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)
Modified by
- Commission Regulation (EU) No 278/2012of 28 March 2012amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32012R0278, March 29, 2012
- Commission Regulation (EU) No 51/2013of 16 January 2013amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed(Text with EEA relevance)Corrigendum to Commission Regulation (EU) No 51/2013 of 16 January 2013 amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed(Official Journal of the European Union L 20 of 23 January 2013), 32013R005132013R0051R(01), January 23, 2013
- Commission Regulation (EU) No 691/2013of 19 July 2013amending Regulation (EC) No 152/2009 as regards methods of sampling and analysis(Text with EEA relevance), 32013R0691, July 20, 2013
- Commission Regulation (EU) No 709/2014of 20 June 2014amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32014R0709, June 27, 2014
- Commission Regulation (EU) 2017/645of 5 April 2017correcting the Latvian language version of Regulation (EC) No 152/2009 laying down the methods of sampling and analysis for the official control of feed(Text with EEA relevance), 32017R0645, April 6, 2017
- Commission Regulation (EU) 2017/771of 3 May 2017amending Regulation (EC) No 152/2009 as regards the methods for the determination of the levels of dioxins and polychlorinated biphenyls(Text with EEA relevance), 32017R0771, May 4, 2017
- Commission Implementing Regulation (EU) 2020/1560of 26 October 2020amending Annex VI to Regulation (EC) No 152/2009 laying down the methods of analysis for the determination of constituents of animal origin for the official control of feed(Text with EEA relevance), 32020R1560, October 27, 2020
Corrected by
- Corrigendum to Commission Regulation (EU) No 51/2013 of 16 January 2013 amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed, 32013R0051R(01), March 6, 2013
Sampling personnel: the samples shall be taken by persons authorised for that purpose by the competent authority. The sample has to be sealed in such a manner as to prevent any access to the sample without breaking or removing the seal. The seal’s mark should be clearly identifiable and clearly visible. Alternatively, the sample can be put in a recipient which can be closed in such a manner that it cannot be opened without irreversibly damaging the receptacle or container, avoiding the re-use of the receptacle or container. Identification of the sample: the sample has to be indelibly marked and must be identified in such a way that there is an unambiguous link to the sampling report. From each aggregate sample at least two final samples are taken: at least one for control (enforcement) and one for the feed business operator (defence). Eventually, one final sample may be taken for reference. In case the complete aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator.
The quantitative requirements in points 5.1 and 5.2 as regards the number of incremental samples are applicable for sampled portion sizes up to a maximum of 500 tonnes and which can be sampled in a representative way. The sampling procedure described is equally valid for quantities larger than prescribed maximum sampled portion size provided that the maximum number of incremental samples given in the tables below is ignored, the number of incremental samples being determined by the square-root formula given in the appropriate part of the procedure (see point 5.3) and the minimum aggregate sample size increased proportionally. This does not prevent a large lot being divided into smaller sublots and each sublot sampled in accordance with the procedure described in points 5.1 and 5.2. The size of the sampled portion must be such that each of its constituent parts can be sampled. For very large lots or sublots (> 500 tonnes) and for lots which are transported or stored in such a way that sampling cannot be done in accordance with the sampling procedure provided for in points 5.1 and 5.2 of this chapter, the sampling procedure as provided for in point 5.3 is to be applied. In case the feed business operator is required by legislation to comply with this Regulation within the frame of a mandatory monitoring system, the feed business operator may deviate from the quantitative requirements as provided for in this chapter to take into account operational characteristics on the condition that the feed business operator has demonstrated to the satisfaction of the competent authority the equivalence of the sampling procedure as regards representativeness and after authorisation from the competent authority. In exceptional cases, if it is not possible to carry out the method of sampling set out as regards the quantitative requirements because of the unacceptable commercial damage to the lot (because of packaging forms, means of transport, way of storage etc.) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 2,5 tonnes | |
| > 2,5 tonnes | √ 20 times the number of tonnes making up the sampled portion |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 2,5 tonnes or ≤ | |
| > 2,5 tonnes or > |
| Size of sampled portion | Minimum number of units from which (at least) one incremental sample has to be taken |
|---|---|
| 1 to 20 units | 1 unit |
| 21 to 150 units | 3 units |
| 151 to 400 units | 5 units |
| > 400 units | ¼ of the √ number of units making up the sampled portion |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 5 tonnes | |
| > 5 tonnes | √ 5 times the number of tonnes making up the sampled portion |
control of aflatoxins, rye ergot, other mycotoxins and harmful botanical impurities in feed materials; control of cross contamination by a constituent, including GM material, or substance for which non-uniform distribution is expected in feed materials.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| < 80 tonnes | See quantitative requirements under point 5.1. The number of incremental samples to be taken has to be multiplied by 2,5. |
| ≥ 80 tonnes |
| A single aggregate sample per sampled portion is required. | ||
|---|---|---|
| Nature of feed | Minimum size of aggregate sample | |
| 6.1. | Loose feed | |
| 6.2. | Packaged feed: | |
| 6.3. | Liquid or semi-liquid feed: | 4 litres |
| 6.4. | Feed blocks or mineral licks: | |
| 6.4.1. | each weighing more than 1 kg | |
| 6.4.2. | each weighing not more than 1 kg | weight of four original blocks or licks |
| 6.5. | Roughage/forage | |
| Solid feed | |
| Liquid or semi-liquid feed |
Each sample shall be put into an appropriate container/receptacle. All necessary precautions shall be taken to avoid any change of composition of the sample, contamination or adulteration which might arise during transportation or storage. In case of the control of constituents or substances uniformly distributed throughout the feed, the aggregate sample can be representatively reduced to at least 2,0 kg or 2,0 litres (reduced sample) Except in the case of roughage or forage with low specific gravity. completely homogenized and divided afterwards into final samples or reduced to at least 2 kg or 2 litres Except in the case of roughage or forage with low specific gravity. 35000 seeds/grains to enable to obtain the final samples for enforcement, defence and reference of at least10000 seeds grain (see footnote (**) in chapter 6 and footnote (*) in chapter 7).
Mix the sieved final sample and collect it in a suitable clean, dry container fitted with an air-tight stopper. Mix again in order to ensure full homogenisation, immediately before weighing out the amount for analysis (test aliquot).
Unless otherwise specified in the methods of analysis, dry the final sample to bring its moisture content down to a level of 8 to 12 %, according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in Part A of Annex III). Then proceed as indicated in section 3.1.1.
Collect the final sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly in order to ensure full homogenisation immediately before weighing out the amount for analysis (test aliquot).
Final samples which cannot be prepared according to one of the above procedures shall be treated by any other procedure which ensures that the amounts weighed out for the analysis (test aliquot) are homogeneous and representative of the final samples.
In case of an examination by visual inspection (without making use of microscope), the whole laboratory sample is used for examination. In case of a microscopic examination, the laboratory may reduce the aggregate sample, or further reduce the reduced sample. The final samples for defence and eventually reference purposes are taken following a procedure equivalent to the procedure followed for the final sample for enforcement. In case the whole aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample.
