**SW-846 Update V 4025 - 1 Revision 1**

**October 2012 METHOD 4025** 

**SCREENING FOR POLYCHLORINATED DIBENZODIOXINS AND P OLYCHLORINATED**  **DIBENZOFURANS (PCDD/PCDFs) BY IMMUNOASSAY**

**SW-846 is not intended to be an analytical training manual. Therefore, method procedures**  **are written based on the assumption that they will be performed by analysts who are formally**  **trained in at least the basic principles of chemica l 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 guida nce 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 procedure for the analysis of polychlorinated dibenzodioxins**  **and polychlorinated dibenzofurans (PCDDs/PCDFs) in soil at 500 ppt (pg/g) using a simple one**  **step liquid phase oxidative cleanup or, at mid to l ow ppt levels, using a two step coupled column**  **cleanup (oxidation and activated carbon binding) ad apted from Method 8290. The analysis**  **method uses a commercially-available enzyme immunoa ssay (EIA) test kit containing a**  **polyclonal antibody specific for PCDDs/PCDFs. The EIA kit is designed for the screening of**  **samples according to their toxic equivalent concent ration (TEQ) by responding to the toxic**  **PCDD/PCDF congeners in approximate correlation with their toxic equivalency factors (TEFs).**  **The test is capable of multiple congener recognitio n and preferentially targets congeners with**  **high TEF values; i.e., those with the highest toxic ity relative to 2,3,7,8-tetrachlorodibenzo-**  p

**** **dioxin (2,3,7,8-TCDD). The final measured EIA resp onse is the sum of the individual congener**  **responses. This response correlates with TEQ becau se the immunoassay cross-reaction profile** 

**for PCDDs/PCDFs correlates with TEF values. See Ta ble 2 for a detailed cross-reaction profile.** 

**1.2 The testing product evaluated for this method e mploys a competitive enzyme**  **immunoassay. It is important to note that this met hod differs from other 4000 series**  **immunoassay methods due to the specific chemistry o f the analyte. The chemistry of dioxin**  **analysis is more difficult, as shown by the cost an d turnaround time differences between**  **dioxin/furan analysis (e.g., Methods 8280/8290) and PCB analysis (e.g., Method 8082). Dioxins**  **are 3 to 4 orders of magnitude less water soluble t han most other 4000 series target analytes.**  **Additionally, sensitivity targets can be orders of magnitude lower than for PCBs and other**  **analytes, so the matrix concentration factors can b e extreme and extracts generally require**  **partial cleanup**

.

**1.3 The PCDD/PCDF congener composition of samples c an be highly variable.**  **Because PCDDs/PCDFs are formed unintentionally by a variety of chemical and combustion**  **processes, samples usually contain a mixture of man y different congeners. Samples from**  **different sources often have very different mixture s of congeners which are consistent within the**  **source. In most samples, the majority of the PCDD/ PCDF mass present does not contribute**  **significantly to the total sample TEQ. Also, in mo st samples, only a few PCDD/PCDF congeners**  **are responsible for the majority of the TEQ. SW-846 Update V 4025 - 2 Revision 1** 

**October 2012 1.4 Detection for the test kit from which this meth**  **od was developed is based upon structure. Strong recognition by the antibody used**  **in this immunoassay kit generally requires**

**both the dioxin/furan core structure and the 2,3,7, 8- chlorination pattern. For example, test**  **response to non-toxic PCDDs/PCDFs is greatly reduce d because of their deviation from this**  **chlorination pattern. Recognition of the PCBs that are most similar to 2,3,7,8-TCDD (PCBs 77**  **and 126) is very limited because the core structure to which the chlorines are attached is not a** 

**dioxin or furan. When both core structure and chlo rination pattern are changed, as for PCB 153,**  **there is no detectable recognition. See Table 2 fo r more detailed information on cross-reactivity.** 

**1.5 The sensitivity submitted by the manufacturer o f this testing product is approx. 4**  **pg of 2,3,7,8-TCDD, based on a standard curve from 3.2 to 100 pg per EIA tube. The ultimate**  **limit of detection in soil and sediment samples is determined by the amount of sample extracted,**  **multiplied by the fraction of that prepared sample extract which is added to the EIA tube. The**  **EIA sensitivity is sufficient for analysis at 10 pp t (pg/g) TEQ using a 5 g sample (or 5 ppt using a**  **10 g sample), prepared using the two step column cl eanup (Refs. 7, 8). Analysis based on the**  **single step oxidative cleanup is designed to provid e screening at 500 ppt. Data supporting this**  **sensitivity are given in Table 1.**

**1.6 This immunoassay is designed to measure sample TEQ by responding to the**  **toxic PCDD/PCDF congeners in correlation with their TEFs. Variation in accuracy among**  **samples may occur solely because of the variability of congener composition noted above. To**  **maximize accuracy, the variability of congener comp osition in the target sample population**  **should be known. Best method performance is achieve d when all samples are from a single**  **group that shares as many properties as possible (c ommon source of contamination, similar**  **congener composition, similar sample matrix, etc.). Data supporting this approach are given in** 

**Ref. 7 and 8. However, without such information it is still possible to use this method for blind**  **screening at mid to low ppt levels. Data supporting this approach are given in Ref. 9.** 

**1.7 Prior to employing this method, analysts are ad vised 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**  **intended flexibility in the choice of methods, apparatus, materials, reagents, and supplies, and o**  **n 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 th at, except where explicitly required 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 s upervision of, personnel**  **appropriately experienced and trained in the perfor mance and interpretation of immunoassay**  **methods. Each analyst must demonstrate the ability to generate acceptable results with this** 

