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)

3.10.Lasalocid sodium standard substance with guaranteed purity, C34H53O8Na (sodium salt of a polyether monocarboxylic acid produced by Streptomyces lasaliensis), E763

3.10.1.Lasalocid sodium stock standard solution, 500 μg/ml

Weigh to the nearest 0,1 mg, 50 mg of lasalocid sodium (point 3.10) into a 100 ml graduated flask, dissolve in acidified methanol (point 3.8), make up to the mark with the same solvent and mix. This solution must be freshly prepared before use.

3.10.2.Lasalocid sodium intermediate standard solution, 50 μg/ml

Pipette 10,0 ml of stock standard solution (point 3.10.1) into a 100 ml graduated flask, make up to the mark with acidified methanol (point 3.8) and mix. This solution must be prepared freshly before use.

3.10.3.Calibration solutions

Into a series of 50 ml graduated flasks transfer 1,0, 2,0, 4,0, 5,0 and 10,0 ml of the intermediate standard solution (point 3.10.2). Make up to the mark with acidified methanol (point 3.8) and mix. These solutions correspond to 1,0, 2,0, 4,0, 5,0 and 10,0 μg of lasalocid sodium per ml respectively. These solutions must be prepared freshly before use.

3.11.Water, equivalent to HPLC grade.

4.1.Ultrasonic bath (or shaking water-bath) with temperature control.

4.2.Membrane filters, 0,45 μm.

4.3.HPLC equipment with injection system, suitable for injecting volumes of 20 μl

4.3.1.Liquid chromatographic column 125 mm × 4 mm, reversed-phase C18, 5 μm packing or equivalent.

4.3.2.Spectrofluorometer with variable wavelength adjustment of excitation and emission wavelengths.

For the performance of the recovery test (point 5.1.2) a blank feed shall be analysed to check that neither lasalocid sodium nor interfering substances are present. The blank feed shall be similar in type to that of the sample and lasalocid sodium or interfering substances shall not be detected.

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of lasalocid sodium, similar to that present in the sample. To fortify at a level of 100 mg/kg, transfer 10,0 ml of the stock standard (point 3.10.1) to a 250 ml conical flask and evaporate the solution to approximately 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 minutes mixing again several times before proceeding with the extraction step (point 5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see point 5.1.1), a recovery test can be performed by means of the standard addition method. In this case the sample to be analysed is fortified with a quantity of lasalocid sodium similar to that already present in the sample. This sample is analysed together with the unfortified sample and the recovery calculated by subtraction.

Weigh to the nearest 0,01 g, from 5 g to 10 g of the sample into a 250 ml conical flask with stopper. Add 100,0 ml of acidified methanol (point 3.8) by pipette. Stopper loosely and swirl to disperse. Place the flask in an ultrasonic bath (point 4.1) at approximately 40 °C for 20 minutes, then remove and cool to room temperature. Allow to stand for about 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (point 4.2) into a suitable vessel. Proceed to the HPLC determination (point 5.3).

Weigh to the nearest 0,001 g about 2 g of the unground premix into a 250 ml graduated flask. Add 100,0 ml of acidified methanol (point 3.8) and swirl to disperse. Place the flask and contents in an ultrasonic bath (point 4.1) at approximately 40 °C for 20 minutes, then remove and cool to room temperature. Dilute to the mark with acidified methanol (point 3.8) and mix thoroughly. Allow to stand for 1 hour until the suspended matter has settled, then filter an aliquot portion through a 0,45 μm membrane filter (point 4.2). Dilute an appropriate volume of the clear filtrate with acidified methanol (point 3.8) to produce a final test solution containing about 4 μg/ml of lasalocid sodium. Proceed to the HPLC determination (point 5.3).

The following conditions are offered for guidance; other conditions may be used, provided they yield equivalent results:

Liquid chromatographic column (point 4.3.1):	125 mm × 4 mm, reversed-phase C18, 5 μm packing or equivalent
Mobile phase (point 3.9):	Mixture of phosphate buffer solution (point 3.7) and methanol (point 3.5), 5 + 95 (V + V)
Flow rate:	1,2  ml/min
Detection wavelengths:	Excitation: 310 nm
	Emission: 419 nm
Injection volume:	20 μl

Check the stability of the chromatographic system, injecting the calibration solution (point 3.10.3) containing 4,0 μg/ml several times, until constant peak heights (or areas) and retention times are achieved.

