LegalizeCommission 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)
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)
Article 1
Sampling for the official control of feed, in particular as regards the determination of constituents, including material which contains or consists of or is produced from genetically modified organisms (GMOs), feed additives as defined by Regulation (EC) No 1831/2003 of the European Parliament and of the Council (1), undesirable substances as defined by Directive 2002/32/EC of the European Parliament and of the Council (2) shall be carried out in accordance with the methods set out in Annex I, with the exception of sampling for the control of microbiological contamination.
The method of sampling set out in Annex I is applicable for the control of feed as regards the determination of pesticide residues as defined in Regulation (EC) No 396/2005 of the European Parliament and of the Council (3) and control of compliance with Regulation (EU) No 619/2011.
Article 2
Preparation of samples for analysis and expression of results shall be carried out in accordance with the methods set out in Annex II.
Article 3
Analysis for the official control of feed shall be carried out using the methods set out in Annex III (Methods of analysis to control the composition of feed materials and compound feed, Annex IV (Methods of analysis to control the level of authorised additives in feed), Annex V (Methods of analysis to control undesirable substances in feed) and Annex VI (Methods of analysis for the determination of constituents of animal origin for the official control of feed).
Article 4
The energy value of compound poultry feed shall be calculated in accordance with Annex VII.
Article 5
The methods of analysis to control illegal presence of no longer authorised additives in feed set out in Annex VIII shall be used for confirmatory purposes.
Article 6
Directives 71/250/EEC, 71/393/EEC, 72/199/EEC, 73/46/EEC, 76/371/EEC, 76/372/EEC, 78/633/EEC, 81/715/EEC, 84/425/EEC, 86/174/EEC, 93/70/EEC, 93/117/EC, 98/64/EC, 1999/27/EC, 1999/76/EC, 2000/45/EC, 2002/70/EC and 2003/126/EC are repealed.
References to the repealed Directives shall be construed as references to this Regulation and shall be read in accordance with the correlation tables in Annex IX.
Article 7
This Regulation shall enter into force on the twentieth day following that of 20th day following its publication in the Official Journal of the European Union.
It shall apply from 26 August 2009.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
ANNEX I
1. PURPOSE AND SCOPE
Samples intended for the official control of feed shall be taken according to the methods described below. Samples thus obtained shall be considered as representative of the sampled portions.
The purpose of representative sampling is to obtain a small fraction from a lot in such a way that a determination of any particular characteristic of this fraction will represent the mean value of the characteristic of the lot. The lot shall be sampled by repeatedly taking incremental samples at various single positions in the lot. These incremental samples shall be combined by mixing to form an aggregate sample from which representative final samples shall be prepared by representative dividing.
If by a visual inspection or based on other relevant information, portions of the feed to be sampled show a difference in quality from the rest of the feed from the same lot, such portions shall be separated from the rest of the feed and treated as a separate sublot. If it is not possible to divide the feed into separate sublots, the feed shall be sampled as one lot. In such cases, mention shall be made of this fact in the sampling report.
Where a feed sampled in accordance with the provisions of this Regulation is identified as not satisfying the EU requirements and is part of a lot of feed of the same class or description, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
Sampling may also include feed offered for sale by feed business operators by means of distance communication in accordance with Article 11(3) of Regulation (EC) No 767/2009 of the European Parliament and of the Council (4). Sampling of feed offered for sale by means of distance communication shall in principle be subject to the points set out in this Annex. Specific aspects of the sampling of distance selling samples are described in point 11.
2. DEFINITIONS
— Lot (or batch): an identified quantity of feed determined to have common characteristics, such as origin, variety, type of packaging, packer, consignor or labelling, and in case of a production process, a unit of production from a single plant using uniform production parameters or a number of such units, when produced in continuous order and stored together.
— Sampled portion: A lot or an identified part of the lot or sublot.
— Sealed sample: a sample sealed in such a manner as to prevent any access to the sample without breaking or removing the seal.
— Incremental sample: A quantity taken from one point in the sampled portion.
— Aggregate sample: An aggregate of incremental samples taken from the same sampled portion.
— Reduced sample: A part of the aggregate sample, obtained from the latter by a process of representative reduction.
— Final sample: A part of the aggregate sample (mixed), of the reduced sample or of the homogenised aggregate sample, depending on the type of control (see point 9.4).
— Laboratory sample: a sample intended for the laboratory (as received by the laboratory) and can be the final, reduced or aggregate sample.
— Distance selling sample: Sample of a lot or batch of feed offered for sale by means of distance communication.
3. GENERAL PROVISIONS
— The samples shall be taken by persons authorised for that purpose by the competent authority.
— For a distance selling sample, a quantity of the feed shall be requested from the feed business operator by the competent authority by means of distance communication.
— The sample has to be sealed in such a manner as to prevent any access to the sample without breaking or removing the seal.
The seal’s mark should be clearly identifiable and clearly visible.
— Identification of the sample: the sample has to be indelibly marked and must be identified in such a way that there is an unambiguous link to the sampling report.
— From each aggregate sample or reduced sample, the following final samples are taken: one for control (enforcement) and one for the feed business operator (defence sample) must be taken. Eventually, one other final sample may be taken for reference. In case the complete aggregate sample is homogenised, the final samples are taken from the homogenised aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator.
— In accordance with Article 15, paragraphs 1 and 2, of Regulation (EU) 2017/625, when it is necessary for the performance of official sampling, feed business operators shall, where required by the competent authorities: — give the staff of the competent authorities access to the equipment under their control, including, when necessary, making available the proper sampling equipment and personal protective equipment, — assist and cooperate with the staff of the competent authorities in order to enable the sampling, including making feed accessible to the staff of the competent authorities.
4. APPARATUS
4.1. The sampling apparatus must be made of materials which cannot contaminate the products to be sampled. Apparatus which is intended to be used multiple times must be easy to clean to avoid any cross-contamination.
4.2. Apparatus recommended for the sampling of solid feed
4.2.1.1.Flat-bottomed sampling shovel with vertical sides.
4.2.1.2.Sampling spear with a long split or compartments. The dimensions of the sampling spear must be appropriate to the characteristics of the sampled portion (depth of container, dimensions of sack, etc.) and to the particle size of the feed.
In case the sampling spear has several apertures, in order to ensure that the sample is taken at the different locations alongside the spear, the apertures should be separated by compartments or sequentially staggered apertures.
Appropriate mechanical apparatus may be used for the sampling of moving feed. The mechanical apparatus shall be considered appropriate when at least the whole section of the flow is sampled.
Sampling of feed in motion (at high flow rates) can be performed by automatic samplers.
If possible and appropriate, apparatus designed to divide the sample into approximately equal parts should be used for the preparation of reduced samples in a representative way.
5. QUANTITATIVE REQUIREMENTS AS REGARDS THE NUMBER OF INCREMENTAL SAMPLES
— The quantitative requirements in points 5.1 and 5.2 as regards the number of incremental samples are applicable for sampled portion sizes up to a maximum of 500 tonnes and which can be sampled in a representative way. The sampling procedure described is equally valid for quantities larger than prescribed maximum sampled portion size provided that the maximum number of incremental samples given in the following tables under points 5.1.1, 5.1.3 and 5.1.5 is ignored, the number of incremental samples being determined by the square-root formula given in the appropriate part of the procedure (see point 5.3) and the minimum aggregate sample size increased proportionally. This does not prevent a large lot being divided into smaller sublots and each sublot sampled in accordance with the procedure described in points 5.1 and 5.2.
— The size of the sampled portion must be such that each of its constituent parts can be sampled.
— For very large lots or sublots (> 500 tonnes) and for lots which are transported or stored in such a way that sampling cannot be done in accordance with the sampling procedure provided for in points 5.1 and 5.2 of this point, the sampling procedure as provided for in point 5.3 is to be applied.
— For distance selling samples, the size of the lot from which the quantity is requested is usually not known by the competent authority. Therefore, the procedure referred to in points 5.1 and 5.2 cannot be used. In this case, the procedure described in point 11 shall be applied.
— In case the feed business operator is required by legislation to comply with this Regulation within the frame of a mandatory monitoring system, the feed business operator may deviate from the quantitative requirements as provided for in this point to take into account operational characteristics on the condition that the feed business operator has demonstrated to the satisfaction of the competent authority the equivalence of the sampling procedure as regards representativeness and after authorisation from the competent authority.
— In exceptional cases, if it is not possible to carry out the method of sampling set out as regards the quantitative requirements because of the unacceptable commercial damage to the lot (because of packaging forms, means of transport, way of storage, etc.) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.