(a) corrected for recovery, the level of recovery being indicated. The correction for recovery is not necessary in case the recovery rate is between 90-110 %. (b) 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 %.
0,2 % in absolute value, for crude protein contents of less than 20 %,1,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,0,4 % in absolute value, for crude protein contents of more than 40 %.
10 %, in relative value, for ammonia contents of less than 1,0 %,0,1 %, in absolute value, for ammonia contents of1,0 % or more.
a 100 ml round-bottomed flask (4.1) for open hydrolysis (5.3.2.3) or, a 250 ml round-bottomed flask (4.1) if a low sodium concentration is required (5.3.3.1) or, a 100 ml bottle fitted with a screw cap (4.2), for closed hydrolysis (5.3.2.4).
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Liquid chromatographic column (4.2): | 125 mm x 4 mm, C |
| Column temperature: | Room temperature |
| Mobile phase (3.22): | |
| Flow rate: | 1 ml/min. |
| Total run time: | approx. 34 min. |
| Detection wavelength: | excitation: 280 nm, emission: 356 nm. |
| Injection volume | 20 μl |
| L | 12 | 12 | 12 |
| n | 50 | 55 | 50 |
| Mean [g/kg] | |||
| s | |||
| r [g/kg] | |||
| CV | |||
| S | |||
| R [g/kg] | |||
| CV |
| L | 12 | 12 |
| n | 55 | 60 |
| Mean [g/kg] | ||
| s | ||
| r [g/kg] | ||
| CV | ||
| S | ||
| R [g/kg] | ||
| CV |
| L | 7 | 7 | 7 | 7 |
| n | 25 | 30 | 30 | 30 |
| Mean [g/kg] | ||||
| s | ||||
| r [g/kg] | ||||
| CV | ||||
| S | ||||
| R [g/kg] | ||||
| CV |
| Liquid chromatographic column: | 125 mm x 4 mm, C | ||
| Column temperature: | 32 | ||
| Mobile phase: | |||
| Gradient program: | 0 min. | 100 % A | 0 % B |
| 15 min. | 100 % A | 0 % B | |
| 17 min. | 60 % A | 40 % B | |
| 19 min. | 60 % A | 40 % B | |
| 21 min. | 100 % A | 0 % B | |
| 33 min. | 100 % A | 0 % B | |
| Flow rate: | |||
| Total run time: | approx. 33 min. | ||
0,2 %, in absolute value, for contents of crude oils and fats lower than 5 %,4,0 % relative to the highest result for contents of 5 % to 10 %,0,4 %, in absolute value, for contents above 10 %.
0,6 % in absolute value for crude fibre contents lower than 10 %,6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
| ml | mg | difference | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|---|---|
| ml | mg | difference | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|---|---|
| + | rice starch |
| + | potato starch |
| + | maize starch |
| + | wheat starch |
| + | barley starch |
| + | oat starch |
| + | other types of starch and starch mixtures in compound feed |
(sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp — molassed, (sugar) beet vinasse, (beet) sugar, citrus pulp, linseed; linseed expeller; linseed extracted, rape seed; rape seed expeller; rape seed extracted; rape seed hulls, sunflower seed; sunflower seed extracted; sunflower seed, partially decorticated, extracted, copra expeller; copra extracted, potato pulp, dehydrated yeast, products rich in inulin (e.g. Chips and meal of Jerusalem artichokes), greaves.
0,5 g for products containing from 50 % to 100 % of carbonates, expressed as calcium carbonate,1 g for products containing from 40 % to 50 % of carbonates, expressed as calcium carbonate, 2 to 3 g for other products.
3 %, relative to the higher result, for phosphorus contents of less than 5 %, 0,15 % in absolute value, for phosphorus contents of 5 % or more.
| Liquid chromatographic column (4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (3.9): | Mixture of methanol (3.3) and water e.g. 980 + 20 (v + v). |
| Flow rate: | 1-2 ml/min. |
| Detector (4.5.2): |
for vitamin A acetate = for vitamin A palmitate = 957 at 326 nm in 2-propanol) for vitamin A alcohol = 
[IU/kg]at saponification (5.2): due to the amount of fat present in the sample, increasing of potassium hydroxide solution amount (3.4) may be necessary, at extraction (5.3): due to the presence of emulsions, adaptation of the water/ethanol 2:1 ratio may be necessary.
| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | 13 | 12 | 13 | 12 | 13 |
| n | 48 | 45 | 47 | 46 | 49 |
| mean [IU/kg] | |||||
| S | 682 | ||||
| r [IU/kg] | |||||
| CV | |||||
| S | |||||
| R [IU/kg] | |||||
| CV | 15 | 20 |
| Liquid chromatographic column (4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (3.8): | Mixture of methanol (3.3) and water e.g. 980 + 20 (v + v). |
| Flow rate: | 1-2 ml/min. |
| Detector (4.5.2) |
= = 
[mg/kg]| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | 12 | 12 | 12 | 12 | 12 |
| n | 48 | 48 | 48 | 48 | 48 |
| mean [mg/kg] | 926 | 315 | |||
| S | 384 | ||||
| r [mg/kg] | |||||
| CV | |||||
| S | 830 | ||||
| R [mg/kg] | |||||
| CV |
iron (Fe): 20 mg/kg copper (Cu): 10 mg/kg manganese (Mn): 20 mg/kg zinc (Zn): 20 mg/kg.
dissolve 1 g of copper in powder form in 25 ml of 6 mol/litre hydrochloric acid (3.2), add 5 ml of hydrogen peroxide (3.6) and make up to one litre with water.
dissolve 1 g of manganese in powder form in 25 ml of 6 mol/litre hydrochloric acid (3.2) and make up to one litre with water.
dissolve 1 g of zinc in strip or leaf form in 25 ml of 6 mol/litre hydrochloric acid (3.2) and make up to one litre with water.
(a) In determining trace elements it is important to be alert to the risks of contamination, particularly by zinc, copper and iron. For this reason, the equipment used in preparing the samples must be free of these metals. To reduce the general risk of contamination, work in a dust-free atmosphere with scrupulously clean equipment and carefully washed glassware. The determination of zinc is particularly sensitive to many types of contamination, e.g. from glassware, reagents, dust, etc. (b) The weight of sample to be ashed is calculated from the approximate trace element content of the feed in relation to the sensitivity of the spectrophotometer used. For certain feed low in trace elements it may be necessary to start with a 10 to 20 g sample and make up the final solution to only 100 ml. (c) Ashing must be carried out in a closed furnace without injection of air or oxygen. (d) The temperature indicated by the pyrometer must not exceed 475 o C.
| μg Fe/ml | 0 | 1 | 2 | 3 | 4 | 5 | |
| ml working standard solution (3.7.1) (1 ml = 100 μg Fe) | 0 | 1 | 2 | 3 | 4 | 5 | |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
| μg Cu/ml | 0 | ||||||
| ml working standard solution (3.8.1) (1 ml = 10 μg Cu) | 0 | 1 | 2 | 4 | 6 | 8 | 10 |
| ml HCl (3.2) | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| μg Mn/ml | 0 | ||||||
| ml working standard solution (3.9.1) (1 ml = 10 μg Mn) | 0 | 1 | 2 | 4 | 6 | 8 | 10 |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
| μg Zn/ml | 0 | ||||||
| ml working standard solution (3.10.1) (1 ml = 10 μg Zn) | 0 | 1 | 2 | 4 | 6 | 8 | |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
Fe: 248,3 nmCu: 324,8 nmMn: 279,5 nmZn: 213,8 nm
5 mg/kg, in absolute value, for contents of the trace element concerned up to 50 mg/kg, 10 % of the higher result for contents of the trace element concerned from 50 and up to 100 mg/kg, 10 mg/kg, in absolute value, for contents of the trace element concerned from 100 and up to 200 mg/kg, 5 % of the higher result for contents of the trace element concerned above 200 mg/kg.