Inject each calibration solution (point 3.10.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

Inject the sample extracts obtained in point 5.2.1 or 5.2.2 several times, using the same volume as taken for the calibration solution and determine the mean peak heights (areas) of the lasalocid sodium peaks.

From the mean peak height (area) produced by injection of the sample solution (point 5.3.3) determine the concentration of lasalocid sodium (μg/ml) by reference to the calibration graph.

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

where:

The lasalocid sodium content, w (mg/kg) in the sample is given by the following formula:

where:

Methods based on spectrofluorometry are less subject to interference than those in which UV detection is used. The identity of the analyte can be confirmed by co-chromatography.

A sample extract (point 5.2.1 or 5.2.2) is fortified by the addition of an appropriate amount of a calibration solution (point 3.10.3). The amount of added lasalocid sodium must be similar to the amount of lasalocid sodium found in the sample extract. Only the height of the lasalocid sodium peak shall be enhanced after taking into account the amount of lasalocid sodium added and the dilution of the extract. The peak width, at half height, must be within ± 10 % of the original peak width produced by the unfortified sample extract.

The difference between the results of two parallel determinations carried out on the same sample must not exceed:

— 15 % relative to the higher value for lasalocid sodium contents from 30 mg/kg to 100 mg/kg,

— 15 mg/kg for lasalocid sodium contents from 100 mg/kg to 200 mg/kg,

— 7,5 % relative to the higher value for lasalocid sodium contents of more than 200 mg/kg.

For the fortified (blank) feed sample, the recovery shall be at least 80 %. For the fortified premixture samples, the recovery shall be at least 90 %.

A collaborative study ()  was arranged in which 2 premixtures (samples 1 and 2) and 5 feeds (samples 3-7) were analysed by 12 laboratories. Duplicate analyses were performed on each sample. The results are given in the following table:

	Sample 1 Chicken premix	Sample 2 Turkey premix	Sample 3 Turkey pellets	Sample 4 Chicken crumbs	Sample 5 Turkey Feed	Sample 6 Poultry Feed A	Sample 7 Poultry Feed B
L	12	12	12	12	12	12	12
n	23	23	23	23	23	23	23
Mean [mg/kg]	5 050	16 200	76,5	78,4	92,9	48,3	32,6
sr [mg/kg]	107	408	1,71	2,23	2,27	1,93	1,75
CVr [%]	2,12	2,52	2,24	2,84	2,44	4,00	5,37
sR [mg/kg]	286	883	3,85	7,32	5,29	3,47	3,49
CVR [%]	5,66	5,45	5,03	9,34	5,69	7,18	10,70
Nominal content [mg/kg]	5 000  (*2)	16 000  (*2)	80 (*2)	105 (*2)	120 (*2)	50 ()	35 ()
(1) Analyst, 1995, 120, p. 2175-2180. (2) Content declared by manufacturer. (1) Feed prepared in the laboratory. L =  number of laboratories n =  number of single results sr =  standard deviation of repeatability sR =  standard deviation of reproducibility CVr =  coefficient of variation of repeatability, % CVR =  coefficient of variation of reproducibility, %.

H. DETERMINATION OF AMPROLIUM HYDROCHLORIDE

1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride monohydrochloride

This method makes it possible to determine the level of amprolium in feed. The detection limit is 1 mg/kg, the limit of quantification is 5 mg/kg.

The sample is extracted with a methanol-water mixture. After dilution with the mobile phase and membrane filtration the content of amprolium is determined by cation exchange high performance liquid chromatography (HPLC) using a UV detector.

3.1.Methanol.

3.2.Acetonitrile, equivalent to HPLC grade.

3.3.Water, equivalent to HPLC grade.

3.4.Sodium dihydrogen phosphate solution, c = 0,1 mol/l

Dissolve 13,80 g of sodium dihydrogen phosphate monohydrate in water (point 3.3) in a 1 000  ml graduated flask, make up to the mark with water (point 3.3) and mix.