**method. SW-846 Update V 4025 - 3 Revision 1** 

**October 2012 2.0 SUMMARY OF METHOD** 

**2.1 Sample preparation and analysis procedures are described in the** 

**manufacturer’s literature. In addition, EIA test k its and sample preparation kits are commercially** 

**available for this method and the kit instructions used to develop this method can be**  **downloaded from the following Web address: www.cap e-tech.com**  **. Using the EIA test kit from which this method was developed, the following step**  **s are followed for the two different extraction and cleanup methods (using sample prepar**  **ation kits from the same manufacturer); Section 2.1.1 for the one step oxidative cleanup an**  **d Section 2.1.2 for the two step column cleanup.** 

**2.1.1 One Step Oxidative Cleanup**

**2.1.1.1 For the less sensitive analysis using the one step oxidative**  **cleanup, sodium sulfate is added to a soil sample a nd mixed. Dimethylformamide (DMF)**  **is added to the soil sample and the soil is extract ed by shaking for two hours. The**  **supernatant DMF extract is removed. DMF extracts a re stable for weeks to months at** 

**room temperature.**

**2.1.1.2 Interferences are removed by chemical oxi dation. Hexane**  **is added to an aliquot of the DMF extract, then tre ated with 15% SO**  **3 in concentrated** 

**H 2**  **SO 4**  **(fuming sulfuric acid). The supernatant hexane is removed and exchanged to a**  **water-miscible organic solvent solution. This hexa ne-based fuming sulfuric acid cleanup**  **is sufficient for most samples, but in certain circ umstances an additional cleanup step**  **may be required. This is the case for samples that contain large amounts of non-volatile**  **aliphatic oils. When the DMF extracts of such soil s are cleaned using fuming sulfuric** 

**acid, the oil is not oxidized, and it remains after evaporation of the hexane, causing a**  **biphasic system when introduced to the EIA first in cubation. Such EIA samples appear**  **opalescent or milky and their results will be inval id because the biphasic system prevents**  **capture of analyte by the antibody. For these samp les, a new aliquot of DMF extract is**  **cleaned by carbon adsorption or a new soil sample i s extracted and cleaned using the**  **more sensitive method (Sec. 2.1.2). In either case , the final solvent in the cleanup**  **procedure is toluene rather than hexane.**

**2.1.2 Two Step Column Cleanup**

**2.1.2.1 For the more sensitive analysis using the two step coupled**  **column cleanup, sodium sulfate is added to a soil s ample and mixed. A mixture of 1:1**  **hexane:acetone (HA) is added to the soil sample and the soil is extracted by shaking for**  **4 to 6 hours. The supernatant HA extract is remove d for immediate processing.** 

**2.1.2.2 Polar interferences are removed by oxida tion and non**  **polar interferences are removed by non-binding duri ng the adsorption of the**  **PCDDs/PCDFs on activated carbon. The two step clea nup is as follows: The HA extract**  **is evaporated to a tetradecane keeper to remove the acetone, which is reactive with**  **concentrated sulfuric acid (H 2**  **SO 4**  **), then the tetradecane solution is diluted with he xane.**  **Polar interferences are removed by chemical oxidati on (Ref. 11) by first pretreating with**  **loose bulk acid silica (conc. sulfuric acid adsorbe d to chromatographic silica) until the**  **liquid phase is clear. The pretreated sample is th en added to an acid silica column**  **coupled to an activated carbon column. Pressure is applied until the entire sample** 

**enters the acid silica column. Hexane is then adde d to the acid silica column and the**  **system is pressurized again to force the sample and additional hexane through the**  **carbon column. The sample and hexane washes are di scarded after passing through** **SW-846 Update V 4025 - 4 Revision 1**

**October 2012 the columns. Non-polar interferences, which are no**  **t reactive with the acid silica, are removed by**  **passing the extract through an activated carbon col umn, which captures the**  **PCDDs/PCDFs while allowing non-aromatic non-polar c ompounds to flow through**  **without binding. The carbon column, which contains the adsorbed PCDDs/PCDFs, is** 

**then removed and washed with 1:1 hexane:toluene in the forward direction, removing**  **more interferences, including the lower chlorinated PCDDs/PCDFs and dioxin-like PCBs**  **(Refs.10, 11). The carbon column is then eluted wi th toluene in the reverse direction.**  **The captured toluene eluate is then exchanged to a water-miscible organic solvent**  **solution. This cleanup is sufficient, in terms of both recovery and interference removal,**  **for analysis of most samples by HRGC-HRMS as well a s by EIA (Ref.12).** 

**2.2 The cleaned sample in hexane or toluene is exch anged to a water-miscible**  **organic solvent solution for EIA analysis. PCDDs/P CDFs have very low volatility and are**  **retained during this solvent exchange in a small vo lume of keeper (Triton X-100 detergent in**  **polyethylene glycol [PEG]) after evaporation of the original solvent. Methanol is added to dilute** 

**this solution and the methanol-PEG-Triton mixture a dded directly to the EIA tubes. It should be**  **noted that the literature value for solubility of 2 ,3,7,8-TCDD in methanol is 10 ppm, which is 5000**  **times higher than the concentration of the highest standard recommended for the kit used to**  **develop this method. Additionally, the solubility of PCDDs/PCDFs in methanol is augmented**  **significantly by the addition of PEG and Triton X-1 00.** 

**2.3 An accurately measured volume of negative contr ol, standard or prepared**  **sample is mixed with an aqueous sample diluent in t est tubes with anti-dioxin antibody** 

**immobilized on the surface. The mixture is incubat ed at the temperature, and for the time,** 

**described in the manufacturer ’**  **s instructions.**

**2.4 After incubation, antibody tubes are washed and 0.5 mL of Horseradish**  **Peroxidase (HRP) Competitor Conjugate is added to e ach tube using a repeater pipettor.**  **Bound PCDDs/PCDFs occupy the dioxin binding sites o f the antibodies in proportion to the**  **PCDD/PCDF content of the sample and prevent binding of the competitor-HRP conjugate. After**  **a short incubation, unbound conjugate is removed an d the test tubes are washed thoroughly.** 

