&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/540/R-07/001
January 2007
Interim Report on the
Evolution and Performance
of the Eichrom Technologies
Procept® Rapid Dioxin Assay
for Soil and Sediment Samples
-------
-------
EPA/540/R-07/001
January 2007
Interim Report on the
Evolution and Performance of the Eichrom
Technologies Procept® Rapid Dioxin Assay
for
Soil and Sediment Samples
Prepared by
Battelle
505 King Avenue
Columbus, Ohio 43201
Contract No. 68-C-00-185
Stephen Billets
Environmental Sciences Division
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89119
-------
Notice
This document was prepared for the U.S. Environmental Protection Agency (EPA) Superfund Innovative
Technology Evaluation Program under Contract No. 68-C-00-185. The document has met the EPA's
requirements for peer and administrative review and has been approved for publication. Mention of
corporation names, trade names, or commercial products does not constitute endorsement or
recommendation for use.
-------
Abstract
A demonstration of screening technologies for determining the presence of dioxin and dioxin-like
compounds in soil and sediment was conducted under the U.S. Environmental Protection Agency's
(EPA's) Superfund Innovative Technology Evaluation Program in Saginaw, Michigan in 2004. The
objectives of the demonstration included evaluating each participating technology's accuracy, precision,
sensitivity, sample throughput, tendency for matrix effects, and cost. The test also included an assessment
of how well the technology's results compared to those generated by established laboratory methods
using high-resolution mass spectrometry (HRMS). The demonstration objectives were accomplished by
evaluating the results generated by each technology from 209 soil, sediment, and extract samples. The test
samples included performance evaluation (PE) samples (i.e., contaminant concentrations were certified or
the samples were spiked with known contaminants) and environmental samples collected from
10 different sampling locations. The PE and environmental samples were distributed to the technology
developers in blind, random order. One of the participants in the original SITE demonstration was
Hybrizyme Corporation, which demonstrated the use of the AhRC PCR™ Kit. The AhRC PCR™ Kit
was a technology that reported the concentration of aryl hydrocarbon receptor (AhR) binding compounds
in a sample, with units reported as Ah Receptor Binding Units (AhRBU). At the time of the original
demonstration, this particular technology was intended for use as a screening tool to rank samples from
those inducing the greatest AhR activity to those inducing the least AhR activity rather than to provide
highly quantitative dioxin concentration in units of toxicity equivalents (TEQ). After the SITE Dioxin
demonstration, this technology was exclusively licensed to Eichrom Technologies. Eichrom focused their
efforts on developing optimal sample preparation procedures for the assay and reporting TEQ values
instead of AhRBU. The technology is now marketed under the trade name Procept® Rapid Dioxin Assay.
The developers and potential users of the technologies provided feedback after the demonstration. There
was significant interest in evaluating the performance of these technologies on a site-specific basis. This
would more closely represent the expected application of the technologies than was the case during the
original demonstration, which targeted technology performance when challenged with a broad range of
sample types. Consequently, a second test (referred to as the "site-specific study") was conducted in
which the developers were given a total of 112 samples that were segregated by site of origin. In contrast
to the original demonstration, in which all sample information was unknown, environmental information
for each site was provided to the developers to more closely represent the background information that
would be available to contractors supporting a site-specific application. Each batch included some
samples previously analyzed as part of the SITE Dioxin Demonstration and some unique samples in
archive that were not used as part of the SITE Dioxin Demonstration, along with replicates and quality
control (QC) samples. Only dioxin and furan concentrations were evaluated in this study. The developers
were given the HRMS data from the SITE Dioxin Demonstration so that they would have the opportunity
to utilize a site-specific calibration and knowledge regarding typical congener patterns at a particular site.
Data analysis focused on analytical performance on a site-specific basis, and included an evaluation of
comparability to the HRMS total dioxin/furan toxicity equivalents (TEQD/F) results, precision on replicate
analyses, and QC sample results.
This report describes the experimental design of the site-specific study, the analytical methods used, and
comparisons of the TEQD/F results from the HRMS data to those reported by Eichrom Technologies
Procept® Rapid Dioxin Assay. The data generated and evaluated during the site-specific study showed
that the TEQ data produced by the Procept® Rapid Dioxin Assay was more comparable to the HRMS
iii
-------
TEQD/F data than was the data reported using the Hybrizyme AhRC PCR™ Kit in the original SITE
demonstration. The Procept® Rapid Dioxin Assay could be used as an effective screening tool to
determine areas of greatest concern for cleanup at a site and could help to minimize the number of more
expensive analyses needed for specific analytes, particularly considering that the cost and the time to
analyze samples is significantly less than that of HRMS analyses.
IV
-------
Contents
Page
Chapter 1 Introduction 1
1.1 SITE MMT Program Description 1
1.2 Background of SITE Dioxin Demonstration 1
1.3 Description of Eichrom Technologies 3
1.4 Overview of the Report 3
Chapter 2 Test Description 4
2.1 Experimental Design 4
2.2 Site Descriptions 5
2.2.1 Soil Sampling Sites 5
2.2.2 Sediment Sampling Sites 6
2.3 Testing Protocol 7
2.4 Data Analysis 7
2.4.1 Comparability 7
2.4.2 Precision 7
Chapter3 Methods 9
3.1 Sample Preparation 10
3.1.1 Procept® Rapid Dioxin Assay Method 12
3.1.2 Characterization HRMS Method 13
3.2 Sample Analysis 14
3.2.1 Procept® Rapid Dioxin Assay Method 14
3.2.2 Characterization HRMS Method 15
3.3 Quality Control 16
3.3.1 Procept® Rapid Dioxin Assay Method 16
3.3.2 Characterization HRMS Method 16
3.4 Data Presentation Results 17
3.4.1 Eichrom Method 17
3.4.2 Characterization HRMS Method 19
3.5 Comparison of Procept® and HRMS methods 19
Chapter 4 Results and Discussion 22
4.1 Evolution of Procept® Rapid Dioxin Assay 22
4.2 Procept® Rapid Dioxin Assay Results 22
4.3 Discussion 22
4.4 Operational Factors 29
4.4.1 Cost of Procept® Rapid Dioxin Assay 29
4.4.2 Cost Comparison to HRMS Methods 30
4.4.3 Availability of Technology 30
4.4.4 Turnaround 31
4.4.5 Training/Ease of Use for Procept® Assay 31
Chapter 5 Conclusions and Future Directions 33
Chapter 6 References 34
-------
List of Tables
Page
Table 2-1. HRMS Holding Time Analysis for Archived Samples 4
Table 2-2. Summary of Site-Specific Study Experimental Design 5
Table 3-1. Summary of HRMS Method Modifications Relative to Traditional EPA Method 1613B 9
Table 3-2. Comparison of Procept® Rapid Dioxin Assay Response Factors to Toxicity
Equivalency Factors (TEF) 18
Table 3-3. Comparison between Procept® Rapid Dioxin Assay and Characterization HRMS Methods 20
Table 4-1. WinonaPost Sample Results 23
Table 4-2. Tittabawassee River Sample Results 24
Table 4-3. Newark Bay Sample Results 25
Table 4-4. Raritan Bay Sample Results 26
Table 4-5. Solutia Sample Results 27
Table 4-6. Hybrizyme AhRBU Results Compared to HRMS by Ranking—Original SITE
Demonstration Data 28
Table 4-7. Capital Equipment Costs for the Procept® Assay 29
Table 4-8. Chemicals and Supplies Cost for Procept® Assay 30
Table 4-9. Estimation of Sample Turnaround Time Using Procept® Assay 31
List of Figures
Page
Figure 1. Procept® Sample Preparation and Cleanup 10
Figure 2. Procept® Assay Procedure 11
Figure 3. Procept® Rapid Dioxin Assay Kit 12
VI
-------
Abbreviations, Acronyms, and Symbols
AhR aryl hydrocarbon receptor
AhRBU aryl hydrocarbon receptor binding units
ASE accelerated solvent extraction
ATSDR Centers for Disease Control's Agency for Toxic Substances and Disease Registry
D/F dioxin/furan
D/QAPP demonstration and quality assurance project plan
DQO Data quality objective
EPA Environmental Protection Agency
ERA Environmental Resource Associates
g gram
GC gas chromatography
GPC gel permeation chromatography
HRMS high-resolution mass spectrometry
ITVR innovative technology verification report
MDEQ Michigan Department of Environmental Quality
MMT Monitoring and Measurement Technology
MS mass spectrometry
NERL National Exposure Research Laboratory
NIST National Institute for Standards and Technology
ORD Office of Research and Development
PAH polynuclear aromatic hydrocarbon
PCB polychlorinated biphenyl
PCDD/F polychlorinated dibenzo-p-dioxin/dibenzofuran
PE performance evaluation
pg picogram
PCR polymerase chain reaction
ppt parts per trillion; picogram/g; pg/g
QA/QC quality assurance/quality control
RPD relative percent difference
RSD relative standard deviation
vii
-------
SITE Superfund Innovative Technology Evaluation
TAT turn around time
TCDD tetrachlorodibenzo-p-dioxin
TCDF tetrachlorodibenzofuran
TEF toxicity equivalency factor
TEQ toxicity equivalent
TEQD/F total toxicity equivalents of dioxins/furans
WHO World Health Organization
Vlll
-------
Acknowledgements
This report was prepared for the U.S. Environmental Protection Agency (EPA) Superfund Innovative
Technology Evaluation (SITE) Program under the direction and coordination of Stephen Billets of the
EPA's National Exposure Research Laboratory (NERL)—Environmental Sciences Division in Las
Vegas, Nevada. The EPA NERL thanks the technology developer, Eichrom Technologies, in particular
Mike Fern and Dan McAlister, for their participation in this study and for their review of this report. The
EPA NERL thanks the following peer reviewers for their review of this report: Andy Grange (US EPA
NERL) and Al Taylor (Michigan Department of Environmental Quality). This report was prepared for the
EPA by Battelle. Acknowledgment is given to Amy Dindal, who was the Battelle Project Manager, and to
Elizabeth Thompson, Joe Tabor, and Mary Schrock, for their contributions to the preparation of this
report.
-------
Chapter 1
Introduction
1.1 SITE MMT Program Description
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
National Exposure Research Laboratory (NERL) contracted with Battelle (Columbus, Ohio) to conduct a
demonstration of monitoring and measurement technologies for dioxin and dioxin-like compounds in soil
and sediment. Testing of screening technologies for dioxin and dioxin-like compounds was conducted as
part of the EPA Superfund Innovative Technology Evaluation (SITE) Monitoring and Measurement
Technology (MMT) Program. The MMT Program evaluates technologies that sample, detect, monitor, or
measure hazardous and toxic substances. These technologies are expected to provide better, faster, or
more cost-effective methods for producing real-time data during site characterization and remediation
efforts than conventional laboratory technologies. The purpose of the SITE MMT Program is to
demonstrate reliable performance by the technologies to provide (1) potential users with a better
understanding of the technologies' performance under well-defined conditions and (2) technology
developers with documented results that will help promote the acceptance and use of their technologies.
