EN ISO 13906:2008

Animal feeding stuffs - Determination of acid detergent fibre (ADF) and acid detergent lignin (ADL) contents (ISO 13906:2008)

ISO 13906:2008 specifies a method for the determination of acid detergent fibre (ADF) insoluble residue and acid detergent lignin (ADL) in all types of animal feeding stuffs. The limit of determination is 1 % mass fraction for ADF and 1,5 % mass fraction for ADL.
A gravimetric routine and reference method is used.

Futtermittel - Bestimmung des Gehalts an Säure-Detergens-Faser (ADF) und Säure-Detergens-Lignin (ADL) (ISO 13906:2008)

Diese Internationale Norm legt ein Verfahren zur Bestimmung des Gehalts an unlöslichen Säure-Detergens-Faserrückständen (ADF) und Säure-Detergens-Lignin (ADL) in allen Futtermittelarten fest. Die Bestimmungs¬grenze für ADF ist 1 % Massenanteil und 1,5 % Massenanteil für ADL.
Ein gravimetrisches Routine- und Referenzverfahren wird angewendet.

Aliments des animaux - Détermination des teneurs en fibres au détergent acide (ADF) et en lignine sulfurique (ADL) (ISO 13906:2008)

L'ISO 13906:2008 spécifie une méthode de détermination de la teneur en résidus fibreux insolubles dans le détergent acide [teneur en ADF (pour «Acid Detergent Fibre»)] et de la teneur en lignine sulfurique [teneur en ADL (pour «Acid Detergent Lignin»)] dans tous types d'aliments des animaux. La limite de détermination est de 1 % en fraction massique pour l'ADF et de 1,5 % en fraction massique pour l'ADL.
Une méthode gravimétrique et une méthode de référence sont utilisées.

Krma - Določevanje vsebnosti v kislem detergentu netopnih vlaken (ADF) in v kislem detergentu netopnega lignina (ADL) (ISO 13906:2008)

General Information

• Status: Published
• Publication Date: 14-Jul-2008
• Withdrawal Date: 30-Jan-2009
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 2 - Composition
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Jul-2008
• Completion Date: 15-Jul-2008
• Ref Project: SIST EN ISO 13906:2008 - Animal feeding stuffs - Determination of acid detergent fibre (ADF) and acid detergent lignin (ADL) contents (ISO 13906:2008)

Overview

EN ISO 13906:2008 (ISO 13906:2008) specifies a gravimetric routine and reference method for determining acid detergent fibre (ADF) and acid detergent lignin (ADL) in all types of animal feeding stuffs. The method is a two‑stage chemical/gravimetric procedure: ADF is measured after treatment with a cationic detergent in 0.5 mol/L sulfuric acid, and ADL is determined by solubilizing remaining cellulose with 72 % (m/m) sulfuric acid to leave lignin. The standard sets the limits of determination at 1 % mass fraction for ADF and 1.5 % mass fraction for ADL.

Key topics and technical requirements

• Principle: two‑stage treatment - removal of acid‑labile carbohydrates and non‑complexed protein (ADF), then cellulose solubilization to isolate lignin (ADL).
• Limits of determination: ADF ≥ 1 %, ADL ≥ 1.5 % (mass fraction).
• Reagents:
  • Acid detergent solution: 20 g cetyl(trimethyl)ammonium bromide per 1 L of 0.5 mol/L H2SO4.
  • 72 % (m/m) sulfuric acid (≈12.00 mol/L) for lignin determination (standardization and density guidance provided).
  • Filtration aid (diatomaceous earth), acetone, n‑octanol (antifoam).
• Apparatus:
  • Analytical balances (0.1 mg readability), mills producing 0.22–0.26 mm mean particle size, air‑ventilated ovens (103 °C or 130 °C for ash/moisture; < 60 °C drying for samples to avoid artefact fibres), reflux units, fritted‑disk crucibles (40–60 µm porosity), incinerator (525 °C ± 15 °C).
• Sample preparation:
  • Follow ISO 6498 for test sample preparation; sampling guidance refers to ISO 6497.
  • Drying < 60 °C for moist materials to prevent artefact fibre and lignin; pre‑extraction recommended for high‑fat samples.
• Quality and precision: gravimetric weighing of residues and ashing are core operations; the standard includes precision/interlaboratory results.

