BS 

EN ISO 17226‑1:2019

BSI Standards Publication

Leather - Chemical determination

of formaldehyde content

Part 1: Method using high performance liquid chromatography

BS EN ISO 17226‑1:2019 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN ISO 17226‑1:2019.
It is identical to ISO 17226‑1:2018. It supersedes BS EN ISO 17226‑1:2008,
which is withdrawn.
The UK participation in its preparation was entrusted to Technical
Committee TCI/69, Footwear & Leather.
A list of organizations represented on this committee can be obtained on
request to its secretary.
This publication does not purport to include all the necessary provisions
of a contract. Users are responsible for its correct application.
© The British Standards Institution 2019
Published by BSI Standards Limited 2019
ISBN 978 0 580 98550 8
ICS 59.140.30
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 28 February 2019.

Amendments/corrigenda issued since publication

Date Text affected
EUROPEAN STANDARD EN ISO 17226‑1
NORME EUROPÉENNE
EUROPÄISCHE NORM February 2019

ICS 59.140.30 Supersedes EN ISO 17226‑1:2008

English Version

Leather - Chemical determination of formaldehyde

content - Part 1: Method using high performance liquid
chromatography (ISO 17226-1:2018)
Cuir - Dosage chimique du formaldéhyde - Partie Leder - Chemische Bestimmung des
1: Méthode par chromatographie en phase liquide Formaldehydgehalts - Teil 1: Verfahren mittels
à haute performance (ISO 17226-1:2018) Hochleistungs-Flüssigkeitschromatographie
(ISO 17226-1:2018)

This European Standard was approved by CEN on 16 December 2018.

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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17226‑1:2019: E
worldwide for CEN national Members
BS EN ISO 17226‑1:2019
EN ISO 17226‑1:2019 (E)

European foreword
This document (EN ISO 17226‑1:2019) has been prepared by Technical Committee ISO/IULTCS
"International Union of Leather Technologists and Chemists Societies" in collaboration with Technical
Committee CEN/TC 289 “Leather” the secretariat of which is held by UNI.
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 August 2019, and conflicting national standards shall
be withdrawn at the latest by August 2019.
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 ISO 17226‑1:2008.
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, 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.

Endorsement notice
The text of ISO 17226‑1:2018 has been approved by CEN as EN ISO 17226‑1:2019 without any
modification.

ii
 BS EN ISO 17226‑1:2019
ISO 17226‑1:2018


Contents Page

Foreword......................................................................................................................................................................................................................................... iv
1 Scope.................................................................................................................................................................................................................................. 1
2 Normative references....................................................................................................................................................................................... 1
3 Terms and definitions...................................................................................................................................................................................... 1
4 Conformance.............................................................................................................................................................................................................. 1
5 Principle......................................................................................................................................................................................................................... 1
6 Reagents......................................................................................................................................................................................................................... 2
6.1 Reagents for the formaldehyde stock solution............................................................................................................. 2
6.2 Reagents for the HPLC method.................................................................................................................................................. 2
7 Apparatus...................................................................................................................................................................................................................... 2
8 Procedures................................................................................................................................................................................................................... 3
8.1 Procedure for the determination of formaldehyde in the stock solution.............................................. 3
8.1.1 Preparation of the formaldehyde stock solution................................................................................... 3
8.1.2 Determination..................................................................................................................................................................... 3
8.2 Procedure for the determination of formaldehyde in leather by the HPLC method................... 3
8.2.1 Sampling and preparation of samples............................................................................................................ 3
8.2.2 Extraction................................................................................................................................................................................ 3
8.2.3 Reaction with DNPH...................................................................................................................................................... 4
8.2.4 Calibration of HPLC........................................................................................................................................................ 4
8.2.5 Calculation of the formaldehyde content in leather samples..................................................... 4
8.2.6 Spiking — determination of recovery rate................................................................................................. 5
9 Expression of results......................................................................................................................................................................................... 5
10 Test report.................................................................................................................................................................................................................... 5
Annex A (informative) Precision: reliability of the chromatographic HPLC method.......................................... 6
Annex B (informative) HPLC conditions............................................................................................................................................................ 7
Bibliography................................................................................................................................................................................................................................. 8

