SIST EN ISO 19040-1:2023

Water quality - Determination of the estrogenic potential of water and waste water - Part 1: Yeast estrogen screen (Saccharomyces cerevisiae) (ISO 19040-1:2018)

This document specifies a method for the determination of the estrogenic potential of water and waste water by means of a reporter gene assay with genetically modified yeast strains Saccharomyces cerevisiae. 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 8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between 120 ng/l and 160 ng/l 17β-estradiol equivalents (EEQ). 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 1: Hefebasierter Östrogentest (Saccharomyces cerevisiae) (ISO 19040-1:2018)

Dieses Dokument legt ein Verfahren zur Bestimmung des estrogenen Potenzials von Wasser und Abwasser mittels eines Reportergen-Tests mit gentechnisch veränderten Hefestämmen von Saccharomyces cerevisiae 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 8 ng/l und 15 ng/l 17β Estradiol-Äquivalenten (EEQ). Diese Werte wurden in einem internationalen Ringversuch ermittelt (siehe Anhang F). Der obere Schwellenwert des dynamischen Bereichs für diesen Test liegt zwischen 120 ng/l und 160 ng/l 17β Estradiol-Äquivalenten (EEQ). Proben, die ein estrogenes Potenzial oberhalb dieser Schwelle zeigen, müssen für eine gültige Quantifizierung verdünnt werden. Eine Extraktion und Anreicherung von Wasserproben können notwendig sein, wenn ihr estrogenes Potenzial 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 oestrogénique de l'eau et des eaux résiduaires - Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces cerevisiae) (ISO 19040-1:2018)

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 à l’aide de souches de levure génétiquement modifiées Saccharomyces cerevisiae. 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 8 ng/l et 15 ng/l d’équivalents 17β-œstradiol (EEQ) sur la base des résultats de l’essai interlaboratoires international (voir l’Annexe F). Le seuil supérieur de la gamme dynamique pour cet essai est compris entre 120 ng/l et 160 ng/l d’équivalent 17β-œstradiol (EEQ). 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 - 1. del: Presejalni preskus s kvasovkami (Saccharomyces cerevisiae) (ISO 19040-1:2018)

Ta dokument opisuje metodo za določevanje estrogenskega potenciala vode in odpadne vode z uporabo poročevalskega gena z gensko spremenjenimi sevi kvasovk Saccharomyces cerevisiae. 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 8 ng/l in 15 ng/l 17β-estradiol ekvivalentov (EEQ) na podlagi rezultatov mednarodnega medlaboratorijskega preskusa (glej dodatek F). Zgornja mejna vrednost dinamičnega razpona za ta preskus je med 120 ng/l in 160 ng/l 17β-estradiol ekvivalentov (EEQ). 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 19040-1:2022 - Water quality - Determination of the estrogenic potential of water and waste water - Part 1: Yeast estrogen screen (Saccharomyces cerevisiae) (ISO 19040-1:2018)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 19040-1:2023
01-junij-2023
Kakovost vode - Določanje estrogenega potenciala vode in odpadne vode - 1. del:
Presejalni preskus s kvasovkami (Saccharomyces cerevisiae) (ISO 19040-1:2018)
Water quality - Determination of the estrogenic potential of water and waste water - Part
1: Yeast estrogen screen (Saccharomyces cerevisiae) (ISO 19040-1:2018)
Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und
Abwasser - Teil 1: Hefebasierter Östrogentest (Saccharomyces cerevisiae) (ISO 19040-
1:2018)
Qualité de l'eau - Détermination du potentiel oestrogénique de l'eau et des eaux
résiduaires - Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces cerevisiae)
(ISO 19040-1:2018)
Ta slovenski standard je istoveten z: EN ISO 19040-1:2022
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 19040-1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 19040-1:2023


EN ISO 19040-1
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 1: Yeast estrogen screen
(Saccharomyces cerevisiae) (ISO 19040-1:2018)
Qualité de l'eau - Détermination du potentiel Wasserbeschaffenheit - Bestimmung des estrogenen
oestrogénique de l'eau et des eaux résiduaires - Partie Potentials von Wasser und Abwasser - Teil 1: Hefe-
1: Essai d'oestrogénicité sur levures (Saccharomyces Estrogenscreening (Saccharomyces cerevisiae) (ISO
cerevisiae) (ISO 19040-1:2018) 19040-1:2018)
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 19040-1:2022 E
worldwide for CEN national Members.