Applications and users

• Routine and reference testing in feed analysis laboratories for quality control and composition labelling.
• Animal nutritionists and feed formulators assessing fibre and lignin contributions to digestibility and ration formulation.
• Regulatory bodies and certification labs verifying compliance with feed standards.
• Research laboratories studying fibre fractions, forage quality, and rumen digestibility.

Related standards

• ISO 6498 - Preparation of test samples (normative reference).
• ISO 6497 - Recommended sampling methods for animal feeding stuffs.
• CEN/TC 327 - Committee on animal feeding stuffs methods of sampling and analysis.

Note: ISO 13906:2008 contains safety warnings - users must follow appropriate laboratory safety when handling concentrated sulfuric acid and other hazardous reagents. Keywords: EN ISO 13906:2008, ISO 13906, ADF, ADL, acid detergent fibre, acid detergent lignin, animal feeding stuffs, gravimetric method, feed analysis.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2008
.UPD'RORþHYDQMHYVHEQRVWLYNLVOHPGHWHUJHQWXQHWRSQLKYODNHQ$')LQY
NLVOHPGHWHUJHQWXQHWRSQHJDOLJQLQD$'/,62
Animal feeding stuffs - Determination of acid detergent fibre (ADF) and acid detergent
lignin (ADL) contents (ISO 13906:2008)
Futtermittel - Bestimmung des Gehalts an Säure-Detergens-Faser (ADF) und Säure-
Detergens-Lignin (ADL) (ISO 13906:2008)
Aliments des animaux - Détermination des teneurs en fibres au détergent acide (ADF) et
en lignine sulfurique (ADL) (ISO 13906:2008)
Ta slovenski standard je istoveten z: EN ISO 13906:2008
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 13906
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2008
ICS 65.120
English Version
Animal feeding stuffs - Determination of acid detergent fibre
(ADF) and acid detergent lignin (ADL) contents (ISO
13906:2008)
Aliments des animaux - Détermination des teneurs en Futtermittel - Bestimmung des Gehalts an Säure-
fibres au détergent acide (ADF) et en lignine sulfurique Detergens-Faser (ADF) und Säure-Detergens-Lignin (ADL)
(ADL) (ISO 13906:2008) (ISO 13906:2008)
This European Standard was approved by CEN on 11 July 2008.
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 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13906:2008: E
worldwide for CEN national Members.

Contents Page
Foreword.3

Foreword
This document (EN ISO 13906:2008) has been prepared by Technical Committee ISO/TC 34 "Agricultural
food products" in collaboration with 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 January 2009, and conflicting national standards shall be withdrawn at
the latest by January 2009.
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 patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 13906:2008 has been approved by CEN as a EN ISO 13906:2008 without any modification.

INTERNATIONAL ISO
STANDARD 13906
First edition
2008-07-15
Animal feeding stuffs — Determination of
acid detergent fibre (ADF) and acid
detergent lignin (ADL) contents
Aliments des animaux — Détermination des teneurs en fibres au
détergent acide (ADF) et en lignine sulfurique (ADL)

Reference number
ISO 13906:2008(E)
©
ISO 2008
ISO 13906:2008(E)
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ii © ISO 2008 – All rights reserved

ISO 13906:2008(E)
Contents Page
Foreword. iv
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Principle.2
5 Reagents.2
6 Apparatus .3
7 Sampling.4
8 Preparation of test sample.4
9 Procedure .4
10 Calculation and expression of results.6
11 Precision.7
12 Test report .9
Annex A (informative) Results of interlaboratory test.10
Annex B (informative) Results of interlaboratory test.14
Bibliography .17

ISO 13906:2008(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 13906 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal
feeding stuffs.
iv © ISO 2008 – All rights reserved

INTERNATIONAL STANDARD ISO 13906:2008(E)

