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31971L0250

First Commission Directive 71/250/EEC of 15 June 1971 establishing Community methods of analysis for the official control of feeding-stuffs

Official Journal L 155 , 12/07/1971 P. 0013 - 0037 Finnish special edition: Chapter 3 Volume 3 P. 0213  Danish special edition: Series I Chapter 1971(II) P. 0423  Swedish special edition: Chapter 3 Volume 3 P. 0213  English special edition: Series I Chapter 1971(II) P. 0480  Greek special edition: Chapter 03 Volume 6 P. 0218  Spanish special edition: Chapter 03 Volume 5 P. 0003  Portuguese special edition Chapter 03 Volume 5 P. 0003

FIRST COMMISSION DIRECTIVE  of 15 June 1971  establishing Community methods of analysis for the official control of feeding-stuffs  (71/250/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 introduction of Community methods of sampling and analysis for the official control of feeding-stuffs, (1) and in particular Article 2 thereof;  Whereas that Directive requires that official controls of feeding-stuffs be carried out using Community methods of sampling and analysis for the purpose of checking compliance with requirements arising under the provisions laid down by law, regulation or administrative action concerning the quality and composition of feeding-stuffs;  Whereas all the necessary methods of analysis should be established as soon as possible ; whereas the establishment of methods for the determination of hydrocyanic acid, calcium, carbonates, crude ash, ash insoluble in HCl, chlorine from chlorides, mustard oil, lactose, potassium, sodium, sugars, theobromine and urea, and the determination of alkaloids in lupins and the estimation of the urease activity of products derived from soya are a first stage in this process;  Whereas the measures provided for in this Directive are in accordance with the Opinion of the Standing Committee for Feeding-stuffs;  HAS ADOPTED THIS DIRECTIVE:    Article 1 The Member States shall require that analyses for official controls of feeding-stuffs as regards their levels of hydrocyanic acid, calcium, carbonates, crude ash, ash insoluble in HCl chlorine from chlorides, mustard oil, lactose, potassium, sodium, sugars, theobromine and urea, and the determination of alkaloids in lupins and the estimation of the urease activity of products derived from soya be carried out using the methods set out in the Annex to this Directive.  (1)OJ No L 170, 3.8.1970, p. 2.    Article 2 The Member States shall, not later than 1 July 1972, bring into force the laws, regulations or administrative provisions necessary to comply with this Directive. They shall forthwith inform the Commission thereof.   Article 3 This Directive is addressed to the Member States.     Done at Brussels, 15 June 1971.  For the Commission  The President  Franco M. MALFATTI     ANNEX METHODS OF ANALYSIS OF THE COMPONENTS OF FEEDING-STUFFS     1. INTRODUCTION  Methods of analysis of the components of feeding-stuffs are generally applicable to all straight and compound feeding-stuffs. However certain feeding-stuffs, by reason of their inherent compositional characteristics, require their own individual methods of analysis. These cases have been provided for under the heading "observations" in the description of methods.  When two or more methods may be used to determine a component of a feeding-stuff, the choice of method shall, except where otherwise indicated, be left to the test laboratory concerned ; the method used must however be indicated in the certificate of analysis.  Preparation of the sample for analysis  It is essential for the chemical analysis to be carried out on a homogeneous sample. It should however be possible to carry out certain macroscopic or microscopic determinations and also the determination of moisture on the sample in the state in which it reaches the laboratory. In order to satisfy these two requirements, the sample shall be divided into two parts. One part shall be taken as it is ; the other shall be prepared as follows for chemical analysis.  Divide the sample by means of a mechanical apparatus or by hand after carefully mixing the whole of it on a clean, dry surface. In the latter case it is advisible to use the method of quartering which consists of taking samples in turn from two opposite sections. Finally, remove for analysis a portion weighing approximately 100 g and if necessary crush it so that the whole sample will pass through a sieve with a round 1 mm mesh. Transfer this sample immediately to a dry container with an air-tight fitting and seal it.  If the sample is very moist it must be pre-dried to bring the moisture content down to a level between 8 and 12 %. To do this, dry the sample at a suitable temperature for an adequate length of time.  Reagents and apparatus  Only special instruments or apparatus or those requiring special standards are mentioned in the description of the methods of analysis. It is not considered necessary to mention all the apparatus or instruments which form part of the everyday equipment of test laboratories.  Also, where water is mentioned in connection with diluting or washing this always means distilled water. Similarly, where a solution of a reagent is mentioned without any other indication, this means a solution in distilled water.  Expression of results  The result given in the certificate of analysis shall be the average value obtained on the basis of at least two tests. Subject to special provisions, it shall be expressed as a percentage of the original sample as it was when it reached the laboratory. The result must not be given to more significant figures than the accuracy of the method of analysis allows.        2. DETERMINATION OF HYDROCYANIC ACID      1. Purpose and scope  This method makes it possible to determine the level of hydrocyanic acid, free and combined in the form of glycosides, in feeding-stuffs and in particular in products derived from flax seed, manioc flour and certain species of beans.           2. Principle  The sample is suspended in water. The hydrocyanic acid is released by the action of enzymes, entrained by steam distillation and collected in a specific volume of acidified silver nitrate solution. The silver cyanide is separated by filtration and the excess silver nitrate is titrated with a solution of ammonium thiocyanate.       3. Reagents      3.1. A suspension of sweet almonds : crush twenty blanched sweet almonds in 100 ml of water at 37 to 40 ºC. Check that there is no hydrocyanic acid in 10 ml of the suspension using sodium picrote paper or by carrying out a blank test as described in the last paragraph of 5.           3.2. 10 % solution (w/v) of sodium acetate, neutral to phenolphthalein.           3.3. Anti-foaming emulsion (e.g. silicone).           3.4. Nitric acid, d : 1 740.           3.5. Silver nitrate solution : 0 702 N.           3.6. Ammonium thiocyanate solution : 0 702 N.           3.7. Saturated solution of ammonium ferric sulphate.           3.8. Ammonia, d : 0 7958.                  4. Apparatus      4.1. Oven with thermostat set at 38 ºC.           4.2. Apparatus for distillation by entrainment in steam fitted with a condenser with a curved extension piece.           4.3. 1000 ml flat-bottomed flasks with ground-glass stoppers.           4.4. Oil bath.           4.5. Burette graduated in 1/20 ml.                  5. Procedure  Weigh 20 g of the sample to the nearest 5 mg, place in 1 litre flat-bottomed flask and add 50 ml of water and 10 ml of sweet almond suspension (3.1.). Stopper the flask and transfer to the oven for sixteen hours at 38 ºC. Next, cool to room temperature and add 80 ml of water, 10 ml of sodium acetate solution (3.2) and a drop of anti-foaming emulsion (3.3).  Connect the flask to the steam distillation apparatus and place in an oilbath which has first been brought to a temperature slightly above 100 ºC. Distil 200 to 300 ml of liquid by passing a powerful current of steam through the flask and gently heating the oil-bath. Collect the distillate in an Erlenmeyer flask protected from the light and containing exactly 50 ml of silver nitrate solution 0 702 N (3.5) and 1 ml of nitric acid (3.4). Make sure that the condenser's extension piece is immersed in the silver nitrate solution.  Transfer the contents of the Erlenmeyer flask to a 500 ml volumetric flask, make up to volume with water, stir and filter. Remove 250 ml of the filtrate, add approximately 1 ml solution of ammonium ferric sulphate (3.7) and back-titrate the excess silver nitrate with the solution of ammonium thiocyanate 0 702 N (3.6) taken from the burette graduated in 1/20 ml.  A blank test may, if required, be carried out by applying the same procedure to 10 ml of sweet almond suspension (3.1), omitting the sample to be analysed.        6. Calculation of results  If the blank test indicates that silver nitrate solution 0.02 N has been consumed, subtract the value of this from the volume consumed by the distillate of the sample. 1 ml of AgNO2 0 702 N corresponds to 0 754 mg of HCN. Express the result as a percentage of the sample.       7. Observation  If the sample contains a large quantity of sulphides (e.g. beans), a black precipitate of silver sulphide is formed which is filtered together with the silver cyanide deposit. The formation of this precipitate causes a loss of silver nitrate solution 0 702 N, the volume of which must be subtracted from the volume used to calculate the HCN content. To do this, proceed as follows:  Treat the deposit left on the filter with 50 ml of ammonia (3.8) in order to dissolve the silver cyanide. Wash the residue in dilute ammonia and then determine its silver content. Convert the value obtained into ml of silver nitrate solution 0 702 N.  The sample's HCN content may also be determined by titrating the acidified ammoniacal filtrate with nitric acid.     3. DETERMINATION OF CALCIUM      1. Purpose and Scope  The method makes it possible to determine the total calcium content of feeding-stuffs.           2. Principle  The calcium is ashed, the ash treated with hydrochloric acid and the calcium precipitated as calcium oxalate. Dissolve the precipitate in sulphuric acid and titrate the oxalic acid which forms with a solution of potassium permangante.           