1978L0633 — EN — 20.12.1983 — 002.001 — 1
Thisdocumentismeantpurelyasadocumentationtoolandtheinstitutionsdonotassumeanyliabilityforitscontents
►B EIGHTCOMMISSIONDIRECTIVE
of15June1978
establishingCommunitymethodsofanalysisfortheofficialcontroloffeedingstuffs
(78/633/EEC)
(OJ L 206, 29.7.1978, p. 43)
Amended by:
Official Journal
No page date
►M1Commission Directive 81/680/EEC of 30 July 1981 L 246 32 29.8.1981
►M2Commission Directive 84/4/EEC of 20 December 1983 L 15 28 18.1.1984
▼B
EIGHTCOMMISSIONDIRECTIVE
of15June1978
establishingCommunitymethodsofanalysisfortheofficialcontrol
offeedingstuffs
(78/633/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Directive 70/373/EEC of 20 July 1970 on the
introduction of Community methods of sampling and analysis for the
official control of feedingstuffs(1), aslas t amended by the Act of
Accession, 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(
2), 71/
393/EEC of 18 November 1971(3), 72/199/EEC of 27 April 1972(4),
73/46/EEC of 5 December 1972(5), 74/203/EEC of 25 March 1974(6),
75/84/EEC of 20 December 1974(7) and 76/372/EEC of 1 March
1976(8) have already established a number of Community methods of
analysis; whereas the progress of work since then makes it advisable to
adopt an eighth 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
1. Member States shall require that analyses for official controls of
feedingstuffs, as regards their content of zinc bacitracin, flavophospho-lipol, iron, copper, manganese and zinc, be carried out in accordancewith the methodsdes cribed in the Annex to thisDirective.
Article 2
Member States shall, on 1 January 1979, bring into force the laws,
regulations or administrative provisions necessary to comply with thisDirective. They shall forthwith notify the Commission thereof.
Article 3
This Directive is addressed to the Member States.1978L0633 — EN — 20.12.1983 — 002.001 — 2
(1) OJ No L 170, 3. 8. 1970, p. 2.
(2) OJ No L 155, 12. 7. 1971, p. 13.
(3) OJ No L 279, 20. 12. 1971, p. 7.
(4) OJ No L 123, 29. 5. 1972, p. 6.
(5) OJ No L 83, 30. 3. 1973, p. 21.
(6) OJ No L 108, 22. 4. 1974, p. 7.
(7) OJ No L 32, 5. 2. 1975, p. 26.
(8) OJ No L 102, 15. 4. 1976, p. 8.▼M1
▼B
▼B
ANNEX
1.DETERMINATIONOFZINCBACITRACIN
— by diffusion in an agar medium —
1. PURPOSE AND SCOPE
The method isfor the determination of zinc bacitracin in feedings tuffs
and premixes. The lower limit of determination is 5 mg/kg (5 ppm)(1).
2. PRINCIPLE
The sample is extracted at pH 2 with a mixture of methanol/water/
hydrochloric acid, and a sodium sulphide solution. The addition of
sodium sulphide is to precipitate any soluble copper salts that may
interfere with the assay. The extract is brought to pH 6,5, concentrated
(where necessary) and diluted. Its antibiotic activity is determined by
measuring the diffusion of zinc bacitracin in an agar medium inoculated
with Micrococcus luteus (flavus). Diffusion is shown by the formation
of zones of inhibition of the micro-organism. The diameter of these
zonesistaken to be in direct proportion to the logarithm of the anti-
biotic concentration over the range of antibiotic concentrations
employed.
3. MICRO-ORGANISM: MICROCOCCUS LUTEUS (FLAVUS) ATCC
10240
3.1.Maintenanceofstockculture
Inoculate tubescontaining s lopesof culture medium (4.1) with Micro-
coccusluteus(flavus ) and incubate for 24 hoursat 30 ºC. Store the
culture in a refrigerator at about 4 ºC. Reinoculate every two weeks.
3.2.Preparationofthebacterialsuspension (a)
Harvest the growth from a recently prepared agar slope (3.1) with 2 to
3 ml of sodium chloride solution (4.3). Use this suspension to inoculate
250 ml of culture medium (4.1) contained in a Roux flask and incubate
for 18 to 20 hours at 30 ºC. Harvest the growth in 25 ml of sodium
chloride solution (4.3) and mix. Dilute the suspension to 1/10 with
sodium chloride solution (4.3). The light transmission of the suspension
must be about 75 %, measured at 650 nm in a 1 cm cell against sodium
chloride solution (4.3). This suspension may be kept for one week at
about 4 ºC.
4. CULTURE MEDIA AND REAGENTS
4.1.Culturemedium (b)
Meat peptone 6,0 g
Tryptone 4,0 gYeast extract 3,0 gMeat extract 1,5 gGlucose 1,0 gAgar 10,0 to 20,0 gWater 1 000 mlpH 6,5 to 6,6 (after sterilization).
4.2.Assaymedium (
b)
Tryptone 10,0 g
Yeast extract 3,0 gMeat extract 1,5 gGlucose 1,0 gAgar 10,0 to 20,0 gTween 80 1 mlWater 1 000 mlpH 6,5 (after sterilization).1978L0633 — EN — 20.12.1983 — 002.001 — 3
(1) 1 mg feedingstuff grade zinc bacitracin is equivalent to 42 international units (i.u.).
