EN ISO 19040-2:2022

SLOVENSKI STANDARD
01-junij-2023
Kakovost vode - Določanje estrogenega potenciala vode in odpadne vode - 2. del:
Presejalni preskus s kvasovkami (A-YES, Arxula adeninivorans) (ISO 19040-
2:2018)
Water quality - Determination of the estrogenic potential of water and waste water - Part
2: Yeast estrogen screen (A-YES, Arxula adeninivorans) (ISO 19040-2:2018)
Wasserbeschaffenheit - Bestimmung des estrogenen Potentials von Wasser und
Abwasser - Teil 2: Hefe-Estrogenscreening (A-YES, Arxula adeninivorans) (ISO 19040-
2:2018)
Qualité de l'eau - Détermination du potentiel oestrogène de l'eau et des eaux résiduaires
- Partie 2: Test d'oestrogénicité (A-YES, Arxula adeninivorans) (ISO 19040-2:2018)
Ta slovenski standard je istoveten z: EN ISO 19040-2:2022
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 19040-2
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 2: Yeast estrogen screen
(A-YES, Arxula adeninivorans) (ISO 19040-2:2018)
Qualité de l'eau - Détermination du potentiel Wasserbeschaffenheit - Bestimmung des estrogenen
oestrogène de l'eau et des eaux résiduaires - Partie 2: Potentials von Wasser und Abwasser - Teil 2: Hefe-
Test d'oestrogénicité (A-YES, Arxula adeninivorans) Estrogenscreening (A-YES, Arxula adeninivorans) (ISO
(ISO 19040-2:2018) 19040-2:2018)
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 19040-2:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 19040-2:2018 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-
2:2022 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 19040-2:2018 has been approved by CEN as EN ISO 19040-2:2022 without any
modification.
INTERNATIONAL ISO
STANDARD 19040-2
First edition
2018-08
Water quality — Determination of
the estrogenic potential of water and
waste water —
Part 2:
Yeast estrogen screen (A-YES, Arxula
adeninivorans)
Qualité de l'eau — Détermination du potentiel oestrogène de l'eau et
des eaux résiduaires —
Partie 2: Test d'oestrogénicité (A-YES, Arxula adeninivorans)
Reference number
ISO 19040-2:2018(E)
©
ISO 2018
ISO 19040-2:2018(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
below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
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Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 4
5 Interferences . 4
6 Apparatus and materials. 4
7 Reagents, media and test strains . 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 .10
8.4 Sampling procedure .10
8.5 Transport of samples .10
8.6 Pretreatment of samples .10
8.7 Storage of samples .11
9 Procedure.11
9.1 Test set up .11
9.1.1 Preparation of the reference dilution series .11
9.1.2 Reactivation of the yeast .11
9.1.3 Negative control .12
9.1.4 Blank replicate .12
9.1.5 Sample dilution .12
9.1.6 Field blank .13
9.1.7 Plate set up .13
9.1.8 Inoculation of the test plate .13
9.2 Measurement .14
9.2.1 Measurement of the reporter gene activity .14
9.2.2 Measurement of the cell density .15
9.3 Calculations .15
9.3.1 Background correction .15
9.3.2 Calculation of the relative growth .16
9.3.3 Calculations for assessment of sample blanks .17
9.3.4 Calculation of the reporter gene induction .19
10 Validity criteria .22
11 Assessment criteria .23
12 Test report .23
13 Verification .23
Annex A (informative) Plate set up .25
Annex B (informative) Lyophilization of Arxula adeninivorans cells .26
Annex C (informative) Scheme of test principle .28
Annex D (informative) Test set up for chemicals and extracts .29
Annex E (informative) Preparation of dilution series .30
Annex F (informative) Performance data .31
Annex G (informative) Statistical assessment .41
ISO 19040-2:2018(E)
Annex H (informative) Calculation of estradiol equivalents.42
Annex I (informative) Alternative test design for EEQ determination .45
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water
— A simplified evaluation for testing of waste water .46
Annex K (informative) Example for statistical evaluation .48
Bibliography .55
iv © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation 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.
INTERNATIONAL STANDARD ISO 19040-2:2018(E)
Water quality — Determination of the estrogenic potential
of water and waste water —
Part 2:
Yeast estrogen screen (A-YES, Arxula adeninivorans)
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 a genetically modified yeast strain Arxula adeninivorans.