(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 225 and 300 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 225 and 300 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 % to 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 apex.
| Sample B (Meal) | Sample C (Pellets) | ||||
|---|---|---|---|---|---|
| On receipt | After two months | On receipt | After two months | ||
| Mean [mg/kg] | ND | ||||
| S | — | ||||
| CV | — | 16 | 18 | 14 | 17 |
| Rec. [%] | 86 | 74 | 88 | 75 | |
650 ml acetonitrile (3.3), 250 ml water (equivalent to HPLC-grade), 50 ml potassium di-hydrogen phosphate solution (3.6), 50 ml di-sodium hydrogen phosphate solution (3.7).
(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 approximately 2 nm; (b) between 250 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 250 and 400 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 % to 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 apex.
| Poultry | Rabbit | |||
|---|---|---|---|---|
| Meal | Pellet | Meal | Pellet | |
| Mean [mg/kg] | ||||
| s | ||||
| CV | ||||
| S | ||||
| CV | ||||
| Recovery [%] | ||||
| Liquid chromatographic column (4.2.1) | 100 mm × | |
| Mobile phase: | Eluent A (3.13.1): | Aqueous solution of ammonium acetate and tetrabutyl-ammonium hydrogen sulphate |
| Eluent B (3.13.2): | acetonitrile | |
| Eluent C (3.13.3): | methanol | |
| Elution mode: |
| |
| Flow rate: | ||
| Injection volume: | 20 μl | |
| Detector wavelength: | 280 nm. | |
[mg/kg]
[mg/kg](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 230 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 230 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.
30 % relative, to the higher value for diclazuril contents from 0,5 mg/kg to2,5 mg/kg,0,75 mg/kg for diclazuril contents between2,5 mg/kg and 5 mg/kg,15 % relative to the higher value for diclazuril contents of more than 5 mg/kg.
| L | 11 | 11 | 11 | 11 | 6 |
| n | 19 | 18 | 19 | 19 | 12 |
| Mean | |||||
| S | |||||
| CV | |||||
| S | |||||
| CV | |||||
| Nominal content (mg/kg) | 100 | 100 | 1 | 1 | 1 |
| Liquid chromatographic column (4.3.1): | 125 mm × 4 mm, reversed-phase C |
| Mobile phase (3.9): | Mixture of phosphate buffer solution (3.7) and methanol (3.5), 5+95 (V+V) |
| Flow rate: | |
| Detection wavelengths: | |
| Excitation: | 310 nm |
| Emission: | 419 nm |
| Injection volume: | 20 μl |
[mg/kg]
[mg/kg]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.
| L | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
| n | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
| Mean [mg/kg] | |||||||
| s | 107 | 408 | |||||
| CV | |||||||
| s | 286 | 883 | |||||
| CV | |||||||
| Nominal content [mg/kg] | 80 | 105 | 120 | 50 | 35 |
| Free gossypol: |  |
| Total gossypol: |  |
15 %, in relative value to the higher level, for gossypol contents of less than 500 ppm, 75 ppm, in absolute value, for contents of not less than 500 ppm and not more than 750 ppm, 10 %, in relative value to the higher value, for contents of more than 750 ppm.
| Congener | TEF value | Congener | TEF value |
|---|---|---|---|
| Dibenzo-p-dioxins ("PCDDs") and Dibenzo-p-furans '("PCDFs") | |||
| 2,3,7,8-TCDD | 1 | ||
| 1,2,3,7,8-PeCDD | 1 | Non-ortho PCBs | |
| 1,2,3,4,7,8-HxCDD | 0,1 | PCB 77 | 0,0001 |
| 1,2,3,6,7,8-HxCDD | 0,1 | PCB 81 | 0,0003 |
| 1,2,3,7,8,9-HxCDD | 0,1 | PCB 126 | 0,1 |
| 1,2,3,4,6,7,8-HpCDD | 0,01 | PCB 169 | 0,03 |
| OCDD | 0,0003 | Mono-ortho PCBs | |
| 2,3,7,8-TCDF | 0,1 | PCB 105 | 0,00003 |
| 1,2,3,7,8-PeCDF | 0,03 | PCB 114 | 0,00003 |
| 2,3,4,7,8-PeCDF | 0,3 | PCB 118 | 0,00003 |
| 1,2,3,4,7,8-HxCDF | 0,1 | PCB 123 | 0,00003 |
| 1,2,3,6,7,8-HxCDF | 0,1 | PCB 156 | 0,00003 |
| 1,2,3,7,8,9-HxCDF | 0,1 | PCB 157 | 0,00003 |
| 2,3,4,6,7,8-HxCDF | 0,1 | PCB 167 | 0,00003 |
| 1,2,3,4,6,7,8-HpCDF | 0,01 | PCB 189 | 0,00003 |
| 1,2,3,4,7,8,9-HpCDF | 0,01 | ||
| OCDF | 0,0003 | ||
"Screening methods" means methods used for selection of those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels or the action thresholds. They shall allow a cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks to consumers. Screening methods shall be based on bioanalytical or GC-MS methods. Results from samples exceeding the cut-off value used to check compliance with the maximum level shall be verified by a full re-analysis from the original sample using a confirmatory method. "Confirmatory methods" means methods that provide full or complementary information enabling the PCDD/Fs and dioxin-like PCBs to be identified and quantified unequivocally at the maximum or in case of need at the action threshold. Such methods utilize gas chromatography/high resolution mass spectrometry (GC-HRMS) or gas chromatography/tandem mass spectrometry (GC-MS/MS).
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.
(a) Screening 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 noncompliance 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. (b) Confirmatory methods Confirmatory methods allow the unequivocal identification and quantification of PCDD/Fs and dioxin-like PCBs present in a sample and provide full information on congener level. 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.
(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 sample intake.
| Screening with bioanalytical or physico-chemical methods | Confirmatory methods | |
|---|---|---|
| False-compliant rate | < 5 % | |
| Trueness | – 20 % to + 20 % | |
| Repeatability (RSD | < 20 % | |
| Intermediate precision (RSD | < 25 % | < 15 % |
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.