3. Reagents  3.6.  3.1.Hydrochloric acid A.R., d : 1 714   3.2. Nitric acid A.R., d : 1 740   3.3. Sulphuric acid A.R., d : 1 713   3.4. Ammonia A.R., d : 0 798   3.5. Cold saturated solution of ammonium oxalate A.R.   3.6. 30 % solution (w/v) of citric acid A.R.   3.7. 5 % solution (w/v) of ammonium chloride A.R.   3.8. 0 704 % solution (w/v) of bromo-cresol green   3.9. Solution of potassium permanganate 0 71 N             4. Apparatus        4.1. Electric muffle-furnace with air circulation and thermostat.               4.2. Platinum, silica or porcelain crucibles for ashing.               4.3. Glass filter crucibles of G4 porosity.                           5. Procedure  Weigh approximately 5 g of the sample (or more if necessary) to the nearest mg, calcine at 550 ºC and transfer the ash to a 250 ml beaker.  Add 40 ml of hydrochloric acid (3.1), 60 ml of water and a few drops of nitric acid (3.2). Bring to the boil and keep at boiling point for thirty minutes. Cool and transfer the solution to a 250 ml volumetric flask. Rinse, bring the volume up to the mark with water, homogenise and filter.  Using a pipette, remove to a 250 ml beaker an aliquot amount containing 10 to 40 mg of calcium according to the assumed calcium content. Add 1 ml of citric acid solution (3.6) and 5 ml of ammonium chloride solution (3.7).  5. Make the volume up to approximately 100 ml with water. Bring to the boil, add eight to ten drops of bromo-cresol green solution (3.8) and 30 ml of a warm solution of ammonium oxalate (3.5). If a precipitate forms, dissolve it by adding a few drops of hydrochloric acid (3.1).  Neutralise very slowly with ammonia (3.4), stirring continuously until a pH reading of 4.4 to 4.6 is obtained (i.e. when the indicator changes colour). Place the beaker in a boiling water bath and keep there for thirty minutes to allow the precipitate which has formed to settle. Remove the beaker from the water bath. Leave it to stand for an hour and filter through a G4 filter crucible.  Wash the beaker and the crucible with water until the excess ammonium oxalate is completely removed (the absence of chloride in the washing water indicates that they have been sufficiently washed).  Dissolve the precipitate on the filter in 50 ml of warm sulphuric acid (3.3). Rinse the crucible with warm water and make the filtrate up to approximately 100 ml. Bring the temperature up to 70-80 ºC and titrate drop by drop with a solution of potassium permanganate (3.9) until a pink colour is obtained which lasts for one minute.           6. Calculation of results  1 ml of potassium permanganate 0 71 N corresponds to 2 7004 mg of calcium. Express the result obtained as a percentage of the sample.           7. Observations        7.1. For very low contents of calcium proceed as follows : filter the calcium oxalate precipitate through an ash-free filter paper. After washing, dry the filter and ash at 550 ºC in a platinum crucible. Redissolve the residue in a few drops of sulphuric acid (3.3), evaporate until dry, calcine again at 550 ºC and weigh. If W is the weight of the calcium sulphate obtained, the calcium content of the aliquot amount taken as a sample = W × 0 72944.               7.2. If the sample consists solely of mineral substances, dissolve in hydrochloric acid without ashing it first. In the case of products such as calcium-aluminium phosphate which are difficult to dissolve in acid, melt as follows by an alkaline process before dissolving ; mix the sample to be analysed thoroughly in a platinum crucible with a mixture five times its weight, consisting of equal amounts of potassium carbonate and sodium carbonate. Heat carefully until the mixture is completely melted. Cool, and dissolve in hydrochloric acid.               7.3. If the magnesium content of the sample is high, precipitate the calcium oxalate a second time.        4. DETERMINATION OF CARBONATES          1. Purpose and Scope  This method makes it possible to determine the amount of carbonates, conventionally expressed as calcium carbonate, in most feeding-stuffs.  However in certain cases (for example, with iron carbonate) a special method must be used.                   2. Principle  The carbonates are decomposed in hydrochloric acid ; the carbon dioxide released is collected in a graduated tube, and its volume compared with that released under the same conditions by a known quantity of calcium carbonate A.R.                   3. Reagents            3.1. Hydrochloric acid, d : 1 710.                       3.2. Calcium carbonate, A.R.                       3.3. Sulphuric acid, approximately 0 71 N, coloured with methyl red.                                          4. Apparatus  Scheibler-Dietrich apparatus (see diagram) or equivalent apparatus.                   5. Procedure  According to the sample's carbonate content, weigh a portion of the sample as shown below:  0 75 g for products containing from 50 to 100 % of carbonates, expressed as calcium carbonate;  1 g for products containing from 40 to 50 % of carbonates, expressed as calcium carbonate;  2 to 3 g for other products.  Place the portion of the sample in the special flask (4) of the apparatus, fitted with a small tube of unbreakable material containing 10 ml of hydrochloric acid (3.1), and connect the flask to the apparatus. Turn the three-way cock (5) so that the tube (1) connects with the outside. Using the mobile tube (2), which is filled with coloured sulphuric acid (3.3) and connected to the graduated tube (1), bring the level of the liquid up to the zero mark. Turn the cock (5) in order to connect up tubes (1) and (3) and check that the level is at zero.  Run the hydrochloric acid (3.1) slowly over the portion of the sample, tilting the flask (4). Make the pressure equal by lowering the tube (2). Shake the flask (4) until the release of carbon dioxide has stopped completely.  Restore pressure by bringing the liquid back to the same level in tubes (1) and (2). After a few minutes, when the volume of gas has become constant, take the reading.  Carry out a control test in the same conditions on 0 75 g of calcium carbonate (3.2).                   6. Calculation of results  The content in grammes of carbonates, expressed as calcium carbonate, as a percentage of the sample, is calculated by using the formula: >PIC FILE= "T0010555">   where:  V = ml of CO2 released by the portion of the sample.  T = ml of CO2 released by 0 75 g of CaCO2 A.R.  W = weight, in grammes, of the portion of the sample.                    7. Observations            7.1. When the portion of the sample weighs more than 2 g, first place 15 ml of distilled water in the flask (4) and mix before beginning the test. Use the same volume of water for the control test.                       7.2. If the apparatus used has a different volume from that of the Scheibler-Dietrich apparatus, the portions taken from the sample and from the control substance and the calculation of the results must be adapted accordingly.      >PIC FILE= "T0010556">    5. DETERMINATION OF CRUDE ASH              1. Purpose and Scope  This method makes it possible to determine the crude ash content of feeding-stuffs.                           2. Principle  The sample is ashed at 550 ºC ; the residue is weighed.                           3. Reagents  20 % solution (w/v) of ammonium nitrate.                           4. Apparatus                4.1. Hot-plate.                               4.2. Electric muffle-furnace with thermostat.                               4.3. Crucibles for ashing made of platinum or an alloy of platinum and gold (10 % Pt, 90 Au), either rectangular (60 × 40 × 25 mm) or circular (diameter : 60 to 75 mm, height : 20 to 25 mm).                                                          5. Procedure  Weigh out to the nearest mg approximately 5 g of the sample (2 75 in the case of products which have a tendency to swell) and place in a crucible for ashing which has first been calcined and tared. Place the crucible on the hot-plate and heat gradually until the substance carbonises. Put the crucible into the muffle-furnace set at 550 ºC ± 5 ºC. Keep at this temperature until white, light grey or reddish ash is obtained which appears to be free from carbonaceous particles. Place the crucible in a desiccator, leave to cool and weigh immediately.                           6. Calculation of results  Calculate the weight of the residue by deducting the tare.  Express the result as a percentage of the sample.                           7. Observations                7.1. The ash of substances which are difficult to ash must be subjected to an initial ashing of at least three hours, cooled and then a few drops of 20 % solution of ammonium nitrate added to it (carefully, to avoid dispersal of the ash or the formation of lumps). Continue calcining after drying in the oven. Repeat the operation as necessary until ashing is complete.                               7.2. In the case of substances resistant to the treatment described under 7.1, proceed as follows : after ashing for three hours, place the ash in warm water and filter through a small, ash-free filter. Ash the filter and its contents in the original crucible. Place the filtrate in the cooled crucible, evaporate until dry, ash and weigh.                               7.3. In the case of oils and fats, weigh accurately a sample of approximately 25 g in a suitably sized crucible. Carbonise by setting light to the substance with a strip of ash-free filter paper. After combustion, moisten with as little water as possible. Dry and ash as described under 5.          6. DETERMINATION OF ASH WHICH IS INSOLUBLE IN HYDROCHOLORIC ACID                  1. Purpose and Scope  This method makes it possible to determine the level in feeding-stuffs of mineral substances which are insoluble in hydrochloric acid. Two methods can be used, depending on the nature of the sample.                    1.1. Method A : applicable to straight organic feeding-stuffs and to most compound feeding-stuffs;                                       1.2. Method B : applicable to mineral compounds and mixtures and to compound feeding-stuffs whose content in substances insoluble in hydrochloric acid, as determined by Method A, is greater than 1 %.                                                                          