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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 are 

formally 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 analysis 

of 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 this 

document. 

 1.1 This method is a bio-analytical screening procedure for dioxin-like compounds in 

soils and sediments. This method is based on the ability of dioxin and related chemicals to 

activate 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 these 

chemicals 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 the 

following 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 the 

chemical-activated luciferase expression or CALUX®

 assay. The most widely studied class of 

compounds 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 biological 

potency of many PCDH compounds are quantified and expressed relative to that of 2,3,7,8-

TCDD, since this is one of the most potent activators of AhR-mediated effects, including gene 

transcription. 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 chlorinated 

dioxins/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-like 

chemicals and the basis of the CALUX®

 bioassay measurement and estimate of TEQ is shown 

in 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 stably 

transfected 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 high 

molecular weight polynuclear aromatic hydrocarbons (PAHs) with the dose-dependent induction 

of 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 a 

means of quantifying for risk assessment purposes the toxicity of a family of chemicals with a 

similar 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 the 

molecule, 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 the 

most 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 detail 

in 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 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 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 step 

 This 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 an 

estimate 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-like 

 chemicals 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 the 

disclaimer statement at the front of the manual and the information in Chapter Two for guidance 

on the responsibilities of the analyst for demonstrating that the techniques employed are 

appropriate 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 with 

organic/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 is 

critical 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 extract 

that 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 the 

amount 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 the 

semi-quantitative mode. The screening mode involves the analysis of a single aliquot of the 

sample and recovery is estimated from the mean of historical recoveries that have been 

obtained for soils/sediment samples. This is considered to be acceptable as the variability of 

recoveries for soils/sediment samples has been relatively small (76.2 +/- 8.5%). Using this 

mode would indicate whether a sample needed to be further analyzed by either the semi-
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quantitative mode or by chemical analysis. The screening mode surrogate recovery involves 

processing two aliquots of the sample, the first for analysis in the DIPS-CALUX bioassay and 

the 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 the 

clean-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 gel 

column. In addition, chrysene, benzo(b)fluoranthene, indeno(1,2,3-cd)pyrene and 

acenaphthylene 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 and 

should 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 other 

halogenated (i.e., brominated and fluorinated) dibenzo-p-dioxins/furans and biphenyls, and 

halogenated 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 2,3,7,8-

TCDD do not measure these structurally similar halogenated dibenzodioxins/furans and 

biphenyls and this may be one of the primary reasons that estimates of TEQ of dioxin-like 

chemicals 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 be 

cytotoxic. The manufacturer has generally found that the sample processing procedures used 

in this method result in removal of most potentially cytotoxic compounds. The manufacturer as 

a standard operating procedure monitors the cells after chemical treatment to determine 

whether cytotoxicity has occurred and which could lead to potential false negative results for 

dioxin-like TEQ. Cell toxicity is identified if cells are found to be detached from the culture plate

or whether they exhibit a major change in morphology. Generally, both cell toxicity and 

chemical 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 components 

that 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 for 

more 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 file 

of 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 the 

reagents and avoid contact with eyes, skin and mucous membranes. All waste materials and 

solutions should be placed in appropriate containers and disposed of according to all governing 

state 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 a 

comprehensive 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 a 

comprehensive 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 and 

may be specified in the regulation or project planning document that requires compliance 

monitoring 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 of 

samples 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 

oC – 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. 

 Samples 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 work 

need 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 and 

systematic 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 to 

document the quality of the data generated. All data sheets and quality control data should be 

maintained 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 data 

of acceptable accuracy and precision for target analytes in a clean matrix. At a 

minimum, 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 a 

demonstration 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 less

than 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 or 

column 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 for 

a standard solution of PCB 126 and a mixture of the 17 dioxin/furan congeners (those 

congeners assigned TEF values for dioxin-like activity by the WHO) that are analyzed on each 

plate (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, the 

data 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 are 

reported as a ratio relative to the 6.25 pg/g point of the 2,3,7,8-TCDD standard curve (near 

middle 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 are 

reanalyzed. Figures 5 and 6 depict the QC charts for the PCDD/PCDF standard mixture for 

2003 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 of 

dioxin 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 least 

squares best fit. Estimation of TEQ values for sample extracts are conducted based on the 

derived 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, are 

reanalyzed 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 with 

either congeners that are radioactively labeled, or with a known amount of an equimolar mixture 

of the unlabeled 17 congeners (see Figure 1). Using the radioactively labeled spike, the 

recovery is the percent of the recovered spike versus the amount added to the sample as 

determined 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 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 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 pg of 2,3,7,8-TCDD is determined from the relative light unit limits of 

detection 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 that 

type of sample. 

 9.9 Standards, 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 and 

standardization 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, 2 

additional QC samples are added to estimate recovery. The dioxin and PCB QC points are 

compared 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-analytical 

methods for dioxins/furans and PCBs. Bioassays do not differentiate between isotopically 

labeled and unlabeled analytes. Therefore, recovery determinations in bioassays can be 

accomplished 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 of 

chemicals, 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 is 

differentially 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- 2,3,7,8-

TCDD 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-like 

chemicals 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 storage 

of the mouse H1L6.1c3 recombinant cell line; preparation of 96-well plates, extraction and 

cleanup 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. 

 11.1.2 Cell 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, and 
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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. 

 11.1.3 Thawing 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 a 

 water bath or an incubator at 37°C until thawed. The cell and freezing media mixture is 

 then transferred to a 50-mL centrifuge tube and 10 to 20 mL of RPMI 1640 is added and 

 centrifuged at 2000 rpm for 10 min. The media is removed from the cells and 5 mL of 

 fresh RPMI 1640 is added. 

 The cell pellet is disrupted by repeated pipetting in the media and placed in a 

25cm2

 culture flask for growth. 

 11.1.4 Growth and splitting cells 

 The cells are first grown in 25-cm2

 flasks to 80% confluence before transfer to a 

 75-cm2 flask, which is also grown to 80% confluence. The media is removed from the 

 flasks 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. 

 11.1.5 Counting cells 

 After trypsinizing and centrifugation of the cells, the cells are counted using a 

hemocytometer. 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 hemocytometer 

for 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 volume 

of 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: 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. 

 11.2 Preparation for testing 

 First 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 
SW-846 Update V 4435 - 11 Revision 1 

 October 2012 

and time of plating and cell concentration. Incubate plate(s) at 37°C in an atmosphere 

supplemented 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) A stock solution of 50 ng/mL 2,3,7,8-TCDD is used to 

 generate an eleven point standard curve. 

 2) Place 4 μL of dimethyl sulfoxide (DMSO) to each 

 of the eleven test tubes. 

 3) 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). 

 4) Transfer 4 μL of the solution in the first tube to the 

 second test tube and mix using a vortex. 

 5) Continue the process of producing two fold dilutions until 

 you have added 4 μL of the mixture in tube 10 to 

 tube 11. Mix tube eleven, and discard 4 μL of the 

 mixture from tube 11. 

 6) This produces a 2,3,7,8-TCDD standard curve composed 

 of 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) Add 4 μL of DMSO to 13-mm tube. 

 2) Add an appropriate sample amount. 

 3) If sample is less than 1 mL, add hexane to sample until 

 1 mL is reached. 

 4) Concentrate samples for 6 minutes, then an additional two 

 minutes to remove any remaining solvent. 

