Commission Regulation (EC) No 152/2009Show full title

[F1 2.1. Light microscopy U.K.

2.1.1. Principle U.K.

The constituents of animal origin which may be present in feed materials and compound feed sent for analysis are identified on the basis of typical and microscopically identifiable characteristics like muscle fibres and other meat particles, cartilage, bones, horn, hair, bristles, blood, feathers, egg shells, fish bones and scales.

2.1.2. Reagents and equipment U.K.

2.1.2.1. Reagents U.K.

2.1.2.1.1. Concentrating agent U.K.

2.1.2.1.1.1. Tetrachloroethylene (specific gravity 1,62) U.K.

2.1.2.1.2. Staining reagent U.K.

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

2.1.2.1.3. Mounting media U.K.

2.1.2.1.3.1. Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v) U.K.

2.1.2.1.3.2. Glycerol (undiluted, viscosity: 1 490 cP) U.K.

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

2.1.2.1.4. Mounting media with staining properties U.K.

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

2.1.2.1.4.2. Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water) U.K.

2.1.2.1.4.3. Fehling’s reagent (prepared before use from equals parts (1/1) of two stock solutions A and B. Solution A: dissolve 6,9 g copper (II) sulphate pentahydrate in 100 ml water. Solution B: dissolve 34,6 g potassium sodium tartrate tetrahydrate and 12 g NaOH in 100 ml water) U.K.

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

2.1.2.1.5. Rinsing agents U.K.

2.1.2.1.5.1. Ethanol ≥ 96 % (technical grade) U.K.

2.1.2.1.5.2. Acetone (technical grade) U.K.

2.1.2.1.6. Bleaching reagent U.K.

2.1.2.1.6.1. Commercial sodium hypochlorite solution (9 - 14 % active chlorine) U.K.

2.1.2.2. Equipment U.K.

2.1.2.2.1. Analytical balance with an accuracy of 0,001 g U.K.

2.1.2.2.2. Grinding equipment: mill - or mortar U.K.

2.1.2.2.3. Sieves with square meshes of 0,25 mm and 1 mm width U.K.

2.1.2.2.4. Conical glass separation funnel with a content of 250 ml with Teflon or ground glass stopcock at the base of the cone. Stopcock opening diameter shall be ≥ 4mm. Alternatively, a conical bottomed settling beaker may be used provided the laboratory has demonstrated that detection levels are equivalent to that obtained using the conical glass separation funnel. U.K.

Separation funnel U.K.

2.1.2.2.5. Stereomicroscope covering at least a 6,5× to 40× final magnification range U.K.

2.1.2.2.6. Compound microscope covering at least a 100× to 400× final magnification range with transmitted light bright field. Polarised light and differential interferential contrast can additionally be used U.K.

2.1.2.2.7. Standard laboratory glassware U.K.

2.1.2.2.8. Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20 × 20 mm), tweezers, fine spatula U.K.

2.1.3. Sampling and sample preparation U.K.

2.1.3.1. Sampling U.K.

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

2.1.3.2. Precautions to be taken U.K.

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

2.1.3.3. Preparation of samples other than fat or oil U.K.

2.1.3.3.1. Sample drying : samples with a moisture content > 14 % shall be dried prior to handling. U.K.

2.1.3.3.2. Sample pre-sieving : it is recommended to pre-sieve at 1 mm pelleted feeds and kernels and to subsequently prepare and analyse the two resulting fractions as distinct samples. U.K.

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

2.1.3.3.4. Extraction and preparation of the sediment : a portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel or conical bottomed settling beaker and 50 ml of tetrachloroethylene shall be added. The portion transferred into the funnel shall be limited to 3 g in case of fishmeal or other pure animal products, mineral ingredients or premixes which generate more than 10 % of sediment. The mixture shall be vigorously shaken for at least 30 s and at least 50 ml more of tetrachloroethylene shall be added cautiously while washing down the inside surface of the funnel to remove any adhering particles. The resulting mixture shall be left to stand for at least 5 minutes before the sediment is separated off by opening the stopcock. U.K.

If a conical bottomed settling beaker is used then the mixture shall be vigorously stirred for at least 15 s and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene. The mixture shall be left to stand for 3 minutes and then stirred again for 15 seconds and any particles adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene. The resulting mixture shall be left to stand for at least 5 minutes and then the liquid fraction is removed and discarded by careful decanting, taking care not to lose any of the sediment.

The sediment shall be dried and subsequently weighed (accurate to 0,001 g). If more than 5 % of the sediment consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined.

2.1.3.3.5. Extraction and preparation of the flotate : after recovery of the sediment with the method described above, two phases should remain in the separation funnel: a liquid one consisting of tetrachloroethylene and a solid one made of floating material. This solid phase is the flotate and shall be recovered by pouring off completely tetrachloroethylene from the funnel by opening the stopcock. By inverting the separation funnel, the flotate shall be transferred into a large Petri dish and air dried in a fumehood. If more than 5 % of the flotate consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined. U.K.

2.1.3.3.6. Preparation of raw material : a portion of at least 5 g of the ground sub-sample shall be prepared. If more than 5 % of the material consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined. U.K.

2.1.3.4. Preparation of samples consisting of fat or oil U.K.

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

• 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 4 000 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 4 000 r.p.m.