For GC-HRMS: In HRMS, the resolution shall typically be greater than or equal to 10000 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.
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 EC 70 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 R 2 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.
square sum parameter, i = index for calibration point i(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 the figure by a bell-shaped curve) at the corresponding mean BEQ value.
(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.
(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.
(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.
| Isotope dilution mass spectrometry | Other techniques | |
|---|---|---|
| Trueness | – 20 to + 20 % | – 30 to + 30 % |
| Intermediate precision (RSD %) | ≤ 15 % | ≤ 20 % |
| Difference between upper and lower-bound calculation | ≤ 20 % | ≤ 20 % |
if the fat is solid, it shall be warmed in a oven until it is liquid. by using a pipette, 40 ml of fat or oil shall be transferred from the bottom of the sample to a centrifugation tube. centrifuge during 10 minutes at 4000 r.p.m.if the fat is solid after centrifugation, it shall be warmed in an oven until it is liquid. repeat the centrifugation during 5 minutes at 4000 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 2.1.3.3.
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.
2.1.5.1. No animal particle of a given nature 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."
2.1.5.2. Between 1 and 5 animal particles of a given nature detected when only one determination has been performed, or between 1 and 10 particles of a given nature detected in case of two determinations (the number of detected particles is below the decision limit established in the standard operating procedures (SOP) of the EU reference laboratory for animal proteins in feedingstuffs (EURL-AP) and published on its website http://eurl.craw.eu/ 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…]. 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…]. 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…]. 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…]. 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 detected when only one determination has been performed, or more than 10 particles of a given nature 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…]." "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…]."
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…]." "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…]."
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.
an extraction blank control, a positive DNA extraction control.
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.
for determining the crude fat content: procedure B of the method for the determination of crude oils and fats, laid down in Part H of Annex III. for determining the starch content: the polarimetric method, laid down in Part L of Annex III.
liquid chromatographic column (4.4.1), HPLC mobile phase: methanol-water mixture (3.3), flow rate: 1 to 1,5 ml/minute,detection wavelength: 265 nm, Injection volume: 20 to 50 μl.
(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 approximately 2 nm; (b) between 220 and 350 nm, the sample and standard spectra recorded at the peak apex on the chromatogram must not be different for those parts of the spectrum within the range 10 % to 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 220 and 350 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 % to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at no observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.
| Blank | Meal 1 | Pellet 1 | Meal 2 | Pellet2 | |
|---|---|---|---|---|---|
| Mean [mg/kg] | ND | ||||
| s | — | ||||
| CV | — | ||||
| s | — | ||||
| CV | — | ||||
| Recovery [%] | — |
| Analytical column (4.3.1) | |
| Mobile Phase (3.4): | water (3.3)-methanol (3.2) mixture, 900 + 100 (V + V) |
| Flow rate: | |
| Detection wavelength: | 380 nm |
| Injection volume: | 20 μl –100 μl |
(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 220 and 400 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 220 and 400 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.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | |
|---|---|---|---|---|
| L | 13 | 10 | 11 | 11 |
| n | 40 | 40 | 44 | 44 |
| mean [mg/kg] | — | |||
| S | — | |||
| S | — | |||
| CV | — | |||
| CV | — | |||
| Nominal content | ||||
| [mg/kg] | — | 15 | 50 | 100 |
| recovery % | — |
| Liquid chromatographic | |
| column (4.1.1): | 125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent |
| Mobile phase (3.6): | Mixture of acetonitrile (3.2), sodium dihydrogen phosphate solution (3.4) and sodium perchlorate solution (3.5), 450+450+100 (v+v+v). |
| Flow rate: | |
| Detection wavelength: | 264 nm |
| Injection volume: | 100 μl |
[mg/kg](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.
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 1000 mg/kg,7,5 % relative to the higher value for amprolium contents of more than1000 mg/kg.
| 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] | — | 188 | |||
| s | — | 178 | 550 | ||
| CVr [%] | — | ||||
| s | — | 266 | 760 | ||
| CV | — | ||||
| nominal content [mg/kg] | — | 50 | 200 |
| Liquid chromatographic | |
| column (4.4.1): | 300 mm × 4 mm, C |
| Mobile phase (3.10): | Mixture of acetate buffer solution (3.9) and acetonitrile (3.2), 825 + 175 (v+v) |
| Flow rate: | |
| Detection wavelength: | 365 nm |
| Injection volume: | 20 μl |
[mg/kg] [mg/kg](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 225 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 225 and 400 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 % to 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 apex.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
|---|---|---|---|---|---|---|
| L | 8 | 8 | 8 | 8 | 8 | 8 |
| n | 15 | 14 | 15 | 15 | 15 | 15 |
| Mean (mg/kg) | ||||||
| Sr (mg/kg) | ||||||
| CVr (%) | ||||||
| SR (mg/kg) | ||||||
| CVR (%) | ||||||
| Nominal content (mg/kg) |
| Premixtures | Preparations | ||||||
|---|---|---|---|---|---|---|---|
| A | B | C | D | A | B | C | |
| L | 7 | 7 | 7 | 7 | 8 | 8 | 8 |
| n | 14 | 14 | 14 | 14 | 16 | 16 | 16 |
| Mean (g/kg) | 104 | ||||||
| Sr (g/kg) | |||||||
| CVr (%) | |||||||
| SR (g/kg) | |||||||
| CVR (%) | |||||||
| Nominal content (g/kg) | 100 | 100 | 100 | ||||
| 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 | — |
| Directive 71/393/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part I | Annex III, part A |
| Annex, part II | Annex III, part E |
| Annex, part III | Annex III, part P |
| Annex, part IV | Annex III, part H |
| Directive 72/199/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part L |
| Annex I, part 2 | Annex III, part C |
| Annex I, part 3 | — |
| Annex I, part 4 | — |
| Annex I, part 5 | Annex V, part A |
| Annex II | — |
| Directive 73/46/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part B |
| Annex I, part 2 | — |
| Annex I, part 3 | Annex III, part I |
| Directive 76/371/EEC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex I |
| Directive 76/372/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 78/633/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | — |
| Annex, part 2 | — |
| Annex, part 3 | Annex IV, part C |
| Directive 81/715/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 84/425/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 86/174/EEC | This Regulation |
|---|---|
| Article 1 | Article 4 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex VII |
| Directive 93/70/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex IV, part D |
| Directive 93/117/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | Annex IV, part E |
| Annex, part 2 | Annex VIII, part A |
| Directive 98/64/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex III, part F |
| Annex, part C | Annex VIII, part B |
| Directive 1999/27/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Article 7 | — |
| Annex, part A | Annex VIII, part C |
| Annex, part B | Annex IV, part F |
| Annex, part C | Annex VIII, part D |
| Directive 1999/76/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex | Annex IV, part G |
| Directive 2000/45/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex IV, part A |
| Annex, part B | Annex IV, part B |
| Annex, part C | Annex III, part G |
| Directive 2002/70/EC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | Articles 2 and 3 |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Annex I | Annex I and Annex V part B(I) |
| Annex II | Annex II and Annex V part B(II) |
| Directive 2003/126/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Annex | Annex VI |
Sampling personnel: the samples shall be taken by persons authorised for that purpose by the competent authority. The sample has to be sealed in such a manner as to prevent any access to the sample without breaking or removing the seal. The seal’s mark should be clearly identifiable and clearly visible. Alternatively, the sample can be put in a recipient which can be closed in such a manner that it cannot be opened without irreversibly damaging the receptacle or container, avoiding the re-use of the receptacle or container. Identification of the sample: the sample has to be indelibly marked and must be identified in such a way that there is an unambiguous link to the sampling report. From each aggregate sample at least two final samples are taken: at least one for control (enforcement) and one for the feed business operator (defence). Eventually, one final sample may be taken for reference. In case the complete aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator.
