SIST EN ISO 19040-3:2023

Water quality - Determination of the estrogenic potential of water and waste water - Part 3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene assay is based on the activation of the human estrogen receptor alpha.
This method is applicable to:
—          fresh water;
—          waste water;
—          aqueous extracts and leachates;
—          eluates of sediments (fresh water);
—          pore water;
—          aqueous solutions of single substances or of chemical mixtures;
—          drinking water;
—          the limit of quantification (LOQ) of this method for the direct analysis of water samples is between 0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction and pre concentration of water samples can prove necessary if their estrogenic potential is below the given LOQ.

Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und Abwasser - Teil 3: In vitro Reportergentest mit humanen Zellen (ISO 19040-3:2018)

Dieses Dokument legt ein Verfahren zur Bestimmung des estrogenen Potentials von Wasser und Abwasser mittels eines Reportergen-Tests unter Verwendung stabil transfizierter, humaner Zellen fest. Dieser Reportergen-Test beruht auf der Aktivierung des humanen Estrogenrezeptors alpha.
Dieses Verfahren ist anwendbar auf:
   Süßwasser;
   Abwasser;
   wässrige Extrakte und Sickerwasser;
   Eluate von Sedimenten (Süßwasser);
   Porenwasser;
   wässrige Lösungen von Einzelsubstanzen oder von chemischen Gemischen;
   Trinkwasser;
Die Bestimmungsgrenze (LOQ) dieser Methode zur direkten Analyse von Wasserproben liegt zwischen 0,3 ng/l und 1 ng/l 17β Estradiol-Äquivalenten (EEQ). Diese Werte wurden in einem internationalen Ringversuch ermittelt (siehe Anhang F). Der obere Arbeitsbereich wurde bis zu einem Gehalt von 75 ng EEQ/l bewertet [basierend auf einem internationalen Ringversuch (siehe Tabelle F.3)]. Proben, die ein estrogenes Potenzial oberhalb dieser Schwelle zeigen, müssen für eine gültige Quantifizierung verdünnt werden. Eine Extraktion und Anreicherung von Wasserproben kann notwendig sein, wenn ihr estrogenes Potential unter der oben angegebenen Bestimmungsgrenze liegt.
WARNUNG —Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortung des Arbeitgebers, angemessene Sicherheits- und Schutzmaßnahmen zu treffen und sicherzustellen.
WICHTIG — Es ist erforderlich, bei den Untersuchungen nach diesem Dokument Fachleute oder Facheinrichtungen einzuschalten.

Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires - Partie 3: Essai in vitro sur cellules humaines avec gène rapporteurns (ISO 19040-3:2018)

Le présent document spécifie une méthode permettant de déterminer le potentiel œstrogénique de l'eau et des eaux résiduaires au moyen d’un essai avec gène rapporteur utilisant des cellules humaines transfectées de façon stable. Cet essai avec gène rapporteur se fonde sur l’activation du récepteur des œstrogènes humains alpha.
Cette méthode est applicable:
—    aux eaux douces;
—    aux eaux résiduaires;
—    aux extraits aqueux et lixiviats;
—    aux éluats de sédiments (eau douce);
—    aux eaux interstitielles;
—    aux solutions aqueuses contenant des substances uniques ou des mélanges chimiques;
—    à l’eau potable;
—    la limite de quantification (LDQ) de cette méthode pour l’analyse directe d’échantillons d’eau est comprise entre 0,3 ng/l et 1 ng/l d’équivalents 17β-œstradiol (EEQ) sur la base des résultats de l’essai interlaboratoires international (voir l’Annexe F). Le domaine de mesure supérieur a été évalué [sur la base des résultats de l’essai interlaboratoires international (voir le Tableau F.3)] jusqu’à un niveau de 75 ng d’EEQ/l. Les échantillons présentant un potentiel œstrogénique supérieur à ce seuil doivent être dilués pour une quantification valable. L’extraction et la préconcentration des échantillons d’eau peuvent s’avérer nécessaires, si leur potentiel œstrogénique est inférieur à la LDQ donnée.

Kakovost vode - Določanje estrogenega potenciala vode in odpadne vode - 3. del: Preskus in vitro na človeških celicah z markerskim genom (ISO 19040-3:2018)

Ta dokument opisuje metodo za določevanje estrogenskega potenciala vode in odpadne vode z uporabo poročevalskega gena s stabilno transfeciranimi človeškimi celicami. Poročevalski gen temelji na aktiviranju človeškega estrogenskega receptorja alfa.
Ta metoda se uporablja za:
–          sladko vodo;
–          odpadno vodo;
–          vodne ekstrakte in izcedne vode;
–          izlužke sedimentov (sladka voda);
–          porno vodo;
–          vodne raztopine posameznih snovi ali kemičnih mešanic;
–          pitno vodo;
–        mejna vrednost kvantifikacije (LOQ) te metode za neposredno analizo vzorcev vode je med 0,3 ng/l in 1 ng/l 17β-estradiol ekvivalentov (EEQ) na podlagi rezultatov mednarodnega medlaboratorijskega preskusa (glej dodatek F). Zgornje delovno območje je ocenjeno [na podlagi rezultatov mednarodnega medlaboratorijskega preskusa (glej preglednico F.3)] do 75 ng EEQ/l. Vzorce, ki kažejo estrogene potence nad to mejo, je treba za veljavno kvantifikacijo razredčiti. Če je estrogenski potencial pod določeno mejno vrednostjo kvantifikacije, je morda potrebna ekstrakcija in predkoncentracija vzorcev vode.

General Information

• Status: Published
• Public Enquiry End Date: 02-Aug-2022
• Publication Date: 11-May-2023
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: KAV - Water quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 05-Dec-2022
• Due Date: 09-Feb-2023
• Completion Date: 12-May-2023
• Ref Project: EN ISO 19040-3:2022 - Water quality - Determination of the estrogenic potential of water and waste water - Part 3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 19040-3:2023
01-junij-2023
Kakovost vode - Določanje estrogenega potenciala vode in odpadne vode - 3. del:
Preskus in vitro na človeških celicah z markerskim genom (ISO 19040-3:2018)
Water quality - Determination of the estrogenic potential of water and waste water - Part
3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)
Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und
Abwasser - Teil 3: In vitro Reportergentest mit humanen Zellen (ISO 19040-3:2018)
Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires
- Partie 3: Essai in vitro sur cellules humaines avec gène rapporteurns (ISO 19040-
3:2018)
Ta slovenski standard je istoveten z: EN ISO 19040-3:2022
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 19040-3:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 19040-3:2023


EN ISO 19040-3
EUROPEAN STANDARD

NORME EUROPÉENNE

September 2022
EUROPÄISCHE NORM
ICS 13.060.70
English Version

Water quality - Determination of the estrogenic potential
of water and waste water - Part 3: In vitro human cell-
based reporter gene assay (ISO 19040-3:2018)
Qualité de l'eau - Détermination du potentiel Wasserbeschaffenheit - Bestimmung des estrogenen
oestrogène de l'eau et des eaux résiduaires - Partie 3: Potentials von Wasser und Abwasser - Teil 3: In vitro-
Essai in vitro sur cellules humaines avec gène Reportergentest mit humanen Zellen (ISO 19040-
rapporteur (ISO 19040-3:2018) 3:2018)
This European Standard was approved by CEN on 19 September 2022.

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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 19040-3:2022 E
worldwide for CEN national Members.

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EN ISO 19040-3:2022 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 19040-3:2023
EN ISO 19040-3:2022 (E)
European foreword
The text of ISO 19040-3:2018 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 19040-
3:2022 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 March 2023, and conflicting national standards shall
be withdrawn at the latest by March 2023.
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 19040-3:2018 has been approved by CEN as EN ISO 19040-3:2022 without any
modification.

3

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SIST EN ISO 19040-3:2023
INTERNATIONAL ISO
STANDARD 19040-3
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 3: In vitro human cell-based
reporter gene assay
Qualité de l'eau — Détermination du potentiel oestrogène de l'eau et
des eaux résiduaires —
Partie 3: Essai in vitro sur cellules humaines avec gène rapporteur
Reference number
ISO 19040-3:2018(E)
©
ISO 2018

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COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

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Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Interferences . 4
5 Principle . 4
6 Apparatus and materials. 4
7 Reagents, cells and media . 5
8 Sampling and samples . 9
8.1 General . 9
8.2 Bottles and material for sampling . 9
8.3 Bottles and material pre-cleaning . 9
8.4 Sampling procedure . 9
8.5 Transport of samples . 9
8.6 Pretreatment of sample .10
8.7 Storage of samples .10
9 Procedure.10
9.1 Cell culture maintenance .10
9.1.1 Freezing cells .10
9.1.2 Starting a new cell culture .10
9.1.3 Culturing cells .11
9.2 Human cell reporter gene assay test procedure .11
9.2.1 Seeding the cells (day 1) .11
9.2.2 Preparation of the E2-reference (day 2) .11
9.2.3 Preparation of the sample dilutions (day 2) .12
9.2.4 Field blank .12
9.2.5 Exposing the cells (day 2) .12
9.2.6 Harvesting the cells (day 3) .13
9.2.7 Measurement of luminescence (day 3) .13
9.3 Data analysis .13
9.3.1 Calculation of the reporter gene induction .13
9.3.2 Calculation of the percentage of maximum response .14
9.3.3 Calculation of the dose-response curve .14
10 Validity criteria .14
10.1 Validity criteria for the assay .14
10.2 Validity criteria for samples.15
11 Assessment criteria .15
12 Test report .15
Annex A (informative) Settings of the luminometer .16
Annex B (informative) Plate setup .17
Annex C (informative) Bioassay characteristics and details .18
Annex D (informative) Test set up for chemicals and extracts .20
Annex E (informative) Preparation of dilution series .22
Annex F (informative) Performance data .23
Annex G (informative) Statistical assessment .33
Annex H (informative) Calculation of 17β-estradiol equivalents .34
© ISO 2018 – All rights reserved iii

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Annex I (informative) Measurement of the lowest ineffective dilution (LID) of waste
water — A simplified evaluation for testing of waste water .36
Bibliography .39
iv © ISO 2018 – All rights reserved

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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 on 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 the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
A list of all parts in the ISO 19040 series can be found on the ISO website.
© ISO 2018 – All rights reserved v

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SIST EN ISO 19040-3:2023
INTERNATIONAL STANDARD ISO 19040-3:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 3: In vitro human cell-based reporter gene assay
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
1 Scope
This document specifies a method for the determination of the estrogenic potential of water and waste
water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene
assay is based on the activation of the human estrogen receptor alpha.
This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water;
— the limit of quantification (LOQ) of this method for the direct analysis of water samples is between
0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international
interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of
the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing
estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction
and pre concentration of water samples can prove necessary if their estrogenic potential is below
the given LOQ.
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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2018 – All rights reserved 1

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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/
3.1
culture medium
nutrients presented in a form and phase (liquid or solidified) which support cellular growth
[SOURCE: ISO 6107-6:2004, 24, modified — “cellular” replaces “microbiological”]
3.2
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water
with dilution water as integral number
Note 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. The corresponding and
smallest possible value of D is 1. In this document, the arrow indicates the transition from initial total volume to
final total volume.
[SOURCE: ISO 6107-6:2004, 28]
3.3
dilution water
sterile water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.4
EC
50
effective concentration of a compound which causes 50 % of an effect
Note 1 to entry: In the sense of the present document the EC is the effective concentration of a compound which
50
induces 50 % of the maximal reporter gene activity which can be achieved by this compound.
3.5
extract
test sample after extraction and possible removal of extraction vehicle
3.6
field blank
container prepared in the laboratory, using reagent water or other blank matrix, and sent with the
sampling personnel for exposure to the sampling environment to verify possible contamination during
sampling
[SOURCE: ISO 11074:2015, 4.5.3]
3.7
induction rate
quotient of the mean value of wells with enhanced reporter gene activity measured on the plates treated
with a dose of the test sample or with a positive control, and the mean value of the corresponding wells
treated with the negative control using the same cells under identical conditions
[SOURCE: ISO 6107-6:2004, 43, modified — “wells with enhanced reporter gene activity measured”
replaces “mutant colonies”; “corresponding wells” replaces “corresponding plates”, “quotient” replaces
“difference”; “cells” replaces “strain”.]
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3.8
limit of quantification
LOQ
lowest value that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.9
lowest ineffective-dilution value
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant
increase in the reporter gene activity compared with the negative control
[SOURCE: ISO 11350:2012, 3.4, modified — “increase in the reporter gene activity” replaces “increase
in the number of revertant wells”]
3.10
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.11
passage number
the number of subcultures from cells in a new culture vessel (cell culture flask or micro titer plate)
3.12
reference compound
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
[SOURCE: ISO 7405:2008, 3.6, 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”.]
3.13
relative light units
RLU
amount of reporter gene activity as measured by light produces using a luminometer, expressed as
relative light units
3.14
reporter gene activity
quantitative activity of a gene attached to the promoter sequence of another gene
3.15
stock culture
frozen culture of cells for the preservation of the characteristics of the cell line
[SOURCE: ISO 21427-2:2006, 13, modified — “the cell line” replaces “V79 cells”]
3.16
subculturing
transfer of part of a cell culture into a new cell culture vessel during cell culture
3.17
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all
preparation steps such as centrifugation, filtration, homogenization, pH adjustment and determination
of ionic strength
[SOURCE: ISO 6107-6:2004, 92]
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4 Interferences
Toxic effects present in the test samples may lead to a reduction of cell viability and hence to a reduction
of the measured cellular response. Consequently, estrogenic effects of a sample may be masked by acute
toxic effects leading to false negative test results (see Clause 9 for further information). In this case, the
sample should be diluted further until no cytotoxicity is observed (see manual of cytotoxicity test used).
The use of inappropriate sampling devices and/or sampling flask may influence the test result because
of the possible adsorption of active compounds on surfaces leading to false negative results. On the
other hand, active compounds could be released into the sample from sampling flasks, especially if
plastic ware is used, and false positive results might be generated. See Clause 7 for more information.
High salinity can cause toxic effects due to the resulting osmotic pressure. The ER(α) CALUX cells
(References [10] to [16]) tolerates a conductivity of the sample up to 34,000 µS/cm (1,0 % w/w salinity).
Bacterial and fungal contaminations can negatively influence the response of the cells. Therefore,
antibiotics are added to the cell culture medium. Contamination of the cells is assessed by visual
observation (microscope) when testing the sample. See Clause 9 for further information.
If filtered samples are tested in order to remove bacteria from the sample solid particles are separated
from the sample also. Thus, substances with estrogenic activity which are adsorbed on particles might
not be detected.
Anti-estrogenic compounds and other non-toxic inhibitory compounds might mask estrogenic effects.
The presence of interfering compounds can be assessed by samples which are spiked with a defined
amount of an estrogenic compound with defined properties (e.g. 17β-estradiol) leading to a known
induction of the test system.
Compounds with estrogenic properties might be present as inactive conjugates. A chemical de-
conjugation can be necessary in order to quantify the overall estrogenic potential of a sample.
5 Principle
Estrogen receptor (ER) - mediated signaling is essential in estrogen action and the mechanism of
estrogen receptor signaling is well established. Upon estrogen binding the estrogen receptor becomes
activated, and binds to recognition sequences in promoter regions of target genes, the so-called
estrogen responsive elements (EREs). These EREs have been linked to a promoter element and a gene
transcribing for the easily measurable protein luciferase. In these cells, the ligand-activated receptor
will activate luciferase transcription, and the transcribed luciferase protein will emit light when a
substrate is added. The signal dose-dependently increases as a result of increasing concentrations of
ligand. The luciferase activity in cellular lysates is measured with a luminometer, allowing reliable,
sensitive and quantitative measurements.
6 Apparatus and materials
Beside the equipment which is usually present in a laboratory for cell culture the following apparatus
and materials are needed. For suitable sampling devices see Clause 8.
6.1 Laminar air flow cabinet, standard: “biological hazard”.
6.2 Water bath, 37 °C.
6.3 CO incubator, 5 % CO , 37 °C, humidity 100 %.
2 2
6.4 Inverted phase-contrast microscope.
6.5 Freezer, at least ≤−18 °C and at ≤−70 °C.
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6.6 Shaking apparatus for micro-test plates.
6.7 Centrifuge.
6.8 Laboratory balance.
6.9 Sterile pipettes, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, glassware or plastics.
6.10 Pipette controller.
2
6.11 Cell culture flasks, 75 cm with filter lids.
6.12 Sterile plastic containers, 12 ml and 50 ml with sterile cap.
6.13 Sterile plates with 12 wells.
6.14 Multi-channel multi-stepper pipette (repeater pipette), including 5 ml and 10 ml tips.
6.15 Pipettes, 1 μl, 50 μl, 200 μl and 1 000 μl, with sterile tips.
6.16 Multi-channel pipettes, up to 50 μl and up to 300 μl.
6.17 Sterile polystyrene 96-well plates, with flat transparent bottom and lid, appropriate for cell
culture, volume 300 μl per well.
6.18 Microplate luminometer with two injectors, for addition of substrate and stop reagent.
6.19 Cell counter or hemacytometer.
6.20 pH meter.
6.21 Cryovials, sterile, 2 ml.
6.22 Liquid nitrogen container for long term cell storage.
6.23 Filter, cellulose acetate, 0,45 µm pore size.
7 Reagents, cells and media
7.1 Reagents
As far as possible, use "reagent grade" chemicals. If (different) hydrates are used that differ from the
compounds specified, ensu
...

SLOVENSKI STANDARD
oSIST prEN ISO 19040-3:2022
01-julij-2022
[Not translated]
Water quality - Determination of the estrogenic potential of water and waste water - Part
3: In vitro human cell-based reporter gene assay (ISO 19040-3:2018)
Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und
Abwasser - Teil 3: In vitro Reportergentest mit humanen Zellen (ISO 19040-3:2018)
Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires
- Partie 3: Essai in vitro sur cellules humaines avec gène rapporteurns) (ISO 19040-
3:2018)
Ta slovenski standard je istoveten z: prEN ISO 19040-3
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 19040-3:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 19040-3:2022
INTERNATIONAL ISO
STANDARD 19040-3
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 3: In vitro human cell-based
reporter gene assay
Qualité de l'eau — Détermination du potentiel oestrogène de l'eau et
des eaux résiduaires —
Partie 3: Essai in vitro sur cellules humaines avec gène rapporteur
Reference number
ISO 19040-3:2018(E)
©
ISO 2018

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oSIST prEN ISO 19040-3:2022
ISO 19040-3:2018(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

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Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Interferences . 4
5 Principle . 4
6 Apparatus and materials. 4
7 Reagents, cells and media . 5
8 Sampling and samples . 9
8.1 General . 9
8.2 Bottles and material for sampling . 9
8.3 Bottles and material pre-cleaning . 9
8.4 Sampling procedure . 9
8.5 Transport of samples . 9
8.6 Pretreatment of sample .10
8.7 Storage of samples .10
9 Procedure.10
9.1 Cell culture maintenance .10
9.1.1 Freezing cells .10
9.1.2 Starting a new cell culture .10
9.1.3 Culturing cells .11
9.2 Human cell reporter gene assay test procedure .11
9.2.1 Seeding the cells (day 1) .11
9.2.2 Preparation of the E2-reference (day 2) .11
9.2.3 Preparation of the sample dilutions (day 2) .12
9.2.4 Field blank .12
9.2.5 Exposing the cells (day 2) .12
9.2.6 Harvesting the cells (day 3) .13
9.2.7 Measurement of luminescence (day 3) .13
9.3 Data analysis .13
9.3.1 Calculation of the reporter gene induction .13
9.3.2 Calculation of the percentage of maximum response .14
9.3.3 Calculation of the dose-response curve .14
10 Validity criteria .14
10.1 Validity criteria for the assay .14
10.2 Validity criteria for samples.15
11 Assessment criteria .15
12 Test report .15
Annex A (informative) Settings of the luminometer .16
Annex B (informative) Plate setup .17
Annex C (informative) Bioassay characteristics and details .18
Annex D (informative) Test set up for chemicals and extracts .20
Annex E (informative) Preparation of dilution series .22
Annex F (informative) Performance data .23
Annex G (informative) Statistical assessment .33
Annex H (informative) Calculation of 17β-estradiol equivalents .34
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Annex I (informative) Measurement of the lowest ineffective dilution (LID) of waste
water — A simplified evaluation for testing of waste water .36
Bibliography .39
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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 on 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 the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
A list of all parts in the ISO 19040 series can be found on the ISO website.
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oSIST prEN ISO 19040-3:2022
INTERNATIONAL STANDARD ISO 19040-3:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 3: In vitro human cell-based reporter gene assay
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
1 Scope
This document specifies a method for the determination of the estrogenic potential of water and waste
water by means of a reporter gene assay utilizing stably transfected human cells. This reporter gene
assay is based on the activation of the human estrogen receptor alpha.
This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water;
— the limit of quantification (LOQ) of this method for the direct analysis of water samples is between
0,3 ng/l and 1 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international
interlaboratory trial (see Annex F). The upper working range was evaluated [based on the results of
the international interlaboratory trial (see Table F.3)] up to a level of 75 ng EEQ/l. Samples showing
estrogenic potencies above this threshold have to be diluted for a valid quantification. Extraction
and pre concentration of water samples can prove necessary if their estrogenic potential is below
the given LOQ.
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 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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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/
3.1
culture medium
nutrients presented in a form and phase (liquid or solidified) which support cellular growth
[SOURCE: ISO 6107-6:2004, 24, modified — “cellular” replaces “microbiological”]
3.2
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water
with dilution water as integral number
Note 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. The corresponding and
smallest possible value of D is 1. In this document, the arrow indicates the transition from initial total volume to
final total volume.
[SOURCE: ISO 6107-6:2004, 28]
3.3
dilution water
sterile water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.4
EC
50
effective concentration of a compound which causes 50 % of an effect
Note 1 to entry: In the sense of the present document the EC is the effective concentration of a compound which
50
induces 50 % of the maximal reporter gene activity which can be achieved by this compound.
3.5
extract
test sample after extraction and possible removal of extraction vehicle
3.6
field blank
container prepared in the laboratory, using reagent water or other blank matrix, and sent with the
sampling personnel for exposure to the sampling environment to verify possible contamination during
sampling
[SOURCE: ISO 11074:2015, 4.5.3]
3.7
induction rate
quotient of the mean value of wells with enhanced reporter gene activity measured on the plates treated
with a dose of the test sample or with a positive control, and the mean value of the corresponding wells
treated with the negative control using the same cells under identical conditions
[SOURCE: ISO 6107-6:2004, 43, modified — “wells with enhanced reporter gene activity measured”
replaces “mutant colonies”; “corresponding wells” replaces “corresponding plates”, “quotient” replaces
“difference”; “cells” replaces “strain”.]
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3.8
limit of quantification
LOQ
lowest value that can be determined with an acceptable level of accuracy and precision
[SOURCE: ISO 15839:2003, 3.18]
3.9
lowest ineffective-dilution value
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant
increase in the reporter gene activity compared with the negative control
[SOURCE: ISO 11350:2012, 3.4, modified — “increase in the reporter gene activity” replaces “increase
in the number of revertant wells”]
3.10
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.11
passage number
the number of subcultures from cells in a new culture vessel (cell culture flask or micro titer plate)
3.12
reference compound
compound with one or more property values that are sufficiently reproducible and well established to
enable the calibration of the measurement method
[SOURCE: ISO 7405:2008, 3.6, 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”.]
3.13
relative light units
RLU
amount of reporter gene activity as measured by light produces using a luminometer, expressed as
relative light units
3.14
reporter gene activity
quantitative activity of a gene attached to the promoter sequence of another gene
3.15
stock culture
frozen culture of cells for the preservation of the characteristics of the cell line
[SOURCE: ISO 21427-2:2006, 13, modified — “the cell line” replaces “V79 cells”]
3.16
subculturing
transfer of part of a cell culture into a new cell culture vessel during cell culture
3.17
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all
preparation steps such as centrifugation, filtration, homogenization, pH adjustment and determination
of ionic strength
[SOURCE: ISO 6107-6:2004, 92]
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4 Interferences
Toxic effects present in the test samples may lead to a reduction of cell viability and hence to a reduction
of the measured cellular response. Consequently, estrogenic effects of a sample may be masked by acute
toxic effects leading to false negative test results (see Clause 9 for further information). In this case, the
sample should be diluted further until no cytotoxicity is observed (see manual of cytotoxicity test used).
The use of inappropriate sampling devices and/or sampling flask may influence the test result because
of the possible adsorption of active compounds on surfaces leading to false negative results. On the
other hand, active compounds could be released into the sample from sampling flasks, especially if
plastic ware is used, and false positive results might be generated. See Clause 7 for more information.
High salinity can cause toxic effects due to the resulting osmotic pressure. The ER(α) CALUX cells
(References [10] to [16]) tolerates a conductivity of the sample up to 34,000 µS/cm (1,0 % w/w salinity).
Bacterial and fungal contaminations can negatively influence the response of the cells. Therefore,
antibiotics are added to the cell culture medium. Contamination of the cells is assessed by visual
observation (microscope) when testing the sample. See Clause 9 for further information.
If filtered samples are tested in order to remove bacteria from the sample solid particles are separated
from the sample also. Thus, substances with estrogenic activity which are adsorbed on particles might
not be detected.
Anti-estrogenic compounds and other non-toxic inhibitory compounds might mask estrogenic effects.
The presence of interfering compounds can be assessed by samples which are spiked with a defined
amount of an estrogenic compound with defined properties (e.g. 17β-estradiol) leading to a known
induction of the test system.
Compounds with estrogenic properties might be present as inactive conjugates. A chemical de-
conjugation can be necessary in order to quantify the overall estrogenic potential of a sample.
5 Principle
Estrogen receptor (ER) - mediated signaling is essential in estrogen action and the mechanism of
estrogen receptor signaling is well established. Upon estrogen binding the estrogen receptor becomes
activated, and binds to recognition sequences in promoter regions of target genes, the so-called
estrogen responsive elements (EREs). These EREs have been linked to a promoter element and a gene
transcribing for the easily measurable protein luciferase. In these cells, the ligand-activated receptor
will activate luciferase transcription, and the transcribed luciferase protein will emit light when a
substrate is added. The signal dose-dependently increases as a result of increasing concentrations of
ligand. The luciferase activity in cellular lysates is measured with a luminometer, allowing reliable,
sensitive and quantitative measurements.
6 Apparatus and materials
Beside the equipment which is usually present in a laboratory for cell culture the following apparatus
and materials are needed. For suitable sampling devices see Clause 8.
6.1 Laminar air flow cabinet, standard: “biological hazard”.
6.2 Water bath, 37 °C.
6.3 CO incubator, 5 % CO , 37 °C, humidity 100 %.
2 2
6.4 Inverted phase-contrast microscope.
6.5 Freezer, at least ≤−18 °C and at ≤−70 °C.
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6.6 Shaking apparatus for micro-test plates.
6.7 Centrifuge.
6.8 Laboratory balance.
6.9 Sterile pipettes, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, glassware or plastics.
6.10 Pipette controller.
2
6.11 Cell culture flasks, 75 cm with filter lids.
6.12 Sterile plastic containers, 12 ml and 50 ml with sterile cap.
6.13 Sterile plates with 12 wells.
6.14 Multi-channel multi-stepper pipette (repeater pipette), including 5 ml and 10 ml tips.
6.15 Pipettes, 1 μl, 50 μl, 200 μl and 1 000 μl, with sterile tips.
6.16 Multi-channel pipettes, up to 50 μl and up to 300 μl.
6.17 Sterile polystyrene 96-well plates, with flat transparent bottom and lid, appropriate for cell
culture, volume 300 μl per well.
6.18 Microplate luminometer with two injectors, for addition of substrate and stop reagent.
6.19 Cell counter or hemacytometer.
6.20 pH meter.
6.21 Cryovials, sterile, 2 ml.
6.22 Liquid nitrogen container for long term cell storage.
6.23 Filter, cellulose acetate, 0,45 µm pore size.
7 Reagents, cells and media
7.1 Reagents
As far as possible, use "reagent grade" chemicals. If (different) hydrates are used that differ from the
compounds specified, ensure that the appropriate mass of the main compound is employed.
7.1.1 Dimethyl sulfoxide (DMSO).
7.1.2 Glycerol for molecular biology, ≥99 %, molecular weight: 92,09 g/mol, CAS: 56-81-5.
7.1.3 17β-estradiol, ≥98 %, (C H O ), molecular weight: 272,38 g/mol, CAS: 50-28-2.
18 24 2
7.1.4 Ethylene-diamine-tetra-acetate (EDTA), CAS: 6381-92-6.
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7.1.5 Trypsine, CAS: 9002-07-7.
7.1.6 Fetal calf serum (FCS).
7.1.7 DMEM/F12 medium with phenol red as pH indicator.
7.1.8 Non-essential amino acids (100x).
7.1.9 Penicillin-streptomycin (100x), concentration used 5 000 units penicillin per ml / 5 000 μg
streptomycin per ml).
7.1.10 Phosphate buffered saline pH 7,2 (PBS), without calcium and magnesium.
7.1.11 Adenosine-5'-triphosphate (ATP), CAS: 34369-07-8.
7.1.12 Trans-1, 2-diaminocyclohexane-N, N, N', N'-tetraacetic acid monohydrate (CDTA),
CAS: 125572-95-4.
7.1.13 Dithiothreitol (DTT), CAS: 3483-12-3.
7.1.14 2-amino-2-(hydroxyl-methyl)-1,3-propanediol (TRIS), CAS: 77-86-1.
7.1.15 Dextran T500.
7.1.16 Activated charcoal.
7.1.17 Coenzyme A, free acid grade, Add CAS: 85-61-0.
7.1.18 D-Luciferin sodium salt, CAS: 103404-75-7.
7.1.19 Magnesium hydroxide carbonate pentahydrate, C H Mg O , CAS: 56378-72-4.
4 2 5 14
7.1.20 Magnesium sulfate, CAS: 7487-88-9.
7.1.21 Sodium bicarbonate, CAS: 144-55-8.
7.1.22 Cell culture medium powder, Sigma, D2902, without phenol red and sodium bicarbonate.
7.1.23 Tricine, CAS: 5704-04-1.
7.1.24 Acetone, (purity p.a.), CAS: 67-64-1.
7.1.25 Sodium hydroxide, molecular weight 40,00 g/mol, CAS: 1310-73-2.
7.1.26 Triton X-100, CAS: 9002-93-1.
7.1.27 Hydrochloric acid solution, 1 M (HCl), molecular weight 36,46 g/mol, CAS: 7647-01-0.
7.2 Water, grade 3, as defined in ISO 3696; water with a conductivity up to 5 µS/cm is acceptable.
If sterile water is needed, autoclave or sterilize by filtration (cellulose acetate, 0,2 µm). Water as
specified here is also used for the stepwise dilution of the test sample.
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7.3 Cell line.
A genetically modified human cell line, expressing the human estrogen receptor (hERα and/or hERβ)
and containing a reporter plasmid for luciferase. More detailed descriptions of the individual cell lines
are given in Annex C.
7.4 Media.
Always use sterile solutions, glassware, etc. Carry out all procedures under aseptic conditions and in the
sterile environment of a laminar flow cabinet (biological hazard standard). If autoclaving is necessary,
always autoclave for 20 min at (121 ± 2) °C. Cover vessels loosely, never seal air tight.
7.4.1 Cell culture medium.
Add 5,4 ml non-essential amino acids (7.1.8), 42 ml FCS (7.1.6) and 1 ml of penicillin-streptomycin
(7.1.9) to a bottle containing 500 ml DMEM/F12 medium with phenol red. Complete cell culture medium
should be kept at 4 °C and stored for no longer than eight weeks.
7.4.2 Freezing medium.
Add 1 ml non-essential amino acids, 10 ml DMSO, 20 ml FCS and 1 ml penicillin-streptomycin (7.1.9) to
68 ml of DMEM/F12 medium with phenol red. Distribute 20 ml batches in a sterile manner over sterile
tubes. Complete freezing medium should be kept at (−20 ± 1) °C until use and kept on ice on use.
7.4.3 Concentrated assay medium.
7.4.3.1 3x concentrated assay medium.
Dissolve the contents of one vial of cell culture medium powder (7.1.22) in 250 ml of water (at room
temperature) and then dissolve 3,7 g sodium bicarbonate (7.1.21) in the same solution. Adjust the
pH to 7,4 using a 1 mol/l HCl or 1 mol/l NaOH solution. Add water to a final volume of 300 ml and
filter sterilize. Add 12 ml of non-essential amino acids (7.1.8), 55 ml stripped serum (7.4.8) and 37 ml
penicillin-streptomycin (7.1.9). Complete media should be kept at (4 ± 1) °C and stored for no longer
than eight weeks.
7.4.3.2 1x concentrated assay medium.
Dilute 100 ml of 3x concentrated assay medium with 200 ml of water and filter sterilize. Complete
media should be kept at (4 ± 1) °C and stored for no longer than eight weeks.
7.4.4 Trypsin solution.
Add 20 ml of trypsin (7.1.5) to 980 ml PBS (7.1.10) and add 0,2 g EDTA (7.1.4). Sterile filter the trypsin
solution and aliquot into 50
...