**SW-846 Update V 4435 - 1 Revision 1**

**October 2012 METHOD 4435** 

**SCREENING FOR DIOXIN-LIKE CHEMICAL ACTIVITY IN SOIL S AND SEDIMENTS** 

**USING THE CALUX ®**  **BIOASSAY AND TEQ DETERMINATIONS**

**SW-846 is not intended to be an analytical trainin g 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 o f 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 gu idance methods which contain general**  **information on how to perform an analytical procedu re or technique which a laboratory can use**  **as a basic starting point for generating its own de tailed standard operating procedure (SOP),**  **either for its own general use or for a specific pr oject application. The performance data**  **included in this method are for guidance purposes o nly, 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 se e Appendix A at the end of this** 

**document.**

**1.1 This method is a bio-analytical screening proc edure for dioxin-like compounds in** 

**soils and sediments. This method is based on the a bility of dioxin and related chemicals to**  **activate the aryl hydrocarbon receptor (AhR), a che mical-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 expre ssion by a chemical or chemical extract**  **provides a measure by which to estimate the relativ e potency and toxic potential of these**  **chemicals and/or extracts with resulting values exp ressed 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 detec tion 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 polychlo**  **rinated diaromatic hydrocarbons (PCDH),**
**such as 2,3,7,8-tetrachlorodibenzo-** p **-dioxin (2,3,7,8-TCDD). The relative toxic and bio logical**  **potency of many PCDH compounds are quantified and e xpressed relative to that of 2,3,7,8-**  **TCDD, since this is one of the most potent activato rs of AhR-mediated effects, including gene**  **transcription. This relative quantification approa ch generates overall potency values as TEQs**  **and the results obtained from using this method pro vide a measure of TEQs in a sample.** 

**1.2 By using the sample processing procedures in t his method and an affinity column**  **(Ref. 42), polychlorinated biphenyls (PCBs) can be separated from chlorinated**  **dioxins/dibenzofurans (PCDDs/PCDFs) making it possi ble 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 bioa ssay for dioxin-like chemicals.** 

**1.3 The AhR-dependent mechanism of the toxic and b iological 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 SW-846 Update V 4435 - 2 Revision 1** 

**October 2012 and other dioxin-like compounds. Once these chemic**  **als 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 seque**  **nce, the dioxin responsive element (DRE), which is present upstream from many genes in**  **cluding 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 fi**  **refly luciferase reporter gene was stably transfected into the mouse Hepa1c1c7 cell line to p**  **roduce the recombinant cell line H1L6.1c3 (6.1 cells). This transformed cell line responds t**  **o 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). Compar**  **ison of these results to a 2,3,7,8-TCDD**

**standard curve for induction allows for determinati on 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 diox ins/dibenzofurans present in the same**  **sample. Using this DIPS-CALUX ®**  **bioassay it is possible to determine the portion o f 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 t he toxicity of a family of chemicals with a**  **similar overall mechanism of toxicity (Ref. 38). T he 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 wit**  **h 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 ne eds of the project. As the sample size**  **increases, the detection limit will decrease. Samp le sizes of 2 – 10 g will typically give a**  **detection limit of less than1 pg/g. Limits of dete ction are determined based on the y-intercept**  **from the Hill Equation and the standard deviation o f 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 detecti on for the plate in picograms of 2,3,7,8-TCDD**  **is determined from the relative light unit limits o f detection using the Hill Equation. The limit of**  **detection for each sample is determined based on th e 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 quantific ation of TEQs in a sample.** 

**1.7.1 Range finding analysis, the first step**

**This step can be performed on all of the three sam ple 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 dilu tion (see Sec. 4.2). This provides an** 

**estimate of the concentration of dioxin-like chemic als 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 EC 50**  **(EC 50**  **is the equivalent concentration at SW-846 Update V 4435 - 3 Revision 1** 

**October 2012 50% of the maximum response of the 2,3,7,8-TCDD sta**  **ndard) value for the 2,3,7,8-TCDD standard curve run on each plate. This completes t**  **he 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 estim ation of the TEQ value of dioxin-like**  **chemicals present in a given sample and allows mea n and standard deviation** 

**calculations.**

**1.8 Prior to employing this method, analysts are a dvised to consult the manufacturer’s**  **instructions for additional information on quality control procedures, development of QC**  **acceptance criteria, calculations, and general guid ance. 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 demonstr ating that the techniques employed are**  **appropriate for the analytes of interest, in the ma trix of interest, and at the levels of concern.** 

**In addition, analysts and data users are advised t hat, 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 me**  **thod 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 t**  **he intended application.**

**1.9 This method is intended to be performed by tra ined 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 manufactu rer. Instructions are provided in**  **the form of standard operating procedures (SOPs), w hich are detailed and thorough. The**  **manufacturer ( http://www.dioxins.com/**  **) also provides comprehensive training which is inc luded**  **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, pipe tting small volumes, and accurately**  **weighing out samples.**

