SIST EN 15621:2017

Animal feeding stuffs: Methods of sampling and analysis - Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES

This European Standard specifies a method for the determination of the elements calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt in animal feeding stuffs by inductively coupled plasma atomic emission spectrometry (ICP-AES) after pressure digestion.
The method was fully statistically tested and evaluated for the elements calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt within the following 11 animal feeds: 2 complete feeds (pig feed, sheep feed), 3 complementary feeds (3 mineral feeds), 1 mineral premixture, 3 feed materials (MgO, phosphate, CaCO3) and 2 feed additives (CuSO4, bentonite).
For potassium and sulphur the HORRAT values were mostly higher than 2. Therefore, for these elements the method is more applicable as a screening method and not for confirmatory purposes.
Other elements like molybdenum, lead, cadmium, arsenic were not fully statistically tested and evaluated within 11 animal feeding stuff samples because these elements did not occur in concentrations higher than the limit of quantification in most of these samples. A single laboratory validation is therefore necessary for the use of this multi element method for these elements.
For the determination of extractable lead in minerals and feeds, containing phyllosilicates (e.g. kaolinite clay) wet digestion with nitric acid should be used.
The method limit of quantification for each element is dependent on the sample matrix as well as on the instrument. The method is not applicable for determination of low concentrations of elements. A limit of quantification of 1 mg/kg should normally be obtained.
NOTE 1   This method can also be used for the determination in products with high content (> 5 %) of the element to be measured, but for this purpose the accuracy of the method has to be checked individually.
NOTE 2   Results of this European Standard EN 15621 may be higher than of EN 15510 because EN 15621 is using pressure digestion mode.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Calcium, Natrium, Phosphor, Magnesium, Kalium, Schwefel, Eisen, Zink, Kupfer, Mangan und Kobalt nach Druckaufschluss mittels ICP-AES

Diese Europäische Norm legt ein Verfahren zur Bestimmung der Elemente Calcium, Natrium, Phosphor, Magnesium, Kalium, Schwefel, Eisen, Zink, Kupfer, Mangan und Kobalt in Futtermitteln mittels Atomemissionsspektrometrie mit induktiv gekoppeltem Plasma (ICPAES) nach Druckaufschluss fest.
Das Verfahren wurde für die Elemente Calcium, Natrium, Phosphor, Magnesium, Kalium, Schwefel, Eisen, Zink, Kupfer, Mangan und Kobalt in den folgenden 11 Tierfuttermitteln vollständig statistisch geprüft und bewertet: 2 Alleinfuttermittel (Schweine-, Schaffutter), 3 Ergänzungsfuttermittel (3 Mineralstoff-mischungen), 1 Mineralstoffvormischung, 3 Einzelfuttermittel (MgO, Phosphat, CaCO3) und 2 Futtermittel-Zusatzstoffe (CuSO4, Bentonit).
Bei Kalium und Schwefel waren die HorRat-Werte meistens höher als 2. Deshalb ist das Verfahren für diese Elemente eher als Screening-Verfahren und nicht für Bestätigungszwecke geeignet.
Die bei anderen Elementen, wie Molybdän, Blei, Cadmium und Arsen, erzielten Ergebnisse wurden im Rahmen von 11 Futtermittelproben nicht vollständig statistisch geprüft und bewertet, da diese Elemente nicht in Konzentrationen vorkamen, die in den meisten dieser Proben höher als die Bestimmungsgrenze waren. Deshalb ist bei Anwendung dieses Mehrelementverfahrens eine einzelne laborspezifische Validierung notwendig.
Zur Bestimmung von extrahierbarem Blei in Mineralstoffen und Futtermitteln, die Schichtsilikate enthalten (z. B. kaolinitischer Ton), sollte Nassaufschluss mit Salpetersäure verwendet werden.
Die Bestimmungsgrenze des Verfahrens ist bei jedem Element sowohl von der Probenmatrix als auch von dem Gerät abhängig. Das Verfahren ist nicht für die Bestimmung von geringen Elementkonzentrationen anwendbar. Eine Bestimmungsgrenze von 1 mg/kg sollte normalerweise erreicht werden.
ANMERKUNG 1   Dieses Verfahren kann ebenfalls zur Bestimmung in Produkten mit hohem Gehalt des zu messenden Elements (> 5 %) angewendet werden, jedoch muss die Fehlergrenze des Verfahrens im Einzelfall geprüft werden.
ANMERKUNG 2   Die Ergebnisse nach dieser Europäischen Norm EN 15621 können höher sein als die nach EN 15510, da EN 15621 die Druckaufschlussmethode verwendet.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dosage du calcium, du sodium, du phosphore, du magnésium, du potassium, du soufre, du fer, du zinc, du cuivre, du manganèse et du cobalt après digestion sous pression par ICP-AES

