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
-
First Commission Directive 71/250/EEC of 15 June 1971 establishing Community methods of analysis for the official control of feedingstuffsOJ L 155, 12.7.1971, p. 13 . Second Commission Directive 71/393/EEC of 18 November 1971 establishing Community methods of analysis for the official control of feedingstuffsOJ L 279, 20.12.1971, p. 7 .Third Commission Directive 72/199/EEC of 27 April 1972 establishing Community methods of analysis for the official control of feedingstuffsOJ L 123, 29.5.1972, p. 6 .Fourth Commission Directive 73/46/EEC of 5 December 1972 establishing Community methods of analysis for the official control of feedingstuffsOJ L 83, 30.3.1973, p. 21 .First Commission Directive 76/371/EEC of 1 March 1976 establishing Community methods of sampling for the official control of feedingstuffsOJ L 102, 15.4.1976, p. 1 .Seventh Commission Directive 76/372/EEC of 1 March 1976 establishing Community methods of analysis for the official control of feedingstuffsOJ L 102, 15.4.1976, p. 8 .Eight Commission Directive 78/633/EEC of 15 June 1978 establishing Community methods of analysis for the official control of feedingstuffsOJ L 206, 29.7.1978, p. 43 .Ninth Commission Directive 81/715/EEC of 31 July 1981 establishing Community methods of analysis for the official control of feedingstuffsOJ L 257, 10.9.1981, p. 38 .Tenth Commission Directive 84/425/EEC of 25 July 1984 establishing Community methods of analysis for the official control of feedingstuffsOJ L 238, 6.9.1984, p. 34 .Commission Directive 86/174/EEC of 9 April 1986 fixing the method of calculation for the energy value of compound poultry-feedOJ L 130, 16.5.1986, p. 53 .Eleventh Commission Directive 93/70/EEC of 28 July 1993 establishing Community methods of analysis for official control of feedingstuffsOJ L 234, 17.9.1993, p. 17 .Twelfth Commission Directive 93/117/EC of 17 December 1993 establishing Community methods of analysis for official control of feedingstuffsOJ L 329, 30.12.1993, p. 54 .Commission Directive 98/64/EC of 3 September 1998 establishing Community methods of analysis for the determination of amino-acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EECOJ L 257, 19.9.1998, p. 14 .Commission Directive 1999/27/EC of 20 April 1999 establishing Community methods of analysis for the determination of amprolium, diclazuril and carbadox in feedingstuffs and amending Directives 71/250/EEC, 73/46/EEC and repealing Directive 74/203/EECOJ L 118, 6.5.1999, p. 36 .Commission Directive 1999/76/EC of 23 July 1999 establishing a Community method of analysis for the determination of lasalocid sodium in feedingstuffsOJ L 207, 6.8.1999, p. 13 .Commission Directive 2000/45/EC of 6 July 2000 establishing Community methods of analysis for the determination of vitamin A, vitamin E and tryptophan in feedingstuffsOJ L 174, 13.7.2000, p. 32 .Commission Directive 2002/70/EC of 26 July 2002 establishing requirements for the determination of levels of dioxins and dioxin-like PCBs in feedingstuffsOJ L 209, 6.8.2002, p. 15 .Commission Directive 2003/126/EC of 23 December 2003 on the analytical method for the determination of constituents of animal origin for the official control of feedingstuffsOJ L 339, 24.12.2003, p. 78 .
