Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)

• 20 % of the sample extracts shall be measured in routine screening without and with 2,3,7,8-TCDD added corresponding to the maximum level or action threshold, to check if the response is possibly suppressed by interfering substances present in the sample extract. The measured concentration of the spiked sample shall be compared to the sum of the concentration of the unspiked extract plus the spiking concentration. If this measured concentration is more than 25 % lower than the calculated (sum) concentration, this is an indication of potential signal suppression and the respective sample shall be submitted to GC-HRMS confirmatory analysis. Results shall be monitored in quality control charts.

• 5.7.5.Quality control on compliant samples:

• Approximately 2 to 10 % of the compliant samples, depending on sample matrix and laboratory experience, shall be confirmed by GC/HRMS.

• 5.7.6.Determination of false-compliant rates from quality control data:

• The rate of false-compliant results from screening of samples below and above the maximum level or the action threshold shall be determined. Actual false-compliant rates shall be below 5 %. When a minimum of 20 confirmed results per matrix/matrix group is available from the quality control of compliant samples, conclusions on the false compliant rate shall be drawn from this database. The results from samples analysed in ring trials or during contamination incidents, covering a concentration range up to for example 2x the maximum level (ML), may also be included in the minimum of 20 results for evaluation of the false-compliant rate. The samples shall cover most frequent congener patterns, representing various sources.

• Although screening assays shall preferentially aim to detect samples exceeding the action threshold, the criterion for determining false-compliant rates is the maximum level, taking into account the expanded measurement uncertainty of the confirmatory method.

• 5.7.7.Potential non-compliant samples from screening shall always be verified by a full re-analysis of the original sample by a confirmatory method of analysis. These samples may also be used to evaluate the rate of false non-compliant results. For screening methods, the rate of false non-compliant results shall be the fraction of results confirmed to be compliant from confirmatory analysis, while in previous screening the sample has been declared to be potentially non-compliant. Evaluation of the advantages of the screening method shall be based on comparison of false-non-compliant samples with the total number of samples checked. This rate shall be low enough to make the use of a screening tool advantageous.

• 5.7.8.Under validation conditions, bioanalytical methods shall provide a valid indication of the TEQ level, calculated and expressed as BEQ.

• Also for bioanalytical methods carried out under repeated conditions, the intra-laboratory RSDr would typically be smaller than under reproducibility conditions (RSDR).

6. Specific requirements for GC-MS methods to be complied with for screening or confirmatory purposes

6.1.   Acceptable differences between upper-bound and lower-bound WHO-TEQ results

The difference between upper-bound level and lower-bound level shall not exceed 20 % for confirmation of exceedance of maximum level or in case of need of action thresholds.

6.2.   Control of recoveries

• 6.2.1.Addition of ¹³C-labelled 2,3,7,8-chlorine-substituted internal PCDD/F standards and of ¹³C-labelled internal dioxin-like PCB standards shall 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/Fs and at least one congener for each of the homologous groups for dioxin-like PCBs shall be added (alternatively, at least one congener for each mass spectrometric selected ion recording function used for monitoring PCDD/Fs and dioxin-like PCBs). In the case of confirmatory methods, all 17 ¹³C-labelled 2,3,7,8-substituted internal PCDD/F standards and all 12 ¹³C-labelled internal dioxin-like PCB standards shall be used.

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

• 6.2.3.For feed of plant origin and feed of animal origin containing less than 10 % fat, the addition of the internal standards shall be mandatory prior to extraction. For feed of animal origin containing more than 10 % fat, the internal standards shall be added either before 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.

• 6.2.4.Prior to GC-MS analysis, 1 or 2 recovery (surrogate) standard(s) shall be added.

• 6.2.5.Control of recovery is required. 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 dibenzo-p-dioxins and dibenzofurans, shall be 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 GC-MS screening methods, the recoveries shall be in the range of 30 to 140 %.

6.3.   Removal of interfering substances

• — Separation of PCDD/Fs 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).

• — Gas-chromatographic separation of isomers shall be < 25 % peak to peak between 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF.

6.4.   Calibration with standard curve

The range of the calibration curve shall cover the relevant range of maximum level or action thresholds.

6.5.   Specific criteria for confirmatory methods

• — For GC-HRMS: In HRMS, the resolution shall typically be greater than or equal to 10 000 for the entire mass range at 10 % valley. Fulfilment of further identification and confirmation criteria as described in internationally recognised standards, for example, in standard EN 16215:2012 (Animal feed – Determination of dioxins and dioxin-like PCBs by GC-HRMS and of indicator PCBs by GC-HRMS) and/or in EPA methods 1613 and 1668 as revised.

• — For GC-MS/MS: Monitoring of at least 2 specific precursor ions, each with one specific corresponding transition product ion for all labelled and unlabelled analytes in the scope of analysis. Maximum permitted tolerance of relative ion intensities of ± 15 % for selected transition product ions in comparison to calculated or measured values (average from calibration standards), applying identical MS/MS conditions, in particular collision energy and collision gas pressure, for each transition of an analyte. Resolution for each quadrupole to be set equal to or better than unit mass resolution (unit mass resolution: sufficient resolution to separate two peaks one mass unit apart) in order to minimise possible interferences on the analytes of interest. Fulfilment of the further criteria as described in internationally recognised standards, for example, in standard EN 16215:2012 (Animal feed – Determination of dioxins and dioxin-like PCBs by GC-HRMS and of indicator PCBs by GC-HRMS) and/or in EPA methods 1613 and 1668 as revised, except the obligation to use GC-HRMS.

7. Specific requirements for bioanalytical methods

Bioanalytical methods are methods based on the use of biological principles like cell-based assays, receptor-assays or immunoassays. This point establishes requirements for bioanalytical methods in general.

A screening method in principle classifies a sample as compliant or suspected to be non-compliant. For this, the calculated BEQ level is compared to the cut-off value (see point 7.3). Samples below the cut-off value are declared compliant, samples equal or above the cut-off value are suspected to be non-compliant, requiring analysis by a confirmatory method. In practice, a BEQ level corresponding to two-thirds of the maximum level may serve as cut-off value provided that a false-compliant rate below 5 % and an acceptable rate for false non-compliant results are ensured. With separate maximum levels for PCDD/Fs and for the sum of PCDD/Fs and dioxin-like PCBs, checking compliance of samples without fractionation requires appropriate bioassay cut-off values for PCDD/Fs. For checking of samples exceeding the action thresholds, an appropriate percentage of the respective action threshold shall suit as cut-off value.

If an indicative level is expressed in BEQs, sample results shall be in the working range and shall exceed the reporting limit (see points 7.1.1 and 7.1.6).