**2.0 SUMMARY OF METHOD**

**2.1 This method is a relatively rapid screening me thod 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 process ing procedure (see Sec. 11). An extract**  **that contains the halogenated dioxins/furans is sep arated 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. E stimation 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, sc reening mode surrogate recovery, and the**  **semi-quantitative mode. The screening mode involve s the analysis of a single aliquot of the**  **sample and recovery is estimated from the mean of h istorical recoveries that have been**  **obtained for soils/sediment samples. This is consi dered to be acceptable as the variability of**  **recoveries for soils/sediment samples has been rela tively small (76.2 +/- 8.5%). Using this**  **mode would indicate whether a sample needed to be f urther analyzed by either the semi-** **SW-846 Update V 4435 - 4 Revision 1**

**October 2012 quantitative mode or by chemical analysis. The scr**  **eening mode surrogate recovery involves processing two aliquots of the sample, the first fo**  **r analysis in the DIPS-CALUX bioassay and the second used for the surrogate spike with radiol**  **abeled 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 fo**  **r determination of recovery with radiolabeled 2,3,7,8-TCDD. The cost of sample analysis is depen**  **dent upon which mode of the DIPS-CALUX bioassay is used for estimation of the levels of sa**  **mple contamination.**

**3.0 DEFINITIONS**

**Refer to Chapter One and the manufacturer's instru ctions for definitions that may be**  **relevant to this procedure. In addition, see Metho d 4000 for glossaries of basic terms.** 

**4.0 INTERFERENCES**

**4.1 Chemical interference**

**The chemicals listed in Table 2 are relatively wea k agonists for the Ah receptor and induce**  **poorly. The results in the table demonstrate the p ercent recovery of each compound in the**  **clean-up method and the amount of carry-over into t he 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 al l PAHs using an acid silica gel**  **column. In addition, chrysene, benzo(b)fluoranthen e, indeno(1,2,3-cd)pyrene and**  **acenaphthylene are the only PAHs we have found to b leed through the clean-up**  **procedure with approximately 5% - 26% (see Table 2) passing through the processing**  **steps. For the analysis of samples that are expect ed to have extremely high levels of**  **PAHs, users should subject the sample to an additio nal acidic-silica cleanup step. Testing**  **the relative activity of the sample after each roun d of cleanup would reveal whether**  **additional AhR active chemicals (i.e., PAHs) are re moved with additional cleanup. Other**  **materials such as silver nitrate, florsil and alumi na can be used in additional cleanup**  **steps. However, due to their rarity in the environ ment, these additional materials are**  **rarely used.**

**4.1.2 AhR receptor interference**

**Compounds that can activate the AhR receptor coul d result in potential** 
**interference with the determination of TEQ due to d ioxins/furans and PCBs. The PAH**  **class of potential interferences do not typically p ass through the cleanup system and**  **should not be potential interfering agents. The cl eanup 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) dibe nzo-p-dioxins/furans and biphenyls, and**  **halogenated naphthalene’s and these appear to have similar toxicological properties as**  **their chlorinated derivatives. Isotope specific di lution methods using high-resolution mass**  **spectroscopy high-resolution gas chromatography (HR GC/MS) for quantifying 2,3,7,8-**  **TCDD do not measure these structurally similar halo genated dibenzodioxins/furans and**  **biphenyls and this may be one of the primary reason s that estimates of TEQ of dioxin-like**  **chemicals by this method are slightly higher than H RGC/MS generated estimates of TEQ.** **SW-846 Update V 4435 - 5 Revision 1**

**October 2012 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 th at the sample processing procedures used**  **in this method result in removal of most potentiall y cytotoxic compounds. The manufacturer as**  **a standard operating procedure monitors the cells a fter chemical treatment to determine**  **whether cytotoxicity has occurred and which could l ead to potential false negative results for**  **dioxin-like TEQ. Cell toxicity is identified if ce lls are found to be detached from the culture plate**  **or whether they exhibit a major change in morpholog y. Generally, both cell toxicity and**  **chemical insolubility can be overcome by sample dil ution. In this method, typically 6 dilutions**  **are run to reduce potential cell toxicity and overc ome 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 diluti ons, 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 wo rk 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 (MSDS s) should be available to all personnel**  **involved in these analyses.**

**5.2 Safety procedures in compliance with good labo ratory practices (GLPs) and OSHA** 

**standards should be maintained at all times. Some reagents may contain hazardous materials**  **such as solvents and acids; therefore, the technici an should use caution when using the**  **reagents and avoid contact with eyes, skin and muco us membranes. All waste materials and**  **solutions should be placed in appropriate container s and disposed of according to all governing**  **state and federal regulations.**

**5.3 This method employs the use of dilute concentr ations of 2,3,7,8-TCDD for the**  **standard curve; and quality control solutions conta in 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 sh ould 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 sup plies.** 