1.2 Background of SITE Dioxin Demonstration
Conventional analytical methods for determining concentrations of dioxin and dioxin-like compounds are
time-consuming and costly. For example, EPA standard methods require solvent extraction of the sample,
processing the extract through multiple cleanup columns, and analyzing the cleaned fraction by gas
chromatography (GC)/high-resolution mass spectrometry (HRMS). Turnaround times for HRMS results
are typically three weeks. Use of these traditional methods for high volume sampling or screening a
contaminated site often is limited by budgetary constraints. The cost of these analyses can range from
$800 to $1,200 per sample, depending on the method selected, the level of quality assurance/quality
control incorporated into the analyses, and reporting requirements. The use of a simple, rapid (i.e., real-
time or near real-time), cost-effective analytical method would allow field personnel to quickly assess the
extent of contamination at a site and could be used to direct or monitor remediation or risk assessment
activities. This data could be used to provide immediate feedback on potential health risks associated with
the site and permit the development of a more focused and cost-effective sampling strategy.
Five technology developers participated in the SITE MMT Dioxin Demonstration in 2004 (referred to as
the "original SITE demonstration" throughout this report). The participating technologies included
immunoassay test kits and aryl hydrocarbon receptor (AhR)-based bioassays. A field demonstration of the
technologies was conducted in Saginaw, Michigan. A test suite of 209 soil, sediment, and extract samples
with a variety of distinguishing characteristics, such as high levels of poly chlorinated biphenyls (PCBs)
and polynuclear aromatic hydrocarbons (PAHs), was analyzed by each developer as described in the
project's demonstration plan (U.S. EPA, 2004). Samples were collected from 10 different sites around the
country with a known variety of dioxin-contaminated soil and sediment. Samples were identified and
supplied through EPA Regions 2, 3, 4, 5, and 7 and the Michigan Department of Environmental Quality
(MDEQ). In addition to providing environmental samples, MDEQ also facilitated access to the field
demonstration site and provided on-site technical and logistical support. The samples were homogenized
and characterized by HRMS prior to use in the original SITE demonstration to ensure a variety of
homogeneous, environmentally derived samples with concentrations over a large dynamic range (< 50 to
-------
> 10,000 picogram/gram [pg/g]) were included. The environmental samples comprised 61% of the test
samples (128 of the 209 samples) included in the original SITE demonstration. Performance evaluation
(PE) samples were obtained from five commercial sources. PE samples consisted of known quantities of
dioxin and dioxin-like compounds. Fifty-eight of the 209 demonstration samples (28%) were PE samples.
Soil or sediment samples were extracted with toluene using Dean Stark Soxhlet extraction, and aliquots
were provided to each of the five study participants to avoid possible variation due to sample
heterogeneity. A total of 23 extracts (11% of the total number of samples) was included in the original
SITE demonstration. For the 209 samples, sample type and sampling site were unknown to the developer
during the analysis in order to challenge the technologies with a variety of matrices and potential
interferences in an unbiased way. During the development of the demonstration plan, the possibility of
identifying the environmental site to the developers was discussed, but the Demonstration Panel (which
included all of the developers and approximately 20 EPA Regional experts) concluded that all sample
analyses should be blind to the developers. Also, all developers refused additional sample information
when it was offered to them prior to the demonstration. An EPA innovative technology verification report
(ITVR) was published for each technology (U.S. EPA, 2005a, b, c, d, e). Each report is posted on the
EPA SITE program Web Site (www.epa.gov/ORD/SITE).
The results of the original SITE demonstration suggested that all of the technologies could be used in
some capacity to screen for sample concentrations above and below threshold values (e.g., less than or
greater than 1,000 pg/g toxicity equivalents (TEQ)). However, none of the tested technologies
demonstrated a significantly high correlation with the HRMS data. After publication of the SITE reports
and dissemination of the information through seminars and conference presentations, subsequent
feedback from the developers and from potential users of the technologies indicated significant interest in
evaluating the performance of these technologies on a site-specific basis. The consensus was that, if the
technology developers had more information about the sample identities (for example, sample site) and
had access to historical analytical information, the results from the screening technologies would be more
highly correlated to the HRMS results. Since this type of information (sample location and dioxin
congeners) would typically be made available during a site characterization, this approach was adopted
and a second study was launched.
The follow-on study (referred to as the "site-specific study") was conducted in May 2006. All past
participants in the original SITE demonstration were invited to participate in the site-specific study, and
three developers did so. The study was conducted in the developer's laboratories, rather than a central
demonstration site, since the experiences of the original SITE demonstration suggested that these were
primarily laboratory-based technologies that could be mobilized in a field environment. The developers
were given a total of 112 samples that were segregated by site and asked to report sample concentration in
terms of total TEQD/F (Only dioxin and furan concentrations were evaluated due to the limited range of
PCB concentrations in the samples that were available for this study). In contrast to the original SITE
demonstration in which all sample information was unknown, environmental information for each site
was provided to the developers. Samples were obtained from archived samples from the original SITE
demonstration. Each batch included some samples previously analyzed as part of the original SITE
demonstration and additional samples in archive along with replicates and one quality control (QC)
sample per site batch. The developers were provided with the HRMS TEQD/F concentration and dioxin
congener data for the QC sample only. This provided the developers with an opportunity to calibrate their
results on a site-specific basis using the HRMS data from the QC sample for each site. The developers
were asked to analyze the QC sample unspiked, then spike the QC sample with a known quantity of
dioxin congeners (which congeners and at what concentration was left to the discretion of the developer)
in duplicate to assess accuracy. Data analysis focused on analytical performance on a site-specific basis,
and included an evaluation of comparability to the HRMS total dioxin/furan toxicity equivalents (TEQD/F)
results, precision on replicate analyses, and QC sample results. One of the participants in the site-specific
-------
study was the Procept® Rapid Dioxin Assay by Eichrom Technologies, and the results for this technology
are described in this report.
1.3 Description of Eichrom Technologies
Founded in 1990 to commercialize chemical separation technology developed at Argonne National
Laboratory, Eichrom Technologies is a provider of products in the areas of radiochemistry, geochemistry,
hazardous metals analysis, and environmental screening. Eichrom supplies a range of ion exchange resins,
from industry standard cation and anion exchange resins, to proprietary chelating resins that have unusual
selectivity for higher valence transition metals. In 2004 Eichrom Europe, a French subsidiary, established
an analytical laboratory to monitor drinking water for gross alpha/beta, tritium and potassium-40. This
lab also performs radionuclide-specific analysis for a variety of alpha- and beta-emitting isotopes,
whether anthropogenic or naturally occurring.
In 2005 Eichrom signed a licensing agreement with Hybrizyme Corporation, which provided exclusive
manufacturing and sales rights in the United States and Europe to Hybrizyme's AhRC PCR® Assay for
poly chlorinated dibenzodioxins and furans (PCDD/F). The Hybrizyme Corporation AhRC PCR® Assay
was a technology that reported the concentration of AhR binding compounds in units reported as AhRBU.
The technology was intended for use as a screening tool to rank samples from those inducing the greatest
AhR activity to those inducing the least AhR activity, rather than to provide highly quantitative TEQ
values. Hybrizyme's goal was a highly portable screening technology that could help determine areas of
greatest concern for cleanup at a site and could help minimize the number of more expensive analyses
needed for specific analytes. Hybrizyme's AhRC PCR® Assay was one of the five technologies evaluated
in the original SITE demonstration. After the original SITE demonstration, Hybrizyme licensed this
technology to Eichrom Technologies. Since that time, Eichrom has focused on developing optimal sample
preparation procedures for the assay and reporting quantitative TEQ values. This technology is now
marketed under the trade name Procept® Rapid Dioxin Assay and is designed for use in analytical
chemistry labs.
1.4 Overview of the Report
This report describes the experimental design of the site-specific study. Detailed methods are provided
for the Procept® Rapid Dioxin Assay and the HRMS methods are also discussed. Correlations between
Eichrom TEQD/F and HRMS TEQD/F results are discussed along with the accuracy and precision of the
test results. Operational factors such as cost comparisons, availability, turnaround times, and ease of use
and training are also reported, although this information was provided by Eichrom and not independently
verified.
-------
Chapter 2
Test Description
2.1 Experimental Design
Samples used in the site-specific study had been collected for the original SITE demonstration from a
variety of dioxin-contaminated soil and sediment sampling locations around the country. Samples were
identified and supplied by EPA Regions 2, 3, 4, 5, and 7 and the MDEQ. A mixture of soil and sediment
samples that would bracket the Centers for Disease Control's Agency for Toxic Substances and Disease
Registry (ATSDR) guidance levels (DeRosa, 1997) were used. The ATSDR decision framework
specifies that sites with TEQ levels between 50 and 1,000 picogram per gram (pg/g) should be further
evaluated and recommends action for levels above 1,000 pg/g (i.e., 1 part per billion (ppb)) TEQ. The
samples were homogenized and characterized by HRMS prior to use in the original SITE demonstration
to ensure inclusion of a variety of homogeneous, environmentally derived samples with concentrations
over a large dynamic range (<50 to >10,000 picogram/gram [pg/g]). Procedures for homogenization and
characterization are described in the demonstration/quality assurance project plan that can be found on the
SITE Program's Web Site (www.epa.gov/ORD/SITE) (U.S. EPA, 2004). Samples included in the site-
specific study experimental design were from five of the ten original SITE demonstration sites and
represented different matrices, congener patterns, and potential interferences. The environmental sites
included in the site-specific study were: Tittabawassee River, Newark Bay, Solutia, Raritan Bay, and
Winona Post. The samples had been stored in a freezer (approximately -20 °C) at Battelle for
approximately three years, since the time when the samples were collected for the original SITE
demonstration. As shown in Table 2-1, one sample from each site was analyzed by HRMS to confirm
that the concentrations had not changed significantly (>20% relative percent difference (RPD)) since the
initial analysis.
Table 2-1. HRMS Holding Time Analysis for Archived Samples
Site
Tittabawassee River
Newark Bay
Solutia
Raritan Bay
Winona
Original
Total TEQD/F (pg/g)
from
Characterization Analysis
3127
38.3
3951
13.8
11259
2006
Total TEQD/F (pg/g)
from
Holding Time Check
2560
36.7
4768
14.3
10156
Relative Percent
Difference
(%)
20
4
19
3
10
Only dioxin/furan (D/F) concentrations were evaluated in this study, as the PCB concentrations in the
available environmental samples ranged from 0.5 to 40 pg/g TEQPCB and most concentrations were
<10 pg/g TEQpCB . Consequently, the dynamic range of the PCB concentrations was inadequate for an
effective evaluation of the technologies. A total of 112 samples were included in this study and evaluated
by each technology. The distribution of samples amongst the five environmental sites and range of
concentrations analyzed are described in Table 2-2. Five or six discrete sampling locations were included
in each site batch. The samples in each site batch included those from sampling locations that were
-------
previously analyzed as part of the original SITE demonstration. In addition, samples from locations
within the site that were in archive that were not utilized as part of the original study were included. Four
replicates of each environmental sample were included to determine analysis precision. Included in the
number of samples per site are one to four replicates of an uncontaminated ("blank") soil matrix in each
site batch. The sample concentrations and identities were unknown to the developers, but the samples
were grouped by site batch, so that the developers knew which samples came from which site. The
HRMS data for one sample from each site, the QC sample, was provided to the developers. For the QC
samples only, the developers had access to all HRMS congener (dioxin, furan, PCB) data and supporting
analytical information (e.g., PAH concentrations) that was available. The intention was that the QC
samples would provide historical analytical information that could be used to calibrate the technology
responses on a site-specific basis. The developers were also asked to spike the QC samples in duplicate
to serve as a matrix spike/matrix spike duplicate. Congener and concentration selection for the spiking
solution were the developer's choice.