2. Principle                    2.1. Method A : the sample is ashed, the ash boiled in hydrochloric acid and the insoluble residue filtered and weighed.                                       2.2. Method B : the sample is treated with hydrochloric acid. The solution is filtered, the residue ashed and the ash thus obtained treated in accordance with Method A.                                                                          3. Reagents                    3.1. Hydrochloric acid 3 N.                                       3.2. 20 % solution (w/v) of trichloroacetic acid.                                       3.3. 1 % solution (w/v) of trichloroacetic acid.                                                                          4. Apparatus                    4.1. Hot plate.                                       4.2. Electric muffle-furnace with thermostat.                                       4.3. Crucibles for ashing made of platinum or an alloy of platinum and gold (10 % Pt, 90 % Au), either rectangular (60 × 40 × 25 mm) or circular (diameter 60 to 75 mm, height : 20 to 25 mm).                                                                          5. Procedure                    5.1. Method A:  Ash the sample using the method described for the determination of crude ash. Ash obtained from that analysis may also be used.  Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid 3 N (3.1). Bring slowly to the boil and boil gently for fifteen minutes. Filter the warm solution through an ash-free filter paper and wash the residue with warm water until the acid reaction is no longer visible. Dry the filter containing the residue and ash in a tared crucible at a temperature of not less than 550 ºC and not more than 700 ºC. Cool in a desiccator and weigh.                                       5.2. Method B  Weigh 5 g of the sample to the nearest mg and place in a 250 to 400 ml beaker. Add 25 ml of water and 25 ml of hydrochloric acid 3 N (3.1) successively, mix and wait for effervescence to cease. Add a further 50 ml of hydrochloric acid 3 N (3.1). Wait for any release of gas to cease then place the beaker in a boiling water bath and keep it there for thirty minutes or longer, if necessary, in order to hydrolyse thoroughly any starch which may be present.   Filter while warm through an ash-free filter and wash the filter in 50 ml of warm water (see observation 7). Place the filter containing the residue in a crucible for ashing, dry and ash at a temperature of not less than 550 ºC and not more than 700 ºC. Place the ash in a 250 to 400 ml beaker using 75 ml of hydrochloric acid 3 N (3.1) ; continue as described in the second subparagraph of 5.1.                                                                          6. Calculation of results  Calculate the weight of the residue by deducting the tare. Express the result as a percentage of the sample.                                   7. Observation  If filtration proves difficult recommence the analysis, replacing the 50 ml of hydrochloric acid 3 N (3.1) by 50 ml of 20 % trichloroacetic acid (3.2) and washing the filter in a warm solution of 1 % trichloroacetic acid (3.3).       7. DETERMINATION OF CHLORINE FROM CHLORIDES                    1. Purpose and Scope  This method makes it possible to determine the amount of chlorine in chlorides which are soluble in water, conventionally expressed as sodium chloride. It is applicable to all feeding-stuffs.                                       2. Principle  The chlorides are dissolved in water. If the product contains organic matter it is clarified. The solution is slightly acidified with nitric acid and the chlorides precipitated in the form of silver chloride by means of a solution of silver nitrate. The excess silver nitrate is titrated with a solution of ammonium thiocyanate, by Volhard's method.                                       3. Reagents                      3.1. Solution of ammonium thiocyanate 0.1 N.                                           3.2. Solution of silver nitrate 0.1 N.                                           3.3. Saturated solution of ammonium ferric sulphate.                                           3.4. Nitric acid, d : 1.38.                                           3.5. Diethyl ether A.R.                                           3.6. Acetone A.R.                                           3.7. Carrez I solution : dissolve in water 21.9 g of zinc acetate, Zn (CH3 COO)2 72H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.                                           3.8. Carrez II solution : dissolve in water 10 76 g of potassium ferrocynaide K4 Fe (CN)6 73H2O. Make up to 100 ml with water.                                           3.9. Active carbon A.R., free from chlorides and not absorbing them.                                                                                4. Apparatus  Mixer (tumbler) : approximately 35 to 40 rpm.                                       5. Procedure                      5.1. Preparation of the solution  According to the nature of the sample, prepare a solution as shown under 5.1.1, 5.1.2 or 5.1.3.  At the same time carry out a blank test omitting the sample to be analysed.                         5.1.1. Samples free from organic matter  Weigh to the nearest mg a sample of not more than 10 g and containing not more than 3 g of chlorine in the form of chlorides. Place with 400 ml of water in a 500 ml volumetric flask at approximately 20 ºC. Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.                                               5.1.2. Samples containing organic matter, excluding the products listed under 5.1.3.  Weigh approximately 5 g of the sample to the nearest mg and place with 1 g of active carbon in a 500 ml volumetric flask. Add 400 ml of water at approximately 20 ºC and 5 ml of Carrez solution I (3.7), stir then add 5 ml of Carrez solution II (3.8). Mix for thirty minutes in the tumbler, bring up to volume, homogenise and filter.                                               5.1.3. Cooked feeding-stuffs, flax cakes and flour, products rich in flax flour and other products rich in mucilage or in colloidal substances (for example, dextrinated starch)  Prepare the solution as described under 5.1.2 but do not filter. Decant (if necessary centrifuge), remove 100 ml of the supernatant liquid and transfer to a 200 ml measuring flask. Mix with acetone (3.6) and bring up to volume with this solvent, homogenise and filter.                                                                                5.2. Titration  Using a pipette, transfer to an Erlenmayer flask from 25 ml to 100 ml of the filtrate (according to the assumed chlorine content) obtained as described under 5.1.1, 5.1.2 or 5.1.3. The aliquot portion must not contain more than 150 mg of chlorine (Cl). Dilute if necessary to not less than 50 ml with water, add 5 ml of nitric acid (3.4.), 20 ml of saturated solution of ammonium ferric sulphate (3.3) and two drops of ammonium thiocyanate solution (3.1) transferred by means of a burette filled up to the zero mark. Using a burette, transfer the silver nitrate solution (3.2) in such a way that an excess of 5 ml is obtained. Add 5 ml of diethyl ether (3.5) and shake hard to coagulate the precipitate.  Titrate the excess silver nitrate with the ammonium thiocyanate solution (3.1) until the reddish-brown tint has lasted for one minute.                                                                                6. Calculation of results  The amount of chlorine (w), expressed as sodium chloride, present in the volume of filtrate taken for titration is calculated by using the following formula:  W = 5 7845 (V1 - V2) mg  where:  V1 = ml of silver nitrate solution C 71 N added  V2 = ml of ammonium thiocyanate solution 0 71 N used for titration.  If the blank test indicates that silver nitrate solution 0 71 N has been consumed deduct this value from the volume (V1 - V2).                                       7. Observations                      7.1. Titration may also be carried out by potentiometry;                                           7.2. In the case of products which are very rich in oils and fats, first de-fat with diethyl ether or light petroleum;                                           7.3. In the case of fish-meal, titration may be carried out by Mohr's method.            8. DETERMINATION OF MUSTARD OIL      1. Purpose and scope  This method makes it possible to determine the amount of mustard oil contained in cakes made from the Brassica and Sinapis species, and in compound feeding-stuffs which contain cakes made from those species, and, expressed as allyl isothiocyanate, which can be steam entrained.           2. Principle  The sample is suspended in water. The mustard oil is released by the action of enzymes, entrained by distillation with ethanol and collected in dilute ammonia. The solution is treated while warm with a given volume of silver nitrate solution, then cooled and filtered. The excess silver nitrate is titrated with a solution of ammonium thiocyanate.           3. Reagents        3.1. White mustard (Sinapis alba).               3.2. 95 to 96 % ethanol (v/v).               3.3. Anti-foaming emulsion (e.g. silicone).               3.4. Ammonia, d : 0 7958.               3.5. Silver nitrate solution 0 71 N.               3.6. Ammonium thiocyanate solution 0 71 N. 3.7. Nitric acid, d : 1 740.               3.8. Saturated solution of ammonium ferric sulphate.                          4. Apparatus        4.1. Flat-bottomed 500 ml flasks with ground-glass stoppers.               4.2. Distilling apparatus fitted with a condenser and with equipment for preventing the entrainment of droplets.                          