5.1. Quantitative requirements as regards incremental samples in relation to the control of substances or products uniformly distributed throughout the feed
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| ≤ 2,5 tonnes | 7 |
| > 2,5 tonnes | √ (20 times the number of tonnes making up the sampled portion) (*1), up to 40 incremental samples |
| (*1) Where the number obtained is a fraction, it shall be rounded up to the next whole number. | |
| Size of sampled portion | Minimum number of incremental samples |
| --- | --- |
| ≤ 2,5 tonnes or ≤ 2 500 litres | 4 (*1) |
| > 2,5 tonnes or > 2 500 litres | 7 (*1) |
| (*1) In case it is not possible to make the liquid homogeneous, the number of incremental samples has to be increased. |
Feed (solid and liquid) can be packaged in bags, sacks, cans, barrels, etc. which are referred to in the following table as units. Large units (≥ 500 kg or litres) have to be sampled in accordance with the provisions foreseen for loose feed (see 5.1.1 and 5.1.2).
| Size of sampled portion | Minimum number of units from which (at least) one incremental sample has to be taken (*1) |
|---|---|
| 1 to 20 units | 1 unit (*2) |
| 21 to 150 units | 3 units (*2) |
| 151 to 400 units | 5 units (*2) |
| > 400 units | ¼ of the √ (number of units making up the sampled portion) (*3), up to 40 units |
| (1) In the case where opening of a unit might affect the analysis (e.g. perishable wet feeds) an incremental sample shall be the unopened unit. (2) For units whose contents do not exceed 1 kg or one litre, an incremental sample shall be the contents of one original unit. (*3) Where the number obtained is a fraction, it shall be rounded up to the next whole number. |
Minimum one block or lick to be sampled per sampled portion of 25 units, up to a maximum of four blocks or licks.
For blocks or licks weighing not more than 1 kg each, an incremental sample shall be the contents of one block or one lick.
| Size of sampled portion | Minimum number of incremental samples (*1) |
|---|---|
| ≤ 5 tonnes | 5 |
| > 5 tonnes | √(5 times the number of tonnes making up the sampled portion) (*2), up to 40 incremental samples |
| (1) It is acknowledged that in certain situations (e.g. silages), it is not possible to take the required incremental samples without causing unacceptable damage to the lot. An alternative method of sampling may be applied in such situations and a guidance for sampling such lots has been elaborated which is available at https://food.ec.europa.eu/system/files/2016-10/animal-feed-guidance_documents_691_2013_en.pdf (2) Where the number obtained is a fraction, it shall be rounded up to the next whole number. |
5.2. Quantitative requirements as regards incremental samples in relation to the control of constituents or substances likely to be distributed non-uniformly in feed
These quantitative requirements as regards incremental samples are to be used in the following situations:
— control of aflatoxins, rye ergot, other mycotoxins and harmful botanical impurities in feed materials,
— control of cross-contamination by a constituent, including GM material, or substance for which non-uniform distribution is expected in feed.
In case the control authority has strong suspicion that such a non-uniform distribution occurs also in case of cross-contamination by a constituent or substance in a compound feed, the quantitative requirements as provided for in the following table can be applied.
| Size of sampled portion | Minimum number of incremental samples |
|---|---|
| < 80 tonnes | See quantitative requirements under 5.1. The number of incremental samples to be taken has to be multiplied by 2,5. |
| ≥ 80 tonnes | 100 |
In the case of large sampled portions (sampled portions > 500 tonnes), the number of incremental samples to be taken = 40 incremental samples + √tonnes in relation to the control of substances or products uniformly distributed throughout the feed or 100 incremental samples + √tonnes in relation to the control of constituents or substances likely to be distributed non-uniformly in feed.
6. QUANTITATIVE REQUIREMENTS AS REGARDS AGGREGATE SAMPLE
A single aggregate sample per sampled portion is required.
| Nature of feed | Minimum size of aggregate sample (1) (2) | |
|---|---|---|
| 6.1. | Loose feed | 4 kg |
| 6.2. | Packaged feed: | 4 kg (*3) |
| 6.3. | Liquid or semi-liquid feed: | 4 litres |
| 6.4. | Feed blocks or mineral licks: | |
| 6.4.1. | each weighing more than 1 kg | 4 kg |
| 6.4.2. | each weighing not more than 1 kg | weight of four original blocks or licks |
| 6.5. | Roughage/forage | 4 kg (*4) |
| (1) In case the sampled feed is of high value, a smaller quantity of aggregate sample can be taken on the condition this is described and documented in the sampling report. (2) In accordance with the provisions of Commission Regulation (EU) No 619/2011 of 24 June 2011 laying down the methods of sampling and analysis for the official control of feed as regards presence of genetically modified material for which an authorisation procedure is pending or the authorisation of which has expired (OJ L 166, 25.6.2011, p. 9), the aggregate sample for the control of the presence of genetically modified material must contain at least 35 000 seeds/grains. This means that for maize the size of the aggregate sample must be at least 10,5 kg and for soybean 7 kg. For other seeds and grains such as barley, millet, oat, rice, rye, wheat and rapeseed, the aggregate sample size of 4 kg corresponds to more than 35 000 seeds/grains. (3) In case of packaged feed, it may also not be possible to achieve the size of 4 kg for the aggregate sample depending on the size of the individual units. (4) In case it concerns roughage/forage with a low specific density (e.g. hay, straw), the aggregate sample should have a minimum size of 1 kg. |
7. QUANTITATIVE REQUIREMENTS AS REGARDS FINAL SAMPLES
Final samples
Analysis of at least one final sample is required. The amount in the final sample for analysis shall be not less than the following:
| Solid feed | 500 g (1) (2) (3) (4) |
|---|---|
| Liquid or semi-liquid feed | 500 ml (*1) |
| (1) In accordance with the provisions of Regulation (EU) No 619/2011, the final sample for the control of the presence of genetically modified material must contain at least 10 000 seeds/grains. This means that for maize the size of the final sample must be at least 3 000 g and for soybean 2 000 g. For other seeds and grains such as barley, millet, oat, rice, rye, wheat and rapeseed, the final sample size of 500 g corresponds to more than 10 000 seeds/grains. (2) In case the size of the aggregate sample is significantly less than 4 kg or litre (see footnotes point 6), also a smaller quantity of final sample can be taken on the condition this is described and documented in the sampling report. (3) In case of sampling pulses, cereal grains and tree nuts for the determination of pesticide residues, the minimum size of the final sample shall be 1 kg in accordance with the provisions of Commission Directive 2002/63/EC of 11 July 2002 establishing Community methods of sampling for the official control of pesticide residues in and on products of plant and animal origin and repealing Directive 79/700/EEC (OJ L 187, 16.7.2002, p. 30). (4) In case of examination by visual inspection or by microscopy, the amount of the final sample for examination shall be 1 kg. |
8. METHOD OF SAMPLING FOR VERY LARGE LOTS OR LOTS STORED OR TRANSPORTED IN A WAY WHEREBY SAMPLING THROUGHOUT THE LOT IS NOT FEASIBLE
8.1. General principles
In case the way of transport or storage of a lot does not enable to take incremental samples throughout the whole lot, sampling of such lots should preferably be done when the lot is in flow.
In the case of large warehouses destined to store feed, operators should be encouraged to install equipment in the warehouse enabling (automatic) sampling across the whole stored lot.
In case of applying the sampling procedures as provided for in this point, the feed business operator or his representative is informed of the sampling procedure. In case this sampling procedure is questioned by the feed business operator or his representative, the feed business operator or his representative shall enable the competent authority to sample throughout the whole lot at the operator’s cost.
8.2. Large lots transported by ship
The sampling of large lots in ships is preferably carried out while the product is in flow (dynamic sampling).
The sampling is to be done per hold (entity that can physically be separated). Holds are however emptied partly one after the other so that the initial physical separation does no longer exist after transfer into storage facilities. Sampling can therefore be performed in function of the initial physical separation or in function of the separation after transfer into the storage facilities.
The unloading of a ship can last for several days. Normally, sampling has to be performed at regular intervals during the whole duration of unloading. It is however not always feasible or appropriate for an official inspector to be present for sampling during the whole operation of unloading. Therefore sampling of part (sampled portion) of the whole lot is allowed to be undertaken. The number of incremental samples is determined by taking into account the size of the sampled portion.
In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
Even if the official sample is taken automatically, the presence of an inspector is necessary. However, in case the automatic sampling is done with preset parameters which cannot be changed during the sampling and the incremental samples are collected in a sealed receptacle, preventing any possible fraud, then the presence of an inspector is only required at the beginning of the sampling, every time the receptacle of the samples needs to be changed and at the end of the sampling.