7.1.   Evaluation of the test response

7.1.1.   General requirements

• — When calculating the concentrations from a TCDD calibration curve, values at the higher end of the curve will show a high variation (high coefficient of variation (CV)). The working range is the area where this CV is smaller than 15 %. The lower end of the working range (reporting limit) shall be set at least by a factor of three above the procedure blanks. The upper end of the working range is usually represented by the EC70 value (70 % of maximal effective concentration), but lower if the CV is higher than 15 % in this range. The working range shall be established during validation. Cut-off values (see point 7.3) shall be well within the working range.

• — Standard solutions and sample extracts shall be tested in triplicate or at least in duplicate. When using duplicates, a standard solution or a control extract tested in four to six wells divided over the plate shall produce a response or concentration (only possible in the working range) based on a CV < 15 %.

7.1.2.   Calibration

7.1.2.1.   Calibration with standard curve

• — Levels in samples shall be estimated by comparison of the test response with a calibration curve of TCDD (or PCB 126 or a PCDD/PCDF/dioxin-like PCB standard mixture) to calculate the BEQ level in the extract and subsequently in the sample.

• — Calibration curves shall contain 8 to 12 concentrations (at least in duplicates), with enough concentrations in the lower part of the curve (working range). Special attention shall be paid to the quality of the curve-fit in the working range. As such, the R2 value is of little or no value in estimating the goodness of fit in non-linear regression. A better fit shall be achieved by minimising the difference between calculated and observed levels in the working range of the curve, for example by minimising the sum of squared residuals.

• — The estimated level in the sample extract shall be subsequently corrected for the BEQ level calculated for a matrix or solvent blank sample (to account for impurities from solvents and chemicals used), and the apparent recovery (calculated from the BEQ level of suitable reference samples with representative congener patterns around the maximum level or action threshold). To perform a recovery correction, the apparent recovery shall be within the required range (see point 7.1.4). Reference samples used for recovery correction shall comply with the requirements laid down in point 7.2.

7.1.2.2.   Calibration with reference samples

Alternatively, a calibration curve prepared from at least four reference samples (see point 7.2.4): one matrix blank, plus three reference samples at 0,5x, 1x and 2x the maximum level or action threshold may be used, eliminating the need to correct for blank and recovery if matrix properties of the reference samples match those of the unknown samples. In this case, the test response corresponding to two-thirds of the maximum level (see point 7.3) may be calculated directly from these samples and used as cut-off value. For checking of samples exceeding the action thresholds, an appropriate percentage of these action thresholds shall suit as cut-off value.

7.1.3.   Separate determination of PCDD/Fs and dioxin-like PCBs

Extracts may be split into fractions containing PCDD/Fs and dioxin-like PCBs, allowing a separate indication of PCDD/Fs and dioxin-like PCB TEQ levels (in BEQ). A PCB 126 standard calibration curve shall preferentially be used to evaluate results for the fraction containing dioxin-like PCBs.

7.1.4.   Bioassay apparent recoveries

The ‘bioassay apparent recovery’ shall be calculated from suitable reference samples with representative congener patterns around the maximum level or action threshold and expressed as percentage of the BEQ level in comparison to the TEQ level. Depending on the type of assay and TEFs ([^E0060]) used, the differences between TEF and REP factors for dioxin-like PCBs can cause low apparent recoveries for dioxin-like PCBs in comparison to PCDD/Fs. Therefore, if a separate determination of PCDD/Fs and dioxin-like PCBs is performed, bioassay apparent recoveries shall be: for dioxin-like PCBs 20 % to 60 %, for PCDD/Fs 50 % to 130 % (ranges apply for the TCDD calibration curve). As the contribution of dioxin-like PCBs to the sum of PCDD/Fs and dioxin-like PCBs can vary between different matrices and samples, bioassay apparent recoveries for the sum of PCDD/Fs and dioxin-like PCBs reflect these ranges and shall be between 30 % and 130 %. Any implication of substantially revised TEF values for the Union legislation for PCDD/Fs and dioxin-like PCBs requires the revision of these ranges.

7.1.5.   Control of recoveries for clean-up

The loss of compounds during the clean-up shall be checked during validation. A blank sample spiked with a mixture of the different congeners shall be submitted to clean-up (at least n = 3) and the recovery and variability checked by a confirmatory method. The recovery shall be within 60 % to 120 % especially for congeners contributing more than 10 % to the TEQ-level in various mixtures.

7.1.6.   Reporting limit

When reporting BEQ levels, a reporting limit shall be determined from relevant matrix samples involving typical congener patterns, but not from the calibration curve of the standards due to low precision in the lower range of the curve. Effects from extraction and clean-up shall be taken into account. The reporting limit shall be set at least by a factor of three above the procedure blanks.

7.2.   Use of reference samples

• 7.2.1.Reference samples shall represent sample matrix, congener patterns and concentration ranges for PCDD/Fs and dioxin-like PCBs around the maximum level or action threshold.

• 7.2.2.A matrix blank, and where it is not possible, a procedure blank, and a reference sample at the maximum level or action threshold shall be included in each test series. These samples shall be extracted and tested at the same time under identical conditions. The reference sample shall show a clearly elevated response in comparison to the blank sample, thus ensuring the suitability of the test. Those samples may be used for blank and recovery corrections.

• 7.2.3.Reference samples chosen to perform a recovery correction shall be representative for the test samples, meaning that congener patterns may not lead to an underestimation of levels.

• 7.2.4.Extra reference samples at e.g. 0,5x and 2x the maximum level or action threshold may be included to demonstrate the proper performance of the test in the range of interest for the control of the maximum level or action threshold. Combined, these samples may be used for calculating the BEQ levels in test samples (see point 7.1.2.2).

7.3.   Determination of cut-off values

The relationship between bioanalytical results in BEQ and results from the confirmatory method in TEQ shall be established, for example by matrix-matched calibration experiments, involving reference samples spiked at 0, 0,5x, 1x and 2x the ML, with 6 repetitions on each level (n = 24). Correction factors (blank and recovery) may be estimated from this relationship but shall be checked in accordance with point 7.2.2.

Cut-off values shall be established for decisions over sample compliance with maximum levels or for the control of action thresholds, if relevant, with the respective maximum levels or action threshold set for either PCDD/Fs and dioxin-like PCBs alone, or for the sum of PCDD/Fs and dioxin-like PCBs. They are represented by the lower end-point of the distribution of bioanalytical results (corrected for blank and recovery) corresponding to the decision limit of the confirmatory method based on a 95 % level of confidence, implying a false-compliant rate < 5 %, and on a RSDR < 25 %. The decision limit of the confirmatory method is the maximum level, taking into account the expanded measurement uncertainty.

The cut-off value (in BEQ) may be calculated in accordance with one of the approaches set out in points 7.3.1, 7.3.2 and 7.3.3. (see Figure 1).

