1976L0372 — EN — 19.08.1981 — 001.001 — 1
Thisdocumentismeantpurelyasadocumentationtoolandtheinstitutionsdonotassumeanyliabilityforitscontents
►B SEVENTHCOMMISSIONDIRECTIVE
of1March1976
establishingCommunitymethodsofanalysisfortheofficialcontroloffeedingstuffs
(76/372/EEC)
(OJ L 102, 15.4.1976, p. 8)
Amended by:
Official Journal
No page date
►M1Commission Directive 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
▼B
SEVENTHCOMMISSIONDIRECTIVE
of1March1976
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(76/372/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to the Council Directive of 20 July 1970 on the intro-
duction of Community methods of sampling and analysis for the
official control of feedingstuffs(1), aslas t amended by the Act of
Accession(2), and in particular Article 2 thereof,
Whereasthat Directive requiresthat official control of feedings tuffsbe
carried out using Community methods of sampling and analysis for thepurpose of checking compliance with requirements arising under theprovisions laid down by law, regulation or administrative action
concerning the quality and composition of feedingstuffs;
Whereas Commission Directives 71/250/EEC of 15 June 1971(
3), 71/
393/EEC of 18 November 1971(4), 72/199/EEC of 27 April 1972(5),
73/46/EEC of 5 December 1972(6), 74/203/EEC of 25 March 1974(7)
and 75/84/EEC of 20 December 1974(8) have already established a
number of Community methods of analysis; whereas the progress of
work since then makes it advisable to adopt a seventh set of methods;
Whereasthe meas uresprovided for in thisDirective are in accordance
with the opinion of the Standing Committee for Feedingstuffs,
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall require that analyses for official controls of
feedingstuffs, as regards their content of aflatoxin B1, be carried out in
accordance with the methodsdes cribed in the Annex to thisDirective.
Article 2
The Member Statess hall, not later than 1 October 1976, bring into
force the laws, regulations or administrative provisions necessary to
comply with this Directive. They shall forthwith notify the Commissionthereof.
Article 3
This Directive is addressed to the Member States.1976L0372 — EN — 19.08.1981 — 001.001 — 2
(1) OJ No L 170, 3. 8. 1970, p. 2.
(2) OJ No L 73, 27. 3. 1972, p. 14.
(3) OJ No L 155, 12. 7. 1971, p. 13.
(4) OJ No L 279, 20. 12. 1971, p. 7.
(5) OJ No L 123, 29. 5. 1972, p. 6.
(6) OJ No L 83, 30. 3. 1973, p. 21.
(7) OJ No L 108, 22. 4. 1974, p. 7.
(8) OJ No L 32, 5. 2. 1975, p. 26.▼M1
▼B
▼B
ANNEX
DETERMINATIONOFAFLATOXINB1
A. ONE-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD
1.Purposeandscope
The method makes it possible to determine the level of aflatoxin B1in the following feedingstuffs: groundnut, copra, linseed, soya,
sesame, babassu palm and maizegerm oil cakes, cereals and cereal
products, pea meal, potato pulp and starch. The lower limit of
determination is0·01 mg/kg (10 ppb).
If the presence of interfering substances hinders the determination,
it is necessary to start the analysis again using method B (two-
dimensional thin layer chromatography).
2.Principle
The sample is subjected to extraction with chloroform. The extract
isfiltered, and an aliquot portion taken and purified by column
chromatography on silica gel. The eluate is evaporated and theresidue redissolved in a specific volume of chloroform or of a
mixture of benzene and acetonitrile. An aliquot portion of thiss olu-
tion iss ubjected to thin-layer chromatography (TLC). The quantity
of aflatoxin B
1isdetermined under UV irradiation of the chromato-
gram, either visually or by fluorodensitometry, by comparison with
known quantitiesof s tandard aflatoxin B1. The identity of the afla-
toxin B1extracted from the feedingstuff must be confirmed by the
procedure indicated.
3.Reagents
NB:All the reagents must be of ‘analytical reagent’ quality unless
otherwise stated.
3.1. Acetone.
3.2. Chloroform, stabilized with 0·5 to 1·0 % of 96 % ethanol (v/v).3.3. N-hexane.3.4. Methanol.3.5. Anhydrousdiethyl ether, free from peroxides .
