METHODS OF ANALYSIS TO CONTROL UNDESIRABLE SUBSTANCES IN FEED
ANNEX VSupplementary provisions
ANNEX V METHODS OF ANALYSIS TO CONTROL UNDESIRABLE SUBSTANCES IN FEED A. DETERMINATION OF FREE AND TOTAL GOSSYPOL 1. Purpose and scope This method makes it possible to determine the levels of free gossypol, total gossypol and chemically related substances in cottonseed, cottonseed meal and cottonseed cake and in compound feed containing these feed materials where more than 20 mg/kg of free gossypol, total gossypol and chemically related substances are present. 2. Principle The gossypol is extracted in the presence of 3-aminopropan-1-ol, either with a mixture of propan-2-ol and hexane, for the determination of free gossypol, or with dimethylformamide, for the determination of total gossypol. The gossypol is converted by aniline into gossypol-dianiline, the optical density of which is measured at 440 nm. 3. Reagents 3.1. Propan-2-ol-hexane mixture: mix 60 parts by volume of propan-2-ol with 40 parts by volume of n -hexane. 3.2. Solvent A: Place in a 1 litre graduated flask approximately 500 ml of propan-2-ol-hexane mixture (3.1), 2 ml of 3-aminopropan-1-ol, 8 ml of glacial acetic acid and 50 ml of water. Make up to volume with the propan-2-ol-hexane mixture (3.1). This reagent is stable for one week. 3.3. Solvent B: Pipette 2 ml of 3-aminopropan-1-ol and 10 ml of glacial acetic acid into a 100 ml graduated flask. Cool to room temperature and make up to volume with N, N-dimethylformamide. This reagent is stable for one week. 3.4. Aniline: If the optical density in the blank test exceeds 0,022 , distil the aniline over zinc dust, discarding the first and last 10 % fractions of the distillate. Refrigerated and stored in a brown, stoppered glass flask, this reagent will keep for several months. 3.5. Standard gossypol solution A: Place 27,9 mg of gossypol acetate in a 250 ml graduated flask. Dissolve and make up to volume with solvent A (3.2). Pipette 50 ml of this solution into a 250 ml graduated flask and make up to volume with solvent A. The gossypol concentration of this solution is 0,02 mg/ml. Leave to stand for one hour at room temperature before use. 3.6. Standard gossypol solution B: Place 27,9 mg of gossypol acetate in a 50 ml graduated flask, Dissolve and make up to volume with solvent B (3.3). The gossypol concentration of this solution is 0,5 mg/ml. Standard gossypol solutions A and B will remain stable for 24 hours if protected from the light. 4. Apparatus 4.1. Mixer (tumbler): approximately 35 r.p.m. 4.2. Spectrophotometer. 5. Procedure 5.1. Test sample The amount of test sample used depends on the presumed gossypol content of the sample. It is preferable to work with a small test sample and a relatively large aliquot part of the filtrate, so as to obtain sufficient gossypol for precise photometric measurement to be possible. For the determination of free gossypol in cottonseed, cottonseed meal and cottonseed cake, the test sample shall not exceed 1 g; for compound feed, it may be as much as 5 g. A 10 ml aliquot part of filtrate is suitable in most cases; it shall contain 50 to 100 μg of gossypol. For the determination of total gossypol, the test sample shall be between 0,5 and 5 g, that a 2 ml aliquot part of filtrate will contain 40 to 200 μg of gossypol. The analysis shall be carried out at a room temperature of about 20 o C. 5.2. Determination of free gossypol Place the test sample in a ground-necked 250 ml flask, the bottom of the flask having been covered with crushed glass. Using a pipette, add 50 ml of solvent A (3.2), stopper the flask and mix for one hour in the mixer. Filter through a dry filter and collect the filtrate in a small ground-necked flask. During filtration, cover the funnel with a watch glass. Pipette identical aliquot parts of filtrate containing 50 to 100 μg of gossypol into each of two 25 ml graduated flasks (A and B). If necessary, make up the volume to 10 ml with solvent A (3.2). Then make the contents of flask (A) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the sample solution. Pipette 10 ml of solvent A (3.2) into each of two other 25 ml graduated flasks (C and D). Make the contents of flask (C) up to volume with the propan-2-ol-hexane mixture (3.1). This solution will be used as a reference solution against which to measure the blank test solution. Add 2 ml of aniline (3.4) to each of flasks (D) and (B). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour. Determine the optical density of the blank test solution (D) by comparison with the reference solution (C), and the optical density of the sample solution (B) by comparison with the reference solution (A), in the spectrophotometer at 440 nm using 1 cm glass cells. Subtract the optical density of the blank test solution from that of the sample solution (= corrected optical density). From this value calculate the free gossypol content as indicated in 6. 5.3. Determination of total gossypol Place a test sample containing 1 to 5 mg of gossypol in a 50 ml graduated flask and add 10 ml of solvent B (3.3). At the same time, prepare a blank test, placing 10 ml of solvent B (3.3) in another 50 ml graduated flask. Heat the two flasks for 30 minutes over a boiling water bath. Cool to room temperature and make the contents of each flask up to volume with the propan-2-ol-hexane mixture (3.1). Homogenise and leave to settle for 10 to 15 minutes, then filter and collect the filtrates in ground-necked flasks. Pipette 2 ml of the sample filtrate into each of two 25 ml graduated flasks, and 2 ml of the blank test filtrate into each of two other 25 ml flasks. Make the contents of one flask from each series up to 25 ml with the propan-2-ol-hexane mixture (3.1). These solutions will be used as reference solutions. Add 2 ml of aniline (3.4) to each of the other two flasks. Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to 25 ml with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour. Determine the optical density as indicated in 5.2 for free gossypol. From this value calculate the total gossypol content as indicated in 6. 6. Calculation of results Results may be calculated either from the specific optical density (6.1), or by reference to a calibration curve (6.2). 6.1. From the specific optical density The specific optical densities, under the conditions described, will be the following: Free gossypol: Total gossypol: The free or total gossypol content of the sample is calculated by using the following formula: where: E = corrected optical density, determined as indicated in 5.2, p = test sample in g, a = aliquot part of the filtrate in ml. 6.2. From a calibration curve 6.2.1. Free gossypol Prepare 2 series of five 25 ml graduated flasks. Pipette aliquots of 2,0, 4,0, 6,0, 8,0 and 10,0 ml of standard gossypol solution A (3.5) into each series of flasks. Make up the volumes to 10 ml with solvent A (3.2). Complete each series with a 25 ml graduated flask containing only 10 ml of solvent A (3.2) (blank test). Make the volume of the flasks in the first series (including the flask for the blank test) up to 25 ml with the propan-2-ol-hexane mixture (3.1) (reference series). Add 2 ml of aniline (3.4) to each flask in the second series (including the flask for the blank test). Heat for 30 minutes over a boiling water bath to develop the colour. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour (standard series). Determine as indicated in 5.2 the optical density of the solutions in the standard series by comparison with the corresponding solutions in the reference series. Trace the calibration curve by plotting the optical densities against the quantities of gossypol (in μg). 6.2.2. Total gossypol Prepare six 50 ml graduated flasks. In the first flask place 10 ml of solvent B (3.3), and in the others 2,0, 4,0, 6,0, 8,0 and 10,0 ml of standard gossypol solution B (3.6) respectively. Make the contents of each flask up to 10 ml with solvent B (3.3). Heat for 30 minutes over a boiling water bath. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1) and homogenise. Place 2,0 ml of these solutions in each of two series of six 25 ml graduated flasks. Make the contents of the flasks in the first series up to 25 ml with the propan-2-ol-hexane mixture (3.1) (reference series). Add 2 ml of aniline (3.4) to each flask in the second series. Heat for 30 minutes over a boiling water bath. Cool to room temperature, make up to volume with the propan-2-ol-hexane mixture (3.1), homogenise and leave to stand for one hour (standard series). Determine as indicated in 5.2 the optical density of the solutions in the standard series by comparison with the corresponding solutions in the reference series. Trace the calibration curve by plotting the optical densities against the quantities of gossypol (in μg). 