The quantitative requirements in points 5.1 and 5.2 as regards the number of incremental samples are applicable for sampled portion sizes up to a maximum of 500 tonnes and which can be sampled in a representative way. The sampling procedure described is equally valid for quantities larger than prescribed maximum sampled portion size provided that the maximum number of incremental samples given in the tables below is ignored, the number of incremental samples being determined by the square-root formula given in the appropriate part of the procedure (see point 5.3) and the minimum aggregate sample size increased proportionally. This does not prevent a large lot being divided into smaller sublots and each sublot sampled in accordance with the procedure described in points 5.1 and 5.2. The size of the sampled portion must be such that each of its constituent parts can be sampled. For very large lots or sublots (> 500 tonnes) and for lots which are transported or stored in such a way that sampling cannot be done in accordance with the sampling procedure provided for in points 5.1 and 5.2 of this chapter, the sampling procedure as provided for in point 5.3 is to be applied. In case the feed business operator is required by legislation to comply with this Regulation within the frame of a mandatory monitoring system, the feed business operator may deviate from the quantitative requirements as provided for in this chapter to take into account operational characteristics on the condition that the feed business operator has demonstrated to the satisfaction of the competent authority the equivalence of the sampling procedure as regards representativeness and after authorisation from the competent authority. In exceptional cases, if it is not possible to carry out the method of sampling set out as regards the quantitative requirements because of the unacceptable commercial damage to the lot (because of packaging forms, means of transport, way of storage etc.) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 2,5 tonnes | |
| > 2,5 tonnes | √ 20 times the number of tonnes making up the sampled portion |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 2,5 tonnes or ≤ | |
| > 2,5 tonnes or > |
| Size of sampled portion | Minimum number of units from which (at least) one incremental sample has to be taken |
|---|---|
| 1 to 20 units | 1 unit |
| 21 to 150 units | 3 units |
| 151 to 400 units | 5 units |
| > 400 units | ¼ of the √ number of units making up the sampled portion |
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 5 tonnes | |
| > 5 tonnes | √ 5 times the number of tonnes making up the sampled portion |
control of aflatoxins, rye ergot, other mycotoxins and harmful botanical impurities in feed materials; control of cross contamination by a constituent, including GM material, or substance for which non-uniform distribution is expected in feed materials.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| < 80 tonnes | See quantitative requirements under point 5.1. The number of incremental samples to be taken has to be multiplied by 2,5. |
| ≥ 80 tonnes |
| A single aggregate sample per sampled portion is required. | ||
|---|---|---|
| Nature of feed | Minimum size of aggregate sample | |
| 6.1. | Loose feed | |
| 6.2. | Packaged feed: | |
| 6.3. | Liquid or semi-liquid feed: | 4 litres |
| 6.4. | Feed blocks or mineral licks: | |
| 6.4.1. | each weighing more than 1 kg | |
| 6.4.2. | each weighing not more than 1 kg | weight of four original blocks or licks |
| 6.5. | Roughage/forage | |
| Solid feed | |
| Liquid or semi-liquid feed |
Each sample shall be put into an appropriate container/receptacle. All necessary precautions shall be taken to avoid any change of composition of the sample, contamination or adulteration which might arise during transportation or storage. In case of the control of constituents or substances uniformly distributed throughout the feed, the aggregate sample can be representatively reduced to at least 2,0 kg or 2,0 litres (reduced sample) Except in the case of roughage or forage with low specific gravity. completely homogenized and divided afterwards into final samples or reduced to at least 2 kg or 2 litres Except in the case of roughage or forage with low specific gravity. 35000 seeds/grains to enable to obtain the final samples for enforcement, defence and reference of at least10000 seeds grain (see footnote (**) in chapter 6 and footnote (*) in chapter 7).
Mix the sieved final sample and collect it in a suitable clean, dry container fitted with an air-tight stopper. Mix again in order to ensure full homogenisation, immediately before weighing out the amount for analysis (test aliquot).
Unless otherwise specified in the methods of analysis, dry the final sample to bring its moisture content down to a level of 8 to 12 %, according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in Part A of Annex III). Then proceed as indicated in section 3.1.1.
Collect the final sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly in order to ensure full homogenisation immediately before weighing out the amount for analysis (test aliquot).
Final samples which cannot be prepared according to one of the above procedures shall be treated by any other procedure which ensures that the amounts weighed out for the analysis (test aliquot) are homogeneous and representative of the final samples.
In case of an examination by visual inspection (without making use of microscope), the whole laboratory sample is used for examination. In case of a microscopic examination, the laboratory may reduce the aggregate sample, or further reduce the reduced sample. The final samples for defence and eventually reference purposes are taken following a procedure equivalent to the procedure followed for the final sample for enforcement. In case the whole aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample.
(a) corrected for recovery, the level of recovery being indicated. The correction for recovery is not necessary in case the recovery rate is between 90-110 %. (b) 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 %.
0,2 % in absolute value, for crude protein contents of less than 20 %,1,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,0,4 % in absolute value, for crude protein contents of more than 40 %.
10 %, in relative value, for ammonia contents of less than 1,0 %,0,1 %, in absolute value, for ammonia contents of1,0 % or more.
a 100 ml round-bottomed flask (4.1) for open hydrolysis (5.3.2.3) or, a 250 ml round-bottomed flask (4.1) if a low sodium concentration is required (5.3.3.1) or, a 100 ml bottle fitted with a screw cap (4.2), for closed hydrolysis (5.3.2.4).