La présente Norme européenne spécifie une méthode de détermination de la teneur en calcium, sodium, phosphore, magnésium, potassium, soufre, fer, zinc, cuivre, manganèse et cobalt présents dans les aliments pour animaux par spectrométrie d’émission atomique à plasma à couplage inductif (ICP AES) après digestion sous pression.
La méthode a été soumise à un essai statistique complet et à une évaluation pour doser les éléments suivants: calcium, sodium, phosphore, magnésium, potassium, soufre, fer, zinc, cuivre, manganèse et cobalt sur 11 aliments pour animaux : 2 aliments complets (un aliment pour porc et un pour ovin), 3 compléments alimentaires pour animaux (3 minéraux pour l’alimentation animale), 1 prémélange minéral, 3 matières minérales destinées aux aliments des animaux (MgO, phosphate, CaCO3) et 2 additifs pour l’alimentation animale (CuSO4, bentonite).
Dans le cas du potassium et du soufre, les valeurs HORRAT sont pour la plupart supérieures à 2. Par conséquent, pour ces éléments, la méthode est applicable en tant que méthode d’évaluation et non pas à des fins de confirmation.
D’autres éléments tels que le molybdène, le plomb, le cadmium et l’arsenic n’ont pas été soumis à un essai statistique complet ni à une évaluation sur les 11 échantillons d’aliments pour animaux, car leur concentration n’était pas supérieure à la limite de quantification dans la plupart des échantillons. Une validation interne par chaque laboratoire est par conséquent nécessaire pour utiliser cette méthode avec ces éléments.
Pour la détermination de la teneur en plomb extractible dans les minéraux et les aliments pour animaux contenant des phyllosilicates (argiles à kaolinite, par exemple), il convient d’utiliser une digestion humide à l’acide nitrique.
La limite de quantification de la méthode pour chacun des éléments est fonction de la matrice de l’échantillon et de l’instrument. Cette méthode ne permet pas la détermination des éléments en faibles concentrations. Il convient de parvenir à une limite de quantification de 1 mg/kg, dans des conditions d’essai normales.
NOTE 1   Cette méthode peut également servir au dosage dans des produits à teneur élevée en éléments (> 5 %), mais à cette fin, l’exactitude de la méthode doit être vérifiée sur une base individuelle.
NOTE 2   Les résultats de la présente Norme européenne EN 15621 peuvent être supérieurs à ceux de l’EN 15510, car l’EN 15621 utilise le mode de digestion sous pression.

Krma: metode vzorčenja in analize - Določevanje kalcija, natrija, fosforja, magnezija, kalija, žvepla, železa, cinka, bakra, mangana in kobalta po razklopu pod tlakom z ICP-AES

Ta evropski standard določa metodo za določevanje kalcija, natrija, fosforja, magnezija, kalija, žvepla, železa, cinka, bakra, mangana in kobalta v krmi z atomsko emisijsko spektrometrijo z induktivno sklopljeno plazmo (ICP-AES) po razklopu pod tlakom.
Metoda je bila v celoti statistično preizkušena in ocenjena za kalcij, natrij, fosfor, magnezij, kalij, žveplo, železo, cink, baker, mangan in kobalt v naslednjih enajstih krmah: dveh celovitih krmah (krmi za prašiče, krmi za ovce), treh dopolnilnih krmah (treh mineralnih krmah), enem mineralnem premiksu, treh sestavinah krme (MgO, fosfatu, CaCO3) in dveh dodatkih za krmo (CuSO4, bentonitu).
Vrednosti HORRAT so bile pri kaliju in žveplu večinoma višje od 2. Zato je pri teh dveh elementih ta metoda bolj ustrezna kot presejalna metoda in ne kot metoda za potrditev.
Drugi elementi, kot so molibden, svinec, kadmij in arzen, niso bili v celoti statistično testirani in ovrednoteni na 11 vzorcih krme, ker se v večini teh vzorcev niso pojavljali v koncentracijah, višjih od meje kvantifikacije. Zato je za uporabo te večelementarne metode pri teh elementih potrebna ena sama laboratorijska validacija.
Za določevanje izločljivega svinca v mineralih in krmah, ki vsebujejo filosilikate (npr. kaolinitna glina), je treba uporabiti postopek mokrega razklopa z dušikovo kislino.
Meja kvantifikacije vseh elementov je pri tej metodi odvisna od matrice vzorca in instrumenta. Metoda se ne uporablja za določevanje nizkih koncentracij elementov. Običajno je treba doseči mejo kvantifikacije 1 mg/kg.
OPOMBA 1   Ta metoda se lahko uporablja tudi za določevanje koncentracije v izdelkih z visoko vsebnostjo (> 5 %) elementa, ki se ga meri, vendar je treba natančnost metode za ta namen preveriti posebej.
OPOMBA 2   Rezultati, pridobljeni skladno s tem evropskim standardom EN 15621, so lahko višji kot rezultati, pridobljeni skladno s standardom EN 15510, ker standard EN 15621 uporablja način razklopa pod tlakom.

General Information

• Status: Published
• Public Enquiry End Date: 19-Dec-2016
• Publication Date: 07-Sep-2017
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 23-Aug-2017
• Due Date: 28-Oct-2017
• Completion Date: 08-Sep-2017
• Ref Project: EN 15621:2017 - Animal feeding stuffs: Methods of sampling and analysis - Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES

Relations

• Replaces: SIST EN 15621:2012 - Animal feeding stuffs - Determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt after pressure digestion by ICP-AES
• Effective Date: 01-Oct-2017

Overview

EN 15621:2017 is a CEN standard that defines a multi-element analytical method for animal feeding stuffs. It specifies sample preparation by pressure digestion (closed-vessel) followed by analysis using inductively coupled plasma atomic emission spectrometry (ICP‑AES). The method covers routine determination of major and trace elements in feeds and feed materials after mineralisation.