6.3. Repeatability The difference between the results of two parallel determinations carried out on the same sample must not exceed: — 15 %, in relative value to the higher level, for gossypol contents of less than 500 ppm, — 75 ppm, in absolute value, for contents of not less than 500 ppm and not more than 750 ppm, — 10 %, in relative value to the higher value, for contents of more than 750 ppm. (B) DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND DIOXIN-LIKE PCBs I. METHODS OF SAMPLING AND INTERPRETATION OF ANALYTICAL RESULTS 1. Purpose and scope The samples intended for the official control of the levels of dioxins (polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF)) and dioxin-like polychlorinated biphenyls (PCBs) ( 1 ) in feed, shall be taken in accordance with the provisions of Annex I. The quantitative requirements in relation to the control of substances or products uniformly distributed throughout the feed as provided for in point 5.A. of the Annex I have to be applied. Aggregate samples thus obtained shall be considered as representative for the lots or sublots from which they are taken. Compliance with maximum levels laid down in Directive 2002/32/EC of the European Parliament and of the Council ( 2 ) shall be established on the basis of the levels determined in the laboratory samples. 2. Compliance of the lot or sublot with the specification The lot is accepted if the analytical result of a single analysis does not exceed the respective maximum level as laid down in Directive 2002/32/EC taking into account the measurement uncertainty. The lot is non-compliant with the maximum level as laid down in Directive 2002/32/EC, if the upperbound ( 3 ) analytical result, confirmed by duplicate analysis ( 4 ) , exceeds the maximum level beyond reasonable doubt taking into account the measurement uncertainty. Measurement uncertainty may be taken into account according to one of the following approaches: — by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot is non-compliant if the measured value minus U is above the maximum level. In case of a separate determination of dioxins and dioxin-like-PCBs the sum of the estimated expanded uncertainty of the separate analytical results of dioxins and dioxin-like PCBs has to be used for the sum of dioxins and dioxin-like PCBs, — by establishing the decision limit (CCα) in accordance with Commission Decision 2002/657/EC ( 5 ) (point 3.1.2.5 of the Annex — the case of substances with established permitted level). A lot is non-compliant if the measured value is equal to or above the CCα. The present interpretation rules apply for the analytical result obtained on the sample for official control. It does not affect the right of Member States to apply national rules to analyses for defence or referee purposes. II. SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF ANALYSIS USED IN OFFICAL CONTROL OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND DIOXIN-LIKE PCBs 1. Objective and field of application These requirements shall be applied where feed materials and feed are analysed for the determination of the dioxins (polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF)) and dioxin-like polychlorinated biphenyls (PCBs). Monitoring for the presence of dioxins in feed can be performed by a strategy involving a screening method in order to select those samples with levels of dioxins and dioxin-like PCBs that are less than 25 % below or exceed the level of interest. The concentration of dioxins in those samples with significant levels needs to be determined/confirmed by a confirmatory method. Screening methods are methods that are used to detect the presence of dioxins and dioxin-like PCBs at the level of interest. These methods have a capacity for a high sample throughput and are used to sift large numbers of samples for potential positives. They are specifically designed to avoid false negatives. Confirmatory methods are methods that provide full or complementary information enabling the dioxins and dioxin-like PCBs to be identified and quantified unequivocally at the level of interest. 