**7.0 REAGENTS AND STANDARDS**

**7.1 Reagent-grade chemicals must be used in all tes ts. Unless otherwise indicated, it**  **is intended that all reagents conform to the specif ications of the Committee on Analytical**  **Reagents of the American Chemical Society, where su ch specifications are available. Other**  **grades may be used, provided it is first ascertaine d 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 contamin ants from plastic containers.** 

**7.2 Refer to Table 4 and to the manufacturer’s web site (http://www.dioxins.com/) for a**  **comprehensive list of recommended reagents and stan dards.** **SW-846 Update V 4435 - 6 Revision 1**

**October 2012 8.0 SAMPLE COLLECTION AND STORAGE** 

**Sample collection, preservation, and storage requi rements may vary by EPA program and**  **may be specified in the regulation or project plann ing document that requires compliance**  **monitoring for a given contaminant. Where such req uirements are specified in the regulation,**  **follow those requirements. In the absence of speci fic regulatory requirements, use the following**  **information as guidance in determining the sample c ollection, preservation and storage**  **requirements.**

**8.1 Sample collection**

**This bioassay testing process employs very small s ample 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 manufacture r, received and logged in via the**  **manufacturer’s electronic tracking system. Tempera ture of the samples and the condition of**  **samples are noted immediately. Any instructions fr om the client are also noted. The samples**  **are then placed in the appropriate storage conditio ns 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** 

**o C – 4**  **o C depending on individual needs of the sample. Fee**  **d, 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 alread y 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. Ex tracts 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 es tablished. However, PCDDs,**  **PCDFs, and PCB have an extremely long half-life and are very stable in environmental**  **matrices. They are essentially nonvolatile. Clien ts may request specific holding times for**  **project-specific applications.**

**9.0 QUALITY CONTROL**

**9.1 Refer to Chapter One for guidance on quality a ssurance (QA) and quality control**  **(QC) protocols. When inconsistencies exist between QC guidelines, method-specific QC**  **criteria take precedence over both technique-specif ic criteria and those criteria given in Chapter**  **One, and technique-specific QC criteria take preced ence over the criteria in Chapter One. Any** **SW-846 Update V 4435 - 7 Revision 1**

**October 2012 effort involving the collection of analytical data**  **should include development of a structured and systematic planning document, such as a Quality Ass**  **urance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which translates project o**  **bjectives and specifications into directions for those that will implement the project and asses**  **s the results. Each laboratory should maintain a formal quality assurance program. The l**  **aboratory should also maintain records to document the quality of the data generated. All da**  **ta 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 1 613. 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 proficie ncy with each sample**  **preparation and determinative method combination it utilizes by generating data**  **of acceptable accuracy and precision for target ana lytes in a clean matrix. At a**  **minimum, such a demonstration will encompass the in itial demonstration of**  **proficiency described in method 3500, using a clean reference matrix. The**  **laboratory must also repeat the demonstration of pr oficiency whenever new staff**  **members are trained or significant changes in instr umentation 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 preparat ion is conducted on a regular basis.**  **Each lot of solvent is tested by evaporating a 5-mL aliquot of the solvent into 4**  μ **L of DMSO. The DMSO solution is suspended in cell culture medi**  **um 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 so**  **lvents to be used. A solvent blank is also included in each batch of samples. The solvent bla**  **nk is treated in the same way as the samples and serves as a control to monitor for cont**  **ribution of activity from any of the solvents or column matrices used in sample preparation.** 

**9.4 Reference samples were prepared from lake sedi ment materials that were finely**  **ground (solids) and analyzed twice by GC/HRMS for d ioxin TEQ (average GC/MS TEQ = 13.6**  ± **6.8 pg/g). The lake sediment material was then spi ked with 30 pg/g 2,3,7,8-TCDD to provide a**  **final dioxin TEQ concentration equivalent to the ac tion level. The reference material was**  **shaken or stirred for three days, aliquoted and an aliquot was analyzed by GC/HRMS. The un**  **spiked 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 un**  **spiked lake sediment reference sample for 2003 and 2004 respectively. Figure 4 depicts the**  **QC chart for the spiked lake sediment reference sam ple.** 

**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 activ ity 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 repor ted 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 referen ce material or either of the standard mixtures** **SW-846 Update V 4435 - 8 Revision 1**

**October 2012 differs by more than two standard deviations from t**  **he moving average or a reference material is below the limits of detection, the plate is declare**  **d invalid and all samples on the plate are reanalyzed. Figures 5 and 6 depict the QC charts f**  **or 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 metho d as a semi-quantitative estimate of** 
**dioxin TEQ**

**In addition to the quality controls used in the sc reening assay, the 2,3,7,8-TCDD standard**  **curve is modeled to a sigmoid curve described by th e four parameter Hill Equation using a least**  **squares best fit. Estimation of TEQ values for sam ple extracts are conducted based on the**  **derived Hill Equation with the following limitation s 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 limi ts, 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**  **14 C labeled**  **2,3,7,8-TCDD recoveries) is conducted using a dupli cate 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). Usin g the radioactively labeled spike, the**  **recovery is the percent of the recovered spike vers us the amount added to the sample as**  **determined by liquid scintillation. For the unlabe led 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 t he QC process please refer to Ref. 6,**  **Appendix B available from the manufacturer’s websit e (**  **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 bioa ssay. 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 stand ard deviation of the DMSO blanks. The limit of**  **detection for the plate in pg of 2,3,7,8-TCDD is de termined from the relative light unit limits of**  **detection using the Hill Equation. The limit of de tection for each sample is determined based on**  **the amount of sample used, the portion of the sampl e extract used and the recovery for that**  **type of sample.**

