SW-846 Update V 4025 - 1 Revision 1 

July 2014 

METHOD 4025 

 SCREENING FOR POLYCHLORINATED DIBENZODIOXINS AND POLYCHLORINATED

 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 chemical analysis and in the use of the subject 

technology. 

In addition, SW-846 methods, with the exception of required method use for the analysis of 

method-defined parameters, are intended to be guidance methods which contain general 

information on how to perform an analytical procedure or technique which a laboratory can use 

as a basic starting point for generating its own detailed standard operating procedure (SOP), 

either for its own general use or for a specific project application. The performance data 

included in this method are for guidance purposes only, and are not intended to be and must not 

be used as absolute QC acceptance criteria for the purposes of laboratory accreditation. 

1.0 SCOPE AND APPLICATION 

For a summary of changes in this version, please see Appendix A at the end of this 

document. 

1.1 This method is a procedure for the analysis of polychlorinated dibenzodioxins 

and polychlorinated dibenzofurans (PCDDs/PCDFs) in soil at 500 ppt (pg/g) using a simple onestep liquid phase oxidative cleanup or, at mid to low ppt levels, using a two step coupled column 

cleanup (oxidation and activated carbon binding) adapted from Method 8290. The analysis 

method uses a commercially-available enzyme immunoassay (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 concentration (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 recognition and preferentially targets congeners with 

high TEF values; i.e., those with the highest toxicity relative to 2,3,7,8-tetrachlorodibenzo-pdioxin (2,3,7,8-TCDD). The final measured EIA response is the sum of the individual congener 

responses. This response correlates with TEQ because the immunoassay cross-reaction profile 

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

1.2 The testing product evaluated for this method employs a competitive enzyme 

immunoassay. It is important to note that this method differs from other 4000 series 

immunoassay methods due to the specific chemistry of the analyte. The chemistry of dioxin 

analysis is more difficult, as shown by the cost and 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 than 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 be extreme and extracts generally require 

partial cleanup. 

1.3 The PCDD/PCDF congener composition of samples can be highly variable. 

Because PCDDs/PCDFs are formed unintentionally by a variety of chemical and combustion 

processes, samples usually contain a mixture of many different congeners. Samples from 

different sources often have very different mixtures 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 most samples, only a few PCDD/PCDF congeners 

are responsible for the majority of the TEQ. 
SW-846 Update V 4025 - 2 Revision 1 

July 2014 

1.4 Detection for the test kit from which this method 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 reduced 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 chlorination pattern are changed, as for PCB 153, 

there is no detectable recognition. See Table 2 for more detailed information on cross-reactivity.

1.5 The sensitivity submitted by the manufacturer of 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 ppt (pg/g) TEQ using a 5 g sample (or 5 ppt using a 

10 g sample), prepared using the two step column cleanup (Refs. 7, 8). Analysis based on the 

single step oxidative cleanup is designed to provide 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 composition in the target sample population 

should be known. Best method performance is achieved when all samples are from a single 

group that shares as many properties as possible (common 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 advised 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 acceptance criteria, calculations, and general 

guidance. Analysts also should consult the disclaimer statement at the front of the manual and 

the information in Chapter Two for guidance on the intended flexibility in the choice of methods, 

apparatus, materials, reagents, and supplies, and on the responsibilities of the analyst for 

demonstrating that the techniques employed are appropriate for the analytes of interest, in the 

matrix of interest, and at the levels of concern. 

In addition, analysts and data users are advised that, except where explicitly required in a 

regulation, the use of SW-846 methods is not mandatory in response to Federal testing 

requirements. The information contained in this method is provided by EPA as guidance to be 

used by the analyst and the regulated community in making judgments necessary to generate 

results that meet the data quality objectives for the intended application. 

1.8 This method is restricted to use by, or under supervision of, personnel 

appropriately experienced and trained in the performance 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 

July 2014 

2.0 SUMMARY OF METHOD 

2.1 Sample preparation and analysis procedures are described in the 

manufacturer’s literature. In addition, EIA test kits 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.cape-tech.com. Using the EIA test kit from 

which this method was developed, the following steps are followed for the two different 

extraction and cleanup methods (using sample preparation kits from the same manufacturer); 

Section 2.1.1 for the one step oxidative cleanup and 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 and mixed. 

Dimethylformamide (DMF) is added to the soil sample and the soil is extracted 

by shaking for two hours. The supernatant DMF extract is removed. DMF 

extracts are stable for weeks to months at room temperature. 

