Water quality - Determination of the growth inhibition effects of waste waters, natural waters and chemicals on the duckweed Spirodela polyrhiza - Method using a stock culture independent microbiotest (ISO 20227:2017)

ISO 20227:2017 specifies a method for the determination of the inhibition of the growth of the first fronds of Spirodela polyrhiza germinated from turions, by substances and mixtures contained in water or waste water, including treated municipal waste water and industrial effluents.
The test is also applicable to pure chemicals and in particular, plant protection products and pesticides.

Wasserbeschaffenheit - Bestimmung der wachstumshemmenden Wirkung von Abwässern, natürlichen Wässern und Chemikalien auf die Wasserlinsenart Spirodela polyrhiza - Verfahren mittels Stammkultur unabhängigem mikrobiologischem Test (ISO 20227:2017)

Qualité de l'eau - Détermination des effets d'inhibition sur la croissance de la lentille d'eau Spirodela polyrhiza par les eaux usées, les eaux naturelles et les produits chimiques - Méthode utilisant un bioessai miniaturisé indépendant d'une culture mère (ISO 20227:2017)

L'ISO 20227 :2017 spécifie une méthode permettant de déterminer l'inhibition de la croissance des premières frondes de Spirodela polyrhiza ayant germé à partir de turions, provoquée par des substances et des préparations contenues dans les eaux ou les eaux résiduaires, y compris les eaux résiduaires urbaines après traitement et les effluents industriels.
L'essai s'applique également aux produits chimiques purs et, en particulier, aux produits phytopharmaceutiques et aux pesticides.

Kakovost vode - Določevanje učinka odpadne vode, naravne vode in kemikalij na zaviranje rasti vodne leče Spirodela polyrhiza - Metoda z neodvisnim mikrobiološkim preskusom z založno kulturo (ISO 20227:2017)

Ta mednarodni standard določa metodo za določevanje zaviranja rasti prvih listov vodne leče Spirodela polyrhiza, ki vzkalijo iz mirujočih brstnih mešičkov, s snovmi in zmesmi, ki jih vsebuje voda ali odpadna voda, vključno s prečiščeno gospodinjsko odpadno vodo in industrijskimi odplakami. Preskus se uporablja tudi za čiste kemikalije, še posebej za izdelke za zaščito rastlin in pesticide.

General Information

Status
Published
Publication Date
04-Jul-2017
Withdrawal Date
30-Jan-2018
Technical Committee
Drafting Committee
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
05-Jul-2017
Due Date
16-Dec-2017
Completion Date
05-Jul-2017

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 20227:2017
01-december-2017
.DNRYRVWYRGH'RORþHYDQMHXþLQNDRGSDGQHYRGHQDUDYQHYRGHLQNHPLNDOLMQD
]DYLUDQMHUDVWLYRGQHOHþH6SLURGHODSRO\UKL]D0HWRGD]QHRGYLVQLP
PLNURELRORãNLPSUHVNXVRP]]DORåQRNXOWXUR ,62
Water quality - Determination of the growth inhibition effects of waste waters, natural
waters and chemicals on the duckweed Spirodela polyrhiza - Method using a stock
culture independent microbiotest (ISO 20227:2017)
Wasserbeschaffenheit - Bestimmung der wachstumshemmenden Wirkung von
Abwässern, natürlichen Wässern und Chemikalien auf die Wasserlinsenart Spirodela
polyrhiza - Verfahren mittels Stammkultur unabhängigem mikrobiologischem Test (ISO
20227:2017)
Qualité de l'eau - Détermination des effets d'inhibition sur la croissance de la lentille
d'eau Spirodela polyrhiza par les eaux usées, les eaux naturelles et les produits
chimiques - Méthode utilisant un bioessai miniaturisé indépendant d'une culture mère
(ISO 20227:2017)
Ta slovenski standard je istoveten z: EN ISO 20227:2017
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 20227:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 20227:2017