This reporter gene assay is based on the activation of the human estrogen receptor alpha.
Arxula adeninivorans is a highly robust and salt- and temperature-tolerant test organism and is
especially suitable for the analysis of samples with high salinity (conductivity up to 70 mS/cm). The test
organism can be cultivated in medium with sodium chloride content up to 20 %.
This method is applicable to:
— fresh water;
— waste water;
— sea water;
— brackish 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
1,5 ng/l and 3 ng/l 17β-estradiol equivalents (EEQ). The upper threshold of the dynamic range for
this test is between 25 ng/l and 40 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.
An international interlaboratory trial for the validation of this document has been carried out. The
results are summarized in Annex F.
NOTE Extraction and pre-concentration of water samples can prove necessary.
ISO 19040-2:2018(E)
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 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
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
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
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
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]
2 © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
3.7
growth rate
proportional rate of increase in cell density
[SOURCE: ISO 10253:2006, 3.2]
3.8
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, and the mean value of the corresponding wells treated with the
negative control using the same strain under identical conditions
Note 1 to entry: Instead of the negative control, the estimated parameter A of the four-parameter model, which
describes the dose response relationship between reference compound and the induction rate, can be used.
[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”.]
3.9
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]
3.10
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”.]
3.11
negative control
dilution water without test sample
[SOURCE: ISO 6107-6:2004, 51]
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
reporter gene activity
quantitative activity of a gene attached to the promoter sequence of another gene
3.14
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]
ISO 19040-2:2018(E)
4 Principle
The A-YES (Arxula Yeast Estrogen Screen) is a reporter gene assay which can be used for the
measurement of the activation of the human estrogen receptor alpha (ERα) in the presence of a sample
containing compounds which cause estrogenic effects. 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.
The human estrogen receptor α is constitutively expressed in the yeast cell under control of a TEF1
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. This activation
of the estrogen receptor is measured by the induction of the reporter gene phyK which encodes the
enzyme phytase. The phyK is fused to an estrogen dependent promoter which contains estrogen
responsive elements (ERE). The ER-dimer binds to the promoter and by this activates the expression
and secretion of the phytase. Finally, the activity of the phytase 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 the cell density and/
or the detection of the cleaved substrate of the reporter enzyme phytase (see Clause 10 for further
information).
Effects of the sample matrix may lead to a reduction or increase of viable cells and to a reduction or
increase of the measurable signal. Estrogenic effects of a sample may be masked by matrix effects
leading to false negative or false positive test results.
High salinity can cause toxic effects due to the resulting osmotic pressure. The conductivity of a sample
is a measure for its salinity. The Arxula adeninivorans yeast tolerates a conductivity of the sample up to
20 % sodium chloride which meets a conductivity of 180 mS/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.
For detailed information about appropriate sampling material that does not influence the test result
see Clause 8.
6 Apparatus and materials
For suitable sampling devices see Clause 8. Use usual laboratory apparatus and glassware if required.
In particular, the following material is needed:
6.1 Temperature- and time-controlled incubator shaker, shaker orbit at least 3 mm, 30 °C to
37 °C with an accuracy of ±1 °C.
If the shaker has no incubation function use a lab shaker with a shaker orbit of at least 3 mm in
combination with an incubator (6.17).
6.2 Lab mini shaker.
4 © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
6.3 Multi-parameter measurement device for pH and conductivity or separate devices for each
parameter.
6.4 Steam sterilizer.
6.5 Centrifuge, with a rotor for 96-well plates up to 1 000 g and a rotor for 2 ml reaction tubes.
6.6 Sterile filters, cellulose acetat, 0,2 µm pore size.
6.7 Single-channel pipettes, nominal volume 10 µl up to 10 000 µl.
6.8 Multi-channel pipettes, nominal volume 100 µl and 300 µl.
6.9 Transparent polystyrene 96-well plates with flat bottom (F-profile, 300 µl) and lid.
6.10 96-deep well plates with at least 1 ml volume with round bottom and square wells.
6.11 Microplate photometer for 96-well plates, for absorbance measurement for wavelength
405 nm ± 20 nm and 630 nm ± 5 nm or alternatively 600 nm ± 20 nm.
