METHOD 4435
SCREENING FOR DIOXIN-LIKE CHEMICAL ACTIVITY IN SOILS AND SEDIMENTS
USING THE CALUX® BIOASSAY AND TEQ DETERMINATIONS
SW-846 is not intended to be an analytical training manual. Therefore, method
procedures are written based on the assumption that they will be performed by analysts who areformally trained in at least the basic principles of chemical analysis and in the use of the subject
technology.
In addition, SW-846 methods, with the exception of required method use for the analysisof method-defined parameters, are intended to be guidance methods which contain general
information on how to perform an analytical procedure or technique which a laboratory can use
as a basic starting point for generating its own detailed standard operating procedure (SOP),
either for its own general use or for a specific project application. The performance data
included in this method are for guidance purposes only, and are not intended to be and must not
be used as absolute QC acceptance criteria for the purposes of laboratory accreditation.
1.0

SCOPE AND APPLICATION

For a summary of changes in this version, please see Appendix A at the end of thisdocument.
1.1
This method is a bio-analytical screening procedure for dioxin-like compounds insoils and sediments. This method is based on the ability of dioxin and related chemicals toactivate the aryl hydrocarbon receptor (AhR), a chemical-responsive DNA-binding protein that is
responsible for producing the toxic and biological effects of these chemicals. Measurement of
the level of activation of AhR-dependent gene expression by a chemical or chemical extract
provides a measure by which to estimate the relative potency and toxic potential of thesechemicals and/or extracts with resulting values expressed as toxic equivalents (TEQs).
Information on a commercially-available genetically-engineered cell line that contains the firefly
luciferase gene under trans-activational control of the AhR (Ref. 41) can be found at thefollowing website: http://www.dioxins.com/.
This cell line can be used for the sensitive detection and relative quantification of AhR
agonists and agonist activity of complex mixtures. The in vitro assay is designated as thechemical-activated luciferase expression or CALUX® assay. The most widely studied class ofcompounds that activate this system is the polychlorinated diaromatic hydrocarbons (PCDH),
such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The relative toxic and biologicalpotency of many PCDH compounds are quantified and expressed relative to that of 2,3,7,8TCDD, since this is one of the most potent activators of AhR-mediated effects, including genetranscription. This relative quantification approach generates overall potency values as TEQs
and the results obtained from using this method provide a measure of TEQs in a sample.
1.2
By using the sample processing procedures in this method and an affinity column(Ref. 42), polychlorinated biphenyls (PCBs) can be separated from chlorinateddioxins/dibenzofurans (PCDDs/PCDFs) making it possible to determine what portion of the total
TEQs of a sample is due to each of these classes of compounds. This is the dioxin/furan- and
PCB-specific (DIPS) analysis or the DIPS-CALUX bioassay for dioxin-like chemicals.
1.3
The AhR-dependent mechanism of the toxic and biological effects of dioxin-likechemicals and the basis of the CALUX® bioassay measurement and estimate of TEQ is shownin Figure 1 (Ref. 13). The AhR receptor complex is capable of binding dioxins, furans, PCBs
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and other dioxin-like compounds. Once these chemicals bind to the AhR, the complex migrates
into the nucleus where it specifically binds to the ARNT protein. The resulting chemical
AhR:ARNT complex then binds to a specific DNA sequence, the dioxin responsive element(DRE), which is present upstream from many genes including that of CYP1A1, and this binding
stimulates expression of the adjacent gene. In the case of the CALUX® assay, a plasmid
containing four DREs immediately upstream of the firefly luciferase reporter gene was stablytransfected into the mouse Hepa1c1c7 cell line to produce the recombinant cell line H1L6.1c3(6.1 cells). This transformed cell line responds to toxic PCDDs, PCDFs and PCBs, and highmolecular weight polynuclear aromatic hydrocarbons (PAHs) with the dose-dependent inductionof firefly luciferase (Refs. 13-16 and 34). Comparison of these results to a 2,3,7,8-TCDD
standard curve for induction allows for determination of the TEQs in a given sample.
1.4
By using sample processing procedures in this method, it is possible to separate
polyhalogenated biphenyls from polyhalogenated dioxins/dibenzofurans present in the same
sample. Using this DIPS-CALUX® bioassay it is possible to determine the portion of the total
TEQ activity in a given sample that is due to each of these classes of compounds (Ref. 6).
1.5

Toxic Equivalents (TEQs)

The concept of TEQs has been promoted by the World Health Organization to provide ameans of quantifying for risk assessment purposes the toxicity of a family of chemicals with asimilar overall mechanism of toxicity (Ref. 38). The family of dioxin-like chemicals (PCDHs)
within this group includes 7 chlorinated dibenzo-p-dioxin congeners with 4 to 8 chlorines on themolecule, 10 chlorinated dibenzofuran congeners with 4 to 8 chlorines on the molecule, and 12
chlorinated biphenyls with 4 to 10 chlorines on the molecule. A list of the dioxin-like chemicals
along with their assigned toxic equivalency factors (TEFs) to scale their toxicity relative to themost toxic congener 2,3,7,8-tetrachlorodibenzo-p-dioxin is shown in Table 1. The relative
potency (REP) values shown on this table and their significance are discussed in greater detailin Ref. 3.
1.6

Limits of detection

Limits of detection can be adjusted to meet the needs of the project. As the sample size
increases, the detection limit will decrease. Sample sizes of 2 – 10 g will typically give a
detection limit of less than1 pg/g. Limits of detection are determined based on the y-intercept
from the Hill Equation and the standard deviation of the DMSO blanks from the bioassay. The
limit of detection for the plate in relative light units is defined as the greater of either 2.5 times
the standard deviation of the DMSO blanks, or the y-intercept plus 2.5 times the standard
deviation of the DMSO blanks. The limit of detection for the plate in picograms of 2,3,7,8-TCDD
is determined from the relative light unit limits of detection using the Hill Equation. The limit ofdetection for each sample is determined based on the amount of sample used, the portion of thesample extract used and the recovery for that type of sample.
1.7

A tiered approach is recommended for quantification of TEQs in a sample.
1.7.1

Range finding analysis, the first stepThis step can be performed on all of the three sample processing procedures
outlined in Sec. 2.2. The first step is to screen the samples by conducting a range finding
analysis on the sample to determine the proper dilution (see Sec. 4.2). This provides anestimate of the concentration of dioxin-like chemicals in the sample that is in the linear
portion of the 2,3,7,8-TCDD calibration curve, for both the dioxin/furan and PCB fractions.
The optimal results are obtained with a dilution of the sample that produces an induction
response from the cells that falls close to EC50 (EC50 is the equivalent concentration at
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50% of the maximum response of the 2,3,7,8-TCDD standard) value for the 2,3,7,8-TCDD
standard curve run on each plate. This completes the sample screen procedure. For
more comprehensive sample analysis refer to Sec. 1.7.2.
1.7.2

Quantification analysis, the second step

The second step for comprehensive analysis is to analyze the three individual
extracts of the sample with a positive response at the optimum dilution (determined as
described in Sec. 2.2); this allows accurate estimation of the TEQ value of dioxin-likechemicals present in a given sample and allows mean and standard deviation
calculations.
1.8
Prior to employing this method, analysts are advised to consult the manufacturer’s
instructions for additional information on quality control procedures, development of QC
acceptance criteria, calculations, and general guidance. Analysts also should consult thedisclaimer statement at the front of the manual and the information in Chapter Two for guidanceon the responsibilities of the analyst for demonstrating that the techniques employed areappropriate for the analytes of interest, in the matrix of interest, and at the levels of concern.
In addition, analysts and data users are advised that, except where explicitly specified in a
regulation, the use of SW-846 methods is not mandatory in response to Federal testing
requirements. The information contained in this method is provided by EPA as guidance to be
used by the analyst and the regulated community in making judgments necessary to generate
results that meet the data quality objectives for the intended application.
1.9
This method is intended to be performed by trained analysts who are familiar withorganic/analytical sample processing techniques and cell culture techniques. Training on the
CALUX bioassay system can be obtained from the manufacturer. Instructions are provided in
the form of standard operating procedures (SOPs), which are detailed and thorough. The
manufacturer (http://www.dioxins.com/) also provides comprehensive training which is included
with licensing the technology. This technology has several steps where attention to detail iscritical to generating acceptable sample results. This includes careful processing of samples
through the extraction and cleanup procedures, pipetting small volumes, and accurately
weighing out samples.
2.0

SUMMARY OF METHOD

2.1
This method is a relatively rapid screening method capable of estimating the TEQs
concentration for dioxin-like chemicals in a sample. The sample is extracted in an organic
solvent and fractionated through the sample processing procedure (see Sec. 11). An extractthat contains the halogenated dioxins/furans is separated from an extract containing the
halogenated biphenyls. These extracts are applied to monolayers of H1L6.1c3 cells and theamount of luciferase induction is measured after 20 to 24 hr. A standard dilution series of
2,3,7,8-TCDD is included on each plate of cells. Estimation of dioxin/2,3,7,8-TCDD-like TEQ
activity present in the sample extract is performed by extrapolation to the 2,3,7,8-TCDD
standard curve by least squares estimates with the 4 parameter Hill Equation.
2.2
There are three modes by which the DIPS-CALUX bioassay is performed. These
are the screening mode with historical recovery, screening mode surrogate recovery, and thesemi-quantitative mode. The screening mode involves the analysis of a single aliquot of thesample and recovery is estimated from the mean of historical recoveries that have beenobtained for soils/sediment samples. This is considered to be acceptable as the variability ofrecoveries for soils/sediment samples has been relatively small (76.2 +/- 8.5%). Using thismode would indicate whether a sample needed to be further analyzed by either the semiSW-846 Update V

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processing two aliquots of the sample, the first for analysis in the DIPS-CALUX bioassay andthe second used for the surrogate spike with radiolabeled 2,3,7,8-TCDD to estimate recovery.
The semi-quantitative mode involves analyzing three aliquots of the sample in the DIPS-CALUX
bioassay and a fourth aliquot of the sample used for determination of recovery with radiolabeled
2,3,7,8-TCDD. The cost of sample analysis is dependent upon which mode of the DIPS-CALUX
bioassay is used for estimation of the levels of sample contamination.
3.0

DEFINITIONS

Refer to Chapter One and the manufacturer's instructions for definitions that may be
relevant to this procedure. In addition, see Method 4000 for glossaries of basic terms.
4.0

INTERFERENCES
4.1

Chemical interference

The chemicals listed in Table 2 are relatively weak agonists for the Ah receptor and induce
poorly. The results in the table demonstrate the percent recovery of each compound in theclean-up method and the amount of carry-over into the dioxin and PCB fractions (Refs. 7, 33, 35
and 37).
4.1.1

PAH interference

PAHs pose little interference in the method since the extraction and cleanup
methodology employed typically removes virtually all PAHs using an acid silica gelcolumn. In addition, chrysene, benzo(b)fluoranthene, indeno(1,2,3-cd)pyrene andacenaphthylene are the only PAHs we have found to bleed through the clean-up
procedure with approximately 5% - 26% (see Table 2) passing through the processing
steps. For the analysis of samples that are expected to have extremely high levels of
PAHs, users should subject the sample to an additional acidic-silica cleanup step. Testing
the relative activity of the sample after each round of cleanup would reveal whether
additional AhR active chemicals (i.e., PAHs) are removed with additional cleanup. Other
materials such as silver nitrate, florsil and alumina can be used in additional cleanup
steps. However, due to their rarity in the environment, these additional materials are
rarely used.
4.1.2

AhR receptor interference

Compounds that can activate the AhR receptor could result in potential
interference with the determination of TEQ due to dioxins/furans and PCBs. The PAH
class of potential interferences do not typically pass through the cleanup system andshould not be potential interfering agents. The cleanup methodology is outlined in Secs.
11.3 and 11.4. The remaining classes of potential interfering compounds are otherhalogenated (i.e., brominated and fluorinated) dibenzo-p-dioxins/furans and biphenyls, andhalogenated naphthalene’s and these appear to have similar toxicological properties as
their chlorinated derivatives. Isotope specific dilution methods using high-resolution mass
spectroscopy high-resolution gas chromatography (HRGC/MS) for quantifying 2,3,7,8TCDD do not measure these structurally similar halogenated dibenzodioxins/furans andbiphenyls and this may be one of the primary reasons that estimates of TEQ of dioxin-likechemicals by this method are slightly higher than HRGC/MS generated estimates of TEQ.