3.6. Mixture of benzene and acetonitrile: 98/2 (v/v).
3.7. Mixture of chloroform (3.2) and methanol (3.4): 97/3 (v/v).3.8. Silica gel, for column chromatography, particle size 0·05 to
0·20 mm.
3.9. Absorbent cotton wool, previously defatted with chloroform, or
glass wool.
3.10. Sodium sulphate, anhydrous, granular.3.11. Inert gas, e.g. nitrogen.3.12. 1 N Hydrochloric acid.
3.13. 50 % (v/v) sulphuric acid.
3.14. Kieselguhr (hyflosupercel), washed in acid.3.15. Silica gel G-HR or equivalent, for TLC.3.16. Standard solution with about 0·1 µg of aflatoxin B
1per ml in
chloroform (3.2) or the benzene/acetonitrile mixture (3.6), prepared
and checked asindicated in Section 7.
3.17. Standard solution for qualitative testing purposes containing about
0·1 µg of aflatoxin B1and B2per ml in chloroform (3.2) or the
benzene/acetonitrile mixture (3.6). These concentrations are given
as a guide. They must be adjusted so as to obtain the same intensity
of fluorescence for both aflatoxins.
3.18. Developing solvents:
3.18.1. Chloroform (3.2)/acetone (3.1): 9/1 (v/v), unsaturated tank;1976L0372 — EN — 19.08.1981 — 001.001 — 3
▼B
3.18.2. Diethyl ether (3.5)/methanol (3.4)/water: 96/3/1 (v/v/v), unsaturated
tank;
3.18.3. Diethyl ether (3.5)/methanol (3.4)/water: 94/4·5/1·5 (v/v/v), satu-
rated tank;
3.18.4. Chloroform (3.2)/methanol (3.4): 94/6 (v/v), saturated tank;
3.18.5. Chloroform (3.2)/methanol (3.4): 97/3 (v/v), saturated tank.
4.Apparatus
4.1. Grinder-mixer.
4.2. Shaking apparatusor magnetic s tirrer.
4.3. Fluted filter papers, Schleicher and Schüll No 588 or equivalent,
diameter: 24 cm
4.4. Glass tube for chromatography (internal diameter: 22 mm, length:
300 mm), with a PTFE cock and a 250-ml reservoir.
4.5. Rotary vacuum evaporator, with a 500-ml round-bottom flask.
4.6. 500-ml conical flasks, with ground-glass stoppers.
4.7. TLC apparatus.
4.8. Glass plates for TLC, 200 × 200 mm, prepared as follows (the
quantities indicated are sufficient to cover five plates). Put 30 g ofsilica gel G-HR (3.15) into a conical flask. Add 60 ml water,
stopper and shake for a minute. Spread the suspension on the plates
so as to obtain a uniform layer 0·25 mm thick. Leave to dry in the
air and then store in a desiccator containing silica gel. At the time
of use, activate the plates by keeping them in the oven at 110 ºC
for one hour.
Ready-to-use plates are suitable if they give results similar to those
obtained with the platesprepared asindicated above.
4.9. Long-wavelength (360 nm) UV lamp. The intensity of irradiation
must make it possible for a spot of 1 ng of aflatoxin B
1to be still
clearly distinguished on a TLC plate at a distance of 10 cm from
the lamp.
4.10. 10 ml graduated tubes with polyethylene stoppers.
4.11. UV spectrophotometer.
4.12. Fluorodensitometer (optional).
5.Procedure
5.1. Preparation of the sample (see under ‘Observations’, Part C, point
1).
Grind the sample so that the whole of it will pass through a sieve
with a 1-mm mesh (in accordance with recommendation ISO R
565).
5.2. Extraction
Put 50 g of ground, homogenized sample into a 500-ml conical
flask (4.6). Add 25 g of Kieselguhr (3.14), 25 ml of water and
250 ml of chloroform (3.2). Stopper the flask, shake or stir for 30
minuteswith the apparatus(4.2) and filter through a fluted filter
paper (4.3). Discard the first 10 ml of the filtrate and then collect
50 ml.
5.3. Column clean-up
Insert into the lower end of a chromatography tube (4.4) a cotton or
glass wool plug (3.9), fill two-thirds of the tube with chloroform
(3.2) and add 5 g of sodium sulphate (3.10).