It should be noted that it was not an objective of the demonstration to accurately characterize the
concentration of dioxins, furans, and PCBs from a specific sampling site. It was, however, an objective to
ensure comparability between technology samples and the HRMS analysis samples. This was
accomplished by homogenizing each matrix, such that all sub-samples of a given matrix had consistent
contaminant concentrations. As a result, homogenized samples were not necessarily representative of
original concentrations at the site.
Table 2-2. Summary of Site-Specific Study Experimental Design
Site
Winona
Tittabawassee River
Newark Bay
Raritan Bay
Solutia
Matrix
Soil
Soil
Sediment
Sediment
Soil
Approximate Range of
Concentrations
(pg/g TEQD/F)
8,000 - 12,000
40- 1,100
15-65
10-15
40 - 4,000
Total number of site-specific study samples
# Samples a
21
24
21
21
25
112
a # samples includes one blank sample per site, except for Tittabawassee River which had four blanks included
2.2 Site Descriptions
This section provides descriptions of each of the soil and sediment sites, including how the sites became
contaminated and approximate dioxin concentrations, as well as the type and concentrations of other
major constituents (such as PCBs, pentachlorophenol (PCP), and PAHs), where known. This information
was provided by the site owners/sample providers (e.g., the EPA, the EPA contractors, and the MDEQ).
2.2.1 Soil Sampling Sites
2.2.1.1 Winona Post
The Winona Post site in Winona, Missouri, was a wood treatment facility that had been remediated.
Contaminants at the site included PCP, dioxin, diesel fuel, and PAHs. Over a period of at least 40 years,
these contaminants were deposited into an on-site drainage ditch and sinkhole. Areas of contaminant
deposition (approximately 8,500 cubic yards of soils/sludge) were excavated in late 200 I/early 2002. This
material was placed into an approximate 2!/2-acre treatment cell located on facility property. During
2002/2003, material at the treatment cell was treated through addition of amendments (high-ammonia
-------
fertilizer and manure) and tilling. Final concentrations achieved in the treatment cell averaged 26
milligrams per kilogram (mg/kg) for PCP and from 8,000 to 10,000 for pg/g TEQD/F. Samples used for
this study from this site were obtained from the treatment cell after these concentrations had been
achieved.
2.2.1.2 Solatia
The chemical production facility at the Solutia site in Nitro, West Virginia, is located along the eastern
bank of the Kanawha River, in Putnam County, West Virginia. The site has been used for chemical
production since the early 1910s. The initial production facility was developed by the U.S. government
for the production of military munitions during the World War I era between 1918 and 1921. The facility
was then purchased by a small private chemical company, which began manufacturing chloride,
phosphate, and phenol compounds at the site. A major chemical manufacturer purchased the facility in
1929 from Rubber Services Company. The company continued to expand operations and accelerated its
growth in the 1940s. A variety of raw materials has been used at the facility over the years, including
inorganic compounds, organic solvents, and other organic compounds, including Agent Orange. Agent
Orange is a mixture of chemicals containing equal amounts of two herbicides: 2,4-D (2,4
dichlorophenoxyacetic acid) and 2,4,5-T (2,4,5 trichlorophenoxyacetic acid). Manufacture of this
chemical herbicide began at the site in 1948 and ceased in 1969. Dioxin contamination in the site soils
was associated with the manufacture of 2,4,5-T, where dioxins are an unintentional by-product. The site
has a dioxin profile from the ppt to low parts per billion (ppb) range. No PCBs or PAHs were identified in
the soil.
2.2.1.3 Tittabawassee River
The MDEQ sampled Tittabawassee River flood plain soils at three sites. The contamination source was
possibly legacy contamination from chemical manufacturing. Individual samples were collected from two
locations at Imerman Park in Saginaw Township. The first sample was taken near the boat launch, and the
second sample was taken in a grassy area near the river bank. Previous analyses from these areas of this
park indicated a range of PCDD/F concentrations from 600 to 2,500 pg/g. Total PCBs from these
previous measurements were in the low parts-per-trillion (ppt) range. Individual samples were collected
from two locations at Freeland Festival Park in Freeland, MI. The first sample was taken above the river
bank, and the second sample was taken near a brushy forested area.
2.2.2 Sediment Sampling Sites
2.2.2.1 Newark Bay
Surrounded by manufacturing industries, Newark Bay is a highly contaminated area with numerous
sources (sewage treatment plants, National Pollutant Discharge Elimination System discharges, and
nonpoint sources). This bay is downstream from a dioxin Superfund site that contains some of the highest
dioxin concentrations in the United States and also is downstream from a mercury Superfund site. The
dioxin concentration in the area sampled for this demonstration was approximately 450 pg/g. Average
PCB concentrations ranged from 300 to 740 ppb. Fine-grained sediments make up 50% to 90% of the
dredged material. Average total organic carbon was about 4%.
2.2.2.2 RaritanBay
Surrounded by industry and residential discharges, Raritan Bay has dioxin contamination, but to a lesser
degree than Newark Bay. No major Superfund sites are located in the vicinity. Dioxin concentration
should be significantly less than in Newark Bay. PCB concentrations are around 250 ppb. The fine-
grained sediment and total organic carbon values were similar to percentages in Newark Bay.
-------
2.3 Testing Protocol
Samples from each of the five sites were sent to each developer in a "site batch", the compositions of
which are shown in Table 2-2. A total of 112 individual samples were provided to the developers.
Samples within each site batch were distributed in blind, random order. Samples from a particular site
were colored-coded (e.g., Site Batch #1 had green labels, Site Batch #2 had blue labels, etc.) to minimize
sample mix-up. Site names for each batch were disclosed to the developers prior to shipment of samples.
As stated earlier, the developers were provided with HRMS data for the QC samples so that they could
perform site-specific calibrations, but no other HRMS data was provided to the developers.
The composition of each site batch is discussed in Section 2.1. Developers were given 25 g of each
sample, except for the QC sample which was distributed in 50 g aliquots. Certified samples or Standard
Reference Materials® were not included in the study since the developers performed site-specific
calibrations. The developers were permitted and encouraged to calibrate their technologies for the D/F
responses at each site based on the HRMS data from the original SITE demonstration. The developers
had to specify the period of time required to analyze the samples.
Eichrom received their samples on March 20, 2006 and reported results to Battelle for all 112 samples on
May 1, 2006. After receiving the HRMS data, Eichrom elected to re-run all of their sample extracts,
using an additional purification step (i.e., a larger silica column and increased elution solvent) and revised
their reported data on June 2, 2006.
2.4 Data Analysis
As for the original SITE demonstration, HRMS analysis was used as the reference method against which
all developer data was compared. Data analysis focused on analytical performance on a site-specific basis
and included an evaluation of comparability to the HRMS total TEQD/F result, precision on replicate
analyses, and QC sample results. Qualitative parameters such as ease of use, cost, and sample throughput
were not assessed during this study, but information was provided by the developer for inclusion in the
report. In addition to the TEQD/F sample results, the developer reported the results from any additional
QC performed (for example: method blanks, positive controls, duplicates, etc.) that were analyzed as part
of their method for the batches of analyses from each site.
2.4.1 Comparability
The percent recovery (R) of the Procept® Rapid Dioxin Assay relative to the HRMS analysis was
calculated from the following equation:
TEQ
Recovery = —x 100 (eqn. 2-1)
where TEQA is the average measured concentration reported by Eichrom and TEQHRMs is the average
HRMS concentration. Acceptable performance is generally in the range of 70 - 130% relative recovery
values.
2.4.2 Precision
The standard deviation (S) of the results for the replicate environmental samples was calculated and used
as a measure of the Procept® Rapid Dioxin Assay's precision. Standard deviation was calculated from the
following equation:
-------
-cy
(eqn 2-2)
where n is the number of replicate samples, Ck is the concentration measured for the kth sample, and C is
the average concentration of the replicate samples. Precision was reported in terms of the relative standard
deviation (RSD) as described in equation 2-3. A method is considered to have acceptable precision if the
RSD values are less than 25%.
RSD
_S
C
xlOO
(eqn. 2-3)
-------
Chapter 3
Methods
This chapter describes the sample preparation, analytical, quality control, and data presentation methods
used by Eichrom Technologies. Additionally the reference HRMS method is discussed. Each section will
describe Eichrom's approach and the HRMS approach, followed by a description of the similarities and
differences between the procedures. The Eichrom approach is described in greater detail than the HRMS
method because it is assumed that the reader will have some basic knowledge of the HRMS method.
Flowcharts of the Eichrom method are presented in Figures 1 and 2. A photo of the Eichrom assay is
provided in Figure 3.
The HRMS method for determining TEQD/F described in this section is the same method that was used to
generate the characterization concentrations prior to the original SITE demonstration. This method was a
modification of EPA Method 1613B (U.S. EPA, 1994). Modifications to Method 1613B are allowed,
provided that method performance specifications can be met. Differences in the method employed and
traditional Method 1613B are summarized in Table 3-1. Both methods are described in detail in the
ITVRs (U.S. EPA, 2005a, b, c, d, e). As stated in Chapter 2, the modified 1613B method was used to
characterize the TEQD/F concentrations prior to the original SITE demonstration as a way to select
samples for use in the demonstration and to ensure that the samples were homogenized to acceptable
reproducibility criteria. This data set is referred to as "characterization" HRMS data. The samples
selected for use in the original SITE demonstration were then analyzed using the traditional 1613B
method. This data set is referred to as "reference" HRMS data. For samples that were analyzed by both
methods, the results were highly correlated (coefficient of determination = 0.99), demonstrating that the
characterization and reference 1613B methods produced comparable results (U.S. EPA, 2005a, b, c, d, e).
Since the characterization HRMS data was generated on all collected samples, including samples that
were and were not used in the original SITE demonstration, the characterization data were used for
comparison with the developer results for the site-specific study.