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4.2

Cell toxicity

Extremely high concentrations of PCDD/PCDF or PCB are not cytotoxic to cells.
However, other contaminants that may be present in sample extracts could potentially becytotoxic. The manufacturer has generally found that the sample processing procedures usedin this method result in removal of most potentially cytotoxic compounds. The manufacturer asa standard operating procedure monitors the cells after chemical treatment to determine
whether cytotoxicity has occurred and which could lead to potential false negative results fordioxin-like TEQ. Cell toxicity is identified if cells are found to be detached from the culture plateor whether they exhibit a major change in morphology. Generally, both cell toxicity andchemical insolubility can be overcome by sample dilution. In this method, typically 6 dilutions
are run to reduce potential cell toxicity and overcome any insolubility issues the sample may
have (1:4, 1:10, 1:100, 1:500, 1:1000 and 1:10,000). For samples that may contain componentsthat are not completely soluble at the above dilutions, the dilutions may be increased to(1:50,000, 1:100,000, 1:200,000, 1:500,000, 1:1,000,000 and 1:2,000,000). See Sec. 11.4 formore information.
5.0

SAFETY

5.1
This method does not address all safety issues associated with its use. The
laboratory is responsible for maintaining a safe work environment and a current awareness fileof OSHA regulations regarding the safe handling of the chemicals listed in this method. A
reference file of material safety data sheets (MSDSs) should be available to all personnel
involved in these analyses.
5.2
Safety procedures in compliance with good laboratory practices (GLPs) and OSHA
standards should be maintained at all times. Some reagents may contain hazardous materials
such as solvents and acids; therefore, the technician should use caution when using thereagents and avoid contact with eyes, skin and mucous membranes. All waste materials andsolutions should be placed in appropriate containers and disposed of according to all governingstate and federal regulations.
5.3
This method employs the use of dilute concentrations of 2,3,7,8-TCDD for the
standard curve; and quality control solutions contain PCB 126, and a solution comprised of all
17 chlorinated dioxin/furan congeners for which the WHO (World Health Organization) has
established dioxin-like TEF values. The analyst should take the appropriate measures when
preparing, handling, and disposing these standards.
6.0

EQUIPMENT AND SUPPLIES

Refer to Table 3 and to the manufacturer’s website (http://www.dioxins.com/) for acomprehensive list of recommended equipment and supplies.
7.0

REAGENTS AND STANDARDS

7.1
Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it
is intended that all reagents conform to the specifications of the Committee on Analytical
Reagents of the American Chemical Society, where such specifications are available. Other
grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination. Reagents should be
stored in glass to prevent the leaching of contaminants from plastic containers.
7.2
Refer to Table 4 and to the manufacturer’s website (http://www.dioxins.com/) for acomprehensive list of recommended reagents and standards.
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8.0

SAMPLE COLLECTION AND STORAGE

Sample collection, preservation, and storage requirements may vary by EPA program andmay be specified in the regulation or project planning document that requires compliancemonitoring for a given contaminant. Where such requirements are specified in the regulation,
follow those requirements. In the absence of specific regulatory requirements, use the following
information as guidance in determining the sample collection, preservation and storage
requirements.
8.1

Sample collection

This bioassay testing process employs very small sample volumes. Therefore, sample
collection procedures must focus on the amounts and procedures necessary to ensure that the
sample is representative of the source.
8.2

Sample receiving and tracking

Samples can be shipped directly to the manufacturer, received and logged in via the
manufacturer’s electronic tracking system. Temperature of the samples and the condition ofsamples are noted immediately. Any instructions from the client are also noted. The samples
are then placed in the appropriate storage conditions prior to processing.
8.3

Sample and extract storage

Biological samples are generally stored in a –70 °C freezer. Food products are stored at 0
C – 4 oC depending on individual needs of the sample. Feed, soils and other non-perishable
samples are usually held at room temperature unless the sample requires or the client requests
other storage conditions.
oSamples may be extracted and analyzed at the same laboratory or samples may be
extracted and then shipped to the manufacturer for analysis. If extracts are shipped to the
manufacturer, the manufacturer performs the sample cleanup to separate the PCDD/PCDF and
the PCBs from the extract. If a cleanup was already performed on the extracts, no further workneed be done on the samples prior to shipping. The extracts must be shipped in sealed vials.
Storage of extracts can be at room temperature. Extracts must also be covered with clean
polytetrafluoroethylene (PTFE)-lined caps to reduce evaporation or clean aluminum foil, if
extracts are dry.
8.4

Holding times

No maximum holding times for extracts have been established. However, PCDDs,
PCDFs, and PCB have an extremely long half-life and are very stable in environmental
matrices. They are essentially nonvolatile. Clients may request specific holding times for
project-specific applications.
9.0

QUALITY CONTROL

9.1
Refer to Chapter One for guidance on quality assurance (QA) and quality control(QC) protocols. When inconsistencies exist between QC guidelines, method-specific QC
criteria take precedence over both technique-specific criteria and those criteria given in Chapter
One, and technique-specific QC criteria take precedence over the criteria in Chapter One. Any
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effort involving the collection of analytical data should include development of a structured andsystematic planning document, such as a Quality Assurance Project Plan (QAPP) or a Sampling
and Analysis Plan (SAP), which translates project objectives and specifications into directions
for those that will implement the project and assess the results. Each laboratory should
maintain a formal quality assurance program. The laboratory should also maintain records todocument the quality of the data generated. All data sheets and quality control data should bemaintained for reference or inspection.
9.2
Parts of the quality control criteria outlined in this method are based on methods for
GC/HRMS such as SW-846 Method 8290 and EPA Method 1613. For a more detailed
description of the QC process please refer to Ref. 3, Appendix B, available from the
manufacturer’s website (http://www.dioxins.com/).
9.2.1

Initial demonstration of proficiency

Each laboratory must demonstrate initial proficiency with each sample
preparation and determinative method combination it utilizes by generating dataof acceptable accuracy and precision for target analytes in a clean matrix. At aminimum, such a demonstration will encompass the initial demonstration of
proficiency described in method 3500, using a clean reference matrix. The
laboratory must also repeat the demonstration of proficiency whenever new staff
members are trained or significant changes in instrumentation are made. See
Method 8000D, Section 9.3 for information on how to accomplish ademonstration of proficiency.
9.3
Monitoring of solvents used in sample preparation is conducted on a regular basis.
Each lot of solvent is tested by evaporating a 5-mL aliquot of the solvent into 4 µL of DMSO.
The DMSO solution is suspended in cell culture medium and exposed to the cells. All solvents
are tested for reactivity in the assay prior to use and a relative light units (RLU) induction of lessthan 2 times background must be obtained for the solvents to be used. A solvent blank is also
included in each batch of samples. The solvent blank is treated in the same way as the
samples and serves as a control to monitor for contribution of activity from any of the solvents orcolumn matrices used in sample preparation.
9.4
Reference samples were prepared from lake sediment materials that were finely
ground (solids) and analyzed twice by GC/HRMS for dioxin TEQ (average GC/MS TEQ = 13.6 ±6.8 pg/g). The lake sediment material was then spiked with 30 pg/g 2,3,7,8-TCDD to provide a
final dioxin TEQ concentration equivalent to the action level. The reference material was
shaken or stirred for three days, aliquoted and an aliquot was analyzed by GC/HRMS. The unspiked reference sample is included in each sample batch and is prepared and analyzed using
the same method as the unknown samples. Figures 2 and 3 show the QC charts for the unspiked lake sediment reference sample for 2003 and 2004 respectively. Figure 4 depicts the
QC chart for the spiked lake sediment reference sample.
9.5
Quality control (QC) charts are maintained for all reference samples as well as fora standard solution of PCB 126 and a mixture of the 17 dioxin/furan congeners (thosecongeners assigned TEF values for dioxin-like activity by the WHO) that are analyzed on eachplate (each of these standard solutions produces a response near the middle of the dose
response curve). These figures are generally reported as a three-month average. However, thedata for these samples can be monitored over longer time periods to insure against longer-term
variation in the assay (Figures 5-8). In QC charts, the results for the standard mixtures arereported as a ratio relative to the 6.25 pg/g point of the 2,3,7,8-TCDD standard curve (nearmiddle of linear range) and the reference materials are reported as the TEQ estimate
determined from the standard curve. If the reference material or either of the standard mixtures
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differs by more than two standard deviations from the moving average or a reference material is
below the limits of detection, the plate is declared invalid and all samples on the plate arereanalyzed. Figures 5 and 6 depict the QC charts for the PCDD/PCDF standard mixture for2003 and 2004 respectively. Figures 7 and 8 depict the QC charts for the PCB 126 for 2003
and 2004 respectively. Figures 9 and 10 depict the 2,3,7,8-TCDD standard curves for 30
samples in 2003 and 2004 respectively.
9.6
Quality control criteria for use of this method as a semi-quantitative estimate ofdioxin TEQ
In addition to the quality controls used in the screening assay, the 2,3,7,8-TCDD standard
curve is modeled to a sigmoid curve described by the four parameter Hill Equation using a leastsquares best fit. Estimation of TEQ values for sample extracts are conducted based on thederived Hill Equation with the following limitations listed (i.e., PCB fraction response must be
less than 50% of 2,3,7,8-TCDD maximal response and dioxin/furan fraction response must be
less than 75%). Any samples that exceed these limits, or are below the limits of detection, arereanalyzed using appropriate sample dilutions.
9.7

Recovery determination

Recovery determination (as described in Sec. 10.2 for 1,2,3,4-TCDD and 14C labeled
2,3,7,8-TCDD recoveries) is conducted using a duplicate sample that has been spiked witheither congeners that are radioactively labeled, or with a known amount of an equimolar mixtureof the unlabeled 17 congeners (see Figure 1). Using the radioactively labeled spike, therecovery is the percent of the recovered spike versus the amount added to the sample asdetermined by liquid scintillation. For the unlabeled spike, subtracting the TEQ for the sample
from the TEQ for the spiked sample and dividing the result by the TEQ for the spike determines
the recovery. For a more detailed description of the QC process please refer to Ref. 6,
Appendix B available from the manufacturer’s website (http://www.dioxins.com/).
9.8

Limits of detection

Limits of detection are determined based on the y-intercept from the Hill Equation and thestandard deviation of the DMSO blanks from the bioassay. The limit of detection for the plate inrelative light units is defined as the greater of 2.5 times the standard deviation of the DMSO
blanks, or the y-intercept plus 2.5 times the standard deviation of the DMSO blanks. The limit ofdetection for the plate in pg of 2,3,7,8-TCDD is determined from the relative light unit limits ofdetection using the Hill Equation. The limit of detection for each sample is determined based on
the amount of sample used, the portion of the sample extract used and the recovery for thattype of sample.
9.9Standards, reagents, solvents and any other materials used in the extraction, clean
up, and analysis, must be properly stored and must never be used beyond the manufacturer’s
and/or supplier’s expiration dates. A standard expiration time of one year is utilized for all
solvents and reagents.
10.0 CALIBRATION AND STANDARDIZATION
Calibration and standardization of each assay is very important to ensure accuracy of any
measurement of any system. The following is a description of the necessary calibration andstandardization procedures for this method. Also see the manufacturer’s instructions for
information that may be relevant.

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10.1

Calibration and standardization of each assay

Calibration and standardization is performed with each assay. An eleven point standard
curve of 2,3,7,8-TCDD (100 pg – 0.0977 pg) is run on each 96-well plate analyzed. Each plate
also contains 7 additional QC calibration points consisting of 4 DMSO solvent controls, 1 media
blank QC, 1 dioxin QC standard (contains all 17 dioxin congeners recognized by the World
Health Organization), and 1 PCB QC standard (80.5 ng/ml PCB 126). For internal recoveries, 2additional QC samples are added to estimate recovery. The dioxin and PCB QC points arecompared to the 6.25 pg point on the standard curve and entered into their respective QC
charts. See Sec. 10.2 for an explanation of the percent recovery points. This gives this method
20 QC calibration points.
10.2

Recovery determinations

Analyses of recovery determinations are very important for quantitative bio-analyticalmethods for dioxins/furans and PCBs. Bioassays do not differentiate between isotopicallylabeled and unlabeled analytes. Therefore, recovery determinations in bioassays can beaccomplished with a surrogate sample spiked with a radiolabeled congener of dioxin. It has
been demonstrated that 1,2,3,4-TCDD, a biologically inactive congener of the dioxin family ofchemicals, can be used as an internal spike to determine recoveries of dioxin-like chemicals.
Samples were spiked with 14C labeled 2,3,7,8-TCDD or 1,2,3,4-TCDD and submitted to
extraction and clean up using the cleanup method in Sec. 11.3.2. The cleanup column isdifferentially eluted to yield a PCB and PCDD/PCDF fraction. The 1,2,3,4-TCDD spiked
samples were resuspended in toluene containing four PCB injection standards, and recoveries
determined by gas chromatography with electron capture detection or scintillation counting.
Average recoveries determined by 1,2,3,4-TCDD with paired samples spiked with 14C- 2,3,7,8TCDD indicated that the recoveries determined by the two methods were very similar, 88.5% (±1.2%) and 87.2% (± 2.4%), respectively (see Figure 1). Recovery determinations were also
verified by HRGC/HRMS. This procedure allows for quantitative determination of dioxin-likechemicals in various sample matrices. For a more detailed description of the QC process refer
to Ref. 6, Appendix B, available from the manufacturer’s website (http://www.dioxins.com/).
11.0 PROCEDURE
This section describes the following procedures used in this method: Growth and storageof the mouse H1L6.1c3 recombinant cell line; preparation of 96-well plates, extraction andcleanup of samples, dosing plates and analysis of the data.
CAUTION: Always use sterile techniques when working with cells.
11.1