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SIST EN ISO 20227:2017


EN ISO 20227
EUROPEAN STANDARD

NORME EUROPÉENNE

July 2017
EUROPÄISCHE NORM
ICS 13.060.70
English Version

Water quality - Determination of the growth inhibition
effects of waste waters, natural waters and chemicals on
the duckweed Spirodela polyrhiza - Method using a stock
culture independent microbiotest (ISO 20227:2017)
Qualité de l'eau - Détermination des effets d'inhibition Wasserbeschaffenheit - Bestimmung der
sur la croissance de la lentille d'eau Spirodela polyrhiza wachstumshemmenden Wirkung von Abwässern,
par les eaux usées, les eaux naturelles et les produits natürlichen Wässern und Chemikalien auf die
chimiques - Méthode utilisant un bioessai miniaturisé Wasserlinsenart Spirodela polyrhiza - Verfahren
indépendant d'une culture mère (ISO 20227:2017) mittels Stammkultur unabhängigem
mikrobiologischem Test (ISO 20227:2017)
This European Standard was approved by CEN on 9 May 2017.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20227:2017 E
worldwide for CEN national Members.

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SIST EN ISO 20227:2017
EN ISO 20227:2017 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 20227:2017
EN ISO 20227:2017 (E)
European foreword
This document (EN ISO 20227:2017) has been prepared by Technical Committee ISO/TC 147 "Water
quality" in collaboration with 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 January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
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.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 20227:2017 has been approved by CEN as EN ISO 20227:2017 without any modification.
3

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SIST EN ISO 20227:2017

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SIST EN ISO 20227:2017
INTERNATIONAL ISO
STANDARD 20227
First edition
2017-06
Water quality — Determination of
the growth inhibition effects of waste
waters, natural waters and chemicals
on the duckweed Spirodela polyrhiza
— Method using a stock culture
independent microbiotest
Qualité de l’eau — Détermination des effets d’inhibition sur la
croissance de la lentille d’eau Spirodela polyrhiza par les eaux usées,
les eaux naturelles et les produits chimiques — Méthode utilisant un
bioessai miniaturisé indépendant d’une culture mère
Reference number
ISO 20227:2017(E)
©
ISO 2017

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test organisms . 3
6 Growth medium . 3
6.1 Preparation of stock solutions . 4
6.2 Preparation of the final concentration of modified Steinberg medium . 4
7 Apparatus . 4
8 Reference chemicals . 5
9 Procedure. 5
9.1 Germination of the Spirodela polyrhiza turions . 5
9.2 Tests on effluents (and waste waters) . 5
9.2.1 Addition of concentrated growth medium to the effluent sample . 5
9.2.2 Preparation of the effluent dilutions . 6
9.2.3 Procedure . 6
9.3 Tests on chemical compounds . 7
9.3.1 Range finding test . 7
9.3.2 Definitive test . 8
9.4 Filling of the test plate with the toxicant dilutions . 9
9.4.1 General. 9
9.4.2 Procedure . 9
9.5 Transfer of the germinated turions in the test cups .10
9.6 Photo of the multiwell at the start of the toxicity test .10
9.7 Incubation of the multiwell .10
9.8 Photo of the multiwell at the end of the toxicity test .11
9.9 Measurement of the area of the first fronds .11
10 Data treatment — Calculation of the growth inhibition .11
11 Validity criterion .12
12 Test sensitivity .12
13 Test with reference chemicals .12
14 Test report .15
Annex A (informative) Spirodela polyrhiza stock culturing for turion production .16
Annex B (informative) Sensitivity of the Spirodela polyrhiza microbiotest .17
Annex C (informative) Performance data .19
Bibliography .20
© ISO 2017 – All rights reserved iii

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SIST EN ISO 20227:2017
ISO 20227:2017(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: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2017 – All rights reserved