3.5.Sodium perchlorate solution, c = 1,6 mol/l

Dissolve 224,74 g of sodium perchlorate monohydrate in water (point 3.3) in a 1 000  ml graduated flask, make up to the mark with water (point 3.3) and mix.

3.6.Mobile phase for HPLC (see observation point 9.1).

Mixture of acetonitrile (point 3.2), sodium dihydrogen phosphate solution (point 3.4) and sodium perchlorate solution (point 3.5), 450 + 450 + 100 (v+v+v). Prior to use filter through a 0,22 μm membrane filter (point 4.3) and degas the solution (e.g. in the ultrasonic bath (point 4.4) for at least 15 minutes).

3.7.Standard substance: pure amprolium, 1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride, E 750 (see point 9.2)

3.7.1.Amprolium stock standard solution, 500 μg/ml

Weigh to the nearest 0,1 mg, 50 mg of amprolium (point 3.7) in a 100 ml graduated flask, dissolve in 80 ml methanol (point 3.1) and place the flask for 10 min in an ultrasonic bath (point 4.4). After ultrasonic treatment bring the solution to room temperature, make up to the mark with water and mix. At a temperature of ≤ 4 °C the solution is stable for 1 month.

3.7.2.Amprolium intermediate standard solution, 50 μg/ml

Pipette 5,0 ml of the stock standard solution (point 3.7.1) into a 50 ml graduated flask, make up to the mark with the extraction solvent (point 3.8) and mix. At a temperature of ≤ 4 °C the solution is stable for 1 month.

3.7.3.Calibration solutions

Transfer 0,5, 1,0 and 2,0 ml of the intermediate standard solution (point 3.7.2) into a series of 50 ml graduated flasks. Make up to the mark with the mobile phase (point 3.6) and mix. These solutions correspond to 0,5, 1,0 and 2,0 μg of amprolium per ml respectively. These solutions must be prepared freshly before use.

3.8.Extraction solvent

Methanol (point 3.1)-water mixture 2 + 1 (v+v).

4.1.HPLC equipment with injection system, suitable for injection volumes of 100 μl

4.1.1.Liquid chromatographic column 125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent.

4.1.2.UV detector with variable wavelength adjustment or diode array detector.

4.2.Membrane filter, PTFE material, 0,45 μm.

4.3.Membrane filter, 0,22 μm.

4.4.Ultrasonic bath.

4.5.Mechanical shaker or magnetic stirrer.

For the performance of the recovery test (point 5.1.2) a blank feed shall be analysed to check that neither amprolium nor interfering substances are present. The blank feed shall be similar in type to that of the sample and amprolium or interfering substances must not be detected.

A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of amprolium, similar to that present in the sample. To fortify at a level of 100 mg/kg, transfer 10,0 ml of the stock standard solution (point 3.7.1) to a 250 ml conical flask and evaporate the solution to approximately 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 min mixing again several times before proceeding with the extraction step (point 5.2).

Alternatively, if a blank feed similar in type to that of the sample is not available (see point 5.1.1), a recovery test can be performed by means of the standard addition method. In this case, the sample to be analysed is fortified with a quantity of amprolium similar to that already present in the sample. This sample is analysed together with the unfortified sample and the recovery can be calculated by subtraction.

Weigh to the nearest 0,01 g, 5–40 g of the sample depending on the amprolium content into a 500 ml conical flask and add 200 ml extraction solvent (point 3.8). Place the flask in the ultrasonic bath (point 4.4) and leave for 15 minutes. Remove the flask from the ultrasonic bath and shake it for 1 h on the shaker or stir on the magnetic stirrer (point 4.5). Dilute an aliquot of the extract with the mobile phase (point 3.6) to an amprolium content of 0,5–2 μg/ml and mix (see observation point 9.3). Filter 5–10 ml of this diluted solution on a membrane filter (point 4.2). Proceed to the HPLC determination (point 5.3).