(a) Other methods may be used provided that it has been established that they give similar
bacterial suspensions.
(b) Any commercial culture medium of similar composition and giving the same results
may be used.▼M2
▼M2
4.3. Sodium chloride solution 0,8 % (w/v): dissolve 8 g sodium chloride in
water and dilute to 1 000 ml; sterilize.
4.4. Mixture of methanol/water hydrochloric acid (4.6):
80/17,5/2,5 (v/v/v).
4.5.Phosphatebuffer,pH6,5:
Potassium hydrogen phosphate K2HPO422,15 g.
Potassium dihydrogen phosphate KH2PO427,85 g.
Water to 1 000 ml.
4.6. Hydrochloric acid (d: 1,18 to 1,19).4.7. Hydrochloric acid (0,1 M).4.8. Sodium hydroxide 1 M solution.
4.9. Sodium sulphide about 0,5 M solution.
4.10. Bromocresol purple solution 0,04 % (w/v): dissolve 0,1 g of bromo-
cresol purple in 18,5 ml of 0,01 M sodium hydroxide solution. Make
up the volume to 250 ml with water and mix.
4.11. Standard substance: zinc bacitracin of known activity (in i. u.).
5. STANDARD SOLUTIONS
Weigh out a quantity of standard zinc bacitracin (4.11) corresponding to
1 050 i.u. (according to the activity indicated). Add 5 ml of 0,1 M
hydrochloric acid (4.7) and leave to stand for 15 minutes. Add 30 ml
of water, adjust the pH to 4,5 with phosphate buffer (4.5) (about 4 ml),
make up to a volume of 50 ml with water and mix well (1 ml = 21 i.u.).
From this solution prepare by successive dilution with phosphate buffer
(4.5) the following solutions:
s
80,42 i.u./ml
s40,21 i.u./ml
s20,105 i.u./ml
s10,0525 i.u./ml
6. PREPARATION OF DIE EXTRACT AND ASSAY SOLUTIONS
6.1.Extraction
6.1.1. Premixes and mineral feeds
Weigh out a quantity of sample of 2,0 to 5,0 g, add 29,0 ml of the
mixture (4.4) and 1,0 ml of sodium sulphide solution (4.9) and shake
briefly. Check that the pH isabout 2. Shake for 10 minutes , add 30 ml
of phosphate buffer (4.5), shake for 15 minutes and centrifuge. Take a
suitable aliquot of the supernatant solution and adjust the pH to 6,5 bymeansof 1 M hydroxide s olution (4.8) with a pH-meter or with the
bromocresol purple solution (4.10) as indicator. Dilute with phosphate
buffer (4.5) to obtain an expected zinc bacitracin content of 0,42 i.u./
ml (= u
8).
6.1.2. Protein concentrates
Weigh out a quantity of sample of 10,0 g, add 49,0 ml of the mixture
(4.4) and 1,0 ml of sodium sulphide solution (4.9) and shake briefly.
Check that the pH isabout 2. Shake for 10 minutes . Add 50 ml of
phosphate buffer (4.5), shake for 15 minutes and centrifuge. Take a
suitable volume of the supernatant solution and adjust the pH to 6,5
by means of 1 M sodium hydroxide solution (4.8) with a pH-meter or
with the bromocresol purple solution (4.10) as indicator. Evaporate to
approximately half volume in a rotary evaporator at a temperature not
exceeding 35 ºC.
Dilute with phosphate buffer (4.5) to obtain an expected zinc bacitracin
content of 0,42 i.u./ml (= u8).
6.1.3. Other feeds
Weigh out a quantity of sample of 10,0 g (20,0 g for an expected zinc
bacitracin content of 5 mg/kg). Add a mixture of 24,0 ml of the mixture
(4.4) and 1,0 ml of sodium sulphide solution (4.9) and homogenize for
10 minutes. Add 25 ml of phosphate buffer (4,5), shake for 15 minutes
and centrifuge. Take 20 ml of the supernatant solution and adjust the
pH to 6.5 by means of 1 M sodium hydroxide solution (4.8) with a
pH-meter or with the bromocresol purple solution (4.10) as indicator.
Evaporate to about 4 ml in a rotary evaporator at a temperature not1978L0633 — EN — 20.12.1983 — 002.001 — 4
▼M2
exceeding 35 ºC. Dilute the residue with phosphate buffer (4.5) to
obtain an expected zinc bacitracin content of 0,42 i.u./ml (= u8).
6.2.Assaysolutions
From solution u8prepare solutions u4(expected content: 0,21 i.u./ml), u2(expected content: 0,105 i.u./ml) and u1(expected content: 0,0525 i.u./
ml) by means of successive dilution (1 + 1) with phosphate buffer (4.5).
7. ASSAY PROCEDURE
7.1.Inoculationoftheassaymedium
Inoculate the assay medium (4.2) with the bacterial suspension (3.2) at
about 50 ºC. By preliminary trials on plates with assay medium (4.2)
determine the quantity of bacterial suspension required to give the
largest and clearest zones of inhibition with the various concentrations
of zinc bacitracin.