 5) Add 400 μL of RPMI 1640 cell culture medium 

 (supplemented with 8% fetal calf serum and 1% 

 penicillin/streptomycin solution to each sample tube and 

 vortex for 15 sec. 

 11.2.4 Dosing plate 

 1) 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. 

 2) Place a piece of absorbent paper in the bio-hood. 

 Remove plate lid and invert the plate and tap it on paper to 

 remove medium. 

 3) After visually inspecting the plate, add 200 μL of sample to 

be tested to each well. When adding the sample, place 

the tip of the pipettor on the wall of the well and pipette 

slowly. This is done so that the cells layer is not disturbed. 

 4) Once all samples have been added, record date/time on 

 plate 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. 
SW-846 Update V 4435 - 12 Revision 1 

 October 2012 

 11.2.5 Cell lysis and luciferase activity measurement 

 After the appropriate incubation time (see Sec. 11.2.1) the plates are removed from 

the 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, 50 

μL of luciferase substrate (luciferin) is automatically injected into each well, the mixture is 

allowed 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 (when 

possible) 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, and 

another 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 with 

toluene. Each extraction of sample with solvent is incubated in an ultrasonic water bath 

for approximately 10 minutes. The three extracts from each sample are filtered, pooled 

and concentrated by vacuum centrifugation. The sample extract is then suspended in 

hexane 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. These 

chemical classes can be differentially eluted first with a mixture of hexane, toluene and 

ethyl 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 is 

concentrated 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 of 

hexane (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 
SW-846 Update V 4435 - 13 Revision 1 

 October 2012 

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 and 

any other AhR active chemicals in the sample extract. To calculate the estimate of PAH, the 

sample 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 (“raw 

results”) at the completion of the Standard Luciferase Assay. This Excel worksheet then 

graphs 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 from 

induced 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 the 

photomultiplier 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). 

 12.1.3 Standard 2,3,7,8-TCDD curve 

 A standard curve for 2,3,7,8-TCDD is included in each experiment. Refer to 

 Sec. 11.2.2 for solution makeup (see also Figures 11 - 14). 
SW-846 Update V 4435 - 14 Revision 1 

 October 2012 

 12.1.4 Four parameter Hill Equation formula 

 Results are calculated using TEQ values for the sample based on a least 

 squares best fit of the standard curve of 2,3,7,8-tetrachlorodibenzo-p-dioxin (see 

 Sec.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 the 

compound 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 or 

slowly increase with increased concentration until a threshold is reached. At this point the 

dose 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 a 

regression analysis with the concentration of 2,3,7,8-TCDD plotted versus relative light 

units (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 of 

concentrations. In order to make a good estimate of the EC50 it would be preferable to 

have at least four data points within this linear region (2 or 3-fold dilution series) (see 

Figures 13 and 14). 

 12.2.3 Calculating EC50 

 The EC50 for a sample is calculated using Microsoft Forecast Function. First, the 

midpoint 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 plotted 

versus 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) (see 

Figures 11 - 14). 
SW-846 Update V 4435 - 15 Revision 1 

 October 2012 

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 users 

of 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 this 

method. 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/sediment 

and 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 (see 

Figures 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 airtight 

box 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 in 

a 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 and 

Figure 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 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. 

 13.6 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 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 
SW-846 Update V 4435 - 16 Revision 1 

 October 2012 

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 with 

a 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 data 

table 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 data 

table 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 each 

individual 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 the 

data 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 and 

company. 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 each 

individual 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 
SW-846 Update V 4435 - 17 Revision 1 

 October 2012 

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 each 

individual 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 for 

all 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 the 

adjusted 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 is 

shown, 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 for 

all 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 the 

quantity 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 of 

environmental 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 the 

source, 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 and 

produces minimal waste pollution. This method can be used and is currently used in 
SW-846 Update V 4435 - 18 Revision 1 

 October 2012 

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 like 

compounds. GLP and OSHA guidelines are followed at all times to prevent any pollution of the 

environment. 

 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 MANAGEMENT 

 The 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 from 

hoods 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 Along 

 the 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. 
SW-846 Update V 4435 - 19 Revision 1 

 October 2012 

8. Clark, G., V. Garry, et al. (2002). "Relationships between exposure to dioxin-like 

 chemicals, 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 in 

 detection 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 Detection 

 of 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 aromatic 

 hydrocarbons, 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 the 

 detection 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 detection 

 of 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. 
SW-846 Update V 4435 - 20 Revision 1 

 October 2012 

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 and 

 Organochlorine 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 CERTIFIED 

 ENVIRONMENTAL REFERENCE MATERIALS USING CALUXTM ASSAY AND HIGH 

 RESOLUTION 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 Belgian 

 Federal 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., and 

 Eckert, 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 - 21 Revision 1 

 October 2012 

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 aromatic 

 hydrocarbons, 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 polyaromatic 

 compounds.” 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 for 

 Environmental 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 Report 

from the Office of Water and Office of Science and Technology, Engineering and 

Analysis 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 - 22 Revision 1 

 October 2012 

TABLE 1 

WHO TEF AND METHOD 4435 REP VALUES 

Compound WHO TEF CALUX REP REP Range

Based on EC50 Values (EC20 - EC50)

Dioxins

TCDD 1 1.00 +/- 0.01

12378-PeCDD 1 0.73 +/- 0.10 0.44 to 1.02

123478-HxCDD 0.1 0.075 +/- 0.014 0.034 to 0.137

123678-HxCDD 0.1 0.098 +/- 0.017 0.043 to 0.183

123789-HxCDD 0.1 0.061 +/- 0.012 0.028 to 0.114

1234678-HpCDD 0.01 0.031 +/- 0.008 0.015 to 0.058

OCDD 0.0001 0.00034 +/- 0.00008 0.00025 to 0.00049

Furans

2378-TCDF 0.1 0.067 +/- 0.010 0.040 to 0.104

12378-PeCDF 0.05 0.14 +/- 0.04 0.14 to 0.15

23478-PeCDF 0.5 0.58 +/- 0.08 0.37 to 0.78

123478-HxCDF 0.1 0.13 +/- 0.02 0.07 to 0.20

123678-HxCDF 0.1 0.14 +/- 0.03 0.10 to 0.19

123789-HxCDF 0.1 0.11 +/- 0.02 0.05 to 0.18

234678-HxCDF 0.1 0.31 +/- 0.06 0.31 to 0.31

1234678-HpCDF 0.01 0.024 +/- 0.007 0.019 to 0.031

1234789-HpCDF 0.01 0.044 +/- 0.010 0.032 to 0.059

OCDF 0.0001 0.0016 +/- 0.0005 0.0003 to 0.0058

PCBs

PCB 77 0.0005 0.0014 +/- 0.0004 0.0012 to 0.0017

PCB 81 0.0001 0.0045 +/- 0.0012 0.0022 to 0.0085

PCB 114 0.0005 0.00014 +/- 0.00002 0.00014 to 0.00017

PCB 126 0.1 0.038 +/- 0.007 0.037 to 0.042

PCB 156 0.0005 0.00014 +/- 0.00002 0.00013 to 0.00019

PCB 169 0.01 0.0011 +/- 0.0003 0.0007 to 0.0017
SW-846 Update V 4435 - 23 Revision 1 