Weigh to the nearest 0,001 g, 1–4 g of the premixture depending on the amprolium content into a 500 ml conical flask and add 200 ml extraction solvent (point 3.8). Place the flask in the ultrasonic bath (point 4.4) and leave for 15 minutes. Remove the flask from the ultrasonic bath and shake it for 1 h on the shaker or stir on the magnetic stirrer (point 4.5). Dilute an aliquot of the extract with the mobile phase (point 3.6) to an amprolium content of 0,5–2 μg/ml and mix. Filter 5–10 ml of this diluted solution on a membrane filter (point 4.2). Proceed to the HPLC determination (point 5.3).

The following conditions are offered for guidance, other conditions may be used provided that they give equivalent results.

Liquid chromatographic column (point 4.1.1):	125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent
Mobile phase (point 3.6):	Mixture of acetonitrile (point 3.2), sodium dihydrogen phosphate solution (point 3.4) and sodium perchlorate solution (point 3.5), 450 + 450 + 100 (v + v + v).
Flow rate:	0,7 –1  ml/min
Detection wavelength:	264 nm
Injection volume:	100 μl

Check the stability of the chromatographic system, injecting several times the calibration solution (point 3.7.3) containing 1,0 μg/ml, until constant peak heights and retention times are achieved.

Inject each calibration solution (point 3.7.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) of the calibration solutions as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

Inject the sample extract (point 5.2) several times using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the amprolium peaks.

From the mean height (area) of the amprolium peaks of the sample solution determine the concentration of the sample solution in μg/ml by reference to the calibration graph (point 5.3.2).

The amprolium content w in mg/kg of the sample is given by the following formula:

in which:

The identity of the analyte can be confirmed by co-chromatography, or by using a diode-array detector by which the spectra of the sample extract (point 5.2) and the calibration solution (point 3.7.3) containing 2,0 μg/ml are compared.

A sample extract (point 5.2) is fortified by addition of an appropriate amount of calibration solution (point 3.7.3). The amount of added amprolium must be similar to the amount of amprolium found in the sample extract.

Only the height of the amprolium peak shall be enhanced after taking into account both the amount added and the dilution of the extract. The peak width, at half of its height, must be within ± 10 % of the original width of the amprolium peak of the unfortified sample extract.

The results are evaluated according to the following criteria:

(a) the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection this is typically within ± 2 nm;

(b) between 210 and 320 nm, the sample and standard spectra recorded at the peak apex of the chromatogram, must not be different for those parts of the spectrum within the range 10-100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte;

(c) between 210 and 320 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10-100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the peak apex.

If one of these criteria is not met, the presence of the analyte has not been confirmed.

The difference between the results of two parallel determinations carried out on the same sample must not exceed:

— 15 % relative to the higher value for amprolium contents from 25 mg/kg to 500 mg/kg,

— 75 mg/kg for amprolium contents between 500 mg/kg and 1 000  mg/kg,

— 7,5 % relative to the higher value for amprolium contents of more than 1 000  mg/kg.

For a fortified (blank) sample the recovery shall be at least 90 %.

A collaborative study was arranged in which three poultry feeds (sample 1-3), one mineral feed (sample 4) and one premix (sample 5) were analysed. The results are given in the following table:

	sample 1 (blank feed)	sample 2	sample 3	sample 4	sample 5
L	14	14	14	14	15
n	56	56	56	56	60
mean [mg/kg]	—	45,5	188	5 129	25 140
sr [mg/kg]	—	2,26	3,57	178	550
CVr [%]	—	4,95	1,90	3,46	2,20
sR [mg/kg]	—	2,95	11,8	266	760
CVR [%]	—	6,47	6,27	5,19	3,00
nominal content [mg/kg]	—	50	200	5 000	25 000
L: number of laboratories n: number of single values sr: standard deviation of repeatability CVr:  coefficient of variation of repeatability sR:  standard deviation of reproducibility CVR:  coefficient of variation of reproducibility.

9.1.If the sample contains thiamine, the thiamine peak in the chromatogram appears shortly before the amprolium peak. Following this method amprolium and thiamine must be separated. If the amprolium and thiamine are not separated by the column (point 4.1.1) used in this method, replace up to 50 % of the acetonitrile portion of the mobile phase (point 3.6) by methanol.