• 7.3.1.Use of the lower band of the 95 % prediction interval at the decision limit of the confirmatory method

Image omitted; see official source

• with:

• BEQDL BEQ corresponding to the decision limit of the confirmatory method, being the maximum level taking into account the expanded measurement uncertainty

• sy,x residual standard deviation

• t α,f = m-2 student factor (α = 5 %, f = degrees of freedom, single-sided)

• m total number of calibration points (index j)

• n number of repetitions on each level

• xi sample concentration (in TEQ) of calibration point i determined by a confirmatory method

• Image omitted; see official source mean of the concentrations (in TEQ) of all calibration samples

• Image omitted; see official source square sum parameter, i = index for calibration point i.

• 7.3.2.Calculation from bioanalytical results (corrected for blank and recovery) of multiple analyses of samples (n ≥ 6) contaminated at the decision limit of the confirmatory method, as the lower endpoint of the data distribution at the corresponding mean BEQ value:

Image omitted; see official source

• with:

• SDR standard deviation of bioassay results at BEQDL, measured under within-laboratory reproducibility conditions.

• 7.3.3.Calculation as mean value of bioanalytical results (in BEQ, corrected for blank and recovery) from multiple analysis of samples (n ≥ 6) contaminated at two-thirds of the maximum level or action threshold, based on the observation that this level will be around the cut-off value determined under point 7.3.1 or point 7.3.2:

• Calculation of cut-off values based on a 95 % level of confidence implying a false-compliant rate < 5 %, and a RSDR < 25 %:

• (1) from the lower band of the 95 % prediction interval at the decision limit of the confirmatory method;

• (2) from multiple analysis of samples (n ≥ 6) contaminated at the decision limit of the confirmatory method as the lower end-point of the data distribution (represented in Figure 1 by a bell-shaped curve) at the corresponding mean BEQ value.

Figure 1

Image omitted; see official source

• 7.3.4.Restrictions to cut-off values:

• BEQ-based cut-off values calculated from the RSDR achieved during validation using a limited number of samples with different matrix/congener patterns may be higher than the TEQ-based maximum levels or action thresholds due to a better precision than attainable in routine when an unknown spectrum of possible congener patterns has to be controlled. In such cases, cut-off values shall be calculated from an RSDR = 25 %, or two-thirds of the maximum level or action threshold shall be preferred.

7.4.   Performance characteristics

• 7.4.1.Since no internal standards can be used in bioanalytical methods, tests on the repeatability of bioanalytical methods shall be carried out to obtain information on the standard deviation within and between test series. Repeatability shall be below 20 % and intra-laboratory reproducibility shall be below 25 %. This shall be based on the calculated levels in BEQ after blank and recovery correction.

• 7.4.2.As part of the validation process, the test shall be shown to discriminate between a blank sample and a level at the cut-off value, allowing the identification of samples above the corresponding cut-off value (see point 7.1.2).

• 7.4.3.Target compounds, possible interferences and maximum tolerable blank levels shall be defined.

• 7.4.4.The percent standard deviation in the response or concentration calculated from the response (only possible in working range) of a triplicate determination of a sample extract may not be above 15 %.

• 7.4.5.The uncorrected results of the reference sample(s) expressed in BEQ (blank and at the maximum level or action threshold) shall be used for evaluation of the performance of the bioanalytical method over a constant time period.

• 7.4.6.Quality control charts for procedure blanks and each type of reference sample shall be recorded and checked to make sure the analytical performance is in accordance with the requirements, in particular for the procedure blanks with regard to the requested minimum difference to the lower end of the working range and for the reference samples with regard to within-laboratory reproducibility. Procedure blanks shall be controlled in a manner to avoid false-compliant results when subtracted.

• 7.4.7.The results from the confirmatory methods of suspected samples and 2 to 10 % of the compliant samples (minimum of 20 samples per matrix) shall be collected and used to evaluate the performance of the screening method and the relationship between BEQ and TEQ. This database may be used for the re-evaluation of cut-off values applicable to routine samples for the validated matrices.

• 7.4.8.Successful method performance may also be demonstrated by participation in ring trials. The results from samples analysed in ring trials, covering a concentration range up to, e.g. 2x maximum level, may be included in the evaluation of the false-compliant rate, if a laboratory is able to demonstrate its successful performance. The samples shall cover most frequent congener patterns, representing various sources.

• 7.4.9.During incidents, the cut-off values may be re-evaluated, reflecting the specific matrix and congener patterns of this single incident.

8. Reporting of the results

8.1.   Confirmatory methods

• 8.1.1.The analytical results shall contain the levels of the individual PCDD/F and dioxin-like PCB congeners and TEQ-values shall be reported as lower-bound, upper-bound 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.

• 8.1.2.The report shall include the method used for extraction of PCDD/Fs and dioxin-like PCBs.

• 8.1.3.The recoveries of the individual internal standards shall be made available in case the recoveries are outside the range referred to in point 6.2.5, in case the maximum level is exceeded (in this case, the recoveries for one of the two duplicate analysis) and in other cases upon request.

• 8.1.4.As the expanded measurement uncertainty is to be taken into account when deciding about the compliance of a sample, this parameter shall 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 the case of a separate determination of PCDD/Fs and dioxin-like-PCBs, the sum of the estimated expanded uncertainty of the separate analytical results of PCDD/Fs and dioxin-like PCBs shall be used for the sum of PCDD/Fs and dioxin-like PCBs.

• 8.1.5.The results shall be expressed in the same units and with at least the same number of significant figures as the maximum levels laid down by Directive 2002/32/EC.

8.2.   Bioanalytical screening methods

• 8.2.1.The result of the screening shall be expressed as ‘compliant’ or ‘suspected to be non-compliant’ (‘suspected’).

• 8.2.2.In addition, an indicative result for PCDD/Fs and/or dioxin-like PCBs expressed in BEQ, and not TEQ, may be given.

• 8.2.3.Samples with a response below the reporting limit shall be expressed as ‘lower than the reporting limit’. Samples with a response above the working range shall be reported as ‘exceeding the working range’ and the level corresponding to the upper end of the working range shall be given in BEQ.

• 8.2.4.For each type of sample matrix, the report shall mention the maximum level or action threshold on which the evaluation is based.

• 8.2.5.The report shall mention the type of the test applied, the basic test principle and the kind of calibration.

• 8.2.6.The report shall include the method used for extraction of PCDD/Fs and dioxin-like PCBs.

• 8.2.7.In case of samples suspected to be non-compliant, the report needs to include a note on the action to be taken. The concentration of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs in those samples with elevated levels has to be determined/confirmed by a confirmatory method.

• 8.2.8.Non-compliant results shall only be reported from confirmatory analysis.

8.3.   Physico-chemical screening methods

• 8.3.1.The result of the screening shall be expressed as ‘compliant’ or ‘suspected to be non-compliant’ (‘suspected’).