7.2.Preparationoftheplates
Diffusion through agar is carried out in plates with the four concentra-
tions of the standard solution (s8,s4,s2and s1) and the four
concentrations of the assay solution (u8,u4,u2and u1). These four
concentrations of extract and standard must necessarily be placed in
each plate. To this effect, select plates big enough to allow at least
eight holeswith a diameter of 10 to 13 mm and not les sthan 30 mm
between centresto be made in the agar medium. The tes t may be
carried out on plates consisting of a sheet of glass with a faced alumi-
nium or plastic ring placed on top, 200 mm in diameter and 20 mm
high.
Pour into the platesa quantity of the medium (4.2) inoculated asin
point 7.1, to give a layer about 2 mm thick (60 ml for a plate of
200 mm diameter), Allow to set in a level position, bore the holes and
place in them exactly measured volumes of assay and standard solu-
tions(between 0,10 and 0,15 ml per hole, according to the diameter).
Apply each concentration at least four times so that each determination
iss ubject to an evaluation of 32 zonesof inhibition.
7.3.Incubation
Incubate the platesfor 16 to 18 hoursat 30 ± 2 ºC.
8. EVALUATION
Measure the diameter of the zones of inhibition to the nearest 0,1 mm.
Record the mean measurements for each concentration on semi-loga-
rithmic graph paper showing the logarithm of the concentrations in
relation to the diametersof the zonesof inhibition. Plot the bes t fit lines
of both the standard solution and the extract, for example as below:
Determine the ‘best fit’ point for the standard lowest level (SL) using
the formula:
(a) SL ¼7s1þ4s2þs4/C02s8
10
Determine the ‘best fit’ point for the standard highest level (SH) using
the formula:
(b) SH ¼7s8þ4s4þs2/C02s1
10
Similarly calculate the ‘best fit’ points for the extract lowest level (UL)
and the extract highest level (UH) by substituting u1,u2,u4and u8for s1,
s2,s4and s8in the above formulae.
Record the calculated SL and SH valueson the s ame graph paper and
join them to give the ‘best fit’ line for the standard solution. Similarly
record UL and UH and join them to give the ‘best fit’ line for the
extract.
In the absence of any interference the lines should be parallel. For prac-
tical purposes the lines can be considered parallel if the values
(SH—SL) and (UH—UL) do not differ by more than 10 % from their
mean value.1978L0633 — EN — 20.12.1983 — 002.001 — 5
▼M2
If the linesare found to be non-parallel either u1and s1or u8and s8may
be discarded and SL, SH, UL and UH calculated, using the alternative
formulae, to give alternative ‘best fit’ lines:
(a′)S L=5s1þ2s2/C0s4
6or5s2þ2s4/C0s8
6
(b′)S H=5s4þ2s2/C0s1
6or5s8þ2s4/C0s2
6
and similarly for UL and UH. The same criteria of parallelism should
be satisfied. The fact that the result has been calculated from three
levelss hould be noted on the final report.
When the linesare cons idered asbeing parallel, calculate the logarithm
(log A) of the relative activity (A) by meansof one of the following
formulae, depending upon whether three or four levelshave been us ed
for the assessment of parallelism.
For four levels
(c) log A ¼u1þu2þu4þu8/C0s1/C0s2/C0s4/C0s8 ðÞ /C2 0;602
u4þu8þs4þs8/C0u1/C0u2/C0s1/C0s2
For three levels
(d) log A ¼u1þu2þu4/C0s1/C0s2/C0s4 ðÞ /C2 0;401
u4þs4/C0u1/C0s1
or
(d′) log A ¼u2þu4þu8/C0s2/C0s4/C0s8 ðÞ /C2 0;401
u8þs8/C0u2/C0s2
Activity of sample extract = activity of relevant standard × A
(u8=s8×A )
If the relative activity isfound to be outs ide the range of 0,5 to 2,0,
then repeat the assay making appropriate adjustments to the extract
concentrations or, if this is not possible, to the standard solutions.
When the relative activity cannot be brought into the required range,
any result obtained must be considered as approximate and this should
be noted on the final report.
When the linesare cons idered asnot being parallel, repeat the determi-
nation. If parallelism is still not achieved, a satisfactory determination
hasnot been obtained.
Express the result in milligrams of zinc bacitracin per kilogram of
feedingstuff.
9. REPEATABILITY
The difference between the results of two determinations carried out on
the same sample by the same analyst should not exceed:
— 2 mg/kg, in absolute value, for contents of zinc bacitracin up to
10 mg/kg,
— 20 % related to the highest value for contents from 10 to 25 mg/kg,— 5 mg/kg, in absolute value, for contents of 25 to 50 mg/kg,
— 10 % related to the highest value for contents above 50 mg/kg.
2.DETERMINATIONOFFLAVOPHOSPHOLIPOLBYDIFFUSIONIN
ANAGARMEDIUM
1. PURPOSE AND SCOPE
The method isfor the determination of flavophos pholipol in feeding-
stuffs, concentrates and premixes. The lower limit of determination is
1 mg/kg (1 ppm).1978L0633 — EN — 20.12.1983 — 002.001 — 6
▼B
▼B
2. PRINCIPLE
The sample is extracted with diluted methanol by heating under reflux.