Cell culture
11.1.1

Culturing the H1L6.1c3 cell line

The recombinant H1L6.1c3 mouse cell line (Ref. 41) is grown at 37 °C in 5% CO2
and 100% humidity. Cells are grown in plastic cell culture flasks containing RPMI 1640
media supplemented with 8% fetal calf serum (FCS) and 1% penicillin/streptomycin. No
external selective pressure (i.e., Geneticin -- G418) is needed to maintain the stable
integration of the DRE-reporter plasmid in the cell line.
11.1.2Cell storage

Cells are stored in liquid nitrogen. After growth, the cells are placed in a freezing
media, consisting of RPMI supplemented with 10% FCS, 1% penicillin/streptomycin, andSW-846 Update V

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8% DMSO. Cells are stored at 1,000,000 – 2,000,000 cells per mL in 2-mL cryogenic
vials. After the cells and freezing media are placed in the vials, they are stored at –70°C
for 24 hr and then transferred to the liquid nitrogen Dewar for long-term storage.
11.1.3Thawing cells

Cells are stored in a liquid nitrogen Dewar in 2-ml cryogenic vials. These vials
are removed from the Dewar and the screw cap loosened slightly to release any excess
gas. The cap is then tightened and the vial is thawed quickly by placing it in either awater bath or an incubator at 37°C until thawed. The cell and freezing media mixture isthen transferred to a 50-mL centrifuge tube and 10 to 20 mL of RPMI 1640 is added andcentrifuged at 2000 rpm for 10 min. The media is removed from the cells and 5 mL offresh RPMI 1640 is added.
The cell pellet is disrupted by repeated pipetting in the media and placed in a
25cm2 culture flask for growth.
11.1.4Growth and splitting cells

The cells are first grown in 25-cm2 flasks to 80% confluence before transfer to a75-cm flask, which is also grown to 80% confluence. The media is removed from theflasks and cells are washed with PBS. Two mL of the trypsin is then added to the flask
and allowed to incubate at 37°C for 5 to 10 min. The flasks are then washed with PBS to
remove all of the cells from the growth surface and pipetted into a 50-mL centrifuge tube,
containing 5 mL of RPMI 1640 medium, and centrifuged at 2000 rpm for 10 min. The
cells are then transferred to two 75-cm2 flasks containing 10 mL RPMI 1640 each.
2

11.1.5Counting cells

After trypsinizing and centrifugation of the cells, the cells are counted using ahemocytometer. After centrifuging the trypsinized cells, the cells are re-suspended in 30
ml of RPMI 1640. Fifteen microliters of this solution is then placed on the hemocytometerfor counting. Four of the quadrants on the hemocytometer are counted: the upper left,
upper right, lower right, and lower left. Determine the average of the 4 counts. The volumeof each square is 10-4 mL, therefore: cells/ml = (average number per large square) x
104/ml. x 1/(dilution). This count is used in the plating of cells (see Sec. 11.2.1)
Example:11.2

Starting dilution: 30 mL
Total count of cells for all four grids: 468
Average of four grids: 117
Average / 75 (equivalent of 75 x 104) = 1.56
1.56 x starting dilution (30 mL) = 46.8
Add 16.8mls (to the original 30 mL dilution) for a total of 46.8mls.
On average, 20 mL are needed for one 96-well plate.

Preparation for testingFirst determine the number of samples, standards, blanks and QC to be tested per batch
and prepare plates with sufficient number of wells to accomplish the test.
11.2.1

Plating cells

Remove a 96-well plate from sterile package. Using the eppendorf multi-pipettor,
pipette 200 µl of cell/media solution to each well (see Sec. 11.1.5). Label plate with date
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and time of plating and cell concentration. Incubate plate(s) at 37°C in an atmospheresupplemented with approximately 5% carbon dioxide. Cells incubate for minimum of 24 hr
before dosing.
11.2.2

Dosing cells – Prepare 2,3,7,8-TCDD standard curve
1)
2)
3)
4)
5)

6)

A stock solution of 50 ng/mL 2,3,7,8-TCDD is used togenerate an eleven point standard curve.
Place 4 µL of dimethyl sulfoxide (DMSO) to eachof the eleven test tubes.
Add 4 µL of the 50 ng/mL 2,3,7,8-TCDD solution to
the first tube and thoroughly mix (25 ng/mL solution).
Transfer 4 µL of the solution in the first tube to thesecond test tube and mix using a vortex.
Continue the process of producing two fold dilutions until
you have added 4 µL of the mixture in tube 10 totube 11. Mix tube eleven, and discard 4 µL of the
mixture from tube 11.
This produces a 2,3,7,8-TCDD standard curve composedof the final concentrations listed in Table 7.

NOTE: The standard curve should be treated the same way as the samples. The
same lot of hexane used to prepare the sample extracts should also be used to
add 1 mL of hexane to each tube in the standard curve.
11.2.3

Dosing cells -- Prepare samples for dosing
1)
2)
3)
4)
5)

11.2.4

Dosing plate
1)
2)

3)

4)

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Add 4 µL of DMSO to 13-mm tube.
Add an appropriate sample amount.
If sample is less than 1 mL, add hexane to sample until
1 mL is reached.
Concentrate samples for 6 minutes, then an additional two
minutes to remove any remaining solvent.
Add 400 µL of RPMI 1640 cell culture medium(supplemented with 8% fetal calf serum and 1%
penicillin/streptomycin solution to each sample tube andvortex for 15 sec.

Remove the 96-well plate of cells that have been
incubated for 20-24 hr, at 37oC in an atmosphere of
approximately 5% carbon dioxide, from the incubator.
Place a piece of absorbent paper in the bio-hood.
Remove plate lid and invert the plate and tap it on paper toremove medium.
After visually inspecting the plate, add 200 µL of sample tobe tested to each well. When adding the sample, place
the tip of the pipettor on the wall of the well and pipetteslowly. This is done so that the cells layer is not disturbed.
Once all samples have been added, record date/time onplate and place the plate in the incubator for required time.
Cell line H1L6.1c3 is incubated with a chemical or extract
for 20-24 hr.
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11.2.5

Cell lysis and luciferase activity measurement

After the appropriate incubation time (see Sec. 11.2.1) the plates are removed fromthe incubator and the media removed. Each well is rinsed with 50 µL PBS. After which
each cell is inspected for cell viability, noting any damaged, morphologically changed or
missing cells. White backing tape is then applied to the bottom and 30 µL of Promega
Lysis buffer is added to each well. The 96-well plate is then shaken for 1 min before
inserting the plate into the microplate luminometer. For analysis of luciferase activity, 50µL of luciferase substrate (luciferin) is automatically injected into each well, the mixture isallowed to incubate for 5 sec followed by quantitation of luciferase activity (light
production) over a 15 sec-time period. The light produced from the cleavage of luciferin by
luciferase is expressed as relative light units (RLUs). These RLUs are then analyzed
using a Microsoft® Excel spread sheet that calculates the TEQ for the dioxin/furan and
PCB fractions.
11.3

Experimental set-up, sample extraction and clean-up
11.3.1

Experimental set up

Samples are first logged in to the manufacturer tracking system and assigned
project numbers and individual sample numbers. Next the number of samples, solvent
blanks, QC’s and recoveries are calculated to determine the number of extractions
necessary.
11.3.2

Sample extraction and clean-up

Samples are extracted using a modification of the Method 8290 extraction method(for soil, sediment, and ash). Three dried aliquots of each sample are ground (whenpossible) and two to ten gram aliquots are placed in solvent cleaned glass vials fitted with
PTFE-lined caps. For the screening process, one aliquot of the sample is used, andanother aliquot is processed for recovery purpose if historical recovery data is not used.
The samples are extracted with a 20% solution of methanol in toluene, then twice withtoluene. Each extraction of sample with solvent is incubated in an ultrasonic water bathfor approximately 10 minutes. The three extracts from each sample are filtered, pooled
and concentrated by vacuum centrifugation. The sample extract is then suspended inhexane and prepared for the clean-up method. The clean-up system consists of two
piggy-backed columns. The first column is an acid silica gel chromatography column used
to remove PAHs. The second column is the XCARB clean-up column. The XCARB
column is an affinity column that binds halogenated dioxins/furans and biphenyls. Thesechemical classes can be differentially eluted first with a mixture of hexane, toluene andethyl acetate solution to elute the PCB fraction. Next, the column is inverted and rinsed
with toluene to elute the dioxin/furan fraction. The eluate from the clean-up method isconcentrated under vacuum. This is then brought up to 4 mL in hexane for dilution
analysis in the bioassay.
11.4

Preparation of cleaned-up samples for analysis

After the samples are passed through the clean-up system and re-suspended in 4 mL ofhexane (see Sec 11.3.2), a range finding is performed on the samples by diluting them (1:4;
1:10; 1:100; 1:500; 1:1000; and 1:10,000). For suspected high level samples, where higher
chlorinated dioxin/furan insolubility may be present, range finding dilutions are increased(1:50,000, 1:100,000, 1:200,000, 1:500,000, 1:1,000,000 and 1:2,000,000) (see Sec 4.2). This
allows for a range finding estimation of the dioxin/furan and PCB content and to look for any
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problems with insolubility of the dioxins/furans and PCBs in the sample. The sample aliquots
are then concentrated under vacuum into DMSO and re-suspended in 400 µL of RPMI 1640
and dosed on the plated cells (see Sec. 11.2.4). The cells are incubated in a humidified CO2
atmosphere for 16-24 hr and then assayed for luciferase activity.
11.5

Alternative sample extraction for detection of PAH content

The experimental set up is identical to the dioxin and PCB analysis (see Sec. 11.3.2). The
only differences from the previous method in Sec. 11.3 are that there is no clean-up step
performed and that the bioassay is only incubated for 4 hr instead of the standard 16-24 hrs.
The sample is extracted, pooled and concentrated and then dosed without clean-up as in Sec.
11.3.2. This will give a total TEQ score for the PAHs in addition to the dioxins/furans, PCBs andany other AhR active chemicals in the sample extract. To calculate the estimate of PAH, thesample is run through the full process and the two results are compared. The PCDD/PCDF and
PCB results are subtracted from the previous results. The result would be the estimate of PAHs
within the sample.
12.0 DATA ANALYSIS AND CALCULATIONS
Data analysis and calculations are performed in an Excel spreadsheet using the RLU data
produced by the luminometer. The following is a description of the procedure for data analysis
and calculations (also see Figures 11-14).
12.1

Exporting RLU data

A Microsoft® Excel spread sheet is used to analyze the data.
12.1.1

Luminometer data

Data from the luminometer is exported to a Microsoft® Excel spreadsheet (“rawresults”) at the completion of the Standard Luciferase Assay. This Excel worksheet thengraphs the data as CALUX® activity in RLUs versus concentration of the test compound(Figures 11 – 14).
12.1.2

Background subtraction

The data collected by the luminometer(“raw results”) includes contributions frominduced expression of luciferase caused by the presence of the test compound, as well as,
contributions by background production of luciferase and the “dark current” from thephotomultiplier tube. In order to identify the component contributed by the induced
expression of luciferase, the results from a blank well is subtracted from the “raw results”.
This blank well contains cells that were exposed to cell culture medium dosed with 1%(v/v) DMSO (see Figure 11).
12.1.3Standard 2,3,7,8-TCDD curve

A standard curve for 2,3,7,8-TCDD is included in each experiment. Refer toSec. 11.2.2 for solution makeup (see also Figures 11 - 14).

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12.1.4

Four parameter Hill Equation formula

Results are calculated using TEQ values for the sample based on a leastsquares best fit of the standard curve of 2,3,7,8-tetrachlorodibenzo-p-dioxin (seeSec.12.3 and Figures 13 and 14).
12.2

EC50 determination

The first step in determining the EC50 of a standard curve is to look at the curve for thecompound and determine if it has a sigmoid shape. If it does not, the standard curve cannot be
used for determining the results of the assay.
12.2.1

Standard curve

At low concentrations a sigmoid curve should remain near the background level orslowly increase with increased concentration until a threshold is reached. At this point thedose response curve should become steeper and remain linear with increasing
concentration. Eventually, the response should reach a maximum. Increasing
concentrations will not result in increased response, as the dose response curve flattens
out.
The response at the higher concentrations of 2,3,7,8-TCDD results in saturation of
the RLU response. Generally, the linear portion of the curve is used to perform aregression analysis with the concentration of 2,3,7,8-TCDD plotted versus relative lightunits (RLUs). The linear portion of the curve generally includes RLU responses between
300 and 8000 RLU units.
12.2.2

Linear portion of standard curve

Generally the linear portion of the sigmoid curve will cover a 50 to 100-fold range ofconcentrations. In order to make a good estimate of the EC50 it would be preferable tohave at least four data points within this linear region (2 or 3-fold dilution series) (seeFigures 13 and 14).
12.2.3Calculating EC50

The EC50 for a sample is calculated using Microsoft Forecast Function. First, themidpoint of the linear portion of the standard curve is determined. Then, the forecast
function is applied to the three data points surrounding the midpoint.
Excel forecast formula: FORECAST (x,known_y's,known_x's)
12.3

Calculation of TEQ -- Least squares, best fit

Least squares, best fit is used to predict the best fit for the data using a four variable Hill
Equation with the natural log of the 2,3,7,8-TCDD concentration in femptograms/tube plottedversus relative light units (RLUs).
The response of a sample is compared to the response of 2,3,7,8-TCDD, corrected for
dilution of the unknown and assigned a relative concentration to 2,3,7,8-TCDD (TEQ) (seeFigures 11 - 14).