**9.9 Standards, reagents, solvents and any other ma terials 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 exp iration time of one year is utilized for all**  **solvents and reagents.**

**10.0 CALIBRATION AND STANDARDIZATION**

**Calibration and standardization of each assay is v ery important to ensure accuracy of any**  **measurement of any system. The following is a desc ription of the necessary calibration and**  **standardization procedures for this method. Also s ee the manufacturer’s instructions for**  **information that may be relevant. SW-846 Update V 4435 - 9 Revision 1** 

**October 2012 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 o n each 96-well plate analyzed. Each plate**  **also contains 7 additional QC calibration points co nsisting of 4 DMSO solvent controls, 1 media**  **blank QC, 1 dioxin QC standard (contains all 17 dio xin congeners recognized by the World**  **Health Organization), and 1 PCB QC standard (80.5 n g/ml PCB 126). For internal recoveries, 2**  **additional QC samples are added to estimate recover y. 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 pe rcent recovery points. This gives this method**  **20 QC calibration points.**

**10.2 Recovery determinations**

**Analyses of recovery determinations are very impor tant for quantitative bio-analytical**  **methods for dioxins/furans and PCBs. Bioassays do not differentiate between isotopically**  **labeled and unlabeled analytes. Therefore, recover y 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 dete rmine recoveries of dioxin-like chemicals.**  **Samples were spiked with 14**  **C labeled 2,3,7,8-TCDD or 1,2,3,4-TCDD and submitte d 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 capt ure detection or scintillation counting.**  **Average recoveries determined by 1,2,3,4-TCDD with paired samples spiked with**  **14 C- 2,3,7,8-**  **TCDD indicated that the recoveries determined by th e two methods were very similar, 88.5% (**  ± **1.2%) and 87.2% (** ± **2.4%), respectively (see Figure 1). Recovery dete rminations were also**  **verified by HRGC/HRMS. This procedure allows for q uantitative determination of dioxin-like**  **chemicals in various sample matrices. For a more d etailed description of the QC process refer**  **to Ref. 6, Appendix B, available from the manufactu rer’s website (**  **http://www.dioxins.com/ ).** 

**11.0 PROCEDURE**

**This section describes the following procedures us ed in this method: Growth and storage**  **of the mouse H1L6.1c3 recombinant cell line; prepar ation of 96-well plates, extraction and**  **cleanup of samples, dosing plates and analysis of t he data.** 

**CAUTION: Always use sterile techniques when working with c**  **ells.**

**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% CO**  **2 and 100% humidity. Cells are grown in plastic cell**  **culture flasks containing RPMI 1640 media supplemented with 8% fetal calf serum (FCS) a**  **nd 1% penicillin/streptomycin. No external selective pressure (i.e., Geneticin -- G41**  **8) 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** **SW-846 Update V 4435 - 10 Revision 1**

**October 2012 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 plac**  **ed in the vials, they are stored at –70°C for 24 hr and then transferred to the liquid nitrog**  **en 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 loose ned slightly to release any excess**  **gas. The cap is then tightened and the vial is th awed 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** 

**25cm 2**  **culture flask for growth.**

**11.1.4 Growth and splitting cells**

**The cells are first grown in 25-cm 2**  **flasks to 80% confluence before transfer to a**

**75-cm 2**  **flask, which is also grown to 80% confluence. The media is removed from the** 

**flasks and cells are washed with PBS. Two mL of t he 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 an d pipetted into a 50-mL centrifuge tube,**  **containing 5 mL of RPMI 1640 medium, and centrifug ed at 2000 rpm for 10 min. The** 

**cells are then transferred to two 75-cm 2**  **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 solut ion is then placed on the hemocytometer**  **for counting. Four of the quadrants on the hemocyt ometer are counted: the upper left,**  **upper right, lower right, and lower left. Determin e the average of the 4 counts. The volume**  **of each square is 10 -4**  **mL, therefore: cells/ml = (average number per larg e square) x** 

**10 4**  **/ml. x 1/(dilution). This count is used in the pla ting 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 10**  **4 )**  **= 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 pla te.** 

**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. Usin g the eppendorf multi-pipettor,** 
**pipette 200** μ **l 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. Incuba**  **te 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 standa rd 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**  μ **L of dimethyl sulfoxide (DMSO) to each**

