1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16279:2012Animal feeding stuffs Determination of fluoridecontent after hydrochloric acidtreatment by ion-sensitiveelectrode method (ISE)BS EN 16279:2012 BRITISH STANDARDNat
2、ional forewordThis British Standard is the UK implementation of EN 16279:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can beobtained on request to its secretary.This publication doe
3、s not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 67000 8ICS 65.120Compliance with a British Standard cannot confer immunity fromlegal obli
4、gations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 July 2012.Amendments issued since publicationDate Text affectedBS EN 16279:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16279 July 2012 ICS 65.120 English Version Animal fe
5、eding stuffs - Determination of fluoride content after hydrochloric acid treatment by ion-sensitive electrode method (ISE) Aliments des animaux - Dtermination de la teneur en fluorure, aprs traitement lacide chlorhydrique, selon la mthode utilisant une lectrode slective dions (ISE) Futtermittel - Be
6、stimmung des Fluoridgehaltes nach Salzsure-Behandlung mit ionensensitiver Elektrode (ISE)This European Standard was approved by CEN on 17 May 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of
7、 a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German)
8、. A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, C
9、yprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
10、Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16279:2012: EBS
11、 EN 16279:2012EN 16279:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Principle 54 Reagents .65 Apparatus .76 Sampling .77 Preparation of test sample 78 Procedure .89 Calculation and expression of results 1010 Precision 1211 Test report . 13Annex A (informative)
12、 Results of the interlaboratory study 14Annex B (informative) Flowchart Determination of fluoride content after hydrochloric acid treatment by ISE method . 15Annex C (informative) Interpolation reading . 16Bibliography . 18BS EN 16279:2012EN 16279:2012 (E) 3 Foreword This document (EN 16279:2012) ha
13、s been prepared by Technical Committee CEN/TC 327 “Animal Feeding stuffs Methods of sampling and analysis”, the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by
14、 January 2013, and conflicting national standards shall be withdrawn at the latest by January 2013. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such p
15、atent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Stand
16、ard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spa
17、in, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16279:2012EN 16279:2012 (E) 4 Introduction Fluorine (F) is one of the most abundant elements in the environment. Animals are exposed to the ionic form of the element, fluoride (F-), which may be present in feeding stuffs. The toxicity of
18、fluoride has already been established by EFSA 6; and its availability greatly depends on the solubility of fluoride compounds. In particular, fluoride compounds with low solubility are poorly absorbed while fluoride ions released from readily soluble compounds are almost completely absorbed from the
19、 gastrointestinal tract by passive diffusion in monogastric species. The extraction procedure of this method involves a mild acid treatment with hydrochloric acid solution of 1 mol/l which should reflect the gastric hydrochloric acid concentration of 0,1 mol/l 0,3 mol/l. BS EN 16279:2012EN 16279:201
20、2 (E) 5 1 Scope This European Standard specifies an Ion-Selective Electrode method (ISE) after hydrochloric acid treatment for the determination of fluoride from animal feeding stuffs. The content of fluoride (F-) corresponds to that of fluorine (F) specified in Commission Regulation (EU) 574/20113.