Check that the upper surface of the sodium sulphate layer is flat,
then add 10 g of silica gel (3.8) in small portions. Stir carefully
after each addition to eliminate air bubbles. Leave to stand for 15
minutes and then carefully add 15 g of sodium sulphate (3.10). Let
the liquid fall until it is just above the upper surface of the sodium
sulphate layer.
Mix the 50 ml of extract collected in 5.2 with 100 ml of n-hexane
(3.3) and quantitatively transfer the mixture to the column. Let the
liquid fall until it is just above the upper surface of the sodium1976L0372 — EN — 19.08.1981 — 001.001 — 4
▼B
sulphate layer. Discard this washing. Then add 100 ml of diethy-
lether (3.5) and again allow it to fall to the upper surface of the
sodium sulphate layer. During these operations see that the rate of
flow is8 to 12 ml per minute and that the column doesnot run dry.
Discard the liquid that comes out. Then elute with 150 ml of the
chloroform/methanol mixture (3.7) and collect the whole of the
eluate.
Evaporate the latter almost to dryness at a temperature not
exceeding 50 ºC and under a stream of inert gas (3.11) with the
rotary evaporator (4.5). Quantitatively transfer the residue, using
chloroform (3.2) or the benzene-acetonitrile mixture (3.6), to a
10 ml graduated tube (4.10). Concentrate the solution under a
stream of inert gas (3.11) and then adjust the volume to 2 ml with
chloroform (3.2) or the benzene/acetonitrile mixture (3.6).
5.4. Thin-layer chromatography
Spot on a TLC plate (4.8), 2 cm from the lower edge and at inter-
vals of 2 cm, the volumes indicated below of the standard solution
and the extract:
— 10, 15, 20, 30 and 40 µl of the standard aflatoxin B1solution
(3.16);
— 10 µl of the extract obtained in 5.3 and, superimposed on the
same point , 20 µl of the standard solution (3.16);
— 10 and 20 µl of the extract obtained in 5.3.
Develop the chromatogram in the dark with one of the developing
solvents (3.18). The choice of the solvent must be made before-
hand, by depositing 25 µl of the qualitative standard solution
(3.17) on the plate and checking that, when developed, aflatoxin
B1and B2are completely separated.
Let the solvents evaporate in the dark and then irradiate with UV
light, placing the plate 10 cm from the lamp (4.9). The spots of
aflatoxin B1give a blue fluorescence.
5.5. Quantitative determinations
Determine either visually or by fluorodensitometry as indicated
below.
5.5.1. Visual measurements
Determine the quantity of aflatoxin B1in the extract by matching
the fluorescence intensity of the extract spots with that of one of
the standard solution spots. Interpolate if necessary. The fluores-
cence obtained by the superimposition of the extract on the
standard solution must be more intense than that of the 10 µl of
extract and there must not be more than one visible spot. If the
fluorescence intensity given by the 10 µl of extract is greater than
that of the 40 µl of standard solution, dilute the extract 10 or 100
timeswith chloroform (3.2) or the benzene/acetonitrile mixture
(3.6) before subjecting it again to thin-layer chromatography.
5.5.2. Measurements by fluorodensitometry
Measure the fluorescence intensity of the aflatoxin B1spots with
the fluorodensitometer (4.12) at an excitation wavelength of
365 nm and an emission wavelength of 443 nm. Determine the
quantity of aflatoxin B1in the extract spots by comparison of their
fluorescence intensities with that of the standard aflatoxin B1spots.
5.6. Confirmation of the identity of aflatoxin B1
Confirm the identity of the aflatoxin B1in the extract by the
processes indicated below.
5.6.1. Treatment with sulphuric acid
Spray sulphuric acid (3.13) on to the chromatogram obtained in 5.4.
The fluorescence of the aflatoxin B1spots must turn from blue to
yellow under UV irradiation.
5.6.2. Two-dimensional chromatography involving the forma-
tion of aflatoxin B1-hemiacetal (aflatoxin B2a)
NB:The operations described below must be carried out following
carefully the diagram in figure 3.