**of the eleven test tubes. 3) Add 4**  μ **L of the 50 ng/mL 2,3,7,8-TCDD solution to the first tube and thoroughly mix (25 ng/mL sol**  **ution). 4) Transfer 4**  μ **L 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 di lutions until**  **you have added 4** μ **L of the mixture in tube 10 to**
**tube 11. Mix tube eleven, and discard 4** μ **L of the**

**mixture from tube 11. 6) This produces a 2,3,7,8-TCDD standard curve c**  **omposed**

**of the final concentrations listed in Table 7.**

**NOTE : The standard curve should be treated the same w**  **ay as the samples. The same lot of hexane used to prepare the sample extra**  **cts should also be used to**

**add 1 mL of hexane to each tube in the standard cur ve.** 

**11.2.3 Dosing cells -- Prepare samples for dosing**

**1) Add 4** μ **L of DMSO to 13-mm tube. 2) Add an appropriate sample amount.**  **3) If sample is less than 1 mL, add hexane to sa mple until** 

**1 mL is reached. 4) Concentrate samples for 6 minutes, then an ad**  **ditional two minutes to remove any remaining solvent.**  **5) Add 400** μ **L 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 b een**  **incubated for 20-24 hr, at 37 o**  **C in an atmosphere of approximately 5% carbon dioxide, from the incub**  **ator. 2) Place a piece of absorbent paper in the bio-h**  **ood. Remove plate lid and invert the plate and tap i**  **t on paper to**

**remove medium. 3) After visually inspecting the plate, add 200**  μ **L of sample to be tested to each well. When adding the sample, pl**  **ace the tip of the pipettor on the wall of the well and**  **pipette slowly. This is done so that the cells layer is no**  **t 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 measure**  **ment**

**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**  μ **L PBS. After which each cell is inspected for cell viability, noting a**  **ny damaged, morphologically changed or missing cells. White backing tape is then applied**  **to the bottom and 30** μ **L of Promega Lysis buffer is added to each well. The 96-well pl**  **ate is then shaken for 1 min before inserting the plate into the microplate luminometer**  **. For analysis of luciferase activity, 50**

μ **L of luciferase substrate (luciferin) is automatica lly injected into each well, the mixture is**  **allowed to incubate for 5 sec followed by quantitat ion of luciferase activity (light**  **production) over a 15 sec-time period. The light p roduced from the cleavage of luciferin by**  **luciferase is expressed as relative light units (RL Us). These RLUs are then analyzed**  **using a Microsoft® Excel spread sheet that calculat es the TEQ for the dioxin/furan and** 

**PCB fractions.**

**11.3 Experimental set-up, sample extraction and cl ean-up** 

**11.3.1 Experimental set up**

**Samples are first logged in to the manufacturer tr acking system and assigned**  **project numbers and individual sample numbers. Nex t the number of samples, solvent**  **blanks, QC’s and recoveries are calculated to deter mine 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 aliquot s of each sample are ground (when**  **possible) and two to ten gram aliquots are placed i n solvent cleaned glass vials fitted with**  **PTFE-lined caps. For the screening process, one al iquot of the sample is used, and**  **another aliquot is processed for recovery purpose i f historical recovery data is not used.**  **The samples are extracted with a 20% solution of me thanol 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 f rom each sample are filtered, pooled**  **and concentrated by vacuum centrifugation. The sam ple extract is then suspended in**  **hexane and prepared for the clean-up method. The c lean-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 cle an-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. T he 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 analysi s** 

**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 perform ed on the samples by diluting them (1:4;**  **1:10; 1:100; 1:500; 1:1000; and 1:10,000). For sus pected high level samples, where higher**  **chlorinated dioxin/furan insolubility may be presen t, 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 an**  **d PCBs in the sample. The sample aliquots**

**are then concentrated under vacuum into DMSO and re -suspended in 400**  μ **L of RPMI 1640 and dosed on the plated cells (see Sec. 11.2.4). T**  **he cells are incubated in a humidified CO 2**  **atmosphere for 16-24 hr and then assayed for lucife rase activity.** 

**11.5 Alternative sample extraction for detection o f 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. 1 1.3 are that there is no clean-up step**  **performed and that the bioassay is only incubated f or 4 hr instead of the standard 16-24 hrs.**  **The sample is extracted, pooled and concentrated an d then dosed without clean-up as in Sec.**  **11.3.2. This will give a total TEQ score for the P AHs in addition to the dioxins/furans, PCBs and**  **any other AhR active chemicals in the sample extrac t. 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 result s. 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 de scription 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 Microso ft® Excel spreadsheet (“raw**  **results”) at the completion of the Standard Lucifer ase 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 pres ence of the test compound, as well as,**  **contributions by background production of luciferas e and the “dark current” from the**  **photomultiplier tube. In order to identify the com ponent 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 s ample 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 EC 50**  **determination**

**The first step in determining the EC 50**  **of a standard curve is to look at the curve for th e**  **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 remai n 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 remai n linear with increasing**  **concentration. Eventually, the response should rea ch a maximum. Increasing**  **concentrations will not result in increased respons e, 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 gene rally 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 o f the EC**  **50 it would be preferable to**  **have at least four data points within this linear r egion (2 or 3-fold dilution series) (see**  **Figures 13 and 14).**