5. Procedure  Weigh 10 g of the sample to the nearest mg, place in a 500 ml flat-bottomed flask and add 2 g of finely ground white mustard (3.1) (an enzyme source) and 200 ml of water at 20 ºC. Stopper the flask and keep at 20 ºC for approximately 2 hours, shaking frequently. Add 40 ml of ethanol (3.2) and one drop of anti-foaming emulsion (3.3). Distil approximately 150 ml and collect the distillate in a 250 ml volumetric flask containing 20 ml of ammonia (3.4), making sure that the end of the condenser is immersed in the liquid. Add to the ammoniacal solution 50 ml of silver nitrate solution 0 71 N (3.5) (or more if necessary), place a small funnel over the volumetric flask and heat the mixture over a boiling water bath for one hour. Leave to cool, bring up to volume with water, stir and filter. Remove 100 ml of the clear filtrate, add 5 ml of nitric acid (3.7) and approximately 5 ml of ammonium ferric sulphate solution (3.8). Titrate the excess silver nitrate on return with the ammonium thiocyanate solution 0 71 N (3.6).  Carry out a blank test by applying the same procedure to 2 g of finely ground white mustard, omitting the sample for analysis.           6. Calculation of results  Subtract the volume of silver nitrate solution 0 71 N consumed in the blank test from that consumed by the sample in solution. The value obtained gives the number of ml of silver nitrate solution 0 71 N consumed by the mustard oil in the sample. 1 ml of AgNO3 0 71 N corresponds to 4 7956 mg of allyl isothiocyanate. Express the result as a percentage of the sample.                   9. DETERMINATION OF LACTOSE      1. Purpose and scope  This method makes it possible to determine the level of lactose in feeding-stuffs containing more than 0 75 % of lactose.           2. Principle  The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.           3. Reagents        3.1. Suspension of Saccharomyces cerevisiae : suspend 25 g of fresh yeast in 100 ml of water The suspension will keep for a maximum period of one week in a refrigerator.               3.2. Carrez solution I : dissolve in water 21 79 g of zinc acetate Zn (CH3 COO)2 72H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.               3.3. Carrez solution II : dissolve in water 10 76 g of potassium ferrocyanide K4 Fe (CN6) 73H2O. Make up to 100 ml with water.               3.4. Luff-Schoorl reagent:  Stirring carefully, pour the citric acid solution (3.4.2) into the sodium carbonate solution (3.4.2). Add the copper sulphate solution (3.4.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the normality of the reagent thus obtained (Cu 0 71 N ; Na2 CO3 2N). The solution's pH should be approximately 9 74.          3.4.1. Copper sulphate solution : dissolve 25 g of copper sulphate A.R. Cu SO4 75H2O, free from iron, in 100 ml of water.                   3.4.2. Citric acid solution : dissolve 50 g of citric acid A.R. C6H6O7 7H2O in 50 ml of water.                   3.4.3. Sodium carbonate solution : dissolve 143 78 g of anhydrous sodium carbonate A.R. in approximately 300 ml of warm water. Leave to cool.                                  3.5. Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.               3.6. 30 % solution (w/v) of sodium iodide.               3.7. Sulphuric acid 6 N.               3.8. Solution of sodium thiosulphate 0 71 N.               3.9. Starch solution : add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10 mg of mercuric iodide as a preservative.                          4. Apparatus  Water bath with thermostat set at 38-40 ºC.           5. Procedure  Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a 100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water bath for thirty minutes and then cool to approximately 35 ºC. Add 5 ml of yeast suspension (3.1) and homogenise. Leave the flask to stand for two hours in a water bath, at a temperature of 38-40 ºC. Cool to approximately 20 ºC.  Add 2 75 ml of Carrez solution I (3.2) and stir for thirty seconds, then add 2 75 ml of Carrez solution II (3.3) and again stir for thirty seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which  preferably contains from 40 to 80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.  Carry out a blank test in the same way with 5 ml of yeast suspension (3.1).  Determine the lactose content according to Luff-Schoorl, as follows : add exactly 25 ml of Luff-Schoorl reagent (3.4) and two granules of pumice stone (3.5). Stir by hand while heating over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:  Add 10 ml of potassium iodide solution (3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid 6 N (3.7). Titrate with sodium thiosulphate solution 0 71 N (3.8) until a dull yellow colour appears, add the starch indicator (3.9) and complete titration.  Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (3.4) and 25 ml of water, after adding 10 ml of potassium iodide solution (3.6) and 25 ml of sulphuric acid 6 N (3.7) without boiling.           6. Calculation of results  Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0 71 N.  Express the result as parts of anhydrous lactose as a percentage of the sample.           7. Observation  For products containing more than 40 % of fermentable sugar, use more than 5 ml of yeast suspension (3.1).    >PIC FILE= "T0010557">        10. DETERMINATION OF POTASSIUM      1. Purpose and scope  This method makes it possible to determine the level of potassium in feeding-stuffs.           2. Principle  The sample is ashed and the ash dissolved in hydrochloric acid. The sodium content of the solution is determined by flame photometry in the presence of caesium chloride and aluminium nitrate. The addition of these substances largely eliminates interference from disturbing elements.           3. Reagents        3.1. Hydrochloric acid A.R., d : 1.12.               3.2. Caesium chloride A.R.               3.3. Aluminium nitrate A 1 (No3)3 79 H2O, general purpose reagent.               3.4. Potassium chloride A.R., anhydrous.               3.5. Loading agent : dissolve in water 50 g of caesium chloride (3.2) and 250 g of aluminium nitrate (3.3), make up to 1 litre with water and homogenise. Store in plastic bottles.               3.6. Standard solution of potassium : dissolve in water 1 7907 g of potassium chloride (3.4), add 5 ml of hydrochloric acid (3.1), make up to 1 litre with water and homogenise. Store in plastic bottles. 1 ml of this solution contains 1 700 mg of potassium                          4. Apparatus        4.1. Platinum, silica or porcelain crucibles for ashing, provided if necessary with lids.               4.2. Electric muffle-furnace with thermostat.               4.3. Flame photometer.                          5. Procedure        5.1. Analysis of sample  As a general rule, weigh out 10 g of the sample to the nearest 10 mg, place in a crucible and ash at 450 ºC for three hours. After cooling transfer the ash quantitatively to a 500 ml graduated flask, using 250 to 300 ml of water and then 50 ml of hydrochloric acid (3.1). When all release of carbon dioxide has ceased, heat the solution and keep at a temperature of about 90 ºC for two hours, stirring occasionally. After cooling to room temperature, make up to the mark with water, shake and filter. Transfer to a 100 ml graduated flask an aliquot part of the filtrate containing a maximum of 1 70 mg of potassium, add 10 70 ml of loading agent (3.5), make up to the mark with water and homogenise. In the case of higher levels of potassium dilute the solution to be analysed in suitable proportions before adding the loading agent.  The table below is given as a guide for a sample of about 10 g. >PIC FILE= "T0010558">   Measure by flame photometry at a wavelength of 768 nm. Calculate the result by means of a calibration curve.                5.2. Calibration curve  Place exactly 10 ml of the standard solution (3.6) in a 250 ml graduated flask, make up to the mark with water and homogenise. Place in 100 ml graduated flasks exactly 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to amounts of potassium of 0 72, 0 74, 0 76, 0 78 and 1 70 mg. Complete the series with a blank flask containing no standard solution. Add 10 ml of loading agent (3.5) to each flask, make up to the mark with water and homogenise. Carry out the measurements as indicated in 5.1. The calibration curve is generally linear up to a potassium concentration of 1 mg in 100 ml of solution.                          6. Calculation of results  Express the result as a percentage of the sample.           7. Observations  It is not always necessary to add loading agent (3.5) in order to eliminate the interference of disturbing elements.                  11. DETERMINATION OF SODIUM      1. Purpose and scope  This method makes it possible to determine the level of sodium in feedings-stuffs.           2. Principle  The sample is ashed and the ash dissolved in hydrochloric acid. The sodium content of the solution is determined by flame photometry in the presence of caesium chloride and aluminium nitrate. The addition of these substances largely eliminates interference from disturbing elements.           3. Reagents        3.1. Hydrochloric acid A.R., d : 1.12.               3.2. Caesium chloride A.R.               3.3. Aluminium nitrate A 1 (NO3)3 79 H2O, general purpose reagent               3.4. Sodium chloride A.R., anhydrous.               3.5. Loading agent : dissolve in water 50 g of caesium chloride (3.2) and 250 g of aluminium nitrate (3.3), make up to 1 litre with water and homogenise. Store in plastic bottles.               3.6. Standard solution of sodium : dissolve in water 2 7542 g of sodium chloride (3.4), add 5 ml of hydrochloric acid (3.1), make up to 1 litre with water and homogenise Store in plastic bottles, 1 ml of this solution contains 1 700 mg of sodium.                          4. Apparatus        4.1. Platinum, silica or porcelain crucibles for ashing, provided if necessary with lids.               4.2. Electric muffle-furnace with thermostat.               4.3. Flame photometer.                          5. Procedure        5.1. Analysis of sample  As a general rule, weigh out 10 g of the sample to the nearest 10 mg, place in a crucible (4.2) and ash at 450 ºC for three hours. Avoid overheating (ignition). After cooling transfer the ash quantitatively to a 500 ml graduated flask, using 250 to 300 ml of water and then 50 ml of hydrochloric acid (3.1). When all release of carbon dioxide has ceased,  heat the solution and keep at a temperature of about 90 ºC for two hours, stirring occasionally. After cooling to room temperature, make up to the mark with water, shake and filter. Transfer to a 100 ml graduated flask an aliquot part of the filtrate containing a maximum of 1 70 mg of sodium, add 10 70 ml of loading agent (3.5), make up to the mark with water and homogenise. In the case of higher levels of sodium, dilute the solution to be analysed in suitable proportions before adding the loading agent.  