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

Introduction
Duckweeds are free-floating higher water plants commonly used in ecotoxicological research for the
assessment of the toxicity of waste waters, natural waters and chemicals (see ISO 20079 and References
[6] to [11] and in particular plant protection products, see Reference [12]).
Duckweeds are fast growing plants, many of which have a cosmopolitan distribution, and they are, hence,
well suited as primary producers for hazard assessment of pollutants in freshwater environments.
Contrary to terrestrial plants, for which bioassays can be started from the “dormant” life stages (seeds),
toxicity tests with duckweeds require continuous culturing and maintenance of live stocks, with the
inherent biological, technical and financial costs.
A few duckweed species, however, produce dormant vegetative buds (turions) which can be stored
for long periods of time, and which can be germinated on demand at the time of performance of the
bioassay.
One of the duckweeds producing turions is Spirodela polyrhiza, and this species was eventually selected
for a simple and practical microbiotest which is independent of the stock culturing and maintenance of
live stocks.
Spirodela polyrhiza was found to be as sensitive to toxicants as the conventional bioassays with
duckweeds.
The microbiotest procedure for this document involves a 3 d germination of the turions, followed by a
3 d toxicity test in a multiwell test plate, with determination of the growth inhibition of the first fronds
via image analysis.
The Spirodela polyrhiza microbiotest is very simple and easy to perform:
a) the assay does not require culturing or maintenance of live stocks of the test species, and can be
performed “anytime, anywhere” by the use of stored turions;
b) stored turions have a shelf life of several months with a high germination success;
c) the microbiotest requires minimal bench and incubation space, and minimal equipment;
d) the area measurements of the first fronds do not need to be made immediately and can be
postponed to an appropriate timing;
e) the area measurements by image analysis are very rapid and precise, and take less than 1 h for a
complete test.
© ISO 2017 – All rights reserved v

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SIST EN ISO 20227:2017

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SIST EN ISO 20227:2017
INTERNATIONAL STANDARD ISO 20227:2017(E)
Water quality — Determination of the growth inhibition
effects of waste waters, natural waters and chemicals on
the duckweed Spirodela polyrhiza — Method using a stock
culture independent microbiotest
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 and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this document be
carried out by suitably trained staff.
1 Scope
This document specifies a method for the determination of the inhibition of the growth of the first
fronds of Spirodela polyrhiza germinated from turions, by substances and mixtures contained in water
or waste water, including treated municipal waste water and industrial effluents.
The test is also applicable to pure chemicals and in particular, plant protection products and pesticides.
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 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
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:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
effective concentration
EC
x
concentration of the test sample at which an effect of x % is measured, if compared to the control
3.2
frond
leaf-like structure which develops from a germinated turion
3.3
growth
increase in biomass over time as the result of proliferation of new tissues
Note 1 to entry: In this test, it refers to the increase in size of the first frond developing from a germinated turion.
© ISO 2017 – All rights reserved 1

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

3.4
growth medium
combination of dilution water and/or nutrient medium used in the test
Note 1 to entry: In this test, it refers to the nutrient medium used for the germination of the turions and the
growth of the fronds.
3.5
inoculum
transfer of a germinated turion with its small frond in all the test wells at the start of the toxicity test
3.6
pure water
deionized or distilled water with a conductivity below 10 µS/cm
[SOURCE: ISO 19827:2016, 3.4]
3.7
root
part of the Spirodela polyrhiza plant that assumes a root-like structure and develops underneath a frond
3.8
stock culture
culture of a single species of duckweed for the production of the turions
3.9
test medium
combination of test sample, dilution water and/or nutrient medium used in the test
[SOURCE: ISO 20079:2005, 3.23]
3.10
test sample
discrete portion of a sample (taken from i.e. receiving water, waste water, dissolved chemical substances
or mixtures, products and compounds) pre-treated according to the needs of this test (e.g. dissolution,
filtering, neutralisation)
3.11
turion
small vegetative bud which develops from a colony of the duckweed under specific environmental
conditions
4 Principle
Turions produced by culturing Spirodela polyrhiza, or taken from test tubes in which they are stored
(see Annex A) are transferred to a Petri dish containing growth medium, and incubated for 3 d at 25 °C
−2 −1
with continuous illumination of at least 6 000 lx (corresponding approximately to 85 µE m s ).
During this time, the turions germinate and produce a small (first) frond (see Figure 1).
One germinated turion with its first frond is then taken from the Petri dish and inoculated into each
cup of a 6 × 8 multiwell test plate which contains the toxicant dilutions and the negative control (each of
which is prepared in growth medium).
2 © ISO 2017 – All rights reserved