 October 2012 

TABLE 2 

CHEMICAL INTERFERENCES 

Compound Acid silica only Dioxin fraction PCB fraction 

% Recovery % Recovery % Recovery 

benzo(a)pyrene 0.002% 0.02% ND 

chrysene 28% 0.2% ND 

acenaphthylene ND 0.2% ND 

benzo(a)anthracene 0.05% 0.005% ND 

benzo(b)fluoranthene 26% 5.7% <0.001% 

benzo(k)fluoranthene <0.001% <0.001% ND 

creosote ND ND ND 

p-cresol ND ND ND 

dibenzo(a,h)anthracene <0.001% ND ND 

1,2-diphenylhydrazine ND ND ND 

Fluorene ND ND ND 

ideno(1,2,3 cd)pyrene 0.6% 2.5% 0.005% 

perylene ND ND ND 

2-phenylindole ND ND ND 

o-cresol ND ND ND 

tryptamine ND ND 3% 
SW-846 Update V 4435 - 24 Revision 1 

 October 2012 

TABLE 3 

RECOMMENDED EQUIPMENT AND SUPPLIES 

Supply

Item Supplier Cat # Substitution

Micropipettor, 0.5-10 μL range VWR 40000-200 or equivalent

Micropipetor 2-20 ml range VWR 40000-202 or equivalent

Micropipettor, 20-200 μL range VWR 40000-204 or equivalent

Micropipetor 100 Š 1000 ml range VWR 40000-208 or equivalent

Multipipettor, repeating - syringe type Fisher 21-380-8 or equivalent

EDP2, 10-100ul Electronic Pipette VWR E2-100 EDP2 or equivalent

Drummond diaphragm pipettor VWR 53498-708 or equivalent

10 ml pippet tips Denville Scientific P-1095-CP or equivalent

200 ml pippet tips Denville Scientific P4114 or equivalent

1000 ml pippet tips Denville Scientific P4026 or equivalent

1.0 ml multipipettor syringes, case of 100 Fisher 21-381-337 or equivalent

10.0 ml multipipettor syringes, case of 100 Fisher 21-381-340 or equivalent

10 ml sterile pipettes, plastic, case of 200 Denville Scientific P1096-CP or equivalent

10 ml graduated glass pippets VWR 53283-776 or equivalent

25 cm2 Tisue Culture Flasks VWR 15708-096 or equivalent

75 cm2 Tissue Culture Flasks VWR 29186-080 or equivalent

scintillation vials, case of 500 Fisher 03-340-129 or equivalent

scintillation vial caps, teflon liner, case of 500 Fisher 03-340-131 or equivalent

25 ml drying tubes, case of 100 VWR 17453-142 or equivalent

10 ml drying tubes, case of 200 VWR 17453-140 or equivalent

13 x 100 mm Test Tubes VWR 60825-414 or equivalent

16 x 125mm Test Tubes VWR 60825-630 or equivalent

50 ml centrifuge Tubes VWR 21020-695 or equivalent

15 ml plastic centrifuge tubes, sterile Denville C-1018 or equivalent

50 ml plastic centri. Tubes VWR 21008-951 or equivalent

13mm test tube racks Fisher 14-809-22 or equivalent

13mm test tube racks for dosing Fisher 14-810-54A or equivalent

16 mm test tube racks Fisher 14-809-24 or equivalent

50 ml test tube racks Fisher 14-809-28 or equivalent

9" Pasteur pipettes VWR 53283-915 or equivalent

pipette bulbs, 2 ml capacity, pack of 72 VWR 56311-062 or equivalent

96 well plates VWR 29444-010 or equivalent

Backing Tape Perkin Elmer 6005199 or equivalent

latex gloves Marsh Bio L6003PF or equivalent

glass wool, 8 micron Fisher 11-388 or equivalent

Benchtop paper, 2 rolls of 20" x 300" VWR 14672-200 or equivalent

Tubing 12mm, CS-25lb VWR 32814-227 or equivalent
SW-846 Update V 4435 - 25 Revision 1 

 October 2012 

TABLE 3 

(cont.) 

RECOMMENDED EQUIPMENT AND SUPPLIES 

Equipment

Item name Supplier Cat # Substitution

Class II biological safety hood and stand Fisher 16-108-99 or equivalent

Water Jacketed Cell culture incubator, with CO2 and temp. 

control Fisher 11-689-4 or equivalent

Centrifuge, low speed, tabletop

with swinging bucket rotor

Centrifuge concentrator with vacuum pump with cold trap Fisher 16-315-45 or equivalent

Shaker for 96 well plates Fisher 14-271-9 or equivalent

Microscope, inverted Fisher 12-561-INV or equivalent

Microscope Fisher 12-561-3M or equivalent

Hemocytometer, cell counter Fisher 02-671-5 or equivalent

Hand tally counter Fisher 07-905-6 or equivalent

Vortex – mixer Fisher 12-814 or equivalent

sonicating water bath Fisher 15-335-30 or equivalent

Vacuum pump with liquid trap (side arm erylenmeyer 

flask) Fisher 01-092-29 or equivalent

Refrigerator/freezer Fisher 13-986-106A or equivalent

-70 celcius freezer Fisher 13-989-187 or equivalent

Liquid Nitrogen dewar Fisher 11-675-92 or equivalent

Luminometer Berthold and dedicated computer Berthold or equivalent

Combustion test kit, CO2 monitoring Fisher 10-884-1 or equivalent

250oC Oven VWR or equivalent

Autoclave VWR 58618-009 or equivalent

High-Capacity top loading Balance 0.01g-4200g VWR 14216-518 or equivalent

Fisher 04-978-50 or equivalent
SW-846 Update V 4435 - 26 Revision 1 

 October 2012 

TABLE 4 

RECOMMENDED REAGENTS AND STANDARDS 

Solvent / Reagent Item Supplier Cat # Substitution

Phosphate buffered saline 1x VWR 45000-446 or equivalent

Trypsin VWR MT 25-054-C1 or equivalent

pen/strep solution VWR 45000-650 or equivalent

Fetal Calf serum Atlanta Biological S11150 or equivalent

RPMI 1640 1X (MOD.) with L-Glutamine medium VWR 45000-396 or equivalent

Cell Culture Lysis Reagent Promega E1531 or equivalent

Luciferase Substrate Solution Promega E1501 or equivalent

70 % ethanol, for cleaning and as coolant for cold trap VWR VW3609-4 or equivalent