The table below is given as a guide for a sample of about 10 g. >PIC FILE= "T0010559">   Measure by flame photometry at a wavelength of 589 nm. Calculate the result by means of a calibration curve.               5.2. Calibration curve  Place exactly 10 ml of the standard solution (3.6) in a 250 ml graduated flask, make up to the mark with water and homogenise. Place in 100 ml graduated flasks exactly 5, 10, 15, 20 and 25 ml of this solution, corresponding respectively to amounts of sodium of 0 72, 0 74, 0 76, 0 78 and 1 70 mg. Complete the series with a blank flask containing no standard solution. Add 10 ml of loading agent (3.5) to each flask, make up to the mark with water and homogenise. Carry out the measurements as indicated in 5.1. The calibration curve is generally linear up to a sodium concentration of 1 mg in 100 ml of solution.                          6. Calculation of results  Express the result as a percentage of the sample.           7. Observations        7.1. For products containing more than 4 % of sodium, it is preferable to ash the substance for two hours in a crucible with a lid. After cooling, add water, bring the ash into suspension by means of a platinum wire, dry and ash again for two hours in the crucible with a lid.               7.2. If the sample consists solely of mineral substances, dissolve without prior ashing.                                 12. DETERMINATION OF SUGAR      1. Purpose and scope  This method makes it possible to determine the amount of reducing sugars and total sugars after inversion, expressed as glucose or where appropriate as sucrose, converting by the factor 0 795. It is applicable to compound feeding-stuffs. Special methods are provided for other feeding-stuffs. Where necessary, lactose should be measured separately and taken into account when calculating the results.            2. Principle  The sugars are extracted in dilute ethanol ; the solution is clarified with Carrez solutions I and II. After eliminating the ethanol, the quantities before and after inversion are determined by the Luff-Schoorl method.           3. Reagents        3.1. 40 % ethanol (v/v) d : 0 7948 at 20 ºC, neutralised to phenolphthalein.               3.2. Carrez solution I : dissolve in water 21 79 g of zinc acetate Zn (CH3 COO)2 72H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.               3.3. Carrez solution II : dissolve in water 10 76 g of potassium ferrocyanide K4Fe (CN6) 73H2O. Make up to 100 ml with water.     3.4. 0 71 % solution (w/v) of methyl orange.               3.5. Hydrochloric acid 4 N.               3.6. Hydrochloric acid 0 71 N.               3.7. Sodium hydroxide solution 0 71 N.               3.8. Luff-Schoorl reagent:  Stirring carefully, pour the citric acid solution (3.8.2) into the sodium carbonate solution (3.8.3). Add the copper sulphate solution (3.8.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the normality of the reagent thus obtained (Cu 0 71 N ; Na2 CO3 2 N). The solution's pH should be approximately 9 74.          3.8.1. Copper sulphate solution : dissolve 25 g of copper sulphate A.R., Cu SO4 75H2O, free from iron, in 100 ml of water.                   3.8.2. Citric acid solution : dissolve 50 g of citric acid A.R., C6H3O7 7H2O in 50 ml of water.                   3.8.3. Sodium carbonate solution : dissolve 143 78 g of anhydrous sodium carbonate A.R. in approximately 300 ml of warm water. Leave to cool.                                  3.9. Sodium thiosulphate solution 0 71 N.               3.10. Starch solution : add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool and if necessary add 10 mg of mercuric iodide as a preservative.               3.11. Sulphuric acid 6 N.     3.12. 30 % solution (w/v) of potassium iodide.               3.13. Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.               3.14. 3-methylbutan-l-ol.                          4. Apparatus  Mixer (tumbler) : approximately 35 to 40 rpm.           5. Procedure        5.1. Extraction of sample  Weigh 2 75 g of the sample to the nearest mg and place in a 250 ml volumetric flask. Add 200 ml of ethanol (3.1) and mix in the tumbler for one hour. Add 5 ml of Carrez solution I (3.2) and stir for one minute. Add 5 ml of Carrez solution II (3.3) and again stir for one minute. Make up to volume with ethanol (3.1), homogenise and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume in order to eliminate most of the ethanol. Transfer the evaporation residue quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to volume with water, homogenise and filter if necessary. This solution will be used to determine the amount of reducing sugars and, after inversion, of total sugars.                5.2. Determination of reducing sugars  Using a pipette, remove not more than 25 ml of the solution containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage content of glucose in the sample.               5.3. Determination of total sugars after inversion  Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric flask. Add a few drops of methyl orange solution (3.4) then, carefully and stirring continuously, add hydrochloric acid 4 N (3.5) until the liquid turns a definite red. Add 15 ml of hydrochloric acid 0 71 N (3.6), immerse the flask in a fast boiling water bath and keep there for thirty minutes. Cool rapidly to approximately 20 ºC and add 15 ml of sodium hydroxide solution 0 71 N (3.7). Make up to 100 ml with water and homogenise. Remove not more than 25 ml containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage of glucose or, where appropriate, sucrose, by multiplying by the factor 0 795.               5.4. Titration by the Luff-Schoorl method  Using a pipette, take 25 ml of Luff-Schoorl reagent (3.8) and transfer to a 300 ml Erlenmeyer flask ; add exactly 25 ml of the clarified sugar solution. Add 2 granules of pumice stone (3.13), heat, stirring by hand, over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask, Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:  Add 10 ml of potassium iodide solution (3.12) and immediately afterwards (carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid 6 N (3.11). Titrate with sodium thiosulphate solution 0 71 N (3.9) until a dull yellow colour appears, add the starch indicator (3.10) and complete titration.  Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (3.8) and 25 ml of water, after adding 10 ml of potassium iodide solution (3.12) and 25 ml of sulphuric acid 6 N (3.11) without boiling.                          6. Calculation of results  Using the table establish the amount of glucose in mg which corresponds to the difference between the values of the two titrations, expressed in mg of sodium thiosulphate 0 71 N.  Express the result as a percentage of the sample.           7. Special procedures        7.1. In the case of feeding-stuffs which are rich in molasses and other feeding-stuffs which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using Carrez 1 (3.2) and II (3.3) reagents as described under 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80 % ethanol (v/v).  Homogenise and filter. Eliminate the ethanol as described under 5.1. If there is no dextrinised starch, bring up to volume with distilled water.               7.2. In the case of molasses and straight feeding-stuffs which are rich in sugar and almost starch-free (carobs, dried beetroot cossettes etc.), weigh out 5 g, place in a 250 ml volumetric flask, add 200 ml of distilled water and mix in the tumbler for one hour, or more if necessary. Clarify using Carrez I (3.2) and II (3.3) reagents as described under 5.1. Bring up to volume with cold water, homogenise and filter. In order to determine the amount of total sugars, continue as described under 5.3.                           8. Observations        8.1. In order to prevent foaming it is advisable to add (irrespective of the volume) approximately 1 ml of 3-methylbutan-l-ol (3.14) before boiling with Luff-Schoorl reagent.               8.2. The difference between the content of total sugars after inversion, expressed as glucose, and the content of reducing sugars, expressed as glucose, multiplied by 0 795, gives the percentage content of sucrose.               8.3. In order to determine the content of reducing sugars, excluding lactose, two methods may be adopted:          8.3.1. for an approximate calculation, multiply by 0 7675 the lactose content established by a different method of analysis and subtact the result obtained from the content of reducing sugars.                   8.3.2. For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying).    In both cases the amount of sugar present is determined by the Luff-Schoorl method and calculated in mg of glucose. One of the values is subtracted from the other and the difference is expressed as a percentage of the sample.  Example  The two volumes taken correspond, for each determination, to a sample of 250 mg.  In the first case 17 ml of sodium thiosulphate solution 0 71 N corresponding to 44 72 mg of glucose is consumed ; in the second, 11 ml, corresponding to 27 76 mg of glucose.  The difference is 16 76 mg of glucose.  The content of reducing sugars (excluding lactose), calculated as glucose, is therefore: >PIC FILE= "T0010560">       >PIC FILE= "T0010561">        13. DETERMINATION OF THEOBROMINE      1. Purpose and scope  This method makes it possible to determine the amount of theobromine in the by-products of the processing of cocoa beans.           2. Principle  The theobromine is extracted with chloroform. The extract is evaporated until dry, dissolved in water and treated with a specific quantity of silver nitrate solution. The nitric acid liberated is titrated with a solution of sodium hydroxide.           3. Reagents        3.1. Chloroform A.R.               3.2. Ammonia, d : 0 7958.               