**2.5 A solution of chromogenic HRP substrate and hyd rogen peroxide is added to**  **the test tubes. Color development is directly prop ortional to enzyme concentration and inversely**  **related to the PCDD/PCDF concentration in the origi nal sample. Stop solution is added to each**  **tube using a repeater pipettor in order to fix the amount of color development.** 

**2.6 The test tubes are analyzed using a tube reader or spectrophotometer to**  **measure the optical density (OD) at 450 nm. The te st is interpreted by measuring the signal**  **produced by a sample and determining the concentrat ion from a dose-response curve** 

**constructed from standards tested at the same time.**

**3.0 DEFINITIONS**

**See Method 4000 for a glossary of basic immunoassay terms and see the glossary at the**  **end of this method for procedure-specific terms. A lso refer to the manufacturer's instructions,**  **Chapter One and Methods 8280 and 8290 for other def initions that may be relevant.** **SW-846 Update V 4025 - 5 Revision 1**

**October 2012 4.0 INTERFERENCES** 

**4.1 The anti-dioxin antibody noted in the kit used to develop this method, binds to**  **different PCDD/PCDF congeners with different affini ties. The specificity of the test is**  **predominantly for PCDDs/PCDFs which contain 3 to 6 chlorines, with a strong preference for the**  **2,3,7,8 chlorinated congeners. Cross reactivity da ta showing test recognition relative to 2,3,7,8-**  **TCDD are given in Table 2. These data demonstrate a very restricted recognition profile,**  **indicating very limited potential for positive inte rference due to specific binding. Negative**  **interference is possible through indirect action on analyte solubilization by substances that do**  **not bind to the analyte-binding sites of the antibo dy. Sample preparation and cleanup**  **procedures required for this method reduce the pote ntial for positive and negative interference**  **by removal of all readily oxidizable organic compou nds. Substances stable to fuming sulfuric**  **acid, that are also extracted by the extraction sol vent, may also cause interference. These can**  **be removed by an additional cleanup step as noted i n 2.1.1.2 and summarized in 2.1.2.2. The**  **more rigorous column cleanup described in 2.1.2.2 r emoves both positive and negative**  **interferences well enough that these samples can be analyzed by Method 8290 (Ref. 10).** 

**4.2 The antibody used in this immunoassay recognize s PCDD/PCDF congeners** 

**based on structure, not mass. Therefore, conventio nal stable isotope labeled internal standards**  **are detected as native material. Typical levels of conventional stable isotope labeled internal** 

**standards cannot be used with this EIA. Consult th e manufacturer’s literature for specific**  **recommendations regarding mass labeled internal sta ndards.** 

**4.3 Non-specific interferences such as sample pH, t emperature, osmolarity,**  **solvents, surfactants, and the presence of metal io ns can affect immunoassay performance.**  **Samples should be tested at the pH and temperature range specified by the testing product**  **manufacturer. Review the product literature with r egard to other potential interferences.** 

**4.4 Storage temperatures may alter the useful life of the testing product reagents**  **and supplies. Follow the manufacturer's directions for storage and use of all reagents and**  **supplies.**

**5.0 SAFETY**

**5.1 This method does not address all safety issues associated with its use. The**  **user is responsible for maintaining a safe work env ironment 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 The commercially-available test kits should onl y be used by properly trained**  **personnel in an appropriate laboratory environment.**

**5.3 Treat PCDDs/PCDFs, solutions that contain PCDDs /PCDFs, and potentially**  **contaminated samples as hazardous materials.**

**5.4 Use gloves, proper protective clothing, and mea ns to contain and handle**  **hazardous material where appropriate.**

**5.5 Obtain (if appropriate) permits pertaining to t he handling, analysis and transport**  **of dioxin-containing materials. SW-846 Update V 4025 - 6 Revision 1** 

**October 2012 5.6 Stop solution is 1N hydrochloric acid. Handle**  **carefully.**

**5.7 Fuming sulfuric acid, concentrated sulfuric aci d, and acid silica are corrosive**  **and hygroscopic; carefully follow handling and stor age instructions.** 

**6.0 EQUIPMENT AND SUPPLIES**

**6.1 Each test product will specify the apparatus an d materials provided, as well as**  **any additional apparatus and materials necessary fo r performance of the test. The**  **immunoassay testing products of SW-846 immunoassay methods were submitted to EPA,**  **evaluated by the Agency, and found to meet the perf ormance specification necessary for**  **inclusion in SW-846. As additional testing product s are evaluated by EPA and found to provide**  **equivalent performance, information will be made av ailable by the Office of Solid Waste**  **regarding those testing products that are capable o f meeting the performance specifications in**  **the methods (see http://www.epa.gov/epaoswer/hazwas te/test/pdfs/kits.pdf). However, the**  **methods will not be revised solely to include infor mation on additional testing products.**  **Descriptions and materials lists for products relev ant to the methods should be given in the** 

**manufacturer’s literature.**

**6.2 Most testing products provide the supplies spec ific to the immunoassay,**  **including the tubes or plates containing the immobi lized antibody, and the immunochemical**  **reagents. Do not mix the equipment, supplies, and reagents from the testing products for**  **different analytes or from the testing products fro m different manufacturers. Testing products**  **contain immunochemical reagents that are evaluated by the manufacturer on a lot-specific**  **basis. Do not mix the reagents from one lot with t hose from another lot unless expressly allowed**  **by the manufacturer. Other equipment that may be r equired, but is not supplied with the testing**  **product, includes common laboratory items such as p recision pipetting devices, vortex mixers,** 

**etc.**

**6.3 Additional information regarding equipment requ irements is located in the** 

**relevant manufacturer’s literature or can be downlo aded from their website or obtained by email.** 

**6.4 The immunoassay testing product listed below ha s been submitted to EPA,**  **evaluated by the Agency, and found to meet the perf ormance specifications necessary for**  **inclusion in SW-846. As additional testing product s are evaluated by EPA and found to provide**  **equivalent performance, information will be made av ailable by the Office of Solid Waste**  **regarding those testing products that are capable o f meeting the performance specifications in**  **this method (see http://www.epa.gov/epaoswer/hazwas te/test/pdfs/kits.pdf). However, this**  **procedure will not be revised solely to include inf ormation on additional testing products.**  **Descriptions and materials lists for products relev ant to this method are given in the** 