6.12 Air-permeable adhesive foil for deep well plates.
6.13 Reaction tubes, 2 ml.
6.14 Test tubes, 15 ml and 50 ml.
6.15 Multi-channel pipette trough.
6.16 Balance, minimum load 1 mg, d = 0,1 mg.
6.17 Incubator, 30 °C to 37 °C with an accuracy of ±1 °C. For the purpose of using the incubator in
combination with a shaker a cooled incubator is required.
7 Reagents, media and test strains
7.1 Reagents
As far as possible, use “reagent grade“-chemicals.
7.1.1 Hydrochloric acid solution, c(HCl) = 1 mol/l, molecular weight 36,46 g/mol, CAS: 7647-01-0.
7.1.2 Sodium hydroxide solution, c(NaOH) = 1 mol/l, molecular weight 40,00 g/mol, CAS: 1310-73-2.
7.1.3 Ethanol, ≥99,8 %, C H OH, molecular weight 46,07 g/mol, CAS: 64-17-5.
2 5
7.1.4 17β-estradiol, ≥98 %, C H O , molecular weight 272,38 g/mol, CAS: 50-28-2.
18 24 2
7.1.5 Maltose monohydrate, >95 %, C H O ·H O, molecular weight 360,32 g/mol, CAS: 6363-53-7.
12 22 11 2
7.1.6 Sodium nitrate, >99 %, NaNO , molecular weight 84,98 g/mol, CAS: 7631-99-4.
ISO 19040-2:2018(E)
7.1.7 Potassium dihydrogen phosphate, ≥99 %, KH PO , molecular weight 136,09 g/mol,
2 4
CAS: 7778-77-0.
7.1.8 Magnesium sulfate pure, MgSO , molecular weight 120,37 g/mol (water free), CAS: 7487-88-9.
7.1.9 Iron(III) chloride hexahydrate, >97 %, FeCl ·6H O, molecular weight 270,29 g/mol,
3 2
CAS: 10025-77-1.
7.1.10 Calcium nitrate, >99 %, Ca(NO ) , molecular weight 164,09 g/mol, CAS: 10124-37-5.
3 2
7.1.11 Calcium D-pantothenate, >98 %, C H CaN O molecular weight 238,27 g/mol, CAS: 137-08-6.
18 32 2 10
7.1.12 Thiamine hydrochloride, >98,5 %, C H Cl N OS, molecular weight 337,27 g/mol,
12 18 2 4
CAS: 67-03-8.
7.1.13 Niacin, >99,5 %, C H NO , molecular weight 123,11 g/mol, CAS: 59-67-6.
6 5 2
7.1.14 Pyridoxine hydrochloride, >99 %, C H NO ·HCl, molecular weight 205,64 g/mol, CAS: 58-56-0.
8 11 3
7.1.15 D-(+)-Biotin, ≥98,5 %, C H N O S, molecular weight 244,31 g/mol, CAS: 58-85-5.
10 16 2 3
7.1.16 Inositol, ≥99 %, C H O , molecular weight 180,16 g/mol, CAS: 87-89-8.
6 12 6
7.1.17 Boric acid, >99,8 %, H BO , molecular weight 61,83 g/mol, CAS: 10043-35-3.
3 3
7.1.18 Copper(II) sulfate pentahydrate, >99,5 %, CuSO ·5H O, molecular weight 249,68 g/mol,
4 2
CAS: 7758-99-8.
7.1.19 Potassium iodide, >99 %, KI molecular weight 166,00 g/mol, CAS: 7681-11-0.
7.1.20 Manganese sulfate monohydrate, >99 %, MnSO ·H O, molecular weight 169,02 g/mol,
4 2
CAS: 10034-96-5.
7.1.21 Zinc sulfate heptahydrate, >99,5 %, ZnSO ·7H O, molecular weight 287,56 g/mol, CAS:
4 2
7446-20-0.
7.1.22 Sodium molybdate dihydrate, >99,5 %, Na MoO ·2H O, molecular weight 241,95 g/mol,
2 4 2
CAS: 10102-40-6.
7.1.23 Cobalt(II) chloride for synthesis, CoCl , molecular weight 129,84 g/mol, CAS: 7646-79-9.
7.1.24 Trisodium citrate dihydrate, >99 %, C H Na O ·2H O, molecular weight 294,10 g/mol,
6 5 3 7 2
CAS: 6132-04-3.
