Commission Directive 98/88/EC of 13 November 1998 establishing guidelines for the microscopic identification and estimation of constituents of animal origin for the official control of feedingstuffs (Text with EEA relevance)
Date: 1998-11-13
Institutions: European Commission
Subjects: analytical chemistry, animal nutrition, animal product, food inspection, health control, scientific apparatus

27. 11. 98

EN

Official Journal of the European Communities

L 318/45

COMMISSION DIRECTIVE 98/88/EC

of 13 November 1998

establishing guidelines for the microscopic identification and estimation of
constituents of animal origin for the official control of feedingstuffs

(Text with EEA relevance)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

by weight
animals other than pets;

for compound feedingstuffs

intended for

Having regard to the Treaty establishing the European
Community,

Having regard to the Council Directive 70/373/EEC of 20
July 1970 on the introduction of Community methods of
sampling and analysis for the official control of feeding-
stuffs (1), as last amended by the Act of Accession of
Austria, Finland and Sweden, and in particular Article 2
thereof,

Whereas Directive 70/373/EEC stipulates that official
controls of
feedingstuffs for the purpose of checking
compliance with the requirements arising under the laws,
regulations and administrative provisions governing their
quality and composition must be carried out using
Community sampling and analysis methods;

Whereas Commission Decision 94/381/EC of 27 June
1994 concerning certain protection measures with regard
to bovine spongiform encephalopathy and the feeding of
mammalian derived protein (2), as amended by Decision
95/60/EC (3) prohibits the feeding of protein derived from
all mammalian tissues to ruminants, with the exception of
certain animal products and by-products;

Whereas Commission Decision 91/516/EEC of
9
September 1991 establishing a list of ingredients whose
use is prohibited in compound feedingstuffs (4), as last
amended by Decision 97/582/EC (5), prohibits the use of
protein derived from mammalian tissue in compound
feedingstuffs for ruminants;

Whereas Council Directive 79/373/EEC of 2 April 1979
on the marketing of compound feedingstuffs (6), as last
amended by Commission Directive 97/47/EC (7), provides
in Article 5c that all
ingredients must be mentioned
where a declaration of the ingredients is provided and
that the listing of ingredients is subject to several rules,
inter alia, the listing of ingredients in descending order

(1) OJ L 170, 3. 8. 1970, p. 2.
(2) OJ L 172, 7. 7. 1994, p. 23.
(3) OJ L 55, 11. 3. 1995, p. 43.
(4) OJ L 281, 9. 10. 1991, p. 23.
(5) OJ L 237, 28. 8. 1997, p. 39.
(6) OJ L 86, 6. 4. 1979, p. 30.
(7) OJ L 211, 5. 8. 1997, p. 45.

Whereas Directive 97/47/EC amending the Annexes to
Council Directives 77/101/EEC (8), 79/373/EEC and 91/
357/EEC (9)
introduces appropriate labelling provisions
with regard to the prohibition of these products on their
use in ruminant feed;

Whereas Member States may have adopted more stringent
in accordance with Article 1(2) of Council
provisions,
Directive 90/667/EEC of 27 November 1990 laying down
the veterinary rules for the disposal and processing of
animal waste, for its placing on the market and for the
prevention of pathogens in feedstuffs of animal or fish
origin and amending Directive 90/425/EEC (10), as last
amended by the Act of Accession of Austria, Finland and
Sweden;

Whereas by microscopic examination the presence of
constituents of animal origin can be established; whereas
bones of terrestrial animals and bones of fish can be
distinguished by microscopic examination; whereas the
possibility of distinguishing, by microscopic examination,
the bones of mammalian origin from bones of poultry
origin depends on the experience of the analyst; whereas
the possibility of estimation of the quantity of constitu-
ents of animal origin depends also largely on the experi-
ence of
the analyst; whereas it may be appropriate
according to the progress of scientific and technological
knowledge,
to combine microscopic examination with
other methods of analysis; whereas the fixing of these
the microscopic examination do not
guidelines
exclude the use, instead or in addition, of methods of
analysis, other than microscopic examination, which have
been proved to be scientifically valid;

for

Whereas it is therefore advisable to lay down the provi-
sions concerning microscopic examination as guidelines;

Whereas the measures provided for in this Directive are
in accordance with the opinion of
the Standing
Committee on Feedingstuffs,

(8) OJ L 32, 3. 2. 1977, p. 1.
(9) OJ L 193, 17. 7. 1991, p. 34.
(10) OJ L 363, 27. 12. 1990, p. 51.

