRONMENTAL PROTECTION AGENCY.
"STORAGE**
PROTECT AGAINST PHYSICAL DAMAGE. STORE IN A COOL. DRY, WELL VENTILATED
LOCATION. AWAY FROM ANY AREA WHERE THE FIRE HAZARD MAY BE ACUTE. OUTSIDE OR
DETACHED STORAGE IS PREFERRED (NFPA 49. HAZARDOUS CHEMICALS DATA, 1975).
STORE AT 4 C, PROTECT FROM LIGHT AND KEEP DRY.
STORE AWAY FROM INCOMPATIBLE SUBSTANCES.
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"DISPOSAL**
PENTACHLOROPHENOL - REGULATORY LEVEL: 100.0 MG/L
MATERIALS WHICH CONTAIN THE ABOVE SUBSTANCE AT OR ABOVE THE REGULATORY
LEVEL MEET THE EPA CHARACTERISTIC OF TOXICITY, AND MUST BE DISPOSED OF IN
ACCORDANCE WITH 40 CFR PART 262. EPA HAZARDOUS WASTE NUMBER D037.
CONDITIONS TO AVOID
MAY BURN BUT DOES NOT IGNITE READILY. PREVENT DISPERSION OF DUST IN AIR. DO
NOT ALLOW SPILLED MATERIAL TO CONTAMINATE WATER SOURCES.
SPILLS AND LEAKS
SOIL-RELEASE:
DIG A PIT, POND, LAGOON OR HOLDING AREA TO CONTAIN LIQUID OR SOLID MATERIAL.
COVER SOLIDS WITH A PLASTIC SHEET TO PREVENT DISSOLVING IN RAIN OR
FIREFIGHTING WATER.
WATER-SPILL:
USE NATURAL DEEP WATER POCKETS, EXCAVATED LAGOONS, OR SAND BAG BARRIERS TO
TRAP MATERIAL AT BOTTOM. USE ACTIVATED CARBON AT 10 TIMES THE SPILLED AMOUNT
IF IT IS DISSOLVED AT 10 PPM OR GREATER CONCENTRATION. REMOVE TRAPPED MATERIAL
WITH SUCTION HOSES. USE MECHANICAL DREDGES OR LIFTS TO REMOVE IMMOBILIZED
MASSES OF POLLUTION AND PRECIPITATES.
THE CALIFORNIA SAFE DRINKING WATER AND TOXIC ENFORCEMENT ACT OF 1986
(PROPOSITION 65) PROHIBITS CONTAMINATING ANY KNOWN SOURCE OF DRINKING WATER
WITH SUBSTANCES KNOWN TO CAUSE CANCER AND/OR REPRODUCTIVE TOXICITY.
OCCUPATIONAL-SPILL:
DO NOT TOUCH SPILLED MATERIAL STOP LEAK IF YOU CAN DO IT WITHOUT RISK. FOR
SMALL SPILLS, TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO
CONTAINERS FOR LATER DISPOSAL. FOR SMALL DRY SPILLS, WITH A CLEAN SHOVEL
PLACE MATERIAL INTO CLEAN, DRY CONTAINER AND COVER. MOVE CONTAINERS FROM
SPILL AREA. FOR LARGER SPILLS, DIKE FAR AHEAD OF SPILL FOR LATER DISPOSAL
KEEP UNNECESSARY PEOPLE AWAY. ISOLATE HAZARD AREA AND DENY ENTRY.
REPORTABLE QUANTITY (RQ): 10 POUNDS
THE SUPERFUND AMENDMENTS AND REAUTHORIZATION ACT (SARA) SECTION 304 REQUIRES
THAT A RELEASE EQUAL TO OR GREATER THAN THE REPORTABLE QUANTITY FOR THIS
SUBSTANCE BE IMMEDIATELY REPORTED TO THE LOCAL EMERGENCY PLANNING COMMITTEE
AND THE STATE EMERGENCY RESPONSE COMMISSION (40 CFR 355.40). IF THE RELEASE OF
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THIS SUBSTANCE IS REPORTABLE UNDER CERCLA SECTION 103. THE NATIONAL RESPONSE
CENTER MUST BE NOTIFIED IMMEDIATELY AT (800) 424-8802 OR (202) 426-2675 IN THE
METROPOLITAN WASHINGTON. D.C. AREA (40 CFR 302.6).
PROTECTIVE EQUIPMENT SECTION
VENTILATION:
PROCESS ENCLOSURE RECOMMENDED TO MEET PUBLISHED EXPOSURE LIMITS.
RESPIRATOR:
THE FOLLOWING RESPIRATORS AND MAXIMUM USE CONCENTRATIONS ARE
RECOMMENDATIONS BY THE U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIOSH
POCKET GUIDE TO CHEMICAL HAZARDS; NIOSH CRITERIA DOCUMENTS OR BY THE U.S.
DEPARTMENT OF LABOR, 29 CFR 1910 SUBPART Z
THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND
IN THE WORK PLACE, MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND
BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND
HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION (NIOSH-MSHA).