Sodium hydroxide VWR JT3722-4 or equivalent

DMSO Sigma-Aldrich 27,043-1 or equivalent

Sulfuric acid, concentrated, 2.5 L, ACS reagent Fisher A300-212 or equivalent

Hexane, optima grade, case of 4-4L bottles Fisher BJ216-4 or equivalent

Toluene, optima grade, case of 4-4L bottles Fisher T291-4LC or equivalent

Methanol, case of 4-4L bottles Fisher A454-4 or equivalent

Acetone, case of 4-4L bottles Fisher A929-4 or equivalent

Ethyl acetate VWR JT9282-3 or equivalent
SW-846 Update V 4435 - 27 Revision 1 

 October 2012 

TABLE 5 

EXAMPLE UNLABELED 17 CONGENERS INTERNAL SPIKE RECOVERIES 

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

Sample 

1234-TCDD by 

GC/ECD 

14C 2,3,7,8-TCDD by 

scintillation counting 

1 76.3% ± 1.1% 91.2% ± 0.9% 

2 95.4% ± 1.7% 84.1% ± 2.9% 

3 91.6% ± 1.8% 89.9% ± 2.4% 

4 90.6% ± 0.3% 83.5% ± 3.4% 

Spike sample

Sample TEQ minus sample pg % Recovery

spiked sample 4.65 1.28 80%

unspiked sample 3.37 - -

equimolar spike 1.61 - -

spiked sample 10.52 8.66 84%

unspiked sample 1.86 - -

equimolar spike 10.28 - -

spiked sample 5.22 5.05 72%

unspiked sample 0.17 - -

equimolar spike 6.98 - -

spiked sample 11.09 10.82 94%

unspiked sample 0.27 - -

equimolar spike 11.45 - -
SW-846 Update V 4435 - 28 Revision 1 

 October 2012 

TCDD

Concentrations Picograms/Tube

pg/ml

250 100

125 50

62.5 25

31.25 12.5

15.625 6.25

7.81 3.13

3.91 1.56

1.95 0.78

0.977 0.391

0.488 0.195

0.244 0.098

TABLE 7 

EXAMPLE STANDARD CURVE CONCENTRATIONS 

TABLE 8 

FIELD TEST COMPARISON of Standard INCUBATOR TO AIRTIGHT BOX

XDS Incubator Airtight box

ID # PCDD / PCDF PCDD / PCDF

Site Sample #1 0.13 0.14

Site Sample #2 46.27 55.58

Site Sample #3 0.13 0.15

Site Sample #4 0.13 0.13

Site Sample #5 523.29 711.11

Site Sample #6 169.79 184.15

Site Sample #7 0.34 0.58

Site Sample #8 97.57 86.67

Site Sample #9 193.02 349.01

Site Sample #10 70.56 142.44

Site Sample #11 95.05 97.51

Site Sample #12 0.56 0.75

TEQ-pg/g
SW-846 Update V 4435 - 29 Revision 1 

 October 2012 

TABLE 9 

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

Sample TEQ-ppt Percent GC/MS

Company Matrix Sample ID mean (pg/g) std error std error (pg/g)

Company-2 Asia Soil Soil-1 2.56 0.30 12% 0.19

Company-2 Asia Soil Soil-2 28.33 3.09 11% 3.80

Company-2 Asia Soil Soil-3 25.63 3.47 14% 12.00

Company-2 Asia Soil Soil-4 352.32 30.53 9% 120.00

Company-2 Asia Soil Soil-5 4034.97 339.88 8% 800.00

Company-2 Asia Soil Soil-6 7306.87 1021.26 14% 2600.00

Company-2 Asia Soil Soil-7 20539.55 4661.64 23% 1200.00

Company-3 USA Soil 17100 A, B 207.51 28.56 14% 63.90

Company-3 USA Soil 17101 A, B 12869.85 1057.71 8% 4852.00

Company-3 USA Soil 17102 A, B 306.05 19.80 6% 84.20

Company-3 USA Soil 17103 A, B 539543.83 42835.93 8% 94330.00

Company-3 USA Soil 17105 A, B 441485.41 79369.82 18% 63113.00

Company-3 USA Soil 17106 A, B 637162.72 29247.40 5% 44411.00

Company-3 USA Soil 17107 A, B 394642.48 19514.21 5% 55581.00

Company-3 USA Soil 17108 A, B 2078549.92 80574.11 4% 333638.00

Company-3 USA Soil 17109 A, B 25574.14 154.39 1% 8547.00

Company-3 USA Soil 17118 A, B 272858.59 2423.72 1% 98560.00

Company-3 USA Soil 17120 A, B 7469.31 299.10 4% 1398.00

Company-3 USA Soil 17126 A, B 790.04 85.95 11% 207.00

Company-3 USA Soil 17128 A, B, C, D, E, F 191.58 10.11 5% 65.00

Company-3 USA Soil 17130 A, B 260510.86 2495.27 1% 10891.00

Company-3 USA Soil 17131 A, B 885605.65 83917.76 9% 115539.00

Company-3 USA Soil 17132 A, B 823636.59 93127.53 11% 115451.00

Company-3 USA Soil 17134 A, B 392784.46 468.45 0% 116792.00

Company-3 USA Soil 17138 A, B 6703.40 172.76 3% 2337.00

Company-3 USA Soil 17149 A, B 459.40 23.40 5% 89.50

Company-1 Asia Soil Soil Sample 1 416.00 32.13 8% 150.00

Company-1 Asia Soil Soil Sample 2 5.65 0.01 0% 1.20

Company-1 Asia Soil Sample No. 2 soil/1185-2 27.87 3.13 11% 7.90

Company-1 Asia Soil Sample No. 3 Soil/ 1185-3 14.41 2.17 15% 7.90

Company-1 Asia Soil Sample No. 4, Soil./ 1185-4 39.90 6.60 17% 21.00

Company-1 Asia Soil Sample No. 6, Soil./ 1185-6 16.98 2.85 17% 2.40

Company-1 Asia Soil Sample No. 7, Soil./ 1185-7 7.67 1.19 16% 6.00

Company-1 Asia Soil Sample No. 8, Soil./ 1185-8 9.98 1.47 15% 6.00

Company-1 Asia Soil Soil Sample 1. 9.05 0.78 9% 0.21

Company-1 Asia Soil Soil Sample 3. 15.22 2.36 15% 0.31

Company-1 Asia Soil Soil Sample 4. 21.82 0.06 0% 3.30

Company-1 Asia Soil Soil Sample 5 4699.31 171.10 4% 4000.00

Company-1 Asia Soil Soil Sample 6 6725.04 525.67 8% 5900.00

Company-1 Asia Soil Soil Sample 14. 24.04 0.43 2% 3.80

Company-4 Europe Soil A 160.00 0.01 0.00% 17.67

Company-4 Europe Soil C 600.00 0.07 0.01% 180.00

Company-4 Europe Soil D 1061.00 0.09 0.01% 323.33

Company-4 Europe Soil B 154.00 0.04 0.02% 19.67

Company-4 Europe Soil C 570.00 0.11 0.02% 496.67

Company-5 USA Soil XDSG 16.28 0.62 4% 8.79

Company-5 USA Soil XDSG30 37.24 1.39 4% 38.83
SW-846 Update V 4435 - 30 Revision 1 

 October 2012 

TABLE 10 

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

Sample TEQ-ppt Percent CALUX GCMS

Company Matrix Sample ID mean (pg/g) std error std error (pgTEQ/10g) (pgTEQ/10g)