2. Background Because environmental and biological samples (including samples of feed materials/feed) in general contain complex mixtures of different dioxin congeners, the concept of Toxic Equivalency Factors (TEFs) has been developed to facilitate risk assessment. These TEFs have been established to express concentrations of mixtures of 2,3,7,8-substituted PCDDs and PCDFs and some non-ortho and mono-ortho chlorine substituted PCBs which possess dioxin-like activity in toxic equivalents (TEQs) of 2,3,7,8-TCDD. The concentrations of the individual substances in a given sample are multiplied by their respective TEF and subsequently summed to give the total concentration of dioxin-like compounds expressed in TEQs. For the purposes of this Regulation only, the accepted specific limit of quantification of an individual congener is the concentration of an analyte in the extract of a sample which produces an instrumental response at two different ions to be monitored with an S/N (signal/noise) ratio of 3:1 for the less sensitive signal and fulfilment of the basic requirements such as e.g. retention time and isotope ratio according to the determination procedure as described in EPA method 1613 revision B. 3. Quality assurance requirements to be complied with for sample preparation The general provisions on the preparation of samples for analysis as laid down in the Annex II are applicable. In addition following requirements have to be complied with: — The samples must be stored and transported in glass, aluminium, polypropylene or polyethylene containers. Traces of paper dust must be removed from the sample container. Glassware shall be rinsed with solvents previously controlled for the presence of dioxins. — Perform a blank analysis by carrying out the entire analytical procedure omitting only the sample. — Sample weight used for the extraction must be sufficient to fulfil the requirements with respect to sensitivity. 4. Requirements for laboratories — Laboratories shall demonstrate the performance of a method in the range of the level of interest, e.g. 0,5x, 1x and 2x the level of interest with an acceptable coefficient of variation for repeated analysis. For details of acceptance criteria, see point 5. — Limit of quantification for a confirmatory method shall be in the range of about one fifth of the level of interest, to make sure that acceptable coefficients of variations are met in the range of the level of interest. — Regular blank controls and spiking experiments or analysis of control samples (preferably, if available, certified reference material) shall be performed as internal quality control measures. — Successful participation in interlaboratory studies that assess laboratory proficiency is the best way to prove the competence in specific analyses. However successful participation in interlaboratory studies for e.g. soil or sewage samples does not necessarily prove the competence also in the field of food or feed samples, which present lower contamination levels. Therefore, the continuous participation in interlaboratory studies for the determination of dioxins and dioxin-like PCBs in the relevant feed/food matrices is mandatory. — Laboratories shall be accredited by a recognised body operating in accordance with ISO Guide 58 to ensure that they are applying analytical quality assurance. Laboratories shall be accredited following the ISO/IEC/17025 standard. 5. Requirements for the analytical procedures for dioxins and dioxin-like PCBS Basic requirements for acceptance of analytical procedures: — High sensitivity and low limits of detection . For PCDDs and PCDFs, detectable quantities have to be in the picogram TEQ (10 -12 g) range because of extreme toxicity of some of these compounds. PCBs are known to occur at higher levels than the PCDDs and PCDFs. For most PCB congeners sensitivity in nanogram (10 -9 g) range is already sufficient. However, for the measurement of the more toxic dioxin-like PCB congeners (in particular non-ortho substituted congeners), the same sensitivity must be reached as for the PCDDs and PCDFs. — High selectivity (specificity) . A distinction is required for PCDDs, PCDFs and dioxin-like PCBs from a multitude of other, coextracted and possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of the analytes of interest. For gas chromatography/mass spectrometry (GC/MS) methods, a differentiation among various congeners is necessary, such as between toxic (e.g. the 17 2,3,7,8-substituted PCDDs and PCDFs and dioxin-like PCBs) and other congeners. Bioassays must be able to determine TEQ values selectively as the sum of PCDDs, PCDFs and dioxin-like PCBs. — High accuracy (trueness and precision) . The determination shall provide a valid and reliable estimate of the true concentration in a sample. High accuracy (accuracy of the measurement: the closeness of agreement between the result of a measurement with the true or assigned value of the measurand) is