Feedingstuffs, 2005

L.N. 75 of 2005

VETERINARY SERVICES ACT

(CAP. 437)

Rules on the Analytical Method for the Determination of

Constituents of Animal Origin for the Official Control of

Feedingstuffs, 2005

IN exercise of the powers conferred by article 25 of the Veterinary

Services Act, the Minister for Rural Affairs and the Environment has

made the following rules>–

(1) The title of these rules is the Rules on the Analytical

Method for the Determination of Constituents of Animal Origin for the

Official Control of Feedingstuffs, 2005.

(2) The scope of these rules is to implement the rules found

under the European Union Commission Directive 2003#126#EEC on

the analytical method for the determination of constituents of animal

origin for the official control of feedingstuffs.

Conditions for the microscopic detection, identification or estimation of constituents of animal origin

in feedingstuffs

1. Objective and field of application

These conditions shall be used when detection of constituents of animal origin (defined as products from

processing bodies and body parts of mammals, poultry and fish) in feedingstuffs is carried out by means of

microscopic examination in the framework of the coordinated inspection programme in the field of animal

nutrition in accordance with Council Directive 95/53/EC. Provided that the methods in this Schedule are

used in all official tests, a second test may also be carried out using variant or alternative methods, in order

to improve the detection of certain types of animal constituents or to specify further the origin of the animal

constituents. Furthermore, a variant protocol may be used when examining certain specific animal

constituents such as plasma or bones in tallow (see also point 9), provided that these analyses are made in

addition to the analyses foreseen in the coordinated inspection programme.

2. Sensitivity

Dependent on the nature of the constituents of animal origin, very small amounts (< 0,1 %) in feedingstuffs

can be detected.

3. Principle

A representative sample, taken in accordance with the provisions laid down in Commission Directive

76/371/EEC of 1 March 1976 establishing Community methods of sampling for the official control of

feedingstuffs (1) which has undergone suitable preparation is used for the identification. The following

protocol is fit for handling feed with low moisture content. Feed with an amount of moisture higher than 14

% shall be dried (condensed) prior to handling. Special feed or feed materials (e.g. fats, oils) need dedicated

treatment (see point 9). The constituents of animal origin are identified on the basis of typical,

microscopically identifiable characteristics (i.e. muscle fibres and other meat particles, cartilage, bones,

horn, hair, bristles, blood, feathers, egg shells, fish bones, scales). The identification has to be done both on

the sieve fraction (6.1) and the concentrated sediment (6.2) of the sample.

Where official analysis of feedingstuffs is carried out with a

view to officially controlling the presence, identification and, or

estimation of the amount of constituents of animal origin in feedingstuffs,

in the framework of the coordinated inspection programme in the field

of animal nutrition in accordance with the European Union Council

Directive 95#53#EC, it shall be carried out in accordance with the

provisions of the Schedule to these rules.

(1) Laboratories carrying out official controls on the presence

Role of laboratories.

of animal constituents in feedingstuffs shall participated periodically in

proficiency testing on the analytical methods.

(2) Laboratory personnel carrying out analyses shall receive

adequate training.

Reagents

4.1. Embedding agent

4.1.1. Chloral hydrate (aqueous, 60 % w/v)

4.1.2. Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v) for sieve fractions

4.1.3. Paraffin oil or glycerol (viscosity: 68-81) for microscopic observations in the sediment

4.2. Rinsing agents

4.2.1. Alcohol, 96 %

4.2.2. Acetone

4.3. Concentrating agent

4.3.1. Tetrachloroethylene (density 1,62)

4.4. Staining reagents

4.4.1. Iodine/potassium iodide solution (Dissolve 2 g potassium iodide in 100 ml water and add 1 g iodine

while shaking frequently)

4.4.2. Alizarin Red (Dilute 2,5 ml 1M hydrocloric acid in 100 ml water and add 200 mg alizarine red to this

solution)

4.4.3. Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml H2O)

4.4.4. Iodine/potassium iodide solution (dissolved in 70 % ethanol)

4.5. Bleaching reagent

4.5.1. Commercial sodium hypochlorite solution (9.6 % active chlorine)

Equipment and accessories

5.1. Analytical balance (accuracy of 0,01 g except for the concentrated sediment: 0,001 g)

5.2. Material for grinding (grinding mill or a mortar, especially for feed containing > 15 % fat on analysis)

5.3. Sieve fitted with sieve mesh with square meshes of width of 0,50 mm maximum

5.4. Separation funnel or conical bottomed settling beaker

5.5. Stereomicroscope (minimum 40' magnification)

5.6. Compound microscope (minimum 400' magnification), transmitted light or polarised light

5.7. Standard laboratory glassware

All equipment shall be thoroughly cleaned. Separation funnels and glassware need washing in a washing

machine. Sieves need cleaning using a brush with stiff hairs.