Company-1 Asia Soil Extract S-74 115.02 16.01 14% 120.00 23.00

Company-1 Asia Soil Extract S-121 74.50 10.37 14% 76.00 6.80

Company-1 Asia Soil Extract S-16 12.23 3.40 28% 12.00 0.92

Company-1 Asia Soil Extract S-61 522.67 73.34 14% 540.00 110.00

Company-1 Asia Soil Extract S-119 55.27 7.56 14% 55.00 5.40

Company-1 Asia Soil Extract S-54 104.50 17.76 17% 110.00 25.00

Company-1 Asia Soil Extract S-120 176.43 30.43 17% 180.00 15.00

Company-1 Asia Soil Extract S-132 847.00 157.42 19% 860.00 150.00

Company-1 Asia Soil Extract S-43 1.21 0.17 14% 1.00 0.07

Company-1 Asia Soil Extract S-17 2.70 0.52 19% 3.00 0.11

Company-1 Asia Soil Extract S-49 89.67 10.74 12% 92.00 12.00

Company-1 Asia Soil Extract S-53 345.28 22.20 6% 360.00 87.00

Company-1 Asia Soil Extract S-18 8.90 0.61 7% 9.00 0.39

Company-1 Asia Soil Extract S-55 82.48 8.22 10% 84.00 8.20

Company-1 Asia Soil Extract S-28 313.50 37.88 12% 310.00 42.00

Company-1 Asia Soil Extract S-42 98.96 10.41 11% 103.00 12.00

Company-1 Asia Soil Extract S-127 682.20 67.87 10% 680.00 110.00

Company-1 Asia Soil Extract S-19 5.97 0.73 12% 6.00 0.26

Company-1 Asia Soil Extract S-7 93.94 12.16 13% 160.00 17.00

Company-1 Asia Soil Extract S-52 102.10 10.46 10% 105.00 18.00

Company-1 Asia Soil Extract S-75 316.10 18.96 6% 320.00 21.00
SW-846 Update V 4435 - 31 Revision 1 

 October 2012 

TABLE 11 

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

Company 

Sample 

Matrix Sample ID 

TEQ-ppt 

mean (pg/g) std error 

Percent 

std error

GC/MS 

(pg/g) 

Company-2 Asia Ash Ash-1 7.34 1.89 26% 2.80 

Company-2 Asia Ash Ash-2 105.70 27.01 26% 60.00 

Company-2 Asia Ash Ash-3 584.55 61.40 11% 400.00 

Company-2 Asia Ash Ash-5 52795.74 9307.38 18% 27000.00 

Company-1-Asia Ash 

Sample No. 9, 

Ash, 1185-9 402.60 14.84 4% 360.00 

Company-1-Asia Ash 

Sample No.10, 

Ash/1185-10 684.47 38.78 6% 470.00 

Company-1-Asia Ash Ash Sample 7 142.83 14.06 10% 89.00 

Company-1-Asia Ash Ash Sample 8 260.60 33.29 13% 71.00 

Company-1-Asia Ash Ash Sample 9 19870.12 983.14 5% 7000.00 

Company-1-Asia Ash Ash Sample 11 1598.46 73.54 5% 1100.00 

Company-4 Europe Ash B 36.00 0.00 0% 32.00 

Company-4 Europe Ash C 523.00 0.02 0% 390.00 

Company-4 Europe Ash B 63240.00 9.11 0% 57500.00 

Company-4 Europe Ash C 545.00 0.04 0% 285.33 
SW-846 Update V 4435 - 32 Revision 1 

 October 2012 

TABLE 12 

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

Sample TEQ-ppt Percent CALUX GCMS

Company Matrix Sample ID mean (pg/g) std error std error (ngTEQ/10g) (ngTEQ/10g)

Company-1 Asia Ash Extract A-20 12721.95 1048.59 8% 12.00 2.80

Company-1 Asia Ash Extract A-17 28261.60 4132.32 15% 18.00 2.20

Company-1 Asia Ash Extract A-23 84.69 5.55 7% 0.10 0.012

Company-1 Asia Ash Extract A-40 193299.28 20703.51 11% 210.00 19.00

Company-1 Asia Ash Extract A-18 1442.06 84.06 6% 1.00 0.22

Company-1 Asia Ash Extract A-66 5862.67 736.10 13% 6.10 1.20

Company-1 Asia Ash Extract A-57 16871.73 1857.26 11% 17.00 1.50

Company-1 Asia Ash Extract A-43 7896.12 334.81 4% 8.60 1.20

Company-1 Asia Ash Extract A-42 4525.55 457.10 10% 4.60 0.85

Company-1 Asia Ash Extract A-50 48462.10 5962.94 12% 34.00 5.00

Company-1 Asia Ash Extract A-56 4646.37 333.91 7% 2.90 0.68

Company-1 Asia Ash Extract A-2 2443.32 188.29 8% 3.00 0.39

Company-1 Asia Ash Extract A'-2 2192.71 143.06 7% 2.20 0.23

Company-1 Asia Ash Extract A'-1 1844.48 187.46 10% 1.10 0.23

Company-1 Asia Ash Extract A'-5 1273.59 114.65 9% 1.30 0.28

Company-1 Asia Ash Extract A-13 128781.60 13106.95 10% 130.00 10.00

Company-1 Asia Ash Extract A-19 22838.28 1799.11 8% 16.00 2.70

Company-1 Asia Ash Extract A-9 60.94 5.96 10% 0.10 0.0038

Company-1 Asia Ash Extract A-59 6812.57 902.82 13% 20.90 2.00

Company-1 Asia Ash Extract A-60 92283.85 12021.15 13% 280.00 20.00

Company-1 Asia Ash Extract A-28 77824.56 4318.94 6% 102.00 6.20

Company-1 Asia Ash Extract A-41 103465.03 15583.39 15% 71.00 8.10

Company-1 Asia Ash Extract A-34 327501.35 27583.75 8% 200.00 26.00

Company-1 Asia Ash Extract A-61 20.66 2.61 13% 0.012 0.0035

Company-1 Asia Ash Extract A-27 56.60 6.85 12% 0.034 0.0061

Company-1 Asia Ash Extract A-45 60374.36 8614.39 14% 60.00 3.10

Company-1 Asia Ash Extract A-14 164.08 17.35 11% 0.10 0.019

Company-1 Asia Ash Extract A-16 47848.74 6204.24 13% 32.00 3.30

Company-1 Asia Ash Extract A-44 3985.64 239.54 6% 4.00 0.53

Company-1 Asia Ash Extract A-75 512.53 24.61 5% 0.30 0.074

Company-1 Asia Ash Extract A-65 458.24 19.23 4% 0.28 0.035

Company-1 Asia Ash Extract A-15 2630.39 200.54 8% 2.70 0.36

Company-1 Asia Ash Extract A-58 5399.43 168.09 3% 6.00 1.40

Company-1 Asia Ash Extract A-48 201.53 11.01 5% 0.12 0.012

Company-1 Asia Ash Extract A-8 14904.29 1335.97 9% 15.00 1.10

Company-1 Asia Ash Extract A-74 781.28 61.79 8% 0.74 0.13

Company-1 Asia Ash Extract A-31 975.80 47.11 5% 0.60 0.11
SW-846 Update V 4435 - 33 Revision 1 

 October 2012 

TABLE 13 

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

Sample TEQ-ppt Percent CALUX GCMS

Company Matrix Sample ID mean(pg/sample) std error std error (ngTEQ/M3) (ngTEQ/M3)