Key topics

• Scope of elements: calcium (Ca), sodium (Na), phosphorus (P), magnesium (Mg), potassium (K), sulphur (S), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and cobalt (Co).
• Validated sample types: 11 matrices were statistically tested, including:
  • 2 complete feeds (pig, sheep)
  • 3 complementary/mineral feeds
  • 1 mineral premixture
  • 3 feed materials (MgO, phosphate, CaCO3)
  • 2 feed additives (CuSO4, bentonite)
• Analytical principle: closed‑vessel pressure digestion to dissolve the sample, followed by ICP‑AES with external calibration or standard addition for quantification.
• Performance and limits:
  • Method precision was established through inter‑laboratory testing.
  • Typical limit of quantification (LOQ) depends on matrix and instrument; a LOQ of about 1 mg/kg is normally achievable.
  • For potassium and sulphur, HORRAT values were often >2 - these elements are better treated as screening rather than confirmatory measurements.
• Elements requiring extra validation: molybdenum (Mo), lead (Pb), cadmium (Cd) and arsenic (As) were not sufficiently present in the test materials; single‑laboratory validation is needed before routine use.
• Special cases: For extractable lead in minerals and feeds containing phyllosilicates (e.g., kaolinite), wet digestion with nitric acid is recommended instead of pressure digestion.
• Safety: Use of concentrated nitric acid and pressure digestion requires appropriate laboratory safety measures.

Applications

• Quality control and routine monitoring in feed testing laboratories
• Regulatory compliance and enforcement testing for mineral and trace element content
• Feed additive and premixture manufacturers verifying formulation content
• Research and method development for multi‑element feed analysis EN 15621:2017 is particularly useful when a multi‑element, digestion‑based ICP‑AES workflow is required for a variety of feed matrices.

Related standards

• EN 15510 (alternative digestion approach; note: EN 15621 results may be higher because of pressure digestion)
• EN ISO 3696 (laboratory water quality)
• EN ISO 6498 (sample preparation guidance for animal feeding stuffs)

Keywords: EN 15621:2017, animal feeding stuffs, ICP‑AES, pressure digestion, determination of calcium sodium phosphorus magnesium potassium sulphur iron zinc copper manganese cobalt, feed analysis, LOQ, HORRAT.

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2017
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SIST EN 15621:2012
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Animal feeding stuffs: Methods of sampling and analysis - Determination of calcium,
sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese
and cobalt after pressure digestion by ICP-AES
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Calcium,
Natrium, Phosphor, Magnesium, Kalium, Schwefel, Eisen, Zink, Kupfer, Mangan und
Kobalt nach Druckaufschluss mittels ICP-AES
Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dosage du calcium,
du sodium, du phosphore, du magnésium, du potassium, du soufre, du fer, du zinc, du
cuivre, du manganèse et du cobalt après digestion sous pression par ICP-AES
Ta slovenski standard je istoveten z: EN 15621:2017
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15621
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2017
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15621:2012
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of calcium, sodium, phosphorus,
magnesium, potassium, sulphur, iron, zinc, copper,
manganese and cobalt after pressure digestion by ICP-AES
Aliments pour animaux : Méthodes d'échantillonnage Futtermittel - Probenahme- und
et d'analyse - Dosage du calcium, du sodium, du Untersuchungsverfahren - Bestimmung von Calcium,
phosphore, du magnésium, du potassium, du soufre, du Natrium, Phosphor, Magnesium, Kalium, Schwefel,
fer, du zinc, du cuivre, du manganèse et du cobalt après Eisen, Zink, Kupfer, Mangan und Kobalt nach
digestion sous pression par ICP-AES Druckaufschluss mittels ICP-AES
This European Standard was approved by CEN on 6 February 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of 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). 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, 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15621:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 6
6 Apparatus . 6
7 Sampling . 7
8 Preparation of the test sample . 7
8.1 General . 7
8.2 Animal feeding stuffs which can be ground as such . 8
8.3 Liquid animal feeding stuffs . 8
9 Procedure. 8
9.1 Pressure digestion – Preparation of the blank test solution and the test solution . 8
9.2 Extractable lead in minerals and feeds containing phyllosilicates (e.g. kaolinite clay)
– extraction with diluted nitric acid . 9
9.3 Calibration . 9
9.4 Determination . 9
Table 1 — Selected emission wavelengths and interferences for determination with ICP-AES . 10
10 Calculation and expression of the results . 11
10.1 General . 11
10.2 External calibration . 11
10.3 Standard addition method with only one addition . 11
10.4 Standard addition method with several additions . 12
10.5 Calculation of the element content in the sample . 12
11 Precision . 13
11.1 Inter-laboratory test . 13
11.2 Repeatability . 13
11.3 Reproducibility . 13
Table 2 — Precision data – Ca, K, Mg, Na, P, S . 14
Table 3 — Precision data – Co, Cu, Fe, Mn, Mo, Zn, Cd, As, Pb . 17
12 Test report . 21
Annex A (informative) Results of the inter-laboratory test . 22
Table A.1 — Statistical results of an inter-laboratory test – Calcium (Ca) . 23
Table A.2 — Statistical results of an inter-laboratory test – Potassium (K) . 24
Table A.3 — Statistical results of an inter-laboratory test – Magnesium (Mg) . 25
Table A.4 — Statistical results of an inter-laboratory test – Sodium (Na) . 26
Table A.5 — Statistical results of an inter-laboratory test – Phosphorus (P) . 27
Table A.6 — Statistical results of an inter-laboratory test – Sulphur (S) . 28
Table A.7 — Statistical results of an inter-laboratory test – Cobalt (Co) . 29
Table A.8 — Statistical results of an inter-laboratory test – Copper (Cu) . 30
Table A.9 — Statistical results of an inter-laboratory test – Iron (Fe). 31
Table A.10 — Statistical results of an inter-laboratory test – Manganese (Mn) . 32
Table A.11 — Statistical results of an inter-laboratory test – Molybdenum (Mo) . 33
Table A.12 — Statistical results of an inter-laboratory test – Zinc (Zn) . 34
Table A.13 — Statistical results of an inter-laboratory test – Cadmium (Cd) . 35
Table A.14 — Statistical results of an inter-laboratory test – Arsenic (As) . 36
Table A.15 — Statistical results of an inter-laboratory test – Lead (Pb) . 37
Annex B (informative) Notes on the detection technique, interferences and quantification,
pressure digestion . 38
B.1 General . 38
B.2 Interferences . 38
B.2.1 General . 38
B.2.2 Spectral line interference . 38
B.2.3 Ionization interferences . 38
B.2.4 Physical interferences . 38
B.3 Quantification and matrix matching . 39
B.3.1 General . 39
B.3.2 Calibration curve . 39
B.3.3 Matrix matching . 39
B.3.4 Standard addition . 39
B.4 Pressure digestion conditions . 40
B.4.1 General . 40
B.4.2 Initial sample mass and acid volumes. 40
B.4.3 Digestion temperature . 40
B.4.4 Digestion time . 40
B.4.5 Digestion solution. 40
B.4.6 Blank solution . 40
Bibliography . 41