Procedure

Pelleted feeds may be pre-sieved if both fractions are analysed as a separate sample.

At least 50 g of the sample shall be treated (ground with care using the suitable grinding equipment (5.2) if

necessary in order to achieve an appropriate structure). From the ground material two representative

portions shall be taken, one for the sieve fraction (at least 5 g) (6.1) and one for the concentrated sediment

(at least 5 g) (6.2). Colouring with staining reagents (6.3) can additionally be applied for the identification.

In order to indicate the nature of the animal proteins and the origin of the particles, a decision support

system such as Aries can be used and reference samples can be documented.

6.1. Identification of constituents of animal origin in the sieve fractions

At least 5 g of the sample is sieved through the sieve (5.3) in two fractions.

The sieve fraction(s) with the large particles (or a representative part of the fraction) is applied as a thin

layer to a suitable support and screened systematically under the stereomicroscope (5.5) at various

magnifications for constituents of animal origin.

Slides made with the sieve fraction(s) with the fine particles are screened systematically under the

compound microscope (5.6) at various magnifications for constituents of animal origin.

6.2. Identification of constituents of animal origin from the concentrated sediment

At least 5 g (accurate to 0,01 g) of the sample shall be transferred into a separation funnel or conical

bottomed settling beaker and treated with at least 50 ml of tetrachloroethylene (4.3.1). The mixture shall be

shaken or stirred repeatedly.

— If a closed separation funnel is used the sediment shall be left to stand for a sufficient time (at least three

minutes) before the sediment is separated off. Shaking shall be repeated and the sediment shall be left to

stand again at least three minutes. The sediment shall be separated off again.

— If an open beaker is used, the sediment shall be left to stand for at least five minutes before the sediment

is separated off.

The total sediment shall be dried and subsequently weighed (accurate to 0,001 g). The weighing is only

necessary in case an estimation is required. If the sediment consists of many large particles it may be sieved

through a sieve (5.3) in two fractions. The dried sediment shall be examined for bone constituents under the

stereomicroscope (5.5) and the compound microscope (5.6).

6.3. Use of embedding agents and staining reagents

The microscopic identification of the constituents of animal origin can be supported by the use of special

embedding agents and staining reagents.

Chloral hydrate (4.1.1): By carefully heating, cell structures can be seen more clearly because starch grains

gelatinise and unwanted cell contents are removed.

Lye (4.1.2): Either sodiumhydroxide or potassiumhydroxide clears the material of the feed, assisting the

detection of muscle fibres, hairs and other keratin structures.

Paraffin oil and glycerol (4.1.3): Bone constituents can be well identified in this embedding agent because

most lacunae remain filled with air and appear as black holes about 5-15 µm.

Iodine/potassium iodide solution (4.4.1): Used for the detection of starch (blue-violet colour) and protein

(yellow-orange colour). Solutions may be diluted if required.

Alizarin red solution (4.4.2): Red/pink colouring of bones, fish bones and scales. Before drying the

sediment (see section 6.2), the total sediment shall be transferred into a glass test tube and rinsed twice with

approximately 5 ml alcohol (4.2.1) (each time a vortex shall be used, the solvent shall be let settle about

one minute and poured off). Before using this staining reagent, the sediment shall be bleached by adding at

least 1 ml sodium hypochlorite solution (4.5.1). The reaction shall be allowed to continue for

10 minutes. The tube shall be filled with water, the sediment shall be let settle two to three minutes, and the

water and the suspended particles shall be poured off. The sediment shall be rinsed twice more with about

10 ml of water (a vortex shall be used, let settle, and the water poured off each time). Two to 10 or more

drops (depending on the amount of residue) of the alizarine red solution shall be added. The mixture shall

be shaken and the reaction shall be let occur a few seconds. The coloured sediment shall be rinsed twice

with approximately 5 ml alcohol (4.2.1) followed by one rinse with acetone (4.2.2) (each time a vortex

shall be used, the solvent shall be let settle about one minute and poured off). The sediment is then ready to

be dried.

Cystin reagent (4.4.3): By carefully heating, cystin-containing constituents (hair, feathers, etc.) become

black-brown.

6.4. Examination in feed possibly containing fishmeal

At least one slide shall be examined from the fine sieve fraction and from the fine fraction of the sediment

under the compound microscope (see sections 6.1 and 6.2).