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EN ISO 19040-1:2022 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 19040-1:2023
EN ISO 19040-1:2022 (E)
European foreword
The text of ISO 19040-1:2018 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-
1:2022 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 19040-1:2018 has been approved by CEN as EN ISO 19040-1:2022 without any
modification.

3

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SIST EN ISO 19040-1:2023
INTERNATIONAL ISO
STANDARD 19040-1
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 1:
Yeast estrogen screen (Saccharomyces
cerevisiae)
Qualité de l'eau — Détermination du potentiel oestrogénique de l'eau
et des eaux résiduaires —
Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces
cerevisiae)
Reference number
ISO 19040-1:2018(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 . 2
4 Principle . 4
5 Interferences . 4
6 Apparatus and materials. 5
7 Reagents, media and test strain . 6
8 Sampling and samples .10
8.1 General .10
8.2 Bottles and material for sampling .10
8.3 Bottles and material pre-cleaning .10
8.4 Sampling procedure .10
8.5 Transport of samples .11
8.6 Pretreatment of samples .11
8.7 Storage of samples .11
9 Procedure.12
9.1 Preparation of cryo-cultures for long-term storage.12
9.2 Overnight culture .12
9.3 Test set up for aqueous samples .12
9.3.1 Preparation .12
9.3.2 Preparation of the reference dilution series .12
9.3.3 Negative control .13
9.3.4 Blank replicate .14
9.3.5 Sample dilution .14
9.3.6 Field blank .14
9.3.7 Plate setup .14
9.3.8 Inoculation of the test plate .14
9.4 Measurement .15
9.4.1 Measurement of the cell density .15
9.4.2 Measurement of the reporter gene activity .16
9.5 Calculation of the corrected absorbance and the reporter gene induction .16
9.6 Calculation of the relative growth .17
9.7 Estimation of the EC of the reference compound by linear interpolation .17
50
10 Validity criteria .17
11 Assessment criteria .18
12 Test report .18
Annex A (normative) Strain selection .19
Annex B (informative) Plate set up .20
Annex C (informative) Scheme of test principle .21
Annex D (informative) Test set up for chemicals and extracts .22
Annex E (informative) Preparation of dilution series .26
Annex F (informative) Performance data .27
Annex G (informative) Use of other yeast strains based on Saccharomyces cerevisiae .40
Annex H (informative) Statistical assessment .43
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Annex I (informative) Calculation of 17β-estradiol equivalents .45
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste
water — A simplified evaluation for testing of waste water .48
Bibliography .50
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.
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SIST EN ISO 19040-1:2023
INTERNATIONAL STANDARD ISO 19040-1:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 1:
Yeast estrogen screen (Saccharomyces cerevisiae)
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 with genetically modified yeast strains Saccharomyces
cerevisiae. 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
8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international
interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between
120 ng/l and 160 ng/l 17β-estradiol equivalents (EEQ). 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
ISO 7027, Water quality — Determination of turbidity
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http: //www .iso .org ./obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
blank replicate
additional replicate that contains no test organism, but is treated in the same way as the other replicates
of a sample
[SOURCE: ISO 10872:2010, 3.5]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[SOURCE: ISO 6107-6:2004, 24]
3.3
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]
3.4
dilution water
water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.5
50 % effect concentration
EC
50
concentration of a compound which causes 50 % of an effect
Note 1 to entry: In the sense of this document, the EC is the concentration of a compound which induces 50 % of
50
the maximal reporter gene activity which can be achieved by this compound.
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]
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3.7
induction rate
quotient of the mean signal measured after exposure to a dose of the test sample or with a positive
control, and the mean signal measured for the negative control using the same experimental conditions
[SOURCE: ISO 6107-6:2004, 43, modified — “corrected absorbance” replaces “mutant colonies”; “wells”
replaces “corresponding plates”, “quotient” replaces “difference”.]
3.8
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing
preculture, in fresh medium
[SOURCE: ISO 6107-6:2004, 44]
3.9
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]