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Official Journal of the European Communities

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HAS ADOPTED THIS DIRECTIVE:

Article 2

Article 1

The Member States shall provide that where, with a view
to officially controlling the identification and/or estima-
tion of the amount of constituents of animal origin in
feedingstuffs, microscopic examination is carried out, it
shall be carried out using the guidelines set out in the
Annex hereto.

In accordance with the requirements posed by the
competent authorities to the analysis, point 7 ‘Calculation
and evaluation' of these guidelines are to be considered as
optional, provided that in the case where the estimation
of the quantity is carried out, the provisions laid down in
point 7 have to be followed.

The fixing of these guidelines, in respect of the procedure
for microscopic examination does not exclude the use,
instead or in addition, of methods of analysis, other than
microscopic examination, which have been scientifically
proved to be valid for the identification and/or estimation
of the amount of constituents of animal origin.

The Member States shall bring into force the laws, regula-
tions or administrative provisions necessary to comply
with the provisions of this Directive, not later than 1
September 1999. They shall forthwith notify the Commis-
sion thereof.

When Member States adopt these provisions, the provi-
sions shall contain a reference to this Directive or shall be
accompanied by such reference at the time of their offi-
cial publication. The procedure for such reference shall be
adopted by the Member States.

Article 3

This Directive shall enter into force on the 20th day
following its publication in the Official Journal of the
European Communities.

This Directive is addressed to the Member States.

Done at Brussels, 13 November 1998.

For the Commission

Franz FISCHLER

Member of the Commission

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Official Journal of the European Communities

L 318/47

ANNEX

Guidelines for the microscopic identification and estimation of constituents of animal origin in
feedstuffs

1.

Objective and field of application

These guidelines should be used where 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 case where the estimation of the quantity of animal constituents is carried out, the provisions
under point 7 of these guidelines have to be followed.

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 constitu-
ents 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.

4.

Reagents (2)

4.1.

Embedding agent

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

4.1.2. Paraffin oil

4.2. Concentrating agent

4.2.1. Tetrachloroethylene (density 1,62)

4.3.

Staining reagents

4.3.1. Bradford reagent

4.3.2. Iodine/potassium iodide solution

4.3.3. Millon reagent

4.3.4. Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml H2

O)

The reagents listed may be replaced by others which produce comparable results.

5.

Equipment and accessories

5.1. Analytical balance (accuracy of 0,001 g)

5.2. Material for grinding (rasp, mill, etc.)

5.3.

Sieve fitted with sieve mesh with square meshes of width 0,1 to 2 mm

5.4.

Stereomicroscope (up to 50Y magnification)

5.5. Compound microscope (up to 400Y magnification), transmitted light/polarised light

5.6.

Standard laboratory glassware

(1) OJ L 102, 15. 4. 1976, p. 1.
(2) The reagents listed are commercially available, if no other indication is given.

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6.

Procedure

At least 10 g of the sample should, if necessary, depending on the nature of the material be treated
(depelletarised or ground with care using the suitable grinding equipment) and then divided into two
representative parts, one of at least 5 g for the sieve fraction (6.1) and one of at least 2 g for the
concentrated sediment (6.2). Colouring with staining reagents (6.3) is recommended for the identifica-
tion.

6.1.

Identification of constituents of animal origin in the sieve fractions

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

The sieve fraction(s) ;0,5 mm (or a representative part of the fraction) is applied as a thin layer to a
suitable support and screened systematically under the stereomicroscope (5.4) at various magnifications
for constituents of animal origin.

Slides made with the sieve fraction(s) : 0,5 mm are screened systematically under the compound
microscope (5.5) at various magnifications for constituents of animal origin.

6.2.

