CEN/TC 327/WG 4 - Heavy metals, trace elements and minerals

This document specifies a procedure for the determination of inorganic arsenic in animal feeding stuffs by anion-exchange HPLC-ICP-MS following water bath extraction.
This method was successfully tested in the range of 0,149 mg/kg to 9,69 mg/kg in the following animal feed matrices: rice meal, seaweed meal, fish meal, grass meal, complete feed (marine-based), complete feed (cereal based) and a synthetic solution.
NOTE   Mineral feed matrices are not included in the scope of this method. It is good to perform a determination of the total arsenic content in such matrices.

This document specifies performance criteria for the selection of single-laboratory validated or collaborative-trial validated methods of analysis of elements and their chemical species in feed. The terms and definition of the relevant parameters for method validation are included. The performance requirements and characteristics are provided. This document may serve as a guide:
-   to assess the quality of new European Standard methods under validation;
-   to review the quality of previous collaborative trials;
-   to confirm the extension of the scope of an already published European Standard applied to other analyte concentrations or matrices; or
-   to evaluate the fitness-for-purpose of single-validated methods.
The performance criteria can apply to methods dedicated to the determination of heavy metals, trace elements, major elements and minerals.

This European Standard specifies a method for the determination of trace elements, heavy metals and other elements in animal feed by inductively coupled plasma mass spectrometry (ICP-MS). The method is used to determine As, Cd, Co, Cu, Fe, Hg, Mn, Mo, Pb, Se, Tl, U and Zn in the extraction solution after pressurized digestion. 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 described is suitable for use in quadrupole instruments equipped either with or without additional technology to reduce molecular ion interferences (e.g. collision or reaction technologies) as well as in high-resolution sector-field systems.
The method was fully statistically tested and evaluated in a collaborative trial comprising eight animal feeding stuff samples for the elements As, Cd, Co, Cu, Fe, Hg, Mn, Mo, Pb, Se, Tl, U and Zn. For elements with a HORRAT value higher than 2 (e.g. mercury, see Annex A) the method is more applicable as a screening method and not for confirmatory purposes. High-resolution sector-field ICP-MS was not tested in the validation ring trial.
The limit of quantification for each element is dependent on the sample matrix as well as the instrument. For the elements Co, Mn, Mo, Pb, Tl, U a limit of quantification of 0,10 mg/kg should normally be obtained, for the elements Fe and Zn 5,0 mg/kg, while for Cd 0,03 mg/kg, Hg 0,04 mg/kg and As 0,05 mg/kg should normally be quantifiable.
Details on the successfully tested working range for each element are described in this standard.

This European standard specifies a method for the determination of iodine in animal feeding stuffs by inductively coupled plasma mass spectrometry (ICP-MS) following extraction with an alkaline solution.
This method was successfully tested in the range of 0,70 to 631 mg/kg in following animal feeds: seaweed meal, mineral premixture, fish meal, plant based ingredient, marine based compound feed and a synthetic iodine solution.

This European Standard specifies the inductively coupled plasma atomic emission spectroscopy (ICP-AES) method for the determination of the elements calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead.
The elements calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead are extracted either in feeds mainly consisting of organic matter after dry ashing and dissolving in hydrochloric acid or in feeds mainly consisting of inorganic matter after wet digestion with hydrochloric acid.
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 was successfully tested for:
-   calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt and molybdenum in the following animal feeding stuffs: 2 complete feeds (pig feed, sheep feed), 1 feed material  (phosphate), 1 mineral premixture and 2 complementary feeds (2 mineral feeds),
-   lead in 2 feed materials (phosphate, CaCO3), 2 feed additives (Bentonite, CuSO4), 1 complementary feed (mineral feed)
The method detection limit for each element is dependent on the sample matrix and the instrument. The method is not applicable for the determination of a low concentration of elements. The limit of quantification should be 3 mg/kg or lower.
This method also applies for the determination in products with high element content (>5 %). For this purpose the accuracy of the method has to be checked individually.
NOTE 1   EN 15621 uses the pressure digestion mode, therefore lower results may be obtained with the described method in this standard.

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.

This European Standard specifies a method for the determination of the elements cadmium and lead in animal feeding stuffs by graphite furnace atomic absorption spectrometry (GF-AAS) after pressure digestion.
The method was successfully tested in the range of 0,015 to 5,65 mg/kg for Cd and 0,18 to 40,3 mg/kg for lead in 11 animal feeds: 2 complete feeds (pig feed, sheep feed), 2 complementary feeds (2 mineral feeds), 1 mineral premixture, 4 feed materials (MgO, 2 phosphates, CaCO3) and 2 feed additives (CuSO4, bentonite).
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 the instrument. For cadmium a limit of quantification of 0,05 mg/kg should normally be obtained while for lead, a limit of quantification of 0,5 mg/kg should be obtained.

This European Standard describes a procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin by Solid Phase Extraction (SPE) and Hydride Generation Atomic Absorption Spectrometry (HG-AAS). The method has been successfully tested in a collaborative trial with a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues < 2). The LOQ of the method is usually approximately 0,1 mg/kg or lower.

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) 574/2011[3].
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 ºC) 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 followed 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 for 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.

This European Standard specifies a method for the determination of mercury in animal feeding stuffs by Cold-Vapour Atomic Absorption Spectrometry (CVAAS) after microwave pressure digestion. The limit of quantification in the test solution should be 0,25 µg/l or lower. Using a test portion of 0,5 g and a volume of the test solution of 25 ml a limit of quantification of 0,0125 mg/kg or lower should be obtained.

This European Standard specifies a method for the determination of total arsenic in animal feeding stuffs by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion. The limit of quantification is 0,5 µg/l of the test solution. Using a test portion of 0,5 g, a volume of the test solution of 25 ml and an aliquot of 5 ml for pre-reduction the limit of quantification is 0,125 mg/kg in the feed material.
NOTE   For feed materials containing organic arsenic species from compounds of marine origin (i.e. arsenobetaine and tetramethylarsine oxide) a higher digestion temperature of the microwave system up to 300 °C may be necessary in order to enable the hydridisation of these arsenic compounds and in order to determine all different kinds of arsenic species in the corresponding feeding stuffs. Alternatively, the digestion procedure of Annex D can be used if the microwave system does not reach higher temperatures up to 300 °C to ensure complete mineralization for HGAAS determination.

This European Standard specifies a method for the determination of selenium in animal feeding stuffs by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion.
The method was successfully tested by an inter-laboratory study of CEN/TC 327/WG 4 in the range of 0,25 mg/kg to 74 mg/kg.
The limit of quantification is 0,5 µg/l of the test solution which corresponds to the calibration standard 2. Using a test portion of 0,5 g and a volume of the test solution of 25 ml after pressure digestion the limit of quantification is calculated as 0,125 mg/kg in the feed material.
NOTE   A lower limit of quantification could be achieved – each laboratory has to prove it.

This European Standard specifies a method for the determination of the minerals calcium, sodium, phosphorus, magnesium, potassium and sulphur and the elements iron, zinc, copper, manganese, 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 within 11 animal feeding stuff samples for the minerals calcium, sodium, phosphorus, magnesium, potassium and sulphur and the elements iron, zinc, copper, manganese and cobalt.
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.
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   This method can also be used for the determination of minerals in products with high mineral content (> 5 %), yet for this purpose other more precise analytical techniques are available.