EN ISO 23196:2023

SLOVENSKI STANDARD
01-februar-2024
Kakovost vode - Izračun biološko ekvivalentnih koncentracij (BEQ) (ISO
23196:2022)
Water quality - Calculation of biological equivalence (BEQ) concentrations (ISO
23196:2022)
Wasserbeschaffenheit - Berechnung biologischer Äquivalenzkonzentrationen (BEQ)
(ISO 23196:2022)
Qualité de l'eau - Calcul des concentrations en équivalent biologique (BEQ) (ISO
23196:2022)
Ta slovenski standard je istoveten z: EN ISO 23196:2023
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 23196
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2023
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Calculation of biological equivalence (BEQ)
concentrations (ISO 23196:2022)
Qualité de l'eau - Calcul des concentrations en Wasserbeschaffenheit - Berechnung biologischer
équivalent biologique (BEQ) (ISO 23196:2022) Äquivalenzkonzentrationen (BEQ) (ISO 23196:2022)
This European Standard was approved by CEN on 21 August 2023.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23196:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 23196:2022 has been prepared by Technical Committee ISO/TC 147 "Water quality” of
the International Organization for Standardization (ISO) and has been taken over as EN ISO 23196:2023
by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
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 2024, and conflicting national standards
shall be withdrawn at the latest by February 2024.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 23196:2022 has been approved by CEN as EN ISO 23196:2023 without any modification.

INTERNATIONAL ISO
STANDARD 23196
First edition
2022-02
Water quality — Calculation of
biological equivalence (BEQ)
concentrations
Reference number
ISO 23196:2022(E)
ISO 23196:2022(E)
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 23196:2022(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Procedure .5
5.1 General . 5
5.2 Procedure for the calculation of biological equivalence (BEQ) concentrations . 5
5.2.1 Assessment of the suitability of the experimental data for the calculation
of a biological equivalence (BEQ) concentration . 5
5.2.2 Fitting of concentration-effect data for the reference compound . 8
5.2.3 Calculation of quality measures for the fit . 9
5.2.4 Normalization of data from the reference compound and samples . 10
5.2.5 Calculation of the x %-effect level of the reference concentration-effect
relationship and the respective RC -value . 11
x
5.2.6 Assessment of the validity of the experimental data for the calculation of a
biological equivalence (BEQ) concentration .12
5.2.7 Calculation of the concentration factor of the sample at x %-effect level by
linear interpolation .12
5.2.8 Calculation of the biological equivalence (BEQ) concentration .13
6 Validity criteria .14
7 Test report .14
Annex A (informative) Illustration of χ statistics using data from Table 1 .15
Bibliography .18
iii
ISO 23196:2022(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.
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 Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
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
INTERNATIONAL STANDARD ISO 23196:2022(E)
Water quality — Calculation of biological equivalence
(BEQ) concentrations
1 Scope
This document specifies the derivation of biological equivalence (BEQ) concentrations for results of in
vitro bioassays which are based on measuring effects on a biological process such as enzyme induction
or cellular growth. The concept described here can be used for any biological assay after the proof of its
applicability.
To derive BEQ concentrations, the effect on a biological process caused by a sample – i.e. the activity
of the sample – is expressed in terms of a concentration of a reference compound which results in an
equivalent effect on the process. The term "sample" used in this document addresses environmental
samples as well as defined mixtures and pure compounds used as test item in a bioassay. BEQ
concentrations can be derived for environmental water samples, extracts of environmental water
samples including tap water or solutions of pure chemicals or mixtures of chemicals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological equivalence concentration
BEQ concentration
concentration of a reference compound (3.6) that causes the same effect as the effect measured in a
sample, a sample dilution or a solution containing one or more chemicals
3.2
concentration-effect relationship
response to a concentration gradient of an environmental sample or a known substance or mixture of
substances which is described by pre-determined diagnostic indicators
[SOURCE: ISO 6107:2021, 3.127, modified — the term "environmental sample" added; Note 1 to entry
has been deleted.]
3.3
concentration factor
CF
ratio of the actual concentration of the sample compared to the original sample taking all enrichment
and dilution steps of the sample into account
ISO 23196:2022(E)
3.4
limit of quantification
LOQ
lowest value of a determinant that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.5
negative control material
well characterized material and/or substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately negative,
non-reactive or minimal response in the test system
Note 1 to entry: In practice, negative controls include blanks, vehicles/solvents and reference materials.
[SOURCE: ISO 7405:2018, 3.4, modified — Note 1 to entry has been deleted.]
3.6
reference compound
RC
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
Note 1 to entry: For the purpose of this document, a reference compound is any well characterized material and/
or substance that, when tested by the procedure described, demonstrates the suitability of the procedure to yield
a reproducible, predictable positive response.
[SOURCE: ISO 7405:2018, 3.5, modified — "compound" replaces "material"; "the calibration of the
measurement method" replaces "use of the material or substance for the calibration of an apparatus,
the assessment of a measurement method or for the assignment of values to materials". Note 1 was
modified to cover only a reference compound resulting in a positive response – otherwise the proposed
concept is not applicable.]
