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Sublot: designated part of a large lot in order to apply the sampling method on that designated part. Each sublot must be physically separated and identifiable.

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Incremental sample: a quantity of material taken from a single place in the lot or sublot.

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Aggregate sample: the combined total of all the incremental samples taken from the lot or sublot.

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Laboratory sample: a representative part/quantity of the aggregate sample intended for the laboratory.

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In case the lot to be sampled contains small fishes (individual fishes weighing < about 1 kg), the whole fish is taken as incremental sample to form the aggregate sample. In case the resulting aggregate sample weighs more than 3 kg, the incremental samples may consist of the middle part, weighing each at least 100 grams, of the fishes forming the aggregate sample. The whole part to which the maximum level is applicable is used for homogenisation of the sample.

The middle part of the fish is where the centre of gravity is. This is located in most cases at the dorsal fin (in case the fish has a dorsal fin) or halfway between the gill opening and the anus.

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In case the lot to be sampled contains larger fishes (individual fishes weighing more than about 1 kg), the incremental sample consists of the middle part of the fish. Each incremental sample weighs at least 100 grams.

For fishes of intermediate size (about 1 to 6 kg) the incremental sample is taken as a slice of the fish from backbone to belly in the middle part of the fish.

For very large fishes (e.g. > about 6 kg), the incremental part is taken from the right side (frontal view) dorso-lateral muscle meat in the middle part of the fish. In case the taking of such a piece of the middle part of the fish would result in a significant economic damage, taking of three incremental samples of at least 350 grams each may be considered as being sufficient, independently of the size of the lot or alternatively an equal part of the muscled meat close to the tail part and the muscle meat close to the head part of one fish may be taken to form the incremental sample being representative for the level of dioxins in the whole fish.

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The provisions of point 4.3 as regards sample constitution are applicable.

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In case a size or weight class/category is predominant (about 80 % or more of the batch), the sample is taken from fishes with the predominant size or weight. This sample is to be considered as being representative for the whole batch.

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In case no particular size or weight class/category predominates, then it must be ensured that the fishes selected for the sample are representative for the consignment. Specific guidance for such cases is provided in ‘Guidance document for the sampling of lots of fish containing whole fishes of different size and/or weight (2).’

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by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sublot is non-compliant if the measured value minus U is above the established permitted 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,

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by establishing the decision limit (CCα) according to the provisions of Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (5) (point 3.1.2.5 of the Annex — the case of substances with established permitted level) a lot or sublot is non-compliant if the measured value is equal to or above the CCα.

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Measures must be taken to avoid cross-contamination at each stage of the sampling and analysis procedure.

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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, certified to be free from dioxins or previously controlled for the presence of dioxins.

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The sample storage and transportation has to be performed in a way that maintains the integrity of the foodstuff sample.

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Insofar as relevant, finely grind and mix thoroughly each laboratory sample using a process that has been demonstrated to achieve complete homogenisation (e.g. ground to pass a 1 mm sieve); samples have to be dried before grinding if moisture content is too high.

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Perform a blank analysis by carrying out the entire analytical procedure omitting only the sample.

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Sample weight used for the extraction must be sufficient to fulfil the requirements with respect to sensitivity.

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The specific sample preparation procedures used for the products under consideration shall be validated according to internationally accepted guidelines.

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In the case of fish, the skin has to be removed as the maximum level applies to muscle meat without skin. However it is necessary that all remaining rests of muscle meat and fat tissue at the inner side of the skin are carefully and completely scraped of from the skin and that these rests of muscle meat and fat tissue are added to the sample to be analysed.

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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 part 5.

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Limit of quantification for a confirmatory method shall be in the range of about one fifth of the level of interest.

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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.

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Laboratory proficiency shall be proved by the continuous successful participation in interlaboratory studies for the determination of dioxins and dioxin-like PCBs in the relevant feed/food matrices.

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In accordance with the provisions of Regulation (EC) No 882/2004, 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 EN ISO/IEC 17025 standard.