PENTACHLOROPHENOL:
5.0 MG/M3- CHEMICAL CARTRIDGE RESPIRATOR WITH ORGANIC VAPOR CARTRIDGE(S)
IN COMBINATION WITH A DUST, MIST, AND FUME FILTER.
SUPPLIED-AIR RESPIRATOR.
SELF-CONTAINED BREATHING APPARATUS.
12.5 MG/M3- SUPPLIED-AIR RESPIRATOR OPERATED IN CONTINUOUS FLOW MODE.
POWERED AIR-PURIFYING RESPIRATOR WITH ORGANIC VAPOR CARTRIDGE(S)
IN COMBINATION WITH A DUST, MIST. AND FUME FILTER.
25.0 MG/M3- CHEMICAL CARTRIDGE RESPIRATOR WITH FULL FACEPIECE AND ORGANIC
VAPOR CARTRIDGE(S) IN COMBINATION WITH A HIGH-EFFICIENCY
PARTICULATE FILTER.
SUPPLIED-AIR RESPIRATOR WITH FULL FACEPIECE.
SELF-CONTAINED BREATHING APPARATUS WITH FULL FACEPIECE.
150 MG/M3- SUPPLIED-AIR RESPIRATOR WITH A FULL FACEPIECE AND OPERATED IN
PRESSURE DEMAND OR OTHER POSITIVE PRESSURE MODE.
ESCAPE- AIR-PURIFYING FULL FACEPIECE RESPIRATOR (GAS MASK) WITH A
CHIN-STYLE OR FRONT- OR BACK-MOUNTED ORGANIC VAPOR CANISTER
HAVING A HIGH-EFFICIENCY PARTICULATE FILTER.
ESCAPE-TYPE SELF-CONTAINED BREATHING APPARATUS.
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FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH
CONDITIONS:
SELF-CONTAINED BREATHING APPARATUS WITH FULL FACEPIECE OPERATED IN
PRESSURE-DEMAND OR OTHER POSITIVE PRESSURE MODE.
SUPPLIED-AIR RESPIRATOR WITH FULL FACEPIECE AND OPERATED IN PRESSURE-DEMAND
OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY
SELF-CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE-DEMAND OR OTHER
POSITIVE PRESSURE MODE.
CLOTHING:
EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE (IMPERVIOUS) CLOTHING AND
EQUIPMENT TO PREVENT ANY POSSIBILITY OF SKIN CONTACT WITH THIS SUBSTANCE.
GLOVES:
EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH
THIS SUBSTANCE.
EYE PROTECTION:
EMPLOYEE MUST WEAR SPLASH-PROOF OR DUST-RESISTANT SAFETY GOGGLES AND A
FACESHIELD TO PREVENT CONTACT WITH THIS SUBSTANCE.
EMERGENCY WASH FACILITIES:
WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE'S EYES AND/OR SKIN MAY BE
EXPOSED TO THIS SUBSTANCE, THE EMPLOYER SHOULD PROVIDE AN EYE WASH FOUNTAIN
AND QUICK DRENCH SHOWER WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE.
AUTHORIZED BY- OCCUPATIONAL HEALTH SERVICES, INC.
CREATION DATE: 03/18/85 REVISION DATE: 10/10/90
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MATERIAL SAFETY DATA SHEET OHS14280
OCCUPATIONAL HEALTH SERVICES. INC. FOR EMERGENCY SOURCE INFORMATION
11 WEST 42ND STREET. 12TH FLOOR CONTACT: 1-615-366-2000
NEW YORK, NEW YORK 10036
1-800-445-MSDS (1-800-445-6737) OR 1-212-789-3535
SUBSTANCE IDENTIFICATION
CAS-NUMBER 67-56-1
RTEC-NUMBER PC1400000
SUBSTANCE: METHYL ALCOHOL
TRADE NAMES/SYNONYMS:
METHANOL: WOOD ALCOHOL: METHYL HYDROXIDE: CARBINOL:
MONOHYDROXYMETHANE: WOOD SPIRIT: WOOD NAPHTHA: METHYLOL: COLONIAL
SPIRIT: COLUMBIAN SPIRIT: PYROXYLIC SPIRIT: BOOSTER FUEL (HENES
PRODUCT CORP.): METHANOL (ELECTROKLEIN) (ROK): METHANOL. SPECTRO
QUALITY (MCB MANF. CHEMIST): COULOMATIC (R) CONDITIONER SOLUTION:
STANDARD WATER IN METHANOL: STCC 4904230: RCRA U154: UN 1230: GH4O:
OHS14280
CHEMICAL FAMILY:
HYDROXYL, ALIPHATIC
MOLECULAR FORMULA: C-H3-O-H MOLECULAR WEIGHT: 32.04
CERCLA RATINGS (SCALE 0-3): HEALTH=3 FIRE=3 REACTIVITY=0 PERSISTENCE=0
NFPA RATINGS (SCALE 0-4): HEALTH=1 FIRE=3 REACTIVITY=0
COMPONENTS AND CONTAMINANTS
COMPONENT: METHYL ALCOHOL (METHANOL) CAS# 67-56-1 PERCENT: 100
OTHER CONTAMINANTS: NONE
EXPOSURE LIMIT:
METHYL ALCOHOL (METHANOL):
200 PPM (260 MG/M3) OSHA TWA (SKIN); 250 PPM (325 MG/M3) OSHA STEL
200 PPM (260 MG/M3) ACGIH TWA (SKIN); 250 PPM (310 MG/M3) ACGIH STEL
200 PPM NIOSH RECOMMENDED 10 HOUR TWA;
800 PPM NIOSH RECOMMENDED 15 MINUTE CEILING
5000 POUNDS CERCLA SECTION 103 REPORTABLE QUANTITY
SUBJECT TO SARA SECTION 313 ANNUAL TOXIC CHEMICAL RELEASE REPORTING
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PHYSICAL DATA
DESCRIPTION: CLEAR. COLORLESS LIQUID WITH A CHARACTERISTIC ALCOHOLIC ODOR.