European foreword
This document (EN 15621:2017) has 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 February 2018, and conflicting national standards
shall be withdrawn at the latest by February 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 15621:2012.
WARNING — The method described in this standard implies the use of reagents that pose a hazard to
health. The standard does not claim to address all associated safety problems. It is the responsibility of
the user of this standard to take appropriate measures for the health and safety protection of the
personnel prior to use of the standard and to ensure that regulatory and legal requirements are
complied with.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
1 Scope
This European Standard specifies a method for the determination of the elements calcium, sodium,
phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt in animal
feeding stuffs by inductively coupled plasma atomic emission spectrometry (ICP-AES) after pressure
digestion.
The method was fully statistically tested and evaluated for the elements calcium, sodium, phosphorus,
magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt within the following 11
animal feeds: 2 complete feeds (pig feed, sheep feed), 3 complementary feeds (3 mineral feeds), 1
mineral premixture, 3 feed materials (MgO, phosphate, CaCO ) and 2 feed additives (CuSO , bentonite).
3 4
For elements with a HORRAT values higher than 2 (e.g. potassium and sulphur, see Annex A), the
method is more applicable as a screening method and not for confirmatory purposes.
Other elements like molybdenum, lead, cadmium, arsenic were not fully statistically tested and
evaluated within 11 animal feeding stuff samples because these elements did not occur in
concentrations higher than the limit of quantification in most of these samples. A single laboratory
validation is therefore necessary for the use of this multi element method for these elements.
For the determination of extractable lead in minerals and feeds, containing phyllosilicates (e.g. kaolinite
clay) wet digestion with nitric acid should be used.
The method limit of quantification for each element is dependent on the sample matrix as well as on the
instrument. The method is not applicable for determination of low concentrations of elements. A limit of
quantification of 1 mg/kg should normally be obtained.
NOTE 1 This method can also be used for the determination of minerals in products with high mineral content
(>5 %). For this purpose the accuracy of the method has to be checked individually. Other more matrix-specific
analytical techniques are also available.
NOTE 2 Results using this European Standard EN 15621 may be higher than those obtained when applying
EN 15510 as pressure digestion is used in EN 15621.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its 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 (ISO 3696)
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
For the purposes of this document the terms and definitions given in the European legislation apply.
4 Principle
For the determination of calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc,
copper, manganese, cobalt, molybdenum, lead, cadmium and arsenic a test portion of the sample is
digested under pressure.
The concentration of the elements is determined by inductively coupled plasma atomic emission
spectrometry (ICP-AES) using external calibration or standard addition technique.
5 Reagents
Use only reagents of recognized analytical grade, and water conforming to grade 2 of EN ISO 3696.
WARNING —The use of this European Standard can involve hazardous materials, operations and
equipment. This standard does not purport 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.
5.1 Nitric acid, concentrated, not less than 65 % (m/m), c(HNO ) = 14,4 mol/l, having a density of
approximately ρ (HNO ) 1,42 g/ml.
5.2 Nitric acid solution of 2 % (v/v), to be prepared: pipette 20 ml nitric acid concentrated (5.1) in a
1 000 ml volumetric flask (6.4) and fill to the mark with water.
5.3 Hydrogen peroxide, not less than 30 % (m/m).
5.4 Element stock solutions
Ca, Na, P, Mg, K, S, Fe, Zn, Cu, Mn, Co, Mo, Cd, Pb, As
c = 1 000 mg/l.
The user should choose a suitable stock solution. Both single-element stock solutions and multi-element
stock solutions with adequate specification stating the acid used and the preparation technique are
commercially available. It is advisable to use certified stock solutions.
NOTE Element stock solutions with concentrations different from 1 000 mg/l are also acceptable.
5.5 Standard solutions
Depending on the scope, different multi-element standard solutions may be necessary. In general, when
combining multi-element standard solutions, their chemical compatibility and the possible hydrolysis of
the components shall be regarded. Spectral interferences from other elements present in multi-element
standards also need to be considered.
Various combinations of elements at different concentrations can be used, provided that the stock
solutions (5.4) are diluted with the same acid and equal concentration as the acid in the test solution to
a range of standards that covers the concentrations of the elements to be determined.