| 5.A. | In relation to the control of substances or products uniformly distributed throughout the feed | |
| 5.A.1. | ||
| 5.A.2. | ||
| 5.A.2.1. | Loose feed: | Minimum number of incremental samples: |
| 5.A.2.1.1. | sampled portions not exceeding |
seven |
| 5.A.2.1.2. | sampled portions exceeding |
√ 20 times the number of metric tons making up the sampled portion |
| 5.A.2.2. | Packaged feed: | Minimum number of packages to be sampled |
| 5.A.2.2.1. | Packages of more than 1 kg: | |
| 5.A.2.2.1.1. | sampled portions of one to four packages | all packages |
| 5.A.2.2.1.2. | sampled portions of 5 to 16 packages | four |
| 5.A.2.2.1.3. | sampled portions of more than 16 packages | √ number of packages making up the sampled portion |
| 5.A.2.2.2. | Packages not exceeding 1 kg | four |
| 5.A.2.3. | Liquid or semi-liquid feed: | Minimum number of containers to be sampled |
| 5.A.2.3.1. | Containers of more than one litre: | |
| 5.A.2.3.1.1. | sampled portions of one to four containers | all containers |
| 5.A.2.3.1.2. | sampled portions of 5 to 16 containers | four |
| 5.A.2.3.1.3. | sampled portions of more than 16 containers | √ number of containers making up the sampled portion |
| 5.A.2.3.2. | Containers not exceeding one litre | four |
| 5.A.2.4. | Feed blocks and mineral licks | |
| 5.A.3. | ||
| 5.A.3.1. | Loose feed | 4 kg |
| 5.A.3.2. | Packaged feed: | |
| 5.A.3.2.1. | packages of more than 1 kg | 4 kg |
| 5.A.3.2.2. | packages not exceeding 1 kg | weight of the contents of four original packages |
| 5.A.3.3. | Liquid or semi-liquid feed: | |
| 5.A.3.3.1. | containers of more than one litre | four litres |
| 5.A.3.3.2. | containers not exceeding one litre | volume of the contents of four original containers |
| 5.A.3.4. | Feed blocks or mineral licks: | |
| 5.A.3.4.1. | each weighing more than 1 kg | 4 kg |
| 5.A.3.4.2. | each weighing not more than 1 kg | weight of four original blocks or licks |
| 5.A.4. | ||
| Solid feed | 500 g | |
| Liquid or semi-liquid feed | 500 ml | |
| 5.B. | In relation to the control of undesirable substances or products likely to be distributed non-uniformly throughout the feed, such as aflatoxins, rye ergot, castor-oil plant and crotalaria in feed materials | |
| 5.B.1. | ||
| 5.B.2. | ||
| 5.B.2.1. | Loose feed: see 5.A.2.1. | |
| 5.B.2.2. | Packaged feed: | Minimum number of packages to be sampled: |
| 5.B.2.2.1. | sampled portions consisting of one to four packages | all packages |
| 5.B.2.2.2. | sampled portions consisting of 5 to 16 packages | four |
| 5.B.2.2.3. | sampled portions consisting of more than 16 packages | √ number of packages making up the sampled portion |
| 5.B.3. | ||
| 5.B.3.1. | Loose feed | |
| Weight of the sampled portion in metric tons: | Minimum number of aggregate samples per sampled portion: | |
| up to 1 | 1 | |
| more than 1 and up to 10 | 2 | |
| more than 10 and up to 40 | 3 | |
| more than 40 | 4 | |
| 5.B.3.2. | Packaged feed | |
| Size of the sampled portion in number of packages: | Minimum number of aggregate samples per sampled portion: | |
| 1 to 16 | 1 | |
| 17 to 200 | 2 | |
| 201 to 800 | 3 | |
| more than 800 | 4 | |
| 5.B.4. | ||
-
(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 % and 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 nm Cu: 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 |
|---|---|
| 2,3,7,8-TCDD | |
| 1,2,3,7,8-PeCDD | |
| 1,2,3,4,7,8-HxCDD | |
| 1,2,3,6,7,8-HxCDD | |
| 1,2,3,7,8,9-HxCDD | |
| 1,2,3,4,6,7,8-HpCDD | |
| OCDD | |
| 2,3,7,8-TCDF | |
| 1,2,3,7,8-PeCDF | |
| 2,3,4,7,8-PeCDF | |
| 1,2,3,4,7,8-HxCDF | |
| 1,2,3,6,7,8-HxCDF | |
| 1,2,3,7,8,9-HxCDF | |
| 2,3,4,6,7,8-HxCDF | |
| 1,2,3,4,6,7,8-HpCDF | |
| 1,2,3,4,7,8,9-HpCDF | |
| OCDF | |
| PCB 77 | |
| PCB 81 | |
| PCB 126 | |
| PCB 169 | |
| PCB 105 | |
| PCB 114 | |
| PCB 118 | |
| PCB 123 | |
| PCB 156 | |
| PCB 157 | |
| PCB 167 | |
| PCB 189 | |
-
by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sub-lot is non-compliant if the measured value minus U is above the maximum level, by establishing the decision limit (CCα) in accordance with point 3.1.2.5 of Annex I to Decision 2002/657/EC. A lot or sub-lot is non-compliant if the measured value is equal to or above the CCα.