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Reference material | Amino Acid | |||
|---|---|---|---|---|
| Threonine | Cyst(e)ine | Methionine | Lysine | |
| Mixed Pig Feed | ||||
| Broiler Compound | ||||
| Protein Concentrate | ||||
| Premixture | — | |||
| Liquid chromatographic column (4.2): | 125 mm x 4 mm, C |
| Column temperature: | Room temperature |
| Mobile phase (3.22): | |
| Flow rate: | 1 ml/min. |
| Total run time: | approx. 34 min. |
| Detection wavelength: | excitation: 280 nm, emission: 356 nm. |
| Injection volume | 20 μl |
| L | 12 | 12 | 12 |
| n | 50 | 55 | 50 |
| Mean [g/kg] | |||
| s | |||
| r [g/kg] | |||
| CV | |||
| S | |||
| R [g/kg] | |||
| CV |
| L | 12 | 12 |
| n | 55 | 60 |
| Mean [g/kg] | ||
| s | ||
| r [g/kg] | ||
| CV | ||
| S | ||
| R [g/kg] | ||
| CV |
| L | 7 | 7 | 7 | 7 |
| n | 25 | 30 | 30 | 30 |
| Mean [g/kg] | ||||
| s | ||||
| r [g/kg] | ||||
| CV | ||||
| S | ||||
| R [g/kg] | ||||
| CV |
| Liquid chromatographic column: | 125 mm x 4 mm, C | ||
| Column temperature: | 32 | ||
| Mobile phase: | |||
| Gradient program: | 0 min. | 100 % A | 0 % B |
| 15 min. | 100 % A | 0 % B | |
| 17 min. | 60 % A | 40 % B | |
| 19 min. | 60 % A | 40 % B | |
| 21 min. | 100 % A | 0 % B | |
| 33 min. | 100 % A | 0 % B | |
| Flow rate: | |||
| Total run time: | approx. 33 min. | ||
0,2 %, in absolute value, for contents of crude oils and fats lower than 5 %,4,0 % relative to the highest result for contents of 5 % to 10 %,0,4 %, in absolute value, for contents above 10 %.
0,6 % in absolute value for crude fibre contents lower than 10 %,6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.
| ml | mg | difference | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|---|---|
| ml | mg | difference | mg | difference | mg | difference | ml |
|---|---|---|---|---|---|---|---|
| + | rice starch |
| + | potato starch |
| + | maize starch |
| + | wheat starch |
| + | barley starch |
| + | oat starch |
| + | other types of starch and starch mixtures in compound feed |
(sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp — molassed, (sugar) beet vinasse, (beet) sugar, citrus pulp, linseed; linseed expeller; linseed extracted, rape seed; rape seed expeller; rape seed extracted; rape seed hulls, sunflower seed; sunflower seed extracted; sunflower seed, partially decorticated, extracted, copra expeller; copra extracted, potato pulp, dehydrated yeast, products rich in inulin (e.g. Chips and meal of Jerusalem artichokes), greaves.
0,5 g for products containing from 50 % to 100 % of carbonates, expressed as calcium carbonate,1 g for products containing from 40 % to 50 % of carbonates, expressed as calcium carbonate, 2 to 3 g for other products.
3 %, relative to the higher result, for phosphorus contents of less than 5 %, 0,15 % in absolute value, for phosphorus contents of 5 % or more.
| Liquid chromatographic column (4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (3.9): | Mixture of methanol (3.3) and water e.g. 980 + 20 (v + v). |
| Flow rate: | 1-2 ml/min. |
| Detector (4.5.2): |
for vitamin A acetate = for vitamin A palmitate = 957 at 326 nm in 2-propanol) for vitamin A alcohol = [IU/kg]at saponification (5.2): due to the amount of fat present in the sample, increasing of potassium hydroxide solution amount (3.4) may be necessary, at extraction (5.3): due to the presence of emulsions, adaptation of the water/ethanol 2:1 ratio may be necessary.
| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | 13 | 12 | 13 | 12 | 13 |
| n | 48 | 45 | 47 | 46 | 49 |
| mean [IU/kg] | |||||
| S | 682 | ||||
| r [IU/kg] | |||||
| CV | |||||
| S | |||||
| R [IU/kg] | |||||
| CV | 15 | 20 |
| Liquid chromatographic column (4.5.1): | 250 mm × 4 mm, C |
| Mobile phase (3.8): | Mixture of methanol (3.3) and water e.g. 980 + 20 (v + v). |
| Flow rate: | 1-2 ml/min. |
| Detector (4.5.2) |
= = [mg/kg]| Premix | Premix feed | Mineral concentrate | Protein feed | Piglet | |
|---|---|---|---|---|---|
| L | 12 | 12 | 12 | 12 | 12 |
| n | 48 | 48 | 48 | 48 | 48 |
| mean [mg/kg] | 926 | 315 | |||
| S | 384 | ||||
| r [mg/kg] | |||||
| CV | |||||
| S | 830 | ||||
| R [mg/kg] | |||||
| CV |
iron (Fe): 20 mg/kg copper (Cu): 10 mg/kg manganese (Mn): 20 mg/kg zinc (Zn): 20 mg/kg.
dissolve 1 g of copper in powder form in 25 ml of 6 mol/litre hydrochloric acid (3.2), add 5 ml of hydrogen peroxide (3.6) and make up to one litre with water.
dissolve 1 g of manganese in powder form in 25 ml of 6 mol/litre hydrochloric acid (3.2) and make up to one litre with water.
dissolve 1 g of zinc in strip or leaf form in 25 ml of 6 mol/litre hydrochloric acid (3.2) and make up to one litre with water.
(a) In determining trace elements it is important to be alert to the risks of contamination, particularly by zinc, copper and iron. For this reason, the equipment used in preparing the samples must be free of these metals. To reduce the general risk of contamination, work in a dust-free atmosphere with scrupulously clean equipment and carefully washed glassware. The determination of zinc is particularly sensitive to many types of contamination, e.g. from glassware, reagents, dust, etc. (b) The weight of sample to be ashed is calculated from the approximate trace element content of the feed in relation to the sensitivity of the spectrophotometer used. For certain feed low in trace elements it may be necessary to start with a 10 to 20 g sample and make up the final solution to only 100 ml. (c) Ashing must be carried out in a closed furnace without injection of air or oxygen. (d) The temperature indicated by the pyrometer must not exceed 475 o C.
| μg Fe/ml | 0 | 1 | 2 | 3 | 4 | 5 | |
| ml working standard solution (3.7.1) (1 ml = 100 μg Fe) | 0 | 1 | 2 | 3 | 4 | 5 | |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
| μg Cu/ml | 0 | ||||||
| ml working standard solution (3.8.1) (1 ml = 10 μg Cu) | 0 | 1 | 2 | 4 | 6 | 8 | 10 |
| ml HCl (3.2) | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| μg Mn/ml | 0 | ||||||
| ml working standard solution (3.9.1) (1 ml = 10 μg Mn) | 0 | 1 | 2 | 4 | 6 | 8 | 10 |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
| μg Zn/ml | 0 | ||||||
| ml working standard solution (3.10.1) (1 ml = 10 μg Zn) | 0 | 1 | 2 | 4 | 6 | 8 | |
| ml HCl (3.2) | 7 | 7 | 7 | 7 | 7 | 7 | 7 |
| + 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml with water | |||||||
Fe: 248,3 nmCu: 324,8 nmMn: 279,5 nmZn: 213,8 nm
5 mg/kg, in absolute value, for contents of the trace element concerned up to 50 mg/kg, 10 % of the higher result for contents of the trace element concerned from 50 and up to 100 mg/kg, 10 mg/kg, in absolute value, for contents of the trace element concerned from 100 and up to 200 mg/kg, 5 % of the higher result for contents of the trace element concerned above 200 mg/kg.