21、 This European Standard is strictly based on several conventions such as those contained in the following example: EXAMPLE 0,5 g test portion for extraction of fluoride from animal feeds by means of an acid treatment with 20 ml of 1 mol/l hydrochloric acid solution at ambient temperature (20 C to 25
22、 C) for 20 min. The pH is controlled and adjusted to 5,5 in the buffered test solution before determination of fluoride by ISE using standard addition technique. The method was successfully tested in an interlaboratory study in concentrations between 100 mg/kg up to 500 mg/kg. If this method is foll
23、owed strictly, then theoretically all concentrations from 40 mg/kg up to 4 000 mg/kg can be analysed within the linear calibration function. Only for concentrations lower than 40 mg/kg is the use of an interpolation technique required instead of standard addition Annex C. The quantification limit fo
24、r fluoride using the conventions of the method including the standard addition technique is 40 mg/kg or lower than 2,5 mg/kg when using interpolation Annex C. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for it
25、s application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods EN ISO 6498, Animal feeding stuffs Guidelines
26、 for sample preparation 3 Principle For the determination of fluoride, a test portion of 0,5 g of the sample is treated with 20 ml of 1 mol/l hydrochloric acid solution for 20 min at ambient temperature (20 C to 25 C). The amount of fluoride extracted from the sample is determined by means of a fluo
27、ride selective electrode. NOTE This ISE method is based upon a potentiometric technique. This means that it is based upon the measurement of a concentration of an analyte present in solution, by means of an ion selective electrode. This electrode has a linear response within a working range of analy
28、te concentrations, which is provided by the calibration curve. Thus, in general, the operator should take an adequate amount of sample to ensure that the final concentration of the analyte is within this working range given by the calibration step. To ensure satisfactory reproducibility of the metho
29、d however, this extraction procedure as a convention method fixes the ratio of the amount of the test portion to the volume of the extraction solvent for all kind of feeds. WARNING The use of this European Standard can involve hazardous materials, operations and equipment. This standard does not pur
30、port to address all the safety problems associated with its use. It is the responsibility of the user of this European Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. BS EN 16279:2012EN 16279:2012 (E) 6 4 Reagents
31、Use only reagents of recognised analytical grade, unless otherwise specified. 4.1 Water, complying with grade 2 in accordance with EN ISO 3696. 4.2 Element stock solution, fluoride (NaF); Fluoride c = 1 000 mg/l. 4.3 Hydrochloric acid (HCl), mass fraction of 37 %, having a density of approximately (
32、HCl) 1,185 g/ml. 4.4 Perchloric acid (HClO4), mass fraction of 70 % to 72 %, having a density of approximately (HClO4) 1,68 g/ml. 4.5 Acetic acid (CH3COOH), glacial, 99 %. 4.6 Sodium acetate trihydrate (CH3COONa 3H2O), 99 %. 4.7 Trisodium citrate dihydrate (C6H5Na3O72H2O), 99 %. 4.8 Hydrochloric aci
33、d solution of 1 mol/l. To prepare: pipette 83 ml hydrochloric acid (4.3) into a 1 000 ml volumetric flask (5.5) and fill to the mark with water (4.1). 4.9 Sodium acetate solution of 3 mol/l. To prepare: dissolve 408 g of sodium acetate trihydrate (4.6) in 750 ml of water (4.1). When the solution rea
34、ches room temperature (20 C to 25 C), adjust the pH to 7,0 with acetic acid (4.5), transfer to a 1 000 ml volumetric flask (5.5) and dilute to the mark with water (4.1). 4.10 Sodium citrate solution. To prepare: dissolve 222 g of trisodium citrate dehydrate (4.7) in 250 ml of water (4.1), add 28 ml
35、perchloric acid (4.4), transfer to a 1 000 ml volumetric flask (5.5) and dilute to the mark with water (4.1). 4.11 Intermediate fluoride solution of 100 mg/l. To prepare: pipette 10 ml from the 1 000 mg/l fluoride stock solution (4.2) into a 100 ml plastic volumetric flask (5.7) and dilute to the ma