necessary to avoid the rejection of a sample analysis result on the basis of poor reliability of the estimate of TEQ. Accuracy is expressed as trueness (difference between the mean value measured for an analyte in a certified material and its certified value, expressed as percentage of this value) and precision (RSD R, relative standard deviation calculated from results generated under reproducibility conditions). Screening methods can comprise bioassays and GC/MS methods; confirmatory methods are high-resolution gas-chromatography/high-resolution mass-spectrometry (HRGC/HRMS) methods. Following criteria have to be complied with on total TEQ value: Screening methods Confirmatory methods False negative rate < 1 % Trueness - 20 % to + 20 % Precision RSD R < 30 % < 15 % 6. Specific requirements for gc/ms methods to be complied with for screening or confirmatory purposes. — Addition of 13 C-labelled 2,3,7,8-chlorine substituted internal PCDD/F standards and of 13 C-labelled internal dioxin-like PCB standards must be carried out at the very beginning or start of the analytical method, e.g. prior to extraction in order to validate the analytical procedure. At least one congener for each of the tetra to octa-chlorinated homologous groups for PCDD/F and at least one congener for each of the homologous groups for dioxin-like PCBs must be added (alternatively, at least one congener for each mass spectrometric selected ion recording function used for monitoring PCDD/F and dioxin-like PCBs). There shall be a clear preference, certainly in case of confirmatory methods, of using all 17 13 C-labelled 2,3,7,8-substituted internal PCDD/F standards and all 12 13 C-labelled internal dioxin-like PCB standards. — Relative response factors shall also be determined for those congeners for which no 13 C-labelled analogue is added by using appropriate calibration solutions. — For feed of plant origin and feed of animal origin containing less than 10 % fat, the addition of the internal standards is mandatory prior to extraction. For feed of animal origin containing more than 10 % fat, the internal standards can be added either before extraction or after fat extraction. An appropriate validation of the extraction efficiency shall be carried out, depending on the stage at which internal standards are introduced and on whether results are reported on a product or fat basis. — Prior to GC/MS analysis, 1 or 2 recovery (surrogate) standard(s) must be added. — Control of recovery is necessary. For confirmatory methods, the recoveries of the individual internal standards must be in the range of 60 % to 120 %. Lower or higher recoveries for individual congeners, in particular for some hepta- and octa- chlorinated dibenzodioxins and dibenzofurans, are acceptable on the condition that their contribution to the TEQ value does not exceed 10 % of the total TEQ value (based on sum of PCDD/F and dioxin-like PCBs). For screening methods, the recoveries must be in the range of 30 % to 140 %. — Separation of dioxins from interfering chlorinated compounds such as non-dioxin-like PCBs and chlorinated diphenyl ethers shall be carried out by suitable chromatographic techniques (preferably with a florisil, alumina and/or carbon column). — Gaschromatographic separation of isomers shall be sufficient (< 25 % peak to peak between 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF). — Determination shall be performed according to EPA Method 1613 revision B: Tetra- through octa-chlorinated dioxins and furans by isotope dilution HRGC/HRMS or another with equivalent performance criteria. — The difference between upperbound level and lower bound level must not exceed 20 % for feed with a dioxin contamination in the range or above the maximum level. For feed with contamination levels well below the maximum level, the difference may be in the range of 25 %-40 %. 