United States Office of Research and EPA/540/R-07/001 

 Environmental Protection Development January 2007 

 Agency Washington, DC 20460 

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. 

ii 
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 PCRTM Kit. The AhRC PCRTM 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 PCRTM 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
 

Chapter 3 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
 

v 
 

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. Winona Post 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 

viii 
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. 

ix 
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., realtime 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 polychlorinated 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 

1
 
> 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 

2
 
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 

polychlorinated 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. 

3
 
 

 

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 sitespecific 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 

Original 

Total TEQD/F (pg/g) 

from 

Characterization Analysis 

2006 

Total TEQD/F (pg/g) 

from 

Holding Time Check 

Relative Percent 

Difference 

(%) 

Tittabawassee River 3127 2560 20 

Newark Bay 38.3 36.7 4 

Solutia 3951 4768 19 

Raritan Bay 13.8 14.3 3 

Winona 11259 10156 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 

4
 
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 Matrix 

Approximate Range of 

Concentrations 

(pg/g TEQD/F) # Samples a 

Winona Soil 8,000 – 12,000 21 

Tittabawassee River Soil 40 – 1,100 24 

Newark Bay Sediment 15 – 65 21 

Raritan Bay Sediment 10 – 15 21 

Solutia Soil 40 – 4,000 25 

Total number of site-specific study samples 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 2001/early 2002. This 

material was placed into an approximate 21⁄2-acre treatment cell located on facility property. During 

2002/2003, material at the treatment cell was treated through addition of amendments (high-ammonia 

5
 
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 Solutia 

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 Raritan Bay 

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 finegrained sediment and total organic carbon values were similar to percentages in Newark Bay. 

6
 
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 A Recovery = × 100 (eqn. 2-1) TEQ HRMS 

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: 

7
 
 1 n 

1

2 

S = (C − C) 2 (eqn 2-2) 

n − ∑ k  1 k =1 

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%. 

S

×1

C 

RSD = 00 (eqn. 2-3) 

8
 
 

 

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 Reference Analysis – Traditional 1613B 

Accelerated solvent extraction with methylene 

chloride 

Soxhlet-Dean Stark extraction with toluene 

2,3,7,8-tetrachlorodibenzofuran (TCDF) 

concentrations not confirmed 

2,3,7,8-TCDF concentrations confirmed 

1 to 10 g was used, depending on what was known 

about the site 

Used extrapolation if calibration range was exceeded 

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 

9
 
 

 

 

Dry Soil Sample at 

110 degrees Celcius 

for a minimum of 12 

hours 

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 

Transfer to 40 mL 

vial, with 5 mL of 

hexane, add 5-10 

grams of 44% 

H2SO4 Silica Gel 

Allow sample to sit 

overnight 

Pretreatment/ 

Extraction 

Pack Silica and 

Florisil columns 

1) 25-50 mL 

hexane 

Column 

Purification 

Na2SO4 

Sulfuric 

Acid, 

AgNO3, 

and 

KOH 

Silicas 

Glass 

Wool 

Silica 

Glass 

Wool 

Na2SO4 

Glass 

Wool 

Florisil 

Glass 

Wool 

1) Waste 

1) 10 mL 

hexane 

(1) (2) (3) (4) (5) (6) 

(7) (8) Waste 

2) Sample + 5-10 g 

 44% H2SO4 Silica 

3) Rinse vial 2x hexane 

4) Rinse column additional

 hexane 

(2) (3) (4) 

7) 10 mL 2% dichloromethane in hexane 

8) 10 mL 5% dichloromethane in hexane 

9) 15 mL 50% dichloromethane in hexane 

(9) Collect in 

20 mL glass 

test tube 

Redissolve in 

0.2-1 mL of 

heptane 

Evaporate 

Figure 1. Procept® 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 50 uL of Capture 

Reagent to each well 

Mix on Plate Shaker 

for 60-90 minutes 

Wash Capture Strips 

(3x Wash) 

Reaction 

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 

Mix on Plate Shaker 

for 30 minutes 

Wash Capture Strips 

(5x Wash) 

Add PCR Reagents 

Cover with adhesive film 

Run PCR 

Figure 2. Procept®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 (Dionex no. 

062819) 

DNase-free water: (Acros no. AC32739)
 

De-ionized water: Milli-Q2 System (or equivalent) 

Potassium Hydroxide: Certified ACS Grade (Fisher 

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 

3.1.1.2.1	 Determination of percent solids 

3.1.1.2.2	 Dry a glass vial at 110o

C for 12 hours; cool in a dessicator for each sample to be 

analyzed. 

3.1.1.2.3	 Weigh 5.0 grams of soil into the dried vial 

3.1.1.2.4	 Dry for a minimum of 12 hours at 110o

C and cool in a dessicator. 