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

Key
1 turion
2 first frond
Figure 1 — Enlargement of a germinated turion with its first frond, in a cup of the test plate
On completion of the inoculations, a photo of the multiwell is taken (at t = 0 h) with a digital camera and
transferred to a computer file.
The multiwell is subsequently incubated for 3 d at (25 ± 1) °C with continuous illumination of minimum
6 000 lx, after which a photo is again taken (at t = 72 h) and transferred to a computer file.
The area of the first frond in each test cup is measured with the aid of an image analysis programme, on
the two photos of the multiwell (i.e. taken at t = 0 h and at t = 72 h).
The growth of the first fronds in the controls and in the test concentrations or dilutions is calculated as
the difference between the t = 72 h areas and the t = 0 h areas, after which the growth inhibition and
the 72 h EC or EC values are determined.
50 x
5 Test organisms
The test species used in this document is the duckweed Spirodela polyrhiza (L.) Schleid.
The test organisms are obtained by germination of (stored) turions.
Turions can be produced in the laboratory according to the procedure described in Annex A.
1)
They can also be purchased from a commercial source .
6 Growth medium
The growth medium (3.4) used for the germination of the turions and the growth of the duckweeds
[2]
during the toxicity test is the modified Steinberg medium which is described and used in ISO 20079
and the OECD guideline for testing chemicals (Reference [8]).
This medium is also used to prepare the toxicant dilutions.
The growth medium is composed of macroelements and microelements of which stock solutions are
prepared according to Table 1 and Table 2 respectively.
1) The turions supplied by MicroBioTests Inc. are an example of a suitable product available commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO
of this product.
© ISO 2017 – All rights reserved 3

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

6.1 Preparation of stock solutions
Prepare the eight stock solutions by adding the prescribed weight of the chemicals to 1 l of pure
water (3.6).
Table 1 — Macroelements stock solutions
Macroelements (50-fold concentrated) g/l
KNO 17,50
3
Stock solution 1 KH PO 4,5
2 4
K HPO 0,63
2 4
Stock solution 2 MgSO ·7H O 5,00
4 2
Stock solution 3 Ca(NO ) ·4H O 14,75
3 2 2
Table 2 — Microelements stock solutions
Microelements (1 000-fold concentrated) mg/l
Stock solution 4 H BO 120,00
3 3
Stock solution 5 ZnSO ·7H O 180,00
4 2
Stock solution 6 Na MoO ·2H O 44,0
2 4 2
Stock solution 7 MnCl ·4H O 180,00
2 2
Stock solution 8 FeCl ·6H O 760,00
3 2
EDTA disodium-dihydrate 1 500,00
Stock solutions 2 and 3, and 4 to 7 may be pooled (taking into account the required concentrations).
6.2 Preparation of the final concentration of modified Steinberg medium
Add 20 ml each of stock solutions 1, 2 and 3 to about 900 ml pure water (3.6) in a 1 l volumetric flask.
Then add 1,0 ml each of stock solutions 4, 5, 6, 7 and 8.
Fill the volumetric flask to 1 000 ml with pure water.
The pH of the growth medium shall be 5,5 ± 0,2 and shall be adjusted with either HCl or NaOH.
Once prepared, the growth medium has a relatively short shelf life and shall be used within two weeks
after preparation.
7 Apparatus
Usual laboratory equipment and in particular the following.
7.1 Temperature-controlled cabinet or room, or incubator, with white fluorescent light providing
continuous uniform illumination of at least 6 000 lx at the surface of the turion germination Petri dish
and the multiwell test plate.
7.2 Lux meter, for the measurement of the light intensity at the surface of the turion germination Petri
dish and the multiwell test plate.
7.3 pH meter, for checking and/or adjustment of the pH of the growth medium.
7.4 Laboratory glassware, for the preparation of the test concentrations (volumetric flasks, graduated
cylinders, pipettes, test tubes).
4 © ISO 2017 – All rights reserved