3.10
lowest ineffective dilution
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”.]
3.11
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.12
overnight culture
culture started late in the afternoon and incubated overnight to be ready during the following morning
for purposes such as the inoculation of a preculture
Note 1 to entry: The procedure for the overnight culture is described in 9.2.
[SOURCE: ISO 6107-6:2004, 54, modified — deleted: "usually about 16 h".]
3.13
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”.]
3.14
reporter gene activity
quantitative activity of a gene attached to the promoter sequence of another gene
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3.15
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[SOURCE: ISO 6107-6:2004, 87]
3.16
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]
4 Principle
The Yeast Estrogen Screen (YES) is a reporter gene assay which can be used for the measurement of
the activation of the human estrogen receptor alpha (hERα) in the presence of a sample containing
compounds which activate the estrogen receptor (ER).
By this means the assay detects the estrogenic activity of the whole sample in its actual state as an
integral measure including possible additive, synergistic and antagonistic mixture-effects on the whole
process of the reporter gene expression.
The basic concept of such assays is explained in References [10] and [11]. The hERα is heterologously
expressed in the yeast cell under control of a copper dependent promoter. The estrogen receptor belongs
to the family of nuclear hormone receptors. If agonists of the estrogen receptor enter the yeast cell, they
bind to the estrogen receptor protein and thus induce its conformational change. As a consequence
two receptor proteins form a receptor dimer which translocates to the nucleus. This activation of the
estrogen receptor is measured by the induction of the reporter gene lacZ which encodes the enzyme
β-galactosidase. The lacZ is fused to a promoter containing estrogen responsive elements (ERE) and
is thus controlled by the activity of the estrogen receptor. The ER-dimer binds to the promoter and
by this activates the expression of the β-galactosidase. Finally, the activity of the β-galactosidase as a
measure for the estrogenic potential of the sample is determined using an appropriate substrate which
is cleaved to a coloured reaction product. The reaction product can be measured photometrically. See
Annex C for a scheme of the test principle.
5 Interferences
Coloured or turbid samples might interfere with the photometric detection of cell density and/or the
detection of the reaction product of the reporter enzyme β-Galactosidase (see Clause 10 for further
information).
Toxic effects of the test sample may lead to a reduction of viable cells and to a reduction of the
measurable signal. Consequently, estrogenic effects of a sample may be masked by acute toxic effects
leading to false negative test results (see Clause 10 for further information).
High salinity can cause toxic effects due to the resulting osmotic pressure. The conductivity of the
sample is a measure for its salinity. The yeast strain constructed by McDonnell et al. (Reference [10])
tolerates a conductivity of the sample up to 34 000 µS/cm.
Bacterial growth in the test wells is assessed by the blank replicate (3.1). See Clause 10 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.
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Due to the high sensitivity of this test avoid any contamination of buffer, media and all reagents used
with compounds exhibiting estrogenic activity to avoid false positive test results.
6 Apparatus and materials
For suitable sampling devices see Clause 8. Usual laboratory apparatus and glassware is required. In
particular, the following material is needed:
6.1 Incubator shaker, temperature- and time-controlled, 30 °C ± 1 °C and 37 °C ± 1 °C.
6.2 pH meter.
6.3 Steam sterilizer.
6.4 Dry sterilizer.
6.5 Centrifuge, with a rotor for 15 ml and 50 ml tubes up to 2 500 g and with a rotor for 96-well plates
up to 2 500 g.
6.6 Rotary mixer.
6.7 Freezer, at least ≤−18 °C and ≤−70 °C.
6.8 Sterile filter, cellulose acetate, 0,2 µm pore size.
6.9 Inoculation loops.
6.10 Multi-channel multistepper pipette (repeater pipette).
6.11 Multi-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
6.12 Spectrophotometer.
6.13 Transparent sterile polystyrene 96-well plates, for suspension cultures with flat bottom and lid.
6.14 Microplate photometer for 96-well plates, for absorbance measurement at 540 nm ± 20 nm or
580 nm ± 20 nm and at 600 nm ± 20 nm.
6.15 Clean bench.
6.16 Petri dishes, diameter approximately 94 mm, height approximately 16 mm.
6.17 Cryogenic vials, sterile, 1 ml, 10 ml.
6.18 Disposable nitrile gloves.
6.19 Air-permeable sealing membranes for 96-well plates.
© ISO 2018 – All rights reserved 5