Table 3-1. Summary of HRMS Method Modifications Relative to Traditional EPA Method 1613B
Characterization Analysis - Modified 1613B
Accelerated solvent extraction with methylene
chloride
2,3,7,8-tetrachlorodibenzofuran (TCDF)
concentrations not confirmed
1 to 10 g was used, depending on what was known
about the site
Used extrapolation if calibration range was exceeded
Reference Analysis - Traditional 1613B
Soxhlet-Dean Stark extraction with toluene
2,3,7,8-TCDF concentrations confirmed
10 g always extracted. High concentration sites
were extracted and then diluted before adding
internal standard
All samples diluted so that peak areas were under
calibration peak areas
-------
'retreatment/
Extraction
Column
Purification
Dry Soil Sample at
110 degrees Celcius
for a minimum of 12
hours
Pack Silica and
Florisil columns
Homogenize sample
and weigh 5 gram
Aliquot for extraction
Extract sample using
approved method, such as
ASE, 30% acetone in toluene,
100 degrees celcius, 1500 psi,
2 five min static steps, 60%
flush volume
Add 2 mL of
dodecane
Evaporate to near
dryness with a stream
of dry air at 60
degrees celcius
Glass
Wool
Na2SO4
Sulfuric
Acid,
AgNO3,
and
KOH
Silicas
Silica
Glass
Wool
'J
\
4)R
h
(2) (3) (4)
/
^7)1
( 8) 1
2) Sample+ 5-10 g
44% H2SO4 Silica
3) Rinse vial 2x hexane
4) Rinse column additional
hexane
Transfer to 40 mL
vial, with 5 mL of
hexane, add 5-10
grams of 44%
H2S04 Silica Gel
Glass
Wool
Na2SO4
Florisil
Glass
Wool
7) 10 mL 2% dichloromethane in hexane
10 mL 5% dichloromethane in hexane
9) 15 mL 50% dichloromethane in hexane
Allow sample to sit
overnight
(1) (2) (3) (4) (5) (6)
(7) (8) Waste
(9) Collect in
20 mL glass
test tube
-I,
^
Figure 1. Precept® Sample Preparation and Cleanup
10
-------
Prepare Capture
Strips
Prepare wash solution
(40 mL in 960 mL
deionized water)
Wash Capture Strips
(3x Wash)
Make Capture Reagent
(40 uL to 600 uL assay
buffer per strip)
Add 5 OuL of Capture
Reagent to each well
Mix on Plate Shaker
for 60-90 minutes
Wash Capture Strips
(3x Wash)
Add 50 uL of Assay
Buffer to Each glass
vial
Add 5-10 uL of
Sample or Standard to
each glass vial
Add 50 uL of thawed
Activation Solution to
each glass vial
Mix on Plate Shaker
for 60 minutes
Add 30 uL from each
glass vial to the
corresponding prepared
capture strip
Add PCR Reagents
Cover with adhesive film
Run PCR
Mix on Plate Shaker
for 30 minutes
Wash Capture Strips
(5x Wash)
Figure 2. Precept®Assay Procedure
11
-------
3.1 Sample Preparation
This section includes the sample extraction and cleanup methods employed.
3.1.1 Procept® Rapid Dioxin Assay Method
3.1.1.1 Reagents
Hexane: CHROMOSOLV Plus for HPLC, 99.9% (Sigma no. 650579)
Heptane: Purification Grade, 99% (Sigma no. 644455)
Methylene chloride: Pesticide Residue Analysis Grade (Acros no. AC61016)
Toluene: CHROMOSOLV Plus for HPLC, 98.5% (Sigma no. 650544)
Acetone: Histological Grade, 99.5% (Sigma no.
534064)
Silica: For Column Chromatography 60 (Fluka no.
60741)
Florisil: 100-200 mesh (Sigma no. 20281)
Sulfuric acid: Reagent Grade, ACS (Acros no.
AC42452)
Diatamaceous earth: Sample Dispersant (Dionexno.
062819)
DNase-free water: (Acros no. AC32739)
De-ionized water: Milli-Q2 System (or equivalent)
Potassium Hydroxide: Certified ACS Grade (Fisher
Figure 3. Procept® Rapid Dioxin Assay Kit
no. P250-1)
Sodium sulfate: Reagent Grade, ACS, anhydrous (Sigma no. 239313)
PCR Master Mix: Brilliant Plus QPCR Core Reagent Kit (Stratagene no. 929540)
Procept® Rapid Dioxin Assay: Eichrom Technologies, Inc.
3.1.1.2 Sample preparation
3.1.
3.1.
3.1.
3.1.
3.1.
.2.1 Determination of percent solids
.2.2 Dry a glass vial at 110°C for 12 hours; cool in a dessicator for each sample to be
analyzed.
.2.3 Weigh 5.0 grams of soil into the dried vial
.2.4 Dry for a minimum of 12 hours at 110°C and cool in a dessicator.
.2.5 Calculate percent solids as follows:
% solids = (weight of sample after drying)/(weight of sample before drying) x 100%
(eqn. 3-1)
12
-------
3.1.1.3 Extraction
Any approved method for the extraction of poly chlorinated dioxins and furans from soil can be used
including Soxhlet and pressurized fluid extraction. The conditions for pressurized fluid extraction with a
Dionex ASE100 used to generate the data in this report are given below.
3.1.1.3.1 Add 5 grams of diatomaceous earth sample dispersant to the dried soil sample
and mix by capping the vial and shaking.
3.1.1.3.2 Place a glass fiber filter into a 34 mL stainless steel extraction cell and add
3 grams of diatomaceous earth sample dispersant.
3.1.1.3.3 Add the sample + dispersant to the extraction cell and fill the remaining volume
with diatomaceous earth sample dispersant. Seal the cell by hand tightening the
top and bottom caps.
3.1.1.3.4 Five minute static step. Flush 30% of the volume of the cell with 3:7 acetone:
toluene. Second 5 minute static step. Flush 30% of the volume of the cell with
3:7 acetone:toluene. Flush for 60 seconds with nitrogen (no solvent).
3.1.1.4 Evaporation and solvent exchange
3.1.1.4.1 Add 2.0 mL of dodecane to the soil extract
3.1.1.4.2 Evaporate the acetone/toluene to approximately 10 mL with a gentle stream of air
while heating at 50°C (sand bath).
3.1.1.4.3 Transfer the extract to a 40 mL glass vial and rinse the 200 mL bottle twice with
5 mL of hexane.
3.1.1.4.4 Complete the solvent evaporation using a gentle stream of air while heating at
50°C (sand bath) and add 5 mL of hexane.
3.1.1.5 Extract cleanup
3.1.1.5.1 Add 5 to 10 grams of 44% H2SO4 silica to the extract and allow the sample to sit
overnight.
3.1.1.5.2 Pack and precondition a multilayer silica column and Florisil column
(immediately prior to use):
Silica column (50 mL glass serological pipette, bottom to top): glass wool
plug, washed silica, 10% AgNO3 Silica, 2% KOH silica, washed silica, 44%
H2SO4 silica, 22% H2SO4 silica, dry Na2SO4, glass wool plug. (See
www.eichrom.com for the most up to date recommended column
compositions and elution parameters)
Florisil column (25 mL glass serological pipette, bottom to top): glass wool
plug, 1.8 to 2.0 grams Florisil (washed by ASE, 50% methylene chloride in
hexane, dried 24 hours at 140°C, cooled and stored in a dessicator),
1.5 grams of dry Na2SO4, glass wool plug.
Silica column prewashed with 25 to 50 mL of hexane
Florisil column prewashed with 10 mL of hexane
3.1.1.5.3 Set up columns in series in a fume hood, with the silica column on top of the
Florisil column and a 150 mL glass beaker beneath the Florisil column.
3.1.1.5.4 Slurry the extract/H2SO4 silica mixture and add it to the top of the silica column.
3.1.1.5.5 Use two portions of 5 mL hexane to complete the transfer of the extract to the
silica column.
3.1.1.5.6 When the solvent level reaches 1 mm above the top of the silica column bed, add
additional hexane to the silica column to complete the dioxin/furan elution.
13
-------
3.1.1.5.7 When the solvent level reaches the top of the silica column bed, remove the silica
column.
3.1.1.5.8 When the solvent level reaches the top of the Florisil column bed, rinse the
column sequentially with 10 mL of 2% (v:v) methylene chloride in hexane and
10 mL of 5% (v:v) methylene chloride in hexane (waste).
3.1.1.5.9 Replace the 150 mL glass beaker with a 20 mL glass test tube and elute the
dioxin and furans with 15 mL of 50% (w:w) methylene chloride in hexane.
3.1.1.5.10 Evaporate the sample to dryness using a gentle stream of air and transfer the
residue to a 2 mL glass vial using methylene chloride.
3.1.1.5.11 Evaporate the sample to dryness using a gentle stream of air and dissolve the
residue in 0.2 to 1.0 mL of heptane.
3.1.1.5.12 Add 0.3 mL of concentrated H2SO4or 0.02 to 0.10 grams of 10% AgNO3 silica,
cap the vial with a PTFE lined cap and equilibrate overnight while mixing on an
orbital plate shaker. (The equilibration with H2SO4 or AgNO3 silica will remove
any PAH compounds accumulated from the reagents used during the extract
cleanup.
3.1.2 Characterization HRMS Method
3.1.2.1 Sample Extraction
Depending on the anticipated levels of dioxins from preliminary information received from each
sampling location, 1 to 10 grams (g) of material were taken for analysis from each aliquot, spiked
with 13Ci2-labeled internal standards, and extracted with methylene chloride using accelerated
solvent extraction techniques. (The accelerated solvent extraction technique is a deviation from
Method 1613B, which calls for a Soxhlet/Dean-Stark extraction with toluene for a total of 16 to
24 hours.)
3.1.2.2 Sample Cleanup
The sample extracts were processed through various cleanup techniques, which included gel
permeation chromatography or acid/base washes, as well as acid/base silica and carbon cleanup
columns. As warranted, based on sample compositions, some samples were put through
additional acid silica cleanup prior to the carbon column cleanup. 13Ci2-labeled recovery
standards were added, then the extracts were concentrated to a final volume of 20 to 50
microliters (\\L).
3.2 Sample Analysis
This section includes the determinative analytical methods employed.
3.2.1 Procept® Rapid Dioxin Assay Method
3.2.1.1 Preparation of Capture Strips
3.2.1.1.1 The wash solution is prepared by diluting 40 mL of the wash solution concentrate
to 1 L with deionized water. The wash solution is then placed into a glass flask
and used to prime the plate washer (BioTek ELx50, new buffer prime).
3.2.1.1.2 The desired number of capture strips are then placed into the orange rack and
washed using the plate washer (3x wash) to remove the protective coating.
3.2.1.1.3 The capture reagent is thawed and diluted in a glass test tube with the assay
buffer (40 (iL of capture reagent to 600 (iL of assay buffer per capture strip).
3.2.1.1.4 Using an eight-channel automatic delivery pipette and 100 \\L barrier pipette tips,
50 \\L of the diluted capture reagent is added to each well of the capture strips.