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13.0 METHOD PERFORMANCE
13.1 Performance data and related information are provided in SW-846 methods only
as examples and guidance. The data do not represent required performance criteria for usersof the methods. Instead, performance criteria should be developed on a project-specific basis,and the laboratory should establish in-house QC performance criteria for the application of thismethod. These performance data are not intended to be and must not be used as absolute QC
acceptance criteria for purposes of laboratory accreditation.
In the case of this method, any test kits used must be able to meet the performance
specifications for the intended application. Also, follow the manufacturer’s instructions for
quality control procedures specific to the test kit used.
13.2

Field studies

Field studies have been conducted at an EPA-sponsored SITE study in Saginaw, MI (April
26 – May 1, 2004) using this method, and a mobile lab was set up for analysis of soil/sedimentand extracts in the field. In addition, pre-site soil/sediment and extracts were tested at the
manufacturer’s facility and the data for the pre-site samples is included in this report (seeFigures 15 - 17). Ninety-six well assay plates containing mouse 6.1 cell line were sealed at the
manufacturer and transported to the site in an airtight box. Extraction and clean-up columns
were also pre-made at the manufacturer and transported to the SITE study location. Samples
were extracted and cleaned up on site in the mobile lab and analyzed using the sealed plates
produced at the manufacturer. The plates had been sealed for 7 days prior to use. An airtightbox was used in the field to see if it could be used in place of a standard humidified CO2
incubator. The analyst can breathe into the specially made valves giving the cells the gas
concentrations needed, or use a 5% CO2 tank if desired. The airtight box can then be placed ina 37 °C water bath for 16 - 20 hr to control temperature during incubation. The comparison
results between the airtight box and the incubator are included in this report (see Sec. 12 andFigure 17). These data are provided for guidance purposes only.
13.3 The following figures demonstrate the correlation between this method and GC/MS
determinations. Figures 15 – 17 represent data from the EPA pre-SITE study samples. Figures
18 – 22 represent samples from the EPA, waste sites, and Asian companies. Figure 23
represents the standard solutions from an international cross-lab validation study. Figure 24
represents the correlation determination for all sample matrices. These data are provided for
guidance purposes only.
13.4 Figure 15 represents the dioxin/furan TEQ comparison of this method to GC/MS for
soil samples for pre-field testing during the EPA SITE field study. These samples were
processed in March of 2004 using the screening technique with a surrogate recovery. These
TEQs were calculated using the WHO 1997 REP values. These data are provided for guidance
purposes only.
13.5 Figure 16 represents the PCB TEQ comparison from this method to GC/MS for soil
samples for pre-field testing during the EPA SITE field study. These samples were processedin March of 2004 using the screening technique with a surrogate recovery. These TEQs were
calculated using the WHO 1997 REP values. These data are provided for guidance purposes
only.
13.6 Figure 17 represents the dioxin/furan TEQ comparison of this method using the 37°C water jacketed incubator to the results obtained using an air tight chamber kept at 37 °C forsoil samples for pre-field testing during the EPA SITE field study. These samples were
processed in March of 2004 using the screening technique with a surrogate recovery. These
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TEQs were calculated using the WHO 1997 REP values. These data are provided for guidance
purposes only.
13.7 Table 8 outlines the TEQ-pg/g for the soil samples for pre-field testing during the
EPA SITE field study. This table represents the dioxin/furan TEQ comparison of this method
using the 37 °C water jacketed incubator to the results obtained using an air tight chamber kept
at 37 °C. These samples were processed in March of 2004 using the screening technique witha surrogate recovery. These TEQs were calculated using the WHO 1997 REP values. These
data are provided for guidance purposes only.
13.8 Figure 18 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for soil samples. The GC/MS data was generated by three different companies. See
Appendix A (available from the manufacturer’s website http://www.dioxins.com/) for the datatable comparing data from this method to GC/MS for each individual sample and company. See
Table 9 for individual sample data correlations. These data are provided for guidance purposes
only.
13.9 Figure 19 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for Ash samples. The GC/MS data was generated by three different companies. See
Appendix A (available from the manufacturer’s website http://www.dioxins.com/) for the datatable comparing data from this method to GC/MS for each individual sample and company. See
Table 11 for individual sample data correlations. These data are provided for guidance
purposes only.
13.10 Figure 20 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for Exhaust extract samples. The GC/MS data was generated by three different
companies. Please see Appendix A (available from the manufacturer’s website
http://www.dioxins.com/) for the data table comparing data from this method to GC/MS for eachindividual sample and company. See Table 13 for individual sample data correlations. These
data are provided for guidance purposes only.
13.11 Figure 21 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for Soil Extract samples. The GC/MS data was generated by three different companies.
See Appendix A (available from the manufacturer’s website http://www.dioxins.com/) for thedata table comparing data from this method to GC/MS for each individual sample and company.
See Table 10 for individual sample data correlations. These data are provided for guidance
purposes only.
13.12 Figure 22 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for Ash Extract samples. The GC/MS data was generated by three different companies.
Please see Appendix A (available from the manufacturer’s website http://www.dioxins.com/) for
the data table comparing data from this method to GC/MS for each individual sample andcompany. See Table 12 for individual sample data correlations. These data are provided for
guidance purposes only.
13.13 Figure 23 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for Standard Solution samples. The GC/MS data was generated by three different
companies. Please see Appendix A (available from the manufacturer’s website
http://www.dioxins.com/) for the data table comparing data from this method to GC/MS for eachindividual sample and company. See Table 14 for individual sample data correlations. These
data are provided for guidance purposes only.
13.14 Figure 24 depicts the dioxin/furan TEQ correlation between this method and
GC/MS for all samples. This shows a high over all correlation between this method and GC/MS
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for all samples with an R2 value of 0.9631. The GC/MS data was generated by five different
companies. See Appendix A (available from the manufacturer’s website
http://www.dioxins.com/) for the data table comparing data from this method to GC/MS for eachindividual sample and company. See Sec. 13.0 for individual sample data correlations. These
data are provided for guidance purposes only.
13.15 Table 9 depicts this method’s dioxin/furan data results with the GC/MS results forall soil samples. See Figure 18 for the associated graph. These data are provided for guidance
purposes only.
13.16 Table 10 depicts this method’s dioxin/furan data results with the GC/MS results for
all Soil Extract samples. Extracts were part of a double-blinded study, in which the original
sample weight was unknown. Original data generated using this method is shown as well as
the adjusted data per 10-gram sample size. See Figure 21 for the associated graph. These
data are provided for guidance purposes only.
13.17 Table 11 depicts this method’s dioxin/furan data results with the GC/MS results for
all Ash samples. See Figure 19 for the associated graph. These data are provided for
guidance purposes only.
13.18 Table 12 depicts this method’s dioxin/furan data results with the GC/MS results for
all Ash Extract samples. Extracts were part of a double blinded, in which the original sample
weight was unknown. Original data generated using this method is shown as well as theadjusted data per 10-gram sample size. See Figure 22 for the associated graph. These data
are provided for guidance purposes only.
13.19 Table 13 depicts this method’s dioxin/furan data results with the GC/MS results for
all Exhaust Extract samples. Extracts were part of a double blinded, in which the original
sample weight and volume of air was unknown. Original data generated using this method isshown, as well as, the adjusted data per cubic meter. See Figure 20 for the associated graph.
These data are provided for guidance purposes only.
13.20 Table 14 depicts this method’s dioxin/furan data results with the GC/MS results forall solution samples. See Figure 23 for the associated graph. These data are provided for
guidance purposes only.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates thequantity and/or toxicity of waste at the point of generation. Numerous opportunities for pollution
prevention exist in laboratory operations. The EPA has established a preferred hierarchy ofenvironmental management techniques that places pollution prevention as the management
option of first choice. Whenever feasible, laboratory personnel should use pollution prevention
techniques to address their waste generation. When wastes cannot be feasibly reduced at thesource, the Agency recommends recycling as the next best option.
14.2 For information about pollution prevention that may be applicable to laboratories
and research institutions consult Less is Better: Laboratory Chemical Management for Waste
Reduction, a free publication available from the American Chemical Society (ACS), Committee
on Chemical Safety,
http://portal.acs.org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_012290.pdf.
14.3 This method is an analytical assay which does not produce any air pollution andproduces minimal waste pollution. This method can be used and is currently used in
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remediation projects around the world to reduce both air and ground pollution. This is done
through monitoring and testing sites suspected of being contaminated with dioxin likecompounds. GLP and OSHA guidelines are followed at all times to prevent any pollution of theenvironment.
14.4 This method produces minimal waste. This waste is contained in biohazard
containers and disposed of in compliance with all state, local and federal regulations. The
method is also used in projects around the world to manage waste problems. Samples heading
for a waste site can be tested for dioxin like compounds prior to disposal to determine if any
further processing needs to be done before disposal. GLP and OSHA guidelines are followed at
all times when performing this method to prevent any pollution of the environment.
15.0 WASTE MANAGEMENTThe Environmental Protection Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and regulations. The Agency urges
laboratories to protect the air, water, and land by minimizing and controlling all releases fromhoods and bench operations, complying with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and hazardous waste regulations, particularly
the hazardous waste identification rules and land disposal restrictions. For further information
on waste management, consult the ACS publication listed in Sec. 14.2.
16.0 REFERENCES
1.

Arrieta, D. E., Ontiveros, C., Denison, M.S., and Washburn, B.S. (2000). "Ah Receptor
Mediated Effects of Particulate Organic Extracts from the Paso Del Morte Airshed Alongthe U.S. - Mexico Border." Organohalogen Compounds 45: 212 - 215.

2.

Behnisch, P. A., Allen, R., Anderson, J., Brouwer, A., Brown, D., Campbell, T. C.,
Goeyens, L., Harrison, R. O., Hoogenboom, R., Overmeire, I.V., Traag, W., and Malisch,
R. (2001). "Harmonised Quality Criteria for Chemical and Bioassays Analyses of
PCDDs/PCDFs in Feed and Food Part 2: General Considerations, Bioassay Methods."
Organohalogen Compounds 45.

3.

Brown, D. J., M. D. Chu, et al. (2001). "Determination of REP Values for the CALUX
bioassay and comparison to the WHO TEF Values." Organohalogen Compounds 53:
211- 214.

4.

Brown, D. J., Goeyens, L., Van Overmeire, I., Chu, M., Murata, H., and Clark, G.C.
(2001). "Quality control criteria implemented for monitoring the CALUX bioassay."
Organohalogen Compounds 54: 32-35.

5.

Brown, D. J., Kishimoto, Y., Ikeno, O., Chu, M., Nomura, J., Murakami, T., and Murata,
H. (2000). "Validation Study for the use of the Dioxin Responsive CALUX Assay for
Analysis of Japanese Ash and Soil Samples." Organohalogen Compounds 45: 200-203.

6.

Brown, D. J., Nakamura, M., Chu, M.D., Denison, M.S., Murata, H., and Clark, G.C.
(2002). "Recovery determinations for bioassay analysis: Condierations and results."
Organohalogen Compounds 58: 357-360.

7.

Brown, D. J., Van Overmeire, I., Goeyens, L., Chu, M.D., Denison, M.S., and Clark, G.C.
(2002). "Elimination of interfering compounds in preparation for analysis by an Ah
receptor based bioassay." Organohalogen Compounds 58: 401-404.

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8.

Clark, G., V. Garry, et al. (2002). "Relationships between exposure to dioxin-likechemicals, testosterone levels, and sex of the children of pesticide applicators."
Organohalogen Compounds 56: 73-76.

9.

Clark, G. C., Brown, D.J., Seidel, S.D., Phelan, D., Denison, M.S. (1999).
"Characterization of the CALUX and GRAB bioassays for sensitivity and specificity indetection of phamacological agents that activate the Ah Receptor signaling system."
Organohalogen Compounds 42: 309-312.

10.

Clark, G. C., Chu, M., Touati, D., Rayfield, B., Stone, J., Cooke, M. (1999). "A Novel
Low-Cost Air Sampling Device (AmbStack Sampler) and Detection System (CALUX
Bioassay) for Measuring Air Emissions of Dioxin, Furan, and PCB on a TEQ Basis
Tested With a Model Industrial Boiler." Organohalogen Compounds 42: 309-312.

11.

Denison, M. S., Nagy, S.R., Clark, G.C., Chu, M., Brown, D.J., Murata, H., Shan, G.,
Sanborn, J.R., and Hammock, B.D. (2001). "Bioanalytical approaches for the Detectionof Dioxin and Related Halogenated Aromatic Hydrocarbons." Organohalogen
Compounds 45.

12.

Denison, M. S., Seidel, S.D., Ziccardi, M., Rogers, W.J., Brown, D.J., and Clark, G.C.
(1999). "Ah receptor-based bioassays for dioxins and related chemicals: Applications
and limitations." Organohalogen Compounds 40: 27-30.

13.

Denison, M.S., Zhao, B., Baston, D.S., Clark, G.C., Murata, H. and Han, D.-H. (2004).
Recombinant Cell Bioassay Systems for the Detection and Relative Quantitation of
Halogenated Dioxins and Related Chemicals, Talanta 63: 1123-1133.

