### SW-846 Update V 4430 - 1 **Revision 1 October 2012**  **METHOD 4430 SCREENING FOR POLYCHLORINATED DIBENZO-**  p **-DIOXINS (PCDDs) AND POLYCHLORINATED DIBENZOFURANS (PCDFs) IN SOILS AND**  **SEDIMENTS USING AN ARYL HYDROCARBON RECEPTOR-BASED PCR ASSAY**  **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 screening procedure for polych**  **lorinated dibenzo-** p **-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) i**  **n soils and sediments. This method uses a commercially-available aryl hydrocarbon receptor**  **(AhR)-based polymerase chain reaction (PCR) assay. The AhR-PCR assay screens samples by**  **their toxicity equivalent quotient (TEQ) by responding to individual PCDD/PCDF congeners in**  **approximate correlation to their toxicity equivalent factors (TEFs). The TEQ measured by the**  **AhR-PCR assay is the sum of the response from the individual congeners. See Table**  **2 for a detailed list of response factors for individual PCDD/PCDF congeners. More information a**  **bout the AhR-PCR assay can be found at www.eichrom.com/dioxin/products.**  **1.2 The AhR-PCR assay employs AhR proteins extracte d from mammalian cells to**  **bind PCDD/PCDFs. Upon binding PCDD/PCDFs, the AhR forms an activated receptor complex**  **with an aryl hydrocarbon nuclear translocator (ARNT ) and a specific DNA response element**  **(DRE). This activated receptor complex is captured onto a microwell in a 96-well plate and**  **isolated from unreacted AhR, ARNT and DRE by washin g on a 96-well plate washer. The level**  **of PCDD/PCDF contamination is then measured indirec tly by amplifying and measuring the**  **DRE using real time PCR. Since the interaction of the AhR with individual PCDD/PCDF**  **congeners is proportional to the TEF values of the individual PCDD/PCDF congeners, the**  **response measured by the AhR-PCR assay correlates v ery well with the TEQ of the sample.**  **1.3 The AhR-PCR assay and the associated sample pre paration method outlined in**  **this document provide a technique for the estimatio n of the total PCDD/PCDF TEQ. The**  **method will not provide concentrations of individua l PCDD/PCDF congeners, as the individual**  **congeners are not separated during the sample prepa ration. During the sample preparation,**  **the PCDD/PCDFs are isolated as a group from potenti ally interfering compounds such as**  **polychlorinated biphenyls (PCB) and polynuclear aro matic hydrocarbons (PAH). A complete list**  **of compounds which have been identified to have sig nificant response on the AhR-PCR assay**  **is provided in Table 2.**

### SW-846 Update V 4430 - 2 **Revision 1 October 2012**  **1.4 The AhR-PCR assay responds to PCDD/PCDF congene rs in proportion to their**  **concentration and TEF value. However, the response factors for individual PCDD/PCDF**  **congeners on the AhR-PCR assay are not identical to the TEF values assigned by the World**  **Health Organization (WHO). Therefore, variation in the accuracy among samples may occur**  **solely because of variability in congener compositi on. A comparison of AhR-PCR response**  **factors and WHO TEF values is provided in Table 2. 1.5 This method details a single extraction techniq**  **ue, pressurized fluid extraction (PFE), and sample preparation method which has been**  **shown to effectively extract the PCDD/PCDFs from soil and sediment samples and isola**  **te the PCDD/PCDFs from most interfering classes of compounds. Other extraction**  **methods, solvent systems or sample clean up methods may be used, provided adequate performan**  **ce of these methods is demonstrated for the analytes of interest from the matrix of int**  **erest. 1.6 The limit of detection submitted by the manufac**  **turer of this testing product is 0.4 pg of 2,3,7,8-tetrachlorodibenzo-**  p **-dioxin (2,3,7,8-TCDD). This detection limit is pr ovided as**  **guidance only, and the limit achievable by a user f or a given sample will be dependent on**  **several factors including the type of sample matrix .**  **1.7 Prior to employing this method, analysts are a dvised to consult the base method**  **for each type of procedure that may be employed in the overall analysis (e.g., Methods 3500,**  **3600, 4000, 5000, and 8000) and the manufacturer’s instructions for additional information on**  **quality control procedures, development of QC accep tance criteria, calculations, and general**  **guidance. Analysts also should consult the disclai mer statement at the front of the manual and**  **the information in Chapter Two for guidance on the responsibilities of the analyst for**  **demonstrating that the techniques employed are appr opriate for the analytes of interest, in the**  **matrix 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. 1.8 This method is restricted to use by, or under t**  **he supervision of, personnel appropriately experienced and trained in the use of**  **general laboratory techniques including sample extraction, column chromatography and polyme**  **rase chain reaction (PCR). Each analyst must demonstrate the ability to generate ac**  **ceptable results with this method. 2.0 SUMMARY OF METHOD**  **2.1 Detailed sample preparation and analysis proced ures for this method are available**  **in the manufacturer’s literature at www.eichrom.com /dioxin/products. A brief description of the**  **key steps is provided below. 2.2 Diatomaceous earth is added to the dry sample a**  **nd mixed. The sample is then extracted using pressurized fluid extraction (PFE).**  **2.3 Following PFE, the solvent is exchanged to hexa ne, and the PCDD/PCDFs are**  **isolated from interfering compounds by column chrom atography using commercially available**  **silica gels and Florisil®. The PCDD/PCDFs are reco vered in 50% methylene chloride:hexane.**  **The sample is concentrated and redissolved in hepta ne.** 