14
-------
3.2.1.1.5 The capture strips are then placed on the plate shaker (Heidolph Titramax 1000
or equivalent, speed set at 900) for 60 to 90 minutes.
3.2.1.2 Reaction of Samples and Standards with Ah-Receptor (Performed while capture strips are
on the plate shaker)
3.2.1.2.1 For each capture strip used, a glass reaction vial is charged with 50 (iL of assay
buffer (8-channel automatic delivery pipette and 100 ^L barrier pipette tips).
3.2.1.2.2 Five (iL of the purified sample extract or standard is added to each glass vial (0.1
to 20 (A automatic delivery pipette and barrier pipette tips).
3.2.1.2.3 For each capture strip used, one vial of the activation solution (stored at -80°C or
in a liquid nitrogen dewar) is thawed and 50 (iL is added to each glass reaction
vial (8-channel automatic delivery pipette and 100 (iL barrier pipette tips).
3.2.1.2.4 The rack of glass reaction vials is placed on the plate shaker for 60 minutes.
3.2.1.3 Addition of reaction mixture to capture strips
3.2.1.3.1 After 60 to 90 minutes on the plate shaker, the capture strips are washed using
the plate washer (3x wash) to remove any excess capture reagent.
3.2.1.3.2 Using the 8-channel automatic delivery pipette and 100 (iL barrier pipette tips,
30 (iL of each solution from the glass reaction vials is added to each
corresponding capture strip.
3.2.1.3.3 The capture strips are placed on the plate shaker for 30 minutes.
3.2.1.3.4 Following 30 minutes on the plate shaker, the capture strips are washed using the
plate washer (5x wash). This takes approximately 15 minutes.
3.2.1.4 Polymerase Chain Reaction (PCR)
3.2.1.4.1 While the capture strips are on the plate washer, the PCR reagents are prepared
per the manufacturers instructions. For each capture strip used, mix 175 (iL of
DNase free water, 140 ^L of PCR master mix and 35 ^L of primer probe solution
in a glass test tube.
3.2.1.4.2 When the 5x wash program is complete, using the 8-channel automatic delivery
pipette and 100 (iL barrier pipette tips, 40 ^L of the PCR reagent is added to each
well of the capture strips.
3.2.1.4.3 The capture strips are sealed using optically clear adhesive film (Applied
Biosystemspartno. 4311971).
3.2.1.4.4 Two optical cover compression pads are placed on top of the sealed capture
strips, and the capture strips are placed in the PCR instrument.
3.2.1.4.5 The quantitative PCR program is run using the following parameters:
Quantification dye: FAM
Reference dye: ROX
Thermal Profile: 2 minutes at 50°C
10 minutes at 95°C
Cycle between 15 seconds at 95°C then 60 seconds at
60°C (40 times)
3.2.2 Characterization HRMS Method
Each extract was analyzed by GC/HRMS in the selected ion monitoring mode at a resolution of 10,000 or
greater. A DB-5 column was used for separation of the seventeen PCDD/F congeners. The instrument
was calibrated for PCDD/F at levels specified in Method 1613B with one additional calibration standard
15
-------
at concentrations equivalent to one-half the level of Method 1613B's lowest calibration point. Method
1613B relative response factor criteria was used for the calibration curve in which the relative response
factors (RRF) were calculated for each analyte at each calibration level (RRF= (summed area of the
native * concentration of the labeled analog)/(summed area of the labeled analog * concentration of the
native)). An average RRF and a percent relative standard deviations (%RSD) were calculated for each
analyte by averaging the calibration levels for that analyte. The % RSD criteria must be below 20% for
the native analytes quantified by isotope dilution and 35% for the labeled analytes quantified by internal
standards. Continuing calibration solutions were monitored at the beginning and end of each 12-hour
analysis. A window-defining and column performance solution was also analyzed at the beginning of
each sequence to verify that all of the 17 PCDD/F isomers were within the acquisition windows and that
there was a 25% valley between 2,3,7,8 TCDF and its closest eluting isomer. PCDD/F data were reported
as both concentration (pg/g dry) and TEQs (pg TEQ/g dry).
3.3 Quality Control
3.3.1 Procept® Rapid Dioxin Assay Method
Since 13C-labeled standards cannot be used to monitor recoveries through the sample preparation method
used for the Procept® Rapid Dioxin Assay, it is important that samples are processed consistently. Also,
it is recommended that a reagent blank and a known sample be processed with each batch of samples. The
reagent blank can be generated by extracting diatomaceous earth or a soil sample known to be free of
dioxin and furan contamination. The known sample can be a sample which has been analyzed for D/F
contamination by HRMS or a blank soil spiked with a known quantity of dioxin and furan standards.
Typical yields for the entire sample preparation method are 80 to 105% for diatomaceous earth spiked
with a mixture of tetra-octa chlorinated dioxins and furans. In addition, for this site-specific study,
Eichrom analyzed the QC sample unspiked as well as spiked in duplicate with a mixture of PCDD/F
standards at a total TEQ value approximately ten times that of the unspiked QC sample. This mixture
contained the following congeners: 2,3,7,8-TCDD (5% by mass); 1,2,3,7,8 PeCDD (5%); 1,2,3,7,8,9-
HxCDD (5%); OCDD (60%); 2,3,7,8-TCDF (10%); 1,2,3,7,8-PeCDF (5%); 2,3,4,7,8-PeCDF (5%); and
1,2,3,4,7,8-HxCDF (5%).
3.3.2 Characterization HRMS Method
The characterization HRMS method followed the Method 1613B QC requirements. Some of the critical
QC criteria included:
• All initial calibrations met the criteria for response factor RSD and minimal signal-to-noise ratio
requirements for the lowest calibration point.
• Continuing calibrations were performed at the beginning and end of every 12-hour analysis
period and were required to meet performance criteria.
• Column performance was checked at the beginning of each 12-hour analytical period and met
method criteria.
• Instrument resolution was documented at the beginning and end of each 12-hour period with one
exception.
• Method 1613B 13C-labeled internal standard was added to each sample prior to extraction to
evaluate sample extraction recovery.
• Method 1613B recovery standard was added to the GC vials and was used to calculate the percent
recoveries for the internal standards and cleanup standards.
• Method 1613B requires that a 13C-labeled cleanup standard be added after sample extraction.
However, the characterization laboratory has demonstrated a consistent quantifiable loss of
analyte with GPC cleanup, therefore a GPC correction factor was applied to the sample weight
extracted and the level of internal standard added to the samples prior to GPC cleanup. The
16
-------
cleanup standard was then added after the GPC step and was used to monitor loss during the
remaining cleanup steps.
• Analysis of one method blank with every extraction batch was required to demonstrate freedom
from contamination.
• One laboratory control spike, an on-going precision and recovery (OPR) sample, was also
processed with every extraction batch. Native and labeled compounds were required to pass the
Method 1613B limits for OPR.
• A decane blank was analyzed after the analysis of the OPR to monitor for carryover.
3.4 Data Presentation Results
3.4.1 Eichrom Method
Whereas HRMS methods measure the concentration of seventeen individual PCDD/F congeners and then
apply atoxicity equivalency factor (TEF) to calculate the TEQ value, the Procept® Rapid Dioxin Assay
measures the total TEQ directly. As shown in Table 3-2, the response factors measured for the seventeen
most toxic PCDD/F congeners on the Procept® Assay are presented along side the World Health
Organization (WHO) 1998 TEF values (van den Berg, 1998) used to calculate TEQ from the HRMS
congener data. Note that the updated WHO 2005 TEF values are presented for comparison, but these
values were not available during the time of the original HRMS analysis, so the WHO 1998 TEF values
were used (van den Berg, 2006). The agreement in magnitude is comparable for some compounds (e.g.,
2,3,7,8-TCDD and 1,2,3,6,7,8-HxCDD), but quite different for others (e.g., 1,2,3,7,8-PCDD and OCDD).
Response factor values (shown in Table 3-2) for some of the non-dioxin/furan compounds, such as the
PAHs, are quite high. For example, indeno-(l,2,3-cd)-pyrene, benzo(k)fluoranthene, benzo(b) fluor-
anthene, dibenzy(a,h)anthracene, and benzo(a)pyrene all have response factors to the Procept® Assay
greater than 0.1. While these response factors are relatively high, the intent of the Procept® Rapid Dioxin
Assay is to remove the PAHs during the sample preparation process. The efficiency of removal of non-
dioxin/furan compounds was not evaluated in this study.
The quantity measured by the Procept® Rapid Dioxin Assay is the Threshold Cycle (Ct) of a polymerase
chain reaction (PCR) growth curve. The Ct value can be converted to a TEQ value by generating a
standard curve of Ct values for solutions of known TEQ. For samples with an unknown or variable ratio
of PCDD/F congeners, the standard curve can be generated using serial dilutions of 2,3,7,8-TCDD. Due
to differences between the WHO TEF values and the Procept® Assay Response factors for individual
congeners, the most accurate results will be achieved by generating a standard curve from serial dilutions
of a mix of PCDD/F standards mimicking the ratio typically observed in the samples. This was the
approach taken for this study, since the samples results were calibrated based on the QC sample results.
The software package for the PCR instrument will typically convert the Ct value of unknown samples to
TEQ based on the standard curve generated for each Procept® Assay. However, this calculation can also
be done independently using Microsoft Excel (or equivalent software) using the Ct value for the unknown
sample extract and a standard curve generated by plotting Ct vs. log TEQ for a series of known standards:
TEQextract=10((ct-b)/m) (eqn3-2)
where Ct is the threshold cycle measured for the unknown sample extract, and m and b are the slope and
y-intercept of the standard curve, respectively. The TEQextract is then used to calculate the TEQ for the soil
sample using the following equation:
TEQSOI1 = (TEQextract • V • DF)/(M • RF)
17
-------
Table 3-2. Comparison of Procept® Rapid Dioxin Assay Response Factors to Toxicity Equivalency
Factors (TEF)
Congener a
2,3,7,8 TCDD
1,2,3,7,8 PCDD
1,2,3,4,7,8 HxCDD
1,2,3,6,7,8 HxCDD
1,2,3,7,8,9 HxCDD
1,2,3,4,6,7,8 HpCDD
1,2,3,4,6,7,8,9 OCDD
2,3,7,8 TCDF
1,2,3,7,8 PCDF
2,3,4,7,8 PCDF
1,2,3,4,7,8 HxCDF
1,2,3,6,7,8 HxCDF
1,2,3,7,8,9 HxCDF
2,3,4,6,7,8 HxCDF
1,2,3,4,6,7,8 HpCDF
1,2,3,4,7,8,9 HpCDF
1,2,3,4,6,7,8,9 OCDF
PCB-81 (3,4,4',5)
PCB-77 (3,3',4,4')
PCB-126 (3,3',4,4',5)
PCB-169 (3,3',4,4',5,5')
PCB-123 (2',3,4,4',5)
PCB-118(2,3',4,4',5)
PCB-114(2,3,4,4',5)
PCB-105 (2,3,3',4,4')
PCB-167 (2,3',4,4',5,5')
PCB-156 (2,3,3',4,4',5)
PCB-157 (2,3,3',4,4',5')
PCB-189 (2,3,3',4,4',5,5')
Indeno( 1 ,2,3 -cd)pyrene
Benzo(k)fluoranthene
Benzo(b)fluoranthene
Dibenzo(a,h)anthracene
Benzo(a)pyrene
Benzo(a)anthracene
Chrysene
Benzo(g,h,i)perylene
WHO 1998 TEF b
1
1
0.1
0.1
0.1
0.01
0.0001
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.0001
0.0001
0.0001
0.1
0.01
0.0001
0.0001
0.0005
0.0001
0.00001
0.0005
0.0005
0.0002
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
WHO 2005 TEF c
1
1
0.1
0.1
0.1
0.01
0.0003
0.1
0.03
0.3
0.1
0.1
0.1
0.1
0.01
0.01
0.0003
0.0003
0.0001
0.1
0.03
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Procept® Assay
Response Factor
1
0.55
0.35
0.1
0.49
0.013
0.0000028
0.06
0.14
0.32
0.39
0.17
0.28
0.1
0.053
0.016
0.00046
0.000045
0.000034
0.014
0.001
0.0000089
<3 x 10'7
0.00001
<3 x 10"7
0.000001
0.000029
0.000043
<3 x 10'7
0.8
0.54
0.59
0.29
0.13
0.054
0.036
0.0038
a Acenaphthylene, anthracene, fluorene, naphthalene, fluoranthene,
phenanthrene, pyrene, acenaphthene, 2-methylnaphthalene,
2-chloronaphthalene, biphenyl, 2,4-dichlorophenol, 3,4-dichlorophenol
and toluene showed no measurable response at 10 ppm.