• 8.3.2.For each type of sample matrix, the report shall mention the maximum level or action threshold on which the evaluation is based.

• 8.3.3.In addition, levels for individual PCDD/F and/or dioxin-like PCB congeners and TEQ-values reported as lower-bound, upper-bound and medium-bound may be given. The results shall be expressed in the same units and with at least the same number of significant figures as the maximum levels laid down by Directive 2002/32/EC.

• 8.3.4.The recoveries of the individual internal standards shall be made available in case the recoveries are outside the range referred to in point 6.2.5, in case the maximum level is exceeded (in this case, the recoveries for one of the two duplicate analysis) and in other cases upon request.

• 8.3.5.The report shall mention the GC-MS method applied.

• 8.3.6.The report shall include the method used for extraction of PCDD/Fs and dioxin-like PCBs.

• 8.3.7.In case of samples suspected to be non-compliant, the report needs to include a note on the action to be taken. The concentration of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs in those samples with elevated levels has to be determined/confirmed by a confirmatory method.

• 8.3.8.Non-compliance can only be decided after confirmatory analysis.

CHAPTER III

SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF ANALYSIS USED IN OFFICAL CONTROL OF THE LEVELS OF NON-DIOXIN-LIKE PCBS IN FEED

1. Field of application

The requirements set out in this Chapter shall be applied where feed is analysed for the official control of the levels of non-dioxin-like PCBs and as regards sample preparation and analytical requirements for other regulatory purposes, which includes the controls performed by the feed business operator to ensure compliance with the provisions of Regulation (EC) No 183/2005.

2. Applicable detection methods

Gas chromatography / Electron Capture Detection (GC-ECD), GC-LRMS, GC-MS/MS, GC-HRMS or equivalent methods.

3. Identification and confirmation of analytes of interest

• 3.1.Relative retention time in relation to internal standards or reference standards (acceptable deviation of +/- 0,25 %).

• 3.2.Gas chromatographic separation of the non-dioxin-like PCBs from interfering substances, especially co-eluting PCBs, in particular if levels of samples are in the range of legal limits and non-compliance is to be confirmed ([^E0061]).

• 3.3.Requirements for GC-MS techniques

• Monitoring of at least the following number of molecular ions or characteristic ions from the molecular cluster:

• (a) two specific ions for HRMS;

• (b) three specific ions for LRMS;

• (c) two specific precursor ions, each with one specific corresponding transition product ion for for MS-MS.

• Maximum permitted tolerances for abundance ratios for selected mass fragments:

• Relative deviation of abundance ratio of selected mass fragments from theoretical abundance or calibration standard for target ion (most abundant ion monitored) and qualifier ion(s): ± 15 %.

• 3.4.Requirements for GC-ECD techniques

• Results exceeding the maximum level shall be confirmed with two GC columns with stationary phases of different polarity.

4. Demonstration of performance of method

The performance of the method shall be validated in the range of the maximum level (0,5 to 2 times the maximum level) with an acceptable coefficient of variation for repeated analysis (see requirements for intermediate precision in point 9).

5. Limit of quantification

The sum of the LOQs ([^E0062]) of non-dioxin-like PCBs shall not be higher than one-third of the maximum level ([^E0063]).

6. Quality control

Regular blank controls, analysis of spiked samples, quality control samples, participation in inter-laboratory studies on relevant matrices.

7. Control of recoveries

• 7.1.Suitable internal standards with physico-chemical properties comparable to analytes of interest shall be used.

• 7.2.Addition of internal standards:

• Addition to products (before extraction and clean-up process).

• 7.3.Requirements for methods using all six isotope-labelled non-dioxin-like PCB congeners:

• (a) results shall be corrected for recoveries of internal standards;

• (b) recoveries of isotope-labelled internal standards shall be between 60 and 120 %;

• (c) lower or higher recoveries for individual congeners with a contribution to the sum of non-dioxin-like PCBs below 10 % are acceptable.

• 7.4.Requirements for methods using not all six isotope-labelled internal standards or other internal standards:

• (a) recovery of internal standard(s) shall be controlled for every sample;

• (b) recoveries of internal standard(s) shall be between 60 and 120 %;

• (c) results shall be corrected for recoveries of internal standards.

• 7.5.The recoveries of unlabelled congeners shall be checked by spiked samples or quality control samples with concentrations in the range of the maximum level. Recoveries for these congeners shall be considered acceptable, if they are between 60 and 120 %.

8. Requirements for laboratories

In accordance with the provisions of Regulation (EU) 2017/625, laboratories shall be accredited by a recognised body operating in accordance with ISO/IEC Guide 58 to ensure that they are applying analytical quality assurance. Laboratories shall be accredited following the EN ISO/IEC 17025 standard. In addition, the principles as described in Technical Guidelines for the estimation of measurement uncertainty and limits of quantification for PCB analysis shall be followed ([^E0064]).

9. Performance characteristics: criteria for the sum of non-dioxin-like PCBs at the maximum level

	Isotope dilution mass spectrometry [^E0065]	Other techniques
Trueness	-20 to 20  %	-30 to 30  %
Intermediate precision (RSD %)	≤ 15  %	≤ 20  %
Difference between upper and lower-bound calculation	≤ 20  %	≤ 20  %

Use of all six ¹³C-labelled analogues as internal standards required.

10. Reporting of the results

• 10.1.The analytical results shall contain the levels of the individual non-dioxin-like PCBs and the sum of those PCB congenersreported as lower-bound, upper-bound 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.

• 10.2.The report shall include the method used for the extraction of PCBs.

• 10.3.The recoveries of the individual internal standards shall be made available in case the recoveries are outside the range referred to in point 7, in case the maximum level is exceeded and in other cases upon request.

• 10.4.As the expanded measurement uncertainty is to be taken into account when deciding about the compliance of a sample, that 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 %.

• 10.5.The results shall be expressed in the same units and with at least the same number of significant figures as the maximum levels laid down by Directive 2002/32/EC.