(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 225 and 300 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 225 and 300 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 % to 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 apex.
| Sample B (Meal) | Sample C (Pellets) | ||||
|---|---|---|---|---|---|
| On receipt | After two months | On receipt | After two months | ||
| Mean [mg/kg] | ND | ||||
| S | — | ||||
| CV | — | 16 | 18 | 14 | 17 |
| Rec. [%] | 86 | 74 | 88 | 75 | |
650 ml acetonitrile (3.3), 250 ml water (equivalent to HPLC-grade), 50 ml potassium di-hydrogen phosphate solution (3.6), 50 ml di-sodium hydrogen phosphate solution (3.7).
(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 approximately 2 nm; (b) between 250 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 250 and 400 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 % to 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 apex.
| Poultry | Rabbit | |||
|---|---|---|---|---|
| Meal | Pellet | Meal | Pellet | |
| Mean [mg/kg] | ||||
| s | ||||
| CV | ||||
| S | ||||
| CV | ||||
| Recovery [%] | ||||
| Liquid chromatographic column (4.2.1) | 100 mm × | |
| Mobile phase: | Eluent A (3.13.1): | Aqueous solution of ammonium acetate and tetrabutyl-ammonium hydrogen sulphate |
| Eluent B (3.13.2): | acetonitrile | |
| Eluent C (3.13.3): | methanol | |
| Elution mode: |
| |
| Flow rate: | ||
| Injection volume: | 20 μl | |
| Detector wavelength: | 280 nm. | |
[mg/kg] [mg/kg](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 230 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 230 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.
30 % relative, to the higher value for diclazuril contents from 0,5 mg/kg to2,5 mg/kg,0,75 mg/kg for diclazuril contents between2,5 mg/kg and 5 mg/kg,15 % relative to the higher value for diclazuril contents of more than 5 mg/kg.
| L | 11 | 11 | 11 | 11 | 6 |
| n | 19 | 18 | 19 | 19 | 12 |
| Mean | |||||
| S | |||||
| CV | |||||
| S | |||||
| CV | |||||
| Nominal content (mg/kg) | 100 | 100 | 1 | 1 | 1 |
| Liquid chromatographic column (4.3.1): | 125 mm × 4 mm, reversed-phase C |
| Mobile phase (3.9): | Mixture of phosphate buffer solution (3.7) and methanol (3.5), 5+95 (V+V) |
| Flow rate: | |
| Detection wavelengths: | |
| Excitation: | 310 nm |
| Emission: | 419 nm |
| Injection volume: | 20 μl |
[mg/kg] [mg/kg]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.
| L | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
| n | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
| Mean [mg/kg] | |||||||
| s | 107 | 408 | |||||
| CV | |||||||
| s | 286 | 883 | |||||
| CV | |||||||
| Nominal content [mg/kg] | 80 | 105 | 120 | 50 | 35 |
| Free gossypol: | |
| Total gossypol: | |
15 %, in relative value to the higher level, for gossypol contents of less than 500 ppm, 75 ppm, in absolute value, for contents of not less than 500 ppm and not more than 750 ppm, 10 %, in relative value to the higher value, for contents of more than 750 ppm.
| Congener | TEF value | Congener | TEF value |
|---|---|---|---|
| Dibenzo-p-dioxins ("PCDDs") and Dibenzo-p-furans '("PCDFs") | |||
| 2,3,7,8-TCDD | 1 | ||
| 1,2,3,7,8-PeCDD | 1 | Non-ortho PCBs | |
| 1,2,3,4,7,8-HxCDD | 0,1 | PCB 77 | 0,0001 |
| 1,2,3,6,7,8-HxCDD | 0,1 | PCB 81 | 0,0003 |
| 1,2,3,7,8,9-HxCDD | 0,1 | PCB 126 | 0,1 |
| 1,2,3,4,6,7,8-HpCDD | 0,01 | PCB 169 | 0,03 |
| OCDD | 0,0003 | Mono-ortho PCBs | |
| 2,3,7,8-TCDF | 0,1 | PCB 105 | 0,00003 |
| 1,2,3,7,8-PeCDF | 0,03 | PCB 114 | 0,00003 |
| 2,3,4,7,8-PeCDF | 0,3 | PCB 118 | 0,00003 |
| 1,2,3,4,7,8-HxCDF | 0,1 | PCB 123 | 0,00003 |
| 1,2,3,6,7,8-HxCDF | 0,1 | PCB 156 | 0,00003 |
| 1,2,3,7,8,9-HxCDF | 0,1 | PCB 157 | 0,00003 |
| 2,3,4,6,7,8-HxCDF | 0,1 | PCB 167 | 0,00003 |
| 1,2,3,4,6,7,8-HpCDF | 0,01 | PCB 189 | 0,00003 |
| 1,2,3,4,7,8,9-HpCDF | 0,01 | ||
| OCDF | 0,0003 | ||
"Screening methods" means methods used for selection of those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels or the action thresholds. They shall allow a cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks to consumers. Screening methods shall be based on bioanalytical or GC-MS methods. Results from samples exceeding the cut-off value used to check compliance with the maximum level shall be verified by a full re-analysis from the original sample using a confirmatory method. "Confirmatory methods" means methods that provide full or complementary information enabling the PCDD/Fs and dioxin-like PCBs to be identified and quantified unequivocally at the maximum or in case of need at the action threshold. Such methods utilize gas chromatography/high resolution mass spectrometry (GC-HRMS) or gas chromatography/tandem mass spectrometry (GC-MS/MS).
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.
(a) Screening 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 noncompliance 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. (b) Confirmatory methods Confirmatory methods allow the unequivocal identification and quantification of PCDD/Fs and dioxin-like PCBs present in a sample and provide full information on congener level. 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.
(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 sample intake.
| Screening with bioanalytical or physico-chemical methods | Confirmatory methods | |
|---|---|---|
| False-compliant rate | < 5 % | |
| Trueness | – 20 % to + 20 % | |
| Repeatability (RSD | < 20 % | |
| Intermediate precision (RSD | < 25 % | < 15 % |
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.
For GC-HRMS: In HRMS, the resolution shall typically be greater than or equal to 10000 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.
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 EC 70 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 R 2 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.
square sum parameter, i = index for calibration point i(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 the figure by a bell-shaped curve) at the corresponding mean BEQ value.
(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.
(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.
(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.
| Isotope dilution mass spectrometry | Other techniques | |
|---|---|---|
| Trueness | – 20 to + 20 % | – 30 to + 30 % |
| Intermediate precision (RSD %) | ≤ 15 % | ≤ 20 % |
| Difference between upper and lower-bound calculation | ≤ 20 % | ≤ 20 % |
if the fat is solid, it shall be warmed in a oven until it is liquid. by using a pipette, 40 ml of fat or oil shall be transferred from the bottom of the sample to a centrifugation tube. centrifuge during 10 minutes at 4000 r.p.m.if the fat is solid after centrifugation, it shall be warmed in an oven until it is liquid. repeat the centrifugation during 5 minutes at 4000 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 2.1.3.3.