The multi-element standard solutions are considered to be stable for several months, if stored in the
dark.
6 Apparatus
Usual laboratory apparatus and, in particular, the following:
NOTE For the determination of sodium in low concentrations it is advisable not to use glassware since
glassware can be a source of sodium contamination.
6.1 Laboratory grinder
6.1.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 0,5 mm, e.g. a
knife mill or equivalent.
6.1.2 Laboratory grinder, capable of grinding to a particle size of less than or equal to 0,1 mm, e.g. a
ball mill or equivalent.
NOTE Elements in the sample (e.g. adsorption, contamination) which have to be analysed.
6.2 Analytical balance, capable of weighing to an accuracy of 1 mg.
6.3 Pressure digestion apparatus, commercially available.
The apparatus shall be tested for safety pressure vessels made of acid-resistant materials and having
holders for the sample of acid-resistant material with low level of contamination by elements to be
determined. Apparatus is available which uses a high-pressure incinerator with or without ambient
autoclave pressure.
Instead of polytetrafluoroethylene (PTFE) holders, it is better to use graduated quartz holders,
fluorinated ethylene propylene (FEP) holders or perfluoroalkoxy (PFA) holders. Quartz is advisable to
be used for decomposition temperatures above 230 °C.
6.4 One-mark volumetric flasks, of capacity 1 000 ml.
6.5 Inductively coupled plasma – Atomic Emission Spectrometer.
The instrument shall be equipped with radial plasma as a minimum requirement; axial plasma is
equally acceptable. Background correction shall also be performed when necessary. Settings of the
working conditions (e.g. viewing height, gas flows, RF or plasma power, sample uptake rate, integration
time, and number of replicates) shall be optimized according the manufacturer’s instructions.
6.6 Freeze drying equipment, capable of freeze-drying liquid animal feeding stuffs.
6.7  Oven for pre-drying, capable to hold a temperature of (70 ± 5) °C
6.8 Breaker, of capacity 250ml
6.9 Electric hot plate, with temperature control
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in EN ISO 6497 [1].
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
8 Preparation of the test sample
8.1 General
Prepare the test sample in accordance with EN ISO 6498:
— The grinding must be done in conditions such that the substance is not appreciably heated and that
no contamination takes place by the grinding tools;
— The operation is to be repeated as many times as is necessary and it must be effected as quickly as
possible in order to prevent any gain or loss of constituents (water);
— The whole ground product is placed in a flask made of e.g. polypropylene, 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 must be thoroughly
mixed for reasons of homogeneity. Since a maximum of 0,5 g of sample is used for the digestion it is
of the utmost importance to have a homogeneous sample in order to take a representative sub
sample.
8.2 Animal feeding stuffs which can be ground as such
Grind the laboratory sample (usually 500 g), using a laboratory grinder (6.1.2) or mortar until a particle
size of 0,5 mm or less has been reached.
8.3 Liquid animal feeding stuffs
8.3.1 General
Liquid feeding stuffs shall be pre-dried according to the procedure described in 8.3.2 or freeze-dried
according to the procedure described in 8.3.3.
8.3.2 Pre-drying
Pre-dry the laboratory sample at a temperature of (70 5) °C during at least 16 h to reduce the
±
moisture content using an oven (6.7). The mass of the sample before and after the pre-drying is to be
determined using an analytical balance (6.2). Grind the pre-dried sample in accordance with 8.2.
8.3.3 Freeze-drying
Freeze-dry the laboratory sample following the instructions of the freeze-drying equipment (6.6). The
mass of the sample before and after the freeze-drying is to be determined using an analytical balance
(6.2). Grind the freeze-dried sample in accordance with 8.2.
Mineral, except mineral products containing crystalline water, e.g. MgCl .6H O, shall be ground using a
2 2
laboratory grinder (6.1.2) or mortar until a particle size of 0,5 mm or less has been reached. Mineral
products containing crystalline water should not be ground.
9 Procedure
9.1 Pressure digestion – Preparation of the blank test solution and the test solution
9.1.1 General
Match the initial sample mass to the capacity of the digestion vessel, with the manufacturer's
instructions being strictly observed for safety reasons. Determine the necessary digestion temperature
and digestion time (EN ISO 13805 [2], see Annex B).
9.1.2 Example of microwave digestion
When using 100 ml vessels, weigh about 0,5 g of the prepared test sample to the nearest 1 mg. Add 3 ml
of nitric acid (5.1) and 0,5 ml of hydrogen peroxide (5.3), seal the digestion vessel and the pressure
holders in the correct manner. Leave to pre-digest outside the microwave for about 30 min. Apply low
microwave energy at the beginning of the digestion and slowly raise the energy to the maximum power,
e.g. start with 100 W, raise up to 600 W within 5 min, hold for 5 min, raise to 1 000 W, hold for 10 min,