**12.2.3 Calculating EC 50** 

**The EC 50**  **for a sample is calculated using Microsoft Forecas t Function. First, the**  **midpoint of the linear portion of the standard curv e is determined. Then, the forecast**  **function is applied to the three data points surrou nding the midpoint.** 

**Excel forecast formula: FORECAST (x,known_y's,kn own_x's)** 

**12.3 Calculation of TEQ -- Least squares, best fit**

**Least squares, best fit is used to predict the bes t fit for the data using a four variable Hill**  **Equation with the natural log of the 2,3,7,8-TCDD c oncentration in femptograms/tube plotted**  **versus relative light units (RLUs).**

**The response of a sample is compared to the respon se of 2,3,7,8-TCDD, corrected for**  **dilution of the unknown and assigned a relative con centration 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 p rovided in SW-846 methods only**  **as examples and guidance. The data do not represen t required performance criteria for users**  **of the methods. Instead, performance criteria shou ld be developed on a project-specific basis,**  **and the laboratory should establish in-house QC per formance 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 accr editation.** 

**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-sponsor ed SITE study in Saginaw, MI (April**  **26 – May 1, 2004) using this method, and a mobile l ab was set up for analysis of soil/sediment**  **and extracts in the field. In addition, pre-site s oil/sediment and extracts were tested at the**  **manufacturer’s facility and the data for the pre-si te samples is included in this report (see**  **Figures 15 - 17). Ninety-six well assay plates con taining mouse 6.1 cell line were sealed at the**  **manufacturer and transported to the site in an airt ight box. Extraction and clean-up columns**  **were also pre-made at the manufacturer and transpor ted 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 use d in place of a standard humidified CO**  **2 incubator. The analyst can breathe into the specia**  **lly made valves giving the cells the gas concentrations needed, or use a 5% CO**  **2 tank if desired. The airtight box can then be pla**  **ced in a 37 °C water bath for 16 - 20 hr to control temper**  **ature 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 p**  **urposes only.**

**13.3 The following figures demonstrate the correlat ion between this method and GC/MS**  **determinations. Figures 15 – 17 represent data fro m 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 internati onal cross-lab validation study. Figure 24**  **represents the correlation determination for all sa mple matrices. These data are provided for**  **guidance purposes only.**

**13.4 Figure 15 represents the dioxin/furan TEQ com parison of this method to GC/MS for**  **soil samples for pre-field testing during the EPA S ITE field study. These samples were**  **processed in March of 2004 using the screening tech nique 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 f rom this method to GC/MS for soil**  **samples for pre-field testing during the EPA SITE f ield 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 da ta are provided for guidance purposes**  **only.**

**13.6 Figure 17 represents the dioxin/furan TEQ com parison 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 S ITE field study. These samples were**  **processed in March of 2004 using the screening tech nique 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 sa mples for pre-field testing during the**  **EPA SITE field study. This table represents the di oxin/furan TEQ comparison of this method**  **using the 37 °C water jacketed incubator to the res ults 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 u sing the WHO 1997 REP values. These**  **data are provided for guidance purposes only.**

**13.8 Figure 18 depicts the dioxin/furan TEQ correl ation between this method and**  **GC/MS for soil samples. The GC/MS data was generat ed by three different companies. See**  **Appendix A (available from the manufacturer’s websi te 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. T hese data are provided for guidance purposes**  **only.**

**13.9 Figure 19 depicts the dioxin/furan TEQ correl ation between this method and**  **GC/MS for Ash samples. The GC/MS data was generate d by three different companies. See**  **Appendix A (available from the manufacturer’s websi te 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 corre lation 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 t he manufacturer’s website**  **http://www.dioxins.com/) for the data table compari ng data from this method to GC/MS for each**  **individual sample and company. See Table 13 for in dividual sample data correlations. These**  **data are provided for guidance purposes only.**

**13.11 Figure 21 depicts the dioxin/furan TEQ corre lation 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 w ebsite 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 correlation s. These data are provided for guidance**  **purposes only.**

**13.12 Figure 22 depicts the dioxin/furan TEQ corre lation 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 manufactu rer’s website http://www.dioxins.com/) for**  **the data table comparing data from this method to G C/MS for each individual sample and**  **company. See Table 12 for individual sample data c orrelations. These data are provided for**  **guidance purposes only.**

**13.13 Figure 23 depicts the dioxin/furan TEQ corre lation between this method and**  **GC/MS for Standard Solution samples. The GC/MS dat a was generated by three different**  **companies. Please see Appendix A (available from t he manufacturer’s website**  **http://www.dioxins.com/) for the data table compari ng data from this method to GC/MS for each**  **individual sample and company. See Table 14 for in dividual sample data correlations. These**  **data are provided for guidance purposes only.**