BOILING POINT: 149 F (65 C) MELTING POINT: -137 F (-94 C)
SPECIFIC GRAVITY: 0.7914 EVAPORATION RATE: 4.6 (BUTYL ACETATE=1)
VISCOSITY: 0.59 CPS @ 20 C SOLUBILITY IN WATER: VERY SOLUBLE
VAPOR DENSITY: 1.11 VAPOR PRESSURE: 97.25 MMHG @ 20 C
ODOR-THRESHOLD: 100 PPM
OTHER SOLVENTS (SOLVENT - SOLUBILITY):
SOLUBLE IN ETHER, BENZENE, ALCOHOL, ACETONE,
CHLOROFORM, ETHANOL, KETONES AND MOST OTHER ORGANIC SOLVENTS.
FIRE AND EXPLOSION DATA
FIRE AND EXPLOSION HAZARD
DANGEROUS FIRE HAZARD WHEN EXPOSED TO HEAT, FLAME, OR OXIDIZERS.
VAPORS ARE HEAVIER THAN AIR AND MAY TRAVEL A CONSIDERABLE DISTANCE TO A SOURCE
OF IGNITION AND FLASH BACK.
VAPOR-AIR MIXTURES ARE EXPLOSIVE.
FLASH POINT: 52 F (11 C) (CC) UPPER EXPLOSION LIMIT: 36.0%
LOWER EXPLOSION LIMIT: 6.0% AUTOIGNITION TEMP.: 725 F (385 C)
FLAMMABILJTY CLASS (OSHA): IB
FIREFIGHTING MEDIA:
DRY CHEMICAL, CARBON DIOXIDE, WATER SPRAY OR ALCOHOL-RESISTANT FOAM
(1990 EMERGENCY RESPONSE GUIDEBOOK, DOT P 5800.5).
FOR LARGER FIRES, USE WATER SPRAY, FOG OR ALCOHOL-RESISTANT FOAM
(1990 EMERGENCY RESPONSE GUIDEBOOK, DOT P 5800.5).
FIREFIGHTING:
MOVE CONTAINER FROM FIRE AREA IF YOU CAN DO IT WITHOUT RISK. DIKE FIRE-CONTROL
WATER FOR LATER DISPOSAL; DO NOT SCATTER THE MATERIAL APPLY COOLING WATER TO
SIDES OF CONTAINERS THAT ARE EXPOSED TO FLAMES UNTIL WELL AFTER FIRE IS OUT.
STAY AWAY FROM ENDS OF TANKS. WITHDRAW IMMEDIATELY IN CASE OF RISING SOUND
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FROM VENTING SAFETY DEVICE OR ANY DISCOLORATION OF TANK DUE TO FIRE. ISOLATE
FOR 1/2 MILE IN ALL DIRECTIONS IF TANK, RAIL CAR OR TANK TRUCK IS INVOLVED IN
FIRE (1990 EMERGENCY RESPONSE GUIDEBOOK, DOT P 5800.5, GUIDE PAGE 28).
EXTINGUISH ONLY IF FLOW CAN BE STOPPED; USE WATER IN FLOODING AMOUNTS AS FOG,
SOLID STREAMS MAY NOT BE EFFECTIVE. COOL CONTAINERS WITH FLOODING QUANTITIES
OF WATER, APPLY FROM AS FAR A DISTANCE AS POSSIBLE. AVOID BREATHING TOXIC
VAPORS, KEEP UPWIND.
TRANSPORTATION
DEPARTMENT OF TRANSPORTATION HAZARD CLASSIFICATION 49 CFR 172.101:
FLAMMABLE LIQUID
DEPARTMENT OF TRANSPORTATION LABELING REQUIREMENTS 49 CFR 172.101 AND
SUBPART E:
FLAMMABLE LIQUID
DEPARTMENT OF TRANSPORTATION PACKAGING REQUIREMENTS: 49 CFR 173.119
EXCEPTIONS: 49 CFR 173.118
TOXICITY
METHYL ALCOHOL (METHANOL):
IRRITATION DATA: 20 MG/24 HOURS SKIN-RABBIT MODERATE; 40 MG EYE-RABBIT
MODERATE; 100 MG/24 HOURS EYE-RABBIT MODERATE.