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

7.5 Petri dishes, diameter 9 cm, with lid, for the germination of the turions.
7.6 Microsieve, 100 µm mesh, for rinsing the stored turions.
7.7 Multiwells, 6 × 8 cups, as test plates.
7.8 Plastic spatula, for the transfer of the germinated turions in the multiwell cups.
7.9 Digital camera, to take a picture of the multiwell with the growing duckweeds.
7.10 Image analysis system, for the measurement of the area of the first fronds.
8 Reference chemicals
8.1 3,5-dichlorophenol, analytical grade > 99 % purity.
8.2 Potassium chloride, KCl, analytical grade > 99 % purity.
9 Procedure
9.1 Germination of the Spirodela polyrhiza turions
When using turions from a culture of Spirodela polyrhiza, place the turions in a Petri dish (7.5) and pour
30 ml growth medium (3.4) into it.
Starting from stored turions, take a tube with stored turions and shake it slightly to re-suspend the
turions.
Pour the contents of the tube in the microsieve (7.6) and rinse with pure water (3.6) to remove the
storage medium.
Put 10 ml growth medium (3.4) in the Petri dish (7.5).
Turn the microsieve upside down and flush all the turions in the Petri dish by pouring 10 ml growth
medium over the surface of the microsieve.
Fill the Petri dish further by adding 10 ml growth medium.
Cover the Petri dish with the transparent lid and place it in the incubator or in the temperature
conditioned room (7.1).
Incubate the Petri dish for 3 d (72 ± 1) h at (25 ± 1) °C, with continuous illumination (at least 6 000 lx at
the surface of the Petri dish).
NOTE Both germination of the turions and the growth of the first fronds are very substantially dependent
on temperature and illumination conditions. It is therefore important that the prescribed values of temperature
and illumination be respected as closely as possible.
9.2 Tests on effluents (and waste waters)
Sampling and samples preparation shall be done according to ISO 5667-16.
9.2.1 Addition of concentrated growth medium to the effluent sample
Transfer about 80 ml effluent in a 100 ml calibrated flask.
© ISO 2017 – All rights reserved 5

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SIST EN ISO 20227:2017
ISO 20227:2017(E)

Add 2 ml of each of the stock solutions 1, 2 and 3, and 100 µl of each of the stock solutions 4, 5, 6, 7 and
8 to the calibrated flask.
Fi
...

SLOVENSKI STANDARD
oSIST prEN ISO 20227:2016
01-julij-2016
.DNRYRVWYRGH'RORþHYDQMHYSOLYRYRGSDGQHYRGHQDUDYQHYRGHLQNHPLNDOLMQD
]DYLUDQMHUDVWLYRGQHOHþH6SLURGHODSRO\UKL]D ,62',6
Water quality - Determination of the growth inhibition effects of waste waters, natural
waters and chemicals on the duckweed Spirodela polyrhiza - Method using a stock
culture independent microbiotest (ISO/DIS 20227:2016)
Wasserbeschaffenheit - Bestimmung der wachstumshemmenden Wirkung von
Abwässern, natürlichen Wässern und Chemikalien auf die Wasserlinsenart Spirodela
polyrhiza - Verfahren mittels Stammkultur unabhängigem mikrobiologischem Test
(ISO/DIS 20227:2016)
Qualité de l'eau - Détermination des effets d'inhibition sur la croissance de la lentille
d'eau Spirodela polyrhiza par les eaux usées, les eaux naturelles et les produits
chimiques - Méthode utilisant un bioessai miniaturisé indépendant d'une culture mère
(ISO/DIS 20227:2016)
Ta slovenski standard je istoveten z: prEN ISO 20227
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 20227:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 20227:2016