cool down for minimum 20 min to 25 min. Treat a blank in the same way.
Dilute the digestion solution accordingly with water. The solution obtained after dilution is called the
test solution. Proceed in accordance with 9.2.
9.1.3 Example of a high pressure digestion
When using a 100 ml vessel, weigh about 0,5 g of the prepared test sample to the nearest 1 mg. Add
3 ml of nitric acid (5.1), seal the digestion vessel and the pressure vessel in the correct manner and heat
from room temperature to 150 °C in 60 min, then to 300 °C in 40 min and keep 300 °C for 90 min before
cooling down. Treat a blank in the same way.
Dilute the digestion solution accordingly with water. The solution obtained after dilution is called the
test solution. Proceed in accordance with 9.2.
9.2 Extractable lead in minerals and feeds containing phyllosilicates (e.g. kaolinite clay)
– extraction with diluted nitric acid
Weigh about 2 g of the prepared test sample to the nearest 1 mg into a beaker of 250 ml. Add 16 ml
diluted nitric acid (1 volume of concentrated nitric acid, not less than 65 % mass fraction with 1 volume
of water). Add about 70 ml of water. Cover the beaker (6.8) with a watch-glass and boil for 30 min on an
electric hot plate with temperature control (6.9). Allow to cool. Transfer the liquid into a 100 ml
volumetric flask (6.4), rinse the beaker and the watch-glass several times with water. Dilute to the mark
with water. After homogenizing, filter through a dry folded filter paper into a dry conical flask. Use the
first portion of the filtrate to rinse the glassware and discard that part. If the determination is not
carried out immediately, the conical flask with the filtrate shall be stoppered. Treat a blank in the same
way.
9.3 Calibration
9.3.1 General
Calibration shall be performed by means of external calibration or standard addition technique. In
general the calibration curve should be linear. Using a nonlinear calibration function is possible if it is
well-described, except when using the standard addition. Appropriate matrix matching of the
calibration solutions shall be performed if an external calibration method is used (see Annex B).
9.3.2 External calibration
The calibration is performed with at least two calibration solutions, of which, one is a blank calibration
solution. If the working range is not linear, the calibration should be performed with a blank calibration
solution and at least three equidistant calibration solutions.
9.3.3 Standard addition technique
The standard addition curve should consist of at least two points, of which, one is an addition. For those
elements whose concentration is near the limit of quantification, the standard addition curve should
consist of at least four points, of which, three are additions. If three additions are used, the
concentration of the highest standard should be 3 to 5 times the concentration in the sample solution.
9.4 Determination
9.4.1 General
Analytical lines, selectivity, limits of detection and quantification, precision, linear working area, and
interferences have to be established before operating the ICP-AES system.
9.4.2 Determination by inductively coupled plasma – atomic emission spectrometry
9.4.2.1 General
Table 1 gives relevant analytical lines and possible interferences for the determination with ICP-AES.
Other wavelengths than those specified in Table 1 can be used (see also Annex B).
Table 1 — Selected emission wavelengths and interferences for determination with ICP-AES
Wavelength of Wavelength of emission
Element Interference Element Interference
emission (nm) (nm)
315,887 Co  257,610 Fe, Mo, Cr
Mn
Ca 317,933 Fe, V  293,306 Al, Fe
393,366   202,030 Al, Fe
Mo
Co 228,616 Ti  204,598
324,754 Ti, Fe  330,237
Cu
327,396   Na 588,995
238,200 Co  589,592 Ar
Fe
259,940   178,287 I
766,490 Mg, Ar  213,618 Cu, Fe, Mo, Zn
K P
769,900   214,914 Cu, Al, Mg
279,079   177,428 Cu
Mg 279,553   181,972
S
285,213 Fe  182,036
188,979   206,200
As Zn
189,042   213,856 P
193,696   Cd 214,438
197,197    226,502
Pb 216,999    228,802
220,353
261,418 Al, Co, Ti
9.4.2.2 External calibration method
Aspirate the blank test solution (9.1), the calibration solutions prepared from the standard solutions
(5.5), and the test solution (9.1) in ascending order separately into the plasma and measure the
emission of the element to be determined using an external calibration (9.4.2.2).
NOTE If the test solution contains residues, decant the solution into the test tube from which the test solution
is aspirated into the ICP. Use the first decanted portion to rinse the test tube.
Perform at least two replicates. Average the values if the values fall within an accepted range. After each
measurement, aspirate water or nitric acid solution (5.2).
9.4.2.3 Standard addition technique
Aspirate the blank test solution (9.1), the test solution (9.1), and the test solution prepared by standard
addition technique (9.4.2.3) in ascending order separately into the plasma, and measure the emission of
the element to be determined.
NOTE If the test solution (9.1) contains residues, decant the solution into the test tube from which the test
solution is aspirated into the ICP. Use the first decanted portion to rinse the test tube.