TOXICITY DATA: 86,000 MG/M3 INHALATION-HUMAN TCLO; 300 PPM INHALATION-HUMAN
TCLO; 64,000 PPM/4 HOURS INHALATION-RAT LC50; 1000 PPM INHALATION-MONKEY
LCLO; 50 GM/M3/2 HOURS INHALATION-MOUSE LCLO; 44.000 MG/M3/6 HOURS
INHALATION-CAT LCLO; 15,800 MG/KG SKIN-RABBIT LD50; 393 MG/KG SKIN-MONKEY
LDLO; 428 MG/KG ORAL-HUMAN LDLO; 143 MG/KG ORAL-HUMAN LDLO; 6422 MG/KG
ORAL-MAN LDLO; 3429 MG/KG ORAL-MAN TDLO; 4 GM/KG ORAL-WOMAN TDLO; 7 GM/KG
ORAL-MONKEY LD50; 5628 MG/KG ORAL-RAT LD50; 7300 MG/KG ORAL-MOUSE LD50;
14.200 MG/KG ORAL-RABBIT LD50; 7500 MG/KG ORAL-DOG LDLO; 9800 MG/KG
SUBCUTANEOUS-MOUSE LD50; 2131 MG/KG INTRAVENOUS-RAT LD50; 4710 MG/KG
INTRAVENOUS-MOUSE LD50; 8907 MG/KG INTRAVENOUS-RABBIT LD50; 7529 MG/KG
INTRAPERITONEAL-RAT LD50; 10,765 MG/KG INTRAPERITONEAL-MOUSE LD50;
1826 MG/KG INTRAPERITONEAL-RABBIT LD50; 868 MG/KG UNREPORTED-MAN LDLO;
MUTAGENIC DATA (RTECS); REPRODUCTIVE EFFECTS DATA (RTECS).
CARCINOGEN STATUS: NONE.
LOCAL EFFECTS: IRRITANT- SKIN, EYE.
ACUTE TOXJCITY LEVEL: SLIGHTLY TOXIC BY INHALATION, DERMAL ABSORPTION,
INGESTION.
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TARGET EFFECTS: NEUROTOXIN; CENTRAL NERVOUS SYSTEM DEPRESSANT.
AT INCREASED RISK FROM EXPOSURE: PERSONS WITH KIDNEY. EYE OR SKIN DISORDERS.
HEALTH EFFECTS AND FIRST AID
INHALATION:
METHYL ALCOHOL (METHANOL):
NARCOTIC/NEUROTOXIN. 25,000 PPM IMMEDIATELY DANGEROUS TO LIFE OR HEALTH.
ACUTE EXPOSURE- MAY CAUSE IRRITATION OF THE MUCOUS MEMBRANES. COUGHING.
OPPRESSION IN THE CHEST, TRACHEITIS, BRONCHITIS, TINNITUS, UNSTEADY
GAIT, TWITCHING, COLIC, CONSTIPATION, NYSTAGMUS, AND BLEPHAROSPASM.
SYMPTOMS FROM OCCUPATIONAL EXPOSURE INCLUDE PARESTHESIAS, NUMBNESS AND
SHOOTING PAINS IN THE HANDS AND FOREARMS. METABOLIC ACIDOSIS, AND EFFECTS
ON THE EYES AND CENTRAL NERVOUS SYSTEM MAY OCCUR AS DETAILED IN ACUTE
INGESTION.
CHRONIC EXPOSURE- REPEATED OR PROLONGED EXPOSURE MAY CAUSE EFFECTS AS IN
ACUTE INGESTION. REPEATED EXPOSURE TO 200-375 PPM CAUSED RECURRENT
HEADACHES IN WORKERS. EXPOSURE FOR 4 YEARS TO 1200-8000 PPM RESULTED IN
MARKED DIMINUTION OF VISION AND ENLARGEMENT OF THE LIVER IN A WORKMAN.
REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS.
FIRST AID- REMOVE FROM EXPOSURE AREA TO FRESH AIR IMMEDIATELY. IF BREATHING
HAS STOPPED, PERFORM ARTIFICIAL RESPIRATION. KEEP PERSON WARM AND AT REST.
TREAT SYMPTOMATICALLY AND SUPPORTIVELY. GET MEDICAL ATTENTION IMMEDIATELY.
SKIN CONTACT:
METHYL ALCOHOL (METHANOL):
IRRITANT/NARCOTIC/NEUROTOXIN.
ACUTE EXPOSURE- CONTACT WITH LIQUID MAY CAUSE IRRITATION. SKIN ABSORPTION
MAY OCCUR AND CAUSE METABOLIC ACIDOSIS AND EFFECTS ON THE EYES AND CENTRAL
NERVOUS SYSTEM AS DETAILED IN ACUTE INGESTION.
CHRONIC EXPOSURE- REPEATED OR PROLONGED CONTACT WITH THE LIQUID MAY CAUSE
DEFATTING OF THE SKIN RESULTING IN ERYTHEMA, SCALING, AND ECZEMATOID
DERMATITIS. CHRONIC ABSORPTION MAY RESULT METABOLIC ACIDOSIS AND EFFECTS
AS DETAILED IN ACUTE INGESTION.