9.2.According to the British Pharmacopoeia, the spectrum of an amprolium solution (c = 0,02 mol/l) in hydrochloric acid (c = 0,1 mol/l) shows maxima at 246 nm and 262 nm. The absorbance shall amount to 0,84 at 246 nm and 0,80 at 262 nm.

9.3.The extract must always be diluted with the mobile phase, because otherwise the retention time of the amprolium peak may shift significantly, due to changes in the ionic strength.

I. DETERMINATION OF NARASIN

The narasin content is to be determined by

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN ISO 14183 Animal feedingstuffs – Determination of monensin, narasin and salinomycin contents – Liquid chromatographic method using post-column derivatisation.

J. DETERMINATION OF NICARBAZIN

The nicarbazin content is to be determined by:

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN 15782 Animal feedingstuffs – Determination of nicarbazin – High-performance liquid chromatographic method.

K. DETERMINATION OF DECOQUINATE

The decoquinate content is to be determined by:

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN 16162 Animal feedingstuffs – Determination of decoquinate by HPLC with fluorescence detection.

L. DETERMINATION OF MONENSIN

The monensin content is to be determined by

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN ISO 14183 Animal feedingstuffs – Determination of monensin, narasin and salinomycin contents – Liquid chromatographic method using post-column derivatisation.

M. DETERMINATION OF SALINOMYCIN

The salinomycin content is to be determined by

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN ISO 14183 Animal feedingstuffs – Determination of monensin, narasin and salinomycin contents – Liquid chromatographic method using post-column derivatisation.

N. DETERMINATION OF SEMDURAMYCIN

The semduramycin content is to be determined by:

— the method of analysis provided for by EN 17299 Animal feeding stuffs: Methods of sampling and analysis – Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography – Tandem Mass Spectrometry detection (LC-MS/MS), or

— the method provided by EN 16158 Animal feedingstuffs – Determination of semduramicin content – Liquid chromatographic method using a ‘tree’ analytical approach.

O. EN STANDARDS

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

ANNEX V

A. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND PCBs

CHAPTER I

METHODS OF SAMPLING AND INTERPRETATION OF ANALYTICAL RESULTS

The samples intended for the official control of the levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), dioxin-like polychlorinated biphenyls (PCBs) (²⁹) and non-dioxin-like PCBs 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 5.1 of Annex I shall be applied. Aggregate samples thus obtained shall be considered representative for the lots or sublots from which they are taken. Compliance with maximum levels laid down by Directive 2002/32/EC shall be established on the basis of the levels determined in the laboratory samples.

For the purposes of this Part, the definitions laid down in Annex I to Commission Implementing Regulation (EU) 2021/808 (³⁰) shall apply.

In addition to those definitions, the following definitions shall apply for the purpose of this Part:

The lot or sublot complies with the maximum level if the analytical result for the sum of PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and PCB 180 (hereafter referred to as non-dioxin-like PCBs) does not exceed the maximum level laid down by Directive 2002/32/EC, taking into account the expanded measurement uncertainty (³¹) The lot or sublot does not comply with the maximum level as laid down by Directive 2002/32/EC, if the mean of two upper-bound (³²) analytical results obtained from duplicate analysis (³³), taking into account the expanded measurement uncertainty, exceeds the maximum level beyond reasonable doubt, i.e. the analysed concentration after deduction of the expanded measurement uncertainty is used to assess compliance.

The expanded measurement uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sublot is non-compliant if the mean of the measured values minus the expanded uncertainty of the mean is above the maximum level.

The rules, mentioned in the paragraphs above under this point, shall apply for the analytical result obtained on the sample for official control. In case of analysis for second expert opinion or reference purposes, the national rules shall apply.

The lot or sublot complies with the maximum level if the result of a single analysis:

— performed by a screening method with a false-compliant rate below 5 %, indicates that the level does not exceed the respective maximum level of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs laid down by Directive 2002/32/EC,

— performed by a confirmatory method, does not exceed the respective maximum level of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs laid down by Directive 2002/32/EC, taking into account the expanded measurement uncertainty.

For screening assays a cut-off value shall be established for decisions on sample compliance with the respective maximum levels set for either PCDD/Fs, or for the sum of PCDD/Fs and dioxin-like PCBs.