3.3. Sodium sulphate A.R., anhydrous.               3.4. Solution of sodium hydroxide 0 71 N.               3.5. Solution of silver nitrate 0 71 N.               3.6. 1 % (w/v) ethanolic solution of phenol red.               3.7. Light petroleum, B.P. 40-60 ºC.                          4. Apparatus  Flat-bottomed 500 ml flasks with ground-glass stoppers.           5. Procedure  Weigh to the nearest mg a sample of not more than 10 g containing not more than 80 mg of theobromine, place in a 500 ml flat-bottomed flask with a ground-glass stopper and add 270 ml of chloroform (3.1) and 10 ml of ammonia (3.2). Stopper the flask and shake vigorously for five minutes. Add 12 g of anhydrous sodium sulphate (3.3), shake again and leave to settle until the following day. Filter into a 500 ml Erlenmeyer and wash the residue with 100 ml of chloroform (3.1). Distil the solvent and eliminate the last traces over a boiling water bath. Redissolve the extract in 50 ml of water and bring to the boil.  Cool, neutralise exactly with the sodium hydroxide solution (3.4) using 0 75 ml of phenol red solution (3.6). Add 20 ml of silver nitrate solution (3.5). Titrate the nitric acid liberated with a solution of sodium hydroxide (3.4) until the indicator changes colour (pH 7 74).           6. Calculation of results  1 ml of 0 71 N NaOH = 18 mg of theobromine.  Express the result as a percentage of the sample.           7. Observations  Products containing more than 8 % of crude fatty matter must first be defatted by a six-hour extraction with light petroleum (B-P. 40-60 ºC).                   14. DETERMINATION OF UREA      1. Purpose and scope  This method makes it possible to determine the level of urea in feeding-stuffs.           2. Principle  The sample is suspended in water with a clarifying agent. The suspension is filtered. The urea content of the filtrate is determined after the addition of 4-dimethylaminobenzaldehyde (4-DMAB) by measuring the optical density at a wavelength of 420 nm,           3. Reagents        3.1. Solution of 4-dimethylaminobenzaldehyde : dissolve 1 76 g of 4-DMAB A.R. in 100 ml of 96 % ethanol and add 10 ml of hydrochloric acid A.R. (d : 1 719). This reagent keeps for a maximum period of two weeks.               3.2. Carrez solution I : dissolve in water 21 79 g of zinc acetate (CH2COO) 72H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.               3.3. Carrez solution II : dissolve in water 10 76 of potassium ferrocyanide, K4 Fe (CN)6 73H2O. Make up to 100 ml with water.               3.4. Active carbon A.R. which does not absorb urea (to be checked).               3.5. 0 71 % solution (w/v) of urea A.R.                          4. Apparatus        4.1. Mixer (tumbler) : approximately 35 to 40 rpm.               4.2. Test tubes : 160 × 16 mm with ground-glass stoppers.               4.3. A spectrophotometer.                          5. Procedure        5.1. Analysis of sample  Weigh out 2 g of the sample to the nearest mg and place with 1 g of active carbon (3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solutions I (3.2) and II (3.3). Mix for thirty minutes in the tumbler. Make up to volume with water, shake and filter.  Remove 5 ml of the transparent colourless filtrates, place in test tubes with ground-glass stoppers, add 5 ml of 4-DMAB solution (3.1) and mix. Place the tubes in a hot-water bath at 20 ºC. After fifteen minutes measure the optical density of the sample solution with the spectrophotometer at 420 nm. Compare with the blank test solution of the reagents.               5.2. Calibration curve  Remove volumes of 1, 2, 4, 5 and 10 ml of the urea solution (3.5), place in 100 ml volumetric flasks and make up the volume with water. Remove 5 ml from each solution, add 5 ml of 4-DMAB solution (3.1) to each of them, homogenise and measure the optical density as shown above in comparison with a control solution containing 5 ml of 4-DMAB and 5 ml of water free from urea. Plot the calibration curve.                          6. Calculation of results  Determine the amount of urea in the sample using the calibration curve.  Express the result as a percentage of the sample.            7. Observations        7.1. In the case of contents of urea exceeding 3 %, reduce the sample to 1 g or dilute the original solution so that there are not more than 50 mg of urea in 500 ml.               7.2. In the case of low contents of urea, increase the sample as long as the filtrate remains transparent and colourless.               7.3. If the sample contains simple nitrogenous compounds such as amino acids, the optical density should be measured at 435 nm.                                 15. DETERMINATION OF LUPIN ALKALOIDS      1. Purpose and scope  This method makes it possible to determine the level of alkaloids in lupin seeds.           2. Principle  The alkaloids are dissolved in a mixture of diethyl ether and chloroform and extracted with hydrochloric acid. The alkaloids are precipitated in silicotungstic acid, the precipitate ashed and the residue weighed.           3. Reagents        3.1. Diethyl ether.               3.2. Chloroform.               3.3. Sodium hydroxide solution 4 N.               3.4. Hydrochloric acid 0 73 N.               3.5. Sodium chloride A.R.               3.6. 10 % (w/v) solution of silicotungstic acid S 7O2 712WO3 726H2O.                          4. Apparatus        4.1. Mechanical stirrer.               4.2. Platinum, silica or porcelain crucibles for ashing.               4.3. Electric muffle-furnace.                          5. Procedure  Weigh 15 g of the sample to the nearest 5 mg and place in a container with a capacity of approximately 200 ml, fitted with a ground-glass stopper (e.g. a separating funnel). Add exactly 100 ml of diethyl ether (3.1) and 50 ml of chloroform (3.2) and then, using a graduated pipette, 10 ml of sodium hydroxide solution (3.3). Shake vigorously to prevent agglomeration. Shake again several times and leave to stand overnight. If the supernatant liquid is not completely clear, add a few drops of water. Filter the etherchloroform layer. Collect 50 ml of filtrate in a 50 ml volumetric flask and transfer quantitatively to a 150 ml separating funnel, using 50 ml of diethyl ether (3.1). Extract three times in succession with 20 ml of hydrochloric acid (3.4), leave to decant and collect the acid extract after each extraction. Collect the acid extracts in a 250 ml beaker and eliminate the last traces of ether and chloroform by warming gently. Add approximately 1 g of sodium chloride (3.5), leave to cool and precipitate the alkaloids in silicotungstic acid solution (3.6). Stir with a mechanical stirrer for thirty minutes. Leave to settle overnight, filter through an ash-free filter and wash the precipitate successively twice with 10 ml and twice with 5 ml of hydrochloric acid (3.4).  Place the filter containing the precipitate in a crucible and ash at 900 ºC. Leave to cool and weigh.           6. Calculation of results  The alkaloid content of the sample is obtained by multiplying the weight of the ash by a factor of 0 72.  Express the result as a percentage of the sample.                  16. ESTIMATION OF THE UREASE ACTIVITY OF PRODUCTS DERIVED FROM SOYA      1. Purpose and scope  This test makes it possible estimate the urease activity of products derived from soya and to show whether these products have been cooked for a sufficient length of time.           2. Principle  Urease activity is estimated by the amount of ammoniacal nitrogen liberated per 1 g of product per minute at 30 ºC from a solution of urea.           3. Reagents        3.1. Hydrochloric acid 0 71 N.               3.2. Solution of sodium hydroxide 0 71 N.               3.3. Loading agent of phosphate 0 705 M, containing, per 1000 ml, 4 745 g of disodium phosphate (Na2HPO4 72H2O) and 3 740 g of monopotassium phosphate (KH2 PO4).               3.4. Freshly-prepared urea loading agent containing 30 70 g of urea per 1000 ml of loading agent (3.3) ; pH 6 79-7 70.                          4. Apparatus        4.1. Potentiometric titration apparatus or high sensitivity pH-meter (0 702 pH) with magnetic stirrer.               4.2. Water bath fitted with thermostat set at 30 ºC exactly.               4.3. Test-tubes with ground-glass stoppers, 150 × 18 mm.                          5. Procedure  Crush about 10 g of the sample (for example in a coffee mill) so that it passes through a sieve with a mesh of 0 72 mm. Weigh out 0 72 g of the crushed sample to the nearest mg, place in a test tube with a ground-glass stopper and add 10 ml of urea loading agent (3.4). Stopper immediately and shake vigorously. Place the tube in a water bath set at 30 ºC exactly and shake vigorously. Place the tube in a water bath set at 30 ºC exactly and keep there for exactly thirty minutes. Immediately add 10 ml of 0 71 N hydrochloric acid (3.1), cool rapidly to 20 ºC and transfer the contents of the tube quantitatively to a titration vessel, rinsing twice with 5 ml of water. Using a glass electrode (4.1) titrate immediately and rapidly to pH 4 77 with the 0 71 N sodium hydroxide solution (3.2) by electrometry.  Carry out a blank test as follows:  Quickly place a sample of 0 72 g weighed to the nearest mg in a test tube with a ground-glass stopper, add 10 ml of 0 71 N hydrochloric acid (3.1) and then 10 ml of urea loading agent (3.4). Cool the tube immediately in ice-water and leave there for thirty minutes. Under the conditions indicated above, transfer the contents of the tube to the titration vessel using the 0 71 N sodium hydroxide solution (3.2) up to pH 4 77.            6. Calculation  The urease activity is calculated by using the formula: >PIC FILE= "T0010562">   where:  a = ml of 0 71 N sodium hydroxide solution consumed by the sample,  b = ml of 0 71 N sodium hydroxide solution consumed in the blank test,  E = weight of sample in g.           7. Observation        7.1. This method is suitable for a urease activity of up to 1 mg of N/g/min at 30 ºC. For more active products, the size of the sample can be reduced to 50 mg.               7.2. Products containing more than 10 % of crude fatty substance must first be defatted cold.