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7 Reagents, media and test strain
7.1 Reagents
As far as possible, use "reagent grade" chemicals.
1)
7.1.1 Yeast nit
...

SLOVENSKI STANDARD
oSIST prEN ISO 19040-1:2022
01-julij-2022
[Not translated]
Water quality - Determination of the estrogenic potential of water and waste water - Part
1: Yeast estrogen screen (Saccharomyces cerevisiae) (ISO 19040-1:2018)
Wasserbeschaffenheit - Bestimmung des östrogenen Potentials von Wasser und
Abwasser - Teil 1: Hefebasierter Östrogentest (Saccharomyces cerevisiae) (ISO 19040-
1:2018)
Qualité de l'eau - Détermination du potentiel oestrogénique de l'eau et des eaux
résiduaires - Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces cerevisiae)
(ISO 19040-1:2018)
Ta slovenski standard je istoveten z: prEN ISO 19040-1
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 19040-1:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 19040-1:2022

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oSIST prEN ISO 19040-1:2022
INTERNATIONAL ISO
STANDARD 19040-1
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 1:
Yeast estrogen screen (Saccharomyces
cerevisiae)
Qualité de l'eau — Détermination du potentiel oestrogénique de l'eau
et des eaux résiduaires —
Partie 1: Essai d'oestrogénicité sur levures (Saccharomyces
cerevisiae)
Reference number
ISO 19040-1:2018(E)
©
ISO 2018

