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Textual Amendments
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, milk globules, lactose crystals, feathers, egg shells, fish bones and scales.]
Textual Amendments
Textual Amendments
A representative sample, taken in accordance with the provisions laid down in Annex I to this Regulation shall be used.]
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.
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.
[F2The sediment shall be collected on a filter paper placed into a funnel to allow the separation of the remaining TCE while avoiding fat deposition into the sediment. The sediment shall be dried. It is recommended to subsequently weigh the sediment (accurate to 0,001 g) to control the sedimentation step. Lastly, the sediment shall be sieved at 0,25 mm and the two resulting fractions shall be examined, unless sieving is not deemed necessary.]
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.
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.
[F2Microscopic slides shall be prepared from the sediment and, depending on the operator’s choice, from either the flotate or the raw material.]
A sufficient number of slides shall be prepared in order to ensure that a complete examination protocol as laid down in point 2.1.4.2 can be carried-out.
Microscopic slides shall be mounted with the adequate mounting medium in accordance with the SOP established by the EURL-AP and published on its website. The slides shall be covered with coverslips.
The prepared microscopic slides shall be observed in accordance with the observation flowcharts laid down in diagrams 1 and 2.
The microscopic observations shall be conducted using the compound microscope on the sediment and, depending on the operator’s choice, either on the flotate or on the raw material. The stereomicroscope may be used in addition to the compound microscope for the coarse fractions. Each slide shall be screened entirely at various magnifications. Precise explanations on how to use the observation flowcharts are detailed by a SOP established by the EURL-AP and published on its website.
The minimum numbers of slides to be observed at each step of the observation flowcharts shall be strictly respected, unless the entire fraction material does not permit to reach the stipulated slide number, for instance when no sediment is obtained. No more than 6 slides per determination shall be used for recording of the number of particles.
When additional slides are prepared on the flotate or the raw material using a more specific mounting medium with staining properties, as laid down in point 2.1.2.1.4, to further characterise structures (e.g. feathers, hairs, muscle or blood particles) which have been detected on slides prepared by other mounting media, as laid down in point 2.1.2.1.3, the number of particles shall be counted based on a number of slides per determination not exceeding 6, including the additional slides with a more specific mounting medium.
In order to facilitate the identification of the particles’ nature and origin, the operator may use support tools like decision support systems, image libraries and reference samples.]
Determinations shall be performed on different sub-samples of 50 g each.
If following the first determination carried out in accordance with the observation flowchart laid down in diagram 1, no animal particles are 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 the first determination carried out in accordance with the observation flowchart laid down in diagram 1, one or more animal particles of a given nature (i.e. terrestrial vertebrate or fish) are detected, and the nature of the particles found confirms the declared content of the sample, no second determination is necessary. If the number of the animal particles of a given nature detected during this first determination is higher than 5, the result of the analysis shall be reported per animal nature using the terminology laid down in point 2.1.5.3. Otherwise, the result of the analysis shall be reported per animal nature using the terminology laid down in point 2.1.5.2.
In other cases, including when no declaration of content has been provided to the laboratory a second determination shall be carried out from a new sub-sample.
If, following the second determination carried out in accordance with the observation flowchart laid down in diagram 2, the sum of the animal particles of a given nature detected over the two determinations is higher than 10, the result of the analysis shall be reported per animal nature using the terminology laid down in point 2.1.5.3. Otherwise, the result of the analysis shall be reported per animal nature using the terminology laid down in point 2.1.5.2.]
When reporting the results, the laboratory shall indicate on which type of material the analysis has been carried-out (sediment, flotate or raw material). The reporting shall clearly indicate how many determinations have been carried-out and if sieving of the fractions prior to slide preparation, in accordance with the last paragraph of point 2.1.3.3.4., was not performed.
The laboratory report shall at least contain information on the presence of constituents derived from terrestrial vertebrates and from fish.
The different situations shall be reported in the following ways.
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.’
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(1)):
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
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.]]