36、rk with water (4.1). 4.12 Intermediate fluoride solution of 10 mg/l. To prepare: pipette 10 ml from the 100 mg/l intermediate fluoride solution (4.11) into a 100 ml plastic volumetric flask (5.7) and dilute to the mark with water (4.1). 4.13 Sodium hydroxide solution of 5 mol/l. To prepare: transfer
37、 NaOH pellets, weighing 200 g, to a 1 000 ml volumetric flask (5.5), which is about 2/3 filled with water (4.1) and contains a magnetic stirring bar (5.4). Stir until dissolved; after cooling down take away the magnet stirrer and fill up to the mark with water (4.1) and shake. NOTE The use of sodium
38、 hydroxide solution of 1 mol/l might be more applicable for a precise pH adjustment. BS EN 16279:2012EN 16279:2012 (E) 7 5 Apparatus Use typical laboratory apparatus with preference for plastic instead of glass for recipients, in order to avoid undesired reactions of fluoride with silicon from glass
39、 in the case of fluoride standard solutions and the test sample solutions. 5.1 Laboratory grinder 5.1.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 0,5 mm. 5.1.2 Laboratory grinder, capable of grinding to a particle size of less than or equal to 0,1 mm. 5.2 An
40、alytical balance, capable of weighing to an accuracy of 0,1 mg. 5.3 Freeze drying equipment, capable of freeze-drying liquid animal feeding stuffs. 5.4 Magnetic stirrer, with polytetrafluoro-ethylene-coated stirring bar. 5.5 One-mark volumetric flasks, of capacities 100 ml, 500 ml and 1 000 ml. 5.6
41、Beakers, of capacities 100 ml and 250 ml. 5.7 One-mark plastic volumetric flasks, of capacities 100 ml and 200 ml. 5.8 Plastic beakers, of capacities 50 ml, 100 ml and 200 ml. 5.9 Fluoride selective electrode. NOTE The use of combined electrodes (i.e. selective and reference in one) is possible. 5.1
42、0 pH combined electrode. 5.11 Reference electrode for fluoride. 5.12 Ion-pH-meter, capable of measuring to an accuracy of 0,1 mV. 6 Sampling Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 6497 2. It is important that the labor
43、atory receives a sample that is truly representative and has not been damaged or changed during transport or storage. 7 Preparation of test sample 7.1 General Prepare the test sample in accordance with EN ISO 6498. The grinding must be performed in conditions such that the substance is not appreciab
44、ly heated. The operation is to be repeated as many times as is necessary and it shall be executed as quickly as possible in order to prevent any gain or loss of constituents (such as water). BS EN 16279:2012EN 16279:2012 (E) 8 The whole ground product is placed in a flask made of e.g. polypropylene,
45、 which can be stoppered and stored in such way to prevent any change in composition. Before any weighing is carried out for the analysis, the whole test sample shall be thoroughly mixed for reasons of homogeneity. 7.2 Animal feeding stuffs which can be ground as such Grind the laboratory sample (usu
46、ally 500 g), using a grinder (5.1.1) or mortar, until a particle size of 0,5 mm or less has been reached. Use e.g. a knife mill or equivalent. 7.3 Liquid animal feeding stuffs 7.3.1 General Liquid feeding stuffs shall be pre-dried according to the procedure described in 7.3.2 or freeze-dried accordi
47、ng to the procedure described in 7.3.3. 7.3.2 Pre-drying Pre-dry the laboratory sample at a temperature of (60 5) C during at least 16 h to reduce the moisture content to a level of 8 % to 12 %. Determine the mass of the sample before and after pre-drying using an analytical balance (5.2) and calcul
48、ate the moisture loss on pre-drying. Grind the pre-dried sample in accordance with 7.1. 7.3.3 Freeze-drying Freeze-dry the laboratory sample following the instructions of the freeze-drying equipment (5.3). The mass of the sample before and after the freeze-drying is to be determined using an analyti
49、cal balance (5.2). Grind the freeze-dried sample in accordance with 7.1. 7.4 Feed materials Feed materials shall be ground using a grinder (5.1.2) or mortar until a particle size of 0,1 mm or less has been reached. Use e.g. a ball mill or equivalent. 8 Procedure 8.1 Extraction: wet extraction 1 mol/l HCl Weigh 0,5 g of the homogenised and (to a particle size of 0,5 mm) ground test sample as test portion to an accuracy of 1 mg into a plastic beaker of e.g. 200 ml volume (5.8). Pi