3.1.1.2.5 Calculate percent solids as follows: 

% solids = (weight of sample after drying)/(weight of sample before drying) x 100% (eqn. 3-1) 

Figure 3. Procept® Rapid Dioxin Assay Kit 

12
 
3.1.1.3 Extraction 

Any approved method for the extraction of polychlorinated 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 50o

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 

50o

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 140o

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 H2SO4 or 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 13C12-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. 13C12-labeled recovery 

standards were added, then the extracts were concentrated to a final volume of 20 to 50 

microliters (μL). 

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 μL of capture reagent to 600 μL of assay buffer per capture strip). 

3.2.1.1.4	 Using an eight-channel automatic delivery pipette and 100 μL barrier pipette tips, 

50 μL 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 μL of assay 

buffer (8-channel automatic delivery pipette and 100 μL barrier pipette tips). 

3.2.1.2.2	 Five μL of the purified sample extract or standard is added to each glass vial (0.1 

to 20 μL 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 -80o

C or 

in a liquid nitrogen dewar) is thawed and 50 μL is added to each glass reaction 

vial (8-channel automatic delivery pipette and 100 μL 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 μL barrier pipette tips, 

30 μL 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 μL of 

DNase free water, 140 μL of PCR master mix and 35 μL 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 μL barrier pipette tips, 40 μL 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 

Biosystems part no. 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 50o

C 

10 minutes at 95o

C 

Cycle between 15 seconds at 95o

C then 60 seconds at 

60o

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 a toxicity 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-(1,2,3-cd)-pyrene, benzo(k)fluoranthene, benzo(b) fluoranthene, 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 nondioxin/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) (eqn 3-2) 

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: 

(eqn 3-3) TEQsoil = (TEQextract · V · DF)/(M · RF) 

17
 
 

 

 

Table 3-2. Comparison of Procept® Rapid Dioxin Assay Response Factors to Toxicity Equivalency 

Factors (TEF) 

Congener a WHO 1998 TEF b WHO 2005 TEF c 

Procept® Assay 

Response Factor 

2,3,7,8 TCDD 1 1 1 

1,2,3,7,8 PCDD 1 1 0.55 

1,2,3,4,7,8 HxCDD 0.1 0.1 0.35 

1,2,3,6,7,8 HxCDD 0.1 0.1 0.1 

1,2,3,7,8,9 HxCDD 0.1 0.1 0.49 

1,2,3,4,6,7,8 HpCDD 0.01 0.01 0.013 

1,2,3,4,6,7,8,9 OCDD 0.0001 0.0003 0.0000028 

2,3,7,8 TCDF 0.1 0.1 0.06 

1,2,3,7,8 PCDF 0.05 0.03 0.14 

2,3,4,7,8 PCDF 0.5 0.3 0.32 

1,2,3,4,7,8 HxCDF 0.1 0.1 0.39 

1,2,3,6,7,8 HxCDF 0.1 0.1 0.17 

1,2,3,7,8,9 HxCDF 0.1 0.1 0.28 

2,3,4,6,7,8 HxCDF 0.1 0.1 0.1 

1,2,3,4,6,7,8 HpCDF 0.01 0.01 0.053 

1,2,3,4,7,8,9 HpCDF 0.01 0.01 0.016 

1,2,3,4,6,7,8,9 OCDF 0.0001 0.0003 0.00046 

PCB-81 (3,4,4',5) 0.0001 0.0003 0.000045 

PCB-77 (3,3',4,4') 0.0001 0.0001 0.000034 

PCB-126 (3,3',4,4',5) 0.1 0.1 0.014 

PCB-169 (3,3',4,4',5,5') 0.01 0.03 0.001 

PCB-123 (2',3,4,4',5) 0.0001 0.00003 0.0000089 

PCB-118 (2,3',4,4',5) 0.0001 0.00003 <3 x 10-7 

PCB-114 (2,3,4,4',5) 0.0005 0.00003 0.00001 

PCB-105 (2,3,3',4,4') 0.0001 0.00003 <3 x 10-7 

PCB-167 (2,3',4,4',5,5') 0.00001 0.00003 0.000001 

PCB-156 (2,3,3',4,4',5) 0.0005 0.00003 0.000029 

PCB-157 (2,3,3',4,4',5') 0.0005 0.00003 0.000043 

PCB-189 (2,3,3',4,4',5,5') 0.0002 0.00003 <3 x 10-7 

Indeno(1,2,3-cd)pyrene N/A N/A 0.8 

Benzo(k)fluoranthene N/A N/A 0.54 

Benzo(b)fluoranthene N/A N/A 0.59 

Dibenzo(a,h)anthracene N/A N/A 0.29 

Benzo(a)pyrene N/A N/A 0.13 

Benzo(a)anthracene N/A N/A 0.054 

Chrysene N/A N/A 0.036 

Benzo(g,h,i)perylene N/A N/A 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 reassayed. 