B. EN STANDARDS

For the application of Article 34(2)(a) of Regulaiton (EU) 2017/625 the following EN standards are relevant:

EN 17194: Animal feeding stuffs: Methods of sampling and analysis – Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1 & B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in feed materials and compound feed by LC-MS/MS

EN 17270: Animal feeding stuffs: Methods of sampling and analysis – Determination of theobromine in feed materials and compound feed, including cocoa derived ingredients, by liquid chromatography

EN 17504 Animal feeding stuffs: Methods of sampling and analysis – Determination of gossypol in cotton seed and feeding stuff by LC-MS/MS

EN 17362 Animal feeding stuffs: Methods of sampling and analysis – Determination of pentachlorophenol (PCP) in feed materials and compound feed by LCMS/MS

EN 16279: Animal feeding stuffs – Determination of fluoride content after hydrochloric acid treatment by ion-sensitive electrode method (ISE)

EN 17053: Animal feeding stuffs: Methods of sampling and analysis – Determination of trace elements, heavy metals and other elements in feed by ICP-MS (multi-method)

EN 15550 Animal feeding stuffs – Methods of sampling and analysis – Determination of cadmium and lead by graphite furnace atomic absorption spectrometry (GF-AAS) after pressure digestion

EN 16206 Animal feeding stuffs – Determination of arsenic by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion (digestion with 65 % nitric acid and 30 % hydrogen peroxide

EN 16277 Animal feeding stuffs – Determination of mercury by cold-vapour atomic absorption spectrometry (CVAAS) after microwave pressure digestion (extraction with 65 % nitric acid and 30 % hydrogen peroxide)

EN 16278 Animal feeding stuffs – Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE)

EN 17374 Animal feeding stuffs: Methods of sampling and analysis – Determination of inorganic arsenic in animal feed by anion-exchange HPLC-ICP-MS

ANNEX VI

METHODS OF ANALYSIS FOR THE DETERMINATION OF CONSTITUENTS OF ANIMAL ORIGIN FOR THE OFFICIAL CONTROL OF FEED

1. PURPOSE AND SCOPE

The determination of constituents of animal origin in feed shall be performed by light microscopy or polymerase chain reaction (PCR) in accordance with the provisions laid down in this Annex.

These two methods make it possible to detect the presence of constituents of animal origin in premixtures, feed materials and compound feed. However, they do not make it possible to calculate the amount of such constituents in premixtures, feed materials and compound feed. Both methods have a limit of detection below 0,1 % (w/w).

The PCR method makes it possible to identify the taxonomic group of constituents of animal origin present in premixtures, feed materials and compound feed.

These methods shall apply for the control of the application of the prohibitions laid down in Article 7(1) of Regulation (EC) No 999/2001 of the European Parliament and of the Council ([^E0066]), Annex IV to that Regulation and Article 11(1) of Regulation (EC) No 1069/2009 of the European Parliament and of the Council ([^E0067]).

Depending on the type of feed being tested, these methods may be used, within one single operational protocol, either on their own or combined together in accordance with the standard operating procedures (‘SOPs’) established by the EU reference laboratory for animal proteins in feedingstuffs (EURL-AP) and published on its website ([^E0068]).

2. METHODS

2.1.   Light microscopy

2.1.1.   Principle

The constituents of animal origin which may be present in premixtures, feed materials and compound feed sent for analysis are identified on the basis of typical and microscopically identifiable characteristics such as muscle fibres and other meat particles, cartilage, bones, horn, hair, bristles, invertebrates cuticular fragments, insect tracheal structures, blood products, milk globules, lactose crystals, feathers, egg shells, fish bones and scales.

Microscopic examinations shall be performed after preparation of samples by sedimentation.

Samples shall be subject to a sedimentation step as follows:

• (a) for the detection of constituents of animal origin other than terrestrial invertebrates, a single Tetrachloroethylene (TCE) sedimentation step as detailed in point 2.1.3.4.3;

• (b) for the detection of constituents of terrestrial invertebrates, a double Petroleum ether/Tetrachloroethylene (PE/TCE) sedimentation step as detailed in point 2.1.3.4.4.

2.1.2.   Reagents and equipment

2.1.2.1.   Reagents

2.1.2.1.1.   Concentrating agent

• — Tetrachloroethylene (specific gravity 1,62).

• — Petroleum ether (PE) boiling point 40– 60 °C (specific gravity 0,65).

2.1.2.1.2.   Staining reagent

• — Alizarin Red solution (dilute 2,5 ml 1M hydrochloric acid in 100 ml water and add 200 mg Alizarin Red to this solution).

2.1.2.1.3.   Mounting media

• — Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v).

• — Glycerol (undiluted, viscosity: 1 490 cP) or a mounting medium with equivalent properties for non-permanent slide preparation.

• — Norland ® Optical Adhesive 65 (viscosity: 1 200 cP) or a resin with equivalent properties for permanent slide preparation.

2.1.2.1.4.   Mounting media with staining properties

• — Lugol solution (dissolve 2 g potassium iodide in 100 ml water and add 1 g iodine while frequently shaking).

• — Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water).

• — Fehling’s reagent (prepared before use from equal parts (1/1) of two-stock solutions A and B: solution A (dissolve 6,9 g copper (II) sulphate pentahydrate in 100 ml water); solution B (dissolve 34,6 g potassium sodium tartrate tetrahydrate and 12 g NaOH in 100 ml water).

• — Tetramethylbenzidine/Hydrogen peroxide (dissolve 1 g 3,3',5,5’ tetramethylbenzidine (TMB) in 100 ml glacial acetic acid and 150 ml water. Before use, mix 4 parts of this TMB solution with 1 part 3 % hydrogen peroxide).

2.1.2.1.5.   Rinsing agents

• — Ethanol ≥ 96 % (technical grade).

• — Acetone (technical grade).

2.1.2.1.6.   Bleaching reagent

• — Commercial sodium hypochlorite solution (9 – 14 % active chlorine).

2.1.2.2.   Equipment

• — Analytical balance with an accuracy of 0,001 g.

• — Grinding equipment: knife or rotor mill. If a rotor mill is used, mill sieves ≤ 0,5 mm shall be prohibited.

• — Sieves with square meshes of 0,25 mm and 1 mm width. With the exception of sample pre-sieving, the diameter of the sieves shall not exceed 10 cm to avoid loss of materials. Calibration of sieves is not required.

• — Conical glass separation funnel with a content of 250 ml with Teflon or ground glass stopcock at the base of the cone. Stopcock opening diameter shall be ≥ 4mm. Alternatively, for single TCE sedimentation only, a conical bottomed settling beaker may be used provided the laboratory has demonstrated that detection levels are equivalent to that obtained using the conical glass separation funnel. Image omitted; see official source Separation funnel

• — Stereomicroscope covering at least a 6,5x to 40x final magnification range.

• — Compound microscope covering at least a 100x to 400x final magnification range with transmitted light bright field. Polarised light, differential interferential contrast can additionally be used.

• — Standard laboratory glassware.

• — Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20x20 mm), tweezers, fine spatula.

• — Laboratory oven.

• — Centrifuge.

• — Filter paper: qualitative cellulose filter (pore size 4-11 μm).

2.1.3.   Sampling and sample preparation

2.1.3.1.   Sampling

A representative sample, taken in accordance with Annex I shall be used.

2.1.3.1.1.   Sample drying

Samples with a moisture content > 14 % shall be dried prior to handling in accordance with Annex III.

2.1.3.1.2.   Sample pre-sieving

In order to collect information on possible environmental contamination of the feed, it is recommended to pre-sieve at 1 mm pelleted feeds and kernels and to subsequently prepare, analyse, and report separately on the two resulting fractions, which must be considered as distinct samples.