7.1.25 Citric acid, >99,5 %, C H O , molecular weight 192,12 g/mol, CAS: 77-92-9.
6 8 7
7.1.26 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP), C H NNa O P·6H O, molecular
6 4 2 6 2
weight 371,14 g/mol, CAS: 333338-18-4.
7.1.27 Sodium hydroxide, ≥99 %, NaOH, molecular weight 40,00 g/mol, CAS: 1310-73-2.
6 © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
7.1.28 Sea salt with the specifications: chloride (Cl) 19 290 mg/l, sodium 10 780 mg/l, sulfate
2 660 mg/l, potassium 420 mg/l, calcium 400 mg/l, carbonate (bicarbonate) 200 mg/l, strontium
8,8 mg/l, boron 5,6 mg/l, bromide 56 mg/l, iodide 0,24 mg/l, lithium 0,3 mg/l, fluoride 1,0 mg/l,
magnesium (Mg) 1 320 mg/l.
7.1.29 Sodium chloride, ≥99 %, NaCl, molecular weight 58,44 g/mol, CAS: 7647-14-5.
7.1.30 Aceton (purity p.a.), C H O, molecular weight 58,08 g/mol, CAS: 67-64-1.
3 6
7.2 Water, grade 3, as defined in ISO 3696; water with conductivity up to 5 µS/cm is acceptable, or
ultrapure water.
If sterile water is needed, autoclave or sterilize by filtration (cellulose acetate, 0,2 µm).
Water as specified here is also used for the preparation of dilution water which is used for the stepwise
dilution of the test sample.
7.3 Test strain
This test strain is derived from Blastobotrys adeninivorans G1214 Syn.: Arxula adeninivorans G1214
(aleu2 aura3::ALEU2), Reference [1]. This strain displays an uracil auxotrophy. To prevent any formations
of antibiotic resistances in environment and to increase acceptance regarding legal requirements the
test organism contains no antibiotic resistance markers.
Genetic modifications: Integration of the selection marker AURA3mm in the plasmid Xplor2-102-hERα-
GAA2(ERE107)-phyK after exchange of the marker ALEU2 promoter-ATRP1m. The selection marker
AURA3mm was isolated from the plasmid pCR4-AURA3mm-13. The sequences of E. coli and Kanamycin
resistant marker were eliminated through restriction digestion. Stable integration of Xplor2-102-hERα-
GAA2(ERE107)-phyK in Arxula adeninivorans genome was achieved by transformation the cassette in
uracil auxotroph mutant of A. adeninivorans G1214 (aleu2 aura3::ALEU2) through recombination with
25S-rDNA.
The yeast cell suspension for determination of the estrogenic potential of aqueous samples is prepared
from lyophilized yeast cells. As the Arxula adeninivorans cells are lyophilized the test can be conducted
under highly standardized conditions and no specific lab equipment for long-term cell cultivation is
required.
The yeast cells are available commercially. Store the lyophilized yeast cells between 4 °C and 8 °C and
follow the manufacturer’s recommendations. After reactivation, the yeast cells can directly be used for
the test, precultivation is not needed for testing.
An alternative to commercially available lyophilized yeast cells are self-made lyophilized yeast cells.
The preparation procedure is described in Annex B. The yeast strain can be isolated from commercially
available lyophilisate.
7.4 Media.
If autoclaving is necessary autoclave for 20 min at 121 °C ± 2 °C. Cover vessels loosely (e.g. with
aluminium foil). Never seal air-tight.
7.4.1 17β-estradiol (E2) stock solution.
Dissolve 10 mg of 17β-estradiol (E2) (7.1.4) in 10 ml ethanol (7.1.3). Store the 17β-estradiol (E2) stock
solution at ≤−18 °C. Store the stock solution no longer than 18 months.
If available certified 17β-estradiol reference standard of equal concentration can be used as stock
solution.
ISO 19040-2:2018(E)
7.4.2 17β-estradiol (E2) working solution.