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

• the remaining impurities shall be used for preparing the sediment as described in point 2.1.3.3.

2.1.3.5. Use of staining reagents U.K.

In order to facilitate the correct identification of the constituents of animal origin, the operator may use staining reagents during the sample preparation in accordance with guidelines issued by the EURL-AP and published on its website.

In case Alizarin Red solution is used to colour the sediment, the following protocol shall apply:

• the dried sediment shall be transferred into a glass test tube and rinsed twice with approximately 5 ml of ethanol (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min 30 s and poured off).

• the sediment shall be bleached by adding at least 1 ml sodium hypochlorite solution. The reaction shall be allowed to continue for 10 min. The tube shall be filled with water, the sediment shall be let settle 2-3 min, and the water and the suspended particles shall be poured off gently.

• the sediment shall be rinsed twice more with about 10 ml of water (a vortex shall be used for 30 s, let settle, and pour off the water each time).

• 2 to 10 drops of the Alizarin Red solution shall be added and the mixture shall be vortexed. The reaction shall be let occur for 30 s and the coloured sediment shall be rinsed twice with approximately 5 ml ethanol followed by one rinse with acetone (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min and poured off).

• the coloured sediment shall be dried.

2.1.4. Microscopic examination U.K.

2.1.4.1. Slide preparation U.K.

Microscopic slides shall be prepared from the sediment and, depending of operator’s choice, from either the flotate or the raw material. In case sieving has been used during the sample preparation, the two resulting fractions (the fine and the coarse one) shall be prepared. Test portions of fractions spread on slides shall be representative of the whole fraction.

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

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

2.1.4.2. Observation protocols for the detection of animal particles in compound feed and feed material U.K.

The prepared microscopic slides shall be observed in accordance with the observation protocols laid down in diagram 1 for compound feed and feed materials other than pure fishmeal, or in diagram 2 for pure fishmeal.

The microscopic observations shall be conducted using the compound microscope on the sediment and, depending of the operator’s choice, either on the flotate or on the raw material. The stereomicroscope may be used in addition to the compound microscope for the coarse fractions. Each slide shall be screened entirely at various magnifications.

The minimum numbers of slides to be observed at each step of the observation protocol shall be strictly respected unless the entire fraction material does not permit to reach the stipulated slide number. No more than 6 slides per determination shall be observed.

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

[X1 Diagram 1 Observation protocol for the detection of animal particles in compound feed and feed material other than fishmeal] U.K.

Editorial Information

X1 Substituted 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 (Official Journal of the European Union L 20 of 23 January 2013).

Diagram 2 Observation protocol for the detection of animal particles in fishmeal U.K.

2.1.4.3. Number of determinations U.K.

If following a first determination carried out in accordance with the observation protocol laid down in diagram 1 or diagram 2 as relevant, no animal particle of a given nature (i.e. terrestrial animal or fish) is detected, no additional determination is necessary and the result of the analysis shall be reported using the terminology laid down in point 2.1.5.1.

If, following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in diagram 2 as relevant, the total number of animal particles of a given nature (i.e. terrestrial animal or fish) detected ranges from 1 to 5, a second determination shall be performed from a new 50 g sub-sample. If, following this second determination, the number of animal particles of this given nature detected ranges from 0 to 5, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.2., else a third determination shall be carried out from a new 50 g sub-sample. Nevertheless, if following the first and the second determination, the sum of the particles of a given nature detected over the two determinations is higher than 15, no additional determination is necessary and the result of the analysis shall be directly reported using the terminology laid down in point 2.1.5.3. If, following the third determination, the sum of the animal particles of a given nature detected over the three determinations is higher than 15, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.3. Otherwise, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.2.

If following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in diagram 2 as relevant, more than 5 animal particles of a given nature (i.e. terrestrial animal or fish) are detected, the result of the analysis shall be reported using the terminology laid down in point 2.1.5.3.

2.1.5. Expression of the results U.K.

When reporting the results, the laboratory shall indicate on which type of material the analysis has been carried-out (sediment, flotate or raw material) and how many determinations have been carried-out.

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

The different situations shall be reported in the following ways.

2.1.5.1. No animal particle of a given nature detected: U.K.

• as far as was discernible using a light microscope, no particle derived from terrestrial animals 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 on average: U.K.

• as far as was discernible using a light microscope, no more than 5 particles derived from terrestrial animals were detected on average per determination in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn…]. This low level presence, being below the limit of detection of the microscopic method, means that a risk of false positive result cannot be excluded.

Or, as relevant,

• as far as was discernible using a light microscope, no more than 5 particles derived from fish were detected on average per determination in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill…]. This low level presence, being below the limit of detection of the microscopic method, means that a risk of false positive result cannot be excluded.

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.

2.1.5.3. More than 5 animal particles of a given nature detected on average U.K.

• as far as was discernible using a light microscope, more than 5 particles derived from terrestrial animals were detected on average per determination in the submitted sample. The particles were identified as … [bone, cartilage, muscle, hair, horn…].

Or, as relevant,

• as far as was discernible using a light microscope, more than 5 particles derived from fish were detected on average per determination in the submitted sample. The particles were identified as … [fishbone, fish scale, cartilage, muscle, otolith, gill…].

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.]

Textual Amendments

F1 Substituted by 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 (Text with EEA relevance).