5.6.2.1. Application of the solutions1976L0372 — EN — 19.08.1981 — 001.001 — 5
▼B
Score two straight lines on the plate (4.8) parallel to two contig-
uous sides (6 cm in from each side) to limit migration of the
solvent fronts. Spot the following solutions on the plate using capil-
lary pipettes or microsyringes:
—on point A: a volume of purified extract of the sample, obtained
in 5.3, containing about 2·5 nm of aflatoxin B1;
—on points B and C: 25 µl of the standard solution (3.16).
5.6.2.2. Development
Develop the chromatogram in direction I, in the dark, using the
developing solvent (3.18.1) (1 cm layer in an unsaturated tank)
until the solvent front reaches the solvent limit line.
Remove the plate from the tank and allow to dry in the dark at
ambient temperature for five minutes. Then spray hydrochloric
acid (3.12) along a band 2·5 cm high, covering pointsA and B
(indicated by the hatched area in figure 3) until it darkens,
protecting the rest of the plate with a glass sheet. Allow to react
for 10 minutesin the dark and dry with a s tream of air at ambient
temperature.
Next, develop the chromatogram in direction II, in the dark, using
the developing solvent (3.18.1) (1 cm layer in an unsaturated tank)
until the solvent front reaches the solvent limit line. Remove the
plate from the tank and allow to dry at ambient temperature.
5.6.2.3. Interpretation of the chromatogram
Examine the chromatogram under UV light (4.9) and check for the
following features.
(a) Appearance of a blue fluorescent spot of aflatoxin B1origi-
nating from the standard solution applied at C (migration in
the direction I).
(b) Appearance of a blue fluorescent spot of unreacted (with the
hydrochloric acid) aflatoxin B1and a more intense blue fluores-
cent spot of aflatoxin B1-hemiacetal, both originating from the
standard solution applied at B (migration in direction II).
(c) Appearance of spots matching those described in (b), origi-
nating from the sample extract applied at A. The position of
these spots is defined first by the migration distance of the afla-
toxin B1from point A in direction I (the same as that travelled
by the standard applied at C), and then by the migration
distances from there in direction II of the aflatoxin B1-hemia-
cetal (same as those travelled by the standard applied at B).
The fluorescence intensities of hemiacetal spots originating
from the extract and from the standard applied at B should
match.
6.Calculationoftheresults
6.1. From the visual measurements
The content in microgramsof aflatoxin B1per kg of sample (ppb)
isgiven by the formula:
S/C1Y/C1V
W /C1X
in which:
Y and X are respectively the volumes in microlitres of the standard
solution of aflatoxin B1(3.16) and of the extract having a similar
intensity of fluorescence;
S = concentration in microgramsof aflatoxin B1per ml in the
standard solution (3.16);
V = final volume of the extract in microlitres, allowing for any
dilution that was necessary;
W = weight in grammes of the sample corresponding to the
volume of extract subjected to column clean-up.1976L0372 — EN — 19.08.1981 — 001.001 — 6
▼B
6.2. From the fluorodensitometric measurements
The content in microgramsof aflatoxin B1per kg of sample is
given by the formula:
S/C1V
W /C1Y
in which:
Y = volume in microlitresof the extract s potted on the plate (10
or 20 µl);
S = quantity in nanogramsof aflatoxin B1in the extract spot
(proportional to the value of Y taken), deduced from the
measurements;
V = final volume of the extract in microlitres, allowing for any
dilution that was necessary;
W = weight in grammes of the sample corresponding to the
volume of extract subjected to column clean-up.
7.Preparationandtestingofthestandardsolution(3.16)
7.1. Determination of the concentration of aflatoxinB1
Prepare a standard solution of aflatoxin B1in the chloroform (3.2)
or the benzene/acetonitrile mixture (3.6) with a concentration of 8
to 10 µg/ml. Determine the absorption spectrum between 330 and
370 nm with the aid of the spectrophotometer (4.11).
Measure the optical density (A) at 363 nm in the case of the
chloroform solution; or at 348 nm in the case of the solution in
the benzene/acetonitrile mixture.
Calculate the concentration in microgramsof aflatoxin B1per ml of
solution from the formulae below:
312 /C1A/C11000
20600for the chloroform solution;
312 /C1A/C11000
19800for the solution in the benzene/acetonitrile mixture.
Dilute asappropriate, away from day light, to obtain a working
standard solution with a concentration of aflatoxin B1of about
0·1 µg/ml. If kept in a refrigerator at 4 ºC, this solution is stable
for two weeks.