3.7
x %-effect concentration
EC
x
concentration at which a specific effect is detected; x is the percentage (e.g. 10, 25, 50) of this effect, e.g.
growth inhibition
[SOURCE: ISO 15952:2018, 3.6]
3.8
x %-effect concentration of a reference compound
RC
x
concentration of a reference compound (3.6) at which a specific effect is detected; x is the percentage
(e.g. 10, 25, 50) of this effect, e.g. growth inhibition
4 Principle
The general idea for the derivation of BEQ concentrations, assessed by the test method, is shown in
Figure 1. The biological activity of a sample is expressed in terms of a concentration of a reference
compound which affects a biological process to the same extent. By this means, BEQ concentrations
allow an indirect quantification of results and a comparison of results obtained by different laboratories.
Furthermore, a robust way to calculate BEQ concentrations is a necessary requirement for a possible
[5],[6],[7],[8]
use of effect-based trigger (EBT) values in regulations .
ISO 23196:2022(E)
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y effect in arbitrary units
1 sample diluted
2 sample concentrated
3 biological equivalence (BEQ) concentration
reference compound
sample
NOTE The measured effect of a sample or a sample dilution is extrapolated to a concentration of a reference
compound which induces the bioassay to the same extend as the sample or sample dilution to derive a biological
equivalence (BEQ) concentration, here an example of an agonistic action is shown.
Figure 1 — Basic principle of the derivation of a biological equivalence (BEQ) concentration
Several different methods for the derivation of BEQ concentrations have been published, but not all
methods are applicable to all kinds of concentration-effect relationships. For a detailed discussion about
advantages and disadvantages of the various mathematical approaches, see References [1],[3],[5],[6].
The method described in this document, termed RC -approach, is reported to be a robust method for
x
[11],[12] [13]
the derivation of BEQ concentrations and outlined as well in OECD 455 .
NOTE In the literature, other terminologies might be found for RC , such as PC . PC describes the
x 10 10
concentration of the positive control that induces a 10 % effect-level. In this document, the term “reference
compound” is used instead of “positive control”. Therefore, RC is used instead of PC.
The approach is illustrated in Figure 2. As a first step of the RC -approach, the concentration-effect
x
relationship of the reference compound is modelled to determine the maximum effect of the assay for
the reference compound. Next, the effect levels are normalized to a percentage scale whereby the effect
ISO 23196:2022(E)
level of the negative control material is defined as 0 % and the maximum effect level of the reference
compound is defined as 100 %. Then, the concentration of the reference compound that affects the
assay to the x %-effect level is calculated (RC ).
x
Key
X concentration of the reference compound, e.g. in ng/l
X1 concentration factor of the sample compared to the original water sample
Y normalized effect in %
RC concentration of the reference compound at 10 % effect level
CF concentration factor of the sample at 10 % effect level with respect to the reference compound
reference compound
sample
NOTE Following data normalization, where 0 % equals the effect in the negative control material and 100 %
is the modelled maximum effect of the reference. The sample concentration factor (CF) and reference compound
concentration (RC) required to reach an effect level of x % (here 10 %) are interpolated from the data using
linear interpolation (dashed line). The BEQ concentration is derived by dividing RC by CF .
x x
Figure 2 — Illustration of the RC -approach for the derivation of a biological equivalence (BEQ)
x
concentration
In a further step, the concentration factor of the sample is determined at which the sample affects the
assay to the selected x %-effect level (CF ). As defined in 3.3, the concentration factor describes the ratio
x
of the actual concentration of the sample compared to the original water sample taking all enrichment
and dilution steps of the sample into account. If a sample is, under consideration of a quantitative
process, enriched 1 000-fold by solid phase extraction, the final concentration factor of the sample after
a 100-fold dilution with, for example, growth medium is 10. If this sample is tested in a series of six
1→2 dilutions, the resulting rounded concentration factors are 10, 5, 2,5, 1,25, 0,62, 0,31 and 0,15. The
biological equivalence (BEQ) concentration of the sample is finally given by the ratio RC /CF .
x x
ISO 23196:2022(E)
5 Procedure
5.1 General
Usually, concentration-effect relationships of reference compounds and samples (see Clause 1) in
reporter gene assays and proliferation assays are sigmoidal. Depending on the individual shape of
the concentration-effect relationship, a suitable mathematical model has to be selected for fitting. In
[14],[15]
general, a five parametric logistic model can be applied as shown in 5.2.
The overall procedure for the calculation of BEQ concentrations consists of the following steps that are
described in detail in 5.2:
— assessment of the suitability of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.1);
— fitting of concentration-effect data for the reference compound (see 5.2.2);
— calculation of quality measures for the fit (see 5.2.3);
— normalization of data from the reference compound and samples (see 5.2.4);
— calculation of the x %-effect level of the reference compound and the respective RC -value (see
x
5.2.5);
— assessment of the validity of the experimental data for the calculation of a biological equivalence
concentration (see 5.2.6);
— calculation of the concentration factor of the sample at the x %-effect level by li
...