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.
2.1.5.1. No animal particle of a given nature 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."
2.1.5.2. Between 1 and 5 animal particles of a given nature detected when only one determination has been performed, or between 1 and 10 particles of a given nature detected in case of two determinations (the number of detected particles is below the decision limit established in the standard operating procedures (SOP) of the EU reference laboratory for animal proteins in feedingstuffs (EURL-AP) and published on its website http://eurl.craw.eu/ 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…]. 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…]. 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…]. 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…]. 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 detected when only one determination has been performed, or more than 10 particles of a given nature 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…]." "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…]."
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…]." "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…]."
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.
an extraction blank control, a positive DNA extraction control.
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.
for determining the crude fat content: procedure B of the method for the determination of crude oils and fats, laid down in Part H of Annex III. for determining the starch content: the polarimetric method, laid down in Part L of Annex III.
liquid chromatographic column (4.4.1), HPLC mobile phase: methanol-water mixture (3.3), flow rate: 1 to 1,5 ml/minute,detection wavelength: 265 nm, Injection volume: 20 to 50 μl.
(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 approximately 2 nm; (b) between 220 and 350 nm, the sample and standard spectra recorded at the peak apex on the chromatogram must not be different for those parts of the spectrum within the range 10 % to 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 220 and 350 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 % to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at no observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.
| Blank | Meal 1 | Pellet 1 | Meal 2 | Pellet2 | |
|---|---|---|---|---|---|
| Mean [mg/kg] | ND | ||||
| s | — | ||||
| CV | — | ||||
| s | — | ||||
| CV | — | ||||
| Recovery [%] | — |
| Analytical column (4.3.1) | |
| Mobile Phase (3.4): | water (3.3)-methanol (3.2) mixture, 900 + 100 (V + V) |
| Flow rate: | |
| Detection wavelength: | 380 nm |
| Injection volume: | 20 μl –100 μl |
(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 220 and 400 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 220 and 400 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.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | |
|---|---|---|---|---|
| L | 13 | 10 | 11 | 11 |
| n | 40 | 40 | 44 | 44 |
| mean [mg/kg] | — | |||
| S | — | |||
| S | — | |||
| CV | — | |||
| CV | — | |||
| Nominal content | ||||
| [mg/kg] | — | 15 | 50 | 100 |
| recovery % | — |
| Liquid chromatographic | |
| column (4.1.1): | 125 mm × 4 mm, cation exchange Nucleosil 10 SA, 5 or 10 μm packing, or equivalent |
| Mobile phase (3.6): | Mixture of acetonitrile (3.2), sodium dihydrogen phosphate solution (3.4) and sodium perchlorate solution (3.5), 450+450+100 (v+v+v). |
| Flow rate: | |
| Detection wavelength: | 264 nm |
| Injection volume: | 100 μl |
[mg/kg](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.
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 1000 mg/kg,7,5 % relative to the higher value for amprolium contents of more than1000 mg/kg.
| 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] | — | 188 | |||
| s | — | 178 | 550 | ||
| CVr [%] | — | ||||
| s | — | 266 | 760 | ||
| CV | — | ||||
| nominal content [mg/kg] | — | 50 | 200 |
| Liquid chromatographic | |
| column (4.4.1): | 300 mm × 4 mm, C |
| Mobile phase (3.10): | Mixture of acetate buffer solution (3.9) and acetonitrile (3.2), 825 + 175 (v+v) |
| Flow rate: | |
| Detection wavelength: | 365 nm |
| Injection volume: | 20 μl |
[mg/kg] [mg/kg](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 225 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 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 225 and 400 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 % to 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 apex.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
|---|---|---|---|---|---|---|
| L | 8 | 8 | 8 | 8 | 8 | 8 |
| n | 15 | 14 | 15 | 15 | 15 | 15 |
| Mean (mg/kg) | ||||||
| Sr (mg/kg) | ||||||
| CVr (%) | ||||||
| SR (mg/kg) | ||||||
| CVR (%) | ||||||
| Nominal content (mg/kg) |
| Premixtures | Preparations | ||||||
|---|---|---|---|---|---|---|---|
| A | B | C | D | A | B | C | |
| L | 7 | 7 | 7 | 7 | 8 | 8 | 8 |
| n | 14 | 14 | 14 | 14 | 16 | 16 | 16 |
| Mean (g/kg) | 104 | ||||||
| Sr (g/kg) | |||||||
| CVr (%) | |||||||
| SR (g/kg) | |||||||
| CVR (%) | |||||||
| Nominal content (g/kg) | 100 | 100 | 100 | ||||
| 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 | — |
| Directive 71/393/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part I | Annex III, part A |
| Annex, part II | Annex III, part E |
| Annex, part III | Annex III, part P |
| Annex, part IV | Annex III, part H |
| Directive 72/199/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part L |
| Annex I, part 2 | Annex III, part C |
| Annex I, part 3 | — |
| Annex I, part 4 | — |
| Annex I, part 5 | Annex V, part A |
| Annex II | — |
| Directive 73/46/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 3 | — |
| Article 4 | — |
| Annex I, part 1 | Annex III, part B |
| Annex I, part 2 | — |
| Annex I, part 3 | Annex III, part I |
| Directive 76/371/EEC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex I |
| Directive 76/372/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 78/633/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | — |
| Annex, part 2 | — |
| Annex, part 3 | Annex IV, part C |
| Directive 81/715/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 84/425/EEC | This Regulation |
|---|---|
| Article 1 | — |
| Article 2 | — |
| Article 3 | — |
| Annex | — |
| Directive 86/174/EEC | This Regulation |
|---|---|
| Article 1 | Article 4 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex VII |
| Directive 93/70/EEC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Annex | Annex IV, part D |
| Directive 93/117/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Annex, part 1 | Annex IV, part E |
| Annex, part 2 | Annex VIII, part A |
| Directive 98/64/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex III, part F |
| Annex, part C | Annex VIII, part B |
| Directive 1999/27/EC | This Regulation |
|---|---|
| Article 1 | Articles 3 and 5 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Article 7 | — |
| Annex, part A | Annex VIII, part C |
| Annex, part B | Annex IV, part F |
| Annex, part C | Annex VIII, part D |
| Directive 1999/76/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex | Annex IV, part G |
| Directive 2000/45/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Annex, part A | Annex IV, part A |
| Annex, part B | Annex IV, part B |
| Annex, part C | Annex III, part G |
| Directive 2002/70/EC | This Regulation |
|---|---|
| Article 1 | Article 1 |
| Article 2 | Articles 2 and 3 |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Annex I | Annex I and Annex V part B(I) |
| Annex II | Annex II and Annex V part B(II) |
| Directive 2003/126/EC | This Regulation |
|---|---|
| Article 1 | Article 3 |
| Article 2 | — |
| Article 3 | — |
| Article 4 | — |
| Article 5 | — |
| Article 6 | — |
| Annex | Annex VI |