**manufacturer’s literature.**

**DF1 High Performance Dioxin/Furan Immunoassay Kit ( CAPE Technologies),**  **www.cape-tech.com. SW-846 Update V 4025 - 7 Revision 1** 

**October 2012 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 spe cifications 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 As with the equipment and supplies, each commer cially available testing**  **product will supply or specify the reagents necessa ry for successful completion of the test. This**  **includes the calibrators (standards) employed in th e immunoassay. Detailed information on**  **reagent requirements is given in the manufacturer ’**  **s literature. As noted in Sec. 6.2, do not mix the equipment, supplies, and reagents from the test**  **ing products for different analytes or from the testing products from different manufacturers. Sto**  **re all reagents and standards according to the manufacturer's instructions, and, where applicable,**  **discard any that are past the expiration date assigned by the manufacturer.** 

**8.0 SAMPLE COLLECTION AND STORAGE**

**Sample collection, preservation, and storage requir ements may vary by EPA program**  **and may be specified in the regulation or project p lanning 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**

**The immunoassay testing products employ very small sample volumes. Therefore,**  **sample collection procedures must focus on the amou nts and procedures necessary to ensure**  **that the sample is representative of the source.**

**The distribution of PCDDs/PCDFs can be extremely he terogeneous. The analyst is**  **responsible for ensuring that soil samples are homo geneous and representative of the area from**  **which they are taken. Subsamples selected for immu noassay analysis should be representative**  **of the samples taken from the site. Water content should be low enough to avoid the presence**  **of standing water in sample containers. If this is not possible, then standing water must**  **be removed before homogenizing for sub-sampling. Addi**  **tionally, visibly wet samples must be pre**  **dried before extraction (such as by placing on clea n paper towels) to guarantee that sodium**  **sulfate remains in excess during the extraction pro cedure. Samples which are insufficiently**  **dried will not be extracted effectively by either D MF or hexane:acetone, potentially giving false**  **negative results. QA samples for verification of a dequate sample drying are essential.** 

**8.2 Extract holding time**

**The sample extracts in DMF are stable for up to thr ee months when stored in a tightly**  **sealed vial at a temperature range of 2 oC - 6 oC ( 36 oF - 43 oF). Extracts are less stable when**  **stored at room temperature. Extracts in hexane:ace tone should not be stored because of their**  **volatility, but can be exchanged to tetradecane or a similar oil such as paraffin oil for storage.**  **Holding times beyond three months may be acceptable as long as it can be substantiated with**  **relevant performance data. SW-846 Update V 4025 - 8 Revision 1** 

**October 2012 8.3 Storage and use of kit** 

**8.3.1 Do not freeze test kit components or expose t hem to temperatures** 

**above 37 oC (99 oF).**

**8.3.2 If desiccant in tube bag is not blue, do not use kit; contact** 

**manufacturer.**

**8.3.3 Do not expose substrate to direct sunlight.**

**8.3.4 If substrate is blue before adding to EIA tub es, do not use; contact** 

**manufacturer.**

**8.3.5 Store all test kit components at 2 oC - 6 oC (36 oF - 43 oF) when not** 

**in use.**

**8.3.6 Storage at ambient temperature (20 oC - 27 oC or 68 oF - 81 oF) on** 

**the day of use or overnight before the day of use i s acceptable. Do**  **not store at ambient temperature for extended perio ds.** 

**8.3.7 Allow all reagents to reach ambient temperatu re (20 oC - 27 oC or 68** 

**oF - 81 oF) before beginning the test. This typica lly requires at least**  **60 min at ambient temperature to warm from recommen ded storage**  **conditions. Warming occurs faster if bottles and t ube bags are** 

**removed from the kit box.**

**8.3.8 Do not use test kit after the expiration date**

**.**

**8.3.9 Do not use components from one test kit with components from a** 

**different test kit.**

**9.0 QUALITY CONTROL**

**9.1 Follow the manufacturer's instructions for the quality control procedures specific**  **to use of the testing product. Also refer to Chapt er One for guidance on quality assurance (QA)**  **and quality control (QC) protocols that may be appl icable. Any effort involving the collection of**  **analytical data should include development of a str uctured and systematic planning document,**  **such as a Quality Assurance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which**  **translates project objectives and specifications in to directions for those that will implement the**  **project and assess the results.**

**9.2 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 . Each time samples are extracted,**  **cleaned up, and analyzed, and when there is a chang e in reagents, a method blank should be**  **prepared and analyzed for the compounds of interest as a safeguard against chronic laboratory**  **contamination. Because of the chemistry of PCDDs/P CDFs, the potential for poor extraction**  **and adsorptive loss of analyte during sample prepar ation procedures is much greater than with** 

**other 4000 series methods. Therefore, demonstratio n of analyte recovery is critical to the** 

**success of this method. SW-846 Update V 4025 - 9 Revision 1** 

**October 2012 9.2.1 Initial demonstration of proficiency** 

**Each laboratory must demonstrate initial proficien cy 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, Sectio n 9.3 for information on how to**  **accomplish a demonstration of proficiency.**

**9.3 Any method blanks, blank or matrix spike sample s, or replicate samples should**  **be subjected to the same analytical procedures (Sec . 11.0) as those used on actual samples.**  **For sample extracts that are cleaned up using this method, the associated quality control**  **samples must also be processed through this cleanup method.** 

**9.4 The commercially available testing product used to develop this method**  **represents a performance-based analytical technique . Therefore, it is imperative that the**  **manufacturer's instructions and specifications be f ollowed closely. Follow the manufacturer's**  **instructions for the testing product being used for the quality control procedures specific to that**  **testing product. The following discussion of quali ty control requirements relies heavily on the**  **analyst's knowledge and understanding of the manufa cturer's instructions.** 