In case the sampling is done in a static way the same procedure as provided for storage facilities (silos) accessible from above has to be applied (see point 8.4.1).
The sampling has to be performed on the accessible part (from above) of the lot/hold. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
8.3. Sampling of large lots stored in warehouses
The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
8.4. Sampling of storage facilities (silos)
The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
Feed stored in such silos cannot be sampled in a static way. Therefore, in case the feed in the silo has to be sampled and there is no possibility to move the consignment, the agreement has to be made with the operator that he or she has to inform the inspector about when the silo will be unloaded in order to enable sampling when the feed is in flow.
Sampling procedure involves the release into a receptacle of a quantity of 50 to 100 kg and taking the sample from it. The size of the aggregate sample corresponds to the whole lot and the number of incremental samples relate to the quantity of the silo released in a receptacle for sampling. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.
8.5. Sampling of loose feed in large closed containers
Such lots can often only be sampled when unloaded. It is in certain cases not possible to unload at the point of import or control and therefore the sampling should take place when such containers are unloaded.
9. INSTRUCTIONS FOR TAKING, PREPARING AND PACKAGING THE SAMPLES
9.1. General
The samples must be taken and prepared without unnecessary delay bearing in mind the precautions necessary to ensure that the product is neither changed nor contaminated. Instruments, surfaces and containers intended to receive samples must be clean and dry.
9.2. Incremental samples
Incremental samples must be taken at random and evenly distributed throughout the whole sampled portion and they must be of approximately equal sizes.
The incremental sample size is at least 100 grams or 25 grammes in case of roughage/forage with low specific density.
In case that in accordance with the rules for the sampling procedure established in point 8 less than 40 incremental samples have to be taken, the size of the incremental samples shall be determined in function of the required size of the aggregate sample to be achieved (see point 6).
In case of sampling of small lots of packaged feed where according to the quantitative requirements a limited number of incremental samples have to be taken, an incremental sample shall be the contents of one original unit whose contents do not exceed 1 kg or one litre.
In case of sampling of packaged feed composed of small units (e.g. < 250 g), the size of the incremental sample depends on the size of the unit.
In case of distance selling samples, the size of the incremental sample depends on the size of the unit and may also contain less than 100 g or 100 ml in individual cases.
Where appropriate, sampling may be carried out when the sampled portion is being moved (loading or unloading).
Having selected the required number of units for sampling as indicated in point 5, part of the contents of each unit shall be removed using a spear or shovel. Where necessary, the samples shall be taken after emptying the units separately.
Having selected the required number of units for sampling as indicated in point 5, the contents shall be homogenised if necessary and an amount taken from each unit.
The incremental samples may be taken when the contents are being discharged.
Having selected the required number of units for sampling as indicated in point 5, samples shall be taken from different levels.
Samples may also be taken when the contents are being discharged but the first fractions shall be discarded.
In either case the total volume taken must not be less than 10 litres.
Having selected the required number of blocks or licks for sampling as indicated in point 5, a part of each block or lick can be taken. In case of suspicion of a non-homogeneous block or lick, the whole block or lick can be taken as sample.
For blocks or licks weighing not more than 1 kg each, an incremental sample shall be the contents of one block or one lick.
9.3. Preparation of aggregate samples
The incremental samples shall be mixed to form a single aggregate sample.
9.4. Preparation of final samples
The material in the aggregate sample shall be carefully mixed (5).
Each sample shall be put into an appropriate container/receptacle. All necessary precautions shall be taken to avoid any change of composition of the sample, contamination or adulteration which might arise during transportation or storage.
In case of the control of constituents or substances uniformly distributed throughout the feed, the aggregate sample can be representatively reduced to at least 2,0 kg or 2,0 litres (reduced sample) (6) preferably either by using a mechanical or automatic divider. For the control of the presence of pesticide residues in pulses, cereal grains and tree nuts, the minimum size of the reduced sample shall be 3 kg. In case the nature of the feed does not allow using a divider or the divider is not available, then the sample can be reduced by the quartering method.
From the aggregate sample or the reduced samples the final samples (for control, defence and possibly reference) shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7.
In case of the control of constituents, including genetically modified material, or substances likely to be distributed non-uniformly in feed, the aggregate sample shall be:
(i) completely homogenised. Afterwards from the homogenised aggregate sample the final samples (for control, defence and possibly reference) shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7; or
(ii) reduced to at least 2 kg or 2 litres (7) by using a mechanical or automatic divider. Only in the case that the nature of the feed does not allow for using a divider, the sample can, if necessary, be reduced by quartering method. For the control of the presence of genetically modified material in the frame of Regulation (EU) No 619/2011, the reduced sample must contain at least 35 000 seeds/grains to enable to obtain the final samples for enforcement, defence and possibly reference of at least 10 000 seeds grain (see footnote (*) in point 6 and footnote () in point 7).
From the reduced sample the final samples shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7.
9.5. Packaging of samples
The containers or packages shall be sealed and labelled in such a manner that they cannot be opened without damaging the seal. The total label must be incorporated in the seal. Alternatively, the sample can be put in a recipient which can be closed in such a manner that it cannot be opened without irreversibly damaging the receptacle or container, avoiding the re-use of the receptacle or container.
9.6. Sending of samples to the laboratory
The sample shall be sent without unnecessary delay to the designated analytical laboratory, together with the information necessary for the analyst.
10. SAMPLING RECORD
A record must be kept of each sample, permitting each sampled portion and its size to be identified unambiguously.
The record shall also mention any deviation of the sampling procedure as provided for in this Regulation.
Besides making the record available to the official control laboratory, the record shall be made available to the feed business operator and/or the laboratory designated by the feed business operator.
11. DISTANCE SELLING SAMPLE
— For a distance selling sample, the feed shall be requested from the feed business operator by means of distance communication techniques. In this case, when requesting feed, the competent authority does not have to identify itself with an official identity to the feed business operator and may use a cover identity.
— The aggregate sample and the final samples of the distance selling sample have to be taken immediately upon receipt of the consignment by persons authorised for this purpose. For generating the aggregate sample, an appropriate number of incremental samples have to be taken randomly and evenly distributed from the total quantity obtained and carefully mixed/homogenised, in accordance, as far as possible, with the principles laid down in point 5 and points 9.2 and 9.3. If the feed is packaged in individual units, at least 4 units should be obtained from which at least one incremental sample has to be taken. Should it be shown on a case-by-case basis that the units obtained come from different lots, the number of units to be sampled has to be reduced and limited to those units originating from the same lot. In case of analysing the distance selling sample for constituents or substances which are non-uniformly distributed in feed, the number of incremental samples has to be at least 2,5 times higher than that for samples analysed for substances uniformly distributed throughout the feed. From the aggregate sample, the corresponding final samples (for control, for defence and possibly reference) are then taken in accordance, as far as feasible, with the principles laid down in point 9.4 and the sampling record indicates that the sample is a distance selling sample. The competent authority then immediately informs the feed business operator of the sampling. The feed business operator is also notified that one sample (for defence) is kept, when possible, at their disposal by the competent authority, in a specified location, for defence purposes or sent to the feed business operator or sent to the laboratory designated by the feed business operator in accordance with the national rules in place. If the sample is sent directly to the official laboratory, the final sample must be prepared and sealed in the laboratory by persons authorised for this purpose or in the presence of persons authorised for this purpose. The sampling record of the distance selling sample has to be sent immediately after the final samples have been formed to the competent authority, which informs the feed business operator of the sampling. It is considered that the quantity supplied by the feed business operator to the competent authority represents a part of a lot of feed of the same class or description. In accordance with Article 15 of Regulation (EC) No 178/2002 of the European Parliament and of the Council (8), if that part of the lot has been identified as not satisfying EU requirements, it shall be presumed, also in the case of a distance selling sample, that all of the feed in that lot is so affected, unless following a detailed assessment (where appropriate in an on-the-spot inspection) there is no evidence that the rest of the lot fails to satisfy the EU requirements.
ANNEX II
A. PREPARATION OF SAMPLES FOR ANALYSIS
1. Purpose
The procedures described in this Annex concern the preparation for analysis of samples, sent to the control laboratories after sampling in accordance with the provisions laid down in Annex I.
The laboratory samples must be prepared in such a way that the amounts weighed out, as provided for in the methods of analysis, are homogeneous and representative of the final samples.
In addition to the procedures described in this Annex, the guidelines for sample preparation as provided for by EN ISO 6498 shall be followed.
2. Precautions to be taken
The sample preparation procedure to be followed is dependent on the methods of analysis to be used and the constituents or substances to be controlled. It is therefore of major importance that that the followed sample preparation procedure be appropriate for the used method of analysis and for constituents or substances to be controlled.