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 Accelerated solvent extraction Extraction solvent and ASE program 

Preparation (ASE) is the extraction 

technique used by both 

methods. 

Eichrom: Sample are mixed with diatomaceous earth and are 

extracted at 1500 psi and 100o

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 Both Eichrom and the HRMS Eichrom: Additional cleanup with Florisil columns stacked under 

Cleanup 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. 

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 Similarities Differences 

Sample 

Analysis 

Both are laboratory-based 

methods which require 

technically trained operators. 

Procept® uses PCR; 1613B uses HRMS 

Quality 

Control 

Both methods include reagent 

blanks, laboratory control 

samples, and matrix 

spike/duplicates performed on 

each batch (20-25) of samples. 

Use of 13C-labeled standards for HRMS methods cannot be used 

for Procept® 

Data 

Presentation 

Results reported in total 

TEQD/F 

Congener specific analysis for HRMS; total TEQD/F result for 

Procept® based on HRMS data for one or more confirmatory or 

quality control samples. 

21
 
 

 

Chapter 4
 

Results and Discussion 

4.1 Evolution of Procept® Rapid Dioxin Assay 

The Hybrizyme Corporation AhRC PCRTM 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 

Analysis Order 

W-13 

Average HRMS 

8648 

Procept® 

8909

 2 W-9 6134

 3 W-17 6723

 4 W-20 3980 

Average 

Standard Deviation (SD) 

Relative standard deviation (RSD) 

% Recovery 

Cell #12 1 W-15 8831 

6436 

2027 

31% 

74% 

2622 

2 W-18 1132 

3 W-5 2844 

4 W-7 4173 

Average 

SD 

2693 

1246 

RSD 46% 

% Recovery 30% 

Cell #2 QC W-1 11,071 12,325

 1 W-10 7204

 2 W-2 8071

 3 W-12 6725 

Average 

SD 

8581 

2557 

RSD 30% 

% Recovery 

Cell #4 1 W-14 11,410 

78% 

9314

 2 W-11 16,339

 3 W-4 11,160

 4 W-21 5506 

Average 

SD 

10,580 

4504 

RSD 43% 

% Recovery 

Cell #8 1 W-16 11,259 

93% 

11,110

 2 W-3 9855

 3 W-19 4668

 4 W-8 13,174 

Average 

SD 

9702 

3624 

RSD 37% 

% Recovery 86% 

ERA Blank W-6 ND 3 

ND = not detected 

23
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-2. Tittabawassee River Sample Results 

 pg/g TEQ 

Sample ID 

ERA Blank 

Replicate 

1 

Analysis Order 

TR-4 

Average HRMS 

ND 

Procept® 

3 

2 TR-16 1 

3 TR-7 11 

4 TR-18 1 

DNR 1 1 TR-3 435 1243

 2 TR-11 394 

3 TR-23 546 

QC QC TR-1 365 

Average 

Standard Deviation (SD) 

Relative standard deviation (RSD) 

% Recovery 

DNR 2 1 TR-13 42 

637 

412 

65% 

146% 

37 

2 TR-19 41 

3 TR-21 3 

4 TR-5 51 

Average 

SD 

33 

21 

RSD 63% 

% Recovery 

FFP 1 1 TR-10 3127 

79% 

4705 

2 TR-20 9486

 3 TR-8 4829

 4 TR-15 4452 

Average 

SD 

5868 

2417 

RSD 41% 

% Recovery 

FFP 2 1 TR-6 1048 

188% 

719 

2 TR-17 1249

 3 TR-14 1193

 4 TR-2 729 

Average 

SD 

972 

288 

RSD 30% 

% Recovery 

IMP 2 1 TR-22 808 

93% 

8556

 2 TR-24 6865

 3 TR-12 1252

 4 TR-9 1871 

Average 

SD 

4636 

3626 

RSD 78% 

% Recovery 

ND = not detected 

574% 

24
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-3. Newark Bay Sample Results 

 pg/g TEQ 

Sample ID 

ERA Blank 

Replicate Analysis Order 

NB-12 

Average HRMS 

ND 

Procept® 

5 

NB 1 1 NB-3 45 92 

2 NB-13 129 

3 NB-6 162 

4 NB-7 92 

Average 

Standard Deviation (SD) 

Relative standard deviation (RSD) 

% Recovery 

119 

34 

29% 

264% 

NB 2 1 NB-14 38 100 

2 NB-16 103 

3 NB-9 58 

4 NB-19 66 

Average 

SD 

RSD 

% Recovery 

NB 3 

Average 

SD 

RSD 

% Recovery 

NB 5 

1 

2 

3 

QC 

1 

2 

3 

4 

NB-8 

NB-5 

NB-21 

NB-1 QC 

NB-17 

NB-2 

NB-11 

NB-20 

32 

16 

82 

23 

28% 

216% 

25 

22 

84 

17 

37 

31 

84% 

116% 

30 

13 

20 

28 

Average 

SD 

RSD 

% Recovery 

NB 6 1 

2 

3 

4 

NB=10 

NB-15 

NB-4 

NB-18 

62 

23 

8 

35% 

143% 

137 

150 

NA 

86 

Average 

SD 

RSD 

% Recovery 

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 

Replicate Analysis Order 

RB-4 

Average HRMS Procept® 

0.2 

RB 1 1 RB-5 14 16.6 

2 RB-14 11.3 

3 RB-16 7.1 

4 RB-20 7.5 

Average 

Standard Deviation (SD) 

Relative standard deviation (RSD) 

% Recovery 

11 

4 

42% 

76% 

RB 2 1 RB-19 12 6.7 

2 RB-3 24.9 

3 RB-18 7.4 

4 RB-9 4.5 

Average 

SD 

11 

9 

RSD 87% 

% Recovery 

RB 4 1 RB-6 15 

91% 

9.2 

2 RB-11 14.4 

3 RB-21 2.7 

4 RB-2 12.4 

Average 

SD 

10 

5 

RSD 53% 

% Recovery 

RB 5 1 RB-17 14 

64% 

4.6 

2 RB-13 3.7 

3 RB-7 12.6 

4 RB-10 8.2 

Average 7 

SD 4 

RSD 56% 

% Recovery 52% 

RB 6 1 RB-15 13 4.7 

2 RB-12 10.2 

3 RB-8 13.0 

QC RB-1 10.4 

Average 10 

SD 3 

RSD 36% 

% Recovery 74% 

ND = not detected 

26
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-5. Solutia Sample Results 

 pg/g TEQ 

Sample ID Replicate Analysis Order Average HRMS Procept® 

ERA Blank S-11 7 

SS 1 1 S05 846 2302

 2 S-9 1096

 3 S-23 1795

 4 S-20 1207 

Average 1600 

Standard Deviation (SD) 560 

Relative standard deviation (RSD) 35% 

% Recovery 189% 

SS 2 1 S-18 48 1289

 2 S-4 1208

 3 S-14 1418

 4 S-22 1255 

Average 1293 

SD 90 

RSD 7% 

% Recovery 2693% 

SS 3 1 S-15 3257 3366

 2 S-2 3736

 3 S-10 4581

 4 S-25 6633 

Average 4579 

SD 1461 

RSD 32% 

% Recovery 141% 

SS 4 1 S-7 1833 2653

 2 S-16 2032

 3 S-13 2461

 QC S-1 2199 

Average 2336 

SD 275 

RSD 12% 

% Recovery 127% 

SS 5 1 S-8 1279 2479

 2 S-17 1405

 3 S-12 2097

 4 S-21 1753 

Average 1934 

SD 460 

RSD 24% 

% Recovery 151% 

SS 6 1 S-24 3951 5226

 2 S-6 4657

 3 S-3 4776

 4 S-19 8552 

Average 5803 

SD 1849 

RSD 32% 

% Recovery 147% 

ND = not detected 

27 
Table 4-6. Hybrizyme AhRBU Results Compared to HRMS by Ranking--Original SITE 

Demonstration Dataa 

Environmental Site 

Hybrizyme Ranking by 

Average AhRBU b 

HRMS Ranking by Total 

Concentration (ng/g) b Did Ranking Agree? 

Newark Bay 1 

4 

2 

3 

1 

2 

4 

3 

No 

Raritan Bay 1 

2 

3 

1 

3 

2 

No 

Solutia 1 

3 

2 

1 

3 

2 

Yes 

Tittabawassee River 3 

2 

1 

3 

1 

2 

No 

Winona Post 1 

2 

3 

1 

2 

3 

Yes 

a U.S. EPA 2005e 

b 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 (< 100 pg/g TEQD/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 of Procept® 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 

 Time Estimate 

Activity Hands On Elapsed 

ASE Extraction 1 hour 6 hours 

Evaporation and Sulfuric Acid Treatment 1 hour Overnight 

Silica and Florisil Columns 3 hours 5 hours 

Evaporation and Sulfuric Acid Treatment 1 hour Overnight 

Procept® Assay 2 hours 4 hours 

Data Analysis 1 hour 1 hour 

Total Man Hours 9 hours 

Per Sample (batch of 20) ~0.5 hours 

Total Elapsed Time: 1 shift > 48 hours 

Total Elapsed Time: 2 shift < 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 eightday 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 PCRTM 

Kit. EPA/540/R-05/005, March. 

van den Berg, M., Birnbaum, L., Bosveld, A. T. C., Brunström, 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