FIRST AID- REMOVE CONTAMINATED CLOTHING AND SHOES IMMEDIATELY. WASH AFFECTED
AREA WITH SOAP OR MILD DETERGENT AND LARGE AMOUNTS OF WATER UNTIL NO
EVIDENCE OF CHEMICAL REMAINS (APPROXIMATELY 15-20 MINUTES). GET MEDICAL
ATTENTION IMMEDIATELY.
EYE CONTACT:
METHYL ALCOHOL (METHANOL):
IRRITANT.
ACUTE EXPOSURE- VAPORS MAY CAUSE IRRITATION. HIGH CONCENTRATIONS HAVE
BEEN REPORTED TO CAUSE VIOLENT INFLAMMATION OF THE CONJUNCTIVA AND
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EPITHELIAL DEFECTS ON THE CORNEA. MILD IRRITATION MAY OCCUR WITH
DILUTE SOLUTIONS; THE UNDILUTED LIQUID HAS PRODUCED MODERATE CORNEAL
OPACITY AND CONJUNCTIVE REDNESS IN RABBITS. APPLICATION OF A DROP
OF METHANOL IN RABBIT EYES CAUSED A MILD REVERSIBLE REACTION, GRADED
3 ON A SCALE OF 1-10 AFTER 24 HOURS.
CHRONIC EXPOSURE- REPEATED OR PROLONGED CONTACT MAY CAUSE CONJUNCTIVITIS.
FIRST AID- WASH EYES IMMEDIATELY WITH LARGE AMOUNTS OF WATER OR NORMAL
SALINE,
OCCASIONALLY LIFTING UPPER AND LOWER LIDS, UNTIL NO EVIDENCE OF CHEMICAL
REMAINS (APPROXIMATELY 15-20 MINUTES). GET MEDICAL ATTENTION IMMEDIATELY.
INGESTION:
METHYL ALCOHOL (METHANOL):
NARCOTIC/NEUROTOXIN.
ACUTE EXPOSURE- MAY CAUSE MILD AND TRANSIENT INEBRIATION AND SUBSEQUENT
DROWSINESS FOLLOWED BY AN ASYMPTOMATIC PERIOD LASTING 8-48 HOURS.
FOLLOWING THE DELAY, COUGHING. DYSPNEA. HEADACHE, DULLNESS, WEAKNESS,
VERTIGO OR DIZZINESS, NAUSEA, VOMITING, OCCASIONAL DIARRHEA, ANOREXIA,
VIOLENT PAIN IN THE BACK. ABDOMEN, AND EXTREMITIES, RESTLESSNESS. APATHY
OR DELIUIUM. AND RARELY, EXCITEMENT AND MANIA MAY OCCUR. RAPID, SHALLOW
RESPIRATION DUE TO METABOLIC ACIDOSIS, COLD AND CLAMMY SKIN, HYPOTENSION,
CYANOSIS, OPISTHOTONOS, CONVULSIONS, MILD TACHYCARDIA, CARDIAC DEPRESSION,
PERIPHERAL NEURITIS, CEREBRAL AND PULMONARY EDEMA, UNCONSCIOUSNESS, AND
COMA ARE POSSIBLE. EFFECTS ON THE EYE MAY INCLUDE OPTIC NEURITIS, BLURRED
OR DIMMED VISION, DILATED, UNRESPONSIVE PUPILS, PTOSIS, EYE PAIN,
CONCENTRIC CONSTRICTION OF VISUAL FIELDS, DIPLOPIA, CHANGE IN COLOR
PERCEPTION, PHOTOPHOBIA, AND OPTIC NERVE ATROPHY. PARTIAL BLINDNESS OR
POSSIBLY DELAYED TRANSIENT OR PERMANENT BLINDNESS MAY OCCUR. BILATERAL
SENSORINEURAL DEAFNESS HAS BEEN REPORTED IN A SINGLE CASE. LIVER, KIDNEY,
HEART, STOMACH, INTESTINAL AND PANCREATIC DAMAGE MAY ALSO OCCUR. DEATH
MAY BE DUE TO RESPIRATORY FAILURE OR RARELY FROM CIRCULATORY COLLAPSE.
AS LITTLE AS 15 ML HAS CAUSED BLINDNESS; THE USUAL FATAL DOSE IS
60-240 ML PROLONGED ASTHENIA AND IRREVERSIBLE EFFECTS ON THE NERVOUS
SYSTEM INCLUDING DIFFICULTY IN SPEECH, MOTOR DYSFUNCTION WITH RIGIDITY,
SPASTICITY, AND HYPOKINESIS HAVE BEEN REPORTED.
CHRONIC EXPOSURE- REPEATED INGESTION MAY CAUSE VISUAL IMPAIRMENT AND
BLINDNESS AND OTHER SYSTEMIC EFFECTS AS DETAILED IN ACUTE INGESTION.