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High sensitivity and low limits of detection. For PCDDs and PCDFs, detectable quantities have to be in the picogram TEQ (10-12g) 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 the nanogram (10-9g) 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.

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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 seventeen 2,3,7,8-substituted PCDDs and PCDFs and dioxin-like PCBs) and other congeners. Bioassays shall be able to determine TEQ values selectively as the sum of PCDDs, PCDFs and dioxin-like PCBs.

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High accuracy (trueness and precision). The determination shall provide a valid estimate of the true concentration in a sample. High accuracy (accuracy of the measurement: the closeness of the 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 (RSDR relative standard deviation calculated from results generated under reproducibility conditions).

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Addition of 13C-labelled 2,3,7,8-chlorine substituted internal PCDD/F standards and of 13C-labelled internal dioxin-like PCB standards must be carried out at the very beginning 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 clear preference, certainly in case of confirmatory methods, of using all 17 13C-labelled 2,3,7,8-substituted internal PCDD/F standards and all 12 13C-labelled internal dioxin-like PCB standard.

Relative response factors shall also be determined for those congeners for which no 13C-labelled analogue is added by using appropriate calibration solutions.

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For foodstuffs of plant origin and foodstuffs of animal origin containing less than 10 % fat, the addition of the internal standards is mandatory prior to extraction. For foodstuffs of animal origin containing more than 10 % fat, the internal standards may 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 product or fat basis.

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Prior to GC/MS analysis, 1 or 2 recovery (surrogate) standard(s) must be added.

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Control of recovery is necessary. For confirmatory methods, the recoveries of the individual internal standards shall 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 shall be in the range of 30 to 140 %.

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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).

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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).

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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.

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The difference between upperbound level and lower bound level shall not exceed 20 % for foodstuffs with a dioxin contamination of about 1 pg WHO-TEQ/g fat (based on the sum of PCDD/PCDF and dioxin-like PCBs). For foodstuffs with a low fat content, the same requirements for contamination levels of about 1 pg WHO-TEQ/g product have to be applied. For lower contamination levels, for example 0,50 pg WHO-TEQ/g product, the difference between upperbound and lowerbound level may be in the range of 25 % to 40 %.

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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.

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Extra reference samples 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.

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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.

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Since no internal standards can be used in bioassays, tests on repeatability shall be carried out to obtain information on the standard deviation within one test series. The coefficient of variation shall be below 30 %.

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For bioassays, the target compounds, possible interferences and maximum tolerable blank levels shall be defined.

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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 part 7.3 of this Annex and for kit-based bioassays in part 7.4 of this Annex.

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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 shall be under 1 %. The rate of false positive samples shall be low enough to make the use of a screening tool advantageous.

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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 % to 10 % of the negative samples). Information on correspondence between bioassay and HRGC/HRMS results shall be made available.

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When performing a bioassay, every test run requires a series of reference concentrations of TCDD or a dioxin/furan/dioxin-like PCB mixture (full dose-response curve with a R2 > 0,95). However, for screening purposes an expanded low level curve for analysing low level samples may be used.

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A TCDD reference concentration (about 3x limit of quantification) on a quality control sheet shall be used for the outcome of the bioassay over a constant time period. An alternative may 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.

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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.

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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 shall be tested.

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The percent standard deviation shall not be above 15 % in a triplicate determination for each sample dilution and not above 30 % between three independent experiments.

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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 above the background (induction factor 10x the solvent blank) calculated from the calibration curve of the day.

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It shall be ensured that the kit-based bioassays have sufficient sensitivity and reliability to be applied for food.

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Manufacturer’s instructions for sample preparation and analyses have to be followed.

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Test kits shall not be used after the expiration date.

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Materials or components designed for use with other kits shall not be used.

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Test kits shall be kept within the specified range of storage temperature and used at the specified operating temperature.

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The limit of detection for immunoassays is determined as 3 x the standard deviation, based on 10 replicate analysis of the blank, to be divided by the slope value of the linear regression equation.

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Reference standards shall be used for tests at the laboratory to make sure that the responsiveness to the standard is within an acceptable range.