-
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/PCDFs and the sum of PCDD/PCDFs 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/PCDFs and the sum of PCDD/PCDFs and dioxin-like PCBs laid down by Directive 2002/32/EC, taking into account the measurement uncertainty.
-
by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %. A lot or sub-lot is non-compliant if the measured value minus U is above the maximum level. In case of a separate determination of PCDD/PCDFs and dioxin-like-PCBs, the sum of the estimated expanded uncertainty of the separate analytical results of PCDD/PCDFs and dioxin-like PCBs shall be used for the sum of PCDD/PCDFs and dioxin-like PCBs, by establishing the decision limit (CCα) in accordance with point 3.1.2.5 of the Annex I to Decision 2002/657/EC. A lot or sub-lot is non-compliant if the measured value is equal to or above the CCα.
-
(a) Selection of those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels, or the action thresholds. This approach may involve a screening method allowing cost-effective high sample-throughput, thus increasing the chance to discover new incidents with high exposure and health risks of consumers. Screening methods may comprise bioanalytical methods and GC/MS methods. Their application should aim at avoiding false-compliant results. The concentration of PCDD/Fs and the sum of PCDD/Fs and dioxin-like PCBs in those samples with significant levels needs to be determined/confirmed by a confirmatory method. (b) Determination of the levels of PCDD/Fs and dioxin-like PCBs in feed samples in the range of low background levels. This is important in order to follow time trends, exposure assessment of the population and to build a database for possible re-evaluation of action and maximum levels. This goal is achieved by confirmatory methods enabling the PCDD/Fs and dioxin-like PCBs to be identified and quantified unequivocally at the level of interest. These methods can be used for confirmation of results obtained by screening methods and for determination of low background levels in feed monitoring. They are also important for establishing congener patterns in order to identify the source of a possible contamination. At present such methods utilise high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS).
-
(a) methods based on the use of biological principles like cell-based assays, receptor-assays or immunoassays, hereinafter bioanalytical methods, which are able to detect the analytes of interest, include a calibration curve, give a yes/no decision for indication for the possible exceedance of the level of interest and allow to report the result as bioanalytical equivalents (BEQ), being an indication of the TEQ value in the sample; (b) physicochemical test (e.g. Gas chromatography-Mass spectrometry/Mass spectrometry (GC-MS/MS) or Gas chromatography/Low resolution mass spectrometry (GC/LRMS)) where the measured method precision characteristics do not meet the requirements for quantitative tests.
| Screening with bioanalytical or physico-chemical methods | Confirmatory methods | |
|---|---|---|
| False-compliant rate |
< 5 % | |
| Trueness | – 20 % to + 20 % | |
| Repeatability (RSD |
< 20 % | |
| Within-laboratory reproducibility (RSD |
< 25 % | < 15 % |
-
The rate of false-compliant results from screening of samples below and above the maximum level or the action threshold shall be determined. Actual false-compliant rates shall be below 5 %. When a minimum of 20 confirmed results per matrix/matrix group is available from the quality control of compliant samples, conclusions on the false-compliant rate shall be drawn from this database. The results from samples analysed in ring trials or during contamination incidents, covering a concentration range up to for example 2 × maximum level (ML), may also be included in the minimum of 20 results for evaluation of the false-compliant rate. The samples shall cover most frequent congener patterns, representing various sources. Although screening assays shall preferentially aim at detecting samples exceeding the action threshold, the criterion for determining false-compliant rates is the maximum level, taking into account the measurement uncertainty of the confirmatory method.
-
Separation of PCDD/PCDFs 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.
-
When calculating the concentrations from a TCDD calibration curve, values at the lower and 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 3 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 8.3) shall be well within the working range. Standard solutions and sample extracts shall be tested at least in duplicate. When using duplicates, a standard solution or a control extract tested in 4 to 6 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 nonlinear 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/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 level of interest). To perform a recovery correction, the apparent recovery shall be within the required range (see point 8.1.4). Reference samples used for recovery correction shall comply with the requirements laid down in point 8.2.