Identification of constituents of animal origin from the concentrated sediment

At least 2 g (accurate to 0,001 g) of the sample are weighed into a test tube or a separating funnel and
treated with at least 15 ml of tetrachloroethylene (4.2.1). After the mixture has been stirred/shaken
repeatedly and left to stand for a sufficient time (at least one minute and no more than two to three
minutes), the sediment is separated off.

The sediment is dried in a fume cupboard and subsequently weighed (accurate to 0,001 g). The
weighing is only necessary in case an estimation is required. Examine the entire dried sediment or part
thereof for bone constituents under the stereomicroscope (5.4) and the compound microscope (5.5).

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):

Paraffin oil (4.1.2):

By carefully heating, cell structures can be seen more clearly
because starch grains gelatinise and unwanted cell contents are
removed.

Bone constituents can be well identified in this embedding agent
because most lacunae remain filled with air and appear as black
holes about 5 to 15 lm.

Bradford reagent (4.3.1):

Is used for the detection of protein (typical blue colour). Dilute
with water approx. 1:4.

Iodine/potassium iodide solution
(4.3.2):

Is used for the detection of starch (blue-violet colour) and protein
(yellow-orange colour). Dilution can be made if required.

Millon reagent (4.3.3):

By carefully heating, the bone constituents become pink.

Cystin reagent (4.3.4):

By carefully heating, cystin-containing constituents (hair, feathers,
etc.) become black-brown.

7.

Calculation and evaluation

In the case where the estimation of the quantity of animal constituents is carried out the provisions
under this point have to be followed.

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 in the microscopic slide by means of the typical
lacunae. The
proportion of constituents of animal origin in the sample material is estimated taking into considera-
tion:

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

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

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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

% fish bone and scale fragments

=

=

S(cid:253)c

W

S(cid:253)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
bone meals 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 the category and origin of fish meal,
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 (%)

=

Estimated content of constituents of fish products (%) =

S(cid:253)c

W(cid:253)f

(cid:253) 100

S(cid:253)d

W(cid:253)f

(cid:253) 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)).

8.

Expression of the result of the examination

The different cases could be reported in the following way:

8.1. As far as was discernible under the microscope, no constituents of animal origin (as defined in point 1)

were found in the submitted sample.

8.2. As far as was discernible under the microscope constituents of animal origin (1) were found in the

submitted sample.

In this case, the reporting of the result of the examination, if required, can be further specified as:

8.2.1. As far as was discernible under the microscope, small amounts of constituents of animal origin (1) were

found in the submitted sample.

8.2.2. Dependent on the experience of the analyst:

— either, as far as was discernible under the microscope, constituents of animal origin (1) were found in
the submitted sample. The content of bone fragments (fish/terrestrial animals — in the case of bone
fragments of terrestrial animals, eventually specified as bone fragments from poultry or mammal-
ians, see remark 9.3) is estimated in an order of magnitude of
. . .%, equal to . . .% of animal
constituents when calculated on the basis of . . .% bone in the animal constituents’ product (=
correction factor f used),

— or, as far as was discernible under the microscope, constituents of animal origin (1) were found in the

submitted sample in measurable quantities.

(1) The type of constituents found, for example bones (fish or terrestrial animals), meat constituents, etc. should be indi-

cated here.

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For the cases under point 8.2, 8.2.1 and 8.2.2, when 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 clause is not necessary in cases where the bone fragments from terrestrial animals have been
specified as bone fragments from poultry or mammalians (see remark 9.3).

9.

9.1.

Remarks

It is recommended in the case of many and big constituents in the concentrated sediment to sieve the
sediment into two fractions (i.e. use of a 320 lm sieve). The fraction with the big constituents can be
examined as a paraffin oil preparation under a stereomicroscope with transmitted light. The fraction
with the fine constituents must be examined under the compound microscope.

9.2.

The concentrated sediment obtained (6.2), can, if necessary, be divided further using a concentrating
agent with a greater density.

9.3. Dependent on the experience of the analyst, the distinction between constituents of mammalian or
poultry origin can be made, making use of specific histological features, by which this distinction can
be made.