2.1.3.2.   Precautions to be taken

In order to avoid laboratory cross-contamination, all reusable equipment shall be carefully cleaned before use. Separation funnel pieces shall be disassembled before cleaning. Separation funnel pieces and glassware shall be pre-washed manually and then washed in a washing machine. Sieves shall be cleaned by using a brush with stiff synthetic hairs. A final cleaning of sieves with acetone and compressed air is recommended after sieving of fatty material like fishmeal.

2.1.3.3.   Preparation of samples consisting of fat or oil

The following protocol shall be followed for the preparation of samples consisting of fat:

• — If the fat is solid, it shall be warmed in an oven until it is liquid.

• — By using a pipette, 40 ml of fat shall be transferred from the bottom of the sample to a centrifugation tube.

• — The sample shall be centrifuged during 10 min at 4 000 r.p.m.

• — If the fat is solid after centrifugation, it shall be warmed in an oven until it is liquid.

• — The centrifugation shall be repeated during 5 min at 4 000 r.p.m.

• — By using a small spoon or a spatula, one half of the decanted impurities shall be transferred to microscopic slides for examination. Glycerol is recommended as mounting medium.

• — The remaining impurities shall be used for preparing the sediment as described in point 2.1.3.4.3, first indent.

The same protocol, with the exception of the first and fourth indents, shall be applied for the preparation of samples consisting of oil.

2.1.3.4.   Preparation of samples other than fat or oil

• 2.1.3.4.1.Sub-sampling and grinding: at least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.

• 2.1.3.4.2.Preparation of raw material: a portion of at least 5 g of the ground sub-sample shall be prepared. It shall be sieved at 0,25 mm and the two resulting fractions shall be examined.

• 2.1.3.4.3.Single TCE sedimentation for the detection of constituents of animal origin other than terrestrial invertebrates.

• — Extraction and preparation of the sediment: A portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel or conical bottomed settling beaker and 50 ml of TCE shall be added. The portion transferred into the funnel shall be limited to 3 g in case of fishmeal or other pure animal products, mineral ingredients or premixtures which generate more than 10 % of sediment. The mixture shall be vigorously shaken for at least 30 s and 50 ml more of TCE shall be added cautiously while washing down the inside surface of the funnel to remove any adhering particles. The resulting mixture shall be left to stand for at least 5 min before the sediment is separated off by opening the stopcock. If a conical bottomed settling beaker is used then the mixture shall be vigorously stirred for at least 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean TCE. The mixture shall be left to stand for 3 min and then stirred again for 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean TCE. The resulting mixture shall be left to stand for at least 5 min and then the liquid fraction is removed and discarded by careful decanting, taking care not to lose any of the sediment. The sediment shall be collected on a filter paper placed into a funnel to allow the separation of the remaining TCE while avoiding fat deposition into the sediment. The sediment shall be dried. It is recommended to subsequently weigh the sediment (accurate to 0,001 g) to control the sedimentation step. Lastly, the sediment shall be sieved at 0,25 mm and the two resulting fractions shall be examined, unless sieving is not deemed necessary.

• — Extraction and preparation of the flotate: After recovery of the sediment with the method described above, two phases shall remain in the separation funnel: a liquid one consisting of TCE and a solid one made of floating material. This solid phase is the flotate and shall be recovered by pouring off completely TCE from the funnel by opening the stopcock. By inverting the separation funnel, the flotate shall be transferred into a large petri dish and air dried in a fume hood. It shall be sieved at 0,25 mm and the two resulting fractions shall be examined.

• — Use of staining reagents: In order to facilitate the correct identification of the constituents of animal origin, the operator may use staining reagents during the sample preparation in accordance with guidelines issued by the EURL-AP and published on its website. In case Alizarin Red solution is used to colour the sediment, the following protocol shall apply: — The dried sediment shall be transferred into a glass test tube and rinsed twice with approximately 5 ml of ethanol (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min 30 s and poured off). — The sediment shall be bleached by adding at least 1 ml sodium hypochlorite solution. The reaction shall be allowed to continue for 10 min. The tube shall be filled with water, the sediment shall be let settle 2-3 min, and the water and the suspended particles shall be poured off gently. — The sediment shall be rinsed twice more with about 10 ml of water (a vortex shall be used for 30 s, let settle, and pour off the water each time). — 2 to 10 drops of the Alizarin Red solution shall be added and the mixture shall be vortexed. The reaction shall be let occur for 30 s and the coloured sediment shall be rinsed twice with approximately 5 ml ethanol followed by one rinse with acetone (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min and poured off). — The coloured sediment shall be dried.

• 2.1.3.4.4.Double PE/TCE sedimentation for the detection of terrestrial invertebrate constituents.

• All steps shall be realised in a conical glass separation funnel of 250 ml as described in point 2.1.2.2, fourth indent.

• — A portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel and submitted first to a single TCE sedimentation as described in point 2.1.3.4.3 including the recovery of the sediment on a filter paper placed on a funnel. This sediment may be used as the one obtained from point 2.1.3.4.3.

• — The small volume of TCE drained together with the sediment shall be transferred into a graduated cylinder. By opening the stopcock of the separation funnel the graduated cylinder has to be filled further until obtaining 30 ml of TCE. Once this volume is achieved, the stopcock shall be closed.

• — This collected volume of TCE shall be substituted by adding a volume of 30 ml of petroleum ether boiling point 40– 60 °C into the separation funnel. The content of the separation funnel shall be mixed thoroughly to obtain a 30 % PE/70 % TCE mixture (with a density of approximately 1,26 g.cm^-3). Allow the material to settle down for 10 min. Two new fractions will segregate: a second sediment and a final flotate (< 1,26 g.cm^-3). The second sediment is to recover in a petri dish (or a filter paper placed on a funnel) by opening the stopcock until only a few solvent mixture and the final flotate remain in the separation funnel. The remaining liquid and the final flotate shall be collected separately on a filter paper placed on a funnel. The wall of the separation funnel shall be rinsed with a flush of PE to collect all material from the final flotate. The final flotate shall be allowed to dry. The final flotate shall be sieved at 0,25 mm and the two resulting fractions shall be examined for the detection of terrestrial invertebrate constituents, unless sieving is not deemed necessary.

2.1.4.   Microscopic examination

2.1.4.1.   Slide preparation

Microscopic slides shall be prepared from the sediment and, depending on operator’s choice, from either the flotate or the raw material. When appropriate, for the detection of terrestrial invertebrate constituents only, slides shall also be prepared from the final flotate obtained as described in point 2.1.3.4.4. The two resulting fractions (the fine and the coarse one) shall be prepared. Test portions of fractions spread on slides shall be representative of the whole fraction.