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oSIST prEN ISO 19040-1:2022
ISO 19040-1:2018(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 . 2
4 Principle . 4
5 Interferences . 4
6 Apparatus and materials. 5
7 Reagents, media and test strain . 6
8 Sampling and samples .10
8.1 General .10
8.2 Bottles and material for sampling .10
8.3 Bottles and material pre-cleaning .10
8.4 Sampling procedure .10
8.5 Transport of samples .11
8.6 Pretreatment of samples .11
8.7 Storage of samples .11
9 Procedure.12
9.1 Preparation of cryo-cultures for long-term storage.12
9.2 Overnight culture .12
9.3 Test set up for aqueous samples .12
9.3.1 Preparation .12
9.3.2 Preparation of the reference dilution series .12
9.3.3 Negative control .13
9.3.4 Blank replicate .14
9.3.5 Sample dilution .14
9.3.6 Field blank .14
9.3.7 Plate setup .14
9.3.8 Inoculation of the test plate .14
9.4 Measurement .15
9.4.1 Measurement of the cell density .15
9.4.2 Measurement of the reporter gene activity .16
9.5 Calculation of the corrected absorbance and the reporter gene induction .16
9.6 Calculation of the relative growth .17
9.7 Estimation of the EC of the reference compound by linear interpolation .17
50
10 Validity criteria .17
11 Assessment criteria .18
12 Test report .18
Annex A (normative) Strain selection .19
Annex B (informative) Plate set up .20
Annex C (informative) Scheme of test principle .21
Annex D (informative) Test set up for chemicals and extracts .22
Annex E (informative) Preparation of dilution series .26
Annex F (informative) Performance data .27
Annex G (informative) Use of other yeast strains based on Saccharomyces cerevisiae .40
Annex H (informative) Statistical assessment .43
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Annex I (informative) Calculation of 17β-estradiol equivalents .45
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste
water — A simplified evaluation for testing of waste water .48
Bibliography .50
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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 19040-1:2022
INTERNATIONAL STANDARD ISO 19040-1:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 1:
Yeast estrogen screen (Saccharomyces cerevisiae)
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 with genetically modified yeast strains Saccharomyces
cerevisiae. 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
8 ng/l and 15 ng/l 17β-estradiol equivalents (EEQ) based on the results of the international
interlaboratory trial (see Annex F). The upper threshold of the dynamic range for this test is between
120 ng/l and 160 ng/l 17β-estradiol equivalents (EEQ). 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
ISO 7027, Water quality — Determination of turbidity
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http: //www .iso .org ./obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
blank replicate
additional replicate that contains no test organism, but is treated in the same way as the other replicates
of a sample
[SOURCE: ISO 10872:2010, 3.5]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[SOURCE: ISO 6107-6:2004, 24]
3.3
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]
3.4
dilution water
water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.5
50 % effect concentration
EC
50
concentration of a compound which causes 50 % of an effect
Note 1 to entry: In the sense of this document, the EC is the concentration of a compound which induces 50 % of
50
the maximal reporter gene activity which can be achieved by this compound.
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]
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3.7
induction rate
quotient of the mean signal measured after exposure to a dose of the test sample or with a positive
control, and the mean signal measured for the negative control using the same experimental conditions
[SOURCE: ISO 6107-6:2004, 43, modified — “corrected absorbance” replaces “mutant colonies”; “wells”
replaces “corresponding plates”, “quotient” replaces “difference”.]
3.8
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing
preculture, in fresh medium
[SOURCE: ISO 6107-6:2004, 44]
3.9
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]
3.10
lowest ineffective dilution
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”.]
3.11
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
3.12
overnight culture