Perform at least two replicates. Average the values if the values fall within an accepted range. After each
measurement, aspirate water or nitric acid solution (5.2).
10 Calculation and expression of the results
10.1 General
Net signal is defined as the number of counts per second at the selected wavelength, corrected for
background contributions.
10.2 External calibration
In the case of a linear calibration curve constructed with one blank calibration solution and one
calibration solution, the calibration function can be described as follows:
S= c×+ba (1)
st st
where
is the net signal of the calibration solution;
S
st
is the concentration, in mg/l, of the calibration
c
st
solution;
a
is the intersection;
is the slope.
b
Calculate the element concentration c , in mg/l, in the test solution (9.1) using the slope and the
f
intersection a found in (1) as follows:
S
fa−
c = (2)
f
b
where
is the net signal of the test solution.
S
f
10.3 Standard addition method with only one addition
In the most simple case of standard addition, where only one addition is made, the element
concentration cf, in mg per l, in the test solution (9.1) is determined as follows:
S ××Vc
0 ss
c = (3)
f
SS−×V
( )
10 f
where
is the concentration, in mg/l, of the standard solution;
c
s
is the volume, in l, of the standard solution added;
V
s
is the volume, in l, of the test solution (9.1) used to prepare the solution without addition;
V
f
is the net signal of the solution without addition;
S
is the net signal after addition.
S
10.4 Standard addition method with several additions
In case of several additions, regression techniques on the linear model of variable y as a function of
variable x, have to be used to determine the element concentration of the test solution (9.1). Generally,
this model can be written as:
y= a+bx× (4)
i i
a
is the intersection;
is the slope;
b
In this particular case of three standard additions,
yS= (for i = 0, 1, 2, 3); (5)
ii
x cV× (for i = 0, 1, 2, 3); (6)
i si
where
is the concentration, in mg/l, of the standard solution;
c
s
are the various volumes, in litres, of the standard solution added;
V
i
are the net signals after the various additions.
S
i
The values of a and b can then be calculated as follows:
ny×− x y
i ∑ ii∑
(7)
b=
nx×− x
( )
∑∑ii
y−×b x
∑∑i i
a= (8)
n
where
n
is the number of solutions measured (n= 4 in case of three additions).
The element concentration c , in mg/l, of the test solution (9.1) can then be found using the following
f
equation:
a
b
c = (9)
f
V
f
where
is the volume, in l, of the test solution (9.1) used to prepare the solution without
V
f
addition.
10.5 Calculation of the element content in the sample
The element content for the minerals Ca, Na, P, Mg, K and S in the sample or mass fraction of element
w , expressed in percentage, is determined using the following equation:
elem
=
cc−
( )
f bi
wV××0,0001 (10)
elem t
m
The element content for the other elements Fe, Zn, Cu, Mn, Co, Mo, Cd, As, Pb in the sample or mass
fraction of element w , expressed in mg/kg, is determined using the following equation:
elem
cc−
( )
f bi
(11)
wV×
elem t
m
where
is the concentration, in mg/l, of the test solution (9.1), as determined using Formula (2) or
c
f
(3) or (9);
is the concentration, in mg/l, of the blank solution;
c
bl
m
is the mass of sample, in kg, taken for the extraction by digestion, and corrected for water
content;
is the total volume, in l, of the test solution (9.1)
V
t
If the test solution (9.1) has been diluted further, take into account the dilution factor.
If the sample has been pre-dried or freeze-dried (8.3), recalculate the result to the fresh weight of the
sample taking into account the loss of moisture during pre-drying or freeze-drying.
11 Precision
11.1 Inter-laboratory test
An inter-laboratory test has been carried out in 2007. Details of inter-laboratory tests on precision of
the method are summarized in Annex A. The values derived from these tests may not be applicable to
concentration ranges and matrices others than those given.
11.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method
on identical test material in the same laboratory by the same operator using the same equipment within
a short interval of time, will in not more than 5 % of the cases be greater than the repeatability limit r
given in Table 2 (Ca, K, Mg, Na, P, S) and Table 3 (Fe, Mn, Cu, Zn, Co, Mo, Cd, As, Pb).
11.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical
test material in different laboratories with different operators using different equipment, will in not
more than 5 % of the cases be greater than the reproducibility limit R given in Table 2 (Ca, Na, Mg, P, K,
S) and Table 3 (Fe, Mn, Cu, Zn, Co, Mo, Cd, As, Pb).
=
=
Table 2 — Precision data – Ca, K, Mg, Na, P, S
Ca
_
Mean, x (%)
Samples r (%) R (%)
Pig feed 1,21 0,17 0,31
Sheep feed 1,12 0,10 0,17
MIN 1 24,1 0,8 2,9
MIN 2 18,1 0,8 2,2
MIN MIX 14,1 0,6 1,8
Com.Premix 4,59 0,53 1,60
CuSO4 < LOQ – –
Phosphate 21,7 0,7 1,9
MgO 1,36 0,11 0,31
CaCO 35,4 1,3 3,5
Bentonite 0,75 0,03 0,10
K
_
Mean, x (%)
Samples r (%) R (%)
Pig feed 0,73 0,06 0,17
Sheep feed 1,26 0,08 0,21
MIN 1 0,06 0,01 0,05
MIN 2 0,37 0,02 0,17
MIN MIX 0,05 0,01 0,04
Com.Premix 0,20 0,02 0,06