A sufficient number of slides shall be prepared in order to ensure that a complete examination protocol as laid down in point 2.1.4.2 can be carried-out.

Microscopic slides shall be mounted with the adequate mounting medium in accordance with the SOP established by the EURL-AP and published on its website. The slides shall be covered with coverslips.

2.1.4.2.   Observation flowchart for the detection of animal particles in compound feed, feed material and premixtures.

The prepared microscopic slides shall be observed in accordance with the observation flowcharts in Diagrams 1 and 2.

The microscopic observations shall be conducted using the compound microscope on the sediment and, depending on the operator’s choice, either on the flotate or on the raw material. Additionally, for the detection of terrestrial invertebrate constituents, observations shall also be conducted on the final flotate obtained as described in point 2.1.3.4.4 in accordance with Diagram 3. The stereomicroscope may be used in addition to the compound microscope for the coarse fractions. Each slide shall be screened entirely at various magnifications. Precise explanations on how to use the flowcharts are detailed by a SOP established by the EURL-AP and published on its website.

The minimum numbers of slides to be observed at each step of the observation flowcharts shall be strictly respected unless the entire fraction material does not permit to reach the stipulated slide number, for instance when no sediment is obtained. No more than 6 slides per determination shall be used for recording of the number of particles.

When additional slides are prepared using a more specific mounting medium with staining properties, as described in point 2.1.2.1.4, on the flotate or the raw material to further characterise structures (e.g. feathers, hairs, muscle or blood particles), which have been detected on slides prepared by other mounting media, as described in point 2.1.2.1.3, the number of particles shall be counted based on a number of slides per determination not exceeding 6, including the additional slides with a more specific mounting medium. The additional slides prepared from the final flotate obtained, as described in point 2.1.3.4.4, for the detection of terrestrial invertebrate constituents shall not be considered for the identification of other natures (terrestrial vertebrates and fish).

In order to facilitate the identification of the particles’ nature and origin, the operator may use support tools like decision support systems, image libraries and reference samples.

Diagram 1

Observation flowchart after single TCE sedimentation for the detection of animal particles other than from terrestrial invertebrates in compound feed, feed materials and premixtures for the first determination

Image omitted; see official source

Diagram 2

Observation flowchart after single TCE sedimentation for the detection of animal particles other than from terrestrial invertebrates in compound feed, feed materials and premixtures for the second determination

Image omitted; see official source

Diagram 3

Observation flowchart after double PE/TCE sedimentation for the detection of terrestrial invertebrates’ constituents in compound feed, feed materials and premixtures

Image omitted; see official source

2.1.4.3.   Number of determinations

Determinations shall be performed on different sub-samples of 50 g each.

If, following the first determination carried out in accordance with the observation flowchart in Diagram 1, or Diagram 3 when appropriate, no animal particles are detected, no additional determination shall be necessary and the result of the analysis shall be reported using the wording set out in point 2.1.5.1.

If, following the first determination carried out in accordance with the observation flowchart in Diagram 1, one or more animal particles of a given nature (i.e. terrestrial vertebrates or fish) are detected and the nature of the particles found confirms the declared content of the sample, no second determination shall be necessary. If the number of the animal particles of a given nature detected during this first determination is higher than 5, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.3. Otherwise, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.2.

If, following the first determination carried out in accordance with the observation flowchart in Diagram 3, more than 5 particles of terrestrial invertebrates are detected, no second determination shall be necessary and the result of the analysis shall be reported using the wording set out in point 2.1.5.3 for this nature.

In all other cases, including when no declaration of content has been provided to the laboratory, a second determination shall be carried out from a new sub-sample. If, following the second determination carried out in accordance with the observation flowchart in Diagram 2, or Diagram 3 when appropriate, the sum of the animal particles of a given nature detected over the two determinations is higher than 10, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.3. Otherwise, the result of the analysis shall be reported per animal nature using the wording set out in point 2.1.5.2.

2.1.5.   Expression of the results

When reporting the results, the laboratory shall indicate on which type of material the analysis has been carried out (sediment, flotate, final flotate or raw material). The reporting shall clearly indicate how many determinations have been carried out and if sieving of the fractions prior to slide preparation, in accordance with point 2.1.3.4.3, first indent, third paragraph, or point 2.1.3.4.4, third indent, was not performed.

The laboratory report shall at least contain information on the presence of constituents derived from terrestrial vertebrates and from fish.

The different situations shall be reported in the following ways.

• 2.1.5.1.No animal particle of a given nature is detected:

• — ‘As far as was discernible using a light microscope, no particle derived from terrestrial vertebrates was detected in the submitted sample.’

• — ‘As far as was discernible using a light microscope, no particle derived from fish was detected in the submitted sample.’

• — ‘As far as was discernible using a light microscope, no particle derived from terrestrial invertebrates was detected in the submitted sample.’

• 2.1.5.2.Between 1 and 5 animal particles of a given nature are detected when only one determination has been performed, or between 1 and 10 particles of a given nature are detected in case of two determinations (the number of detected particles is below the decision limit established in the SOP of the EURL-AP and published on its website):

• When only one determination has been performed:

• — ‘As far as was discernible using a light microscope, no more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

• — ‘As far as was discernible using a light microscope, no more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

• When two determinations have been performed:

• — ‘As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

• — ‘As far as was discernible using a light microscope, no more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

• — ‘As far as was discernible using a light microscope, no more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)]. This low level presence is below the decision limit established for this microscopic method.’

• Additionally:

• — In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.

• — When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.

• 2.1.5.3.More than 5 animal particles of a given nature are detected when only one determination has been performed, or more than 10 particles of a given nature are detected in case of two determinations:

• When only one determination has been performed:

• — ‘As far as was discernible using a light microscope, more than 5 particles derived from terrestrial vertebrates were detected in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)].’

• — ‘As far as was discernible using a light microscope, more than 5 particles derived from fish were detected in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)].’

• — ‘As far as was discernible using a light microscope, more than 5 particles derived from terrestrial invertebrates were detected in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)].’

• When two determinations have been performed:

• — ‘As far as was discernible using a light microscope, more than 10 particles derived from terrestrial vertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn, other (please specify as appropriate)].’

• — ‘As far as was discernible using a light microscope, more than 10 particles derived from fish were detected over the two determinations in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill, other (please specify as appropriate)].’

• — ‘As far as was discernible using a light microscope, more than 10 particles derived from terrestrial invertebrates were detected over the two determinations in the submitted sample. The particles were identified as … [cuticle fragments, mouthparts, muscles, tracheal structures, other (please specify as appropriate)].’

• Additionally:

• — In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction may be the sign of an environmental contamination.

• — When only animal particles which cannot be categorised as either terrestrial vertebrates or fish are detected (e.g. muscle fibres), the report shall mention that only such animal particles were detected and that it cannot be excluded that they originate from terrestrial vertebrates.