All the necessary operations must be performed in such a way as to avoid as far as possible contamination of the sample and changes of its composition.
Grinding, mixing and sieving shall be carried out without delay with minimal exposure of the sample to the air and light. Mills and grinders likely to appreciably heat the sample shall not be used.
Manual grinding is recommended for feed which are particularly sensitive to heat. Care shall also be taken to ensure that the apparatus itself is not a source of contamination.
Homogenisation of the sample by preparing a slurry by high shear mixing with water has proven to provide in certain cases more homogeneous sub-samples than dry homogenisation/grinding, in particular in case of heterogeneously distributed chemical substances. However also homogenisation by sufficient dry grinding might provide homogeneous subsamples.
In certain cases, such as for the determination of rye ergot, harmful botanical impurities, etc., the homogenisation of the sample cannot be done by grinding but by sufficiently mixing the sample.
If the preparation cannot be carried out without significant changes in the moisture content of the sample, determine the moisture content before and after preparation according to the method laid down in Part A of Annex III.
3. Procedure
The test aliquot is taken from the final homogenised sample. Coning and quartering is not recommended because this might provide test aliquots with high splitting error.
— Mix the final sample and collect it in a suitable clean, dry container fitted with an air-tight stopper. Mix again in order to ensure full homogenisation, immediately before weighing out the amount for analysis (test aliquot).
— Unless otherwise specified in the methods of analysis, dry the final sample to bring its moisture content down to a level of 8 to 12 %, according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in Part A of Annex III). Then proceed as indicated in point 3.1.1.
— Collect the final sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly in order to ensure full homogenisation immediately before weighing out the amount for analysis (test aliquot).
— Final samples which cannot be prepared according to one of the above procedures shall be treated by any other procedure which ensures that the amounts weighed out for the analysis (test aliquot) are homogeneous and representative of the final samples.
— In case of an examination by visual inspection (without making use of microscope), the whole aggregate or final sample is used for examination.
— In case of a microscopic examination, the laboratory may reduce the aggregate sample, or further reduce the reduced sample. The final samples for defence and possibly reference purposes are taken following a procedure equivalent to the procedure followed for the final sample for enforcement.
— In case the whole aggregate sample is homogenised, the final samples are taken from the homogenised aggregate sample.
— For the determination of rye ergot and harmful botanical impurities, the final sample has to be divided into 2 subsamples of equal weight of approximately 500 grams. One subsample is examined. In case the result of the subsamples is equal or below 50 % (analytical threshold) of the maximum level, the sample is compliant with the maximum level. If the result is above 50 % of the maximum level, another subsample needs to be examined and the average of the result of the 2 subsamples is used for checking compliance with the maximum level.
4. Storage of samples
Samples must be stored at a temperature that will not alter their composition. Samples intended for the analysis of vitamins or substances which are particularly sensitive to light shall be stored in such conditions that the sample is not adversely affected by light.
B. PROVISIONS RELATING TO REAGENTS AND APPARATUS USED IN METHODS OF ANALYSIS
1.Unless otherwise specified in the methods of analysis, all analytical reagents must be analytically pure (a.p.). When trace analysis is carried out, the purity of the reagents must be checked by a blank test. Depending upon the results obtained, further purification of the reagents may be required.
2.Any operation involving preparation of solutions, dilution, rinsing or washing, mentioned in the methods of analysis without indication as to the nature of the solvent or diluent employed, implies that water must be used. As a general rule, water shall be demineralised or distilled. In particular cases, which are indicated in the methods of analysis, it must be submitted to special procedures of purification.
3.In view of the equipment normally found in control laboratories, only those instruments and apparatus which are special or require specific usage are referred to in the methods of analysis. They must be clean, especially when very small amounts of substances have to be determined.
C. APPLICATION OF METHODS OF ANALYSIS AND EXPRESSION OF THE RESULTS
1. Extraction procedure
Several methods determine a specific extraction procedure. As a general rule, other extraction procedures than the procedure referred to in the method can be applied on the condition that the used extraction procedure has been proven to have the equivalent extraction efficiency for the matrix analysed as the procedure mentioned in the method.
2. Clean-up procedure
Several methods determine a specific clean-up procedure. As a general rule, other clean-up procedures than the procedure referred to in the method can be applied on the condition that the used clean-up procedure has been proven to result in equivalent analytical results for the matrix analysed as the procedure mentioned in the method.
3. Number of determinations
In case of the analysis of undesirable substances, if the result of the first determination is significantly (> 50 %) lower than the specification to be controlled, no additional determinations are necessary, on the condition that the appropriate quality procedures are applied. In other cases a duplicate analysis (second determination) is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The mean of the two determinations, is used for further assessment.
In case of the control of minimum or maximum levels of feed additives, if the results of the first determination is above the minimum level or below the maximum level no additional determinations are necessary, on the condition that the appropriate quality procedures are applied. In other cases, a duplicate analysis (second determination) is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The mean of the two determinations is used for further assessment.
In case of the control of the declared content of a substance or ingredient, if the result of the first determination confirms the declared content, i.e. the analytical result falls within the acceptable range of variation of the declared content, no additional determinations are necessary, on the condition that the appropriate quality procedures are applied. In other cases a duplicate analysis (second determination) is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The mean of the two determinations, is used for further assessment (the average analytical result falls or not within the acceptable range of variation of the declared content).
In some cases this acceptable range of variation is defined in legislation such as in Regulation (EC) No 767/2009 and Regulation (EU) 2019/4 of the European Parliament and of the Council (9).
4. Reporting of the method of analysis used
The analysis report shall mention the method of analysis used.
5. Reporting of the analytical result
The analytical result shall be expressed in the manner laid down in the method of analysis to an appropriate number of significant figures and shall be corrected, if necessary, to the moisture content of the final sample prior to preparation.
Most regulatory levels (e.g. maximum level, minimum level) in EU animal feed legislation are established relative to a feed with a moisture content of 12 %. Therefore, in these cases, in order to assess the analytical result measured on the sample against the regulatory level, the analytical result first needs to be divided by the dry matter content of the sample (in %) multiplied by 88, as indicated in the following formula:
where:
In addition, if the following conditions are met:
— the result of the analysis is significantly (> 50 %) lower or higher than the labelling information/specification to be controlled (depending on whether the labelling information/specification is a maximum or a minimum level),
— the moisture content of the sampled feed is known and it can be determined that correction to the moisture content will not change the assessment, then, on the condition that the appropriate quality procedures are applied and the analysis serves only the purpose of checking compliance with legal provisions, the correction to the moisture content might be omitted (e.g. in cases there is no specification or regulatory level), unless it is required for interpretation.
If the analytical result is corrected to the moisture content, the corresponding measurement uncertainty must also be corrected in the same procedure.
In case of the determination of rye ergot or harmful botanical impurities by visual/microscopic examination correction to the moisture content is not necessary.
6. Analytical measurement uncertainty and recovery rate in case of analysis of undesirable substances
As regards undesirable substances within the meaning of Directive 2002/32/EC, a product intended for animal feed shall be considered as non-compliant with the established maximum content, if the analytical result as a mean of two independent determinations, relative to a feed with a moisture content of 12 %, is deemed to exceed the maximum content taking into account expanded analytical measurement uncertainty using a coverage factor of 2 which gives a level of confidence of approximately 95 % and correction for recovery. This means, in order to assess compliance, the analysed concentration is used after being corrected for recovery and after deduction of the expanded analytical measurement uncertainty. This procedure is only applicable in cases where the method of analysis enables the estimation of the expanded analytical measurement uncertainty and correction for recovery (e.g. not required in case of visual/microscopic examination).
If the analytical result of the sample taken for defence exceeds the maximum content (without taking into account the expanded analytical measurement uncertainty), this confirms the non-compliance established with the control sample, in the absence of specific national rules on this.
The analytical result shall be reported as follows (in so far the method of analysis used enables to estimate the expanded analytical measurement uncertainty):
(a) corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. The correction for recovery is not necessary in case the recovery rate is between 90-110 %;
(b) as ‘x +/- U’, whereby x is the analytical result and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 (10) which gives a level of confidence of approximately 95 %.
However, if the result of the analysis is significantly (> 50 %) lower than the specification to be controlled, and on the condition that the appropriate quality procedures are applied and the analysis serves only the purpose of checking compliance with legal provisions, the reporting of the recovery rate and expanded analytical measurement uncertainty might be omitted (e.g. in cases there is no specification or regulatory level), unless the measurement uncertainty is required for interpretation.