 2.1.1.2 Interferences are removed by chemical oxidation. Hexane 

is added to an aliquot of the DMF extract, then treated with 15% SO3 in 

concentrated H2SO4 (fuming sulfuric acid). The supernatant hexane is removed 

and exchanged to a water-miscible organic solvent solution. This hexanebased fuming sulfuric acid cleanup is sufficient for most samples, but in certain 

circumstances 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 soils 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 incubation. Such EIA samples appear 

opalescent or milky and their results will be invalid because the biphasic system 

prevents capture of analyte by the antibody. For these samples, a new aliquot 

of DMF extract is cleaned by carbon adsorption or a new soil sample is 

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 sample 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 removed for 

immediate processing. 

2.1.2.2 Polar interferences are removed by oxidation and nonpolar interferences are removed by non-binding during the adsorption of the 

PCDDs/PCDFs on activated carbon. The two step cleanup is as follows: The 

HA extract is evaporated to a tetradecane keeper to remove the acetone, which 

is reactive with concentrated sulfuric acid (H2SO4), then the tetradecane 

solution is diluted with hexane. Polar interferences are removed by chemical 

oxidation (Ref. 11) by first pretreating with loose bulk acid silica (conc. sulfuric 

acid adsorbed to chromatographic silica) until the liquid phase is clear. The 

pretreated sample is then added to an acid silica column coupled to an 

activated carbon column. Pressure is applied until the entire sample enters the 
SW-846 Update V 4025 - 4 Revision 1 

July 2014 

acid silica column. Hexane is then added 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 discarded after 

passing through the columns. Non-polar interferences, which are not reactive 

with the acid silica, are removed by passing the extract through an activated 

carbon column, which captures the PCDDs/PCDFs while allowing non-aromatic 

non-polar compounds 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 with 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 as 

by EIA (Ref.12). 

2.2 The cleaned sample in hexane or toluene is exchanged to a water-miscible 

organic solvent solution for EIA analysis. PCDDs/PCDFs have very low volatility and are 

retained during this solvent exchange in a small volume 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 added 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-100. 

 

2.3 An accurately measured volume of negative control, standard or prepared 

sample is mixed with an aqueous sample diluent in test tubes with anti-dioxin antibody 

immobilized on the surface. The mixture is incubated 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 each tube using a repeater pipettor. 

Bound PCDDs/PCDFs occupy the dioxin binding sites of 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 and the test tubes are washed thoroughly. 

2.5 A solution of chromogenic HRP substrate and hydrogen peroxide is added to 

the test tubes. Color development is directly proportional to enzyme concentration and inversely 

related to the PCDD/PCDF concentration in the original 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 test is interpreted by measuring the signal 

produced by a sample and determining the concentration 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. Also refer to the manufacturer's instructions, 

Chapter One and Methods 8280 and 8290 for other definitions that may be relevant. 
SW-846 Update V 4025 - 5 Revision 1 

July 2014 

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 affinities. 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 data 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 interference 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 antibody. Sample preparation and cleanup 

procedures required for this method reduce the potential for positive and negative interference 

by removal of all readily oxidizable organic compounds. Substances stable to fuming sulfuric 

acid, that are also extracted by the extraction solvent, may also cause interference. These can 

be removed by an additional cleanup step as noted in 2.1.1.2 and summarized in 2.1.2.2. The 

more rigorous column cleanup described in 2.1.2.2 removes 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 recognizes PCDD/PCDF congeners 

based on structure, not mass. Therefore, conventional 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 the manufacturer’s literature for specific 

recommendations regarding mass labeled internal standards. 

4.3 Non-specific interferences such as sample pH, temperature, osmolarity, 

solvents, surfactants, and the presence of metal ions 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 regard 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 environment and a current awareness file of 

OSHA regulations regarding the safe handling of the chemicals listed in this method. A 

reference file of material safety data sheets (MSDSs) should be available to all personnel 

involved in these analyses. 

5.2 The commercially-available test kits should only 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 means to contain and handle 

hazardous material where appropriate. 

5.5 Obtain (if appropriate) permits pertaining to the handling, analysis and transport 

of dioxin-containing materials. 
SW-846 Update V 4025 - 6 Revision 1 

July 2014 

5.6 Stop solution is 1N hydrochloric acid. Handle carefully. 

5.7 Fuming sulfuric acid, concentrated sulfuric acid, and acid silica are corrosive 

and hygroscopic; carefully follow handling and storage instructions. 