14.

Denison, M.S. Nagy, S.R., Ziccardi, M., Clark, G.C., Chu, M., Brown, D.J., Shan, G.,
Sugawara, Y., Shirley J. Gee, S.J., James Sanborn, J. and Hammock, B.D. (2002)
Bioanalytical approaches for the detection of dioxin and related halogenated aromatichydrocarbons, in: Technology-Driven Biomarkers Development and Application in
Environmentally-Associated Diseases, Wilson, D. and W. Suk, W., eds., pp. 483-494,
Lewis Press, Boca Raton, FL.

15.

Garrison, P.M., Tullis, K., Aarts, J.M.M.J.G., Brouwer, A. and Giesy, J.P. and Denison,
M.S. (1996) Species-specific recombinant cell lines as bioassay systems for thedetection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals, Fund. Appl. Toxicol. 30,
194-203.

16.

Han, D.-H., Nagy, S.R. and Denison, M.S. (2004) Comparison of recombinant cell
Bioassays for the detection of Ah receptor agonists, Biofactors 20, 11-22.

17.

Han, D., Nagy, S.R., and Denison, M.S. (2002). "Recombinant cell lines for the detectionof dioxins and Ah Receptor ligands- Not all assays are created equal." Organohalogen
Compounds 58: 421-424.

18.

Hoogenboom, R., Portier, L., Ontenk, C., Polman, T., Hamers, A., and Traag, W. (2000).
"Determination of Dioxins and Planar PCBs in Fish Using the GC/MS and CALUX
Bioassay." Organohalogen Compounds 45: 180 - 184.

19.

Hooper, K., G. Clark, et al. (2002). "Using the CALUX Assay to select high-TCDD
mothers in Southern Kazakhstan and map time-course of TCDD elimination via breast
milk." Organohalogen Compounds 58: 377-380.

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20.

Hooper, K., Hayward, D., Anderson, M., Farland, W., Lucier, G., Clark, G. (2000).
"CALUX results correlate with GC/MS/MS data from Kazakhstan breast milk samples."Organohalogen Compounds 45: 236-239.

21.

Huwe, J. K., Clark, G., Chu, A., and Garry, V. (2003). "CALUX and High Resolution
GC/MS Analysis of Dioxin-Like Compounds in Chlorophenoxy Pesticide Formulations."
Organohalogen Compounds 60 - 65.

22.

Kayama, F., Hammatsu, A., Sagisaka, K., Brown, D., Clark, G., and Suzuki, T. (2001).
"CALUX assay is applicable to human epidemiological studies." Organohalogen
Compounds 54: 48-50.

23.

Kayama, F., Horiguchi, H., Kunisue, T., Tanabe, S., Fukatsu, H., Yamazaki, N.,
Nakamura, M., Yabushita, H., Fujino, J., and Clark, G. (2003). "Dioxin andOrganochlorine Pesticide Concentrations of Breast Milk in China and Japan."
Organohalogen Compounds 60 -65.

24.

Kayama, F., Horiguchi, H., Oguma, E., Fujino, J., Yabushita, H., Brown, D., Clark, G.
(2002). "Regional differences of blood dioxins and organchlorine pesticides
concentrations of Japanese female farmers.-Application of CALUX assay for
epidemiological study-." Organohalogen Compounds 55: 275-278.

25.

Nakamura T, Nakamura M, Suzuki S, Takahashi M, Fujino J, Yabushita H, Yamamoto
T, Brown DJ, Nakai K, Satoh H (2002)" A COMPARATIVE ANALYSIS OF CERTIFIEDENVIRONMENTAL REFERENCE MATERIALS USING CALUXTM ASSAY AND HIGHRESOLUTION GC/MS". Organohalogen Compounds 58: 381-384.

26.

Overmeire, I. V., Carbonnelle, S., Loco, J.V., Roos, P., Brown, D., Chu, M., Clark, G.,
and Goeyens, L. (2002). "Validation of the CALUX Bioassay: Quantitative Screening
Approach." Organohalogen Compounds 58: 353 - 356.

27.

Overmeire, I. V., Chu, M., Brown, D., Clark, G., Carbonnelle, S. and Goeyens, L. (2001).
"Application of the CALUX Bioassay for the Determination of Low TEQ Values in Milk
Samples." Organohalogen Compounds 45.

28.

Overmeire, I. V., Goeyens, L., Beernaert, H., Srebrink, S., De Poorter, G., Baeyens, W.,
Clark, G., Chu, M., Chu, A., Chu, D., Morris, R., and Brown, D. (2000). "A Comparitive
Study of GC-HRMS and CALUX TEQ Determinations in Food Samples by the BelgianFederal Ministries of Public Health and Agriculture." Organohalogen Compounds 45: 196- 200.

29.

Pence, L. M., Archer, J.C., Bluhm, Earnheart, C., Clark, G., Chu, A., Lovell, R., andEckert, J. (2003). "Inter-Laboratory CALUX Comparison Data at Low Levels with
Traditional GC-HRMS Confirmations." Organohalogen Compounds 60 - 65.

30.

Shaw, S. D., Brenner, D., Mahaffey, C.A., Guise, S.D., Perkins, C.R., Denison, M.S.,
and Waring, G.T. (2003). "Persistent Organic Pollutants (POPs) and Immune Function in
US Atlantic Coast Harbor Seals." Organohalogen Compounds 60 - 65.

31.

Warner M., E., B., Patterson, D.G., Clark, G., Turner, W.E., Bonsignore, L., Mocarelli, P.,
and Gerthoux, P.M. (2003). "Comparison of the CALUX Bioassay and ID-HRGC/HRMS
as Measures of Dioxin Toxic Equivalents in Serum." Organohalogen Compounds 60 65.

SW-846 Update V

4435 - 20

Revision 1
July 2014

32.

Windal, I., Schroijen, C., Wouwe, N.V., Carbonnelle, S., Overmeire, I.V., Brown, D.,
Clark, G., Baeyens, W., and Goeyens, L. (2003). "Non Additive Interactions in CALUX."
Organohalogen Compounds 60 - 65.

33.

Windal, I., I. Van Overmeire, et al. (2002). "Variables that affect TEQ determinations by
CALUX and GC-HRMS." Organohalogen Compounds 58: 389-392.

34.

Ziccardi, M.H., Gardner, I.A. and Denison, M.S. (2002) Application of the luciferase
recombinant cell culture bioassay system for the analysis of polycyclic aromatichydrocarbons, Environ. Toxicol. & Chem. 21, 2027-2033.

35.

Windal, I., Dennison, M. S, Birnbaum L. S., Van Wouwe, N., Baeyens, W. Goeyens L.
(2005). “Chemically Activated Luciferase Gene Expression (CALUX) Cell Bioassay
Analysis for the Estimation of Doxin-Like Activity: Critical Parameters of the CALUX
Procedure that Impact Assay Results, Environ.” Sci. Technol., 3, 7357-7364.

36.

Behnisch, P.A., Hosoe, K., Sakai, S-i. (2003). “Brominated dioxin-like compounds in vitro
assessment in comparison to classical dioxin-like compounds and other polyaromaticcompounds.” Environment International 29, 861-877.

37.

Schroijen, C.,Windal, I., Goeyens, L, Baeyens, W. (2004). “Study of the interference
problems of dioxin-like chemicals with the bio-analytical method CALUX.” Talanta 63,
1261-1268.

38.

Van den Berg et al., (2006). “The 2005 World Health Organization Reevaluation of
Human and Mammalian Toxic Equivalency Factors for Dioxins and Dioxin-Like
Compounds.” Toxicological Sciences 93(2):223-241.

39.

Brown, D.J., Orelien, J., Gordon, J.D., Chu, A.C., Chu, M.D., Nakamura, M., Handa, H.,
Kayama, F., Denison, M.S., and Clark, G.C. (2007) “Mathematical Model Developed forEnvironmental Samples: Prediction of GC/MS Dioxin TEQ from XDS-CALUX Bioassay
Data,” accepted Environmental Science and Technology Nov. 2007.

40.

USEPA, “Report on the Comparison of Cell Based Assays with Mass Spectrometry
Methods for the Analysis of PCDDs/PCDFs and PCBs in Biosolids. U.S. EPA Reportfrom the Office of Water and Office of Science and Technology, Engineering andAnalysis Division on Biosolids, June 2002.

41.

U.S. patent # 5,854,010

42.

U.S. Patent # 6,720,431

17.0

TABLES, DIAGRAMS, FLOW CHARTS, AND VALIDATION DATA
The following pages contain the tables and figures referenced by this method.

SW-846 Update V

4435 - 21

Revision 1
July 2014

TABLE 1
WHO TEF AND METHOD 4435 REP VALUES

CompoundDioxins
TCDD
12378-PeCDD
123478-HxCDD
123678-HxCDD
123789-HxCDD
1234678-HpCDD
OCDD
Furans2378-TCDF
12378-PeCDF
23478-PeCDF
123478-HxCDF
123678-HxCDF
123789-HxCDF
234678-HxCDF
1234678-HpCDF
1234789-HpCDF
OCDF
PCBs
PCB 77
PCB 81
PCB 114
PCB 126
PCB 156
PCB 169

SW-846 Update V

WHO TEFCALUX REP
Based on EC50 Values

REP Range(EC20 - EC50)

1
1
0.1
0.1
0.1
0.01
0.00011.00 +/- 0.01
0.73 +/- 0.10
0.075 +/- 0.014
0.098 +/- 0.017
0.061 +/- 0.012
0.031 +/- 0.008
0.00034 +/- 0.00008

0.44 to 1.02
0.034 to 0.137
0.043 to 0.183
0.028 to 0.114
0.015 to 0.058
0.00025 to 0.00049

0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.0001

0.067 +/- 0.010
0.14 +/- 0.04
0.58 +/- 0.08
0.13 +/- 0.02
0.14 +/- 0.03
0.11 +/- 0.02
0.31 +/- 0.06
0.024 +/- 0.007
0.044 +/- 0.010
0.0016 +/- 0.0005

0.040 to 0.104
0.14 to 0.15
0.37 to 0.78
0.07 to 0.20
0.10 to 0.19
0.05 to 0.18
0.31 to 0.31
0.019 to 0.031
0.032 to 0.059
0.0003 to 0.0058

0.00050.00010.00050.1
0.00050.01

0.0014 +/- 0.0004
0.0045 +/- 0.0012
0.00014 +/- 0.00002
0.038 +/- 0.007
0.00014 +/- 0.00002
0.0011 +/- 0.0003

0.0012 to 0.0017
0.0022 to 0.0085
0.00014 to 0.00017
0.037 to 0.042
0.00013 to 0.00019
0.0007 to 0.0017

4435 - 22

Revision 1
July 2014

TABLE 2
CHEMICAL INTERFERENCES

Compoundbenzo(a)pyrene
chryseneacenaphthylene
benzo(a)anthracene
benzo(b)fluoranthenebenzo(k)fluoranthenecreosotep-cresoldibenzo(a,h)anthracene
1,2-diphenylhydrazine
Fluorene
ideno(1,2,3 cd)pyrene
perylene
2-phenylindoleo-cresol
tryptamine

SW-846 Update V

Acid silica only
% Recovery
0.002%
28%
ND
0.05%
26%
<0.001%ND
ND<0.001%
ND
ND
0.6%
ND
ND
ND
ND

Dioxin fraction% Recovery
0.02%
0.2%
0.2%
0.005%
5.7%<0.001%
ND
ND
ND
ND
ND
2.5%
ND
ND
ND
ND

4435 - 23

PCB fraction% Recovery
ND
ND
ND
ND
<0.001%
ND
ND
ND
ND
ND
ND
0.005%
ND
ND
ND
3%

Revision 1
July 2014

TABLE 3
RECOMMENDED EQUIPMENT AND SUPPLIESSupplyItem
Micropipettor, 0.5-10 µL range
Micropipetor 2-20 ml range
Micropipettor, 20-200 µL range
Micropipetor 100 Š 1000 ml range
Multipipettor, repeating - syringe type
EDP2, 10-100ul Electronic Pipette
Drummond diaphragm pipettor
10 ml pippet tips
200 ml pippet tips
1000 ml pippet tips
1.0 ml multipipettor syringes, case of 100
10.0 ml multipipettor syringes, case of 100
10 ml sterile pipettes, plastic, case of 200
10 ml graduated glass pippets
25 cm2 Tisue Culture Flasks
75 cm2 Tissue Culture Flasks
scintillation vials, case of 500
scintillation vial caps, teflon liner, case of 500
25 ml drying tubes, case of 100
10 ml drying tubes, case of 200
13 x 100 mm Test Tubes
16 x 125mm Test Tubes
50 ml centrifuge Tubes
15 ml plastic centrifuge tubes, sterile
50 ml plastic centri. Tubes
13mm test tube racks
13mm test tube racks for dosing
16 mm test tube racks
50 ml test tube racks
9" Pasteur pipettes
pipette bulbs, 2 ml capacity, pack of 72
96 well plates
Backing Tapelatex gloves
glass wool, 8 micron
Benchtop paper, 2 rolls of 20" x 300"
Tubing 12mm, CS-25lb