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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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www.iso.
org/iso/foreword.html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 289, Leather, in collaboration with the Chemical Test Commission of the
International Union of Leather Technologists and Chemists Societies (IUC Commission, IULTCS), in
accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This method is technically similar to the Colorimetric Section of the method IUC 19 which was declared
an official method at the IULTCS Delegates meeting on 31st May 2003 in Cancún, Mexico.
IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to
further the advancement of leather science and technology. IULTCS has three Commissions, which
are responsible for establishing international methods for the sampling and testing of leather. ISO
recognizes IULTCS as an international standardizing body for the preparation of test methods
for leather.
This second edition cancels and replaces the first edition (ISO 17226‑1:2008), which has been
technically revised as follows:
— the former Clause 2 has become Clause 4, a new Clause 3, Terms and definitions, inserted and
subsequent clauses renumbered;
— 6.1.1, 6.2.1, 7.4, 7.6, 8.2.2, 8.2.3, 8.2.4.1, 8.2.4.2 and 8.2.6 have been technically modified;
— the recommended HPLC conditions, previously in 7.2.4, have been moved to an informative Annex B.
A list of all parts in the ISO 17226 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv  © ISO 2018 – All rights reserved

 BS EN ISO 17226‑1:2019

INTERNATIONAL STANDARD ISO 17226-1:2018(E)



Leather - Chemical determination of

formaldehyde content —
Part 1:
Method using high performance liquid chromatography

1 Scope
This document specifies a method for the determination of free and released formaldehyde in leathers.
This method, based on high performance liquid chromatography (HPLC), is selective and not sensitive
to coloured extracts and is intended to be used for precise quantification of formaldehyde.
The formaldehyde content is taken to be the quantity of free-formaldehyde and formaldehyde extracted
through hydrolysis contained in a water extract from the leather under standard conditions.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 2418, Leather — Chemical, physical and mechanical and fastness tests — Sampling location
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4044, Leather — Chemical tests — Preparation of chemical test samples
ISO 4684, Leather — Chemical tests — Determination of volatile matter

3 Terms and definitions

No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/

4 Conformance
When compared with ISO 17226‑2, the two analytical methods should give similar trends but not
necessarily the same absolute result. Therefore, in cases of dispute, the method in this document shall
be used in preference to ISO 17226‑2. See Annex A for additional information.

5 Principle
The process is selective. Formaldehyde is separated and quantified as a derivative from other aldehydes
and ketones by liquid chromatography. Detected is the free-formaldehyde and formaldehyde which is
hydrolysed during extraction to yield free-formaldehyde.

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The sample is eluted with a detergent solution at 40 °C. The eluate is mixed with
2,4-dinitrophenylhydrazine, whereby aldehydes and ketones react to give the respective hydrazones.
These are separated by means of a reversed-phase HPLC method, detected at (355 ± 5) nm
and quantified.

6 Reagents
Use only reagents of recognized analytical grade, unless otherwise stated. The water shall be grade 3 in
accordance with ISO 3696. All solutions are aqueous solutions.

6.1 Reagents for the formaldehyde stock solution

6.1.1 Formaldehyde solution, approximately 37 % (mass fraction).

Certified solutions of formaldehyde or formaldehyde-2,4-DNPH are commercially available. When these

solutions are used, the procedure in 8.1.2 is not required.

6.1.2 Iodine solution, 0,05 mol/l, i.e. 12,68 g iodine per litre.

6.1.3 Sodium hydroxide solution, 2,0 mol/l.

6.1.4 Sulfuric acid solution, 2,0 mol/l.

6.1.5 Sodium thiosulfate solution, 0,1 mol/l.

6.1.6 Starch solution, 1 %, i.e. 1 g in 100 ml water.

6.2 Reagents for the HPLC method

6.2.1 Sodium dodecylsulfonate or sodium dodecylsulfate (detergent solution), 0,1 %, 1 g in

1 000 ml water.

6.2.2 Dinitrophenylhydrazine (DNPH) solution, consisting of 0,3 g DNPH

(2,4-dinitrophenylhydrazine) dissolved in 100 ml concentrated o-phosphoric acid (85 % mass fraction).
(DNPH recrystallized from 25 % mass fraction, acetonitrile in water.)

6.2.3 Acetonitrile HPLC grade.

7 Apparatus
Use the usual laboratory equipment and, in particular, the following:

7.1 Volumetric flasks, of capacities 10 ml, 500 ml and 1 000 ml.

7.2 Erlenmeyer flasks, of capacities 100 ml and 250 ml.

7.3 Strainer with glass fibre filter, GF8 (or glass filter strainer G 3, diameter 70 mm to 100 mm).

7.4 Water bath, thermostatically controlled to (40  ±  1)  °C, fitted with a flask shaker, frequency
(50 ± 10) min−1.

7.5 Thermometer, with 0,1 °C graduations over the range 10 °C to 50 °C.

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 BS EN ISO 17226‑1:2019
ISO 17226‑1:2018


7.6 HPLC system with UV detection, (355 ± 5) nm or other validated apparatus.

7.7 Membrane filter, polyamide, 0,45 µm.

7.8 Analytical balance, weighing to an accuracy of 0,1 mg.

8 Procedures

8.1 Procedure for the determination of formaldehyde in the stock solution

8.1.1 Preparation of the formaldehyde stock solution

Pipette 5 ml of the formaldehyde solution (6.1.1) into a 1  000  ml volumetric flask (7.1) containing
approximately 100  ml water and then fill the flask with demineralized water up to the mark. This
solution is the formaldehyde stock solution.

8.1.2 Determination

Pipette 10 ml from this solution into a 250 ml Erlenmeyer flask (7.2) and mix with the 50 ml iodine
solution (6.1.2). Add sodium hydroxide (6.1.3) until it turns yellow. Allow it to react for (15 ± 1) min at
18 °C to 26 °C and then add 15 ml of sulfuric acid (6.1.4) while swirling.
After adding 2 ml of starch solution (6.1.6), titrate the excess iodine with sodium thiosulfate (6.1.5)
until the colour changes. Make three individual determinations. Titrate at least two blank solutions in
the same manner.

(V0 − V1 ) × c 1 × M FA
ρ FA =
2
where
ρFA is the concentration of the formaldehyde stock solution, in milligrams per 10 ml (mg/10 ml);
V0 is the titre of the thiosulfate solution for the blank solution, in millilitres (ml);
V1 is the titre of the thiosulfate solution for the sample solution, in millilitres (ml);
MFA is the relative molecular mass of formaldehyde, 30,02 g/mol;
c1 is the concentration of the thiosulfate solution, in moles per litre (mol/l).

8.2 Procedure for the determination of formaldehyde in leather by the HPLC method

8.2.1 Sampling and preparation of samples

Sample in accordance with ISO 2418. If sampling in accordance with ISO 2418 is not possible (e.g. leathers
from finished products like shoes, garments), provide details about sampling together with the test
report. Cut leather in accordance with ISO 4044.
If the result is to be presented on the basis of dry substance, then test a further sample of the same
leather in accordance with ISO 4684 so that the moisture content can be calculated.

8.2.2 Extraction

Weigh approximately (2 ± 0,1) g of leather pieces to the nearest 0,01 g into a 100 ml glass Erlenmeyer flask.
Add 50 ml of detergent solution (6.2.1) (previously preheated at 40 °C) and fit the Erlenmeyer flask with
a glass stopper. Shake the contents of the flask in the water bath (7.4) for (60 ± 2) min at (40 ± 1) °C.
Immediately filter the warm extract solution by vacuum (use not less than 50 mbar) through a glass
fibre filter (7.3) into a flask. Cool the filtrate, in a closed flask, down to room temperature (18 °C to 26 °C).

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Do not modify the leather/solution ratio. The extraction and analysis should be performed within the
same working day.

8.2.3 Reaction with DNPH

Pipette 4,0 ml of acetonitrile (6.2.3), a 5,0 ml aliquot of the filtered eluate (8.2.2) and 0,5 ml of DNPH
solution (6.2.2) into a 10 ml volumetric flask (7.1). Fill the volumetric flask with demineralized water
up to the mark and shake it briefly by hand to mix the components. Allow it to stand for at least 60 min,
but not more than a maximum of 180 min. After filtering through a membrane filter (7.7), analyse the
sample using HPLC.
For a high content of formaldehyde (>100 mg/kg), make aliquots smaller than 5 ml up to 5 ml with
water. Example of the procedure when formaldehyde content is approximately 500 mg/kg: pipette
4,0 ml of acetonitrile (6.2.3), a 0,5 ml aliquot of the filtered eluate (8.2.2), add 4,5 ml water and 0,5 ml of
DNPH solution (6.2.2) into a 10 ml volumetric flask (6.1). Then follow the procedure as described in the
previous paragraph.
See Annex B for additional information.

8.2.4 Calibration of HPLC

8.2.4.1 Calibration with formaldehyde stock solution

Pipette 1 ml of the formaldehyde stock solution obtained in 8.1.1, with a precisely known formaldehyde
content, into a 500 ml volumetric flask (7.1), pre-filled with approximately 100 ml water. Mix together
and fill to the mark with water, then mix again. This solution is the standard solution for calibration
purposes, i.e. the standard solution is approximately 2 µg formaldehyde/ml.
In each of six 10-ml volumetric flasks (7.1), insert 4 ml acetonitrile (6.2.3), then add a concentration
series of 0,25 ml; 1,0 ml; 2,0 ml; 3,0 ml; 4,0 ml; and 5,0 ml, respectively, of the standard solution. These
solutions cover the formaldehyde concentration range of 0,1  μg/ml to 2,0  μg/ml. (This corresponds
to a formaldehyde in leather concentration range of 5 mg/kg to 100 mg/kg leather under the given
conditions.)
Immediately upon addition of the formaldehyde solution (6.1.1), mix each flask and add 0,5 ml DNPH
solution (6.2.2). Fill the flasks up to the mark with demineralized water and mix. After at least 60 min
and not more than 180  min, analyse the samples using HPLC after filtration through a membrane
filter (7.7).
Plot the concentrations in micrograms per 10 ml in a calibration graph against the measured
formaldehyde derivative peak area. x-axis: concentration in micrograms per 10 ml, y-axis: peak area.

8.2.4.2 Calibration with derivatised DNPH-formaldehyde

In each of six 10-ml volumetric flasks (7.1), insert 4 ml acetonitrile (6.2.3), then add an adequate
quantity of derivatized DNPH-formaldehyde to ensure you have 1,0 μg; 4,0 μg; 8,0 μg; 12,0 μg; 16,0 μg;
and 20,0 μg, respectively, of formaldehyde.
Fill the flasks up to the mark with demineralized water and mix. After at least 60 min and not more
than 180  min, analyse the samples using HPLC after filtration through a membrane filter (7.7). For
the calibration plot a graph of the formaldehyde derivative peak area versus the concentration in
micrograms per 10 ml.

8.2.5 Calculation of the formaldehyde content in leather samples

ρS × F
wF =
m
where

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wF is the concentration of formaldehyde in the sample in milligrams per kilogram (mg/kg) rounded
to 0,01 mg/kg;
ρS is the concentration of formaldehyde obtained from the calibration graph in micrograms per
10 ml (µg/10 ml);
F is the dilution factor in millilitres (ml);
m is the mass of leather weighed in grams (g).

8.2.6 Spiking — determination of recovery rate

Determination of the recovery rate is not mandatory. If necessary the following procedure can be used.
Put 4 ml acetonitrile (6.2.3) into a 10  ml volumetric flask (7.1) and add an aliquot of 2,5 ml of the
filtrate, obtained as described in 8.2.2. Then add an accurately determined volume of the formaldehyde
standard solution to give an almost equal concentration to that found in the sample.
Further treat this solution following the procedure described in 8.2.3 and determine ρS2 following the
same procedure. Carry out the determination and report the value in the test report.

( ρ S2 − 0 , 5 ρ S ) × 100
RR =
ρ FA 1
where
ρS2 is the concentration of formaldehyde obtained from the calibration graph in micrograms per
10 ml (µg/10 ml);
ρS is the concentration of formaldehyde in the non-spiked sample in micrograms per 10 ml (µg/10 ml);
ρFA1 is the spiked quantity of formaldehyde in micrograms per 10 ml (µg/10 ml);
RR is the recovery rate in percent, rounded off to 0,1 %.

9 Expression of results
Express the formaldehyde concentration to the nearest 0,1 mg/kg based on the mass of the leather
sample tested.
If the results are to be reported on the basis of dry substance, multiply the results above by the factor
100/(100 − w), where w is the moisture content in percent (%) according to ISO 4684. If the results are
presented on the basis of dry substance, clearly mention this in the test report.

10 Test report
The test report shall include the following:
a) reference to this document (i.e. ISO 17226‑1);
b) type, origin and designation of the analysed leather sample and the sampling method used;
c) the analytical procedure used;
d) the analytical results for the formaldehyde content;
e) any deviations from the analytical procedure, particularly any additional steps performed;
f) the date of the test;
g) if the results are determined on the basis of the dry substance this shall be reported.

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BS EN ISO 17226‑1:2019
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Annex A
(informative)

Precision: reliability of the chromatographic HPLC method

The following data were obtained in a collaborative trial with 10 laboratories on leather samples with
unknown levels of formaldehyde.

Table A.1 — Reliability data of the chromatographic HPLC method

Leather sample Mean formaldehyde Repeatability Reproducibility Recovery rate
content
r R
mg/kg mg/kg mg/kg %
A 7,65 1,27 3,13 94
B 17,69 3,82 7,97 96
C 28,69 5,40 11,42 91
D 102,16 20,82 64,33 94

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Annex B
(informative)

HPLC conditions

Flow rate: 1,0 ml/min

Mobile phase: acetonitrile/water, 60:40

Separation column: C18 reversed phase column with pre-column (1 cm, RP18)

UV detection wavelength: (355 ± 5) nm

Injection volume: 20 µl

NOTE A Merck 100, CH 18,2 (highly coated, 12 % C) column is an example of a suitable separation column
which is commercially available.1)

1) A Merck 100, CH 18,2 column is an example of a suitable product available commercially. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.

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Bibliography

[1] ISO 17226‑2, Leather — Chemical determination of formaldehyde content — Part 2: Method using
colorimetric analysis

8  © ISO 2018 – All rights reserved

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