6.0 EQUIPMENT AND SUPPLIES 

6.1 Each test product will specify the apparatus and materials provided, as well as 

any additional apparatus and materials necessary for 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 performance specification necessary for 

inclusion in SW-846. As additional testing products are evaluated by EPA and found to provide 

equivalent performance, information will be made available by the Office of Solid Waste 

regarding those testing products that are capable of meeting the performance specifications in 

the methods (see http://www.epa.gov/epaoswer/hazwaste/test/pdfs/kits.pdf). However, the 

methods will not be revised solely to include information on additional testing products. 

Descriptions and materials lists for products relevant to the methods should be given in the 

manufacturer’s literature. 

6.2 Most testing products provide the supplies specific to the immunoassay, 

including the tubes or plates containing the immobilized 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 from 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 those from another lot unless expressly allowed 

by the manufacturer. Other equipment that may be required, but is not supplied with the testing 

product, includes common laboratory items such as precision pipetting devices, vortex mixers, 

etc. 

6.3 Additional information regarding equipment requirements is located in the 

relevant manufacturer’s literature or can be downloaded from their website or obtained by email. 

6.4 The immunoassay testing product listed below has been submitted to EPA, 

evaluated by the Agency, and found to meet the performance specifications necessary for 

inclusion in SW-846. As additional testing products are evaluated by EPA and found to provide 

equivalent performance, information will be made available by the Office of Solid Waste 

regarding those testing products that are capable of meeting the performance specifications in 

this method (see http://www.epa.gov/epaoswer/hazwaste/test/pdfs/kits.pdf). However, this 

procedure will not be revised solely to include information on additional testing products. 

Descriptions and materials lists for products relevant 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 

July 2014 

7.0 REAGENTS AND STANDARDS 

7.1 Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, 

it is intended that all reagents conform to the specifications of the Committee on Analytical 

Reagents of the American Chemical Society, where such specifications are available. Other 

grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity 

to permit its use without lessening the accuracy of the determination. Reagents should be 

stored in glass to prevent the leaching of contaminants from plastic containers. 

7.2 As with the equipment and supplies, each commercially available testing 

product will supply or specify the reagents necessary for successful completion of the test. This 

includes the calibrators (standards) employed in the 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 testing products for different analytes or from the 

testing products from different manufacturers. Store 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 requirements may vary by EPA program 

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

monitoring for a given contaminant. Where such requirements are specified in the regulation, 

follow those requirements. In the absence of specific regulatory requirements, use the following 

information as guidance in determining the sample collection, preservation, and storage 

requirements. 

8.1 Sample collection 

The immunoassay testing products employ very small sample volumes. Therefore, 

sample collection procedures must focus on the amounts and procedures necessary to ensure 

that the sample is representative of the source. 

The distribution of PCDDs/PCDFs can be extremely heterogeneous. The analyst is 

responsible for ensuring that soil samples are homogeneous and representative of the area from 

which they are taken. Subsamples selected for immunoassay 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. Additionally, visibly wet samples must be predried before extraction (such as by placing on clean paper towels) to guarantee that sodium 

sulfate remains in excess during the extraction procedure. Samples which are insufficiently 

dried will not be extracted effectively by either DMF or hexane:acetone, potentially giving false 

negative results. QA samples for verification of adequate sample drying are essential. 

8.2 Extract holding time 

The sample extracts in DMF are stable for up to three 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:acetone 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 

July 2014 

8.3 Storage and use of kit 

8.3.1 Do not freeze test kit components or expose them 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 tubes, 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 is acceptable. Do 

not store at ambient temperature for extended periods. 

8.3.7 Allow all reagents to reach ambient temperature (20 oC - 27 oC or 68 

oF - 81 oF) before beginning the test. This typically requires at least 

60 min at ambient temperature to warm from recommended storage 

conditions. Warming occurs faster if bottles and tube 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 Chapter One for guidance on quality assurance (QA) 

and quality control (QC) protocols that may be applicable. Any effort involving the collection of 

analytical data should include development of a structured and systematic planning document, 

such as a Quality Assurance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which 

translates project objectives and specifications into directions for those that will implement the 

project and assess the results. 

9.2 Initially, before processing any samples, the analyst should demonstrate that all 

parts of the equipment in contact with the sample and 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 change 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/PCDFs, the potential for poor extraction 

and adsorptive loss of analyte during sample preparation procedures is much greater than with 

other 4000 series methods. Therefore, demonstration of analyte recovery is critical to the 

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

July 2014 

 9.2.1 Initial demonstration of proficiency 

Each laboratory must demonstrate initial proficiency with each sample 

preparation and determinative method combination it utilizes by generating data of 

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

such a demonstration will encompass the initial demonstration of proficiency described in 

method 3500, using a clean reference matrix. The laboratory must also repeat the 

demonstration of proficiency whenever new staff members are trained or significant 

changes in instrumentation are made. See Method 8000D, Section 9.3 for information 

on how to accomplish a demonstration of proficiency. 