Company-1 Asia Exhaust Extracts G1 6581.26 622.42 9% 16.00 3.3

Company-1 Asia Exhaust Extracts G7 75.53 4.38 6% 0.29 0.036

Company-1 Asia Exhaust Extracts G8 21436.70 829.11 4% 68.50 9.20

Company-1 Asia Exhaust Extracts G9 548320.73 29207.76 5% 2900.00 210.00

Company-1 Asia Exhaust Extracts G10 605.72 42.02 7% 1.50 0.30

Company-1 Asia Exhaust Extracts G17 457.44 13.64 3% 3.70 0.28

Company-1 Asia Exhaust Extracts G18 46.06 1.64 4% 0.15 0.012

Company-1 Asia Exhaust Extracts G19 5875.83 390.38 7% 17.00 2.30

Company-1 Asia Exhaust Extracts G20 8164.75 269.70 3% 24.00 3.50

Company-1 Asia Exhaust Extracts G24 1728.17 115.02 7% 6.10 0.75

Company-1 Asia Exhaust Extracts G25 2009.68 120.16 6% 7.80 1.10

Company-1 Asia Exhaust Extracts G36 1981.81 132.78 7% 6.20 0.65

Company-1 Asia Exhaust Extracts G37 19927.99 1227.69 6% 100.00 8.30

Company-1 Asia Exhaust Extracts G38 34.94 3.79 11% 0.115 0.0089

Company-1 Asia Exhaust Extracts G40 533.58 15.08 3% 1.90 0.38

Company-1 Asia Exhaust Extracts G41 8002.56 539.02 7% 30.00 5.30

Company-1 Asia Exhaust Extracts G42 2410.17 178.49 7% 7.60 0.90

Company-1 Asia Exhaust Extracts G43 2958.76 252.68 9% 10.80 1.40

Company-1 Asia Exhaust Extracts G48 1477.56 20.27 1% 3.60 0.66

Company-1 Asia Exhaust Extracts G50 212.27 12.76 6% 1.50 0.19

Company-1 Asia Exhaust Extracts G51 2465.49 141.12 6% 7.60 0.83

Company-1 Asia Exhaust Extracts G58 173086.48 9011.13 5% 800.00 44.00

Company-1 Asia Exhaust Extracts G62 34.30 10.53 31% 0.10 0.017

Company-1 Asia Exhaust Extracts G66 191.14 26.82 14% 0.75 0.11

Company-1 Asia Exhaust Extracts G67 1186.26 9.92 0.84% 9.00 1.90

Company-1 Asia Exhaust Extracts G70 36803.65 1739.43 5% 300.00 38.00

Company-1 Asia Exhaust Extracts G77 4548.33 389.91 9% 29.60 4.60

Company-1 Asia Exhaust Extracts G80 4457.20 350.10 8% 16.00 4.40

Company-1 Asia Exhaust Extracts G91 21.57 2.46 11% 0.30 0.0032

Company-1 Asia Exhaust Extracts G96 2381.29 240.40 10% 7.70 0.90

Company-1 Asia Exhaust Extracts G97 3941.68 302.96 8% 20.00 3.50

Company-1 Asia Exhaust Extracts G98 219387.37 16146.81 7% 900.00 69.00

Company-1 Asia Exhaust Extracts G99 2886.31 176.12 6% 16.00 2.40

Company-1 Asia Exhaust Extracts G100 1361.66 111.82 8% 7.10 0.97

Company-1 Asia Exhaust Extracts G105 66.99 4.26 6% 0.23 0.023

Company-1 Asia Exhaust Extracts G107 19.87 1.52 8% 0.032 0.00045

Company-1 Asia Exhaust Extracts G109 34.32 3.21 9% 0.13 0.013

Company-1 Asia Exhaust Extracts G113 33.91 4.52 13% 0.117 0.014

Company-1 Asia Exhaust Extracts G119 255.60 34.76 14% 1.60 0.30

Company-1 Asia Exhaust Extracts G120 329.13 17.24 5% 0.87 0.21

Company-1 Asia Exhaust Extracts G121 746.96 55.48 7% 2.80 0.80

Company-1 Asia Exhaust Extracts G129 1478.34 158.49 11% 8.60 2.40

Company-1 Asia Exhaust Extracts G131 804.38 42.69 5% 4.20 0.95

Company-1 Asia Exhaust Extracts G132 1263.64 66.85 5% 4.20 1.10

Company-1 Asia Exhaust Extracts G133 1359.57 144.41 11% 4.80 0.88

Company-1 Asia Exhaust Extracts G134 2162.18 99.76 5% 7.00 2.40

Company-1 Asia Exhaust Extracts G135 1563.29 74.61 5% 7.30 2.80

Company-1 Asia Exhaust Extracts G136 1170.80 36.58 3% 6.70 1.60
SW-846 Update V 4435 - 34 Revision 1 

 October 2012 

TABLE 14 

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

Sample TEQ-ppt Percent GC/MS

Company Matrix Sample ID mean (pg/ul) std error std error (pg/ul)

Company-4 Europe solution F 448.34 N/A N/A 229.67

Company-4 Europe solution H 8.39 N/A N/A 3.34

Company-4 Europe solution Standard Solution L 381.00 35.04 9% 160.33
SW-846 Update V 4435 - 35 Revision 1 

 October 2012 

FIGURE 1 

SCHEMATIC REPRESENTATION OF THE BIOCHEMICAL PROCESSES INVOLVED IN 

DIOXIN TOXICITY AND TEQ ESTIMATIONS BY METHOD 4435 
SW-846 Update V 4435 - 36 Revision 1 

 October 2012 

FIGURE 2 

LAKE SEDIMENT REFERENCE SAMPLE QC CHART FOR 2003 

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

The dots represent the individual assays. 

XDS Solid QC Scatter Chart

(2003)

-10

0

10

20

30

40

50

60

70

80

90

0 50 100 150 200

# of analysis

pg/g TEQ
SW-846 Update V 4435 - 37 Revision 1 

 October 2012 

FIGURE 3 

LAKE SEDIMENT REFERENCE SAMPLE QC CHART FOR 2004 

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

The dots represent the individual assays. 

XDS Solid QC Scatter Chart

(2004) 

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70 80

# of analysis

pg/g TEQ
SW-846 Update V 4435 - 38 Revision 1 

 October 2012 

FIGURE 4 

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

 

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

The dots represent the individual assays. 

XDS Spiked Solid QC Scatter Chart

(30ppt 2378 TCDD)

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80

# of analysis

pg/g TEQ
SW-846 Update V 4435 - 39 Revision 1 

 October 2012 

FIGURE 5 

QC CHARTS FOR DIOXIN/FURAN STANDARD MIXTURE FOR 2003 

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

The dots represent the individual assays. 

Mix DX Standard QC

(2003)

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

200%

0 20 40 60 80 100 120 140 160

Number of assays

% Ratio
SW-846 Update V 4435 - 40 Revision 1 

 October 2012 

FIGURE 6 

QC CHARTS FOR DIOXIN/FURAN STANDARD MIXTURE FOR 2004 

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

The dots represent the individual assays. 

Mix DX Standard QC

(2004)

0%

20%

40%

60%

80%

100%

120%

140%

0 20 40 60 80 100 120

Number of assays

% Ratio
SW-846 Update V 4435 - 41 Revision 1 

 October 2012 

FIGURE 7 

QC CHARTS FOR PCB 126 STANDARD FOR 2003 

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

The dots represent the individual assays. 

PCB 126 Standard QC

(2003)

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

0 20 40 60 80 100 120 140 160

Number of assays

% Ratio
SW-846 Update V 4435 - 42 Revision 1 

 October 2012 

FIGURE 8 

QC CHARTS FOR PCB 126 STANDARD FOR 2004 

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

The dots represent the individual assays. 