### SW-846 Update V 4430 - 3 **Revision 1 October 2012**  **2.4 An accurately measured volume of blank, sample, standard or recovery standard**  **in heptane is added to a glass vial containing a mi xture of AhR, ARNT and DRE and mixed for**  **one hour. 2.5 An accurately measured volume of the AhR, ARNT,**  **DRE that has been equilibrated with blank, sample, standard or recove**  **ry standard for one hour is then added to a plastic microwell and mixed for 30 min. PCDD/PCDF-**  **AhR-ARNT-DRE complexes bind to the walls of the microwell, and excess AhR, ARNT and DR**  **E are removed by rinsing on a 96-well plate washer.**  **2.6 PCR reagents are added to the microwell and the amount of DRE, which is**  **proportional to the TEQ of the sample, is measured using PCR. The response from the PCR is**  **related to TEQ by a dose-response curve generated f rom 2,3,7,8-TCDD standards. The PCR**  **can analyze up to 38 samples in duplicate, with bla nks, standards and recovery standards**  **simultaneously in 90 min. 3.0 DEFINITIONS**  **See the glossary at the end of this method for pro cedure-specific terms. Also, refer to**  **Chapter One, Methods 8280 and 8290, and the manufac turer’s instructions for definitions that**  **may be relevant to this procedure. 4.0 INTERFERENCES**  **4.1 Solvents, reagents, glassware and other sample processing hardware may yield**  **artifacts and/or interferences to sample analysis. All of these materials must be demonstrated**  **to be free from interferences under the conditions of the analysis by analyzing method blanks.**  **Specific selection of reagents and purification of solvents by distillation in all-glass systems may**  **be necessary. Refer to each method to be used for specific guidance on quality control**  **procedures and to Chapter Four and Method 8290 for general guidance on the cleaning of**  **glassware. 4.2 The AhR used in this method binds to the sevent**  **een PCDD/PCDF congeners with established TEF values, and other aryl hydrocarbon**  **compounds, such as PCBs, PAHs, brominated and mixed brominated/chlorinated dibenzo**  **-** p **-dioxins and furans, PCDD/PCDF congeners without established TEF values and other**  **compounds similar in structure to 2,3,7,8- TCDD. The highest response is observed for 2,3,7,8**  **-TCDD and compounds of similar structure and degree of chlorination. A complete list of com**  **pounds for which the response of the AhR PCR assay has been observed is given in Table 2.**  **4.3 The AhR used in this method binds to PCDD/PCDF and similar molecules based**  **on structure, not mass. Therefore, 13**  

### Carbon- or other stable isotope-labeled standards a **re detected to the same degree as native compounds. T**  **hese labeled standards cannot be used as internal standards with this method.**  **4.4 High levels (10 6**  

### x that of the desired analyte) of selected PAH com **pounds and high molecular weight aliphatic hydrocarbons have b**  **een shown to inhibit the response of the AhR-PCR assay. However, it is unlikely that high l**  **evels of these compounds will remain with the PCDD/PCDF fraction if the sample preparation in**  **structions provided by the manufacturer are followed.** 

### SW-846 Update V 4430 - 4 **Revision 1 October 2012**  **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**  **) should be available to all personnel involved in these analyses.**  **5.2 The AhR-PCR assay should only be used by proper ly trained personnel in an**  **appropriate laboratory environment. Personnel shou ld wear appropriate personal protective**  **equipment, including safety glasses, lab coat and g loves.**  **5.3 WARNING: PCDD/PCDF standards, solutions containing PCDD/PC**  **DFs and potentially contaminated samples should be treated**  **as hazardous materials. 5.4 Handle all organic solvents (toluene, acetone,**  **hexane, heptane and methylene chloride) in a fume hood.**  **5.5 Silica gels and Florisil should be treated as r espiratory hazards and treated with**  **the appropriate care. 5.6 Silica gels impregnated with sulfuric acid, pot**  **assium hydroxide and silver nitrate should be treated with care as they can cause skin**  **irritation, discoloration and/or burns. 5.7 Some test components are stored in freezers (-2**  **0 oC and -80 oC). Care should be taken to avoid direct contact of frozen components**  **with skin. 6.0 EQUIPMENT AND SUPPLIES**  **6.1 This section does not list all common laborator y glassware (e.g., beakers and**  **flasks). A complete list of the equipment and supp lies can be found in the manufacturer’s**  **literature (www.eichrom.com/dioxin/products). 6.2 The AhR-PCR assay requires a real-time PCR inst**  **rument with FAM and ROX detection capabilities. Please see the manufacture**  **r’s literature for a complete list of compatible models.**  **6.3 Other significant equipment required for the Ah R-PCR assay includes a 96-well**  **platewasher (Biotek Elx50 or equivalent), a 96-well plateshaker (Heidolph Titramax 1000 or**  **equivalent), a refrigerator/freezer (≤6 oC/-20 oC) and a -80 oC freezer (or liquid nitrogen vial**  **storage Dewar) for storage of the test kit componen ts.**  **6.4 The manufacturer will supply or specify equipme nt, apparatus and materials**  **necessary for the successful completion of the test . Do not mix the equipment, supplies or**  **reagents from other test kits. The AhR-PCR assay c ontains reagents that are evaluated by the**  **manufacturer on a lot to lot basis. Do not mix the reagents from multiple lots, unless expressly**  **allowed by the manufacturer. 6.5 Incorporation of apparatus for the capture of e**  **vaporated solvents during concentration procedures may be required by Federal**  **, State or local municipality regulations that govern air emissions of volatile organics. EP**  **A recommends the incorporation of this type of**