CuSO < LOQ – –
Phosphate 0,09 0,01 0,05
MgO 0,15 0,02 0,11
CaCO 0,33 0,04 0,29
Bentonite 0,07 0,01 0,05
Mg
_
Mean, x (%)
Samples r (%) R (%)
Pig feed 0,18 0,01 0,02
Sheep feed 0,35 0,02 0,05
MIN 1 3,75 0,13 0,85
MIN 2 2,85 0,14 0,40
MIN MIX 12,2 0,6 2,1
Com.Premix 0,13 0,01 0,05
CuSO < LOQ – –
Phosphate 0,70 0,03 0,11
MgO 48,9 2,2 6,2
CaCO 0,30 0,02 0,09
Bentonite 0,70 0,06 0,91
Na
_
Mean, x (%)
Samples r (%) R (%)
Pig feed 0,24 0,02 0,05
Sheep feed 0,30 0,02 0,05
MIN 1 6,90 0,29 1,25
MIN 2 8,88 0,26 1,68
MIN MIX 11,9 0,7 1,6
Com.Premix 0,96 0,05 0,15
CuSO < LOQ – –
Phosphate 0,12 0,01 0,03
MgO < LOQ – –
CaCO < LOQ – –
Bentonite 1,20 0,06 0,19
P
_
Mean, x (%)
Samples r (%) R (%)
Pig feed 0,51 0,04 0,71
Sheep feed 0,69 0,04 0,10
MIN 1 3,37 0,27 0,63
MIN 2 2,16 0,14 0,30
MIN MIX 3,83 0,13 0,76
Com.Premix 0,15 0,02 0,07
CuSO < LOQ – –
Phosphate 21,9 1,2 2,3
MgO 0,047 0,004 0,014
CaCO 0,023 0,003 0,007
Bentonite 0,020 0,003 0,012
S
_
Samples Mean, x (%) r (%) R (%)
Pig feed 0,19 0,02 0,04
Sheep feed 0,29 0,02 0,06
MIN 1 0,78 0,06 0,37
MIN 2 0,17 0,01 0,15
MIN MIX 0,35 0,04 0,10
Com.Premix 2,64 0,26 0,67
CuSO 12,1 0,4 2,6
Phosphate 0,98 0,08 0,44
MgO < LOQ – –
CaCO 0,39 0,02 0,32
Bentonite 0,22 0,02 0,06
Table 3 — Precision data – Co, Cu, Fe, Mn, Mo, Zn, Cd, As, Pb
Co
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed < LOQ – –
Sheep feed 1,85 0,17 1,05
MIN 1 24,0 1,3 15,0
MIN 2 13,6 1,0 6,2
MIN MIX 47,4 9,2 36,4
Com.Premix 21 540 1 988 3 252
CuSO < LOQ – –
Phosphate < LOQ – –
MgO 2,65 0,78 2,29
CaCO 5,75 0,77 3,44
Bentonite 1,46 0,36 1,66
Cu
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed 22,6 10,1 11,6
Sheep feed 11,6 1,9 5,4
MIN 1 947 59 209
MIN 2 574 30 156
MIN MIX 850 77 335
Com.Premix 2 024 123 397
CuSO 264 900 13 920 60 750
Phosphate 11,6 1,5 6,1
MgO 28,4 6,6 14,1
CaCO 6,26 1,35 7,80
Bentonite 6,17 2,10 7,80
Fe
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed 386 67 115
Sheep feed 277 47 96
MIN 1 2 512 202 669
MIN 2 3 205 287 860
MIN MIX 3 240 431 895
Com.Premix 15 940 1 938 3 942
CuSO 24,2 6,1 12,7
Phosphate 2 074 63 380
MgO 21 280 1,018 4 067
CaCO 6 939 423 1 565
Bentonite 11 390 1 093 13 790
Mn
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed 89,6 13,1 36,9
Sheep feed 95,8 8,4 21,5
MIN 1 2 444 163 663
MIN 2 2 542 150 662
MIN MIX 2 143 177 491
Com.Premix 15 590 1 940 6 526
CuSO < LOQ – –
Phosphate 118 2 39
MgO 718 38 206
CaCO 1 509 56 402
Bentonite 88,0 8,7 12,0
Mo
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed < LOQ – –
Sheep feed < LOQ – –
MIN 1 < LOQ – –
MIN 2 < LOQ – –
MIN MIX < LOQ – –
Com.Premix 17,920 2 351 6 483
CuSO < LOQ – –
Phosphate < LOQ – –
MgO < LOQ – –
CaCO < LOQ – –
Bentonite < LOQ – –
Zn
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed 127 12 47
Sheep feed 155 14 46
MIN 1 6 844 525 1 595
MIN 2 5 440 348 1 144
MIN MIX 3 789 247 940
Com.Premix 10 310 611 1 559
CuSO < LOQ – –
Phosphate 220 12 86
MgO < LOQ – –
CaCO 11,5 2,7 10,8
Bentonite 83,5 19,5 33,0
Cd
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed < LOQ – –
Sheep feed < LOQ – –
MIN 1 37,9 1,7 19,9
MIN 2 < LOQ – –
MIN MIX < LOQ – –
Com.Premix < LOQ – –
CuSO < LOQ – –
Phosphate 4,90 0,40 1,96
MgO < LOQ – –
CaCO < LOQ – –
Bentonite < LOQ – –
As
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed < LOQ – –
Sheep feed < LOQ – –
MIN 1 < LOQ – –
MIN 2 < LOQ – –
MIN MIX < LOQ – –
Com.Premix < LOQ – –
CuSO < LOQ – –
Phosphate < LOQ – –
MgO < LOQ – –
CaCO 9,42 0,99 4,82
Bentonite < LOQ – –
Pb
_
Mean, x
Samples r (mg/kg) R (mg/kg)
(mg/kg)
Pig feed < LOQ – –
Sheep feed < LOQ – –
MIN 1 < LOQ – –
MIN 2 < LOQ – –
MIN MIX < LOQ – –
Com.Premix < LOQ – –
CuSO < LOQ – –
Phosphate 3,97 0,84 3,32
MgO < LOQ – –
CaCO < LOQ – –
Bentonite 35,6 3,8 15,3
12 Test report
The test report shall contain at least the following information:
a) the test method used, with reference to this European Standard;
b) all information necessary for the complete identification of the sample;
c) any particular points observed in the course of the test;
d) all operation details not specified in this document, or regarded as optional, together with details of
any incidents which might have affected the results;
e) the results obtained of the determination, expressed as mass fraction of animal feeding stuff w ,
elem
in percentage for the minerals or in mg/kg for the trace elements and heavy metals
Annex A
(informative)
Results of the inter-laboratory test
An inter-laboratory comparison has been organized by Technische Universität München, Research
Center for Nutrition and Food Sciences, Bioanalytic Weihenstephan in 2007 with 20 participating
laboratories and 11 different animal feeding stuffs, including 2 complete feeds (pig feed, sheep feed), 3
complementary feeds (3 mineral feeds), 1 mineral premixture, 3 feed materials (MgO, phosphate,
CaCO ) and 2 feed additives (CuSO , bentonite). The samples were homogenized centrally and
3 4
distributed to the participants. The tests yielded the data given in Tables A.1 and A.15. Repeatability
and reproducibility were calculated according to ISO 5725-1 [3].
Details are given only in the final report [4].
Table A.1 — Statistical results of an inter-laboratory test – Calcium (Ca)
Sample No. 1 2 3 4 5
...