REPRODUCTIVE EFFECTS HAVE BEEN REPORTED IN ANIMALS.
FIRST AID- IF INGESTION OF METHANOL IS DISCOVERED WITHIN 2 HOURS, GIVE
SYRUP OF IPECAC. LAVAGE THOROUGHLY WITH 2-4 L OF TAP WATER WITH SODIUM
BICARBONATE (20 G/L) ADDED. GET MEDICAL ATTENTION IMMEDIATELY. LAVAGE
SHOULD BE PERFORMED BY QUALIFIED MEDICAL PERSONNEL (DREISBACH, HANDBOOK
OF POISONING, 12TH ED.).
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ANTIDOTE:
THE FOLLOWING ANTIDOTE(S) HAVE BEEN RECOMMENDED. HOWEVER, THE DECISION AS TO
WHETHER THE SEVERITY OF POISONING REQUIRES ADMINISTRATION OF ANY ANTIDOTE
AND ACTUAL DOSE REQUIRED SHOULD BE MADE BY QUALIFIED MEDICAL PERSONNEL
METHANOL POISONING:
GIVE ETHANOL, 50% (100 PROOF), 1.5 ML/KG ORALLY INITIALLY, DILUTED TO NOT MORE
THAN 5% SOLUTION, FOLLOWED BY 0.5-1.0 ML/KG EVERY 2 HOURS ORALLY OR
INTRAVENOUSLY FOR 4 DAYS IN ORDER TO REDUCE METABOLISM OF METHANOL AND TO
ALLOW TIME FOR ITS EXCRETION. BLOOD ETHANOL LEVEL SHOULD BE IN THE RANGE OF
1-1.5 MG/ML (DREISBACH, HANDBOOK OF POISONING, 12TH ED.). ANTIDOTE SHOULD
BE ADMINISTERED BY QUALIFIED MEDICAL PERSONNEL
ORAL OR INTRAVENOUS ADMINISTRATION OF 4-METHYLPYRAZOLE INHIBITS ALCOHOL
DEHYDROGENASE AND HAS BEEN USED EFFECTIVELY AS AN ANTIDOTE FOR METHANOL OR
ETHYLENE GLYCOL POISONING (ELLENHORN AND BARCELOUX, MEDICAL TOXICOLOGY).
REACTIVITY SECTION
REACTIVITY:
STABLE UNDER NORMAL TEMPERATURES AND PRESSURES.
INCOMPATIBILITIES:
METHYL ALCOHOL (METHANOL):
ACETYL BROMIDE: VIOLENT REACTION WITH FORMATION OF HYDROGEN BROMIDE.
ALKYLALUMINUM SOLUTIONS: VIOLENT REACTION.
ALUMINUM: CORRODES.
BARIUM PERCHLORATE: DISTILLATION YIELDS HIGHLY EXPLOSIVE ALKYL PERCHLORATE.
BERYLLIUM HYDRIDE: VIOLENT REACTION. EVEN AT -196 C.
BROMINE: VIGOROUSLY EXOTHERMIC REACTION.
CALCIUM CARBIDE: VIOLENT REACTION.
CHLORINE: POSSIBLE IGNITION AND EXPLOSION HAZARD.
CHLOROFORM AND SODIUM HYDROXIDE: EXPLOSIVE REACTION.
CHROMIUM TRIOXIDE (CHROMIC ANHYDRIDE): POSSIBLE IGNITION.
CYANURIC CHLORIDE: VIOLENT REACTION.
DICHLOROMETHANE: POSSIBLE IGNITION AND EXPLOSION.
DIETHYL ZINC: POSSIBLE IGNITION AND EXPLOSION.
HYDROGEN PEROXIDE + WATER: EXPLOSION HAZARD.
IODINE + ETHANOL + MERCURIC OXIDE: EXPLOSION HAZARD.
LEAD: CORRODES.
LEAD PERCHLORATE: EXPLOSION HAZARD.
MAGNESIUM: VIOLENT REACTION.
MAGNESIUM (POWDERED): MIXTURES ARE CAPABLE OF DETONATION.
METALS: INCOMPATIBLE.
NICKEL: POSSIBLE IGNITION IN THE PRESENCE OF NICKEL CATALYST.
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NITRIC ACID (CONCENTRATED): MIXTURES OF GREATER THAN 25% ACID MAY DECOMPOSE
VIOLENTLY.
OXIDIZERS (STRONG): FIRE AND EXPLOSION HAZARD.
PERCHLORIC ACID: EXPLOSION HAZARD.
PHOSPHOROUS TR1OXIDE: POSSIBLE VIOLENT REACTION AND IGNITION.
PLASTICS, RUBBER, COATINGS: MAY BE ATTACKED.
POTASSIUM: POSSIBLE DANGEROUS REACTION.
POTASSIUM HYDROXIDE + CHLOROFORM: EXOTHERMIC REACTION.
POTASSIUM TERT-BUTOXIDE: FIRE AND EXPLOSION HAZARD.
SODIUM + CHLOROFORM: POSSIBLE EXPLOSION.
SODIUM HYPOCHLORITE: EXPLOSION HAZARD.