### SW-846 Update V 4430 - 5 **Revision 1 October 2012**  **reclamation system as a method to implement an emis sions reduction program. Solvent**  **recovery is a means to conform with waste minimizat ion and pollution prevention initiatives.**  **7.0 REAGENTS AND STANDARDS 7.1 Reagent-grade or pesticide-grade chemicals must**  **be used in all tests. Unless otherwise indicated, it is intended that all reagen**  **ts conform to the specifications of the committee of Analytical Reagents of the American Ch**  **emical Society, where such specifications are available. Other grades may be used, provided**  **it is first ascertained that the reagent is of sufficiently high purity to permit its use without**  **lessening the accuracy of the determination. Reagents should be stored in glass to prevent the l**  **eaching of contaminants from plastic containers.**  **7.2 Extraction solvents -- This method has been val idated using a 3:7 mixture of**  **acetone and toluene for the extraction solvent and a pressurized fluid extraction system. Other**  **solvent systems or another extraction apparatus may also be used provided that they have**  **been shown to provide reproducibly high recovery of the analytes of interest from the matrix of**  **interest. The choice of extraction solvent and app aratus will depend on the analytes of interest**  **and the matrix of interest, and no single solvent o r apparatus is universally applicable to all**  **analytes or matrices . Whatever solvent system and apparatus is employe**  **d, including those specifically listed in this method, the analyst mus**  **t demonstrate adequate performance for the analytes of interest, at the levels of interest, fr**  **om the matrix of interest. See Sec. 9.2.1 of this method for more information about how to demonstrat**  **e adequate performance. . 7.3 Detailed information on reagent requirements i**  **s given in the manufacturer’s literature (www.eichrom.com/dioxin/products). Do n**  **ot mix the equipment, supplies or reagents from different testing products or different lots o**  **f the AhR-PCR assay. Store all reagents and standards according to the manufacturer’s instructi**  **ons, and discard any reagents that are past the expiration date assigned by the manufacturer.**  **8.0 SAMPLE COLLECTION, PRESERVATION AND STORAGE Sample collection, preservation and storage requir**  **ements may vary by EPA program and may be specified in a regulation or project plannin**  **g 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 The AhR-PCR assay requires small sample masses (typically 5-10 g). Sample**  **collection procedures should ensure that the sample is representative of the source. The**  **distribution of PCDD/PCDFs in the sample can be het erogeneous. Samples should be**  **homogenized to ensure that any subsamples are repre sentative of the whole sample. Water**  **content should be low enough to avoid standing wate r in the sample containers and ideally**  **should be removed before homogenizing and subsampli ng.**  **8.2 Sample extracts are stable for up to three mont hs when refrigerated (≤6 oC) and**  **stored in tightly sealed vials. Longer storage tim es may be possible if validated with**  **performance data. 8.3 Store all test kit components as outlined in th**  **e manufacturer’s literature.**

### SW-846 Update V 4430 - 6 **Revision 1 October 2012**  **8.4 Some components must be thawed before use. Unl ess otherwise instructed by the**  **manufacturer, thaw components immediately prior to use to near room temperature. Do not use**  **hot water to thaw components as this can lead to de creased performance of the test.**  **8.5 Do not use test kits or components after the ex piration date.**  **8.6 Do not use test kit components with components from a different lot of the test kit**  **or with components from test kits from different ma nufacturers.**  **9.0 QUALITY CONTROL 9.1 Refer to Chapter One for guidance on quality as**  **surance (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 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 Refer to Methods 3500, 3600, 3620 and 3630 for QC procedures to ensure the**  **proper operation of the various extraction and samp le preparation techniques. Any more**  **specific QC procedures provided in this method will supersede those noted in Methods 3500,**  **3600, 3620 or 3630.**

**9.2.1 Initial demonstration of proficiency Each laboratory must demonstrate initial proficienc**  **y with each sample preparation and determinative method combination it utilizes by**  **generating data of acceptable accuracy and precision for target analytes in a cle**  **an matrix. At a minimum, such a demonstration will encompass the initial demonstrat**  **ion of proficiency described in method 3500, using a clean reference matrix. The laborato**  **ry must also repeat the demonstration of proficiency whenever new staff members are train**  **ed or significant changes in instrumentation are made. See Method 8000D, Sec. 9**  **.3 for information on how to accomplish a demonstration of proficiency.**  **9.3 Initially, before processing any samples, the a nalyst should demonstrate that all**  **parts of the equipment in contact with the sample a nd reagents are interference-free. This is**  **accomplished through the analysis of a method blank . As a continuing check, each time**  **samples are extracted, cleaned up, and analyzed, an d when there is a change in reagents, a**  **method blank should be prepared and analyzed for th e compounds of interest as a safeguard**  **against chronic laboratory contamination. 9.4 Any method blanks, matrix spike samples, or rep**  **licate samples should be subjected to the same analytical procedures (Sec. 1**  **1.0) as those used on actual samples. For sample extracts that are cleaned up using this meth**  **od, the associated quality control samples must also be processed through this cleanup method.**  **9.5 The commercially available testing product used to develop this method represents**  **a performance-based analytical technique. Therefor e, it is imperative that the manufacturer's** 

### SW-846 Update V 4430 - 7 **Revision 1 October 2012**  **instructions and specifications be followed closely . Follow the manufacturer's instructions for**  **the testing product being used for the quality cont rol procedures specific to that testing product.**  **The following discussion of quality control require ments relies heavily on the analyst's**  **knowledge and understanding of the manufacturer's i nstructions.**  **9.6 Stringent quality assurance protocols should be maintained throughout each stage**  **of the testing procedure; i.e., sample extraction, sample preparation, and immunoassay**  **analysis. Various QA actions check for failure at each of these points in the process. Duplicate,**  **check samples, standard reference materials, and ot her QA samples and methods can and**  **should be used with this kit, with the exception of conventional isotope labelled internal**  **standards. The AhR-PCR assay recognizes PCDD/PCDF congeners based on structure and**  **not on mass. Therefore, conventional isotope-label led internal standards are detected as native**  **material and cannot be used with this method. 9.7 As outlined above, routine quality control proc**  **edures associated with this method include the measurement of standards, assay blanks,**  **method blanks and recovery standards. It is recommended that all measurements be performed i**  **n duplicate and that all measurements be performed as part of the same batch of samples. A**  **batch of samples includes up to 38 field samples prepared and analyzed at the same time, alo**  **ng with standards, assay blanks, method blanks and recovery standards.**  **9.8 Additional quality control considerations 9.8.1 Do not use test kit components past their exp**  **iration dates. 9.8.2 Do not mix the equipment, supplies or r**  **eagents from test kits from different manufacturers or from different lots of t**  **he test kit. 9.8.3 Use the test components within the storage te**  **mperature and operating temperature limits specified by the manufacturer.**  **9.8.4 The AhR-PCR assay identifies and measures PCD D/PCDF congeners**  **based on structure, not mass. Therefore isotope-la beled internal standards are**  **detected as native material and cannot be used with this method.**  **10.0 CALIBRATION AND STANDARDIZATION See the manufacturer’s instructions for informatio**  **n on calibration and standardization. 11.0 PROCEDURE**  **Follow the manufacturer’s instructions for the tes t kit being used. Instructions can be**  **found on the test kit package insert or at www.eich rom.com/dioxin/products.**  **12.0 DATA ANALYSIS AND CALCULATIONS Follow the instructions provided by the manufactur**  **er for the calculation of all testing product results. For each batch of samples, use th**  **e calibration curve generated concurrently with that AhR-PCR assay run.** 