PCB 126 Standard QC

(2004)

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

200%

0 20 40 60 80 100 120

Number of assays

% Ratio
SW-846 Update V 4435 - 43 Revision 1 

 October 2012 

FIGURE 9 

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

2378 TCDD Control Standard

(2003)

0

2000

4000

6000

8000

10000

12000

1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03

pg/tube

RLU
SW-846 Update V 4435 - 44 Revision 1 

 October 2012 

FIGURE 10 

2,3,7,8-TCDD STANDARD CURVES WITH ERROR BARS FOR 2004 
SW-846 Update V 4435 - 45 Revision 1 

 October 2012 

FIGURE 11 

TEMPLATE FOR 96 WELL PLATE 

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 (or 

client I.D.#); and RLU result) 

2 3 4 5 6 7 8 9 10 11

20 hour exposure

A

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

B 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

C 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

D 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

E 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

F TCDD TCDD control Soil Soil Method Blank Soil Soil Soil Soil

6480 43 1 4174 6419 398 1533 3026 863 1642

2,3,7,8 TCDD Negative Control Negative Control XDS-2 XDS-4 XDS-7 XDS-2 XDS-4 XDS-2 XDS-4

3.13E+00 DMSO DMSO 1:10 1:10 1:10 1:100 1:100 1:500 1:500

G TCDD control control Soil Soil Method Blank Soil Soil Soil Soil

3819 1 1 4422 5719 583 1824 3212 825 1358

H
SW-846 Update V 4435 - 46 Revision 1 

 October 2012 

FIGURE 12 

RAW DATA FROM THE LUMINOMETER, CORRECTED BY BACKGROUND DMSO 

SUBTRACTION 

Refer to template (Figure 11) for well descriptions. 

Table 1

A

B 14620 2833 1110 1747 8340 1108 1036 5446 1004 2903

C 13814 1665 936 1807 8060 4517 1030 4613 1018 2765

D 12440 1203 8848 2131 9021 4975 1142 4873 1112 2890

E 10149 879 8777 4630 6596 1219 2355 3721 1300 2601

F 7247 810 849 4941 7186 1166 2300 3793 1630 2409

G 4586 726 726 5189 6486 1350 2591 3979 1592 2125

H

1 2 3 4 5 6 7 8 9 10 11 12

Table 2

A

B 13853 2066 1 980 7573 341 269 4679 237 2136

C 13047 898 1 1040 7293 3750 263 3846 251 1998

D 11673 436 8081 1363 8254 4208 375 4106 345 2123

E 9382 112 8010 3863 5829 452 1588 2954 532 1834

F 6480 43 1 4174 6419 398 1533 3026 863 1642

G 3819 1 1 4422 5719 583 1824 3212 825 1358

H

1 2 3 4 5 6 7 8 9 10 11 12

Average background 767

Averages

Raw Data

Raw Data - Blank
SW-846 Update V 4435 - 47 Revision 1 

 October 2012 

FIGURE 13 

BACKGROUND CORRECTED CALCULATIONS USING THE RLU DATA (FIGURE 12) TO 

PRODUCE THE RESULTS FOR THE REPORT (FIGURE 14), USING THE 4 PARAMETER 

HILL EQUATION 

THE MODEL:

RLU = (v*(d^n))/(d^n + k^n) '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

n 9.8105504

v 15042.549

b 100.78584

To obtain predicted Ln(TCDD) concentrations from

To 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 your

from the Tools menu. Be sure that the 'Target Cell' predicted concentrations based on the standard curve

is set to D43 , the 'Equal To' option is set to min ,

and the 'By Changing Cells' option is set to B8:B11 Observed 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 1363.44 7.048033 1.150593

25000.00 10.13 11672.91 3862.51 8.039923 3.102374

12500.00 9.43 9381.71 4174.31 8.128638 3.390179

6250.00 8.74 6480.18 4421.78 8.196694 3.628932

3125.00 8.05 3818.58 7572.58 8.980196 7.944188

1562.50 7.35 2066.24 7293.31 8.912427 7.423655

781.25 6.66 897.58 8253.98 9.147964 9.395293

390.63 5.97 435.78 5828.58 8.557404 5.205153

195.31 5.27 111.84 6418.84 8.701257 6.010462

97.66 4.58 42.98 5718.64 8.530309 5.06601

341.11 5.908263 0.368066

Pred Res Res^2 3749.98 8.006966 3.001795

13919.75 66.58 4432.26 4208.18 8.138066 3.422292

13036.99 -9.78 95.73 451.84 6.145653 0.466684

11568.32 -104.59 10939.57 398.44 6.040917 0.420278

9357.55 -24.16 583.75 582.71 6.353161 0.574305

6580.49 100.31 10061.56 268.71 5.693441 0.296914

3887.57 68.99 4759.79 263.24 5.674053 0.291212

1936.94 -129.30 16719.11 374.64 5.988836 0.39895

853.06 -44.52 1981.92 1588.18 7.178723 1.311232

365.49 -70.28 4939.97 1533.11 7.148216 1.271835

180.61 68.76 4728.28 1824.38 7.300454 1.480972

120.90 77.93 6072.35 4679.31 8.265665 3.888056

6.53E+04 3845.64 8.035017 3.087192

4105.58 8.109386 3.325534

2953.78 7.754997 2.333203

3026.31 7.779599 2.391317

3211.64 7.84077 2.542161

237.11 5.572525 0.263098

250.98 5.628357 0.278205

344.51 5.916868 0.371247

532.44 6.280019 0.533799

862.51 6.669828 0.78826

825.18 6.633984 0.760506

2136.31 7.443251 1.708295

1998.31 7.382232 1.607173

2122.91 7.437461 1.698432

1833.51 7.304898 1.487569

1641.58 7.207478 1.349485

1357.78 7.044507 1.146544

0.00 #NUM! #NUM!

0.00 #NUM! #NUM!

0.00 #NUM! #NUM!

0.00 #NUM! #NUM!

0.00 #NUM! #NUM!

Raw Data and Predicted Curve

0

2000

4000

6000

8000

10000

12000

14000

16000

4 6 8 10 12

Ln(TCDD)

RLU
SW-846 Update V 4435 - 48 Revision 1 

 October 2012 

FIGURE 14 

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

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 the 

sample 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 the 

standard error and percent error are calculated to determine precision. 

pg/tube RLU PCB QC 6.25pg Ratio

1.00E+02 13853 High Level 8081 6480 0.80

5.00E+01 13047 Low Level

2.50E+01 11673 DX QC 6.25pg Ratio

1.25E+01 9382 High Level 8010 6480 0.81

6.25E+00 6480 Low Level

3.13E+00 3819 DMSO mean

1.56E+00 2066 726 853

7.81E-01 898 1110

3.91E-01 436 849

1.95E-01 112 726

9.77E-02 43

Sample identity fraction RLU TEQ, pg/tube ppt/sample corrected mean std error % error

XDS-1 Soil 1:10 980 =calculation!J19-AVERAGE(E$34:E$36) =E19/(2/10) =F19/(67/100) 3.69 0.64 17%