culture started late in the afternoon and incubated overnight to be ready during the following morning
for purposes such as the inoculation of a preculture
Note 1 to entry: The procedure for the overnight culture is described in 9.2.
[SOURCE: ISO 6107-6:2004, 54, modified — deleted: "usually about 16 h".]
3.13
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”.]
3.14
reporter gene activity
quantitative activity of a gene attached to the promoter sequence of another gene
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3.15
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[SOURCE: ISO 6107-6:2004, 87]
3.16
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]
4 Principle
The Yeast Estrogen Screen (YES) is a reporter gene assay which can be used for the measurement of
the activation of the human estrogen receptor alpha (hERα) in the presence of a sample containing
compounds which activate the estrogen receptor (ER).
By this means the assay detects the estrogenic activity of the whole sample in its actual state as an
integral measure including possible additive, synergistic and antagonistic mixture-effects on the whole
process of the reporter gene expression.
The basic concept of such assays is explained in References [10] and [11]. The hERα is heterologously
expressed in the yeast cell under control of a copper dependent promoter. The estrogen receptor belongs
to the family of nuclear hormone receptors. If agonists of the estrogen receptor enter the yeast cell, they
bind to the estrogen receptor protein and thus induce its conformational change. As a consequence
two receptor proteins form a receptor dimer which translocates to the nucleus. This activation of the
estrogen receptor is measured by the induction of the reporter gene lacZ which encodes the enzyme
β-galactosidase. The lacZ is fused to a promoter containing estrogen responsive elements (ERE) and
is thus controlled by the activity of the estrogen receptor. The ER-dimer binds to the promoter and
by this activates the expression of the β-galactosidase. Finally, the activity of the β-galactosidase as a
measure for the estrogenic potential of the sample is determined using an appropriate substrate which
is cleaved to a coloured reaction product. The reaction product can be measured photometrically. See
Annex C for a scheme of the test principle.
5 Interferences
Coloured or turbid samples might interfere with the photometric detection of cell density and/or the
detection of the reaction product of the reporter enzyme β-Galactosidase (see Clause 10 for further
information).
Toxic effects of the test sample may lead to a reduction of viable cells and to a reduction of the
measurable signal. Consequently, estrogenic effects of a sample may be masked by acute toxic effects
leading to false negative test results (see Clause 10 for further information).
High salinity can cause toxic effects due to the resulting osmotic pressure. The conductivity of the
sample is a measure for its salinity. The yeast strain constructed by McDonnell et al. (Reference [10])
tolerates a conductivity of the sample up to 34 000 µS/cm.
Bacterial growth in the test wells is assessed by the blank replicate (3.1). See Clause 10 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.
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Due to the high sensitivity of this test avoid any contamination of buffer, media and all reagents used
with compounds exhibiting estrogenic activity to avoid false positive test results.
6 Apparatus and materials
For suitable sampling devices see Clause 8. Usual laboratory apparatus and glassware is required. In
particular, the following material is needed:
6.1 Incubator shaker, temperature- and time-controlled, 30 °C ± 1 °C and 37 °C ± 1 °C.
6.2 pH meter.
6.3 Steam sterilizer.
6.4 Dry sterilizer.
6.5 Centrifuge, with a rotor for 15 ml and 50 ml tubes up to 2 500 g and with a rotor for 96-well plates
up to 2 500 g.
6.6 Rotary mixer.
6.7 Freezer, at least ≤−18 °C and ≤−70 °C.
6.8 Sterile filter, cellulose acetate, 0,2 µm pore size.
6.9 Inoculation loops.
6.10 Multi-channel multistepper pipette (repeater pipette).
6.11 Multi-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
6.12 Spectrophotometer.
6.13 Transparent sterile polystyrene 96-well plates, for suspension cultures with flat bottom and lid.
6.14 Microplate photometer for 96-well plates, for absorbance measurement at 540 nm ± 20 nm or
580 nm ± 20 nm and at 600 nm ± 20 nm.
6.15 Clean bench.