### SW-846 Update V 4430 - 8 **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 kit 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 Table 1 depicts response on the AhR-PCR assay**  **for 2,3,7,8-TCDD over 1 year for four different laboratories along with QA acceptanc**  **e ranges. These data are provided for guidance purposes only.**  **13.3 Table 2 depicts cross-reactivity on the AhR-PC R assay relative to 2,3,7,8-TCDD.**  **These data are provided for guidance purposes only. 13.4 Table 3 summarizes correlation data for AhR-PC**  **R and GC-HRMS for spiked sand and soil samples. These data are provided for guid**  **ance purposes only. 13.5 Table 4 summarizes correlation data for AhR-PC**  **R and GC-HRMS for real-world soil and sediment samples from US EPA Superfund Sit**  **es. These data are provided for guidance purposes only.**  **13.6 Table 5 summarizes typical false positive and false negative rates for AhR-PCR**  **near the action level of 50 pg/g TEQ. These data a re provided for guidance purposes only.**  **13.7 Table 6 summarizes intra-lot variation on the AhR-PCR assay. These data are**  **provided for guidance purposes only. 13.8 Table 7 summarizes inter-lot variation on the**  **AhR-PCR assay. These data are provided for guidance purposes only.**  **14.0 POLLUTION PREVENTION 14.1 Pollution prevention encompasses any technique**  **that reduces or eliminates the quantity and/or toxicity of waste at the point of g**  **eneration. Numerous opportunities for pollution prevention exist in laboratory operation. 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 tha**  **t 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.**

### SW-846 Update V 4430 - 9 **Revision 1 October 2012**  **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. Dioxins and Furans in Soil and Sediment, Eichrom Method DFS01,**  **www.eichrom.com/dioxin/products. 2. M. Van den Berg, L. S. Birnbaum, M. Denison, M.**  **De Vito, W. Farland, M. Feeley, H. Fiedler, H. Hakansson, A. Hanberg, L. Haws, M. Rose**  **, S. Safe, D. Schrenk, C. Tohyama, A. Tritscher, J. Tuomisto, M. Tysklind, N. Walker,**  **and R. E. Peterson; “The 2005 World Health Organization Reevaluation of Human and Mamma**  **lian Toxicity Equivalency Factors for Dioxins and Dioxin-like Compounds,”**  Toxicol. Sci. 2006 **,** 93[2] **, 223-241. 3. R. L. Allen, J. J. Willey; “Novel Real-Time PCR**  **Assay for the Detection of Dioxins and Furans,”**  Organohalogen Compd. 2002 **,** 58 **, 341-343. 4. D. R. McAlister, M. J. Fern, R. L.; Allen, K. S**  **akata, B. Le Bizec, P. Marchand; “Sample Preparation for the Measurement of Polychlorinated**  **Dioxins and Furans by the Procept Rapid Dioxin Assay,”**  Organohalogen Compnd. 2006 **,** 68 **, 940-943. 5. Y. Z. Gu, J. B. Hogenesch, C. A. Bradfield; “Th**  **e PAS Superfamily: Sensors of Environmental and Developmental Signals,”**  Annu. Rev. Pharmacol. Toxicol. 2000 **,** 40 **, 519-561.**  **6. D. R. McAlister, M. J. Fern; “Soil Screening at 50 pg/g Using a Novel Aryl Hydrocarbon**  **Receptor-based PCR Assay,”** Organohalogen Compd **.,** 2007 **,** 69 **, 1230-1233. 7. D. R. McAlister, M. J. Fern; “Cross-reactivity**  **of Compounds on the Procept Rapid Dioxin Assay,”**  Organohalogen Compd **.,** 2007 **,** 69 **, 1226-1229. 8. S. Heid, R. Pollenz, H. Swanson; “Role of Heat**  **Shock Protein 90 Dissociation in Mediating Agonist-induced Activation of the Aryl Hydrocarbon**  **Receptor,”** Mol. Pharmacol. 2000 **,** 57[1] **, 82-92. 9.**  Fed. Reg. 2006 **,** 71 **, 78441, Update to ATSDR Policy Guideline for Dioxi ns and Dioxin**  **Like Compounds in Residential Soil. 10. C. A. Foy, H. C. Parkes; “Emerging Homogeneous**  **DNA-based Technologies in the Clinical Laboratory,”**  Clin. Chem. 2000 **,** 47(6) **, 990-1000. 11. D. R. McAlister, M. J. Fern, R. L. Allen; “Rap**  **id Aryl Hydrocarbon Receptor-based Polymerase Chain Reaction Screening Assay for Polyc**  **hlorinated Dibenzo-** p **-Dioxins and Furans in Soil and Sediment,”**  Talanta **,** 2007 **, in press, doi.org/10.1016/j.talanta.2007.08.005.** 

### SW-846 Update V 4430 - 10 **Revision 1 October 2012**  **17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION D ATA**  **The following pages contain the tables referenced by this method. A glossary is provided**  **after the tables.**

### SW-846 Update V 4430 - 11 **Revision 1 October 2012**  Eichrom Eichrom Multi-lab Multi-lab Multi-lab Acceptable Historical Multi-lab Site 2 Site 3 Site 4 Range **Ct range 0-5000 5.5 5.8 5.7 5.0 4.9 >4.5 R**  **2** 

### (78-5000) **0.980 0.984 0.992 0.984 0.994 >0.97 average SD of replicates 0.20 0.20 0.20 0.25 0.13 <0.30**  **number of curves generated 25 6 5 4 3** *** Data are from 43 determinations of 2,3,7,8-TCDD R esponse over more than one year for 7 point standar**  **d curve + blank in heptane (5000, 2500, 1250, 625, 31**  **3, 156, 78 and 0 pg 2,3,7,8-TCDD/mL heptane) genera ted**  **in four different laboratories. The assay detection limit, approximated from the lowest standard conce**  **ntration, 78 pg/mL, which lies >3**  σ **from the assay blank, is 0.4 pg 2,3,7,8-TCDD per w ell. Actual achievable method**  **lower limits of quantitation will depend on several factors including sample size, extract volume and**  **recoveries of analytes through extraction and clean-up steps.** 