7. Analytical measurement uncertainty and recovery rate in case of analysis of content of feed additives
In order to check compliance with authorised minimum and maximum content of feed additives, the presence of a feed additive shall be considered as non-compliant with the established minimum and maximum content, if the analytical result as mean of two independent determinations, relative to a feed with a moisture content of 12 %, is deemed to:
— exceed the maximum content taking into account expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after deduction of the expanded analytical measurement uncertainty,
— be lower than the minimum content taking into account the expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after the addition of the expanded analytical measurement uncertainty.
If the analytical result of the sample taken for defence exceeds the maximum content (without taking into account the expanded analytical measurement uncertainty), this confirms the non-compliance established with the control sample, in the absence of specific national rules on this.
The analytical result shall be reported as follows (in so far the method of analysis used enables to estimate the expanded analytical measurement uncertainty):
(a) corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. The correction for recovery is not necessary in case the recovery rate is between 90-110 %;
(b) as ‘x +/- U’, whereby x is the analytical result (mean of two determinations) and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 (11) which gives a level of confidence of approximately 95 %.
ANNEX III
A. DETERMINATION OF MOISTURE
1. Purpose and scope
This method makes it possible to determine the moisture content of feed. In case of feed containing volatile substances, such as organic acids, it is to be observed that also a significant number of volatile substances is determined together with the moisture content.
It does not cover the analysis of milk products as feed materials and compound feed composed predominantly of milk products, the analysis of animal and vegetable fats and oils or the analysis of the oil seeds and oleaginous fruit.
The determination of moisture content in oilseeds is to be determined by the method as provided for by EN ISO 665 Determination of moisture and volatile matter content, with the understanding that soybeans have to be ground before determination of moisture content.
2. Principle
The sample is desiccated under specified conditions which vary according to the nature of the feed. The loss in weight is determined by weighing. It is necessary to carry out preliminary drying when dealing with solid feed which has high moisture content.
3. Apparatus
3.1.Crusher of non-moisture-absorbing material which is easy to clean, allows rapid, even crushing without producing any appreciable heating, prevents contact with the outside air as far as possible and meets the requirements laid down in 4.1.1 and 4.1.2 (e.g. hammer or water-cooled micro-crushers, collapsible cone mills, slow motion or cog-wheeled crushers).
3.2.Analytical balance, accurate to 1 mg.
3.3.Dry containers of non-corrodible metal or of glass with lids ensuring airtight closure; working surface allowing the test sample to be spread at about 0,3 g/cm2.
3.4.Electrically heated isothermal oven (± 2 °C) properly ventilated and ensuring rapid temperature regulation (12).
3.5.Adjustable electrically heated vacuum oven fitted with an oil pump and either a mechanism for introducing hot dried air or a drying agent (e.g. calcium oxide).
3.6.Desiccator with a thick perforated metal or porcelain plate, containing an efficient drying agent.
4. Procedure
NB:The operations described in this section must be carried out immediately after opening the packages of samples. Analysis must be carried out at least in duplicate.
Take at least 50 g of the sample. If necessary, crush or divide in such a way as to avoid any variation in moisture content (see point 6).
Take at least 50 g of the sample. Grind into particles of which at least 50 % will pass through a 0,5 mm mesh sieve and will leave no more than 10 % reject on a 1 mm round-meshed sieve.
Take about 25 g of the sample, weigh to the nearest 10 mg, add an appropriate quantity of anhydrous sand weighed to the nearest 10 mg and mix until a homogeneous product is obtained.
Dry a container (point 3.3) with its lid in the oven set at 103 °C for 30 min +/- 1 min. Remove from the oven and allow to cool to ambient temperature in the desiccator (point 3.6).
Weigh the container with its lid to the nearest 1 mg. Weigh into the weighed container, to the nearest 1 mg, about 5 g of the sample and spread evenly. Place the container, without its lid, in the oven preheated to 103 °C. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible. Leave to dry for four hours reckoned from the time when the oven temperature returns to 103 °C. Open the oven, replace the lid on the container immediately, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 3.6) and weigh to the nearest 1 mg.
For feed composed predominantly (> 50 %) of oils and fats of animal and plant origin, dry in the oven for an additional 30 minutes at 103 °C. The difference between the two weighings must not exceed 0,1 % of moisture.
Weigh the container with its lid to the nearest 0,5 mg. Weigh into the weighed container, to the nearest 1 mg, about 5 g of the crushed sample and spread evenly. Place the container, without its lid, in the oven preheated to 130 °C. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible. Leave to dry for two hours reckoned from the time when the oven temperature returns to 130 °C. Open the oven, replace the lid on the container immediately, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 3.6) and weigh to the nearest 1 mg.
Weigh the container with its lid to the nearest 0,5 mg. Weigh into the weighed container, to the nearest 1 mg, about 5 g of the sample and spread evenly. Place the container, without its lid, in the vacuum oven (point 3.5) preheated to between 80 °C and 85 °C. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible.
Bring the pressure up to 100 Torr and leave to dry for four hours at this pressure, either in a current of hot, dry air or using a drying agent (about 300 g for 20 samples). In the latter instance, disconnect the vacuum pump when the prescribed pressure has been reached. Reckon drying time from the moment when the oven temperature returns to 80 °C to 85 °C. Carefully bring the oven back to atmospheric pressure. Open the oven, replace the lid on the container immediately, remove the container from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 3.6) and weigh to the nearest 1 mg. Dry for an additional 30 minutes in the vacuum oven at 80 °C to 85 °C and reweigh. The difference between the two weighings must not exceed 0,1 % of moisture.
It is necessary to partially dry ‘wet’ feeds with a mass fraction of less than 85 % dry matter (e.g. forages, total mixed rations, (non)-liquid feed) prior to fine grinding in order to analyse their stable substances; for unstable substances, partial drying is not possible.
Partial drying can be performed using either a forced-air oven or a microwave oven or by freeze drying. With the exception of partial drying by freeze drying, the aim is to dry the feed while keeping sample temperature below 60 °C so that chemical composition is minimally affected. Drying at temperatures greater than 60 °C causes chemical changes in the feed (e.g. protein degradation). The dried feed shall be equilibrated at room temperature for about 15 minutes before measuring partial dry matter so as to minimise the potential change in moisture that can occur during grinding and storage. Drying at temperatures lower than 60 °C does not remove all of the water from the feed; therefore, (initial) partial drying does not represent the total dry matter of the feed. Following drying, the subsample is ground and analysed for (final) dry matter of the partially dry sample (the remaining 3 % to 15 % moisture) when other chemical constituents are determined.
Therefore, a two-step procedure for determining dry matter is recommended. First determine the partial dry matter content (if less than 85 % dry matter), then determine the remaining dry matter content on a ground test sample and multiply partial dry matter by the remaining dry matter to determine the total dry matter content.
5. Calculation of results
The moisture content (X), as a percentage of the sample, is calculated by using the following formulae:
where:
where:
The difference between the results of two parallel determinations carried out on the same sample shall not exceed 0,2 % of the absolute value of moisture, except for wet pet food and dog chews, where the difference shall not exceed 0,5 % of the absolute value of moisture.
6. Observation
If crushing proves necessary and if this is seen to alter the moisture content of the product, the results of the analysis of the components of the feed must be corrected on the basis of the moisture content of the sample in its initial state.
B. DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE FATS AND OILS
1. Purpose and scope
This method makes it possible to determine the water and volatile substances content of animal and vegetable fats and oils.
2. Principle
The sample is dried to constant weight (loss in weight between two successive weighings must be less than or equal to 1 mg) at 103 °C. The loss in weight is determined by weighing.
3. Apparatus
3.1.Flat-bottomed dish, of a corrosion-resistant material, 8 to 9 cm in diameter and approximately 3 cm high.
3.2.Thermometer with a strengthened bulb and expansion tube at the top end, graduated from approximately 80 °C to at least 110 °C, and approximately 10 cm in length.
3.3.Sand bath or electric hot-plate.
3.4.Desiccator, containing an efficient drying agent.
3.5.Analytical balance.
4. Procedure
Weigh out to the nearest mg approximately 20 g of the homogenised sample into the dry, weighed dish (point 3.1) containing the thermometer (point 3.2). Heat on the sand bath or hot-plate (point 3.3), stirring continuously with the thermometer, so that the temperature reaches 90 °C in about 7 minutes.
Reduce the heat, watching the frequency with which bubbles rise from the bottom of the dish. The temperature must not exceed 105 °C. Continue to stir, scraping the bottom of the dish, until bubbles stop forming.