XDS-1 Soil 1:10 1040 0.43 2.15 3.22

XDS-1 Soil 1:10 1363 0.66 3.32 4.95

XDS-2 Soil 1:10 3863 2.62 6.54 9.76 10.77 0.57 5%

XDS-2 Soil 1:10 4174 2.90 7.26 10.83

XDS-2 Soil 1:10 4422 3.14 7.85 11.72

XDS-3 Soil 1:10 7573 7.46 18.64 27.82 28.98 2.20 8%

XDS-3 Soil 1:10 7293 6.94 17.34 25.88

XDS-3 Soil 1:10 8254 8.91 22.27 33.24

XDS-4 Soil 1:10 5829 4.72 11.80 17.60 18.43 1.10 6%

XDS-4 Soil 1:10 6419 5.52 13.81 20.61

XDS-4 Soil 1:10 5719 4.58 11.45 17.09

XDS QC-1 Matrix QC sample 1:10 341 -0.12

XDS QC-2 Martix spike sample 1:10 3750 2.51

XDS QC-3 Soil QC sample 1:10 4208 2.94 29.35 43.81

XDS-5 Method Blank 1:10 452 0.47

XDS-6 Method Blank 1:10 398 0.42

XDS-7 Method Blank 1:10 583 0.57

XDS-1 Soil 1:100 269 0.30 14.85 22.16 24.55 2.61 11%

XDS-1 Soil 1:100 263 0.29 14.56 21.73

XDS-1 Soil 1:100 375 0.40 19.95 29.77

XDS-2 Soil 1:100 1588 1.31 32.78 48.93 50.55 2.39 5%

XDS-2 Soil 1:100 1533 1.27 31.80 47.46

XDS-2 Soil 1:100 1824 1.48 37.02 55.26

XDS-3 Soil 1:100 4679 3.89 97.20 145.08 128.12 8.86 7%

XDS-3 Soil 1:100 3846 3.09 77.18 115.19

XDS-3 Soil 1:100 4106 3.33 83.14 124.09

XDS-4 Soil 1:100 2954 2.33 58.33 87.06 90.38 2.32 3%

XDS-4 Soil 1:100 3026 2.39 59.78 89.23

XDS-4 Soil 1:100 3212 2.54 63.55 94.86

XDS-1 Soil 1:500 237 0.26 65.77 98.17 113.50 12.62 11%

XDS-1 Soil 1:500 251 0.28 69.55 103.81

XDS-1 Soil 1:500 345 0.37 92.81 138.53

XDS-2 Soil 1:500 532 0.53 66.72 99.59 129.51 15.04 12%

XDS-2 Soil 1:500 863 0.79 98.53 147.06

XDS-2 Soil 1:500 825 0.76 95.06 141.89

XDS-3 Soil 1:500 2136 1.71 213.54 318.71 311.81 6.01 2%

XDS-3 Soil 1:500 1998 1.61 200.90 299.85

XDS-3 Soil 1:500 2123 1.70 212.30 316.87

XDS-4 Soil 1:500 1834 1.49 185.95 277.53 247.74 18.48 7%

XDS-4 Soil 1:500 1642 1.35 168.69 251.77

XDS-4 Soil 1:500 1358 1.15 143.32 213.91

2,3,7,8-TCDD Standard

0

2000

4000

6000

8000

10000

12000

14000

16000

4.00E+00 6.00E+00 8.00E+00 1.00E+01 1.20E+01

Ln(TCDD)

RLU
SW-846 Update V 4435 - 49 Revision 1 

 October 2012 

FIGURE 15 

PRE-FIELD TEST COMPARISON FOR DIOXIN / FURAN 

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. 

Pre-SITE Soil Samples 

(PCDD/PCDF)

R

2

 = 0.942

0.1

1

10

100

1000

10000

100000

1.00E-02 1.00E+00 1.00E+02 1.00E+04

GCMS pg/g

CALUX by XDS pg/g
SW-846 Update V 4435 - 50 Revision 1 

 October 2012 

FIGURE 16 

PRE-FIELD TEST COMPARISON FOR PCBs 

Pre-SITE Soil Samples 

(PCB)

R

2

 = 0.8105

0.1

1

10

100

1000

10000

100000

1.00E-02 1.00E+00 1.00E+02 1.00E+04 1.00E+06

GCMS pg/g

CALUX by XDS pg/g

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 4435 - 51 Revision 1 

 October 2012 

FIGURE 17 

PRE-FIELD TEST COMPARISON FOR INCUBATOR TO AIRTIGHT BOX 

Incubator vs Airtight box

R

2

 = 0.9945

0.1

1

10

100

1000

1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03

Airtight box pg/g

Incubator pg/g
SW-846 Update V 4435 - 52 Revision 1 

 October 2012 

FIGURE 18 

SOIL SAMPLE DATA COMPARISON 

Soil Samples

R

2

 = 0.9522

0.1

1

10

100

1000

10000

100000

1000000

10000000

1.00E-01 1.00E+01 1.00E+03 1.00E+05 1.00E+07

GCMS pg/g

CALUX pg/g
SW-846 Update V 4435 - 53 Revision 1 

 October 2012 

FIGURE 19 

ASH SAMPLE DATA COMPARISON 

Ash Samples

R

2

 = 0.9811

1

10

100

1000

10000

100000

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05

GCMS pg/g

CALUX by XDS pg/g
SW-846 Update V 4435 - 54 Revision 1 

 October 2012 

FIGURE 20 

EXHAUST EXTRACT SAMPLE DATA COMPARISON 

Exhaust Extracts

R

2

 = 0.9373

0.001

0.01

0.1

1

10

100

1000

10000

1.00E-04 1.00E-02 1.00E+00 1.00E+02 1.00E+04

GCMS ng TEQ/M3

CALUX by XDS ng TEQ/M3
SW-846 Update V 4435 - 55 Revision 1 

 October 2012 

FIGURE 21 

SOIL EXTRACT SAMPLE DATA COMPARISON 

Soil Extracts

R

2

 = 0.9723

1.00

10.00

100.00

1000.00

1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02 1.00E+03

GCMS pgTEQ/10g

CALUX by XDS 

pgTEQ/10g
SW-846 Update V 4435 - 56 Revision 1 

 October 2012 

FIGURE 22 

ASH EXTRACT SAMPLE DATA COMPARISON 

Ash Extracts

R

2

 = 0.9686

0.01

0.1

1

10

100

1000

1.00E-03 1.00E-02 1.00E-01 1.00E+00 1.00E+01 1.00E+02

GCMS ngTEQ/10g

CALUX by XDS 

ngTEQ/10g
SW-846 Update V 4435 - 57 Revision 1 

 October 2012 

FIGURE 23 

STANDARD SOLUTION SAMPLES DATA COMPARISON 

 

Standard Solutions

R

2

 = 0.9984

1.00

10.00

100.00

1000.00

1.00E+00 1.00E+01 1.00E+02 1.00E+03

GCMS pg/ul

CALUX by XDS pg/ul
SW-846 Update V 4435 - 58 Revision 1 

 October 2012 

FIGURE 24 

ALL SAMPLE MATRICES DATA COMPARISON 

Total PCDD / PCDF

R

2

 = 0.9631

0.001

0.01

0.1

1

10

100

1000

10000

100000

1000000

10000000

1.00E-04 1.00E-02 1.00E+00 1.00E+02 1.00E+04 1.00E+06

GCMS TEQ/matrix

CALUX by XDS TEQ/martix
SW-846 Update V 4435 - 59 Revision 1 

 October 2012 

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 2007.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 May 

2012. 

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).