Table 1. Standard Curve Metrics (Ct vs pg/mL 2,3,7, 8-TCDD)*



### These data are provided for guidance purposes only.

### SW-846 Update V 4430 - 12 **Revision 1 October 2012**  AhR-PCR AhR-PCR Structure compound Response compound Response Structure 2,3,7,8 TCDD 1 TEF(1) 2,3,7,8 TCDF 0.06 TEF (0.1) 1,2,3,7,8 PCDD 0.6 TEF (1) 1,2,3,7,8 PCDF 0.1 TEF (0.03) 1,2,3,4,7,8 HxCDD 0.4 TEF (0.1) 1,2,3,4,7,8 HxCDF 0.4 TEF (0.1)
1,2,3,6,7,8 HxCDD 0.1 TEF (0.1) 1,2,3,6,7,8 HxCDF 0.2 TEF (0.1) 1,2,3,7,8,9 HxCDD 0.5 TEF (0.1) 1,2,3,7,8,9 HxCDF 0.3 TEF (0.1) 1,2,3,4,6,7,8 HpCDD 0.01 TEF (0.01) 1,2,3,4,6,7,8 HpCDF 0.05 TEF (0.01)
1,2,3,4,6,7,8,9 OCDD 0.000003 TEF (0.0003) 1,2,3,4,6,7,8,9 OCDF 0.0005 TEF (0.0003)
2,3,4,7,8 PCDF 0.3 TEF (0.3) 1,2,3,4,7,8,9 HpCDF 0.02 TEF (0.01) 2,3,4,6,7,8 HxCDF 0.1 TEF (0.1)
AhR-PCR AhR-PCR Structure compound Response compound Response Structure PCB-77 (3,3',4,4') 0.00003 TEF (0.0001) PCB-126 (3,3',4,4',5) 0.01 TEF (0.1) PCB-81 (3,4,4',5) 0.00005 TEF (0.0003) PCB-156 (2,3,3',4,4',5) 0.00003 TEF (0.00003) PCB-105 (2,3,3',4,4') <3 x 10-7 TEF (0.00003) PCB-157 (2,3,3',4,4',5') 0.00004 TEF (0.00003) PCB-114 (2,3,4,4',5) 0.00001 TEF (0.00003) PCB-167 (2,3',4,4',5,5') 0.000001 TEF (0.00003) PCB-118 (2,3',4,4',5) <3 x 10-7 TEF (0.00003) PCB-169 (3,3',4,4',5,5') 0.001 TEF (0.03) PCB-123 (2',3,4,4',5) 0.000009 TEF (0.00003) PCB-189 (2,3,3',4,4',5,5') <3 x 10-7 TEF (0.00003) Table 2. Cross-reactivity on the AhR-PCR Assay Response for Chlorinated Dioxins and Furans on AhR- PCR Assay

Response for Polychlorinated Biphenyls on AhR-PCR A ssay O O C l

C l C l C l

O

O Cl Cl Cl Cl C l

C l C l

C l O C l C l C l C l

O O Cl Cl Cl Cl C l

C l O Cl C l Cl C l C l

O O Cl Cl Cl Cl C l

C l C l

O O C l Cl Cl C l C l

O

O Cl Cl Cl Cl C l

C l

O O C l Cl Cl C l C l C l

O C l

Cl C l Cl C l

O C l Cl C l Cl C l C l O Cl Cl Cl Cl C l

C l

O Cl Cl Cl Cl C l

C l

O Cl

C l Cl C l C l C l

O Cl

Cl Cl Cl C l

C l C l

O Cl

Cl Cl Cl C l

C l C l O Cl

Cl Cl Cl C l

C l C l

C l

Cl C l Cl C l

Cl Cl C l

C l
Cl Cl C l C l C l

Cl Cl C l C l

C l Cl Cl

C l

C l C l

Cl Cl C l C l C l Cl Cl C l C l C l

Cl Cl C l C l C l

C l

Cl Cl C l C l

C l

C l
Cl Cl C l C l

C l C l Cl Cl C l

C l C l C l

Cl Cl C l

C l C l C l C l 

### These data are provided for guidance purposes only.

### Table 2. Cross-reactivity on the AhR-PCR Assy (cont inued)



### SW-846 Update V 4430 - 13 **Revision 1 October 2012**  AhR-PCR analogous AhR-PCR Structure compound Response chloro-compound Response Structure 2,3,7,8-TBrDD 0.3 2,3,7,8-TCDD 1.0 1-Br-2,3,4,6,7,8,9-HpCDD 0.00004 1,2,3,4,6,7,8,9-OCDD 0.000003 3-Br-2,7,8-TriCDF 0.4 2,3,7,8-TCDF 0.1 2-Br-3,6,7,8,9-PCDD 0.05 1,2,3,4,7,8-HxDD 0.4
1-Br-2,3,7,8-TCDF 0.3 1,2,3,7,8-PCDF 0.1 1-Br-2,3,7,8-TCDD 0.4 1,2,3,7,8-PCDD 0.6
1-Br-2,3,6,7,8,9-HxCDD 0.06 1,2,3,4,6,7,8-HpCDD 0.01 2,3-Br-7,8-DiCDD 0.4 2,3,7,8-TCDD 1.0 2-Br-1,3,7,8-TCDD 0.6 1,2,3,7,8-PCDD 0.6 2-Br-7,8-DiCDD <10 -6 2,3,7-TriCDD N/A 2-Br-3,7,8-TriCDD 0.5 2,3,7,8-TCDD 1.0 2,3,6,7-TCl-xanthene 0.7 2,3,7,8-TCDD 1.0 2,3,7,8-TCl-thiophene 0.2 2,3,7,8-TCDF 0.12,3,7-Cl-8-methyl-dibenzo-pdioxin 0.3 2,3,7,8-TCDD 1.0 Response for Bromo/chloro Dioxins and Furans on AhR -PCR Assay