9.3 Any method blanks, blank or matrix spike samples, 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 followed 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 quality control requirements relies heavily on the 

analyst's knowledge and understanding of the manufacturer's instructions. 

9.5 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 other 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 recognizes 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 with this method include the 

analyses of standards, matrix blank and spike samples, laboratory control samples, method 

blanks, and duplicate or replicate analyses (as specified 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 analyses specified by the manufacturer as well 

as all additional quality control tests specified in this procedure. 

9.7 Other quality control considerations: 

9.7.1 Do not use testing products past their expiration date. 

9.7.2 Do not mix the equipment, supplies, and reagents 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 temperature and 

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

July 2014 

10.0 CALIBRATION AND STANDARDIZATION 

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

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 products and the reagents and 

supplies, the specifics of the required calculations may vary by manufacturer. Some testing 

products may provide measuring devices such as optical density readers or spectrophotometers 

and may include software for performing all the necessary calculations. Other testing products 

may require the analyst to plot results manually, using graph paper that may or may not be 

provided with the testing product, and determine sample 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 specific to each determination, whether in hard 

copy or in electronic form, should be retained by the laboratory to substantiate the results. 

12.2 Follow the manufacturer's instructions regarding calculation of all testing product 

results. For each batch of samples, use the calibration curve generated concurrently with that 

EIA run. 

13.0 METHOD PERFORMANCE 

13.1 Performance data and related information should 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 meet the performance specifications for the 

intended application. However, required performance 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 various PCDD/PCDF and PCB 

congeners relative to 2,3,7,8-TCDD. 

13.5 Tables 3 and 4 present correlation data between 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 

July 2014 

14.0 POLLUTION PREVENTION 

14.1 Pollution prevention encompasses any technique that reduces or eliminates the 

quantity and/or toxicity of waste at the point of generation. Numerous opportunities for pollution 

prevention exist in laboratory operations. The EPA has established a preferred hierarchy of 

environmental management techniques that places pollution prevention as the management 

option of first choice. Whenever feasible, laboratory personnel should use pollution prevention 

techniques to address their waste generation. When wastes cannot be feasibly reduced at the 

source, the Agency recommends recycling as the next best option. 

 14.2 For information about pollution prevention that may be applicable to laboratories 

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

Reduction, a free publication available from the American Chemical Society (ACS), Committee 

on Chemical Safety,

http://portal.acs.org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_012290.pdf. 

15.0 WASTE MANAGEMENT 

The Environmental Protection Agency requires that laboratory waste management 

practices be conducted consistent with all applicable rules and regulations. The Agency urges 

laboratories to protect the air, water, and land by minimizing and controlling all releases from 

hoods and bench operations, complying with the letter and spirit of any sewer discharge permits 

and regulations, and by complying with all solid and hazardous waste regulations, particularly the 

hazardous waste identification rules and land disposal restrictions. For further information on 

waste management, consult the ACS publication listed in Sec. 14.2. 

16.0 REFERENCES 

1. DF1 High Performance Dioxin/Furan Immunoassay Kit (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., Measurement of PCDD/F TEQ by 

Immunoassay: Concept Development and Validation. Organohalogen Compounds, vol. 

5, Stockholm, Sweden (1998). 

4. Harrison, Robert, O., Carlson, Robert, E., Measurement 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 Environmental Applications, American 

Chemical Society, Washington D.C. (1997). 

6. Van den Berg, et. al., “The 2005 World Health Organization Re-evaluation of Human and 

 Mammalian Toxic Equivalency Factors (TEFs) of Dioxins and Dioxin-like Compounds,” 

 Toxicological Sciences, 93(2): 223-241, July 7, 2006. 
SW-846 Update V 4025 - 12 Revision 1 

July 2014 

7. “Correlation between EIA Results and GC-MS TEQ,” revised Figure 2 from document 

listed in Ref. 8. (www.cape-tech.com, Tech References). 

8. “Performance of the CAPE Technologies DF1 Dioxin/Furan Immunoassay Kit for Soil 

and Sediment Samples,” EPA 540/R-08/002, February 2008. (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; Dioxin/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.cape-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 Analysis of Dioxins/Furans, Dioxin-like 

PCBs and PBDES,” (www.cape-tech.com) 

13. Immunoassay Sample Prep Kit SP3-60, CAPE Technologies (www.cape-tech.com, 

Material Lists). 