SW-846 Update V

Supplier

Cat #Substitution

VWR
VWR
VWR
VWR
FisherVWR
VWR
Denville Scientific
Denville Scientific
Denville ScientificFisherFisherDenville Scientific
VWR
VWR
VWR
FisherFisher
VWR
VWR
VWR
VWR
VWR
Denville
VWR
FisherFisherFisherFisherVWR
VWR
VWR
Perkin Elmer
Marsh Bio
Fisher
VWR
VWR

40000-200
40000-202
40000-204
40000-208
21-380-8
E2-100 EDP2
53498-708
P-1095-CP
P4114
P4026
21-381-337
21-381-340
P1096-CP
53283-776
15708-096
29186-080
03-340-129
03-340-131
17453-142
17453-140
60825-414
60825-630
21020-695
C-101821008-951
14-809-22
14-810-54A
14-809-24
14-809-28
53283-915
56311-062
29444-010
6005199
L6003PF
11-38814672-200
32814-227

or equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalent4435 - 24

Revision 1
July 2014

TABLE 3(cont.)
RECOMMENDED EQUIPMENT AND SUPPLIES

Equipment
Item nameClass II biological safety hood and standWater Jacketed Cell culture incubator, with CO 2 and temp.
control
Centrifuge, low speed, tabletop
with swinging bucket rotor
Centrifuge concentrator with vacuum pump with cold trap
Shaker for 96 well plates
Microscope, inverted
Microscope
Hemocytometer, cell counter
Hand tally counter
Vortex – mixer
sonicating water bath
Vacuum pump with liquid trap (side arm erylenmeyer
flask)
Refrigerator/freezer-70 celcius freezer
Liquid Nitrogen dewar
Luminometer Berthold and dedicated computer
Combustion test kit, CO 2 monitoringo

250 C Oven
AutoclaveHigh-Capacity top loading Balance 0.01g-4200g

SW-846 Update V

Supplier

Cat #Substitution

Fisher16-108-99

or equivalent

Fisher

11-689-4

or equivalent

FisherFisherFisherFisherFisherFisherFisherFisherFisher

04-978-50
16-315-45
14-271-9
12-561-INV
12-561-3M
02-671-5
07-905-6
12-81415-335-30

or equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalent

FisherFisherFisherFisherBerthold
Fisher

01-092-29
13-986-106A
13-989-187
11-675-92
10-884-1

or equivalentor equivalentor equivalentor equivalentor equivalentor equivalent

58618-009
14216-518

or equivalentor equivalentor equivalent

VWR
VWR
VWR

4435 - 25

Revision 1
July 2014

TABLE 4
RECOMMENDED REAGENTS AND STANDARDS

Solvent / Reagent Item
Phosphate buffered saline 1x
Trypsinpen/strep solution
Fetal Calf serum

Supplier

Cat #Substitution

VWR
VWR
VWR

45000-446

or equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentor equivalentAtlanta Biological

RPMI 1640 1X (MOD.) with L-Glutamine medium

VWR
Cell Culture Lysis Reagent
Promega
Luciferase Substrate Solution
Promega
70 % ethanol, for cleaning and as coolant for cold trap
VWR
Sodium hydroxide
VWR
Sigma-Aldrich
DMSO
FisherSulfuric acid, concentrated, 2.5 L, ACS reagent
FisherHexane, optima grade, case of 4-4L bottles
FisherToluene, optima grade, case of 4-4L bottles
FisherMethanol, case of 4-4L bottles
FisherAcetone, case of 4-4L bottles
Ethyl acetate
VWR

SW-846 Update V

4435 - 26

MT 25-054-C1

45000-650
S1115045000-396
E1531
E1501
VW3609-4
JT3722-4
27,043-1
A300-212
BJ216-4
T291-4LC
A454-4
A929-4
JT9282-3

Revision 1
July 2014

TABLE 5
EXAMPLE UNLABELED 17 CONGENERS INTERNAL SPIKE RECOVERIES

Samplespiked sample
unspiked sample
equimolar spike
spiked sample
unspiked sample
equimolar spike
spiked sample
unspiked sample
equimolar spike
spiked sample
unspiked sample
equimolar spike

Spike sample
minus sample pg
1.28
8.66
5.05
10.82
-

TEQ
4.65
3.37
1.61
10.52
1.86
10.28
5.22
0.17
6.98
11.09
0.27
11.45

% Recovery
80%
84%
72%
94%
-

TABLE 6
COMPARISON OF 1,2,3,4-TCDD RECOVERIES DETERMINED BY GC/ECD AND 14C
LABELED 2,3,7,8-TCDD RECOVERIES DETERMINED BY SCINTILLATIONS COUNTING

14

Sample1234-TCDD by
GC/ECDC 2,3,7,8-TCDD by
scintillation counting

1
2
3
4

76.3% ± 1.1%
95.4% ± 1.7%
91.6% ± 1.8%
90.6% ± 0.3%

91.2% ± 0.9%
84.1% ± 2.9%
89.9% ± 2.4%
83.5% ± 3.4%

SW-846 Update V

4435 - 27

Revision 1
July 2014

TABLE 7
EXAMPLE STANDARD CURVE CONCENTRATIONSTCDDConcentrations
pg/ml
250
125
62.5
31.25
15.625
7.81
3.91
1.95
0.977
0.488
0.244

Picograms/Tube
100
50
25
12.5
6.25
3.13
1.56
0.78
0.391
0.195
0.098

TABLE 8FIELD TEST COMPARISON of Standard INCUBATOR TO AIRTIGHT BOX

XDS
ID #
Site Sample #1
Site Sample #2
Site Sample #3
Site Sample #4
Site Sample #5
Site Sample #6
Site Sample #7
Site Sample #8
Site Sample #9
Site Sample #10
Site Sample #11
Site Sample #12

SW-846 Update V

TEQ-pg/g
Incubator
Airtight box
PCDD / PCDF
PCDD / PCDF
0.13
0.14
46.27
55.58
0.13
0.15
0.13
0.13
523.29711.11
169.79184.15
0.34
0.58
97.57
86.67
193.02349.01
70.56
142.44
95.05
97.51
0.56
0.75

4435 - 28

Revision 1
July 2014

TABLE 9
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR SOIL SAMPLES

Company
Company-2 Asia
Company-2 Asia
Company-2 Asia
Company-2 Asia
Company-2 Asia
Company-2 Asia
Company-2 Asia
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-3 USA
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-4 Europe
Company-4 Europe
Company-4 Europe
Company-4 Europe
Company-4 Europe
Company-5 USA
Company-5 USA

SampleMatrix
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil

SW-846 Update V

Sample ID
Soil-1
Soil-2
Soil-3
Soil-4Soil-5Soil-6
Soil-717100 A, B
17101 A, B
17102 A, B
17103 A, B
17105 A, B
17106 A, B
17107 A, B
17108 A, B
17109 A, B
17118 A, B
17120 A, B
17126 A, B
17128 A, B, C, D, E, F
17130 A, B
17131 A, B
17132 A, B
17134 A, B
17138 A, B
17149 A, B
Soil Sample 1
Soil Sample 2
Sample No. 2 soil/1185-2
Sample No. 3 Soil/ 1185-3
Sample No. 4, Soil./ 1185-4
Sample No. 6, Soil./ 1185-6
Sample No. 7, Soil./ 1185-7
Sample No. 8, Soil./ 1185-8
Soil Sample 1.
Soil Sample 3.
Soil Sample 4.
Soil Sample 5
Soil Sample 6
Soil Sample 14.
A
C
D
B
C
XDSG
XDSG30

TEQ-ppt
mean (pg/g)
2.56
28.33
25.63
352.324034.977306.8720539.55
207.5112869.85
306.05539543.83
441485.41
637162.72
394642.48
2078549.92
25574.14
272858.59
7469.31790.04
191.58260510.86885605.65823636.59
392784.46
6703.40459.40416.00
5.65
27.87
14.41
39.90
16.98
7.67
9.98
9.05
15.22
21.82
4699.316725.04
24.04
160.00600.001061.00
154.00570.0016.28
37.24

4435 - 29

std error
0.30
3.09
3.47
30.53
339.881021.264661.6428.56
1057.7119.80
42835.93
79369.82
29247.40
19514.2180574.11
154.392423.72299.1085.95
10.11
2495.27
83917.76
93127.53
468.45172.76
23.40
32.13
0.01
3.13
2.17
6.60
2.85
1.19
1.47
0.78
2.36
0.06
171.10525.67
0.43
0.01
0.07
0.09
0.04
0.11
0.62
1.39

Percent
std error
12%
11%
14%
9%
8%
14%
23%
14%
8%
6%
8%
18%
5%
5%
4%
1%
1%
4%
11%
5%
1%
9%
11%
0%
3%
5%
8%
0%
11%
15%
17%
17%
16%
15%
9%
15%
0%
4%
8%
2%
0.00%
0.01%
0.01%
0.02%
0.02%
4%
4%

GC/MS
(pg/g)
0.19
3.80
12.00
120.00800.002600.00
1200.00
63.90
4852.00
84.20
94330.00
63113.00
44411.00
55581.00
333638.00
8547.00
98560.00
1398.00
207.0065.00
10891.00
115539.00
115451.00
116792.00
2337.00
89.50
150.00
1.20
7.90
7.90
21.00
2.40
6.00
6.00
0.21
0.31
3.30
4000.00
5900.00
3.80
17.67
180.00323.3319.67
496.67
8.79
38.83

Revision 1
July 2014

TABLE 10
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR SOIL EXTRACTS

Company
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia

SampleMatrixSoil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract
Soil Extract

SW-846 Update V

TEQ-ppt
Sample ID
S-74
S-121
S-16
S-61
S-119
S-54
S-120
S-132
S-43
S-17
S-49
S-53
S-18
S-55
S-28
S-42
S-127
S-19
S-7
S-52
S-75

mean (pg/g)

115.0274.50
12.23
522.6755.27
104.50176.43847.00
1.21
2.70
89.67
345.288.90
82.48
313.5098.96
682.205.97
93.94
102.10316.10std error
16.01
10.37
3.40
73.34
7.56
17.76
30.43
157.42
0.17
0.52
10.74
22.20
0.61
8.22
37.88
10.41
67.87
0.73
12.16
10.46
18.96

4435 - 30

Percent
std error
14%
14%
28%
14%
14%
17%
17%
19%
14%
19%
12%
6%
7%
10%
12%
11%
10%
12%
13%
10%
6%

CALUX
(pgTEQ/10g)120.0076.00
12.00
540.0055.00
110.00180.00860.00
1.00
3.00
92.00
360.009.00
84.00
310.00103.00680.00
6.00
160.00105.00320.00

GCMS(pgTEQ/10g)
23.00
6.80
0.92
110.00
5.40
25.00
15.00
150.00
0.07
0.11
12.00
87.00
0.39
8.20
42.00
12.00
110.00
0.26
17.00
18.00
21.00

Revision 1
July 2014

TABLE 11
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR ASH SAMPLES

Company

SampleMatrixCompany-2 Asia
Company-2 Asia
Company-2 Asia
Company-2 Asia

Ash
Ash
Ash
Ash

Company-1-Asia

AshCompany-1-Asia
Company-1-Asia
Company-1-Asia
Company-1-Asia
Company-1-Asia
Company-4 Europe
Company-4 Europe
Company-4 Europe
Company-4 Europe

Ash
Ash
Ash
Ash
Ash
Ash
Ash
Ash
Ash

SW-846 Update V

Sample ID
Ash-1
Ash-2
Ash-3
Ash-5
Sample No. 9,Ash, 1185-9
Sample No.10,
Ash/1185-10
Ash Sample 7
Ash Sample 8
Ash Sample 9
Ash Sample 11
B
C
B
C

TEQ-ppt
mean (pg/g)

std error

Percent
std error

GC/MS(pg/g)

7.34
105.70584.5552795.74

1.89
27.01
61.40
9307.38

26%
26%
11%
18%

2.80
60.00
400.0027000.00

402.6014.84

4%360.00684.47142.83260.6019870.12
1598.4636.00
523.0063240.00
545.00

38.78
14.06
33.29
983.14
73.54
0.00
0.02
9.11
0.04

6%
10%
13%
5%
5%
0%
0%
0%
0%

470.0089.00
71.00
7000.00
1100.00
32.00
390.0057500.00
285.334435 - 31

Revision 1
July 2014

TABLE 12
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR ASH EXTRACTS

Company
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia

SampleMatrixAsh Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract
Ash Extract

SW-846 Update V

TEQ-ppt
Sample ID mean (pg/g)
A-20
12721.95
A-1728261.60
A-23
84.69
A-40
193299.28
A-18
1442.06
A-66
5862.67
A-57
16871.73
A-43
7896.12A-424525.55A-50
48462.10
A-56
4646.37
A-2
2443.32A'-22192.71
A'-1
1844.48A'-51273.59
A-13
128781.60
A-1922838.28
A-9
60.94
A-59
6812.57
A-60
92283.85
A-28
77824.56
A-41
103465.03
A-34
327501.35
A-61
20.66
A-27
56.60
A-45
60374.36
A-14
164.08A-1647848.74
A-44
3985.64A-75
512.53A-65
458.24A-15
2630.39A-58
5399.43
A-48
201.53A-814904.29
A-74
781.28A-31
975.80std error
1048.594132.325.55
20703.51
84.06
736.101857.26
334.81457.105962.94333.91
188.29143.06
187.46114.6513106.95
1799.115.96
902.8212021.15
4318.9415583.39
27583.75
2.61
6.85
8614.3917.35
6204.24
239.5424.61
19.23
200.54168.0911.01
1335.97
61.79
47.11