O O C l

C l C l C l O O B r

B r B r B r

O O C l

C l C l C l

O

O C l

C l C l C l O

O C l

C l C l C l O

O C l C l C l O O C l B r C l O O C l Cl Cl C l C l O O C l Br Cl C l C l O O Cl Cl Cl Cl C l

C l C l

C l O O Cl Cl Cl Cl B r

C l C l

C l

O O Cl Cl Cl Cl C l

C l

O O C l Cl Cl C l C l O O C l Cl Cl C l B r

O O Cl Cl Cl Cl C l

C l C l O C l

C l C l C l O C l

C l C l B r

O Cl C l Cl C l C l

O Cl

C l Cl C l B r

O

O C l

C l B r B r O

O Cl

Cl Br Cl C l

C l

O O C l

C l B r C l

O C l C l C l C l

O

O C l C l C l O O C l

C l C l C l O C l C l C l C l S C l

C l C l C l O

O Cl Cl Cl Cl B r

C l C l



### These data are provided for guidance purposes only.

### Table 2. Cross-reactivity on the AhR-PCR Assy (cont inued)



### SW-846 Update V 4430 - 14 **Revision 1 October 2012**  AhR-PCR AhR-PCR Structure Compound Response Compound Response Structure aldrin no measurable

response from 2000 ppt to 2000 ppm Endosulfan II no measurable response from 2000 ppt to 2000 ppm alpha-BHC no measurable response from 2000 ppt to 2000 ppm Endosulfan sulfate no measurable response from 2000 ppt to 2000 ppm beta-BHC no measurable response from 2000 ppt to 2000 ppm Endrin no measurable response from 2000 ppt to 2000 ppm
delta-DHC no measurable response from 2000 ppt to 2000 ppm Endrin Aldehyde no measurable response from 2000 ppt to 2000 ppm
p,p'-DDE no measurable response from 2000 ppt to 2000 ppm Endrin Ketone no measurable response from 2000 ppt to 2000 ppm p,p'-DDD no measurable response from 2000 ppt to 2000 ppm Heptachlor no measurable response from 2000 ppt to 2000 ppm p,p'-DDT no measurable response from 2000 ppt to 2000 ppm Heptachlor epoxide no measurable response from 2000 ppt to 2000 ppm Dieldrin no measurable response from 2000 ppt to 2000 ppm Methoxychlor no measurable response from 2000 ppt to 2000 ppm Endosulfan I no measurable response from 2000 ppt to 2000 ppm Response of Pesticide Compounds on AhR-PCR Assay

**Cl Cl**  **Cl Cl** 

**Cl Cl** 

**Cl Cl** 

**Cl Cl** 
**Cl Cl**

**Cl**

**Cl Cl**  **Cl Cl**

**Cl**
**Cl Cl** 

**Cl Cl**  **Cl**

**Cl**

**Cl Cl**

**Cl Cl** 

**Cl Cl**

**Cl Cl** 

**Cl Cl**  **Cl Cl**  **Cl**

**O Cl**  **Cl Cl**  **Cl**

**Cl Cl** 

**S O**  **O O**  **Cl**

**Cl Cl**  **Cl Cl** 

**Cl S**  **O O**  **O Cl** 

**Cl Cl**  **Cl Cl** 

**Cl**

**S O** 
**O O**  **O Cl** 

**Cl Cl**  **Cl**
**Cl**

**Cl**

**Cl Cl**  **Cl Cl** 

**Cl Cl**  **O**

**Cl**

**Cl Cl**
**Cl Cl**  **Cl O**  **H**

**Cl**

**Cl Cl**
**Cl Cl**  **Cl O**  **Cl**
**Cl Cl**  **Cl Cl** 

**Cl Cl** 

**O**

**Cl Cl**  **Cl Cl**  **Cl Cl**  **Cl**

**Cl Cl**  **Cl O**  **O**



### These data are provided for guidance purposes only.

### Table 2. Cross-reactivity on the AhR-PCR Assy (cont inued)



### SW-846 Update V 4430 - 15 **Revision 1 October 2012**  AhR-PCR AhR-PCR structure compound Response compound Response Structure Indeno(1,2,3-cd)pyrene 0.8 Benzo(a)pyrene 0.1 Benzo(k)fluoranthene 0.5 Benzo(a)anthracene 0.05 Benzo(b)fluoranthene 0.6 Chrysene 0.04
Dibenzo(ah)anthracene 0.3 Benzo(ghi)perylene 0.004 acenaphthylene No response at 200 ppt to 10 ppm anthracene No response at 200 ppt to 10 ppm fluorene No response at

200 ppt to 10 ppm naphthalene No response at 200 ppt to 10 ppm fluoranthene No response at 200 ppt to 10 ppm phenanthrene No response at 200 ppt to 10 ppm pyrene No response at

200 ppt to 10 ppm acenaphthene No response at 200 ppt to 10 ppm 2-methylnaphthalene No response at 200 ppt to 10 ppm 2-chloronaphthalene No response at 200 ppt to 10 ppm biphenyl No response at

200 ppt to 10 ppm 2,4-dichlorophenol No response at 200 ppt to 10 ppm 3,4-dichlorophenol No response at 200 ppt to 10 ppm toluene No response at 200 ppt to 10 ppm triphenylene 0.001 cyclopenta[c,d]pyrene 0.0002 2,2'-binaphthyl 0.05

Response of PAH Compounds on AhR-PCR Assay

**C l** 
**OH C**  **l Cl** 
**OH Cl**  **C l** 



### These data are provided for guidance purposes only.

### Table 2. Cross-reactivity on the AhR-PCR Assy (cont inued)



### SW-846 Update V 4430 - 16 **Revision 1 October 2012**  AhR-PCR AhR-PCR structure compound Response compound Response Structure dibenzo-p-dioxin < 10 -6 1,2,3-TriCDD 0.006 dibenzo-p-furan < 10 -6 2,3,7-TriCDD 0.08
2-MCDD < 10 -6 1,2,7,8-TCDD 0.9
2,3-DiCDD < 10 -6 1,3,6,8-TCDD 0.001
2,7-DiCDD < 10 -6 1,2,3,4-TCDD 0.007
1,7,8-TriCDD 0.004 1,2,4,7,8-PCDD 0.3 Response of non-2,3,7,8 Dioxins/Furans on AhR-PCR A ssay
O O
O O O C l