7. Screening methods of analysis 7.1. Introduction Different analytical approaches can be performed using a screening method: a pure screening approach and a quantitative approach. Screening approach The response of samples is compared to that of a reference sample at the level of interest. Samples with a response less than the reference are declared negative, those with a higher response are suspected positives. Requirements: — A blank and a reference sample(s) have to be included in each test series, which is extracted and tested at the same time under identical conditions. The reference sample must show a clearly elevated response in comparison to a blank. — Additional reference samples at 0,5x and 2x the level of interest shall be included to demonstrate the proper performance of the test in the range of interest for the control of the level of interest. — When testing other matrices, the suitability of the reference sample(s) has to be demonstrated, preferentially by including samples shown by HRGC/HRMS to contain a TEQ level around that of the reference sample or else a blank spiked at this level. — Since no internal standards can be used in bioassays, tests on repeatability are very important to obtain information on the standard deviation within one test series. The coefficient of variation must be below 30 %. — For bioassays, the target compounds, possible interferences, and maximum tolerable blank levels shall be defined. Quantitative approach The quantitative approach requires standard dilution series, duplicate or triplicate clean up and measuring as well as blank and recovery controls. The result may be expressed as TEQ, thereby assuming that the compounds responsible for the signal correspond to the TEQ principle. This can be performed by using TCDD (or a dioxin/furan/dioxin-like PCB standard mixture) to produce a calibration curve to calculate the TEQ level in the extract and thus in the sample. This is subsequently corrected for the TEQ level calculated for a blank sample (to account for impurities from solvents and chemicals used), and a recovery (calculated from the TEQ level in a quality control sample around the limit of interest). It is essential to note that part of the apparent recovery loss may be due to matrix effects and/or differences between the TEF values in the bioassays and the official TEF values set by WHO. 7.2. Requirements for methods of analysis used for screening — GC/MS methods of analysis and bioassays may be used for screening. For GC/MS methods the requirements as laid down in point 6 are to be used. For cell based bioassays specific requirements are laid down in point 7.3 and for kit-based bioassays in point 7.4. — Information on the number of false-positive and false-negative results of a large set of samples below and above the maximum level or action level is necessary, in comparison to the TEQ content as determined by a confirmatory method of analysis. Actual false negative rates must be under 1 %. The rate of false positive samples shall be low enough to make the use of a screening tool advantageous. — Positive results have always to be confirmed by a confirmatory method of analysis (HRGC/HRMS). In addition, samples from a wide TEQ-range shall be confirmed by HRGC/HRMS (approximately 2 %-10 % of the negative samples). Information on correspondence between bioassay and HRGC/HRMS results shall be made available. 7.3. Specific requirements for cell based bioassays — When performing a bioassay, every test run requires a series of reference concentration of TCDD or a dioxin/furan mixture (full dose-response curve with a R 2 > 0,95). However, for screening purposes, an expanded low level curve for analysing low-level samples could be used. — A TCDD reference concentration (about 3 x limit of quantification) on a quality control sheet shall be used for the outcome of the bioassay over a constant time period. An alternative could be the relative response of a reference sample in comparison to the TCDD calibration line since the response of the cells may depend on many factors. — Quality control (QC) charts for each type of reference material shall be recorded and checked to make sure the outcome is in accordance with the stated guidelines. — In particular for quantitative calculations, the induction of the sample dilution used must be within the linear portion of the response curve. Samples above the linear portion of the response curve must be diluted and re-tested. Therefore, at least 3 or more dilutions at one time are recommended to be tested. — The per cent standard deviation shall not be above 15 % in a triplicate determination for each sample dilution and not above 30 % between three independent experiments. — The limit of detection may be set as 3x the standard deviation of the solvent blank or of the background response. Another approach is to apply a response that is above the background (induction factor 5x the solvent blank) calculated from the calibration curve of the day. The limit of quantification may be set as 5 to 6x the standard deviation