14. Sample Prep Starter Kit SP3-ST, CAPE Technologies (www.cape-tech.com, Material 

Lists). 

17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 

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

July 2014 

TABLE 1 

 SENSITIVITY AND REPRODUCIBILITY OF THE EIA STANDARD 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 standards over ten months. No sample 

matrix was present. A total of 41 tests were run in 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, defined as the I50 or the concentration giving 

50% of the negative control OD, was 21.9±7.4 pg/tube (mean±SD). 
SW-846 Update V 4025 - 14 Revision 1 

July 2014 

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 

July 2014 

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 Response curves were prepared for each congener as noted. The percent crossreactivity = (((2,3,7,8-TCDD 

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

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

July 2014 

TABLE 3 

CORRELATION BETWEEN IMMUNOASSAY SCREENING ANALYSIS AND TEQ AS 

DETERMINED BY HIGH RESOLUTION GAS CHROMATOGRAPHY-HIGH 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 

July 2014 

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 and analyzed following the manufacturer’s instructions 

for the DF1 High Performance Dioxin/Furan Immunoassay Kit (CAPE Technologies). An empirically 

determined calibration factor of 1.07 was applied to 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 immunoassay samples which appeared heterogeneous 

or contained visible precipitate during the first EIA incubation were interpreted as giving invalid results. 

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 

July 2014 

TABLE 4 

 CORRELATION BETWEEN IMMUNOASSAY SCREENING ANALYSIS AND TEQ AS 

DETERMINED BY HIGH RESOLUTION GAS CHROMATOGRAPHY-HIGH 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 Immunoassay 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. Several 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 variation for the 13 pairs of duplicate ppt values was 

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

July 2014 

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 

July 2014 

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 soil 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 original 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 replicate ppt values were scored as positive if equal to 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. Initial 

data for sample 46 (*) indicated false negatives at the high spike level (individual EIA replicates were 

401, 366, 366, and 332 ppt). The unusually dark appearance of the acid phase after the first oxidation 

indicated that a second oxidation would be required to reduce the interferences to the same level as the 

other samples. The analysis of sample 46 was repeated using a second oxidation of the first hexane 

supernatant (plus a fresh aliquot of DMF). The appearance of the second phase matched the other 

samples. The individual EIA replicates of 1872 and 1318 ppt gave a correct positive interpretation (the 

corresponding twice oxidized DMF controls were 1068 and 846 ppt).
SW-846 Update V 4025 - 21 Revision 1 

July 2014 

TABLE 6 

EXAMPLE ACCURACY AND PRECISION DATA a

A. Summary of quantitative ppt data from DMF controls 

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 extracts (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 (all 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 precision from the false positive/false negative 

experiment of Table 5. Data are based on four parameter curves and calculated ppt values for each of 

5 runs on 5 separate days. The accuracy and precision shown in parts A and B support the semiquantitative screening method described here. These data also support quantitative use of the test in

certain situations with sufficient quality assurance samples. Note that the data of section B include

variation among the 14 different soils, as well as the intrinsic method variability. The precision data 

shown in parts C and D support screening analysis based on un-replicated sample oxidation and unreplicated EIA tubes for both standards and samples. 
SW-846 Update V 4025 - 22 Revision 1 

July 2014 

GLOSSARY OF TERMS 

Congeners Compounds containing different numbers and positions of chlorination (or other 

single atom substituents) on the same base structure. For example, dioxin 

congeners all contain the same dibenzo-p-dioxin nucleus, but are chlorinated to 

different levels and in different ring positions. 

Keeper A high boiling material used to keep analyte 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 system for EIA analysis. 

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

aryl hydrocarbons relative to the most toxic congener, 2,3,7,8-tetrachlorodibenzop-dioxin (2,3,7,8 TCDD). 

TEQ Toxicity equivalent concentration - A toxicity 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 congeners 

having assigned TEF values. 

 
SW-846 Update V 4025 - 23 Revision 1 

July 2014 

Appendix A: 

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

1. Improved overall method formatting for consistency with new SW-846 methods style 

guidance. The format was updated to Microsoft Word .docx. 

2. The revision number was changed to 1 and the date published was changed to July 

2014. 

3. Additional information provided concerning method sensitivity (Sec. 1.5) and cleanup 

procedures (Sec. 2.0). 

4. The Reference section (16) has been updated to include more current documentation for 

the method. 

5. Minor editorial and technical revisions were made 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).