The lot or sublot does not comply with the maximum level as laid down by Directive 2002/32/EC if the mean of two upper-bound (³⁴) analytical results obtained from duplicate analysis, (³⁵) using a confirmatory method, taking into account the expanded measurement uncertainty, exceeds the maximum level beyond reasonable doubt, i.e. the analysed concentration after deduction of the expanded measurement uncertainty is used to assess compliance.

The expanded measurement uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sublot is non-compliant if the mean of the measured values minus the expanded uncertainty of the mean is above the maximum level.

The sum of the estimated expanded uncertainties 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.

The rules, mentioned in the paragraphs above under this point, shall apply for the analytical result obtained on the sample for official control. In case of analysis for defence or reference purposes, the national rules shall apply.

Action thresholds serve as a tool for the selection of samples in those cases where it is necessary to identify a source of contamination and to take measures for its reduction or elimination. Screening methods shall establish the appropriate cut-off values for selection of those samples. Where significant efforts are necessary to identify a source and to reduce or eliminate the contamination, it is appropriate to confirm exceedance of the action thresholds by duplicate analysis using a confirmatory method and taking into account the expanded measurement uncertainty (³⁶).

CHAPTER II

SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF ANALYSIS USED IN OFFICAL CONTROL OF THE LEVELS OF DIOXINS (PCDD/FS) AND DIOXIN-LIKE PCBS IN FEED

The requirements set out in this Chapter shall be applied where feed is analysed for the official control of the levels of 2,3,7,8-substituted PCDD/Fs and 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 of the European Parliament and of the Council (³⁷).

Monitoring for the presence of PCDD/Fs and dioxin-like PCBs in feed may be performed with two different types of analytical methods:

The goal of screening methods is to select those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels or the action thresholds. Screening methods shall ensure cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks of consumers. Their application shall aim to avoid false-compliant results. They may comprise bioanalytical and GC-MS methods.

Screening methods compare the analytical result with a cut-off value, providing a yes/no decision over the possible exceedance of the maximum level or action threshold. The concentration of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs in samples suspected to be non-compliant with the maximum level shall be determined or confirmed by a confirmatory method.

In addition, screening methods may give an indication of the levels of PCDD/Fs and dioxin-like PCBs present in the sample. In case of application of bioanalytical screening methods the result is expressed as Bioanalytical Equivalents (BEQ), whereas in case of application of physico-chemical GC-MS methods it is expressed as Toxic Equivalents (TEQ). The numerically indicated results of screening methods are suitable for demonstrating compliance or suspected non-compliance or exceedance of action thresholds and give an indication of the range of levels in case of follow-up by confirmatory methods. They are not suitable for purposes such as evaluation of background levels, estimation of intake, following of time trends in levels or re-evaluation of action thresholds and maximum levels.

Confirmatory methods allow the unequivocal identification and quantification of PCDD/Fs and dioxin-like PCBs present in a sample and provide full information on the level of individual congeners. Therefore, those methods allow the control of maximum levels and action thresholds, including the confirmation of results obtained by screening methods. Furthermore, results may be used for other purposes such as determination of low background levels in feed monitoring, following of time trends, exposure assessment and building of a database for possible re-evaluation of action thresholds and maximum levels. They are also important for establishing congener patterns in order to identify the source of a possible contamination. Such methods utilise GC-HRMS. For confirming compliance or non-compliance with the maximum level, also GC-MS/MS can be used.

For calculation of TEQ concentrations, the concentrations of the individual substances in a given sample shall be multiplied by their respective Toxic Equivalency Factor (TEF) (see footnote 1 of Chapter I) and subsequently summed to give the total concentration of dioxin-like compounds expressed as TEQs.

For the purposes of this Part A, the accepted specific limit of quantification of an individual congener means the lowest content of the analyte that can be measured with reasonable statistical certainty, fulfilling the identification 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.

The limit of quantification of an individual congener may be identified as:

(a) the concentration of an analyte in the extract of a sample which produces an instrumental response at two different ions to be monitored with a S/N (signal/noise) ratio of 3:1 for the less intensive raw data signal; or

(b) if for technical reasons the signal-to-noise calculation does not provide reliable results, the lowest concentration point on a calibration curve that gives an acceptable (≤ 30 %) and consistent (measured at least at the start and at the end of an analytical series of samples) deviation to the average relative response factor calculated for all points on the calibration curve in each series of samples. The limit of quantification (LOQ) is calculated from the lowest concentration point taking into account the recovery of internal standards and the sample intake.