Animal feeding stuffs — Determination of acid detergent fibre
(ADF) and acid detergent lignin (ADL) contents
WARNING — The use of this International Standard may involve hazardous materials, operations and
equipment. This International Standard does not purport to address any safety risks associated with
its use. It is the responsibility of the user of this International Standard to establish appropriate safety
and health practices and determine the applicability of local regulatory limitations prior to use.
1 Scope
This International Standard specifies a method for the determination of acid detergent fibre (ADF) insoluble
residue and acid detergent lignin (ADL) in all types of animal feeding stuffs. The limit of determination is 1 %
mass fraction for ADF and 1,5 % mass fraction for ADL.
A gravimetric routine and reference method is used.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 6498, Animal feeding stuffs — Preparation of test samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
acid detergent fibre content
ADF content
mass fraction of fibrous residue obtained after treatment with cationic detergent in 0,5 mol/l sulfuric acid,
primarily consisting of cellulose, lignin and insoluble protein complexes
NOTE The ADF mass fraction is expressed as a percentage.
3.2
acid detergent lignin content
ADL content
mass fraction of residue remaining after cellulose and other organic matter is solubilized by 72 % mass
fraction (12,00 mol/l) sulfuric acid
NOTE The ADL mass fraction is expressed as a percentage.
ISO 13906:2008(E)
4 Principle
ADF is determined in the first stage of the method.
Cationic detergent solution is used to remove acid-labile carbohydrates, protein that is not complexed into
Maillard products (heat damaged), and fats. The remaining fibrous residue is primarily cellulose and lignin
(plant products) or insoluble protein complexes (animal products and heat-damaged feeds). The residue is
weighed for the determination of ADF.
In the second stage, the remaining residue is solubilized by 72 % mass fraction (12,00 mol/l) sulfuric acid,
leaving the lignin (ADL) which is determined gravimetrically.
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or deionized
water or water of at least equivalent purity.
5.1 Acid detergent solution. Add 20 g cetyl(trimethyl)ammonium bromide (technical grade) to 1 l of
0,5 mol/l sulfuric acid, previously standardized. Agitate to aid dissolution.
5.2 Sulfuric acid, 72 % mass fraction (12,00 mol/l).
Standardize sulfuric acid (H SO ) to a relative density of 1,634 at 20 °C or 12,00 mol/l as follows.
2 4
Calculate the mass, in grams, of acid, m(H SO ), and the mass, in grams, of water, m(H O), needed to
2 4 2
prepare 1 000 ml of solution using Equations (1) and (2):
100××98,08 12
m(H SO )= (1)
w(H SO )
where w(H SO ) is the assay mass fraction of sulfuric acid, expressed as a percentage.
2 4
mm(H O)=×−(1000 1,634) (H SO ) (2)
where 1,634 is the relative density of 72 % mass fraction sulfuric acid.
Weigh water into a 1 000 ml volumetric flask and add the calculated amount of sulfuric acid slowly with
occasional swirling. Cool the flask in a water bath while adding the required mass of acid. Cool to 20 °C and
verify the volume. The meniscus should be within 0,5 cm of the calibration mark at 20 °C. If volume is too
large, remove 5 ml water and add 4,55 ml sulfuric acid. If volume is too small, remove 1,5 ml and add 2,5 ml
water. Repeat if necessary.
1)
5.3 Filtration aid, diatomaceous earth .
5.4 Acetone, technical grade.
5.5 n-Octanol, antifoaming agent.

1) Celite, acid washed, and Celite 545 AW are examples of suitable products available commercially. This information is
given for the convenience of users of this International Standard, and does not constitute an endorsement of these
products by ISO. Alternative products may be used if they can be demonstrated to give comparable results.
2 © ISO 2008 – All rights reserved

ISO 13906:2008(E)
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Analytical balance.
6.1.1 Analytical balance, capable of weighing to the nearest 1 mg, with readability to 0,1 mg.
6.1.2 Analytical balance, with a measuring range up to 2 500 g, with readability to 1 g, for weighing sulfuric
acid (5.2).
6.2 Mill, a cyclone mill or cutter mill or rotary mill or equivalent, giving a mean particle size of 0,22 mm to
0,26 mm.
6.3 Drying oven.
6.3.1 Air-ventilated oven, capable of operating at 103 °C ± 2 °C or 130 °C ± 2 °C.
6.3.2 Air-ventilated oven, capable of being maintained at 60 °C ± 2 °C.
To speed up the drying of moist samples without creating artefact fibres, a vacuum oven, maintained at
60 °C ± 2 °C may also be used.
6.4 Refluxing apparatus, with individual heating units and cold water condensers. Any conventional
apparatus suitable for crude fibre or amylase-treated neutral detergent fibre (aNDF) determinations is
acceptable. Calibrate heating unit settings so that 50 ml of water boils in 4 min to 5 min when using cold water
2)
condensers. A Fibertec -type apparatus can be used and should boil 50 ml of water within 10 min.
NOTE This setting can be expected to result in significant particle movement during refluxing.
6.5 Fritted-disk crucibles. Coarse porosity (pore size 40 µm to 60 µm) crucibles, 40 ml to 50 ml capacity,
2)
or Fibertec P2 (pore size 40 µm to 100 µm, 26 ml to 28 ml capacity). Clean new crucibles and ash at
525 °C ± 15 °C for 1 h. Clean crucibles after each use by ashing at 525 °C ± 15 °C for 3 h, removing ash by
inverting in a detergent solution and sonicating for 7 min to 10 min. Rinse crucibles in hot water, and soak in
water at room temperature for at least 30 min.
Occasionally test filtration rate as follows. Fill each crucible with 50 ml of distilled water (25 ml for
2)
Fibertec P2 crucibles) and record the time required to drain completely without vacuum [should be
2)
180 s ± 60 s for Gooch or 75 s ± 30 s for P2]. If drain time is < 100 s (or < 30 s for P2), discard crucible. If it
is < 120 s (or < 45 s for P2), check for cracks in fritted disk. If filtration takes > 240 s (or > 105 s for P2), clean
crucible with acid or alkaline cleaning solution (Reference [4]). If cleaning does not improve filtration rate,
discard crucible.
2)
6.6 Vacuum filter manifold. Suitable apparatus [e.g. Fibertec -type cold extraction unit] that allows
adequate soaking of fibrous residues.
6.7 Incineration furnace, 525 °C ± 15 °C.
6.8 Reflux beakers. As an alternative to a reflux apparatus (6.4), 600 ml Berzelius beakers with
condensers, e.g. made from 500 ml round-bottom flasks, may be used.