**9.5 Stringent quality assurance protocols should be maintained throughout each**  **stage of the testing procedure; i.e., sample extrac tion, 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 antibody in this immunoassay recogn izes PCDD/PCDF congeners based on** 

**structure and not on mass. Therefore, conventional isotope-labelled internal standards are** 

**detected as native material and cannot be used with this method.** 

**9.6 Routine quality control procedures associated w ith this method include the**  **analyses of standards, matrix blank and spike sampl es, laboratory control samples, method**  **blanks, and duplicate or replicate analyses (as spe cified by the manufacturer). All of these**  **analyses must be conducted simultaneously, e.g., as part of the same batch of samples. A**  **batch of samples consists of up to 20 field samples prepared and analyzed at the same time, or**  **the maximum number of samples that can be analyzed along with the standards, controls, and**  **other analyses specified by the manufacturer using a single testing product, whichever is fewer.**  **The batch must include any duplicate or replicate a nalyses specified by the manufacturer as well**  **as all additional quality control tests specified i n this procedure.** 

**9.7 Other quality control considerations**

**9.7.1 Do not use testing products past their expira tion date.** 

**9.7.2 Do not mix the equipment, supplies, and reage nts from the testing** 

**products for different analytes or from the testing products from** 

**different manufacturers.**

**9.7.3 Use the testing products within the storage t emperature and** 

**operating temperature limits specified by the manufacturer.** **SW-846 Update V 4025 - 10 Revision 1**

**October 2012 10.0 CALIBRATION AND STANDARDIZATION** 

**See the manufacturer ’**  **s instructions for information on calibration and s tandardization.** 

**11.0 PROCEDURE**

**Follow the manufacturer’s instructions for the test kit being used.** 

**12.0 DATA ANALYSIS AND CALCULATIONS**

**12.1 As with the specific formats of the testing pr oducts and the reagents and**  **supplies, the specifics of the required calculation s may vary by manufacturer. Some testing**  **products may provide measuring devices such as opti cal density readers or spectrophotometers**  **and may include software for performing all the nec essary calculations. Other testing products**  **may require the analyst to plot results manually, u sing graph paper that may or may not be**  **provided with the testing product, and determine sa mple results by interpolation from a standard**  **curve. Whichever approach is used, the laboratory records (bench notes, etc.) should clearly**  **indicate how the results were obtained. Records sp ecific to each determination, whether in hard**  **copy or in electronic form, should be retained by t he laboratory to substantiate the results.** 

**12.2 Follow the manufacturer's instructions regardi ng calculation of all testing product**  **results. For each batch of samples, use the calibr ation curve generated concurrently with that** 

**EIA run.**

**13.0 METHOD PERFORMANCE**

**13.1 Performance data and related information shoul d be provided by the**  **manufacturer in the package insert.**

**13.2 In the case of this method (which may be used in either the field or the**  **laboratory), any test kits used must be able to mee t the performance specifications for the**  **intended application. However, required performanc e criteria for a particular testing product may** 

**be included in the manufacturer ’**  **s instructions.**

**13.3 Table 1 provides data of accumulated responses for 2,3,7,8-TCDD standards**  **over ten months, including QA acceptance ranges.**

**13.4 Table 2 summarizes the cross reactivity of var ious PCDD/PCDF and PCB**  **congeners relative to 2,3,7,8-TCDD.**

**13.5 Tables 3 and 4 present correlation data betwee n GC/MS and immunoassay for**  **soil samples from two contaminated sites.**

**13.6 Table 5 presents data from screening analysis of negative soil extracts spiked at**  **levels near the 500 pg/g target level.**

**13.7 Table 6 summarizes the accuracy and precision data from the screening**  **analysis noted in Sec. 13.4. SW-846 Update V 4025 - 11 Revision 1** 

**October 2012 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 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 that may be applicable to laboratories** 

**and research institutions consult** Less is Better: Laboratory Chemical Management for Waste

Reduction **, a free publication available from the American Ch emical Society (ACS), Committee**  **on Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229**  **0/pdf/WPCP_012290.pdf.**

**15.0 WASTE MANAGEMENT**

**The Environmental Protection Agency requires that l aboratory 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 dispo sal restrictions. For further information on**  **waste management, consult the ACS publication listed in Sec. 14.2.** 

**16.0 REFERENCES**

**1. DF1 High Performance Dioxin/Furan Immunoassay Ki t (CAPE Technologies),**  **www.cape-tech.com.**

**2. CAPE Technologies Application Notes and related product literature,**  **www.cape-tech.com.**

**3. Carlson, Robert, E., Harrison, Robert, O., Measu rement of PCDD/F TEQ by**  **Immunoassay: Concept Development and Validation. Organohalogen Compounds, vol.**  **5, Stockholm, Sweden (1998).**

**4. Harrison, Robert, O., Carlson, Robert, E., Measu rement of PCDD/F TEQ by**  **Immunoassay: Demonstration Using Real World Samples , Organohalogen Compounds,**  **vol. 5, Stockholm, Sweden (1998).**

**5. Harrison, Robert, O., Role of Quality Assurance in Immunoassay Methods Used for Field**  **Screening. Immunochemical Technology for Environme ntal Applications, American**  **Chemical Society, Washington D.C. (1997).**

**6. Van den Berg, et. al., “The 2005 World Health Or ganization Re-evaluation of Human and**  **Mammalian Toxic Equivalency Factors (TE Fs) of Dioxins and Dioxin-like Compounds,”**  **Toxicological Sciences, 93(2): 223-241 , July 7, 2006.** **SW-846 Update V 4025 - 12 Revision 1**

**October 2012 7. “Correlation between EIA Results and GC-MS TEQ,”**  **revised Figure 2 from document listed in Ref. 8. (www.cape-tech.com, Tech Referenc**  **es).**