After centrifuging, the extract is purified (where necessary) by treat-
ment with ion exchange resins and diluted. Its antibiotic activity is
determined by measuring the diffusion of flavophospholipol in an agar
medium inoculated with Staphylococcus aureus. Diffusion is shown by
the formation of zonesof inhibition of the micro-organis m. Thediameter of these zones is taken to be in direct proportion to the loga-
rithm of the antibiotic concentration over the range of antibiotic
concentrationsemployed.
3. MICRO-ORGANISM: STAPHYLOCOCCUS AUREUS ATCC 6538 P
3.1.Maintenanceofstockculture
Inoculate Staphylococcusaureusonto agar s lopesof culture medium
(4.1). Incubate for 24 hoursat 37 ºC, s tore in a refrigerator at about
4 ºC and reinoculate every month onto agar slopes.
3.2.Preparationofthebacterialsuspension (
a)
Set aside two tubes containing the stock culture (3.1) and reinoculate
them weekly. Incubate for 24 hoursat 37 ºC and s tore in a refrigerator
at about 4 ºC.
24 hours before the assay, inoculate with this growth two to four tubes
containing slopes of culture medium (4.1). Incubate for 16 to 18 hours
at 37 ºC. Make a suspension of the growth in the sodium chloride solu-
tion (4.3). The light transmission of the suspension must be about 40 %,
measured at 578 nm in a 1 cm cell against sodium chloride solution
(4.3).
4. CULTURE MEDIA AND REAGENTS
4.1.Culturemedium (b)
Meat peptone 6·0 g
Tryptone 4·0 g
Yeast extract 3·0 g
Meat extract 1·5 gGlucose 1·0 g
Agar 15·0 g
Water 1 000 mlpH 6·5 (after sterilization)
4.2.Assaymedium
4.2.1. Base layer (
c)
Meat peptone 6·0 g
Yeast extract 3·0 g
Meat extract 1·5 g
Agar 10·0 gWater 1 000 ml
pH 6·5 (after sterilization)
4.2.2. Seed layer
Asfor point 4.1, with the addition of 2·0 g of s ilicone anti-foaming
emulsion(
d).
4.3. Sodium chloride solution 0·4 % (w/v): dissolve 4 g of sodium choride
a.p. in water and dilute to 1 000 ml; sterilize.
4.4. Methanol, pure.
4.5. Methanol 50 % (v/v): dilute 500 ml of methanol (4.4) with 500 ml of
water.
4.6. Methanol 80 % (v/v): dilute 800 ml of methanol (4.4) with 200 ml of
water.1978L0633 — EN — 20.12.1983 — 002.001 — 7
(a) Other methods may be used provided that it has been established that they give similar
bacterial suspensions.
(b) Any commercial culture medium of similar composition and giving the same results
may be used, e.g. oxoid antibiotic medium 1 (CM 327) with an addition of oxoid agar
No 3 (L 13).
(c) Any commercial culture medium of similar composition and giving the same results
may be used, e.g. oxoid antibiotic medium 2 (CM 335) with an addition of oxoid agarNo 3 (L 13).
(
d) e.g. SE 2 from Wacker Chemie GmbH, Munich.
▼B
4.7. Tris(hydroxymethyl) aminomethane a.p.
4.8. Potassium chloride methanolic solution 1·5 % (w/v): dissolve 1·5 g of
potassium chloride a.p. in 20 ml of water, make up the volume to
100 ml with methanol (4.4).
4.9. Cation exchanger: Dowex 50 W×8, 20 to 50 mesh, Na form (cat. Serva
No 41600) or equivalent.
4.10. Anion exchanger: Dowex 1×2, 50 to 100 mesh, Cl form (cat. Serva
No 41010) or equivalent. Before use, keep for 12 to 14 hours in 80 %
methanol (4.6).
4.11. Glass wool.4.12. pH indicator paper (pH 6·6 to 8·1).
4.13. Ascorbic acid.
4.14. Standard substance: flavophospholipol of known activity.
5. APPARATUS
5.1. Glass tube for chromatography, internal diameter: 9 mm, length: 150 to
200 mm, fitted with a stopcock at the narrowed part of the lower end
and a ground-glass joint (to connect with the dropping funnel (5.2)) at
the upper end.
5.2. Dropping funnel 250 ml, fitted with a stopcock and a ground-glass
joint.
5.3. 250 ml conical flask with ground-glass joint.5.4. Reflux condenser with ground-glass joint.
6. STANDARD SOLUTIONS
Dissolve an accurately weighed quantity of the standard substance
(4.14) in 50 % methanol (4.5) and dilute to give a stock solution
containing 100 µ g flavophospholipol per millilitre. Stored in stoppered
flasks at 4 ºC this solution is stable for up to two months.
From this stock solution prepare by successive dilution with 50 %
methanol (4.5) the following solutions:
S
80·2 µg/ml
S40·1 µg/ml
S20·05 µg/ml
S10·025µg/ml
7. PREPARATION OF THE EXTRACT
7.1.Extraction
7.1.1. Concentrates, premixes and mineral feeds
Weigh out a quantity of sample of 2·0 to 5·0 g and add about 150 mg
of ascorbic acid (4.13). Homogenize with 150 ml of 50 % methanol
(4.5) in a conical flask (5.3) and adjust the pH to 8·1 to 8·2 with about
400 mg of tris(hydroxymethyl)aminomethane (4.7). Check the pH with
indicator paper (4.12). Allow to stand for 15 minutes, then readjust the
pH to 8·1 to 8·2 with tris(hydroxymethyl)aminomethane (4.7) and boil
for 10 minutes under reflux (5.4) with constant stirring. Allow to cool,
centrifuge the mixture and decant the extract.