Dilute 17β-estradiol (E2) stock solution (7.4.1) 1→100 by adding 10 µl of the 17β-estradiol (E2) stock
solution (7.4.1) to 990 µl ethanol (7.1.3) and mix well. Make a further 1→10 dilution by adding 100 µl of
the first dilution to 900 µl ethanol (7.1.3) and mix well. The final concentration is 1 mg/l. The working
solution shall be aliquoted in order to avoid thawing and freezing of the working solution. Store the
17β-estradiol (E2) working solution at ≤−18 °C. Store the working solution no longer than six months.
NOTE Pipetting of organic solvents requires the usage of adequate calibrated pipettes or other suitable
liquid handling equipment.
7.4.3 Maltose solution.
Dissolve 20 g of maltose monohydrate (7.1.5) in 70 ml ultrapure water (7.2). Fill the maltose solution up
to 100 ml with ultrapure water (7.2). Autoclave the solution. Store the maltose solution at 2 °C to 8 °C.
Store the maltose solution no longer than six months.
7.4.4 Salt solution for yeast minimal medium.
Dissolve 3,7 g of NaNO (7.1.6), 8,4 g KH PO (7.1.7) and 1 g of MgSO (7.1.8) in 70 ml ultrapure
3 2 4 4
water (7.2). Fill up to 100 ml with ultrapure water (7.2). Autoclave the solution. Store the salt solution
for yeast minimal medium at 2 °C to 8 °C. Store the salt solution no longer than six months.
7.4.5 Salt solution for saline yeast minimal medium.
Dissolve 28 g of NaCl (7.1.29), 3,7 g of NaNO (7.1.6), 8,4 g KH PO (7.1.7) and 1 g of MgSO (7.1.8) in
3 2 4 4
70 ml ultrapure water (7.2). Fill up to 100 ml with ultrapure water (7.2). For the analysis of sea water
samples and brackish water samples the 28 g of NaCl may be replaced by 28 g of a salt composition
similar to the salt composition of sea water. Autoclave the solution. Store the salt solution for saline
yeast minimal medium at 2 °C to 8 °C no longer than six months.
7.4.6 Micronutrient solution.
Weigh the following chemicals separately:
0,05 g H BO (7.1.17), 0,01 g CuSO ·5H O (7.1.18), 0,01 g KI (7.1.19), 0,04 g MnSO ·H O (7.1.20),
3 3 4 2 4 2
0,04 g ZnSO ·7H O (7.1.21), 0,02 g Na MoO ·2H O (7.1.22), 0,01 g CoCl (7.1.23).
4 2 2 4 2 2
Merge the chemicals and dissolve them in 100 ml ultrapure water (7.2). Autoclave the solution.
Precipitation after autoclaving has no influence on quality. Shake the solution before using it. Store the
micronutrient solution at 2 °C to 8 °C. Store the micronutrient solution no longer than 12 months.
7.4.7 FeCl solution.
Weigh 0,2 g of Fe(III)Cl hexahydrate (7.1.9) and dissolve it in 20 ml ultrapure water (7.2). Sterilize the
FeCl solution by filtration (0,2 µm). Store the FeCl solution at 2 °C to 8 °C. Store the FeCl solution no
3 3 3
longer than six months.
7.4.8 Ca(NO ) solution.
3 2
Weigh 10 g of Ca(NO ) (7.1.10) and dissolve it in 10 ml of ultrapure water (7.2). Sterilize the Ca(NO )
3 2 3 2
solution by filtration (0,2 µm). Store the Ca(NO ) solution at 2 °C to 8 °C. Store the Ca(NO ) solution no
3 2 3 2
longer than six months.
7.4.9 Vitamin mix.
Weigh the following chemicals separately:
8 © ISO 2018 – All rights reserved

ISO 19040-2:2018(E)
0,2 g Calcium-D-panthothenate (7.1.11), 0,2 g thiamin hydrochloride (7.1.12), 0,05 g niacin (7.1.13),
0,02 g biotin (7.1.15), 0,2 g pyridoxin hydrochloride (7.1.14), 2 g inositol (7.1.16).
Merge and dissolve the vitamins in 50 ml ultrapure water (7.2). Sterilize the vitamin mix by
filtration (0,2 µm). Store the vitamin mix at 2 °C to 8 °C no longer than six months.
7.4.10 Yeast minimal medium with maltose.
Pipette 1 ml of FeCl solution (7.4.7), 1 ml of Ca(NO ) solution (7.4.8), 1 ml of micronutrient
3 3 2
solution (7.4.6) and 0,5 ml vitamin mix (7.4.9) to 96,5 ml of the salt solution (7.4.4). Add 100 ml maltose
solution (7.4.3).
