- Latest available (Revised)
- Original (As adopted by EU)
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)
When the UK left the EU, legislation.gov.uk published EU legislation that had been published by the EU up to IP completion day (31 December 2020 11.00 p.m.). On legislation.gov.uk, these items of legislation are kept up-to-date with any amendments made by the UK since then.
Legislation.gov.uk publishes the UK version. EUR-Lex publishes the EU version. The EU Exit Web Archive holds a snapshot of EUR-Lex’s version from IP completion day (31 December 2020 11.00 p.m.).
This version of this Regulation was derived from EUR-Lex on IP completion day (31 December 2020 11:00 p.m.). It has not been amended by the UK since then. Find out more about legislation originating from the EU as published on legislation.gov.uk.
Revised legislation carried on this site may not be fully up to date. At the current time any known changes or effects made by subsequent legislation have been applied to the text of the legislation you are viewing by the editorial team. Please see ‘Frequently Asked Questions’ for details regarding the timescales for which new effects are identified and recorded on this site.
The determination of constituents of animal origin in feed shall be performed by light microscopy or polymerase chain reaction (PCR) in accordance with the provisions laid down in this Annex.
These two methods make it possible to detect the presence of constituents of animal origin in feed materials and compound feed. However, they do not make it possible to calculate the amount of such constituents in feed materials and compound feed. Both methods have a limit of detection below 0,1 % (w/w).
The PCR method makes it possible to identify the taxonomic group of constituents of animal origin present in feed materials and compound feed.
These methods shall apply for the control of the application of the prohibitions laid down in Article 7(1) and Annex IV to Regulation (EC) No 999/2001 and in Article 11(1) of Regulation (EC) No 1069/2009.
Depending on the type of feed being tested, these methods may be used, within one single operational protocol, either on their own or combined together in accordance with the standard operating procedures (SOP) established by the EU reference laboratory for animal proteins in feedingstuffs (EURL-AP) and published on its website(1).
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.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A representative sample, taken in accordance with the provisions laid down in Annex I 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Deoxyribonucleic acid (DNA) fragments of animal origin which may be present in feed materials and compound feed are detected by a genetic amplification technique through PCR, targeting species-specific DNA sequences.
The PCR method first requires a DNA extraction step. The amplification step shall be applied afterwards to the so-obtained DNA extract, in order to detect the animal species targeted by the assay.
Only reagents approved by the EURL-AP and published on its website shall be used.
Only primers and probes with sequences of oligonucleotides validated by the EURL-AP shall be used(2).
Only Master Mix solutions which do not contain reagents susceptible to lead to false results due to presence of animal DNA shall be used(3).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A representative sample, taken in accordance with the provisions laid down in Annex I, shall be used.
The preparation of laboratory samples up to DNA extraction shall comply with the requirements set out in Annex II. At least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.
The sample preparation shall be performed in a room different from the ones dedicated to DNA extraction and to genetic amplification reactions as described by ISO 24276.
Two test portions of at least 100 mg each shall be prepared.
The DNA extraction shall be performed on each test portion prepared using the SOP established by the EURL-AP and published on its website.
Two extraction controls shall be prepared for each extraction series as described by ISO 24276.
an extraction blank control,
a positive DNA extraction control.
The genetic amplification shall be performed using the methods validated for each species requiring identification. These methods are laid down in the SOP established by the EURL-AP and published on its website. Each DNA extract shall be analysed at least at two different dilutions in order to evaluate inhibition.
Two amplification controls shall be prepared per species target as described by ISO 24276.
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.
When reporting the results, the laboratory shall indicate at least the weight of the test portions used, the extraction technique used, the number of determinations carried-out and the limit of detection of the method.
Results shall not be interpreted and reported if the positive DNA extraction control and the positive DNA target controls do not provide positive results for the target under assay while the amplification reagent control is negative.
In case results from the two test portions are not consistent, at least the genetic amplification step shall be repeated. If the laboratory suspects that the DNA extracts can be the cause of the inconsistency, a new DNA extraction and a subsequent genetic amplification shall be performed before interpreting the results.
The final expression of the results shall be based on the integration and the interpretation of the results of the two test portions in accordance with the SOP established by the EURL-AP and published on its website.
A negative result shall be reported as follows:
No DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).
A positive result shall be reported as follows:
DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted by the assay).’
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).
http://eurl.craw.eu/
The list of these primers and probes for each animal species targeted by the assay is available on the EURL-AP website.
Examples of Master Mixes that are functional are available on the EURL-AP website.
Latest Available (revised):The latest available updated version of the legislation incorporating changes made by subsequent legislation and applied by our editorial team. Changes we have not yet applied to the text, can be found in the ‘Changes to Legislation’ area.
Original (As adopted by EU): The original version of the legislation as it stood when it was first adopted in the EU. No changes have been applied to the text.
Geographical Extent: Indicates the geographical area that this provision applies to. For further information see ‘Frequently Asked Questions’.
Show Timeline of Changes: See how this legislation has or could change over time. Turning this feature on will show extra navigation options to go to these specific points in time. Return to the latest available version by using the controls above in the What Version box.
Access essential accompanying documents and information for this legislation item from this tab. Dependent on the legislation item being viewed this may include:
This timeline shows the different versions taken from EUR-Lex before exit day and during the implementation period as well as any subsequent versions created after the implementation period as a result of changes made by UK legislation.
The dates for the EU versions are taken from the document dates on EUR-Lex and may not always coincide with when the changes came into force for the document.
For any versions created after the implementation period as a result of changes made by UK legislation the date will coincide with the earliest date on which the change (e.g an insertion, a repeal or a substitution) that was applied came into force. For further information see our guide to revised legislation on Understanding Legislation.
Use this menu to access essential accompanying documents and information for this legislation item. Dependent on the legislation item being viewed this may include:
Click 'View More' or select 'More Resources' tab for additional information including: