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)

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 estrogenen 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) dieses Verfahrens 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.

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 (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
• Publication Date: 20-Sep-2022
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 21-Sep-2022
• Due Date: 16-Oct-2023
• Completion Date: 21-Sep-2022
• Ref Project: 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)

Standards Content (Sample)

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
ISO 19040-3:2018(E)
© 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
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Published in Switzerland
ii © ISO 2018 – All rights reserved

ISO 19040-3:2018(E)
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 .
...