**8. “Performance of the CAPE Technologies DF1 Dioxin /Furan Immunoassay Kit for Soil**  **and Sediment Samples,” EPA 540/R-08/002, February 2 008. (www.cape-tech.com, Tech**  **References).**

**9. “Technologies for Monitoring and Measurement of Dioxin and Dioxin-like Compounds in**  **Soil and Sediment (CAPE Technologies LLC, DF1; Diox in/Furan Immunoassay Kit; PCB**  **TEQ Immunoassay Kit),” EPA 540/R-05/004, March 2005 . (www.cape-tech.com).** 

**10. Technical Note TN-005, CAPE Technologies (www.c ape-tech.com, Technical Notes)** 

**11. Application Note AN-008, CAPE Technologies (www .cape-tech.com, Application Notes)** 

**12. “An Efficient and Green Cleanup System for Anal ysis of Dioxins/Furans, Dioxin-like**  **PCBs and PBDES,” (www.cape-tech.com)**

**13. Immunoassay Sample Prep Kit SP3-60, CAPE Techno logies (www.cape-tech.com,**  **Material Lists).**

**14. Sample Prep Starter Kit SP3-ST, CAPE Technologi es (www.cape-tech.com, Material**  **Lists).**



### 17.0 **TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA**

**The pages to follow contain the tables referenced b y this method.** **SW-846 Update V 4025 - 13 Revision 1**

**October 2012 TABLE 1** 

**SENSITIVITY AND REPRODUCIBILITY OF THE EIA STANDAR D CURVE**  **a**

**Standard Number 1**  **2 3**  **4**

**ng/mL 2378-TCDD in standard (50 mL per EIA tube) 0.064**  **0.2 0.64**  **2**

**pg 2378-TCDD per EIA tube 3.2**  **10 32**  **100**

**mean percent of negative control (%NC) 87**  **66 41**  **29**

**standard deviation (SD) 6**  **7 7**  **6**

**range of mean±2SD 74-99**  **51-80 27-55**  **17-40**

**a Data are accumulated responses for 2,3,7,8-TCDD sta**  **ndards over ten months. No sample matrix was present. A total of 41 tests were run i**  **n four different labs. The detection limit, which is** 

**approximated by the I85 or the concentration giving 85% of the negative control OD, was 3.9**  **± 1.4**  **pg/tube (mean±SD). The midpoint of the curve, defi ned as the I50 or the concentration giving**  **50% of the negative control OD, was 21.9±7.4 pg/tub e (mean±SD).** **SW-846 Update V 4025 - 14 Revision 1**

**October 2012 TABLE 2** 

**SPECIFICITY OF THE EIA a** 

**Compound Percent Crossreactivity** 

**Toxic Dioxin Congeners**

**2,3,7,8-TCDD 100** 

**1,2,3,7,8-PeCDD 105** 

**1,2,3,4,7,8-HxCDD 1.6** 

**1,2,3,6,7,8-HxCDD 7.9** 

**1,2,3,7,8,9-HxCDD 39** 

**1,2,3,4,6,7,8-HpCDD 0.7** 

**OCDD <0.001** 

**Toxic Furan Congeners**

**2,3,7,8-TCDF 20** 

**1,2,3,7,8-PeCDF 4.6** 

**2,3,4,7,8-PeCDF 17** 

**1,2,3,4,7,8-HxCDF 0.4** 

**1,2,3,6,7,8-HxCDF 1.0** 

**1,2,3,7,8,9-HxCDF 3.3** 

**2,3,4,6,7,8-HxCDF 4.9** 

**1,2,3,4,6,7,8-HpCDF 0.02** 

**1,2,3,4,7,8,9-HpCDF 0.9** 

**OCDF <0.001** 

**Other PCDD/PCDF Congeners**

**2,3-dichlorodibenzo-p-dioxin 0.13** 

**2,7-dichlorodibenzo-p-dioxin 0.003** 

**2,3-dichlorodibenzofuran 0.02** 

**2,7-dichlorodibenzofuran <0.002** 

**2,3,7-trichlorodibenzo-p-dioxin 24** 

**2,3,8-trichlorodibenzofuran 0.26** 

**1,2,3,4-TCDD <0.001** 

**1,2,3,4-TCDF <0.001** 

**1,3,6,8-TCDD 0.05** 

**1,3,6,8-TCDF 0.007** 

**PolyChlorinated Biphenyls**

**3,3',4,4' (PCB 77) 0.4** 

**3,3',4,4',5 (PCB 126) 0.5** 

**2,2',4,4',5 (PCB 153) <0.1** 

**3,3',4,4',5,5' (PCB 169) <0.1** 

**Aroclor 1254 <0.1** **SW-846 Update V 4025 - 15 Revision 1**

**October 2012 TABLE 2**  **(Continued)**

**Compound Percent Crossreactivity** 

**Other PCDD/PCDF Congeners**

**2,3-dichlorodibenzo-p-dioxin 0.13** 

**2,7-dichlorodibenzo-p-dioxin 0.003** 

**2,3-dichlorodibenzofuran 0.02** 

**2,7-dichlorodibenzofuran <0.002** 

**2,3,7-trichlorodibenzo-p-dioxin 24** 

**2,3,8-trichlorodibenzofuran 0.26** 

**1,2,3,4-TCDD <0.001** 

**1,2,3,4-TCDF <0.001** 

**1,3,6,8-TCDD 0.05** 

**1,3,6,8-TCDF 0.007** 

**PolyChlorinated Biphenyls**

**3,3',4,4' (PCB 77) 0.4** 

**3,3',4,4',5 (PCB 126) 0.5** 

**2,2',4,4',5 (PCB 153) <0.1** 

**3,3',4,4',5,5' (PCB 169) <0.1** 

**Aroclor 1254 <0.1** 

**a R**  **esponse curves were prepared for each congener as n oted. The percent crossreactivity = (((2,3,7,8-TCD**  **D**