7.1.2. Other feeds
Weigh out a quantity of samle of 5·0 to 30·0 g containing at least 30 µ g
of flavophospholipol. Homegenize with 150 ml of 50 % methanol (4.5)
in a conical flask (5.3) and adjust the pH to 8·1 to 8·2 with about
400 mg of tris(hydroxymethyl)aminomethane (4.7). Check the pH with
indicator paper (4.12). Allow to stand for 15 minutes, then readjust the
pH to 8·1 to 8·2 with tris(hydroxymethyl)aminomethane (4.7) and boil
for 10 minutes under reflux (5.4) with constant stirring. Allow to cool,
centrifuge the mixture and decant the extract.
7.2.Purification (this stepmay be omitted for concentrates, premixes
andmineralfeeds)
Mix 110 ml of the extract with 11 g of the cation exchanger (4.9), boil
for one minute under reflux (5.4) with constant stirring. Separate the
cation exchanger by centrifugation or filtration. Mix 100 ml of the1978L0633 — EN — 20.12.1983 — 002.001 — 8
▼B
extract with 150 ml of methanol (4.4) and store the solution for 12 to
15 hoursat 4 ºC. Filter off the flocculent mas swhile cold.
Insert a glass wool plug (4.11) at the bottom end of a glass tube (5.1),
pour into the tube 5 ml of the anion exchanger (4.10) and wash the
column with 100 ml of 80 % methanol (4.6). Using the funnel (5.2),
transfer to the column a volume of filtrate of at least 100 ml which is
expected to contain 16 µ g of flavophospholipol (200 ml for a 30 g
sample of feedingstuff at 1 ppm). Where necessary, before application
to the column, dilute the filtrate with 80 % methanol (4.6) to obtain an
expected flavophospholipol content of 16 µ g/100 ml. Adjust the flow
rate to about 2 ml/minute. Discard the effluent. Then wash the column
with 50 ml of 80% methanol (4.6) and discard the effluent.
Elute the flavophospholipol with the methanolic solution of potassium
chloride (4.8) keeping the flow rate to about 2 ml/minute. Collect
50 ml of the eluate in a graduated flask, add 30 ml of water and mix.
This solution should have a flavophospholipol content of 0·2 µg/ml (=
U8).
7.3.Assaysolutions
Where necessary (i.e. when the purification step has been omitted),
dilute the extract obtained in point 7.1.1 with 50 % methanol (4.5) to
obtain an expected flavophospholipol content of 0·2 µ g/ml (= U8).
From solution U8prepare solutions U4(expected content: 0·1 µg/ml), U2(expected content: 0·05 µ g/ml) and U1(expected content: 0·025 µ g/ml)
by means of successive dilution (1 + 1) with 50 % methanol (4.5).
8. ASSAY PROCEDURE
8.1.Inoculationoftheassaymedium
Inoculate the assay medium (4.2.2) with the bacterial suspension (3.2)
at about 50 ºC. By preliminary trials on plates with assay medium
(4.2.2) determine the quantity of bacterial suspension required to give
the largest and clearest zones of inhibition with the various concentra-
tionsof flavophos pholipol (about 30 ml/litre).
8.2.Preparationoftheplates
Diffusion through agar is carried out in plates with the four concentra-
tions of the standard solution (S8,S4,S2,S1) and the four concentrations
of the assay solution (U8,U4,U2,U1). These four concentrations of
extract and standard must necessarily be placed in each plate. To thisef-
fect, select plates big enough to allow at least eight holes with a
diameter of 10 to 13 mm and not less than 30 mm between centres to
be made in the agar medium. The test may be carried out on plates
consisting of a sheet of glass with a faced aluminium or plastic ring
placed on top, 200 mm in diameter and 20 mm high.
Pour into the platesa quantity of the medium (4.2.1) to give a layer
about 1·5 mm thick (45 ml for a plate of 200 mm diameter). Allow to
set in a level position and then over-layer with a quantity of the
medium (4.2.2) inoculated asin point 8.1 to give a layer 1 mm thick
(30 ml for a plate of 200 mm diameter). Allow to set again in a level
position, bore the holes and place in them exactly measured volumes of
assay and standard solutions (between 0·10 and 0·15 ml per hole,
according to the diameter).
Apply each concentration at least four times so that each determination
iss ubject to an evaluation of 32 zonesof inhibition.
8.3.Incubation
Icubate the platesfor 16 to 18 hoursat 28 to 30 ºC.
9. EVALUATION
Measure the diameter of the zones of inhibition to the nearest 0·1 mm.
Record the mean measurements for each concentration on semi-loga-
rithmic graph paper showing the logarithm of the concentrations in
relation to the diametersof the zonesof inhibition. Plot the bes t fit lines
of both the standard solution and the extract, for example as below.