Where the label indicates that the ingredients include fishmeal, or if the presence of fishmeal is suspected

or detected in the initial examination, at least two additional slides of the fine sieve fraction from the

original sample, and the total sediment fraction shall be examined.

Calculation and evaluation

Member States shall ensure that the procedures described in this point are used where an official analysis is

carried out with a view to estimating the amount (and not simply the presence) of animal constituents.

The calculation can only be made if the constituents of animal origin contain bone fragments.

Bone fragments of terrestrial warm-blooded species (i.e. mammals and birds) can be distinguished from the

different types of fish bone on the microscopic slide by means of the typical lacunae. The proportion of

constituents of animal origin in the sample material is estimated taking into consideration

— the estimated proportion (weight %) of bone fragments in the concentrated sediment and

— the proportion (weight %) of bone in the constituents of animal origin.

The estimate has to be based on at least three (if possible) slides and at least five fields per slide. In

compound feedingstuffs, the concentrated sediment as a rule contains not only terrestrial animal bone and

fish bone fragments, but also other particles of high specific weight, e.g. minerals, sand, lignified plant

fragments and the like.

7.1. Estimated value of the percentage of bone fragments

% terrestrial bone fragments = (S × c)/W

% fish bone and scale fragments = (S × d)/W

(S = sediment weight (mg), c = correction factor (%) for the estimated portion of terrestrial animal bones in

the sediment, d = correction factor (%) for the estimated portion of fish bones and scale fragments in the

sediment, W = weight of the sample material for the sedimentation (mg)).

7.2. Estimated value of constituents of animal origin

The proportion of bone in animal products can vary greatly. (The percentage of bone in the case of

bonemeals is of the order of 50 to 60 % and in the case of meat meals of the order of 20 to 30 %; in the case

of fish meals bone and scale contents vary according to the category and origin of the fishmeal, normally in

the order of 10 to 20 %).

If the type of animal meal present in the sample is known, it is possible to estimate the content

Estimated content of constituents of terrestrial animal products (%) = (S × c)/(W × f) × 100

Estimated content of constituents of fish products (%) = (S × d)/(W × f) × 100

(S = sediment weight (mg), c = correction factor (%) for the estimated portion of terrestrial animal bone

constituents in the sediment, d = correction factor (%) for the estimated portion of fish bones and scale

fragments in the sediment, f = correction factor for the proportion of bone in the constituents of animal

origin in the sample examined, W = weight of the sample material for the sedimentation (mg)).

Expression of the result of the examination

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

and from fishmeal. The different cases shall be reported in the following way

8.1. With regard to the presence of constituents derived from terrestrial animals

— As far as was discernible using a microscope, no constituents derived from terrestrial animals were

found in the submitted sample, or

— As far as was discernible using a microscope, constituents derived from terrestrial animals were found in

the submitted sample.

8.2. With regard to the presence of fishmeal

— As far as was discernible using a microscope, no constituents derived from fish were found in the

submitted sample, or

— As far as was discernible using a microscope, constituents derived from fish were found in the submitted

sample.

In case constituents derived from fish or terrestrial animals are found, the report of the examination result,

if required, can further indicate an estimation of the amount of constituents detected (x %, < 0,1 %, 0,1-0,5

%, 0,5-5 % or > 5 %), further specification of the type of terrestrial animal if possible and the animal

constituents identified (muscle fibres, cartilage, bones, horn, hair, bristles, feathers, blood, egg shells, fish

bones, scales).

For the case where the amount of animal ingredients is estimated the correction factor f used shall be

mentioned.

For the cases where bone constituents from terrestrial animals are identified, the report shall contain the

additional clause

‘The possibility that the above constituents are derived from mammals cannot be excluded.’

This additional clause is not necessary in cases where the bone fragments from terrestrial animals have

been specified as bone fragments from poultry or mammals.

Optional protocol for analysing fat or oil

The following protocol may be used for the analysis of fat or oil

— If the fat is solid, it is warmed for example in a microwave oven until it is liquid.

— Using a pipette, 40 ml of fat is transferred from the bottom of the sample to a centrifugation tube.

— Centrifuge for 10 minutes at 4 000 rpm.

— If the fat is solid after centrifugation, it is warmed once more in an oven until it is liquid. Repeat the

centrifugation for five minutes at 4 000 rpm.

— Using a small spoon or a spatula, one half of the decanted impurities is transferred to a small petri dish

or a microscopic slide for microscopic identification of a possible content of animal constituents (meat

fibres, feathers, bone fragments). As an embedding agent for microscopy, paraffin oil or glycerol is

recommended.

— The remaining impurities are used for sedimentation as described in point 6.2.

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