In order to ensure complete elimination of moisture, reheat several times to 103 °C ± 2 °C, cooling to 93 °C between successive heatings. Then leave to cool to room temperature in the desiccator (point 3.4) and weigh. Repeat this operation until the loss in weight between two successive weighings no longer exceeds 2 mg.
NB:An increase in the weight of the sample after repeated heating indicates an oxidation of the fat, in which case calculate the result from the weighing carried out immediately before the weight began to increase.
5. Calculation of results
The moisture content (X), as a percentage of the sample, is given by the following formula:
where:
Results lower than 0,05 % must be recorded as ‘lower than 0,05 %’.
The difference in moisture between the results of two parallel determinations carried out on the same sample must not exceed 0,1 %, in absolute value.
C. DETERMINATION OF THE NITROGEN CONTENT AND CALCULATION OF CRUDE PROTEIN CONTENT
1. Purpose and scope
This method makes it possible to determine the crude protein content of feed on the basis of the nitrogen content, determined according to the Kjeldahl method (14).
2. Principle
The sample is digested by sulphuric acid in the presence of a catalyst. The acid solution is made alkaline with sodium hydroxide solution. The ammonia is distilled and collected in a measured quantity of sulphuric acid, the excess of which is titrated with a standard solution of sodium hydroxide.
Alternatively, the liberated ammonia is distilled into an excess of boric acid solution, followed by titration with hydrochloric acid or sulphuric acid solution.
3. Reagents
3.1.Potassium sulphate.
3.2.Catalyst: copper (II) oxide CuO or copper (II) sulphate pentahydrate, CuSO4 5H2O.
3.3.Granulated zinc.
3.4.Sulphuric acid, ρ20 = 1,84 g/ml.
3.5.Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,25 mol/l.
3.6.Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,10 mol/l.
3.7.Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,05 mol/l.
3.8.Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of ethanol, σ = 95-96 % (v/v).
3.9.Sodium hydroxide solution (Technical grade may be used) β = 40 g/100 ml (m/v: 40 %).
3.10.Sodium hydroxide, standard volumetric solution c(NaOH) = 0,25 mol/l.
3.11.Sodium hydroxide, standard volumetric solution c(NaOH) = 0,10 mol/l.
3.12.Granulated pumice stone, washed in hydrochloric acid and ignited.
3.13.Acetanilide (m.p. = 114 °C, N-content = 10,36 %).
3.14.Sucrose (nitrogen free).
3.15.Boric acid (H3BO3).
3.16.Methyl red indicator solution: dissolve 100 mg methyl red in 100 ml ethanol or methanol.
3.17.Bromocresol green solution: dissolve 100 mg bromocresol green in 100 ml ethanol or methanol.
3.18.Boric acid solution (10 g/l to 40 g/l depending on the apparatus used)
When colorimetric end-point detection is applied, methyl red and bromocresol indicators must be added to the boric acid solutions. If 1 litre of the boric acid solution is prepared, before adjusting to volume, 7 ml methyl red indicator solution (point 3.16) and 10 ml bromocresol green solution (point 3.17) shall be added.
Dependent on the water used, the pH of the boric acid solution might differ from batch to batch. The pH of the boric acid solution has to be between 4,3 and 4,7. Often an adjustment with a small volume of alkali is necessary to obtain a positive blank
Note:The addition of about 3 ml to 4 ml of NaOH (point 3.11) into 1 litre of 10 g/l boric acid usually gives good adjustments. Store the solution at room temperature and protect the solution from light and sources of ammonia fumes during storage.
3.19.Hydrochloric acid standard volumetric solution c(HCl) = 0,10 mol/l.
Note: Other concentrations of volumetric solutions (points 3.5, 3.6, 3.7, 3.10, 3.11 and 3.19) can be used, if this is corrected for in the calculations. The concentrations shall always be expressed to four decimal places.
4. Apparatus
Apparatus suitable for performing digestion, distillation and titration according to the Kjeldahl procedure.
5. Procedure
Weigh 1 g of the sample to the nearest 0,001 g and transfer the sample to the flask of the digestion apparatus. Add 15 g of potassium sulphate (point 3.1), an appropriate quantity of catalyst (point 3.2) (0,3 to 0,4 g of copper (II) oxide or 0,9 to 1,2 g of copper (II) sulphate pentahydrate), 25 ml of sulphuric acid (point 3.4) and if required, a few granules of pumice stone (point 3.12) and mix.
Heat the flask moderately at first, swirling from time to time if necessary until the mass has carbonised and the foam has disappeared; then heat more intensively until the liquid is boiling steadily. Heating is adequate if the boiling acid condenses on the wall of the flask. Prevent the sides from becoming overheated and organic particles from sticking to them.
When the solution becomes clear and light green continue to boil for another two hours, then leave to cool.
Add carefully enough water to ensure complete dissolution of the sulphates. Allow to cool and then add a few granules of zinc (point 3.3), if required. Proceed according to point 5.2.1 or 5.2.2.
Place in the collecting flask of the distillation apparatus an exactly measured quantity of 25 ml of sulphuric acid (point 3.5) or (point 3.7) depending on the presumed nitrogen content. Add a few drops of methyl red indicator (point 3.8).
Connect the digestion flask to the condenser of the distillation apparatus and immerse the end of the condenser in the liquid contained in the collecting flask to a depth of at least 1 cm (see observation point 8.3). Slowly pour 100 ml of sodium hydroxide solution (point 3.9) into the digestion flask without loss of ammonia (see observation point 8.1). Heat the flask until the ammonia has distilled over.
Where titration of the ammonia content of the distillate is performed manually, the procedure mentioned below applies. Where the distillation unit is fully automated to include titration of the ammonia content of the distillate, follow the manufacturer’s instructions for operation of the distillation unit.
Place a collecting flask containing 25 ml to 30 ml of the boric acid solution (point 3.18) under the outlet of the condenser in such a way that the delivery tube is below the surface of the excess boric acid solution. Adjust the distillation unit to dispense 50 ml of sodium hydroxide solution (point 3.9). Operate the distillation unit in accordance with the manufacturer’s instructions and distil off the ammonia liberated by the addition of the sodium hydroxide solution. Collect distillate in the boric acid receiving solution. The amount of distillate (time of steam distillation) depends on the amount of nitrogen in the sample. Follow the instructions of the manufacturer.
Note: In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are performed automatically.
Proceed according to point 5.3.1 or 5.3.2.
Titrate the excess sulphuric acid in the collecting flask with sodium hydroxide solution (point 3.10 or 3.11) depending on the concentration of the sulphuric acid used, until the end point is reached.
Titrate the contents of the collecting flask with the hydrochloric acid standard volumetric solution (point 3.19) or with the sulphuric acid standard volumetric solution (point 3.6) using a burette and read the amount of titrant used.
When colorimetric end-point detection is applied, the end-point is reached at the first trace of pink colour in the contents. Estimate the burette reading to the nearest 0,05 ml. An illuminated magnetic stirrer plate or a photometric detector may aid visualisation of the end-point.
This can be done automatically using a steam distiller with automatic titration.
Follow the manufacturers’ instructions for operation of the specific distiller or distiller/titrator.
Note: When an automatic titration system is used, titration begins immediately after distillation starts and the 1 % boric acid solution (point 3.18) is used.
Where a fully automatic distillation unit is employed, the automatic titration of the ammonia can also be carried out with end-point detection using a potentiometric pH system.
In this case an automatic titrator with a pH-meter is used. The pH-meter shall be calibrated properly in the range of pH 4 to pH 7 following normal laboratory pH-calibration procedures.
The pH end-point of the titration is reached at pH 4,6, being the steepest point in the titration curve (inflection point).
To confirm that the reagents are free from nitrogen, carry out a blank test (digestion, distillation and titration) using 1 g of sucrose (point 3.14) in place of the sample.
6. Calculation of results
Calculations are performed according to point 6.1 or 6.2.
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
7. Verification of the method
The difference between the results of two parallel determinations carried out on the same sample must not exceed:
— 0,4 % in absolute value, for crude protein contents of less than 20 %,
— 2,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,
— 0,8 % in absolute value, for crude protein contents of more than 40 %.
The difference between the results of two determinations carried out on the same sample in different laboratories must not exceed:
— 1,8 % in absolute value, for crude protein contents of less than 20 %,
— 9,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,
— 3,6 % in absolute value, for crude protein contents of more than 40 %.
Carry out the analysis (digestion, distillation and titration) on an appropriate quantity of acetanilide (point 3.13) (e.g. 0,2 to 0,3 g) in the presence of 1 g of sucrose (point 3.14); 1 g acetanilide consumes 14,80 ml of sulphuric acid (point 3.5). Recovery must be at least 99 %.