O

O C l C l

O

O C l C l

O O C l

Cl C l O O C l Cl C l O

O C l C l C l

O O Cl C l C l Cl

O O C l Cl

C l Cl O

O C l
Cl Cl
C l O

O C l Cl
C l Cl Cl
AhR-PCR AhR-PCR structure compound Response compound Response Structure bis-(2-ethylhexyl)phthalate < 7 x 10 -7 diethylphthalate < 7 x 10 -7

di-n-butylphthalate < 7 x 10 -7 dimethylphthalate < 7 x 10 -7

butylbenzylphthalate < 7 x 10 -7 di-n-octylphthalate < 7 x 10 -7
C 10 H 22 decane < 9 x10 -7 eicosane < 9 x10 -7 C 20 H 42 C 12 H 26 dodecane < 9 x10 -7 docosane < 9 x10 -7 C 22 H 246 C 14 H 30 tetradecane < 9 x10 -7 tetracosane < 9 x10 -7 C 24 H 50 C 16 H 34 hexadecane < 9 x10 -7 hexacosane < 9 x10 -7 C 26 H 54 C 18 H 38 octadecane < 9 x10 -7 octacosane < 9 x10 -7 C 28 H 58 octamethylcyclotetrasiloxane no measurable response from 2000 ppt to 2000 ppm benzophenone < 1 x10 -6 Response of Miscellaneous Compounds on AhR-PCR Assa y

**O Si**  **Si O Si** 

**O Si**  **O O**  **O O** 

**O O**  **O O** 

**O O**  **O O** 

**O**

**O O**  **O**

**O O**  **O O** 

**O**

**O O**  **O**

**O**

O 

### These data are provided for guidance purposes only.

### SW-846 Update V 4430 - 17 **Revision 1 October 2012**  GC-MS AhR-PCR 50pg/g GC-MS AhR-PCR 50pg/g TEQ TEQ screen TEQ TEQ screen **0.03 0.6 CN 0.03 0.4 CN 0.03 0.4 CN 0.03 0.3 CN**  **0.03 0.8 CN 0.03 0.5 CN 0.03 0.4 CN 0.03 0.5 CN**  **0.03 0.6 CN 0.03 0.4 CN 0.03 0.6 CN 0.03 0.3 CN**  **5 6.9 CN 5 4.8 CN 5 5.9 CN 5 3.6 CN**  **5 4.6 CN 5 6.0 CN 5 4.9 CN 5 3.7 CN**  **5 4.7 CN 5 3.3 CN 5 4.5 CN 5 3.1 CN**  **25 22 CN 25 30 CN 25 29 CN 25 22 CN**  **25 29 CN 25 26 CN 25 28 CN 25 21 CN**  **25 35 CN 25 32 CN 25 22 CN 25 28 CN**  **50 45 FN 50 54 CP 50 50 CP 50 48 FN**  **50 52 CP 50 66 CP 50 31 FN 50 39 FN**  **50 62 CP 50 37 FN 50 76 CP 50 29 FN**  **75 80 CP 75 74 CP 75 90 CP 75 59 CP**  **75 103 CP 75 54 CP 75 82 CP 75 75 CP**  **75 83 CP 75 84 CP 75 52 CP 75 65 CP**  **500 610 CP 500 534 CP 500 652 CP 500 305 CP**  **500 627 CP 500 409 CP 500 372 CP 500 467 CP**  **500 646 CP 500 483 CP 500 497 CP 500 306 CP**  **1000 1275 CP 1000 939 CP 1000 1006 CP 1000 898 CP**  **1000 1078 CP 1000 771 CP 1000 710 CP 1000 1007 CP**  **1000 720 CP 1000 937 CP 1000 1040 CP 1000 930 CP**  **1500 1126 CP 1500 1214 CP 1500 1224 CP 1500 1187 CP**  **1500 1570 CP 1500 1168 CP 1500 876 CP 1500 961 CP**  **1500 1584 CP 1500 1234 CP 1500 1538 CP 1500 1293 CP**  **CP = Correct Positive, CN = Correct Negative, FP = False Positive, FN = False Negative**  Table 3. Correlation of AhR-PCR with GC-HRMS for Sp iked sand and loamy soil

sand loamy soil

### SW-846 Update V 4430 - 18 **Revision 1 October 2012**  GC-HRMS AhR-PCR 50pg/g GC-HRMS AhR-PCR 50pg/g GC-HRMS AhR- PCR 50pg/g TEQ (pg/g) TEQ (pg/g) screen TEQ (pg/g) TEQ (pg/g) scree n TEQ (pg/g) TEQ (pg/g) screen 0.03 0.6 CN 0.03 2.1 CN 0.03 0.7 CN 14 14 CN 16 22 CN 0.03 1 CN 14 11 CN 16 18 CN 0.03 1 CN 14 6.8 CN 16 17 CN 0.03 1 CN 14 14 CN 16 19 CN 42 62 FP 12 8.8 CN 32 32 CN 42 21 CN 12 3.9 CN 32 37 CN 42 26 CN 12 11 CN 32 35 CN 42 21 CN 12 10 CN 32 25 CN 435 435 CP 15 15 CN 38 29 CN 435 637 CP 15 10 CN 38 36 CN 435 406 CP 15 10 CN 38 43 CN 435 255 CP 15 10 CN 38 26 CN 808 597 CP 14 15 CN 45 53 FP 808 2383 CP 14 8.4 CN 45 40 CN 808 423 CP 14 15 CN 45 44 CN 808 1276 CP 14 9.3 CN 45 36 CN 1048 1246 CP 13 14 CN 62 61 CP 1048 1136 CP 13 10 CN 62 55 CP 1048 1237 CP 13 10 CN 62 45 FN 1048 877 CP 13 10 CN 62 37 FN 3127 1548 CP 3127 995 CP 3127 5578 CP 3127 3083 CP GC-HRMS AhR-PCR 50pg/g GC-HRMS AhR-PCR 50pg/g TEQ (pg/g) TEQ (pg/g) screen TEQ (pg/g) TEQ (pg/g) scree n 0.03 6.2 CN 0.03 5.2 CN 48 359 FP 8648 16393 CP 48 465 FP 8648 6684 CP 48 479 FP 8648 9972 CP 48 371 FP 8648 12430 CP 846 721 CP 8831 9889 CP 846 667 CP 8831 10716 CP 846 541 CP 8831 9183 CP 846 466 CP 8831 19528 CP 1279 859 CP 11071 11018 CP 1279 1201 CP 11071 17503 CP 1279 599 CP 11071 20696 CP 1279 1123 CP 11071 28013 CP 1833 1833 CP 11259 33839 CP 1833 1938 CP 11259 8139 CP 1833 1516 CP 11259 7410 CP 1833 1243 CP 11259 26151 CP 3257 4541 CP 11410 15053 CP 3257 3083 CP 11410 15297 CP 3257 2944 CP 11410 7100 CP 3257 2692 CP 11410 12760 CP 3951 4631 CP 3951 3935 CP 3951 3214 CP 3951 3352 CP Solutia Soils Winona Post Soils

Table 4. Correlation of GC-HRMS and AhR-PCR Assay f or Real World Soils Raritan Bay Sediment Newark Bay Sediment Tittabawasse River Soil

### SW-846 Update V 4430 - 19 **Revision 1 October 2012**  

### GC-MS Procept Screen at 50 pg/g GC-MS Procept Screen at 50 pg/g TEQ (pg/g) TEQ (pg/g) Result TEQ (pg/g) TEQ (pg/g) Resul t 25 22 True Negative 25 30 True Negative 25 29 True Negative 25 22 True Negative 25 29 True Negative 25 26 True Negative 25 28 True Negative 25 21 True Negative 25 35 True Negative 25 32 True Negative 25 22 True Negative 25 28 True Negative 75 80 True Positive 75 74 True Positive 75 90 True Positive 75 59 True Positive 75 103 True Positive 75 54 True Positive 75 82 True Positive 75 75 True Positive 75 83 True Positive 75 84 True Positive 75 52 True Positive 75 65 True Positive Table 5. Example False Positive and Negative Rates Near Action Level (50pg/g) Spiked Sand Spiked Soil

### SW-846 Update V 4430 - 20 **Revision 1 October 2012**  Spiked Sample 1 

### Mean Procept Standard (pg/g TEQ) Response (ppt) 2 

### Deviation 2 

### % RSD 2,3 

### % Recovery 4 

### Blank 0.8 0.1 17 N/A **5.0 6.3 0.7 12 126 25 26 5.0 20 104**  **50 46 10 22 91 75 70 15 21 93**  **500 720 130 18 143 1000 1400 260 19 138**  **1500 1900 460 24 127 1**  

### Sand matrix spiked with 7 Dibenzo- p **-Dioxin/Furan Standards (tetra-hexachloro) 2**  

### Eight measurements at each concentration on single lot of test kit on same day **3** 

### Relative standard deviation (standard deviation*100%/mean value) **4** 

### (Mean AhR-PCR Response)*100%/Spike Concentration (pg/g TEQ)

Table 6. Same lot variation of Measurement

### SW-846 Update V 4430 - 21 **Revision 1 October 2012**  Spiked Sample 1 mean mean mean Inter-lot Mean AhR-PCR Inter-lot (pg/g TEQ) lot 101806 2 lot 01106 2 lot 033006 2 Response (pg/g TEQ) % RSD 3 **Blank 0.8 0.3 0.4 0.5 61 5.0 6.3 8.4 8.2 7.6 15**  **25 26 32 36 31 16 50 46 51 43 46 9**  **75 70 62 83 72 15 500 720 560 620 630 13**  **1000 1400 1200 1400 1300 9 1500 1900 1400 1500 1600 17**  **1 Sand matrix spiked with 7 Dibenzo-**  p **-Dioxin/Furan Standards (tetra-hexachloro) 2**  **Mean of Four to Eight measurements on each lot**  **3** **Relative standard deviation (standard deviation*100** **%/mean value)** Table 7. Variation of Measurement Between 3 Lots

### SW-846 Update V 4430 - 22 **Revision 1 October 2012**  **GLOSSARY 2,3,7,8-TCDD - 2,3,7,8-tetrachlorodibenzo-**  p **-dioxin AhR (aryl hydrocarbon receptor) -- A chemical-respo**  **nsive DNA-binding protein that is responsible for producing the toxic and biological**  **effects of dioxin and related chemicals ARNT - Aryl hydrocarbon receptor nuclear translocat**  **or protein Congener - Compounds containing different numbers a**  **nd position of chlorine atoms (or other substituent) on the same base structure.**  **Ct (threshold cycle) - This is the output of the re al-time PCR instrument. Threshold cycle**  **corresponds to the number of PCR temperature cycles at which the measured fluorescence of a**  **sample well exceeds a threshold value. The thresho ld value is set by the user, or software, to**  **optimize the linearity of the standard curve. Comp aring the Ct of an unknown sample to an**  **appropriate standard curve allows the analyst to re late Ct to concentration or TEQ.**  **DRE - DNA response element PAHs - Polycyclic aromatic hydrocarbons**  **PCBs - Polychlorinated biphenyls PCDD/PCDFs - Polychlorinated dibenzo-**  p **-dioxins and polychlorinated dibenzofurans. PCR (polymerase chain reaction) - A technique emplo**  **yed to replicate and measure the concentration of DNA fragments.**  **TEF (toxicity equivalent factor) - Toxicity values established by the World Health Organization**  **(WHO) for chlorinated dibenzo-** p **-dioxins and furans and polychlorinated biphenyls r elative to**  **2,3,7,8-tetrachlorodibenzo-** p **-dioxin. TEQ (toxicity equivalent quotient) - The sum of the**  **concentration of dioxin congeners multiplied by their TEF value. TEQ = ∑(congener concentration**  **) x (TEF).**

### SW-846 Update V 4430 - 23 **Revision 1 October 2012**  

## Appendix A: **Summary of Revisions to Method 4430 (as compared to previous Revision 0, December 2007)**  **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. Sec. 9.2.1 was added to discuss the need for ini**  **tial demonstration of proficiency (IDP).**