Avis juridique important

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32001L0047

Commission Directive 2001/47/EC of 25 June 2001 amending Annex I to Council Directive 91/414/EEC concerning the placing of plant protection products on the market to include Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) as an active substance

Official Journal L 175 , 28/06/2001 P. 0021 - 0023

Commission Directive 2001/47/ECof 25 June 2001amending Annex I to Council Directive 91/414/EEC concerning the placing of plant protection products on the market to include Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) as an active substanceTHE COMMISSION OF THE EUROPEAN COMMUNITIES,Having regard to the Treaty establishing the European Community,Having regard to Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market(1), as last amended by Commission Directive 2001/28/EC(2), and in particular Article 6(1) thereof,Whereas:(1) In accordance with Article 6(2) of Directive 91/414/EEC (hereinafter referred to as "the Directive") Belgium received on 18 May 1994 an application from Thermo Trilogy Corporation, ("the applicant") for the inclusion of the active substance Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) in Annex I to the Directive.(2) In accordance with the provisions of Article 6(3) of the Directive, the Commission confirmed in its Decision 97/164/EC(3) that the dossier submitted for Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) could be considered as satisfying, in principle, the data and information requirements of Annex II and, for a plant protection product containing this active substance, of Annex III to the Directive.(3) In accordance with Article 5(1) of the Directive, an active substance should be included in Annex I for a period not exceeding 10 years if it may be expected that neither the use of, or residues from, plant protection products containing the active substance will have any harmful effects on human or animal health or on groundwater or any unacceptable influence on the environment.(4) For Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874), the effects on human health and the environment have been assessed, in accordance with the provisions of Article 6(2) and (4) of the Directive, for the uses proposed by the applicant. Belgium acting as nominated rapporteur Member State submitted a draft assessment report concerning the substance to the Commission on 9 December 1997.(5) The draft assessment report has been reviewed by the Member States and the Commission within the Standing Committee on Plant Health. This review was finalised on 27 April 2001 in the format of the Commission review report for Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874). If this review report has to be updated to take account of technical and scientific developments, the conditions for the inclusion of DPX KE 459 Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) in Annex I to the Directive will also need to be amended in accordance with the Directive.(6) The dossier and the information from the review of Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) were also submitted to the Scientific Committee on Plants for opinion on 16 December 1999. This Committee has given its opinion on 30 November 2000(4).(7) It has appeared from the various examinations made that plant protection products containing the active substance concerned may be expected to satisfy, in general, the requirements laid down in Article 5(1)(a), (b) and (3) of the Directive, in particular with regard to the uses which were examined and detailed in the Commission review report. It is therefore appropriate to include the active substance concerned in Annex I, in order to ensure that in all Member States the authorisations of plant protection products containing the active substance concerned can be granted in accordance with the provisions of the said Directive.(8) After inclusion, a reasonable period is necessary to permit Member States to implement the provisions of the Directive on plant protection products containing Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) and in particular to review, existing provisional authorisations or to grant, by the end of this period at the latest, new authorisations in accordance with the provisions of the Directive. A longer period may also be required for plant protection products containing Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) and other active substances included in Annex I.(9) As the uniform principles have still to be adopted for micro-organisms, it is appropriate that the Member States should apply the general provisions of Article 4 of the Directive when granting authorisations. It is also appropriate to provide Member States a reasonable period of time to re-evaluate authorisations granted in the light of the uniform principles after their adoption.(10) It is appropriate to provide that the finalised review report (except for confidential information in the meaning of Article 14 of the Directive) is kept available or made available by the Member States for consultation by any interested parties.(11) The measures provided for in this Directive are in accordance with the opinion of the Standing Committee on Plant Health delivered on 27 April 2001,HAS ADOPTED THIS DIRECTIVE:Article 1The table in Annex I to Directive 91/414/EEC shall be amended to include the entry in respect of Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) set out in the Annex hereto.Article 21. Member States shall bring into force the laws, regulations and administrative provisions necessary to comply with this Directive, at the latest by 31 December 2001. They shall forthwith inform the Commission thereof.When Member States adopt those provisions, they shall contain a reference to this Directive or shall be accompanied by such a reference on the occasion of their official publication. Member States shall determine how such reference is to be made.2. However, with regard to the review of existing provisional authorisations granted in the light of the review report, the provisional authorisations shall be withdrawn and where appropriate, replaced by a full authorisation by 30 November 2002.3. However, with regard to the application of the uniform principles, Member States shall re-examine authorisations granted as soon as possible after their adoption and at the latest within a period of 12 months from the date of adoption of such principles.4. However, for plant protection products containing Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) together with another active substance which is in Annex I to Directive 91/414/EEC, the period referred to in paragraph 1 is extended to the extent that a longer implementation period is provided for by the provisions laid down in the Directive amending Annex I to Directive 91/414/EEC to include that other substance in the Annex.5. Member States shall keep available the review report for Paecilomyces fumosoroseus (Apopka strain 97, PFR 97 or CG 170, ATCC20874) (except for confidential information within the meaning of Article 14 of the Directive) for consultation by any interested parties or shall make it available to them on specific request.Article 3This Directive shall enter into force on 1 July 2001.Article 4This Directive is addressed to the Member States.Done at Brussels, 25 June 2001.For the CommissionDavid ByrneMember of the Commission(1) OJ L 230, 19.8.1991, p. 1.(2) OJ L 113, 24.4.2001, p. 5.(3) OJ L 64, 5.3.1997, p. 17.(4) Opinion of the Scientific Committee on Plants regarding the evaluation Paecilomyces fumosoroseus in the context of Council Directive 91/414/EEC concerning the placing of plant protection products on the market. SCP/PAECIL/002 final dated 11 December 2000.ANNEXENTRY TO BE INSERTED IN THE TABLE IN ANNEX I TO DIRECTIVE 91/414/EEC>TABLE>


title: Commission Directive 2006/16/EC of  7 February 2006  amending Council Directive 91/414/EEC to include oxamyl as active substance   (Text with EEA relevance)

text:
               8.2.2006

               EN

               Official Journal of the European Union

               L 36/37

      COMMISSION DIRECTIVE 2006/16/EC
   of 7 February 2006
   amending Council Directive 91/414/EEC to include oxamyl as active substance
   (Text with EEA relevance)
   THE COMMISSION OF THE EUROPEAN COMMUNITIES,
   Having regard to the Treaty establishing the European Community,
   Having regard to Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market (1), and in particular Article 6(1) thereof,
   Whereas:

               (1)

               Commission Regulations (EC) No 451/2000 (2) and (EC) No 703/2001 (3) lay down the detailed rules for the implementation of the second stage of the programme of work referred to in Article 8(2) of Directive 91/414/EEC and establish a list of active substances to be assessed, with a view to their possible inclusion in Annex I to Directive 91/414/EEC. That list includes oxamyl.

               (2)

               For oxamyl, the effects on human health and the environment have been assessed in accordance with the provisions laid down in Regulations (EC) No 451/2000 and (EC) No 703/2001 for a range of uses proposed by the notifier. Moreover, those Regulations designate the Rapporteur Member States which have to submit the relevant assessment reports and recommendations to the European Food Safety Authority (EFSA) in accordance with Article 8(1) of Regulation (EC) No 451/2000. For oxamyl the Rapporteur Member State was Ireland and all relevant information was submitted on 25 August 2003.

               (3)

               The assessment report has been peer reviewed by the Member States and the EFSA within its Working Group Evaluation and presented to the Commission on 14 January 2005 in the format of the EFSA Scientific Report for oxamyl (4). This report has been reviewed by the Member States and the Commission within the Standing Committee on the Food Chain and Animal Health and finalised on 15 July 2005 in the format of the Commission review report for oxamyl.

               (4)

               It has appeared from the various examinations made that plant protection products containing oxamyl may be expected to satisfy, in general, the requirements laid down in Article 5(1)(a) and (b) of Directive 91/414/EEC, in particular with regard to the uses which were examined and detailed in the Commission review report. It is therefore appropriate to include oxamyl in Annex I, in order to ensure that in all Member States the authorisations of plant protection products containing this active substance can be granted in accordance with the provisions of that Directive.

               (5)

               Without prejudice to that conclusion, it is appropriate to obtain further information on certain specific points. Article 6(1) of Directive 91/414/EEC provides that inclusion of a substance in Annex I may be subject to conditions. Therefore it is appropriate to require that oxamyl should be subjected to further testing for confirmation of the risk assessment for some issues and that such studies should be presented by the notifier.

               (6)

               The experience gained from previous inclusions in Annex I to Directive 91/414/EEC of active substances assessed in the framework of Commission Regulation (EEC) No 3600/92 (5) has shown that difficulties can arise in interpreting the duties of holders of existing authorisations in relation to access to data. In order to avoid further difficulties it therefore appears necessary to clarify the duties of the Member States, especially the duty to verify that the holder of an authorisation demonstrates access to a dossier satisfying the requirements of Annex II to that Directive. However, this clarification does not impose any new obligations on Member States or holders of authorisations compared to the directives which have been adopted until now amending Annex I.

               (7)

               A reasonable period should be allowed to elapse before an active substance is included in Annex I in order to permit Member States and the interested parties to prepare themselves to meet the new requirements which will result from the inclusion.

               (8)

               Without prejudice to the obligations defined by Directive 91/414/EEC as a consequence of including an active substance in Annex I, Member States should be allowed a period of six months after inclusion to review existing authorisations of plant protection products containing oxamyl to ensure that the requirements laid down by Directive 91/414/EEC, in particular in its Article 13 and the relevant conditions set out in Annex I, are satisfied. Member States should vary, replace or withdraw, as appropriate, existing authorisations, in accordance with the provisions of Directive 91/414/EEC. By derogation from the above deadline, a longer period should be provided for the submission and assessment of the complete Annex III dossier of each plant protection product for each intended use in accordance with the uniform principles laid down in Directive 91/414/EEC.

               (9)

               It is therefore appropriate to amend Directive 91/414/EEC accordingly.

               (10)

               The measures provided for in this Directive are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health,

         HAS ADOPTED THIS DIRECTIVE:
   Article 1
   Annex I to Directive 91/414/EEC is amended as set out in the Annex to this Directive.
   Article 2
   Member States shall adopt and publish, by 31 January 2007 at the latest, the laws, regulations and administrative provisions necessary to comply with this Directive. They shall forthwith communicate to the Commission the text of those provisions and a correlation table between those provisions and this Directive.
   They shall apply those provisions from 1 February 2007.
   When Member States adopt those provisions, they shall contain a reference to this Directive or be accompanied by such a reference on the occasion of their official publication. Member States shall determine how such reference is to be made.
   Article 3
   1.   Member States shall in accordance with Directive 91/414/EEC, where necessary, amend or withdraw existing authorisations for plant protection products containing oxamyl as an active substance by 31 January 2007.
   By that date, they shall in particular verify that the conditions in Annex I to that Directive relating to oxamyl are met, with the exception of those identified in part B of the entry concerning that active substance, and that the holder of the authorisation has, or has access to, a dossier satisfying the requirements of Annex II to that Directive in accordance with the conditions of Article 13 of that Directive.
   2.   By derogation from paragraph 1, for each authorised plant protection product containing oxamyl as either the only active substance or as one of several active substances all of which were listed in Annex I to Directive 91/414/EEC by 30 July 2006 at the latest, Member States shall re-evaluate the product in accordance with the uniform principles provided for in Annex VI to Directive 91/414/EEC, on the basis of a dossier satisfying the requirements of Annex III to that Directive and taking into account part B of the entry in Annex I to that Directive concerning oxamyl. On the basis of that evaluation, they shall determine whether the product satisfies the conditions set out in Article 4(1)(b), (c), (d) and (e) of Directive 91/414/EEC.
   Following that determination Member States shall:

               (a)

               in the case of a product containing oxamyl as the only active substance, where necessary, amend or withdraw the authorisation by 30 July 2010 at the latest; or

               (b)

               in the case of a product containing oxamyl as one of several active substances, where necessary, amend or withdraw the authorisation by 30 July 2010 or by the date fixed for such an amendment or withdrawal in the respective Directive or Directives which added the relevant substance or substances to Annex I to Directive 91/414/EEC, whichever is the latest.

         Article 4
   This Directive shall enter into force on 1 August 2006.
   Article 5
   This Directive is addressed to the Member States.

      Done at Brussels, 7 February 2006.

            For the Commission

         Markos KYPRIANOU

            Member of the Commission

      (1)  OJ L 230, 19.8.1991, p. 1. Directive as last amended by Commission Directive 2006/6/EC (OJ L 12, 18.1.2006, p. 21).

      (2)  OJ L 55, 29.2.2000, p. 25. Regulation as last amended by Regulation (EC) No 1044/2003 (OJ L 151, 19.6.2003, p. 32).

      (3)  OJ L 98, 7.4.2001, p. 6.

      (4)  EFSA Scientific Report (2005) 26, 1-78, Conclusion on the peer review of the pesticide risk assessment of the active substance Oxamyl (finalised: 14 January 2005).

      (5)  OJ L 366, 15.12.1992, p. 10. Regulation as last amended by Regulation (EC) No 2266/2000 (OJ L 259, 13.10.2000, p. 27).

      ANNEX
      The following entry shall be added at the end of the table in Annex I to Directive 91/414/EEC

                     No

                     Common name, identification numbers

                     IUPAC name

                     Purity (1)

                     Entry into force

                     Expiration of inclusion

                     Specific provisions

                     ‘117

                     Oxamyl
                     CAS No 23135-22-0
                     CIPAC No 342

                     N,N-dimethyl-2-methylcarbamoyloxyimino-2-(methylthio) acetamide

                     970 g/kg

                     1 August 2006

                     31 July 2016

                     PART A
                     Only uses as nematicide and insecticide may be authorised.
                     PART B
                     For the implementation of the uniform principles of Annex VI, the conclusions of the review report on oxamyl, and in particular Appendices I and II thereto, as finalised in the Standing Committee on the Food Chain and Animal Health on 15 July 2005 shall be taken into account. In this overall assessment,

                                 —

                                 Member States must pay particular attention to the protection of birds and mammals, earthworms, aquatic organisms, surface water, and groundwater in vulnerable situations.

                           Conditions of authorisation should include risk mitigation measures, where appropriate.

                                 —

                                 Member States must pay particular attention to the operator safety. Conditions of authorisation should include protective measures, where appropriate.

                           The concerned Member States shall request the submission of further studies to confirm the risk assessment for ground water contamination in acidic soils, birds and mammals and earthworms. They shall ensure that the notifiers at whose request oxamyl has been included in this Annex provide such studies to the Commission within two years from the entry into force of this Directive.’

         (1)  Further details on identity and specification of active substance are provided in the review report.