8. Observations
8.1.Apparatus may be of the manual, semi-automatic or automatic type. If the apparatus requires transference between the digestion and distillation steps, this transfer must be carried out without loss. If the flask of the distillation apparatus is not fitted with a dropping funnel, add the sodium hydroxide immediately before connecting the flask to the condenser, pouring the liquid slowly down the side.
8.2.If the digest solidifies, recommence the determination using a larger amount of sulphuric acid (point 3.4) than that specified in point 5.1.
8.3.For products with a low nitrogen content, the volume of sulphuric acid (point 3.7) to be placed in the collecting flask may be reduced, if necessary, to 10 or 15 ml and made up to 25 ml with water.
8.4.For routine analysis, alternative methods of analysis can be applied for the determination of crude protein but the Kjeldahl method described in this Part C is the reference method. The equivalence of the results obtained with the alternative method (e.g. DUMAS) compared to the reference method must be demonstrated for each matrix individually. As the results obtained with an alternative method, even after having verified the equivalency, might deviate slightly from the results obtained with the reference method, it is necessary to mention in the analytical report the method of analysis used for the determination of crude protein.
D. DETERMINATION OF UREA
1. Purpose and scope
This method makes it possible to determine the level of urea used as feed additive in ruminant feed.
2. Principle
The sample is suspended in water with a clarifying agent. The suspension is filtered. The urea content of the filtrate is determined after the addition of 4-dimethylaminobenzaldehyde (4-DMAB) by measuring the optical density at a wavelength of 420 nm.
3. Reagents
3.1.Solution of 4-dimethylaminobenzaldehyde: dissolve 1,6 g of 4-DMAB in 100 ml of 96 % ethanol and add 10 ml of hydrochloric acid (ρ20 1,19 g/ml). This reagent keeps for a maximum period of two weeks.
3.2.Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn(CH3COO)2 2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.
3.3.Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide, K4Fe(CN)6 3H2O. Make up to 100 ml with water.
3.4.Active carbon which does not absorb urea (to be checked).
3.5.Urea, 0,1 % solution (w/v).
4. Apparatus
4.1.Mixer (tumbler): approximately 35 to 40 rpm.
4.2.Test tubes: 160 × 16 mm with ground-glass stoppers.
4.3.Spectrophotometer.
5. Procedure
Weigh out 2 g of the sample to the nearest mg and place with 1 g of active carbon (point 3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solution I (point 3.2), mix for approximately 30 seconds and add 5 ml of Carrez solution II (point 3.3). Mix for thirty minutes in the tumbler. Make up to volume with water, shake and filter.
Remove 5 ml of the transparent colourless filtrates, place in test tubes with ground-glass stoppers, add 5 ml of 4-DMAB solution (point 3.1) and mix. Place the tubes in a water bath at 20 °C (+/– 4 °C). After fifteen minutes measure the optical density of the sample solution with the spectrophotometer at 420 nm. Compare with the blank test solution of the reagents.
Remove volumes of 1, 2, 4, 5 and 10 ml of the urea solution (point 3.5), place in 100 ml volumetric flasks and make up the volume with water. Remove 5 ml from each solution, add 5 ml of 4-DMAB solution (point 3.1) to each of them, homogenise and measure the optical density as shown above in comparison with a control solution containing 5 ml of 4-DMAB and 5 ml of water free from urea. Plot the calibration curve.
6. Calculation of results
Determine the amount of urea in the sample using the calibration curve.
Express the result in mg urea per kg sample.
7. Evaluation of the method
The difference between the results of two determinations carried out on the same sample in the same laboratory and by the same operator must not exceed:
— At 420 nm: — 50 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 5 000 mg/kg, — 25 % relative to the higher value, for urea contents from 5 000 mg/kg to lower than 7 000 mg/kg, — 20 % relative to the higher value, for urea contents of 7 000 mg/kg or more.
— At 435 nm: — 40 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 5 000 mg/kg, — 25 % relative to the higher value, for urea contents from 5 000 mg/kg to lower than 9 000 mg/kg, — 5 % relative to the higher value, for urea contents of 9 000 mg/kg or more.
The difference between the results of two determinations carried out on the same sample in different laboratories and/or by different operators must not exceed:
— At 420 nm: — 3 000 mg/kg, in absolute value, for urea contents from 3 000 mg/kg to lower than 12 000 mg/kg, — 4 500 mg/kg, in absolute value, for urea contents of 12 000 mg/kg or more.
— At 435 nm — 50 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 8 000 mg/kg, — 25 % relative to the higher value, for urea contents of 8 000 mg/kg or more.
8. Results of a collaborative study
An EU interlaboratory comparison exercise was organised in which 18 laboratories took part. Five positive ruminant compound feed samples (in Tables 1 and 2 referred to as MAT) were analysed (1 analysis) in blind duplicates while one blank compound ruminant feed was analysed once.
Calculations for repeatability (r) and reproducibility (R) limits as defined by international guidelines were carried out after the removal of outliers using Analysis of Variance of the valid values.
The calculated method performance figures (repeatability, reproducibility) are presented in the following tables. Over all tested samples including the blank material, no false positives or false negatives were found.
| MAT 2 | MAT 5 | MAT 3 | MAT 4 | MAT 6 | |
|---|---|---|---|---|---|
| Sheep | Cattle | Sheep | Sheep | Cattle | |
| Target mass fraction (mg kg-1) | 3 000 | 5 000 | 7 001 | 9 036 | 11 000 |
| Average mass fraction. (mg kg-1) | 4 241 | 6 993 | 7 830 | 9 962 | 12 071 |
| Reproducibility standard deviation sR (mg kg-1) | 1 141 | 1 303 | 985 | 994 | 1 711 |
| Repeatability standard deviation sr (mg kg-1) | 723 | 601 | 549 | 712 | 737 |
| Reproducibility relative standard deviation RSDR (%) | 27 | 19 | 13 | 10 | 14 |
| Repeatability relative standard deviation RSDr (%) | 17 | 9 | 7 | 7 | 6 |
| Limit of reproducibility, R [R = 2,8 × sR] | 3 195 | 3 649 | 2 759 | 2 784 | 4 790 |
| Limit of repeatability, r [r = 2,8 × sr] | 2 024 | 1 684 | 1 536 | 1 994 | 2 064 |
| MAT 2 | MAT 5 | MAT 3 | MAT 4 | MAT 6 | |
| --- | --- | --- | --- | --- | --- |
| Sheep | Cattle | Sheep | Sheep | Cattle | |
| Target mass fraction (mg kg-1) | 3 000 | 5 000 | 7 001 | 9 036 | 11 000 |
| Average mass fraction. (mg kg-1) | 4 101 | 6 467 | 7 890 | 10 062 | 11 642 |
| Reproducibility standard deviation sR (mg kg-1) | 706 | 1 194 | 675 | 745 | 1 378 |
| Repeatability standard deviation sr (mg kg-1) | 570 | 628 | 613 | 196 | 167 |
| Reproducibility relative standard deviation RSDR (%) | 17 | 18 | 9 | 7 | 12 |
| Repeatability relative standard deviation RSDr (%) | 14 | 10 | 8 | 2 | 1 |
| Limit of reproducibility, R [R = 2,8 × sR] | 1 977 | 3 344 | 1 889 | 2 087 | 3 859 |
| Limit of repeatability, r [r = 2,8 × sr] | 1 596 | 1 759 | 1 715 | 549 | 467 |
9. Observations
9.1.In the case of contents of urea exceeding 3 %, reduce the sample to 1 g or dilute the original solution so that there are not more than 50 mg of urea in 500 ml.
9.2.In the case of low contents of urea, increase the sample as long as the filtrate remains transparent and colourless.
9.3.The above results from collaborative trials do not indicate a significant difference in precision between urea measured at 420 nm or at 435 nm.
E. DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHAN)
The methods of analysis to be used for the determination of amino acids (except tryptophan) are:
— EN ISO 13903 Animal feeding stuffs – Determination of amino acids content,
— EN ISO 17180 Animal feeding stuffs – Determination of lysine, methionine, and threonine in commercial amino acid products and premixtures (15),
— the method of analysis as described in points 1 to 10 hereafter.
1. Purpose and scope
This method makes the determination possible of free (synthetic and natural) and total (peptide bound and free) amino acids in feed materials, compound feeds and premixtures containing less than 10 % (16) of each amino acid, using an amino acid analyser. It is applicable to the following amino acids: cyst(e)ine, methionine, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine and valine.
The method does not distinguish between the various salts of amino acids and it cannot differentiate between D and L forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids.
2. Principle
The free amino acids are extracted with diluted hydrochloric acid. Co-extracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm.