7.2. Testing of chromatographic purity
Spot on a plate (4.8) 5 µl of the standard solution of aflatoxin B1containing 8 to 10 µg/ml (7.1). Develop the chromatogram asindi-
cated in 5.4. In UV light the chromatogram should show only one
spot and no fluorescence must be perceptible in the original deposit
zone.
8.Repeatability
The difference between the results of two parallel determinations
carried out on the same sample by the same analyst should not
exceed:
— 25 % related to the highest result for contents of aflatoxin B1from 10 and up to 20 µg/kg;
— 5 µg, in absolute value, for contents greater than 20 and up to
50 µg/kg;
— 10 % related to the highest value for contents above 50 µg/kg.
9.Reproducibility
See under ‘Observations’, Part C, point 2.
B TWO-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD
1.Purposeandscope
The method makes it possible to determine the level of aflatoxin B1in feedingstuffs not falling within the scope of method A. The1976L0372 — EN — 19.08.1981 — 001.001 — 7
▼B
lower limit of determination is0·01 mg/kg (10 ppb). The method is
not applicable to feedingstuffs containing citrus pulp.
2.Principle
The sample is subjected to extraction with chloroform. The extract
isfiltered, and an aliquot portion taken and purified by column
chromatography on silica gel. The eluate is evaporated and the
residue redissolved in a specific volume of chloroform or of a
mixture of benzene and acetonitrile. An aliquot portion of thiss olu-
tion is subjected to two-dimensional thin-layer chromatography
(TLC). The quantity of aflatoxin B1isdetermined under UV irra-
diation of the chromatogram, either visually or by
fluorodensitometry, by comparison with known quantities of stan-
dard aflatoxin B1. The identity of the aflatoxin B1extracted from
the feedingstuff must be confirmed by the procedure indicated.
3.Reagents
NB:All the reagents must be of ‘analytical reagent’ quality unless
otherwise stated.
3.1. Acetone.
3.2. Chloroform, stabilized with 0·5 to 1 % of 96 % ethanol (v/v).3.3. N-hexane.
3.4. Methanol.
3.5. Anhydrousdiethyl ether, free from peroxides .
3.6. Mixture of benzene and acetonitrile: 98/2 (v/v).
3.7. Mixture of chloroform (3.2) and methanol (3.4): 97/3 (v/v).
3.8. Silica gel, for column chromatography, particle size 0·05 to
0·20 mm.
3.9. Absorbent cotton wool, previously defatted with chloroform, or
glass wool.
3.10. Sodium sulphate, anhydrous, granular.
3.11. Inert gas, e.g. nitrogen.
3.12. 1 n-hydrochloric acid.
3.13. Kieselguhr, (hyflosupercel), washed in acid.
3.14. Silica gel G-HR or equivalent, for TLC.
3.15. Developing solvents.
3.15.1. Diethyl ether (3.5)/methanol (3.4)/water: 94/4.5/1.5 (v/v/v), satu-
rated tank.
3.15.2. Chloroform (3.2)/acetone (3.1): 9/1 (v/v), unsaturated tank.
3.16. Standard solution with about 0·1 µg aflatoxin B
1per ml in chloro-
form (3.2) or the benzene/acetonitrile mixture (3.6), prepared and
checked asdes cribed in point 7 of method A.
4.Apparatus
See under point 4 of method A.
5.Procedure
5.1. Preparation of the sample
See under points5.1, 5.2 and
5.3 of method A./C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C1505.2. Extraction
5.3. Column clean-up
5.4. Two dimensional TLC
5.4.1. Application of the solutions (follow the diagram in figure 1)
Score two straight lines on a plate (4.8) parallel to two contiguous
sides (5 and 6 cm from each side respectively), to limit migration
of the solvent fronts. Spot the following solutions on the plate
using capillary pipettes or micro-syringes:
—on point A , 20 µl of the purified sample extract obtained in 5.3;1976L0372 — EN — 19.08.1981 — 001.001 — 8
▼B
—on point B , 20 µl of the standard solution (3.16);
—on point C , 10 µl of the standard solution (3.16);
—on point D , 20 µl of the standard solution (3.16);
—on point E , 40 µl of the standard solution (3.16).