SODIUM METHOXIDE + CHLOROFORM: VIOLENT REACTION.
SULFURIC ACID: FIRE AND EXPLOSION HAZARD.
ZINC: EXPLOSION HAZARD.
DECOMPOSITION:
THERMAL DECOMPOSITION PRODUCTS MAY INCLUDE TOXIC OXIDES OF CARBON.
POLYMERIZATION:
HAZARDOUS POLYMERIZATION HAS NOT BEEN REPORTED TO OCCUR UNDER NORMAL
TEMPERATURES AND PRESSURES.
STORAGE-DISPOSAL
OBSERVE ALL FEDERAL, STATE AND LOCAL REGULATIONS WHEN STORING OR DISPOSING
OF THIS SUBSTANCE. FOR ASSISTANCE, CONTACT THE DISTRICT DIRECTOR OF THE
ENVIRONMENTAL PROTECTION AGENCY.
"STORAGE**
STORE IN ACCORDANCE WITH 29 CFR 1910.106.
STORE AWAY FROM INCOMPATIBLE SUBSTANCES.
"DISPOSAL**
DISPOSAL MUST BE IN ACCORDANCE WITH STANDARDS APPLICABLE TO GENERATORS OF
HAZARDOUS WASTE, 40 CFR 262. EPA HAZARDOUS WASTE NUMBER U154.
CONDITIONS TO AVOID
AVOID CONTACT WITH HEAT, SPARKS, FLAMES OR OTHER IGNITION SOURCES. VAPORS MAY
BE EXPLOSIVE. MATERIAL IS POISONOUS; AVOID INHALATION OF VAPORS OR CONTACT
WITH SKIN. DO NOT ALLOW MATERIAL TO CONTAMINATE WATER SOURCES.
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SPILLS AND LEAKS
SOIL-RELEASE:
DIG HOLDING AREA SUCH AS LAGOON, POND OR PIT FOR CONTAINMENT.
DIKE FLOW OF SPILLED MATERIAL USING SOIL OR SANDBAGS OR FOAMED BARRIERS SUCH
AS POLYURETHANE OR CONCRETE.
AIR-RELEASE:
APPLY WATER SPRAY TO KNOCK DOWN VAPORS.
WATER-SPILL:
ALLOW SPILLED MATERIAL TO AERATE.
LIMIT SPILL MOTION AND DISPERSION WITH NATURAL BARRIERS OR OIL SPILL CONTROL
BOOMS.
USE SUCTION HOSES TO REMOVE TRAPPED SPILL MATERIAL
OCCUPATIONAL-SPILL:
SHUT OFF IGNITION SOURCES. DO NOT TOUCH SPILLED MATERIAL. STOP LEAK IF YOU
CAN DO IT WITHOUT RISK. USE WATER SPRAY TO REDUCE VAPORS. FOR SMALL SPILLS.
TAKE UP WITH SAND OR OTHER ABSORBENT MATERIAL AND PLACE INTO CONTAINERS FOR
LATER DISPOSAL FOR LARGER SPILLS, DIKE FAR AHEAD OF SPILL FOR LATER
DISPOSAL NO SMOKING, FLAMES OR FLARES IN HAZARD AREA! KEEP UNNECESSARY PEOPLE
AWAY; ISOLATE HAZARD AREA AND DENY ENTRY.
REPORTABLE QUANTITY (RQ): 5000 POUNDS
THE SUPERFUND AMENDMENTS AND REAUTHORIZATION ACT (SARA) SECTION 304 REQUIRES
THAT A RELEASE EQUAL TO OR GREATER THAN THE REPORTABLE QUANTITY FOR THIS
SUBSTANCE BE IMMEDIATELY REPORTED TO THE LOCAL EMERGENCY PLANNING COMMITTEE
AND THE STATE EMERGENCY RESPONSE COMMISSION (40 CFR 355.40). IF THE RELEASE OF
THIS SUBSTANCE IS REPORTABLE UNDER CERCLA SECTION 103, THE NATIONAL RESPONSE
CENTER MUST BE NOTIFIED IMMEDIATELY AT (800) 424-8802 OR (202) 426-2675 IN THE
METROPOLITAN WASHINGTON, D.C. AREA (40 CFR 302.6).
PROTECTIVE EQUIPMENT SECTION
VENTILATION:
PROVIDE LOCAL EXHAUST OR PROCESS ENCLOSURE VENTILATION TO MEET THE PUBLISHED
EXPOSURE LIMITS. VENTILATION EQUIPMENT MUST BE EXPLOSION-PROOF.
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RESPIRATOR:
THE FOLLOWING RESPIRATORS AND MAXIMUM USE CONCENTRATIONS ARE
RECOMMENDATIONS
BY THE U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIOSH POCKET GUIDE TO
CHEMICAL HAZARDS; NIOSH CRITERIA DOCUMENTS OR BY THE U.S. DEPARTMENT OF
LABOR, 29 CFR 1910 SUBPART Z.