Determine the ‘best fit’ point for the standard lowest level (SL) using
the formula:
(a) SL ¼7S1þ4S2þS4/C02S8
101978L0633 — EN — 20.12.1983 — 002.001 — 9
▼B
(b) SH ¼7S8þ4S4þS2/C02S1
10
Similarly calculate the ‘best fit’ points for the extract lowest level (UL)
and the extract highest level (UH) by substituting U1,U2,U4and U8for
S1,S2,S4and S8in the above formulae.
Record the calculated SL and SH valueson the s ame graph paper and
join them to give the ‘best fit’ line for the standard solution. Similarly
record UL and UH and join them to give the ‘best fit’ line for the
extract.
In the absence of any interference the lines should be parallel. For prac-
tical purposes the lines can be considered parallel if the values (SH-SL)
and (UH-UL) do not differ by more than 10 % from their mean value.
If the linesare found to be non-parallel, either U1and S1or U8and S8may be discarded and SL, SH, UL and UH calculated, using the alter-
native formulae, to give alternative ‘best fit’ lines:
(a′)S L¼5S1þ2S2/C0S4
6or5S2þ2S4/C0S8
6
(b′)S H¼5S4þ2S2/C0S1
6or5S8þ2S4/C0S2
6
and similarly for UL and UH. The alternative best fit lines should be
checked for parallelism as before. The fact that the result has been
calculated from three levelss hould be noted on the final report.
When the lines are considered as being parallel, calculate the logarithm
of the relative activity (log. A) by meansof one of the following
formulae.
For four levels
(c) log :A¼U1þU2þU4þU8/C0S1/C0S2/C0S4/C0S8 ðÞ /C2 0/C1602
U4þU8þS4þS8/C0U1/C0U2/C0S1/C0S2
For three levels
(d) log :A¼U1þU2þU4/C0S1/C0S2/C0S4 ðÞ /C2 0/C1401
U4þS4/C0U1/C0S1
or
(d′) log :A¼U2þU4þU8/C0S2/C0S4/C0S8 ðÞ /C2 0/C1401
U8þS8/C0U2/C0S2
Real activity = supposed activity × relative activity.
When the lines are considered as not being parallel, repeat the determi-
nation. If parallelism is still not achieved, calculate the logarithm of the
relative activity (log. A) by meansof formula (c). The res ult obtained
must however be considered as approximate and this should be noted in
the final report.
10. REPEATABILITY
The difference between the results of two determinations carried out on
the same sample by the same analyst should not exceed:
0·5 mg/kg, in absolute value, for contents of flavophospholipol from 1
and up to 2 mg/kg;
25 % related to the highest value for contents greater than 2 and up to
10 mg/kg;
20 % related to the highest value for contents greater than 10 and up to
25 mg/kg;
5 mg/kg, in absolute value, for contents greater than 25 and up to
50 mg/kg;
10 % related to the highest value for contents above 50 mg/kg.1978L0633 — EN — 20.12.1983 — 002.001 — 10
▼B
3.DETERMINATION OF THE TRACE ELEMENTS IRON, COOPER,
MANGANESEANDZINC
1. PURPOSE AND SCOPE
The method makes it possible to determine the trace elements iron,
copper, manganese and zinc in feedingstuffs. The lower limits of deter-
mination are:
iron (Fe): 20 mg/kg
copper (Cu): 10 mg/kg
manganese (Mn): 20 mg/kg
zinc (Zn): 20 mg/kg
2. PRINCIPLE
The sample is brought into solution in hydrochloric acid after destruc-
tion of organic matter, if any. The elementsiron, copper, manganes e
and zinc are determined, after appropriate dilution, by atomic absorp-
tion spectrometry.
3. REAGENTS
IntroductorycommentsFor preparation of the reagents and analytical solutions use water free
from the cationsto be determined, obtained either by double dis tilling
water in a borosilicate glass or quartz still or by double treatment on
ion exchange resin.
The reagents must be of at least analytical grade (a.p.). Freedom from
the element to be determined must be checked in a blank experiment. If
necessary, the reagents must be further purified.
In place of the standard solutions decribed below, commercial standard
solutions may be used provided that they are guaranteed and have been
checked before use.
3.1. Hydrochloric acid a.p. (d:1·19).3.2. Hydrochloric acid a.p. (6 N).3.3. Hydrochloric acid a.p. (0·5 N).3.4. Hydrofluoric acid 38 to 40 % (v/v) having an iron content of less than
1 mg Fe/litre and a residue after evaporation of less than 10 mg (as
sulphate)/litre.
3.5. Sulphuric acid a.p. (d: 1·84).
3.6. Hydrogen peroxide a.p. (approximately 100 volumesof oxygen (30 %
by weight) ).
3.7. Standard iron solution (1 000 µg Fe/ml) prepared as follows: dissolve
1 g of iron wire a.p. in 200 ml of 6 N hydrochloric acid (3.2), add
16 ml of hydrogen peroxide (3.6) and make up to one litre with water.
3.7.1. Working standard iron solution (100 µg Fe/ml) prepared by diluting the
standard solution (3.7) 1 + 9 with water.
3.8. Standard copper solution (1 000 µg Cu/ml) prepared as follows:
dissolve 1 g of copper in powder form (a.p.) in 25 ml of 6 N hydro-
chloric acid (3.2), add 5 ml of hydrogen peroxide (3.6) and make up
to one litre with water.