Bioanalytical screening methods will not give results at the congener level but merely an indication (³⁸) of the TEQ level, expressed in BEQ to acknowledge the fact that not all compounds present in a sample extract that produce a response in the test may fulfill or meet all requirements of the TEQ-principle.

Screening and confirmatory methods may only be applied for control of a certain matrix if the methods are sensitive enough to detect levels reliably at the action threshold or maximum level.

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

3.2.The samples shall be stored and transported in glass, aluminum, polypropylene or polyethylene containers suitable for storage without any influence on the levels of PCDD/Fs and dioxin-like PCBs in the samples. Traces of paper dust shall be removed from the sample container.

3.3.The sample storage and transportation shall be performed in a way that maintains the integrity of the feed sample.

3.4.Insofar as relevant, each laboratory sample shall be finely grinded and mixed thoroughly using a process that has been demonstrated to achieve complete homogenisation (for example, ground to pass a 1 mm sieve). Samples shall be dried before grinding if the moisture content is too high.

3.5.Control of reagents, glassware and equipment for possible influence of TEQ- or BEQ-based results shall be carried out.

3.6.A blank analysis shall be performed by carrying out the entire analytical procedure omitting only the sample.

3.7.For bioanalytical methods, all glassware and solvents used in analysis shall be tested to be free of compounds that interfere with the detection of target compounds in the working range. Glassware shall be rinsed with solvents or heated at temperatures suitable to remove traces of PCDD/Fs, dioxin-like compounds and interfering compounds from its surface.

3.8.Sample quantity used for the extraction shall be sufficient to fulfill the requirements with respect to a sufficiently low working range including the concentrations of maximum levels or action threshold.

3.9.The specific sample preparation procedures used for the products under consideration shall follow internationally accepted guidelines, i.e. EN ISO 6498.

4.1.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. The principles as described in the Technical Guidelines for the estimation of measurement uncertainty and limits of quantification for PCDD/F and PCB analysis shall be followed (³⁹).

4.2.Laboratory proficiency shall be proven by the continuous successful participation in inter-laboratory studies for the determination of PCDD/Fs and dioxin-like PCBs in relevant feed matrices and concentration ranges.

4.3.Laboratories applying screening methods for the routine control of samples shall establish a close cooperation with laboratories applying the confirmatory method, both for quality control and confirmation of the analytical result of suspected samples.

For PCDD/Fs, detectable quantities shall be in the upper femtogram (10^-15 g) range because of extreme toxicity of some of these compounds. For most PCB congeners a limit of quantification in the nanogram (10^-9 g) range is already sufficient. For the measurement of the more toxic dioxin-like PCB congeners (in particular non-ortho-substituted congeners), the lower end of the working range shall reach the low picogram (10^-12 g) levels. For all other PCB congeners a limit of quantification in the nanogram (10^-9 g) range is sufficient.

5.2.1.A distinction is required between PCDD/Fs and dioxin-like PCBs and 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 GC-MS methods, a differentiation among various congeners is required, such as between toxic (for example, the seventeen 2,3,7,8-substituted PCDD/Fs, and twelve dioxin-like PCBs) and other congeners.

5.2.2.Bioanalytical methods shall be able to detect the target compounds as the sum of PCDD/Fs, and/or dioxin-like PCBs. Sample clean-up shall aim at removing compounds causing false non-compliant results or compounds that may decrease the response, causing false compliant results.

5.3.1.For GC-MS methods, the determination shall provide a valid estimate of the true concentration in a sample. High accuracy is required to avoid the rejection of a sample analysis result on the basis of poor reliability of the determined TEQ level. Accuracy is expressed as trueness (difference between the mean value measured for an analyte in a certified material and its certified value, expressed as a percentage of this value) and precision (RSDR relative standard deviation calculated from results generated under reproducibility conditions).