2) Example of a suitable product available commercially. This information is given for the convenience of users of this
International Standard, and does not constitute an endorsement of this product by ISO.
ISO 13906:2008(E)
7 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not a part of the method specified in this International Standard. A recommended sampling
method is given in ISO 6497.
8 Preparation of test sample
Prepare the test sample in accordance with ISO 6498.
Reduce laboratory sample to approximately 100 g (dry weight equivalent) and place half in a moisture-tight,
sealed container for total moisture determination. Dry remaining wet (> 15 % mass fraction moisture) materials
to < 15 % mass fraction moisture in an air-ventilated oven (6.3.2) at < 60 °C. Drying at < 60 °C is necessary to
prevent formation of artefact fibre and lignin. Grind dry (< 15 % mass fraction moisture) test samples using a
mill (6.2).
Grinding segregates materials with the most fibrous matter grinding through the screen last. Do not discard
material in the grinder, but add it to the ground test sample.
Pre-extraction is required for test samples containing > 10 % mass fraction fat, and is recommended for test
samples containing > 5 % mass fraction fat.
Weigh test samples into previously tared, oven-dried crucibles, place crucible on filtering manifold, extract four
times with 30 ml to 40 ml of acetone, allowing the material to soak in acetone for 3 min to 5 min each time,
vacuum to remove all traces of acetone, air-dry for 10 min to 15 min, and transfer residue to a reflux beaker
for fibre analysis. Use the same crucible to collect the fibre residue for each test portion after acid detergent
extraction.
2)
For the Fibertec -type apparatus, place the crucible in a cold extraction unit and fill the crucible with 25 ml
acetone (5.4). Leave for 3 min to 5 min and filter by applying vacuum. Repeat three times.
NOTE To simplify filtration, 1,00 g of filtration aid (5.3) can be added to the crucible before the sample.
9 Procedure
9.1 ADF
9.1.1 General
Dry empty crucibles in an oven (6.3.1) at 103 °C ± 2 °C for > 4 h [1 h if moved from the incineration furnace
(6.7)] and record tare mass, m .
9.1.2 Conventional apparatus
Weigh, into a Berzelius beaker (6.8), a test portion of dried or as-received material of mass 1 000 mg ± 2 mg,
and record the mass as m .
Materials with > 15 % mass fraction moisture should have the mass adjusted to provide an equivalent mass of
dry matter. If conversion of results to the dry matter basis is required, weigh a test portion for moisture
determination of the test sample at the same time.
Immediately before refluxing, add 100 ml of acid detergent solution (5.1) at room temperature. Heat to boiling
over 5 min to 10 min and, if necessary, reduce heat slightly and add 2 drops to 4 drops of n-octanol (5.5) to
avoid foaming, but provide moderate particle agitation. After 5 min to 10 min of refluxing, rinse down the sides
of the beaker using a fine stream of acid detergent solution (add < 5 ml). Reflux for 60 min ± 5 min from the
time when the solution has reached the boiling point.
4 © ISO 2
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