The procedure chosen depends on the amino acids under investigation. Cyst(e)ine and methionine must be oxidised to cysteic acid and methionine sulphone respectively prior to hydrolysis. Tyrosine must be determined in hydrolysates of unoxidised samples. All the other amino acids listed in point 1 (Purpose and scope) can be determined in either the oxidised or unoxidised sample.
Oxidation is performed at 0 °C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulphite. The oxidised or unoxidised sample is hydrolysed with hydrochloric acid (point 3.20) for 23 hours. The hydrolysate is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin using photometric detection at 570 nm (440 nm for proline).
3. Reagents
Double distilled water or water of equivalent quality must be used (conductivity < 10 μS).
3.1.Hydrogen peroxide, w (w/w) = 30 %.
3.2.Formic acid, w (w/w) = 98–100 %.
3.3.Phenol.
3.4.Sodium disulphite.
3.5.Sodium hydroxide.
3.6.5-Sulfosalicylic acid dihydrate.
3.7.Hydrochloric acid, density approximately 1,18 g/ml.
3.8.tri-Sodium citrate dihydrate.
3.9.2,2'-Thiodiethanol (thiodiglycol).
3.10.Sodium chloride.
3.11.Ninhydrin.
3.12.Light petroleum, boiling range 40–60 °C.
3.13.Norleucine, or other compound suitable for use as internal standard.
3.14.Nitrogen gas (< 10 ppm oxygen).
3.15.1-Octanol.
3.16.Amino acids.
3.16.1.Standard substances of the amino acids listed under point 1 (Purpose and scope). Pure compounds containing no water of crystallisation. Dry under vacuum over P2O5 or H2SO4 for 1 week prior to use.
3.16.2.Cysteic acid.
3.16.3.Methionine sulphone.
3.17.Sodium hydroxide solution, c = 7,5 mol/l:
Dissolve 300 g NaOH (point 3.5) in water and make up to 1 litre.
3.18.Sodium hydroxide solution, c = 1 mol/l:
Dissolve 40 g NaOH (point 3.5) in water and make up to 1 litre.
3.19.Formic acid – phenol solution:
Mix 889 g formic acid (point 3.2) with 111 g water and add 4,73 g phenol (point 3.3).
3.20.Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l:
Add 1 g phenol (point 3.3) to 492 ml HCl (point 3.7) and make up to 1 litre with water.
3.21.Extraction mixture, c = 0,1 mol HCl/l containing 2 % thiodiglycol: Take 8,2 ml HCl (point 3.7), dilute with approximately 900 ml water, add 20 ml thiodiglycol (point 3.9) and make up to 1 litre with water (do not mix points 3.7 and 3.9 directly).
3.22.5-Sulfosalicylic acid, ß = 6 %:
Dissolve 60 g 5-sulfosalicylic acid (point 3.6) in water and make up to 1 l with water.
3.23.Oxidation mixture (Performic acid – phenol):
Mix 0,5 ml hydrogen peroxide (point 3.1) with 4,5 ml formic acid-phenol solution (point 3.19) in a small beaker. Incubate at 20–30 °C for 1 hour in order to form performic acid, then cool on an ice-water bath (15 min) before adding to the sample.
Caution: Avoid contact with skin and wear protective clothing.
3.24.Citrate buffer, c = 0,2 mol Na+/l, pH 2,20:
Dissolve 19,61 g sodium citrate (point 3.8), 5 ml thiodiglycol (point 3.9), 1 g phenol (point 3.3) and 16,50 ml HCl (point 3.7) in approximately 800 ml water. Adjust pH to 2,20. Make up to 1 litre with water.
3.25.Elution buffers, prepared according to conditions for the analyser used (point 4.9).
3.26.Ninhydrin reagent, prepared according to conditions for the analyser used (point 4.9).
3.27.Standard solutions of amino acids. These solutions shall be stored below 5 oC.
3.27.1.Stock standard solution of amino acids (point 3.16.1).
c = 2,5 μmol/ml of each in hydrochloric acid.
May be obtained commercially.
3.27.2.Stock standard solution of cysteic acid and methionine sulphone, c = 1,25 μmol/ml.
Dissolve 0,2115 g cysteic acid (point 3.16.2) and 0,2265 g methionine sulphone (point 3.16.3) in citrate buffer (point 3.24) in a 1 litre graduated flask and make up to mark with citrate buffer. Store below 5 °C for not more than 12 months. This solution is not used if the stock standard solution (point 3.27.1) contains cysteic acid and methionine sulphone.
3.27.3.Stock standard solution of the internal standard e.g. norleucine, c = 20 μmol/ml.
Dissolve 0,6560 g norleucine (point 3.13) in citrate buffer (point 3.24) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 °C for no more than 6 months.
3.27.4.Calibration solution of standard amino acids for use with hydrolysates, c = 5 nmol/50 μl of cysteic acid and methionine sulphone and c = 10 nmol/50 μl of the other amino acids. Dissolve 2,2 g sodium chloride (point 3.10) in 100 ml beaker with 30 ml citrate buffer (point 3.24). Add 4,00 ml stock standard solution of amino acids (point 3.27.1), 4,00 ml stock standard solution of cysteic acid and methionine sulphone (point 3.27.2) and 0,50 ml stock standard solution of internal standard (point 3.27.3) if used. Adjust pH to 2,20 with sodium hydroxide (point 3.18).
Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (point 3.24) and mix.
Store below 5 °C for not more than 3 months.
See also observations point 9.1.
3.27.5.Calibration solution of standard amino acids for use with hydrolysates prepared according to point 5.3.3.1 and for use with extracts (point 5.2). The calibration solution is prepared according to point 3.27.4 but omitting sodium chloride.
Store below 5 °C for not more than 3 months.
4. Apparatus
4.1.100 or 250 ml round-bottomed flask fitted with a reflux condenser.
4.2.100 ml borosilicate glass bottle with screw cap with rubber/teflon liner (e.g. Duran, Schott) for use in the oven.
4.3.Oven with forced ventilation and a temperature regulator with an accuracy better than ± 2 oC.
4.4.pH-meter (three decimal places).
4.5.Membrane filter (0,22 μm).
4.6.Centrifuge.
4.7.Rotary vacuum evaporator.
4.8.Mechanical shaker or magnetic stirrer.
4.9.Amino acid analyser or HPLC equipment with ion exchange column, device for ninhydrin, post column derivatisation and photometric detector.
The column is filled with sulfonated polystyrene resins capable of separating the amino acids from each other and from other ninhydrin-positive materials. The flow in the buffer and ninhydrin lines is provided by pumps having a flow stability of ± 0,5 % in the period covering both the standard calibration run and the analysis of the sample.
With some amino acid analysers hydrolysis procedures can be used in which the hydrolysate has a sodium concentration of c = 0,8 mol/l and contains all the residual formic acid from the oxidation step. Others do not give a satisfactory separation of certain amino acids if the hydrolysate contains excess formic acid and/or high sodium ion concentrations. In this case the volume of acid is reduced by evaporation to approx. 5 ml after the hydrolysis and prior to pH adjustment. The evaporation shall be performed under vacuum at 40 °C maximum.
5. Procedure
The sample is ground to pass through a 0,5 mm sieve. Samples high in moisture must be either air-dried at a temperature not exceeding 50 °C or freeze dried prior to grinding. Samples with a high fat content shall be extracted with light petroleum (point 3.12) prior to grinding.
Weigh to the nearest 0,2 mg an appropriate amount (1-5 g) of the prepared sample (point 5.1), into a conical flask and add 100,0 ml of extraction mixture (point 3.21). Shake the mixture for 60 min using a mechanical shaker or a magnetic stirrer (point 4.8). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a 100 ml beaker.
Add 5,0 ml of sulfosalicylic acid solution (point 3.22), with stirring and continue to stir with the aid of magnetic stirrer for 5 min. Filter or centrifuge the supernatant in order to remove any precipitate. Place 10,0 ml of the resulting solution into a 100 ml beaker and adjust the pH to 2,20 using sodium hydroxide solution (point 3.18), transfer to a volumetric flask of appropriate volume using citrate buffer (point 3.24), and make up to the mark with the buffer solution (point 3.24).
If an internal standard is being used add 1,00 ml of internal standard (point 3.27.3) for each 100 ml final solution and make up to the mark with the buffer solution (point 3.24).
Proceed to the chromatography step according to point 5.4.
If the extracts are not being examined the same day, they must be stored below 5 °C.
Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (point 5.1) into:
— a 100 ml round-bottomed flask (point 4.1) for open hydrolysis (point 5.3.2.3), or
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