b van den Berg, 1998
c van den Berg, 2006
18
-------
where TEQextract is the TEQ value measured for the sample extract, V is the volume, in milliliters, of
solvent in which the extract was dissolved, DF is the dilution factor, M is the dry weight, in grams, of the
sample which was processed to generate the extract and RF is the recovery factor calculated by
processing a known sample through the sample preparation method, which in this case was the QC
sample supplied with each sample batch.
The DF is only needed when multiple dilutions of the sample must be analyzed to achieve the range of
quantification needed by the user. For a single dilution of a sample extract, the calibration curve for the
Procept® Assay is normally linear over just greater than 2 orders of magnitude (for example 1 to
200 ppt). If the user wishes to measure a wider range of TEQ values, the sample must be diluted and re-
assayed.
3.4.2 Characterization HRMS Method
The concentrations of the seventeen individual PCDD/F congeners were calculated in pg/g dry weight,
based on the calibration curve. The World Health Organization's 1998 TEF (van den Berg, 1998) were
then applied to the concentrations and summed to calculate the total TEQD/F value for each sample. (Note
that at the time of the original HRMS analysis, the WHO 2005 TEF values were not available.)
3.5 Comparison of Procept® and HRMS methods
The steps involved in the Procept® and HRMS methods are compared and contrasted in detail in Table 3-
3. In this study, both the Procept® Rapid Dioxin Assay and characterization HRMS extraction methods
employed accelerated solvent extraction (ASE), although the extraction solvents were different (Eichrom
used 30% acetone in toluene; the characterization method used methylene chloride). The reference
HRMS method used toluene extraction by Dean-Stark Soxhlet extraction. The Procept® method also
states that Soxhlet extraction can be used. Sample cleanup for the Procept® Rapid Dioxin Assay and
HRMS methods were similar, utilizing a series of silica and Florisil columns although the specific types,
sizes, and volumes of extraction solvents varied between the methods. The difference between the
Procept® assay and HRMS methods is most significant in the analytical step. The 1613B methods utilize
HRMS, which is a laboratory-based, expensive analysis that allows for congener-specific analysis. The
Procept® assay is analyzed using PCR which can be a field portable or laboratory-based instrument
(although the extensive preparation and cleanup procedures described in Sections 3.1 and 3.2 suggest that
this method fundamentally is a laboratory-based technique). Both HRMS and PCR techniques require a
technically trained operator. The level of QC method criteria are much more stringent and involved for
the HRMS methods, but some common QC techniques (blanks, laboratory control samples, matrix
spikes) are applied in both techniques. The same data units, TEQD/F, are reported by both Procept® and
HRMS, but the values are derived by different methods. The Procept® value is a total TEQ value that is
calibrated based on the assay's response to the site-specific QC sample and therefore requires some
percentage of HRMS confirmatory analyses (either concurrently or based on historical site information).
The HRMS value is the sum of the 17 PCDD/F congener concentrations multiplied by the WHO TEF.
19
-------
Table 3-3. Comparison between Procept® Rapid Dioxin Assay and Characterization HRMS
Methods
Method
Step
Similarities
Differences
Sample
Preparation
Accelerated solvent extraction
(ASE) is the extraction
technique used by both
methods.
Extraction solvent and ASE program
Eichrom: Sample are mixed with diatomaceous earth and are
extracted at 1500 psi and 100°C for two 5 minute static steps and
60% flush volume using 30% acetone in toluene and the extract
collected in 200 mL glass bottles. Samples are concentrated by
nitrogen blow down in a sand bath.
HRMS: Samples are mixed with Hydromatrix and are extracted at
2000 psi and 125°C for a 7 minute heat time, a static time of 10
minutes and a flush volume of 60% using methylene chloride.
Purge time is set to 120 seconds, and there are three static cycles.
Samples are concentrated by TurboVap.
Sample
Cleanup
Both Eichrom and the HRMS
methods use a series of silica
columns eluted with 50 mL
hexane.
Acid silica in the silica
columns is 44% w/w.
Both methods use serological
pipettes for multilayer acid
silica columns.
Eichrom: Additional cleanup with Florisil columns stacked under
multilayer acid/base silica columns. Florisil columns are eluted
with 15 mL of 50:50 (w/w) methylene chloride: hexane to remove
dioxins and furans.
HRMS: Additional cleanup methods are used including Gel
Permeation Chromatography (GPC), acid/base back extraction,
carbon column and silica and alumina columns.
GPC- Extracts are brought to approximately 2 mL in methylene
chloride. Each extract is transferred with four 1-mL aliquots of
methylene chloride rinses to a GPC vial that has been pre-marked
at 7 mL. Methylene chloride is added to the GPC vial to bring the
total volume to the 7 mL mark on the vial. The extract is eluted
according to the calibration data.
Acid/Base back extraction- Extracts must not contain any
Methylene chloride. The extracts are partitioned against 30mL of
sulfuric acid solution and shaken for 2 minutes and the aqueous
portion is discarded. The acid washing is repeated using 20-mL of
sulfuric acid until no color is visible in the aqueous layer, to a
maximum of four washings. The extract is partitioned against 20
mL of sodium chloride solution in the same way as with acid. The
aqueous layer is discarded. The extract is partitioned against 15
mL of potassium hydroxide in the same way as with acid. The
base washing is repeated until no color is visible in the aqueous
layer, to a maximum of four washings. The partitioning is
repeated against sodium chloride solution two times and the
aqueous layer is discarded each time.
Acid silica columns- HRMS uses only the 44% (w/w) in the silica
columns. Eichrom used a layer of 22% w/w as well a layer of
44% (w/w). Both labs use 20-50 mL of hexane as the elution
solvent
Alumina columns- Alumina columns are stacked under the
multilayer acid/base silica columns so that the eluant can drip
directly onto the pre-rinsed alumina columns. 40 mL hexane:
methylene cholride (50:50) is the final elution solvent.
Final cleanup column used is 20% carbon: celite with 40 mL of
toluene as the final elution solvent
20
-------
Method
Step
Sample
Analysis
Quality
Control
Data
Presentation
Similarities
Both are laboratory -based
methods which require
technically trained operators.
Both methods include reagent
blanks, laboratory control
samples, and matrix
spike/duplicates performed on
each batch (20-25) of samples.
Results reported in total
TEQD/F
Differences
Procept® uses PCR; 1613B uses HRMS
Use of 13C-labeled standards for HRMS methods cannot be used
for Procept®
Congener specific analysis for HRMS; total TEQD/F result for
Procept® based on HRMS data for one or more confirmatory
quality control samples.
or
21
-------
Chapter 4
Results and Discussion
4.1 Evolution of Procept® Rapid Dioxin Assay
The Hybrizyme Corporation AhRC PCR™ Kit was a technology that measured the concentration of AhR
binding compounds in units reported as AhRBU. At the time of the original SITE demonstration, this
particular test was intended for use as a screening tool to rank samples from those inducing the greatest
AhR activity to those inducing the least AhR activity, rather than to provide highly accurate TEQ. The
developer's goal was a highly portable screening technology to help determine areas of greatest concern
for cleanup at a site and to help minimize the number of more expensive analyses needed for specific
analytes. In March 2005, Hybrizyme licensed this technology to Eichrom Technologies. Eichrom focused
on optimization of the sample preparation, which involved a more involved, laboratory-based approach.
Eichrom analyzed the 112 samples for this study and reported results within one month. After reviewing
the initial results, Eichrom determined that an additional purification step would improve the accuracy
and precision of the results. Eichrom did not re-extract all 112 samples, but rather took the existing
extracts through the additional purification step and re-assayed the samples by PCR. The results from
Eichrom's second attempt at the site-specific study samples are evaluated in this report. While the
Eichrom method has improved significantly from Hybrizyme's generic screening tool for AhR
compounds to a quantitative technology for TEQD/F, the method is still being optimized. Eichrom expects
to complete optimization of this method by the end of 2006.
4.2 Procept® Rapid Dioxin Assay Results
Eichrom's reported results for the site-specific study are summarized by site in Tables 4-1 through 4-5.
4.3 Discussion
An example of the results reported by Hybrizyme in the original SITE demonstration is shown in Table 4-
6. The Hybrizyme technology did not report TEQ concentrations, but rather was intended to serve as an
indicator of high levels of AhR binding compounds. Once Eichrom obtained exclusive licensing of the
technology, Eichrom's focus was on the sample preparation aspect, since it was apparent that TEQ data
could not be derived using Hybrizyme's method (i.e., a quick extraction using a cocktail of solvents
followed by an acid-wash cleanup). Eichrom arrived at a sample preparation procedure that was similar to
the HRMS method, but used essentially the same detection assay as was performed with the Hybrizyme
method.
The results shown in Tables 4-1 through 4-5 demonstrate the comparability to the HRMS results and the
precision of the method. For the Winona Post samples (Table 4-1), the percent recovery (%R) values
were 30%, 74%, 78%, 86%, and 93%. This indicated that all but one sample set (Cell #12) were reported
with results that were fairly consistent with the HRMS method. These results also indicated that the
sample results reported by Eichrom were usually lower than the HRMS, with all %R values less than
100%. The relative standard deviation (RSD) values were between 30% and 46%. Note that all of the
sample concentrations at this site were the highest among the five sites (approximately 10,000 pg/g
TEQD/F).