2.2.   PCR

2.2.1.   Principle

Deoxyribonucleic acid (DNA) fragments of animal origin which may be present in feed materials and compound feed are detected by a genetic amplification technique through PCR, targeting species-specific DNA sequences.

The PCR method first requires a DNA extraction step. The amplification step shall be applied afterwards to the so-obtained DNA extract, in order to detect the animal species targeted by the assay.

2.2.2.   Reagents and equipment

2.2.2.1.   Reagents

2.2.2.1.1.   Reagents for DNA extraction step

Only reagents approved by the EURL-AP and published on its website shall be used.

2.2.2.1.2.   Reagents for genetic amplification step

2.2.2.1.2.1.   Primers and probes

Only primers and probes with sequences of oligonucleotides validated by the EURL-AP shall be used ([^E0069]).

2.2.2.1.2.2.   Master Mix

Only Master Mix solutions which do not contain reagents susceptible to lead to false results due to presence of animal DNA shall be used ([^E0070]).

2.2.2.1.2.3.   Decontamination reagents

2.2.2.1.2.3.1.   Hydrochloric acid solution (0,1 N)

2.2.2.1.2.3.2.   Bleach (solution of sodium hypochlorite at 0,15 % of active chlorine)

2.2.2.1.2.3.3.   Non-corrosive reagents for decontaminating costly devices like analytical balances (e.g. DNA Erase^TM of MP Biomedicals)

2.2.2.2.   Equipment

2.2.2.2.1.   Analytical balance with an accuracy of 0,001 g

2.2.2.2.2.   Grinding equipment

2.2.2.2.3.   Thermocycler enabling real-time PCR

2.2.2.2.4.   Microcentrifuge for microfuge tubes

2.2.2.2.5.   Set of micropipettes allowing to pipet from 1 μl up to 1 000 μl

• 2.2.2.2.6. Standard molecular biology plastic-ware: microfuge tubes, filtered plastic tips for micropipettes, plates suitable for the thermocycler.

• 2.2.2.2.7. Freezers to store samples and reagents

2.2.3.   Sampling and sample preparation

2.2.3.1.   Sampling

A representative sample, taken in accordance with the provisions laid down in Annex I, shall be used.

2.2.3.2.   Sample preparation

The preparation of laboratory samples up to DNA extraction shall comply with the requirements set out in Annex II. At least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.

The sample preparation shall be performed in a room different from the ones dedicated to DNA extraction and to genetic amplification reactions as described by ISO 24276.

Two test portions of at least 100 mg each shall be prepared.

2.2.4.   DNA extraction

The DNA extraction shall be performed on each test portion prepared using the SOP established by the EURL-AP and published on its website.

Two extraction controls shall be prepared for each extraction series as described by ISO 24276.

• — an extraction blank control,

• — a positive DNA extraction control.

2.2.5.   Genetic amplification

The genetic amplification shall be performed using the methods validated for each species requiring identification. These methods are laid down in the SOP established by the EURL-AP and published on its website. Each DNA extract shall be analysed at least at two different dilutions in order to evaluate inhibition.

Two amplification controls shall be prepared per species target as described by ISO 24276.

• — a positive DNA target control shall be used for each plate or series of PCR assays,

• — an amplification reagent control (also called no template control) shall be used for each plate or series of PCR assays.

2.2.6.   Interpretation and expression of results

When reporting the results, the laboratory shall indicate at least the weight of the test portions used, the extraction technique used, the number of determinations carried-out and the limit of detection of the method.

Results shall not be interpreted and reported if the positive DNA extraction control and the positive DNA target controls do not provide positive results for the target under assay while the amplification reagent control is negative.

In case results from the two test portions are not consistent, at least the genetic amplification step shall be repeated. If the laboratory suspects that the DNA extracts can be the cause of the inconsistency, a new DNA extraction and a subsequent genetic amplification shall be performed before interpreting the results.

The final expression of the results shall be based on the integration and the interpretation of the results of the two test portions in accordance with the SOP established by the EURL-AP and published on its website.

2.2.6.1.   Negative result

A negative result shall be reported as follows:

No DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).

2.2.6.2.   Positive result

A positive result shall be reported as follows:

DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).

ANNEX VII

METHOD OF CALCULATING THE ENERGY VALUE OF POULTRY FEED

1. METHOD OF CALCULATION AND EXPRESSION OF ENERGY VALUE

The energy value of compound poultry feed must be calculated in accordance with the following formula set out on the basis of the percentages of certain analytical components of the feed. This value is to be expressed in megajoules (MJ) of metabolisable energy (ME), corrected for nitrogen, per kilogram of compound feed:

MJ/kg of ME = 0,1551 × % crude protein + 0,3431 × % crude fat + 0,1669 × % starch + 0,1301 × % total sugar (expressed as sucrose).

2. TOLERANCES APPLICABLE TO DECLARED VALUES

If the official inspection reveals a discrepancy (increased or reduced energy value of the feed) between the result of the inspection and the declared energy value, a tolerance of 0,4 MJ/kg of ME shall be permitted.

3. EXPRESSION OF RESULT

After application of the above formula, the result obtained must be given to one decimal place.

4. SAMPLING AND ANALYSIS METHODS

Sampling of the compound feed and determination of the content of analytical components indicated in the method of calculation must be performed in accordance with the Union sampling methods and analysis methods for the official control of feed respectively.

The following are to be applied:

• — for determining the crude fat content: procedure B of the method for the determination of crude oils and fats, laid down in Part G of Annex III,

• — for determining the starch content: the polarimetric method, laid down in Part K of Annex III

METHOD OF CALCULATING THE ENERGY VALUE IN FEED MATERIALS AND COMPOUND FEED FOR CATS AND DOGS

The energy value in feed materials and compound feed for cats and dogs is to be calculated in accordance with EN 16967 Animal feeding stuffs: Methods of sampling and analysis – Predictive equations for metabolisable energy in feed materials and compound feed (pet food) for cats and dogs including dietetic food.

ANNEX IX

CORRELATION TABLES REFERRED TO IN ARTICLE 6

1. Directive 71/250/EEC

Directive 71/250/EEC	This Regulation
Article 1 first subparagraph	Article 3
Article 1 second subparagraph	Article 2
Article 2	—
Article 3	—
Annex, part 1	Annex II
Annex, part 2	—
Annex, part 3	—
Annex, part 4	Annex III, part O
Annex, part 5	Annex III, part M
Annex, part 6	Annex III, part N
Annex, part 7	Annex III, part Q
Annex, part 9	Annex III, part K
Annex, part 10	—
Annex, part 11	—
Annex, part 12	Annex III, part J
Annex, part 14	Annex III, part D
Annex, part 16	—

2. Directive 71/393/EEC