3.8.1. Working standard copper solution (10 µg Cu/ml) prepared by diluting
the standard solution (3.8) 1 + 9 with water and then diluting the
resulting solution 1 + 9 with water.
3.9. Standard manganese solution (1 000 µg Mn/ml) prepared as follows:
dissolve 1 g of manganese in powder form (a.p.) in 25 ml of 6 N hydro-
chloric acid (3.2) and make up to one litre with water.
3.9.1. Working standard manganese solution (10 µg Mn/ml) prepared by
diluting the standard solution (3.9) 1 + 9 with water and then diluting
the resulting solution 1 + 9 with water.
3.10. Standard zinc solution (1 000 µg Zn/ml) prepared as follows: dissolve
1 g of zinc in strip or leaf form (a.p.) in 25 ml of 6 N hydrochloric
acid (3.2) and make up to one litre with water.1978L0633 — EN — 20.12.1983 — 002.001 — 11
▼B
3.10.1. Working standard zinc solution (10 µg Zn/ml) prepared by diluting the
standard solution (3.10) 1 + 9 with water and then diluting the resulting
solution 1 + 9 with water.
3.11. Lanthanum choride solution prepared as follows: dissolve 12 g of
lanthanum oxide in 150 ml of water, add 100 ml of 6 N hydrochloric
acid (3.2) and make up to one litre with water.
4. APPARATUS
4.1. Muffle furnace with temperature regulation and recorder.4.2. Glassware must be of resistant borosilicate type and it is recommended
to use apparatus which is reserved exclusively for trace element deter-
minations.
4.3. Platinum crucible and (optional) quartz crucible.4.4. Atomic absorption spectrophotometer meeting the requirements of the
method with regard to sensitivity and precision in the required range.
5. PROCEDURE
5.1.Samplescontainingorganicmatter
5.1.1. Ashing and preparation of the solution for analysis (*)
(i) Place 5 to 10 g of sample weighed to the nearest 0·2 mg in a quartz
or platinium crucible (4.3) (see Note (b)), dry in an oven at 105 ºC
and introduce the crucible into the cold muffle furnace (4.1). Close
the furnace (see Note (c)) and gradually raise the temperature to
450 to 475 ºC over about 90 minutes. Maintain this temperature
for 4 to 16 hours(e.g. overnight) to remove carbonaceousmaterial
and then open the furnace and allow to cool (see Note (d)).
Wash the crucible out with a total of about 5 ml of hydrochloric
acid (3.1) and add the latter slowly and carefully to the beaker
(there may be a vigorousreaction due to CO
2formation). Add
hydrochloric acid (3.1) dropwise with agitation until all efferves-
cence has stopped. Evaporate to dryness, occasionally stirring with
a glass rod.
Next add 15 ml of 6 N hydrochloric acid (3.2) to the residue
followed by about 120 ml of water. Stir with the glass rod, which
should be left in the beaker, and cover the beaker with a watch-
glass. Bring gently to the boil and maintain at boiling point until
no more ash can be seen to dissolve. Filter on ash-free filter paper
and collect the filtrate in a 250 ml volumetric flask. Wash the
beaker and filter with 5 ml of hot 6 N hydrochloric acid (3.2) and
twice with boiling water. Fill the volumetric flask up to the mark
with water (HCl concentration about 0·5 N).
(ii) If the residue in the filter appears black (carbon), put it back in the
furnace and ash again at 450 to 475 ºC. This ashing, which only
requiresa few hours(about three to five hours ), iscomplete when
the ash appears white or nearly white. Dissolve the residue with
about 2 ml of hydrochloric acid (3.1), evaporate to dryness and
add 5 ml of 6 N hydrochloric acid (3.2). Heat, filter the solution
into the volumetric flask and make up to the mark with water
(HCl concentration about 0·5 N).
Notes:
(a) In determining trace elementsit isimportant to be alert to the ris ks
of contamination, particularly by zinc, copper and iron. For this
reason, the equipment used in preparing the samples must be free
of these metals.1978L0633 — EN — 20.12.1983 — 002.001 — 12
(*) Green fodder (fresh or dried) is liable to contain large amounts of vegetable silica,
which may retain trace elements and must be removed. For samples of these feeding-stuffs, therefore, the following modified procedure must be followed.
Carry out operation 5.1.1 (i) asfar asthe filtration. Was h the filter paper containing
the insoluble residue twice with boiling water and place it in a platinum crucible (4.3).Ignite in the muffle furnace (4.1) at a temperature below 550 ºC until all carbonaceousmaterial hascompletely dis appeared. Allow to cool, add a few dropsof waterfollowed by 10 to 15 ml of hydrofluoric acid (3.4) and evaporate to dryness at about
150 ºC. If any silica remains in the residue, redissolve it in a few millilitres of hydro-
fluoric acid (3.4) and evaporate to dryness. Add five drops of sulphuric acid (3.5) andheat until no more white fumesare given off. After the addition of 5 ml of 6 N hydro-chloric acid (3.2) and about 30 ml of water, heat, filter the solution into the 250 mlvolumetric flask and make up to the mark with water (HCl concentration about 0·5 N).
Proceed then with the determination from point 5.1.3.