22
-------
Table 4-1. Winona Post Sample Results
pg/g TEQ
Sample ID
Cell #10
Replicate
1
2
3
4
Analysis Order
W-13
W-9
W-17
W-20
Average
Standard Deviation (SD)
Relative standard deviation (RSD)
% Recovery
Cell #12
Average
SD
RSD
% Recovery
Cell #2
Average
SD
RSD
% Recovery
Cell #4
Average
SD
RSD
% Recovery
Cell #8
Average
SD
RSD
% Recovery
ERA Blank
1
2
o
5
4
QC
1
2
3
1
2
o
3
4
1
2
o
3
4
W-15
W-18
W-5
W-7
W-l
W-10
W-2
W-12
W-14
W-ll
W-4
W-21
W-16
W-3
W-19
W-8
W-6
Average HRMS Procept®
8648 8909
6134
6723
3980
6436
2027
31%
74%
8831 2622
1132
2844
4173
2693
1246
46%
30%
11,071 12,325
7204
8071
6725
8581
2557
30%
78%
11,410 9314
16,339
11,160
5506
10,580
4504
43%
93%
11,259 11,110
9855
4668
13,174
9702
3624
37%
86%
ND 3
ND = not detected
23
-------
Table 4-2. Tittabawassee River Sample Results
pg/g TEQ
Sample ID
ERA Blank
DNR1
Replicate
1
2
3
4
1
2
3
QC
Analysis Order
TR-4
TR-16
TR-7
TR-18
TR-3
TR-11
TR-23
QCTR-1
Average
Standard Deviation (SD)
Relative standard deviation (RSD)
% Recovery
DNR2
Average
SD
RSD
% Recovery
FFP1
Average
SD
RSD
% Recovery
FFP2
Average
SD
RSD
% Recovery
IMP 2
Average
SD
RSD
% Recovery
1
2
3
4
1
2
o
6
4
1
2
3
4
1
2
o
3
4
TR-13
TR-19
TR-21
TR-5
TR-10
TR-20
TR-8
TR-15
TR-6
TR-17
TR-14
TR-2
TR-22
TR-24
TR-12
TR-9
Average HRMS Procept®
ND 3
1
11
1
435 1243
394
546
365
637
412
65%
146%
42 37
41
3
51
33
21
63%
79%
3127 4705
9486
4829
4452
5868
2417
41%
188%
1048 719
1249
1193
729
972
288
30%
93%
808 8556
6865
1252
1871
4636
3626
78%
574%
ND = not detected
24
-------
Table 4-3. Newark Bay Sample Results
pg/g TEQ
Sample ID
ERA Blank
NB 1
Replicate
1
2
o
6
4
Analysis Order
NB-12
NB-3
NB-13
NB-6
NB-7
Average
Standard Deviation (SD)
Relative standard deviation (RSD)
% Recovery
NB2
Average
SD
RSD
% Recovery
NB3
Average
SD
RSD
% Recovery
NB5
Average
SD
RSD
% Recovery
NB6
Average
SD
RSD
% Recovery
1
2
3
4
1
2
o
5
QC
1
2
o
3
4
1
2
3
4
NB-14
NB-16
NB-9
NB-19
NB-8
NB-5
NB-21
NB-1QC
NB-17
NB-2
NB-11
NB-20
NB=10
NB-15
NB-4
NB-18
Average HRMS Procept®
ND 5
45 92
129
162
92
119
34
29%
264%
38 100
103
58
66
82
23
28%
216%
32 25
22
84
17
37
31
84%
116%
16 30
13
20
28
23
8
35%
143%
62 137
150
NA
86
124
34
27%
200%
ND = not detected
NA = Eichrom did not report data for this sample due to a broken sample vial
25
-------
Table 4-4. Raritan Bay Sample Results
pg/g TEQ
Sample ID
ERA Blank
RB 1
Replicate
1
2
o
3
4
Analysis Order
RB-4
RB-5
RB-14
RB-16
RB-20
Average
Standard Deviation (SD)
Relative standard deviation (RSD)
% Recovery
RB2
Average
SD
RSD
% Recovery
RB4
Average
SD
RSD
% Recovery
RB5
Average
SD
RSD
% Recovery
RB6
Average
SD
RSD
% Recovery
1
2
3
4
1
2
o
3
4
1
2
o
3
4
1
2
3
QC
RB-19
RB-3
RB-18
RB-9
RB-6
RB-11
RB-21
RB-2
RB-17
RB-13
RB-7
RB-10
RB-15
RB-12
RB-8
RB-1
Average HRMS Procept®
0.2
14 16.6
11.3
7.1
7.5
11
4
42%
76%
12 6.7
24.9
7.4
4.5
11
9
87%
91%
15 9.2
14.4
2.7
12.4
10
5
53%
64%
14 4.6
3.7
12.6
8.2
7
4
56%
52%
13 4.7
10.2
13.0
10.4
10
o
5
36%
74%
ND = not detected
26
-------
Table 4-5. Solatia Sample Results
pg/g TEQ
Sample ID
ERA Blank
SSI
Replicate
1
2
3
4
Analysis Order
S-ll
805
S-9
S-23
S-20
Average
Standard Deviation (SD)
Relative standard deviation (RSD)
% Recovery
SS2
Average
SD
RSD
% Recovery
SS3
Average
SD
RSD
% Recovery
SS4
Average
SD
RSD
% Recovery
SS5
Average
SD
RSD
% Recovery
SS6
Average
SD
RSD
% Recovery
1
2
3
4
1
2
3
4
1
2
3
QC
1
2
3
4
1
2
3
4
S-18
S-4
S-14
S-22
S-15
S-2
S-10
S-25
S-7
S-16
S-13
S-l
S-8
S-17
S-12
S-21
S-24
S-6
S-3
S-19
Average HRMS Procept®
7
846 2302
1096
1795
1207
1600
560
35%
189%
48 1289
1208
1418
1255
1293
90
7%
2693%
3257 3366
3736
4581
6633
4579
1461
32%
141%
1833 2653
2032
2461
2199
2336
275
12%
127%
1279 2479
1405
2097
1753
1934
460
24%
151%
3951 5226
4657
4776
8552
5803
1849
32%
147%
ND = not detected
27
-------
Table 4-6. Hybrizyme AhRBU Results Compared to HRMS by Ranking-Original SITE
Demonstration Data3
Environmental Site
Newark Bay
Raritan Bay
Solutia
Tittabawassee River
Winona Post
Hybrizyme Ranking by
Average AhRBU b
1
4
2
3
1
2
o
6
i
o
J
2
o
J
2
1
1
2
o
J
HRMS Ranking by Total
Concentration (ng/g) b
1
2
4
3
1
3
2
1
3
2
3
1
2
1
2
3
Did Ranking Agree?
No
No
Yes
No
Yes
U.S.EPA2005e
Ranking of sample numbers within a site from low to high. Total concentration includes D/F and PCBs.
For the Tittabawassee River samples (Table 4-2), the percent recovery (%R) values were 79%, 93%,
146%, 188%, and 574%. This indicated that all but two sample sets were reported with results that were
consistently higher than the HRMS method. The RSD values were between 30% and 78%.
For the Newark Bay samples (Table 4-3), the percent recovery (%R) values were 116%, 143%, 200%,
216%, and 264%. This indicated all of the sample sets were reported with results that were consistently
higher than the HRMS method. The RSD values were between 27% and 35%, with the exception of one
set, which had an RSD value of 84%. Note that all of the sample concentrations at this site were relatively
low(<100pg/gTEQD/F).
For the Raritan Bay samples (Table 4-4), the percent recovery (%R) values were 52%, 64%, 74%, 76%,
and 91%. This indicated that all of the sample sets were reported with results that were consistently lower
than the HRMS method. The RSD values were between 36% and 87%.
For the Solutia samples (Table 4-5), the percent recovery (%R) values were 127%, 141%, 147%, 151%,
189%, and 2,693%. This indicated that all of the sample sets were reported with results that were
consistently higher than the HRMS method. The RSD values were between 7% and 35%.
Procept® results for the eight uncontaminated ("blank") samples that were included in the experimental
design were reported with TEQD/F values between 0.2 and 11. None of these were reported as non-detects
by the Procept® method.
Overall there was no significant pattern of positive or negative bias relative to the HRMS method results,
since the percent recovery values were both above and below 100%, but three of the five sites had
consistent results within the site (either all >100% or all < 100%). This evaluation also demonstrated the
need for a site-specific factor (based on the QC sample results) to convert the raw data generated by the
Procept® method into TEQD/F data. This suggests that the need for independent HRMS confirmatory
analysis would be appropriate at a level of 5% at the least; presumably, more comparability to HRMS
28
-------
would be obtained with a greater percentage of HRMS confirmation analyses, but this was not evaluated
in this study.
4.4 Operational Factors
Operational factors such as cost, availability of the technology, turnaround time, and training are
described in this section. This information was provided by Eichrom Technologies and not evaluated
independently by Battelle or EPA.
4.4.1 Cost ofProcept® Rapid Dioxin Assay
The costs of running the Procept® Assay can be divided into three categories: capital equipment
necessary to run the sample preparation and the assay itself, chemicals and supplies for the sample
preparation and the assay, and labor necessary to perform the analysis. Labor costs are not described in
this report, but costs for the other categories are described in this section.
Capital Equipment: Table 4-7 summarizes all of the pieces of equipment necessary to prepare samples
and run the Procept® Assay. A range of estimated purchase prices is also shown. The total estimated
acquisition costs to purchase all pieces of equipment new (new setup cost) would be in the range of
$65,000 to $100,000. In practice, however, laboratories currently involved in dioxin analysis would
already possess equipment related to sample preparation and storage and, as a result, they would not need
to purchase all equipment in order to perform the Procept® Assay. Among the pieces of equipment likely
to be owned already by a dioxin laboratory are an ASE system or a Soxhlet extraction system, a
refrigerator/freezer (-20 °C), and a top loading balance. The acquisition cost of pieces of equipment
specific to the Procept® Assay (addition to existing setup) range from $37,000 to $43,000.
Table 4-7. Capital Equipment Costs for the Procept® Assay
Capital Equipment Cost Incremental
Accelerated Solvent Extraction Instrument $25,000 - $50,000
PCR Instrument $30,000 - $35,000 $30,000 - $35,000
Plate Washer $3,000 - $4,000 $3,000 - $4,000
Plate Shaker $1,000 - $1,500 $1,000 - $1,500
Automatic Delivery Pipets $2,000 $2,000
Refrigerator/Freezer (-2°C) $2,000 - $4,000
Liquid Nitrogen Dewar $600 $600
Top Loading Balance $ 1,000
$65,000 - $100,000 $37,000 - $43,000
Chemicals and Supplies: The largest cost in this category is the cost of the Procept® Assay Kit. The list
price of $2,400 is for a kit based on a 96-well plate. The number of samples that can be analyzed depends
on several factors related to the data quality objectives (DQO) of the laboratory. The DQOs will drive
decisions on the number of replicates of each sample and standard to be analyzed, as well as the necessity
for other "QC" samples like blanks, spikes, etc. Assuming that each sample is analyzed in duplicate and
that 16 wells of each plate are reserved to standards and other quality control samples, one kit will yield
40 analytical determinations, at a kit cost of $60. Table 4-8 includes the price of other disposable
chemicals and supplies necessary for sample preparation and for running the kit itself. Total per sample
cost for consumables is approximately $25, plus the cost of the kit.