**13.14 Figure 24 depicts the dioxin/furan TEQ corre lation 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 R**  **2 value of 0.9631. The GC/MS data was generated by**  **five different companies. See Appendix A (available from the manu**  **facturer’s website http://www.dioxins.com/) for the data table compari**  **ng data from this method to GC/MS for each individual sample and company. See Sec. 13.0 for i**  **ndividual sample data correlations. These data are provided for guidance purposes only.** 

**13.15 Table 9 depicts this method’s dioxin/furan d ata 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 Fig ure 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 d ouble 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. Origi nal data generated using this method is**  **shown, as well as, the adjusted data per cubic mete r. 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 associ ated graph. These data are provided for**  **guidance purposes only.**

**14.0 POLLUTION PREVENTION**

**14.1 Pollution prevention encompasses any techniqu e that reduces or eliminates the**  **quantity and/or toxicity of waste at the point of g eneration. Numerous opportunities for pollution**  **prevention exist in laboratory operations. The EPA has established a preferred hierarchy of**  **environmental management techniques that places pol lution prevention as the management**  **option of first choice. Whenever feasible, laborat ory 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 th at 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 Ch emical Society (ACS), Committee**  **on Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229**  **0/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 bot**  **h air and ground pollution. This is done through monitoring and testing sites suspected of b**  **eing 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 was te is contained in biohazard**  **containers and disposed of in compliance with all s tate, 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 comp ounds 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 an y pollution of the environment.** 

**15.0 WASTE MANAGEMENT**

**The Environmental Protection Agency requires that laboratory waste management**  **practices be conducted consistent with all applicab le 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 lett er and spirit of any sewer discharge permits**  **and regulations, and by complying with all solid an d hazardous waste regulations, particularly** 

**the hazardous waste identification rules and land d isposal restrictions. For further information**  **on waste management, consult the ACS publication li sted in Sec. 14.2.** 

**16.0 REFERENCES**

**1. Arrieta, D. E., Ontiveros, C., Denison, M.S., an d Washburn, B.S. (2000). "Ah Receptor**  **Mediated Effects of Particulate Organic Extracts f rom the Paso Del Morte Airshed Along**  **the U.S. - Mexico Border." Organohalogen Compounds 45: 212 - 215.** 

**2. Behnisch, P. A., Allen, R., Anderson, J., Brouwe r, A., Brown, D., Campbell, T. C.,**  **Goeyens, L., Harrison, R. O., Hoogenboom, R., Over meire, I.V., Traag, W., and Malisch,**  **R. (2001). "Harmonised Quality Criteria for Chemic al and Bioassays Analyses of** 

**PCDDs/PCDFs in Feed and Food Part 2: General Consi derations, Bioassay Methods."**  **Organohalogen Compounds 45.**

**3. Brown, D. J., M. D. Chu, et al. (2001). "Determi nation of REP Values for the CALUX**  **bioassay and comparison to the WHO TEF Values." Or ganohalogen Compounds 53:** 

**211- 214.**

**4. Brown, D. J., Goeyens, L., Van Overmeire, I., Ch u, 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 Di oxin Responsive CALUX Assay for**  **Analysis of Japanese Ash and Soil Samples." Organo halogen 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 anal ysis: Condierations and results."**  **Organohalogen Compounds 58: 357-360.**

**7. Brown, D. J., Van Overmeire, I., Goeyens, L., Ch u, M.D., Denison, M.S., and Clark, G.C.**  **(2002). "Elimination of interfering compounds in p reparation 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). "Relationshi**  **ps between exposure to dioxin-like chemicals, testosterone levels, and sex of the chi**  **ldren 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 t he 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) an d Detection System (CALUX**  **Bioassay) for Measuring Air Emissions of Dioxin, F uran, and PCB on a TEQ Basis**  **Tested With a Model Industrial Boiler." Organohalo gen 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). "Bioanaly tical approaches for the Detection**  **of Dioxin and Related Halogenated Aromatic Hydroca rbons." Organohalogen**  **Compounds 45.**

**12. Denison, M. S., Seidel, S.D., Ziccardi, M., Rog ers, W.J., Brown, D.J., and Clark, G.C.**  **(1999). "Ah receptor-based bioassays for dioxins a nd related chemicals: Applications**  **and limitations." Organohalogen Compounds 40: 27-3 0.** 

**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 Detectio n 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 diox in and related halogenated aromatic**  **hydrocarbons, in: Technology-Driven Biomarkers Dev elopment and Application in**  **Environmentally-Associated Diseases, Wilson, D. an d 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 line s as bioassay systems for the**  **detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-l ike 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 agonist s, 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 K azakhstan 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 Chlorop henoxy 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 epidemiologica l 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.-Applica tion of CALUX assay for**  **epidemiological study-." Organohalogen Compounds 5 5: 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 COMPARATIV E ANALYSIS OF CERTIFIED** 

**ENVIRONMENTAL REFERENCE MATERIALS USING CALUX TM**  **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 B ioassay: 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 Determi nation of Low TEQ Values in Milk**  **Samples." Organohalogen Compounds 45.**