of the solvent blank or of the background response or to apply a response that is clearly above the background (induction factor 10x the solvent blank) calculated from the calibration curve of the day. 7.4. Specific requirements for kit based bioassays — It shall be ensured that the kit-based bioassays have sufficient sensitivity and reliability to be applied for feed. — Manufacturer's instructions for sample preparation and analyses have to be followed. — Test kits shall not be used after the expiration date. — Materials or components designed for use with other kits shall not be used. — Test kits shall be kept within the specified range of storage temperature and used at the specified operating temperature. — The limit of detection for immunoassays is determined as the sum of the mean and 3x the standard deviation, based on 10 replicate analysis of the blank, to be divided by the slope value of the linear regression equation. — Reference standards shall be used for tests at the laboratory to make sure that the responsiveness to the standard is within an acceptable range. 8. Reporting of results In so far as the used analytical procedure makes it possible, the analytical results shall contain the levels of the individual PCDD/F and PCB congeners and the analytical results have to be reported as lowerbound, upperbound and medium-bound in order to include a maximum of information in the reporting of the results and thereby enabling the interpretation of the results according to specific requirements. The report shall also include the lipid content of the sample as well the method used for lipid extraction. The recoveries of the individual internal standards must be made available in case the recoveries are outside the range mentioned in point 6, in case the maximum level is exceeded and in the other cases upon request. As the uncertainty of the measurement is to be taken into account when deciding about the compliance of a sample, this parameter shall also be made available. Thus, analytical results shall be reported as x +/- U whereby x is the analytical result and U is the expanded measurement uncertainty using a coverage factor of 2 which gives a level of confidence of approximately 95 %. In case of a separate determination of dioxins and dioxin-like-PCBs the sum of the estimated expanded uncertainty of the separate analytical results of dioxins and dioxin-like PCBs has to be used for the sum of dioxins and dioxin-like PCBs. If the uncertainty of measurement would be taken into account by applying a CCα (as described in I.2 of this Part B) this parameter shall be reported. ( 1 ) Table of TEF (= toxic equivalency factors) for dioxins, furans and dioxin-like PCBs: Congener TEF value Congener TEF value Dibenzo-p-dioxins (‘PCDDs’) ‘Dioxin-like’ PCBs: 2,3,7,8-TCDD 1 1,2,3,7,8-PeCDD 1 Non-ortho PCBs 1,2,3,4,7,8-HxCDD 0,1 PCB 77 0,0001 1,2,3,6,7,8-HxCDD 0,1 PCB 81 0,0001 1,2,3,7,8,9-HxCDD 0,1 PCB 126 0,1 1,2,3,4,6,7,8-HpCDD 0,01 PCB 169 0,01 OCDD 0,0001 Mono-ortho PCBs PCB 105 0,0001 Dibenzofurans (‘PCDFs’) PCB 114 0,0005 2,3,7,8-TCDF 0,1 PCB 118 0,0001 1,2,3,7,8-PeCDF 0,05 PCB 123 0,0001 2,3,4,7,8-PeCDF 0,5 PCB 156 0,0005 1,2,3,4,7,8-HxCDF 0,1 PCB 157 0,0005 1,2,3,6,7,8-HxCDF 0,1 PCB 167 0,00001 1,2,3,7,8,9-HxCDF 0,1 PCB 189 0,0001 2,3,4,6,7,8-HxCDF 0,1 1,2,3,4,6,7,8-HpCDF 0,01 1,2,3,4,7,8,9-HpCDF 0,01 OCDF 0,0001 Abbreviations used: «T» = tetra; «Pe» = penta; «Hx» = hexa; «Hp» = hepta; «O» = octa; «CDD» = chlorodibenzo-p-dioxin; «CDF» = chlorodibenzofuran; «CB» = chlorobiphenyl. ( 2 ) OJ L 140, 30.5.2002, p. 10 . ( 3 ) The concept of ‘upperbound’ requires using the limit of quantification for the contribution of each non-quantified congener to the Toxic Equivalent (TEQ). The concept of ‘lowerbound’ requires using zero for the contribution of each non-quantified congener to the TEQ. The concept of ‘mediumbound’ requires using half of the limit of quantification calculating the contribution of each non-quantified congener to the TEQ. ( 4 ) The duplicate analysis is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The first analysis, taking into account the measurement uncertainty is used for verification of compliance. In case the analysis is performed in the frame of a dioxin contamination incident, confirmation by duplicate analysis might be omitted in the case the samples selected for analysis are through traceability linked to the dioxin contamination incident. ( 5 ) OJ L 221, 17.8.2002, p. 8 .