4435 - 32

Percent
std error
8%
15%
7%
11%
6%
13%
11%
4%
10%
12%
7%
8%
7%
10%
9%
10%
8%
10%
13%
13%
6%
15%
8%
13%
12%
14%
11%
13%
6%
5%
4%
8%
3%
5%
9%
8%
5%

CALUX
(ngTEQ/10g)
12.00
18.00
0.10
210.00
1.00
6.10
17.00
8.60
4.60
34.00
2.90
3.00
2.20
1.10
1.30
130.00
16.00
0.10
20.90
280.00102.0071.00
200.00
0.012
0.034
60.00
0.10
32.00
4.00
0.30
0.28
2.70
6.00
0.12
15.00
0.74
0.60

GCMS
(ngTEQ/10g)
2.80
2.20
0.012
19.00
0.22
1.20
1.50
1.20
0.85
5.00
0.68
0.39
0.23
0.23
0.28
10.00
2.70
0.0038
2.00
20.00
6.20
8.10
26.00
0.00350.0061
3.10
0.019
3.30
0.53
0.074
0.035
0.36
1.40
0.012
1.10
0.13
0.11

Revision 1
July 2014

TABLE 13
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR EXHAUST EXTRACTS

Company
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia
Company-1 Asia

SampleTEQ-ppt
MatrixSample ID mean(pg/sample) std error
Exhaust Extracts
G16581.26622.42
Exhaust Extracts
G7
75.53
4.38
Exhaust Extracts
G821436.70
829.11
Exhaust Extracts
G9548320.73
29207.76
Exhaust Extracts
G10
605.72
42.02
Exhaust Extracts
G17457.44
13.64
Exhaust Extracts
G18
46.06
1.64
Exhaust Extracts
G195875.83
390.38
Exhaust Extracts
G208164.75
269.70Exhaust Extracts
G241728.17115.02
Exhaust Extracts
G25
2009.68
120.16
Exhaust Extracts
G36
1981.81132.78
Exhaust Extracts
G37
19927.99
1227.69
Exhaust Extracts
G38
34.94
3.79
Exhaust Extracts
G40
533.58
15.08
Exhaust Extracts
G418002.56539.02
Exhaust Extracts
G422410.17178.49Exhaust Extracts
G432958.76
252.68
Exhaust Extracts
G481477.56
20.27
Exhaust Extracts
G50
212.27
12.76
Exhaust Extracts
G51
2465.49
141.12
Exhaust Extracts
G58
173086.48
9011.13
Exhaust Extracts
G62
34.30
10.53
Exhaust Extracts
G66
191.14
26.82
Exhaust Extracts
G671186.26
9.92
Exhaust Extracts
G7036803.65
1739.43
Exhaust Extracts
G774548.33
389.91
Exhaust Extracts
G804457.20350.10Exhaust Extracts
G91
21.57
2.46
Exhaust Extracts
G962381.29240.40Exhaust Extracts
G973941.68302.96
Exhaust Extracts
G98
219387.37
16146.81
Exhaust Extracts
G99
2886.31176.12
Exhaust Extracts
G100
1361.66111.82
Exhaust Extracts
G105
66.99
4.26
Exhaust Extracts
G107
19.87
1.52
Exhaust Extracts
G109
34.32
3.21
Exhaust Extracts
G113
33.91
4.52
Exhaust Extracts
G119
255.6034.76
Exhaust Extracts
G120
329.13
17.24
Exhaust Extracts
G121
746.96
55.48
Exhaust Extracts
G1291478.34
158.49Exhaust Extracts
G131
804.38
42.69
Exhaust Extracts
G1321263.64
66.85
Exhaust Extracts
G133
1359.57
144.41
Exhaust Extracts
G1342162.18
99.76
Exhaust Extracts
G135
1563.29
74.61
Exhaust Extracts
G136
1170.80
36.58

SW-846 Update V

4435 - 33

Percent
std error
9%
6%
4%
5%
7%
3%
4%
7%
3%
7%
6%
7%
6%
11%
3%
7%
7%
9%
1%
6%
6%
5%
31%
14%
0.84%
5%
9%
8%
11%
10%
8%
7%
6%
8%
6%
8%
9%
13%
14%
5%
7%
11%
5%
5%
11%
5%
5%
3%

CALUX
(ngTEQ/M3)
16.00
0.29
68.50
2900.00
1.50
3.70
0.15
17.00
24.00
6.10
7.80
6.20
100.00
0.115
1.90
30.00
7.60
10.80
3.60
1.50
7.60
800.00
0.10
0.75
9.00
300.00
29.60
16.00
0.30
7.70
20.00
900.00
16.00
7.10
0.23
0.032
0.13
0.117
1.60
0.87
2.80
8.60
4.20
4.20
4.80
7.00
7.30
6.70

GCMS
(ngTEQ/M3)
3.3
0.036
9.20
210.00
0.30
0.28
0.012
2.30
3.50
0.75
1.10
0.65
8.30
0.0089
0.38
5.30
0.90
1.40
0.66
0.19
0.83
44.00
0.017
0.11
1.90
38.00
4.60
4.40
0.0032
0.90
3.50
69.00
2.40
0.97
0.023
0.00045
0.013
0.014
0.30
0.21
0.80
2.40
0.95
1.10
0.88
2.40
2.80
1.60

Revision 1
July 2014

TABLE 14
COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR SOLUTION SAMPLES

Company
Company-4 Europe
Company-4 Europe
Company-4 Europe

SW-846 Update V

SampleMatrixsolutionsolutionsolution

TEQ-ppt
Sample ID mean (pg/ul) std error
F
448.34
N/A
H
8.39
N/A
Standard Solution L
381.0035.044435 - 34

Percent
std error
N/A
N/A
9%

GC/MS
(pg/ul)229.673.34
160.33

Revision 1
July 2014

FIGURE 1
SCHEMATIC REPRESENTATION OF THE BIOCHEMICAL PROCESSES INVOLVED INDIOXIN TOXICITY AND TEQ ESTIMATIONS BY METHOD 4435

SW-846 Update V

4435 - 35

Revision 1
July 2014

FIGURE 2
LAKE SEDIMENT REFERENCE SAMPLE QC CHART FOR 2003
XDS Solid QC Scatter Chart(2003)
90
80
70

pg/g TEQ

60
50
40
30
20
10
0
-10
0

50

100

150

200

# of analysis

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 36

Revision 1
July 2014

FIGURE 3
LAKE SEDIMENT REFERENCE SAMPLE QC CHART FOR 2004
XDS Solid QC Scatter Chart(2004)
50
45
40

pg/g TEQ

35
30
25
20
15
10
5
0
0

10

20

30

40

50

60

70

80

# of analysis

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 37

Revision 1
July 2014

FIGURE 4
LAKE SEDIMENT REFERENCE SAMPLE QC CHART SPIKED WITH 30 pg/g 2,3,7,8-TCDD

XDS Spiked Solid QC Scatter Chart(30ppt 2378 TCDD)
80
70

pg/g TEQ

60
50
40
30
20
10
0
0

10

20

30

40

50

60

70

80

# of analysis

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 38

Revision 1
July 2014

FIGURE 5
QC CHARTS FOR DIOXIN/FURAN STANDARD MIXTURE FOR 2003

Mix DX Standard QC(2003)
200%
180%
160%

% Ratio

140%
120%
100%
80%
60%
40%
20%
0%
0

20

40

60

80

100

120

140

160

Number of assays

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 39

Revision 1
July 2014

FIGURE 6
QC CHARTS FOR DIOXIN/FURAN STANDARD MIXTURE FOR 2004

Mix DX Standard QC(2004)
140%
120%% Ratio

100%

80%
60%

40%
20%

0%
0

20

40

60

80

100

120

Number of assays

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 40

Revision 1
July 2014

FIGURE 7
QC CHARTS FOR PCB 126 STANDARD FOR 2003

PCB 126 Standard QC(2003)
180%
160%
140%

% Ratio

120%
100%
80%
60%
40%
20%
0%
0

20

40

60

80

100

120

140

160

Number of assays

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 41

Revision 1
July 2014

FIGURE 8
QC CHARTS FOR PCB 126 STANDARD FOR 2004

PCB 126 Standard QC(2004)
200%
180%
160%

% Ratio

140%
120%
100%
80%
60%
40%
20%
0%
0

20

40

60

80

100

120

Number of assays

The middle line is the mean and the two outer lines are 2 standard deviations from the mean.
The dots represent the individual assays.

SW-846 Update V

4435 - 42

Revision 1
July 2014

FIGURE 9
2,3,7,8-TCDD STANDARD CURVES WITH ERROR BARS FOR 2003

2378 TCDD Control Standard(2003)
12000
10000

RLU

8000
6000
4000
2000
0
1.00E-02

1.00E-01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

pg/tubeSW-846 Update V

4435 - 43

Revision 1
July 2014

FIGURE 10
2,3,7,8-TCDD STANDARD CURVES WITH ERROR BARS FOR 2004

SW-846 Update V

4435 - 44

Revision 1
July 2014

FIGURE 11
TEMPLATE FOR 96 WELL PLATE
2

3

4

5

6

7

8

9

10

11

20 hour exposure

A

B

C

D

E

F

2,3,7,8 TCDD

2,3,7,8 TCDD

Negative Control

XDS-1

XDS-3

XDS QC-1

XDS-1

XDS-3

XDS-1

XDS-3

1.00E+02

1.56E+00

DMSO

1:10

1:10

1:10

1:100

1:100

1:500

1:500

TCDD

TCDD

control

Soil

Soil

Matrix QC sample

Soil

Soil

Soil

Soil

13853

2066

1

980

7573

341

269

4679

237

2136

2,3,7,8 TCDD

2,3,7,8 TCDD

Negative Control

XDS-1

XDS-3

XDS QC-2

XDS-1

XDS-3

XDS-1

XDS-3

5.00E+01

7.81E-01

No DMSO

1:10

1:10

1:10

1:100

1:100

1:500

1:500

TCDD

TCDD

control

Soil

Soil

Martix spike sample

Soil

Soil

Soil

Soil

13047

898

1

1040

7293

3750

263

3846

251

1998

2,3,7,8 TCDD

2,3,7,8 TCDD

Postive Control

XDS-1

XDS-3

XDS QC-3

XDS-1

XDS-3

XDS-1

XDS-3

2.50E+01

3.91E-01

High-level PCB-QC

1:10

1:10

1:10

1:100

1:100

1:500

1:500

TCDD

TCDD

control

Soil

Soil

Soil QC sample

Soil

Soil

Soil

Soil

11673

436

8081

1363

8254

4208

375

4106

345

2123

2,3,7,8 TCDD

2,3,7,8 TCDD

Postive Control

XDS-2

XDS-4

XDS-5

XDS-2

XDS-4

XDS-2

XDS-4

1.25E+01

1.95E-01

High-level DX-QC

1:10

1:10

1:10

1:100

1:100

1:500

1:500

TCDD

TCDD

control

Soil

Soil

Method Blank

Soil

Soil

Soil

Soil

9382

112

8010

3863

5829

452

1588

2954

532

1834

2,3,7,8 TCDD

2,3,7,8 TCDD

Negative Control

XDS-2

XDS-4

XDS-6

XDS-2

XDS-4

XDS-2

XDS-4

6.25E+00

9.77E-02

DMSO

1:10

1:10

1:10

1:100

1:100

1:500

1:500

TCDD

TCDD

control

Soil

Soil

Method Blank

Soil

Soil

Soil

Soil

6480

43

1

4174

6419

398

1533

3026

863

1642

XDS-2

XDS-4

XDS-7

XDS-2

XDS-4

XDS-2

XDS-4

1:10

1:10

1:10

1:100

1:100

1:500

1:500

2,3,7,8 TCDD

G

Negative Control Negative Control

3.13E+00

DMSO

DMSO

TCDD

control

control

Soil

Soil

Method Blank

Soil

Soil

Soil

Soil

3819

1

1

4422

5719

583

1824

3212

825

1358

H

Includes 2,3,7,8-TCDD standard curve, 4 DMSO controls, 1 No DMSO control, 2 positive control
QC points; and samples (including THE MANUFACTURER I.D.#; sample dilution; matrix (orclient I.D.#); and RLU result)

SW-846 Update V

4435 - 45

Revision 1
July 2014

FIGURE 12
RAW DATA FROM THE LUMINOMETER, CORRECTED BY BACKGROUND DMSOSUBTRACTION

Raw Data

Table 1
A
B
C
D
E
F
G
H
1

14620

2833

1110

1747

8340

1108

1036

5446

1004

2903

13814

1665

936

1807

8060

4517

1030

4613

1018

2765

12440

1203

8848

2131

9021

4975

1142

4873

1112

2890

10149

879

8777

4630

6596

1219

2355

3721

1300

2601

7247

810

849

4941

7186

1166

2300

3793

1630

2409

4586

726

726

5189

6486

1350

2591

3979

1592

2125

2

3

4

5

6

7

8

9

10

11

Averages
Raw
Data - Blank

Table 2
A
B
C
D
E
F
G
H
1

12

13853

2066

1

980

7573

341

269

4679

237

2136

13047

898

1

1040

7293

3750

263

3846

251

1998

11673

436

8081

1363

8254

4208

375

4106

345

2123

9382

112

8010

3863

5829

452

1588

2954

532

1834

6480

43

1

4174

6419

398

1533

3026

863

1642

3819

1

1

4422

5719

583

1824

3212

825

1358

2

3

4
5
6
Average background

8

9

10

11

7
767

12

Refer to template (Figure 11) for well descriptions.