**28. Overmeire, I. V., Goeyens, L., Beernaert, H., S rebrink, 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 F ood Samples by the Belgian**  **Federal Ministries of Public Health and Agricultur e." 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 Compari son 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 Pollu tants (POPs) and Immune Function in**  **US Atlantic Coast Harbor Seals." Organohalogen Com pounds 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 CALU X 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., Carbonn**  **elle, S., Overmeire, I.V., Brown, D., Clark, G., Baeyens, W., and Goeyens, L. (2003). "N**  **on Additive Interactions in CALUX." Organohalogen Compounds 60 - 65.** 

**33. Windal, I., I. Van Overmeire, et al. (2002). "V ariables that affect TEQ determinations by**  **CALUX and GC-HRMS." Organohalogen Compounds 58: 38 9-392.** 

**34. Ziccardi, M.H., Gardner, I.A. and Denison, M.S. (2002) Application of the luciferase**  **recombinant cell culture bioassay system for the a nalysis of polycyclic aromatic**  **hydrocarbons, Environ. Toxicol. & Chem. 21, 2027-2 033.** 

**35. Windal, I., Dennison, M. S, Birnbaum L. S., Van Wouwe, N., Baeyens, W. Goeyens L.**  **(2005). “Chemically Activated Luciferase Gene Expr ession (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-ana lytical method CALUX.” Talanta 63,** 

**1261-1268.**

**38. Van den Berg et al., (2006). “The 2005 World He alth 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 Technol ogy 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 CO 2 

### 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, CO 2 

### monitoring Fisher 10-884-1 or equivalent

250 o 

### C 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 RECOV ERIES** 

**TABLE 6**

**COMPARISON OF 1,2,3,4-TCDD RECOVERIES DETERMINED BY GC/ECD AND**  **14 C**  **LABELED 2,3,7,8-TCDD RECOVERIES DETERMINED BY SCINT ILLATIONS COUNTING** 



## Sample **1234-TCDD by**

**GC/ECD 14**  

## C 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 AIRT IGHT 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 SOI L 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 SOI L 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% 1 100.00
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 EXH AUST 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% 16.0 0 3.3 Company-1 Asia Exhaust Extracts G7 75.53 4.38 6% 0.29 0.03 6
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 0.0 12
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% 6.1 0 0.75 Company-1 Asia Exhaust Extracts G25 2009.68 120.16 6% 7.8 0 1.10 Company-1 Asia Exhaust Extracts G36 1981.81 132.78 7% 6.2 0 0.65
Company-1 Asia Exhaust Extracts G37 19927.99 1227.69 6% 1 00.00 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% 7.6 0 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% 7.6 0 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% 3 00.00 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% 1.6 0 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% 4.2 0 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% 7.0 0 2.40 Company-1 Asia Exhaust Extracts G135 1563.29 74.61 5% 7.3 0 2.80
Company-1 Asia Exhaust Extracts G136 1170.80 36.58 3% 6.7 0 1.60**SW-846 Update V 4435 - 34 Revision 1**

**October 2012 TABLE 14** 

**COMPARISON OF METHOD 4435 AND GC/MS RESULTS FOR SOL UTION 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 PROCESS ES 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 analysispg/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 analysispg/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 analysispg/g TEQ**SW-846 Update V 4435 - 39 Revision 1**

**October 2012 FIGURE 5** 

**QC CHARTS FOR DIOXIN/FURAN STANDARD MIXTURE FOR 200 3** 

**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 200 4** 

**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 20 03** 



### 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/tubeRLU**SW-846 Update V 4435 - 44 Revision 1**

**October 2012 FIGURE 10** 

2,3,7,8-TCDD STANDARD CURVES WITH ERROR BARS FOR 20 04**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 contro ls, 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 BACKGRO UND 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 DAT A (FIGURE 12) TO**  **PRODUCE THE RESULTS FOR THE REPORT (FIGURE 14), USI NG 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 calculat ion 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 Met hod 4435 to GC/MS for soil**  **samples for pre-field testing during the EPA SITE f ield study. These samples were**  **processed in March of 2004 using the screening tech nique with a surrogate recovery.**  **These TEQs were calculated using the WHO 1997 REP v alues.** 



### 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/gCALUX 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/gCALUX by XDS pg/g

**Represents the PCB TEQ comparison of the Method 443 5 to GC/MS for soil samples for pre**  **field testing during the EPA SITE field study. The se samples were processed in March of 2004**  **using the screening technique with a surrogate reco very. 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/gIncubator 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/gCALUX 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/gCALUX 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/M3CALUX 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/10gCALUX by XDSpgTEQ/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/10gCALUX by XDSngTEQ/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/ulCALUX 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/matrixCALUX 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 consisten cy with new SW-846 methods style**  **guidance. The format was updated to Microsoft Word 2007.docx.** 

**2. Minor editorial and technical revisions were mad e throughout to improve method clarity.**  **3. The revision number was changed to 1 and the dat e 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).**