-
8.3.1. Use of the lower band of the 95 % prediction interval at the GC/HRMS decision limit:Cut-off value = BEQ DL – sy,x * tα,f=m–2 1/ n + 1/m +x i x – 2 /Q xx with: BEQ DL BEQ corresponding to the GC/HRMS decision limit, being the maximum level including measurement uncertainty s y,x residual standard deviation t α,f = m–2 Student factor (α = 5 %, f = degrees of freedom, single-sided) m total number of calibration points (index j) n number of repetitions on each level x i GC/HRMS sample concentration (in TEQ) of calibration point i 
mean of the concentrations (in TEQ) of all calibration samples 
square sum parameter, i = index for calibration point i 8.3.2. Calculation from bioanalytical results (corrected for blank and recovery) of multiple analyses of samples (n ≥ 6) contaminated at the GC/HRMS decision limit, as the lower end-point of the data distribution at the corresponding mean BEQ value:Cut-off value = BEQ DL – 1,64 × SDR With: SD R standard deviation of bioassay results at BEQ DL , measured under within-laboratory reproducibility conditions8.3.3. Calculation as mean value of bioanalytical results (in BEQ, corrected for blank and recovery) from multiple analysis of samples (n ≥ 6) contaminated at 2/3 the level of interest, based on the observation that this level will be around the cut-off value determined under point 8.3.1 or point 8.3.2: Figure 1 
8.3.4. Restrictions to cut-off values: BEQ-based cut-off values calculated from the RSD R achieved during validation using a limited number of samples with different matrix/congener patterns may be higher than the TEQ-based levels of interest due to a better precision than attainable in routine when an unknown spectrum of possible congener patterns has to be controlled. In such cases, cut-off values shall be calculated from an RSDR = 25 %, or two thirds of the level of interest shall be preferred.
-
(a) two specific ions for HRMS; (b) two specific ions of m/z > 200 or three specific ions of m/z > 100 for LRMS; (c) 1 precursor and 2 product ions for MS-MS.
| Relative intensity of qualifier ion(s) compared to target ion | ||
|---|---|---|
| > 50 % | ± 10 % | ± 20 % |
| > 20 % to 50 % | ± 15 % | ± 25 % |
| > 10 % to 20 % | ± 20 % | ± 30 % |
| ≤ 10 % | ± 50 % |
± 50 % |
-
(a) results shall be corrected for recoveries of internal standards; (b) recoveries of isotope-labelled internal standards shall be between 50 and 120 %; (c) lower or higher recoveries for individual congeners with a contribution to the sum of the six indicator 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.
| Trueness | – 30 to + 30 % |
|---|---|
| Intermediate precision (RSD%) | ≤ 20 % |
| Difference between upper- and lower-bound calculation | ≤ 20 % |
-
If a closed separation funnel is used the sediment shall be left to stand for a sufficient time (at least 3 minutes) before the sediment is separated off. Shaking shall be repeated and the sediment shall be left to stand again at least 3 minutes. The sediment shall be separated off again. If an open beaker is used, the sediment shall be left to stand for at least 5 minutes before the sediment is separated off.
-
the estimated proportion (weight %) of bone fragments in the concentrated sediment, and the proportion (weight %) of bone in the constituents of animal origin.
| (S = | sediment weight (mg), c = correction factor (%) for the estimated portion of terrestrial animal bones in the sediment, d = correction factor (%) for the estimated portion of fish bones and scale fragments in the sediment, W = weight of the sample material for the sedimentation (mg)). |
| (S = | sediment weight (mg), c = correction factor (%) for the estimated portion of terrestrial animal bone constituents in the sediment, d = correction factor (%) for the estimated portion of fish bones and scale fragments in the sediment, f = correction factor for the proportion of bone in the constituents of animal origin in the sample examined, W = weight of the sample material for the sedimentation (mg)). |
-
as far as was discernible using a microscope, no constituents derived from terrestrial animals were found in the submitted sample, or as far as was discernible using a microscope, constituents derived from terrestrial animals were found in the submitted sample.
-
as far as was discernible using a microscope, no constituents derived from fish were found in the submitted sample, or as far as was discernible using a microscope, constituents derived from fish were found in the submitted sample.
-
If the fat is solid, it is warmed for example in a microwave oven until it is liquid. By using a pipette, 40 ml of fat is transferred from the bottom of the sample to a centrifugation tube. Centrifugate during 10 minutes at 4000 r.p.m.If the fat is solid after centrifugation, it is warmed once more 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 is transferred to a small Petri dish or a microscopic slide for microscopic identification of a possible content of animal constituents (meat fibres, feathers, bone fragments, …). As embedding agent for microscopy paraffin oil or glycerol is recommended. The remaining impurities are used for sedimentation as described in point 6.2.
-
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 |