5.3.2.For bioanalytical methods, the bioassay apparent recovery shall be determined. Bioassay apparent recovery means the BEQ level calculated from the TCDD or PCB 126 calibration curve corrected for the blank and then divided by the TEQ level determined by the confirmatory method. It aims at correcting factors like the loss of PCDD/Fs and dioxin-like compounds during the extraction and clean-up steps, co-extracted compounds increasing or decreasing the response (agonistic and antagonistic effects), the quality of the curve fit, or differences between the TEF values and the Relative Potency (REP) values. The bioassay apparent recovery is calculated from suitable reference samples with representative congener patterns around the level of interest.

5.4.1.Laboratories shall demonstrate the performance of a method in the range of the maximum level, for example, 0,5x, 1x and 2x the maximum level with an acceptable coefficient of variation for repeated analysis, during the validation procedure and during routine analysis.

5.4.2.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. Quality control charts for blank controls, spiking experiments or analysis of control samples shall be recorded and checked to make sure the analytical performance is in accordance with the requirements.

5.5.1.For a bioanalytical screening method, the establishment of the limit of quantification (LOQ) is not an indispensable requirement but the method shall prove that it can differentiate between the blank and the cut-off value. When providing a BEQ level, a reporting level shall be established to deal with samples showing a response below this level. The reporting level shall be demonstrated to be different from procedure blank samples at least by a factor of three, with a response below the working range. It shall therefore be calculated from samples containing the target compounds around the required minimum level, and not from an S/N ratio or an assay blank.

5.5.2.The LOQ for a confirmatory method shall be about one fifth of the maximum level.

For reliable results from confirmatory or screening methods, the following criteria shall be met in the range of the maximum level for the TEQ or BEQ value, respectively, whether determined as total TEQ or total BEQ (as the sum of PCDD/Fs and dioxin-like PCBs) or separately for PCDD/Fs and dioxin-like PCBs:

	Screening with bioanalytical or physico-chemical methods	Confirmatory methods
False-compliant rate (*1)	< 5  %
Trueness		-20  % to 20  %
Repeatability (RSDr)	< 20  %
Intermediate precision (RSDR)	< 25  %	< 15  %
(*1) With respect to the maximum levels.

5.7.1.Both GC-MS and bioanalytical methods may be used for screening. For GC-MS methods the requirements laid down in point 6 shall be met. For cell based bioanalytical methods specific requirements are laid down in point 7.

5.7.2.Laboratories applying screening methods for the routine control of samples shall establish a close cooperation with laboratories applying the confirmatory method.

5.7.3.Performance verification of the screening method is required during routine analysis, by analytical quality control and ongoing method validation. There shall be a continuous programme for the control of compliant results.

5.7.4.Check on possible suppression of the cell response and cytotoxicity:

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).

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.

6.2.1.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 %.

— 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.

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

— 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.

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).

— 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 %.

— 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.

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.

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.

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 (⁴⁰) 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.

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.

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.

7.2.1.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).

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

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

mean of the concentrations (in TEQ) of all calibration samples

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:

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.

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.

7.4.1.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.

8.1.1.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.

8.2.1.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.

8.3.1.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

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.

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

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 (⁴¹).

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.

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).

The sum of the LOQs (⁴²) of non-dioxin-like PCBs shall not be higher than one-third of the maximum level (⁴³).

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

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 %.

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 (⁴⁴).

	Isotope dilution mass spectrometry (*1)	Other techniques
Trueness	-20 to 20  %	-30 to 30  %
Intermediate precision (RSD %)	≤ 15  %	≤ 20  %
Difference between upper and lower-bound calculation	≤ 20  %	≤ 20  %
(*1) Use of all six 13C-labelled analogues as internal standards required.

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:

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 (⁴⁵), Annex IV to that Regulation and Article 11(1) of Regulation (EC) No 1069/2009 of the European Parliament and of the Council (⁴⁶).

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 (⁴⁷).

2. METHODS

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.

— Tetrachloroethylene (specific gravity 1,62).

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

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

— 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.

— 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).

— Ethanol ≥ 96 % (technical grade).

— Acetone (technical grade).

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

— 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. 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).

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

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

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.

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.

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.

2.1.3.4.1.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.

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.

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.

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.