**I50)** ) **(congener I50)) x 100). Values are typically base d on 2 to 4 independent curves, each containing at** 

**least 4 concentrations. SW-846 Update V 4025 - 16 Revision 1** 

**October 2012 TABLE 3** 

**CORRELATION BETWEEN IMMUNOASSAY SCREENING ANALYSIS AND TEQ AS** 

**DETERMINED BY HIGH RESOLUTION GAS CHROMATOGRAPHY-HI GH RESOLUTION**  **MASS SPECTROMETRY (HRGC-HRMS) a** 

**Sample ID ppt TEQ**  **EIA Result**

**1 94338**  **CP**

**2 1528119**  **CP**

**3 234492**  **CP**

**4 822885**  **CP**

**5 73750**  **CP**

**6 4733**  **CP**

**7 39**  **CN**

**8*** **6278 CP** 

**9 276**  **FP**

**10 2390**  **CP**

**11 2101**  **CP**

**12 30**  **CN**

**13 14**  **CN**

**14 210**  **CN**

**15 5860**  **CP**

**16 2191**  **CP**

**17 343**  **FP**

**18 18**  **CN**

**19 25**  **CN**

**20 70**  **CN**

**21 599**  **CP**

**22 217**  **CN**

**23 27**  **CN**

**24 43**  **CN**

**25 18**  **CN**

**26 18**  **CN**

**27 14**  **CN**

**28 26**  **CN**

**29 13**  **CN**

**30 14**  **CN**

**31 11**  **CN**

**32 579**  **CP**

**33 220**  **CN**

**34 13**  **CN**

**35 1501**  **CP SW-846 Update V 4025 - 17 Revision 1** 

**October 2012 TABLE 3**  **(Continued)**

**Sample ID ppt TEQ**  **EIA Result**

**36 3702**  **CP**

**37 248**  **CN**

**38 216**  **FP**

**39 1088**  **CP**

**40 1210**  **CP**

**41 13**  **CN**

**42 451**  **CN**

**43 16**  **FP**

**44 105**  **CN**

**45 16**  **CN**

**46 10**  **CN**

**47 211**  **FP**

**48 1725**  **CP**

**49 41**  **CN**

**50 551**  **CP**

**51 622**  **CP**

**52*** **26856 CP** 

**53 2122**  **CP**

**54 24**  **CN**

**55 31**  **CN**

**56 31**  **CN**

**a Fifty six soil samples from Site 1 were prepared an**  **d analyzed following the manufacturer ’**  **s instructions for the DF1 High Performance Dioxin/Furan Immunoass**  **ay Kit (CAPE Technologies). An empirically determined calibration factor of 1.07 was applied t**  **o all EIA results before semiquantitative scoring. Results by category are 29 correct negative (CN), 0**  **false negative (FN), 22 correct positive (CP), and 5**  **false positive (FP) (91% correct, 9% FP). Two immu noassay samples which appeared heterogeneous**  **or contained visible precipitate during the first E IA incubation were interpreted as giving invalid re**  **sults. These samples were put through an additional carbon**  **column cleanup for oil removal, were analyzed** **again by EIA, and the latter results reported (*).****SW-846 Update V 4025 - 18 Revision 1**

**October 2012 TABLE 4** 

**CORRELATION BETWEEN IMMUNOASSAY SCREENING ANALYSIS AND TEQ AS** 

**DETERMINED BY HIGH RESOLUTION GAS CHROMATOGRAPHY-HI GH RESOLUTION**  **MASS SPECTROMETRY (HRGC-HRMS) a** 

**ppt by GC-MS EIA run 1**  **EIA run 2**

**2730 CP**  **FN 

** 470** **FP FP** 

**65 CN**  **CN**

**1400 CP**  **CP**

**70 CN** 

**4900 CP**  **CP**

**245 FP** 

**7300 CP**  **CP**

**365 CN** 

**360 CN**  **CN**

**140000 CP**  **CP**

**7000 CP** 

**33 CN**  **CN**

**990 CP**  **CP**

**4100 CP**  **CP**

**205 CN** 

**360 CN**  **CN**

**65 CN**  **CN**

****** **actual value with no calibration adjustment was 497 ppt (run 1 was 630 ppt)** 

**8 CN, 7 CP, 2 FP, 1 FN (**) from 18 samples**

**a Eighteen soil samples from Site 2 were prepared and**  **analyzed following the manufacturer ’**  **s instructions for the DF1 High Performance Dioxin/Furan Immunoass**  **ay Kit (CAPE Technologies). No calibration adjustment factor was applied to EIA results before**  **semiquantitative scoring. None of these samples required additional cleanup before EIA analysis. S**  **everal days later, aliquots of 13 of the 18 DMF extracts were oxidized and analyzed in a second EIA**  **and the concentrations were calculated based on**

**a new standard curve. The mean coefficient of vari ation for the 13 pairs of duplicate ppt values was** 

**35%. SW-846 Update V 4025 - 19 Revision 1** 

**October 2012 TABLE 5** 

**EXAMPLE FALSE POSITIVE/FALSE NEGATIVE RATES a** 

**Spike Level**

**250 ppt spike 1000 ppt spike** 

**Oxidation Replicate Oxidation Replicate** 

**Sample # EIA Replicate**  **1 2**  **1 2** 

**13 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**18 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**19 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**25 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**27 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**28 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**29 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**30 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**31 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**34 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**41A 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**41B 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP**

**45 1**  **CN CN**  **CP CP** 

**2 CN**  **CN CP**  **CP SW-846 Update V 4025 - 20 Revision 1** 

**October 2012 TABLE 5**  **(Continued)**

**Spike Level**

**250 ppt spike 1000 ppt spike** 

**Oxidation Replicate Oxidation Replicate** 

**Sample # EIA Replicate**  **1 2**  **1 2** 

**46*** **1 CN** * **CN*** FN* FN*

**2 CN** * **CN*** FN* *FN *

**46 (oxid. x 2) 1**  **CP**

**2 CP** 

**Summary of FN/FP results (based on repeat oxidation data for sample 46):**  **Correct Negatives: 52 Correct Positives: 54 False Positives: 0 False Negatives: 0** 