SW-846 Update V

4435 - 46

Revision 1
July 2014

FIGURE 13BACKGROUND CORRECTED CALCULATIONS USING THE RLU DATA (FIGURE 12) TO
PRODUCE THE RESULTS FOR THE REPORT (FIGURE 14), USING THE 4 PARAMETERHILL EQUATION
THE MODEL:RLU = (v*(d^n))/(d^n + k^n)

RLU

'd' is the natural logarithm of TCDD concentration"v" is the limiting value of the RLU response as TCDD concentration increases"k" is the dose at which the response is 50% of maximum"n" is a parameter that determines sigmoidal shape of curve"b" is the interecept parameter
Initial Values (replaced with final estimates by 'Solver')k
8.9922483
n9.8105504v
15042.549
b
100.78584
To obtain predicted Ln(TCDD) concentrations fromTo fit standard curve, paste TCDD and RLU data
observed RLU data, paste observed RLU values
into the framed columns below. Then select 'Solver;
into the framed cells below. These are yourfrom the Tools menu. Be sure that the 'Target Cell'
predicted concentrations based on the standard curvemin ,is set to D43 , the 'Equal To' option is set to
B8:B11and the 'By Changing Cells' option is set toObserved Predicted
Response Ln(TCDD)
pg
TCDD
Ln(TCDD) RLU
980.11
6.77386 0.874681
100000.00
11.51
13853.18
1039.78
6.822278 0.918074
50000.00
10.82
13046.78
1363.447.048033 1.150593
25000.00
10.1311672.91
3862.518.039923 3.102374
12500.00
9.43
9381.714174.318.128638 3.390179
6250.00
8.74
6480.184421.78
8.196694 3.628932
3125.00
8.05
3818.587572.58
8.980196 7.944188
1562.507.35
2066.24
7293.318.912427 7.423655
781.25
6.66
897.588253.98
9.147964 9.395293
390.635.97
435.785828.58
8.557404 5.205153
195.315.27
111.846418.848.701257 6.010462
97.66
4.58
42.98
5718.648.530309
5.06601
341.115.908263 0.368066
Pred
Res
Res^2
3749.98
8.006966 3.001795
13919.75
66.58
4432.264208.18
8.138066 3.422292
13036.99
-9.78
95.73
451.846.145653 0.466684
11568.32-104.5910939.57
398.446.040917 0.420278
9357.55-24.16583.75582.716.353161 0.574305
6580.49
100.31
10061.56
268.71
5.693441 0.296914
3887.5768.99
4759.79
263.245.674053 0.291212
1936.94-129.30
16719.11
374.645.988836
0.39895
853.06-44.52
1981.92
1588.18
7.178723 1.311232
365.49-70.284939.971533.117.148216 1.271835
180.6168.76
4728.28
1824.387.300454 1.480972
120.9077.93
6072.35
4679.318.265665 3.888056
6.53E+04
3845.648.035017 3.087192
4105.58
8.109386 3.325534
2953.787.754997 2.333203
Raw Data and Predicted Curve3026.317.779599 2.391317
3211.647.84077 2.542161
237.11
5.572525 0.263098
16000
250.985.628357 0.278205
14000
344.515.916868 0.371247
532.44
6.280019 0.533799
12000
862.51
6.669828
0.78826
10000
825.186.633984 0.760506
8000
2136.317.443251 1.708295
1998.317.382232 1.607173
6000
2122.91
7.437461 1.698432
4000
1833.517.304898 1.487569
1641.58
7.207478 1.349485
2000
1357.787.044507 1.146544
0
0.00
#NUM!
#NUM!
0.00
#NUM!
#NUM!
4
6
8
10
12
0.00
#NUM!
#NUM!
Ln(TCDD)
0.00
#NUM!
#NUM!
0.00
#NUM!
#NUM!

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FIGURE 14
REPORT PRODUCED FROM THE RAW DATA AND CALCULATIONS (FIGURES 12 AND 13)

RLU
13853
13047
11673
9382
6480
3819
2066
898
436
112
43

2,3,7,8-TCDD Standard

16000

High Level
Low Level

14000
12000

RLU

pg/tube1.00E+02
5.00E+01
2.50E+01
1.25E+01
6.25E+00
3.13E+00
1.56E+00
7.81E-01
3.91E-01
1.95E-01
9.77E-02

High Level
Low Level

10000
8000

PCB QC
8081

6.25pg
6480

Ratio
0.80

DX QC
8010

6.25pg
6480

Ratio
0.81

DMSO
726
1110
849
726

6000
4000
2000
0
4.00E+00

6.00E+00

8.00E+00

1.00E+01

mean
853

1.20E+01

Ln(TCDD)
Sampleidentity
XDS-1
Soil
XDS-1
Soil
XDS-1
Soil
XDS-2
Soil
XDS-2
Soil
XDS-2
Soil
XDS-3
Soil
XDS-3
Soil
XDS-3
Soil
XDS-4
Soil
XDS-4
Soil
XDS-4
Soil
XDS QC-1 Matrix QC sample
XDS QC-2 Martix spike sample
XDS QC-3 Soil QC sample
Method Blank
XDS-5
Method Blank
XDS-6
Method Blank
XDS-7
XDS-1
Soil
XDS-1
Soil
XDS-1
Soil
XDS-2
Soil
XDS-2
Soil
XDS-2
Soil
XDS-3
Soil
XDS-3
Soil
XDS-3
Soil
XDS-4
Soil
XDS-4
Soil
XDS-4
Soil
XDS-1
Soil
XDS-1
Soil
XDS-1
Soil
XDS-2
Soil
XDS-2
Soil
XDS-2
Soil
XDS-3
Soil
XDS-3
Soil
XDS-3
Soil
XDS-4
Soil
XDS-4
Soil
XDS-4
Soil

fraction
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:10
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:100
1:500
1:500
1:500
1:500
1:500
1:500
1:500
1:500
1:500
1:500
1:500
1:500

RLU
TEQ, pg/tube
ppt/sample correctedmean
980 =calculation!J19-AVERAGE(E$34:E$36) =E19/(2/10) =F19/(67/100)
3.69
1040
0.43
2.15
3.22
1363
0.66
3.32
4.95
3863
2.62
6.54
9.76
10.77
4174
2.90
7.26
10.83
4422
3.14
7.85
11.72
7573
7.46
18.64
27.82
28.98
7293
6.94
17.34
25.88
8254
8.91
22.27
33.24
5829
4.72
11.80
17.60
18.43
6419
5.52
13.81
20.61
5719
4.58
11.45
17.09
341
-0.12
3750
2.51
43.81
4208
2.94
29.35
452
0.47
398
0.42
583
0.57
269
0.30
14.85
22.16
24.55
263
0.29
14.56
21.73
375
0.40
19.95
29.77
1588
1.31
32.78
48.93
50.55
1533
1.27
31.80
47.46
1824
1.48
37.02
55.26
4679
3.89
97.20
145.08128.12
3846
3.09
77.18
115.19
4106
3.33
83.14
124.09
2954
2.33
58.33
87.06
90.38
3026
2.39
59.78
89.23
3212
2.54
63.55
94.86
237
0.26
65.77
98.17
113.50
251
0.28
69.55
103.81
345
0.37
92.81
138.53
532
0.53
66.72
99.59
129.51
863
0.79
98.53
147.06
825
0.76
95.06
141.892136
1.71
213.54318.71
311.811998
1.61
200.90299.852123
1.70
212.30316.87
1834
1.49
185.95277.53247.741642
1.35
168.69251.771358
1.15
143.32213.91

std error% error
0.64
17%

0.57

5%

2.20

8%

1.10

6%

2.61

11%

2.39

5%

8.86

7%

2.32

3%

12.62

11%

15.04

12%

6.01

2%

18.48

7%

The formula under “TEQ pg/tube” is the TEQ calculation using the data from the calculation(Figure 13). The formula under “pg/g/sample” takes into account the weight (or volume) of thesample and the dilution the sample was analyzed at. The formula under “corrected” takes into
account the percent recovery. The corrected values are then averaged for a “mean” and thestandard error and percent error are calculated to determine precision.

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July 2014

FIGURE 15
PRE-FIELD TEST COMPARISON FOR DIOXIN / FURANCALUX by XDS pg/g

100000
10000

Pre-SITE Soil Samples(PCDD/PCDF)
R 2 = 0.942

1000
100
10
1
0.1
1.00E-02

1.00E+00

1.00E+02

1.00E+04

GCMS pg/g

Represents the dioxin / furan TEQ comparison of Method 4435 to GC/MS for soil
samples for pre-field testing during the EPA SITE field study. These samples were
processed in March of 2004 using the screening technique with a surrogate recovery.
These TEQs were calculated using the WHO 1997 REP values.

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FIGURE 16
PRE-FIELD TEST COMPARISON FOR PCBs

Pre-SITE Soil Samples(PCB)
CALUX by XDS pg/g

10000010000

R2 = 0.8105

1000
100
10
1
0.1
1.00E-02

1.00E+00

1.00E+02
GCMS pg/g

1.00E+04

1.00E+06

Represents the PCB TEQ comparison of the Method 4435 to GC/MS for soil samples for prefield testing during the EPA SITE field study. These samples were processed in March of 2004
using the screening technique with a surrogate recovery. These TEQs were calculated using
the WHO 1997 REP values.

SW-846 Update V

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July 2014

FIGURE 17
PRE-FIELD TEST COMPARISON FOR INCUBATOR TO AIRTIGHT BOX
Incubator vs Airtight box
1000
2

R = 0.9945

Incubator pg/g

100

10

1

0.1
1.00E-01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

Airtight box pg/g

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FIGURE 18
SOIL SAMPLE DATA COMPARISON

Soil Samples
10000000CALUX pg/g

1000000

R2 = 0.9522

100000
10000
1000
100
10
1
0.1
1.00E-01

1.00E+01

1.00E+03

1.00E+05

1.00E+07

GCMS pg/g

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FIGURE 19
ASH SAMPLE DATA COMPARISONCALUX by XDS pg/g

Ash Samples
10000010000

R2 = 0.9811

1000
100
10
1
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05
GCMS pg/g

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FIGURE 20
EXHAUST EXTRACT SAMPLE DATA COMPARISON

Exhaust Extracts

CALUX by XDS ng TEQ/M3

10000
1000

R2 = 0.9373

100
10
1
0.1
0.01
0.001
1.00E-04

1.00E-02

1.00E+00

1.00E+02

1.00E+04

GCMS ng TEQ/M3

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FIGURE 21
SOIL EXTRACT SAMPLE DATA COMPARISON

Soil Extracts
1000.00CALUX by XDS
pgTEQ/10g

R 2 = 0.9723
100.0010.00

1.00
1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03
GCMS pgTEQ/10g

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FIGURE 22
ASH EXTRACT SAMPLE DATA COMPARISONAsh Extracts

CALUX by XDS
ngTEQ/10g

1000
100

R 2 = 0.9686

10
1
0.1
0.01
1.00E-03

1.00E-02

1.00E-01

1.00E+00 1.00E+01 1.00E+02

GCM S ngTEQ/10g

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FIGURE 23
STANDARD SOLUTION SAMPLES DATA COMPARISON

Standard Solutions
CALUX by XDS pg/ul

1000.00
R 2 = 0.9984
100.0010.00

1.00
1.00E+00

1.00E+01

1.00E+02

1.00E+03

GCM S pg/ul

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FIGURE 24
ALL SAMPLE MATRICES DATA COMPARISON

Total PCDD / PCDFCALUX by XDS TEQ/martix

10000000
2
1000000
R = 0.9631
100000
10000
1000
100
10
1
0.1
0.01
0.001
1.00E-04
1.00E-02

1.00E+00

1.00E+02

1.00E+04

1.00E+06

GCMS TEQ/matrix

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Appendix A:
Summary of Revisions to Method 4435 (as compared to previous Revision 0, February 2008)
1. Improved overall method formatting for consistency with new SW-846 methods style
guidance. The format was updated to Microsoft Word .docx.
2. Minor editorial and technical revisions were made throughout to improve method clarity.
3. The revision number was changed to 1 and the date published was changed to July
2014.
4. This appendix was added showing changes from the previous revision.
5. Section 9.2.1 was added to discuss the need for initial demonstration of proficiency(IDP).

SW-846 Update V

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Revision 1
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