THE SPECIFIC RESPIRATOR SELECTED MUST BE BASED ON CONTAMINATION LEVELS FOUND
IN THE WORK PLACE, MUST NOT EXCEED THE WORKING LIMITS OF THE RESPIRATOR AND
BE JOINTLY APPROVED BY THE NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND
HEALTH AND THE MINE SAFETY AND HEALTH ADMINISTRATION (NIOSH-MSHA).
METHYL ALCOHOL (METHANOL):
2000 PPM- ANY SUPPUED-AIR RESPIRATOR.
ANY SELF-CONTAINED BREATHING APPARATUS.
5000 PPM- ANY SUPPUED-AIR RESPIRATOR OPERATED IN A CONTINUOUS FLOW MODE.
10.000 PPM- ANY SELF-CONTAINED BREATHING APPARATUS WITH A FULL FACEPIECE.
ANY SUPPUED-AIR RESPIRATOR WITH A FULL FACEPIECE.
ANY SUPPLIED-AIR RESPIRATOR WITH A TIGHT-FITTING FACEPIECE
OPERATED IN A CONTINUOUS FLOW MODE.
25,000 PPM- ANY SUPPLIED-AIR RESPIRATOR WITH A FULL FACEPIECE AND OPERATED
IN A PRESSURE-DEMAND OR OTHER POSITIVE PRESSURE MODE.
ESCAPE- ANY APPROPRIATE ESCAPE-TYPE SELF-CONTAINED BREATHING APPARATUS.
FOR FIREFIGHTING AND OTHER IMMEDIATELY DANGEROUS TO LIFE OR HEALTH
CONDITIONS:
SELF-CONTAINED BREATHING APPARATUS WITH FULL FACEPIECE OPERATED IN
PRESSURE-DEMAND OR OTHER POSITIVE PRESSURE MODE.
SUPPUED-AIR RESPIRATOR WITH FULL FACEPIECE AND OPERATED IN PRESSURE-DEMAND
OR OTHER POSITIVE PRESSURE MODE IN COMBINATION WITH AN AUXILIARY
SELF-CONTAINED BREATHING APPARATUS OPERATED IN PRESSURE-DEMAND OR OTHER
POSITIVE PRESSURE MODE.
CLOTHING:
EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE (IMPERVIOUS) CLOTHING AND
EQUIPMENT TO PREVENT REPEATED OR PROLONGED SKIN CONTACT WITH THIS SUBSTANCE.
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GLOVES:
EMPLOYEE MUST WEAR APPROPRIATE PROTECTIVE GLOVES TO PREVENT CONTACT WITH
THIS SUBSTANCE.
EYE PROTECTION:
EMPLOYEE MUST WEAR SPLASH-PROOF OR DUST-RESISTANT SAFETY GOGGLES TO PREVENT
EYE CONTACT WITH THIS SUBSTANCE.
EMERGENCY EYE WASH: WHERE THERE IS ANY POSSIBILITY THAT AN EMPLOYEE'S EYES MAY
BE EXPOSED TO THIS SUBSTANCE, THE EMPLOYER SHOULD PROVIDE AN EYE WASH
FOUNTAIN WITHIN THE IMMEDIATE WORK AREA FOR EMERGENCY USE.
AUTHORIZED BY- OCCUPATIONAL HEALTH SERVICES, INC.
CREATION DATE: 09/25/84 REVISION DATE: 10/09/90
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APPENDIX J
CALCULATION OF MATRIX SPIKE PERCENT RECOVERY
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CALCULATION OF MATRIX SPIKE PERCENT RECOVERY
The rationale and calculation of matrix spike recovery is as follows:
1. Calculate the change in concentration (A.) expected due to the addition of the matrix
(internal standard) spike.
2. Measure the concentration in the original solution (C0) and the concentration in the
spiked solution (CJ.
3. The percent recovery is the percent of the change in concentration relative to the
expected change, or
(C C )
Percent recovery = 100 x — - -
For example:
The original measure of the concentration of penta is 20 ppb = C0
The spike is added to increase the concentration by 15 ppb = A,
The measured concentration of the spiked sample is 34 ppb = Cs
The percent recovery = 100 x ' - " — ' = 93%
15
Therefore,
Percent recovery . [sample + spike) - [unspiked sample] x 10Q
[spike added]
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GLOSSARY OF IMMUNOASSAY TERMS
antibody ~ a receptor protein (immunoglobulin) produced in response to an antigen that binds
specifically to form an antigen-antibody complex
antigen - a large complex molecule that has a distinctive shape or functional group that can
induce the formation of specific antibodies and can react to form an antigen-antibody
complex
chromogenic substrate - compound that is converted to a colored end product when acted upon
by an enzyme.
hapten - a small antigenic molecule (e.g., POP) that cannot induce an antibody response unless it
is covalently bound to a carrier molecule forming a hapten-carrier conjugate.
immunoassay - a physical assay based on the reversible interaction of a specific antibody with a
target compound or compounds.
monoclonal antibody ~ a homogeneous antibody population derived from one specific antibody-
producing cell.
polyclonal antiserum - a heterogeneous population of antibodies varying in antigenic specificity
and affinity that is derived from several antibody-producing cells.
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