The yeast minimal medium with maltose is five-fold concentrated. Store the yeast minimal medium at
2 °C to 8 °C. Store the yeast minimal medium no longer than six months.
7.4.11 Saline yeast minimal medium with maltose.
Pipette 1 ml of FeCl solution (7.4.7), 1 ml of Ca(NO ) solution (7.4.8), 1 ml of micronutrient
3 3 2
solution (7.4.6) and 0,5 ml vitamin mix (7.4.9) to 96,5 ml of the salt solution for saline yeast minimal
medium (7.4.5). Add 100 ml maltose solution (7.4.3).
The saline yeast minimal medium with maltose is five-fold concentrated. Store the saline yeast minimal
medium at 2 °C to 8 °C. Store the saline yeast minimal medium no longer than six months.
7.4.12 Substrate buffer.
Weigh 10,35 g of trisodiumcitrate dihydrate (7.1.24) and 12,45 g of citric acid (7.1.25) separately.
Dissolve every reagent in 60 ml of ultrapure water (7.2). Merge the two solutions and fill up to 200 ml
with ultrapure water (7.2). Autoclave the substrate buffer and cool down before using it. Store the
substrate buffer at 2 °C to 8 °C. Store the substrate buffer no longer than six months.
7.4.13 Phytase substrate solution.
Weigh at least 15 mg of 4-Nitrophenyl phosphate disodium salt hexahydrate (7.1.26) and dissolve it in
equivalent volume substrate buffer. A volume of 15 ml phytase substrate solution is sufficient for two
test plates. Prepare phytase substrate solution always fresh and use within 2 h.
7.4.14 Developer.
Dissolve 24 g of solid sodium hydroxide (7.1.27) in 200 ml ultrapure water (7.2). Autoclave the sodium
hydroxide solution and cool down before using it. Store the developer at room temperature. Store the
developer solution no longer than six months.
8 Sampling and samples
8.1 General
This document describes specific requirements for the sampling with respect to the determination of
estrogenic activity in water samples. For general information about sampling consider ISO 5667-16.
8.2 Bottles and material for sampling
Use clean glass bottles (borosilicate glass) with polytetrafluoroethylene (PTFE)-lined caps. To avoid
photo-degradation of compounds of interest, use amber glass bottles. If transparent glass bottles are
used, wrap the bottles in aluminium foil or store them in a dark container.
Alternatively, bottles made from aluminium or stainless steel (both uncoated) may be used. Assess that
a material different from borosilicate glass does not affect results.
ISO 19040-2:2018(E)
8.3 Bottles and material pre-cleaning
After the routine cleaning procedure, additionally clean the bottles and the caps as follows: rinse the
clean bottles and the caps three times with a minimum amount of acetone (7.1.30). Let the residual
acetone evaporate (e.g. drying oven). Close the bottles immediately after drying.
Rinse all glassware, spatulas etc. getting in contact with the sample three times with a minimum
amount of acetone (7.1.30). Let the residual acetone evaporate.
8.4 Sampling procedure
Use disposable nitrile-gloves during sampling. Do not use any hand-cream prior to sampling and avoid
skin contact with the sample. Use material from glass, PTFE, aluminium or stainless steel only.
Fill the bottles completely. Consider possible expansion of the sample due to a change of temperature. If
the samples are to be frozen as part of their preservation, the bottles shall not be completely filled. This
is in order to prevent breakage which may arise from expansion of ice during the freezing and thawing
process.
Do not stabilize the samples with chemicals.
Either cool down the samples to 2 °C to 8 °C or freeze the samples at ≤−18 °C.
8.5 Transport of samples
Deliver the samples to the laboratory as soon as possible after sampling.
During transport keep the sample container frost- and break-proof, protected from exposure to light,
temperature increase and external contamination.
Cooling or freezing procedures shall be applied to the samples in order to increase the time period
available for transport and storage. Cooling should commence as soon as possible after sampling for
instance in cool boxes with ice, frozen gel packs, or cooling elements. A cooling device in the transport
vehicle is also suitable. A cooling temperature during transport of 2 °C to 8 °C has been found suitable.
The suggested cooling temperature applies to the surround
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