**a False positive/false negative data for negative soi**  **l extracts spiked near the 500 ppt decision level. Fourteen site 1 soil extracts ranging from 10 to 26**  **ppt by GC-MS were spiked with 2,3,7,8-TCDD at levels equivalent to 250 and 1000 ppt in the origin**  **al sample. Extracts were oxidized and analyzed by EIA following the manufacturer**  **’ s instructions for the DF1 High Performance Dioxin/**  **Furan Immunoassay Kit (CAPE Technologies). Results for each run were**  **calculated according to the manufacturer**

= **s instructions. Controls consisting of DMF spiked at**  **both levels were processed with each run and used to correct for spike recovery. Individual EIA repl**  **icate ppt values were scored as positive if equal t o or**  **greater than 500 ppt and were scored as negative if less than 500 ppt. Each semi-quantitative score**  **below corresponds to one EIA tube. Sample 41A and 41B were field duplicates that appeared different**  **based on gross color and particle size. Extract 46 was nearly black in color and totally opaque. Ini**  **tial** **data for sample 46 (*) indicated false negatives at** **the high spike level (individual EIA replicates we re**  **401, 366, 366, and 332 ppt). The unusually dark ap pearance of the acid phase after the first oxidatio**  **n indicated that a second oxidation would be required**  **to reduce the interferences to the same level as t he**  **other samples. The analysis of sample 46 was repea ted using a second oxidation of the first hexane**  **supernatant (plus a fresh aliquot of DMF). The app earance of the second phase matched the other**  **samples. The individual EIA replicates of 1872 and 1318 ppt gave a correct positive interpretation (t**  **he corresponding twice oxidized DMF controls were 1068**  **and 846 ppt). SW-846 Update V 4025 - 21 Revision 1** 

**October 2012 TABLE 6** 

**EXAMPLE ACCURACY AND PRECISION DATA a** 

**A. Summary of quantitative ppt data from DMF contr ols** 

**spike level 2**  **1000 ppt spike**

**number of runs (2 or 4 EIA replicates within each run)**  **4 5** 

**mean±SD of within run means (ppt) 271±74**  **957±174**

**coefficient of variation of ppt 27%**  **18%**

**B. Summary of quantitative ppt data from spiked ex tracts (sample 46 data includes only 2x** 

**oxidation**

**spike level 250 ppt spike**  **1000 ppt spike**

**number of individual EIA replicates 54**  **56**

**overall mean±SD (ppt) 266±61**  **984±356**

**coefficient of variation of ppt 23%**  **36%**

**C. Coefficients of variation for ppt within runs (a ll EIA replicates at each spike level for each**  **run)**

**spiked extracts DMF controls** 

**number of runs 9**  **9**

**mean of within run %cv values 18%**  **15%**

**D. Summary of EIA replicate precision (both spike levels combined)** 

**spiked extracts DMF controls** 

**number of pairs of EIA replicates 57**  **11**

**mean of all EIA replicate %cv values for**

**ppt 10%**  **13%**

**a Summary of quantitative data on accuracy and precis**  **ion from the false positive/false negative experiment of Table 5. Data are based on four para**  **meter curves and calculated ppt values for each of 5 runs on 5 separate days. The accuracy and precis**  **ion shown in parts A and B support the semi quantitative screening method described here. Thes**  **e data also support quantitative use of the test in certain situations with sufficient quality assuranc**  **e samples. Note that the data of section B include variation among the 14 different soils, as well as**  **the intrinsic method variability. The precision da ta**  **shown in parts C and D support screening analysis b ased on un-replicated sample oxidation and un**  **replicated EIA tubes for both standards and samples**

**. SW-846 Update V 4025 - 22 Revision 1** 

**October 2012 GLOSSARY OF TERMS** 

**Congeners Compounds containing different numbers a nd positions of chlorination (or other** 

**single atom substituents) on the same base structur e. For example, dioxin**  **congeners all contain the same dibenzo-p-dioxin nuc leus, but are chlorinated to**  **different levels and in different ring positions.**

**Keeper A high boiling material used to keep analyt e in solution during sample** 

**evaporation. In GC methods, typically tetradecane or similar hydrocarbon. In this**  **method, a combination of detergent and high-boiling glycol used for exchanging**  **sample from a non-polar solvent to an aqueous syste m for EIA analysis.** 

**TEF Toxicity equivalency factor - Toxicity values given to several of the halogenated** 

**aryl hydrocarbons relative to the most toxic congen er, 2,3,7,8-tetrachlorodibenzo**  **p-dioxin (2,3,7,8 TCDD).**

**TEQ Toxicity equivalent concentration - A toxicit y weighted concentration which accounts** 

**for both the concentration of individual congeners and their different TEF values.** 

**Calculated as TEQ =** 3 **(congener concentration x congener TEF) for all co ngeners**  **having assigned TEF values. SW-846 Update V 4025 - 23 Revision 1** 

**October 2012 Appendix A:** 

**Summary of Revisions to Method 4025 (as compared to previous Revision 0, October 2002)** 

**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. The revision number was changed to 1 and the dat e published was changed to May** 

**2012. 3. Additional information provided concerning metho**  **d sensitivity (Sec. 1.5) and cleanup procedures (Sec. 2.0).**  **4. The Reference section (16) has been updated to i nclude more current documentation for** 

**the method.**

**5. Minor editorial and technical revisions were mad e throughout to improve method clarity.**  **6. This appendix was added showing changes from the previous revision.** 

**7. Section 9.2.1 was added to discuss the need for initial demonstration of proficiency**  **(IDP).**