6.16 Petri dishes, diameter approximately 94 mm, height approximately 16 mm.
6.17 Cryogenic vials, sterile, 1 ml, 10 ml.
6.18 Disposable nitrile gloves.
6.19 Air-permeable sealing membranes for 96-well plates.
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7 Reagents, media and test strain
7.1 Reagents
As far as possible, use "reagent grade" chemicals.
1)
7.1.1 Yeast nitrogen base without amino acids .
7.1.2 α-D-Glucose, anhydrous, C H O , molecular weight 180,15 g/mol, CAS: 50-99-7.
6 12 6
7.1.3 Adenine, C H N , molecular weight 135,13 g/mol, CAS: 73-24-5.
5 5 5
7.1.4 l-arginine, C H N O , molecular weight 174,20 g/mol, CAS: 74-79-3.
6 14 4 2
7.1.5 l-aspartic acid, C H NO , molecular weight 133,10 g/mol, CAS: 56-84-8.
4 7 4
7.1.6 l-glutamic acid monosodium salt hydrate, C H NNaO ·H O, molecular weight (anhydrous)
5 8 4 2
169,11 g/mol, CAS: 142-47-2 (anhydrous basis).
7.1.7 l-histidine-HCl, C H N O ·HCl·H O, molecular weight 209,6 g/mol, CAS: 5934-29-2.
6 9 3 2 2
7.1.8 l-isoleucine, C H NO , molecular weight 131,17 g/mol, CAS: 73-32-5.
6 13 2
7.1.9 l-leucine, C H NO , molecular weight 131,17 g/mol, CAS: 61-90-5.
6 13 2
7.1.10 l-lysine-HCl, C H N O ·HCl, molecular weight 182,65 g/mol, CAS: 657-27-2.
6 14 2 2
7.1.11 l-methionine, C H NO S, molecular weight 149,21 g/mol, CAS: 63-68-3.
5 11 2
7.1.12 l-phenylalanine, C H NO , molecular weight 165,19 g/mol, CAS: 63-91-2.
9 11 2
7.1.13 l-serine, C H NO , molecular weight 105,09 g/mol, CAS: 56-45-1.
3 7 3
7.1.14 l-threonine, C H NO , molecular weight 119,12 g/mol, CAS: 72-19-5.
4 9 3
7.1.15 l-tyrosine, C H NO , molecular weight 181,19 g/mol, CAS: 60-18-4.
9 11 3
7.1.16 l-valine, C H NO , molecular weight 117,15 g/mol, CAS: 72-18-4.
5 11 2
7.1.17 Copper(II) sulfate pentahydrate, CuSO ·5H O, molecular weight 249,69 g/mol, CAS: 7758-99-8.
4 2
7.1.18 Ampicillin sodium salt, C H N NaO S, molecular weight 371,39 g/mol, CAS: 69-52-3.
16 18 3 4
7.1.19 Streptomycin sulfate salt, C H N O ·1,5H SO , molecular weight 728,69 g/mol,
21 39 7 12 2 4
CAS: 3810-74-0.
7.1.20 Agar for microbiology, (C H O )n, CAS: 9002-18-0.
12 18 9
1) Yeast nitrogen base without amino acids contains a nitrogen source such as ammonium sulfate, vitamins and
trace elements which are required for growth of yeast cells. Yeast nitrogen base without amino acids is used for the
selection of yeast strains depending on requirements for carbon sources and amino acids.
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7.1.21 Hydrochloric acid solution, 1 M (HCl), molecular weight 36,46 g/mol, CAS: 7647-01-0.
7.1.22 Sodium hydroxide, NaOH, molecular weight 40,00 g/mol, CAS: 1310-73-2.
7.1.23 Ethanol, ≥99,8 %, CH CH OH, molecular weight 46,07 g/mol, CAS: 64-17-5.
3 2
7.1.24 Glycerol for molecular biology, ≥99 %, HOCH CH(OH)CH OH, molecular weight 92,09 g/mol,
2 2
CAS: 56-81-5.
7.1.25 17β-Estradiol, ≥98 %, C H O , molecular weight 272,38 g/mol, CAS: 50-28-2.
18 24 2
7.1.26 Disodium hydrogen phosphate dihydrate, Na HPO ·2H O, molecular weight 177,99 g/mol,
2 4 2
CAS: 10028-24-7.
7.1.27 Sodiumdihydrogen phosphate monohydrate, NaH PO ⋅H O, molecular weight 137,99 g/mol,
2 4 2
CAS: 10049-21-5.
7.1.28 Potassium chloride, KCl, molecular weight 74,55 g/mol, CAS: 7447-40-7.
7.1.29 Magnesium sulfate heptahydrate, MgSO ·7H O, molecular weight 246,47 g/mol,
4 2
CAS: 10034-99-8.
7.1.30 Chlorophenolred-β-D-galactopyranoside (CPRG), C H Cl O S, molecular weight 585,41 g/
25 22 2 10
mol, CAS: 99792-79-7.
7.1.31 Lyticase from Arthrobacter luteus lyophilized powder, ≥2 000 units/mg protein,
CAS: 37340-57-1.
7.1.32 DL-Dithiothreitol, HSCH CH(OH)CH(OH)CH SH, molecular weight 154,25 g/mol, CAS: 3483-
2 2
12-3.
7.1.33 Sodium dodecyl sulfate, CH (CH ) OSO Na, molecular weight 288,38 g/mol, CAS: 151-21-3.
3 2 11 3
7.1.34 Aceton (puriss p.a.), CH COCH , molecular weight 58,08 g/mol, CAS: 67-64-1.
3 3
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.
7.3 Test strain.
The generation of the test strain is described in References [10] and [11]. It is derived from the strain
Saccharomyces cerevisiae BJ3505 (protease deficient, MATα, PEP4::HIS3, prb-1-delta1.6R, HIS3-delta200,
lys2-801, trp1-delta101, ura3-52gal2can1). This strain harbours two plasmids. The construction of
these plasmids is described in Reference [10]. The plasmid YEPE10 contains the CUP1::hER fusion which
encodes the human estrogen receptor α cloned from the MCF-7 human cell lineage under the control
of the metallothionein pr
...