29
-------
Table 4-8. Chemicals and Supplies Cost for Procept® Assay
Chemicals Amount per Sample
Toluene 100 mL
Acetone 30 mL
Heptane 100 mL
Hexane 50 mL
Methylene Chloride 10 mL
Florisil 2 grams
Silica 25 grams
Sulfuric Acid 25 grams
Potassium Hydroxide 1 gram
Sodium Chloride 10 grams
Sodium Sulfate Anhydrous 4 grams
Nitrogen Gas 1 tank/200 samples
Diatomaceous Earth 10 grams
PCR Reagents
Deionized Water
DNA-ase Free Water
Disposable Supplies Amount per Sample
0.5 - 20 uL Barrier Pipet Tips 3
0.1 - 100 uL Barrier Pipet Tips 3
0.1 - 200 uL Barrier Pipet Tips 1
100 - 1,000 uL Barrier Pipet Tips 1
Glass Transfer Pipets 2
Glass Test Tube 1
2 mL Glass Vial w/PTFE-Lined Cap 3
Glass Column (25 mL seralogical pipet) 1
Glass Column (50 mL seralogical pipet) 1
Glass Wool 0.1 grams
Chemical and Supplies Cost: $25/Sample
4.4.2 Cost Comparison to HRMS Methods
This section presents the costs associated with the HRMS Method 1613B used to analyze the soil and
sediment samples for dioxins and furans. Typical costs of these analyses can range from $800 to $1,200
per sample, depending on the method selected, the level of quality assurance/quality control incorporated
into the analyses, and reporting requirements. Note that the HRMS cost per sample estimate includes
everything to generate the sample result, where the costs listed for Eichrom in Section 4.4.1 include the
consumables and capital equipment, but not the labor involved with the sample analysis.
4.4.3 Availability of Technology
Eichrom provides the Procept® Dioxin Assay as a kit that is available for purchase. Typical customers for
this technology would include analytical laboratories. The manufacturing and quality control systems of
this product are established and routine. Kits are available in three sizes: (1) a full 96-well format, (2) a
half size kit with sufficient reagents for 48 wells, and (3) a one-fourth size kit with reagents for 24 wells.
30
-------
The list price for a kit based on a 96-well plate is $2,400. The 48-well kit is $1,400, and the 24-well kit is
$800.
4.4.4 Turnaround
The various steps of the sample preparation and Procept® assay are summarized in Table 4-9 with the
amount of time that should be required to perform the step, both in terms of labor hours and in terms of
elapsed time. The labor involved to perform the sample preparation and run the Procept® Assay itself
have been estimated assuming a batch of 20 samples is processed simultaneously. The kit itself can
accommodate larger batch sizes (up to 40 samples as indicated above.) The actual batch size chosen by a
laboratory would depend on its staffing level and available equipment. The assumption of a batch size of
20 is based on Eichrom's experience with the ASE system and with the number of silica/Florisil column
set ups that can fit inside a laboratory fume hood. Larger batch sizes would not require proportionally
more labor or elapsed time. Approximately one-half of a labor hour per sample is necessary to perform
the extraction, sample prep, and analysis. The elapsed time (or turnaround time, TAT) is a little more
difficult to gauge. In Eichrom's experience with a single analyst working one shift, it takes slightly
longer than 48 hours to complete the analysis. In laboratories where staffing is available for longer than
an 8-hour work day, samples can be processed in less than 48 hours. The major constraint on TAT is the
overnight sulfuric acid treatments, which are required to reduce background response for low level
samples. Further optimization of this part of the sample preparation could result in significantly faster
turnaround times.
Table 4-9. Estimation of Sample Turnaround Time Using Procept® Assay
Activity
ASE Extraction
Evaporation and Sulfuric Acid Treatment
Silica and Florisil Columns
Evaporation and Sulfuric Acid Treatment
Procept® Assay
Data Analysis
Total Man Hours
Per Sample (batch of 20)
Total Elapsed Time: 1 shift
Total Elapsed Time: 2 shift
Time
Hands On
1 hour
1 hour
3 hours
1 hour
2 hours
1 hour
9 hours
~0.5 hours
Estimate
Elapsed
6 hours
Overnight
5 hours
Overnight
4 hours
1 hour
> 48 hours
< 48 hours
In comparison, a batch of 20 samples by the HRMS methods for a laboratory operating one shift generally
takes 1 day for the ASE extraction, 1 day for GPC cleanup, two days for layered silica and carbon column
cleanup, one day for final concentration and solvent exchange (a conservative total of 5 days for
preparation) and a total of three days for sample analysis by HRMS, resulting in a total estimated eight-
day TAT for a batch of 20 samples. Turnaround time could increase with a more rigorous QA review.
Quicker than typical TATs for Method 1613B usually involves additional cost on a per sample basis.
4.4.5 Training/Ease of Use for Procept® Assay
The Procept® Assay is designed for use in analytical chemistry laboratories that currently perform dioxin
testing. The sample preparation used is a simplified version of the typical silica/alumina/carbon column
procedure that is widely used. Samples are extracted using toluene/acetone in a Soxhlet or ASE system.
31
-------
Slightly smaller silica and Florisil columns are used and the carbon column is omitted. An additional step
not typically employed in the EPA procedures (e.g., 1613B) is the use of sulfuric acid in batch contact
with the dioxin (hexane) phase at two points in the procedure. However, all the steps in the sample
preparation are easily carried out by any trained laboratory technician.
The Procept® Assay requires the use of multi-channel pipettes, a plate washer, plate shaker, a liquid
nitrogen dewar and a real-time PCR instrument. These are items perhaps not typically used in a dioxin
laboratory. All but the PCR require minimal training that can be accomplished in a matter of minutes or
hours.
The real-time PCR instrument is a combination of a thermocycler to amplify and a detector to measure
the fluorescence of each sample well in the 96-well plate. Software in the system determines the cycle in
which the fluorescence crosses a "threshold" (Ct.) The Ct values are plotted versus TEQ for a set of
TCDD standards. The TEQ for each sample is calculated by fitting its Ct on the standard curve. The
training required for this instrument is typical of what is necessary to learn to operate any automated piece
of laboratory equipment, such as an atomic absorption spectrometer or an inductively-coupled plasma
instrument.
32
-------
Chapter 5
Conclusions and Future Directions
As stated earlier, the Procept® Rapid Dioxin Assay has been licensed to Eichrom Technologies for
approximately one year. Since obtaining the technology from Hybrizyme, Eichrom has made significant
modifications to the front-end sample preparation procedures to improve the robustness of the method and
to make the method more quantitative than the method that was being used by Hybrizyme. Eichrom
participated in the site-specific study with a method that was an interim stage of development, nearing
optimization, but not at the point of finalization of the method. Eichrom plans to continue working on the
sample preparation and cleanup, such that the technology can produce results that are more highly
correlated with HRMS methods with site-specific calibration. Eichrom plans to complete the method
optimization by the end of 2006. It is unknown what impact the complete re-extraction of solid samples
using the additional purification step would have had on the comparability and precision of the results;
Eichrom also intends to investigate this further.
Eichrom anticipates that this technology will mostly be used by analytical laboratories prior to the more
expensive HRMS analysis, given its lower cost and quicker analysis time. Prior to HRMS analyses, the
Procept® Assay may be useful as a screening technique to provide gross estimates (none, a little, or a lot
of toxicity) of the TEQD/F present at a site. With site-specific calibration of the Procept® results using a
one-point (e.g., quality control sample) HRMS result, it is a potential tool for providing an estimate of
total TEQD/F concentrations.
Eichrom has stated that future work will also involve evaluation of the Procept® assay's response to
brominated and chloro-bromo D/F congeners, since it is speculated that these congeners will have some
response to the assay due to the structural similarity to the chlorinated D/F congeners. However the
degree of response relative to the chlorinated D/F congeners is unknown and needs to be investigated.
Future work might also include evaluation of response factors for other compounds (e.g., 2,4-D) and
evaluation of the effectiveness of removal of non-dioxin/furan compounds during the sample cleanup
process.
33
-------
Chapter 6
References
De Rosa, Christopher T., et al. 1997. Dioxin and dioxin like compounds in soil, Part 1: ATSDR Interim
Policy Guideline. Toxicology and Industrial Health, Vol. 13, No. 6, pp. 759 768.
U.S. EPA, 1994. EPA Method 1613B. Dioxins, Tetra- thru Octa-(CDDs) and Furans (CDFs),
EPA/821/B-94-005, 40 Code of Federal Regulations Part 136, Appendix A, October 1994.
U.S. EPA, 2004. Demonstration and Quality Assurance Project Plan, EPA/600/R-04/036, April.
U.S. EPA, 2005a. Innovative Technology Verification Report, Xenobiotic Detection Systems, CALUX®
by XDS. EPA/540/R-05/001, July.
U.S. EPA, 2005b. Innovative Technology Verification Report, Wako Pure Chemical Industries, Dioxin
ELISA Kit. EPA/540/R-05/002, March.
U.S. EPA, 2005c. Innovative Technology Verification Report, Abraxis, Coplanar PCB ELISA Kit.
EPA/540/R-05/003, March.
U.S. EPA, 2005d. Innovative Technology Verification Report, CAPE Technologies, DF1 Dioxin/Furan
Immunoassay Kit and PCB TEQ Immunoassay Kit. EPA/540/R-05/004, March.
U.S. EPA, 2005e. Innovative Technology Verification Report, Hybrizyme Corporation, AhRC PCR™
Kit. EPA/540/R-05/005, March.
van den Berg, M., Birnbaum, L., Bosveld, A. T. C., Brunstrom, B., Cook, P., Feeley, M., Giesy, J. P.,
Hanberg, A., Hasagawa, R., Kennedy, S. W., Kubiak, T., Larsen, J. C., van Leeuwen, F. X. R., Liem, A.
K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes,
M., Waern, F., and Zacharewski, T. 1998. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs
for humans and wildlife. Environmental Health Perspectives 106: 775-792.
van den Berg, M., Birnbaum, L., Denison, M., De Vito, M., Farland, W., Feeley, M., Fiedler, H.,
Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D., Tohyama, C., Tritscher, A.,
Tuomisto, J., Tysklind, M., Walker, N., and Peterson, R. 2006. The 2005 Word Health Organization
Reevaluation of Human and Mammalian Toxic Equivalency Factors for Dioxins and Dioxin-Like
Compounds. Toxicological Sciences 93(2): 223-241.
34
------- |