Dry in a slow stream of air or inert gas (3.11). The spots obtained
must have a diameter of about 5 mm.
5.4.2. Development (follow the diagram in figure 1)
Develop the chromatogram in direction I, in the dark, using the
developing solvent (3.15.1) (1-cm layer in a saturated tank) until
the solvent front reaches the solvent limit line. Remove the plate
from the tank and allow to dry in the dark at ambient temperature
for 15 minutes.
Then develop the chromatogram in direction II, in the dark, using
the developing solvent (3.15.2) (1-cm layer in an unsaturated tank)
until the solvent front reaches the solvent limit line. Remove the
plate from the tank and allow to dry, in the dark, at ambient
temperature.
5.4.3. Interpretation of the chromatogram (follow the diagram in
figure 2)
Irradiate the chromatogram with UV light by placing the plate
10 cm from the (4.9). Locate the position of the blue fluorescent
spots B, C, D and E of the aflatoxin B1from the standard solution.
Project two imaginary lines passing through these spots and at right
angles to the development directions. The intersection P of these
linesisthe location in which to expect to find the aflatoxin B1spot originating from the sample extract applied at A (figure 1).However, the actual location of the aflatoxin B
1spot may be at a
point Q at the intersection of two imaginary straight lines forming
an angle of about 100º between them and passing through spots B
and C respectively. Determine the quantity of aflatoxin B1in the
sample extract as indicated in 5.5.
5.4.4. Supplementary chromatography
Score two straight lines on a new plate (4.8) parallel to two contig-
uous sides, as indicated in the diagram in figure 1, and apply on
point A (see figure 1) 20 µl of the purified sample extract obtained
in 5.3 and, superimposed on it, 20 µl of the standard solution
(3.16). Develop asindicated in 5.4.2. Irradiate the chromatogram
with UV light (4.9) and check that:
— the aflatoxin B1spots from the extract and the standard solution
are superimposed,
— the fluorescence of this spot is more intense than that of the
aflatoxin B1, spot developed at Q on the first plate.
5.5. Quantitative determinations
Determine either visually or by fluorodensitometry as indicated
below.
5.5.1. Visual measurements
Determine the quantity of aflatoxin B1in the extract by matching
the fluorescence intensity of the extract spot with that of spots C,
D and E of the standard solution. Interpolate if necessary. If the
fluorescence intensity given by the 20 µl of extractis greater than
that of the 40 µl of standard solution, dilute the extract 10 or 100
timeswith chloroform (3.2) or the benzene/acetonitrile mixture
(3.6) before subjecting it again to thin-layer chromatography.
5.5.2. Measurements by fluorodensitometry
Measure the fluorescence intensity of the aflatoxin B1spots with
the fluorodensitometer (4.12), using an excitation wavelength of
365 nm and an emission wavelength of 443 nm
Determine the quantity of aflatoxin B1in the extract spot by
comparison of its fluorescence intensity with that of spots C, D
and E of the standard solution.
5.6. Confirmation of the identity of aflatoxin B1
See under point 5.6 of method A.1976L0372 — EN — 19.08.1981 — 001.001 — 9
▼B
6.Calculationoftheresults
See under point 6 of method A.
7.Repeatability
See under point 8 of method A.
8.Reproducibility
See under ‘Observations’, Part C, point 2.
C. OBSERVATIONS CONCERNING METHODS A AND B
1.Defatting
Samplescontaining more than 5 % fatsmus t be defatted with light petroleum
(bp 40 to 60 ºC) after the preparation indicated in 5.1.
In such cases, the analytical results must be expressed in terms of the weight
of the non-defatted sample.
2.Reproducibilityoftheresults
The reproducibility of the results, i.e. the variation between the results
obtained by two or more laboratories on the same sample has been estimated
at:
± 50 % of the mean value for mean valuesof aflatoxin B1from 10 and up to
20 µg/kg;
± 10 µg/kg on the mean value for mean valuesgreater than 20 and up to
50 µg/kg;
± 20 % of the mean value for mean valuesabove 50 µg/kg.1976L0372 — EN — 19.08.1981 — 001.001 — 10
▼B
Appendix to Annex
Figure 1
Figure 21976L0372 — EN — 19.08.1981 — 001.001 — 11
▼B
Figure 31976L0372 — EN — 19.08.1981 — 001.001 — 12