▼B
To reduce the general risk of contamination, work in a dust-free
atmosphere with scrupulously clean equipment and carefully
washed glassware. The determination of zinc is particularly sensi-
tive to many types of contamination, e.g. from glassware, reagents,
dust, etc.
(b) The weight of sample to be ashed is calculated from the approxi-
mate trace element content of the feedingstuff in relation to the
sensitivity of the spectrophotometer used. For certain feedingstuffs
low in trace elements it may be necessary to start with a 10 to 20 g
sample and make up the final solution to only 100 ml.
(c) Ashing must be carried out in a closed furnace without injection of
air or oxygen.
(d) The temperature indicated by the pyrometer must not exceed
475 ºC.
5.1.2. Spectrophotometric determination
5.1.2.1. Preparation of calibration solutions
For each of the elementsto be determined, prepare from the working
standard solutions given in points 3.7.1, 3.8.1, 3.9.1 and 3.10.1 a range
of calibration solutions, each calibration solution having an HCl
concentration of about 0·5 N and (in the cases of iron, manganese and
zinc) a lanthanum chloride concentration equivalent to 0·1 % La (w/v).
The trace element concentrations selected must lie within the range of
sensitivity of the spectrophotometer used. The tables below show, by
way of example, the compositions of typical ranges of calibration solu-
tions; depending, however, on the type and sensitivity of
spectrophotometer used it may be necessary to select other concentra-
tions.
Iron
µ g Fe/ml 0 0·5 1 2 3 4 5
ml working stan-
dard solution
(3.7.1)
(1 ml = 100 µg Fe) 0 0·5 1 2 3 4 5
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml
with water.
Copper
µ g Cu/ml 0 0·1 0·2 0·4 0·6 0·8 0·1
ml working stan-
dard solution
(3.8.1)
(1 ml = 10 µg Cu) 0 1 2 4 6 8 10
+ ml 6 N HCl (3.2) 8 8 8 8 8 8 8
Make up to 100 ml with water.
Manganese
µ g Mn/ml 0 0·1 0·2 0·4 0·6 0·8 1·0
ml working stan-
dard solution
(3.9.1)
(1 ml = 10 µ g
Mn)012468 1 0
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml lanthanum chloride solution (3.11) and make up to 100 ml with
water.1978L0633 — EN — 20.12.1983 — 002.001 — 13
▼B
Zinc
µg Zn/ml 0 0·05 0·1 0·2 0·4 0·6 0·8
ml working stan-
dard solution
(3.10.1)
(1 ml = 10 µg Zn) 0 0·5 1 2 4 6 8
+ ml 6 N HCl (3.2) 7 7 7 7 7 7 7
+ 10 ml of lanthanum chloride solution (3.11) and make up to 100 ml
with water.
5.1.2.2. Preparation of solution for analysis
For the determination of copper, the solution prepared from point 5.1.1
can normally be used directly. If necessary to bring its concentration
within the range of the calibration solutions, an aliquot portion may be
pipetted into a 100 ml volumetric flask and made up to the mark with
0·5 N hydrochloric acid (3.3).
For the determination of iron, manganese and zinc, pipette an aliquot
portion of the solution prepared from point 5.1.1 into a 100 ml volu-
metric flask, add 10 ml of lanthanum chloride solution (3.11) and
make up to the mark with 0·5 N hydrochloric acid (3.3) (see also point
8 ‘Observation’).
5.1.2.3. Blank experiment
The blank experiment must include all the prescribed steps of the
procedure except that the sample material is omitted.
The calibration solution ‘0’ must not be used as the blank.
5.1.2.4. Measurement of the atomic absorption
Measure the atomic absorption of the calibration solutions and of the
solution to be analyzed using an oxidizing air-acetylene flame at the
following wavelengths:
Fe: 248·3 nm
Cu: 324·8 nmMn: 279·5 nmZn: 213·8 nm
Carry out each measurement four times.
5.2.Mineralfeedingstuffs
If the sample contains no organic matter, prior ashing is unnecessary.
Proceed as described in point 5.1.1 (i) starting from the second para-
graph. Evaporation with hydrofluoric acid may be omitted.
6. CALCULATION OF RESULTS
Using a calibration curve, calculate the trace element concentration in
the solution to be analyzed and express the result in milligrams of trace
element per kilogram of sample (ppm).
7. REPEATABILITY
The difference between the results of zwo parallel determinations
carried out on the same sample by the same analyst should not exceed:
—5′mg/kg, in absolute value, for contents of the trace element
concerned up to 50 mg/kg;
— 10 % of the higher result for contents of the trace element
concerned from 50 and up to 100 mg/kg;
— 10 mg/kg, in absolute value, for contents of the trace element
concerned from 100 and up to 200 mg/kg;
— 5 % of the higher result for contents of the trace element concerned
above 200 mg/kg.
8. OBSERVATION
The presence of large quantities of phosphates may interfere with the
determination of iron, manganese and zinc. Such interference must be1978L0633 — EN — 20.12.1983 — 002.001 — 14
▼B
corrected by addition of lanthanum chloride solution (3.11). If,
however, in the sample the weight ratioCaþMg
P2, addition of
lanthanum chloride solution (3.11) to the solution for analysis and to
the calibration solutions may be omitted.1978L0633 — EN — 20.12.1983 — 002.001 — 15