---------------------- Page: 2 ----------------------
oSIST prEN ISO 20227:2016
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20227
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:
2016-04-28 2016-07-27
Water quality — Determination of the growth inhibition
effects of waste waters, natural waters and chemicals on
the duckweed Spirodela polyrhiza — Method using a stock
culture independent microbiotest
Qualité de l’eau — Détermination des effets d’inhibition de la croissance des eaux usées, des eaux
naturelles et des produits chimiques sur la lentille d’eau Spirodela polyrhiza — Méthode utilisant un
microbiotest indépendant de la culture mère
ICS: 13.060.70
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the International Organization for
Standardization (ISO), and processed under the ISO lead mode of collaboration
as defined in the Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member
bodies for a parallel five month enquiry.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 20227:2016(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
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©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2016

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COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Contents Page
Foreword . iv
Introduction . iv
1 Scope. 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle. 2
5 Test organisms. 3
6 Growth medium. 3
7 Apparatus . 4
8 Reference chemicals. 5
9 Procedure . 5
10 Data treatment – Calculation of the growth inhibition. 13
11 Validity criterion. 14
12 Test sensitivity . 14
13 Test with reference chemicals . 14
14 Test report . 16
Annex A (informative) Spirodela polyrhiza stock culturing for turion production . 17
Annex B (informative) Sensitivity of the Spirodela polyrhiza microbiotest. 18
Annex C (informative) Performance data. 21
Bibliography. 22


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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 meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO's adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
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Introduction
Duckweeds are free-floating higher water plants commonly used in ecotoxicological research for the
assessment of the toxicity of waste waters, natural waters and chemicals (see ISO 20079 and References
[1] to [6] and in particular of plant protection products see Reference [7]).
Duckweeds are fast growing plants, many of which have a cosmopolitan distribution, and they are
hence well suited as primary producers for hazard assessment of pollutants in freshwater
environments.
Contrary to terrestrial plants, for which bioassays can be started from the "dormant" life stages (seeds),
toxicity tests with duckweeds require continuous culturing and maintenance of live stocks, with the
inherent biological, technical and financial costs.
A few duckweed species, however, produce "dormant vegetative buds" (turions) which can be stored
for long periods of time, and which can be germinated "on demand" at the time of performance of the
bioassay.
One of the duckweeds producing turions is Spirodela polyrhiza, and this species was eventually selected
for a simple and practical microbiotest which is independent of the stock culturing and maintenance of
live stocks.
Spirodela polyrhiza was found to be as sensitive to toxicants as the conventional bioassays with
duckweeds.
The microbiotest procedure for this International Standard involves a 3 d germination of the turions,
followed by a 3 d toxicity test in a multiwell test plate, with determination of the growth inhibition of
the first fronds via image analysis.
The Spirodela polyrhiza microbiotest is very simple and easy to perform and has several advantages
over the conventional duckweed tests:
1) the assay does not require culturing or maintenance of live stocks of the test species, and can
be performed "anytime, anywhere" by use of stored turions;
2) stored turions have a shelf life of several months with a high germination success;
3) the microbiotest requires minimal bench and incubation space, and minimal equipment;
4) the test does not require manipulation of the organisms during or at the end of the test;
5) the area measurements of the first fronds do not need to be made immediately and can be
postponed to an appropriate timing;
6) the area measurements by image analysis are very rapid and precise, and take less than one
hour for a complete test.

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DRAFT INTERNATIONAL STANDARD ISO/DIS 20227:2016(E)

Water quality — Determination of the growth inhibition
effects of waste waters, natural waters and chemicals on the
duckweed Spirodela polyrhiza — Method using a stock culture
independent microbiotest
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 and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this document be
carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination of the inhibition of the growth of
the first fronds of Spirodela polyrhiza germinated from turions, by substances and mixtures contained in
water or waste water, including treated municipal waste water and industrial effluents.
The test is also applicable to pure chemicals and in particular plant protection products and pesticides.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 20079, Water quality — Determination of the toxic effect of water constituents and waste water on
duckweed (Lemna minor) — Duckweed growth inhibition test
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
ECx
calculated concentration (of a substance) or dilution (of an aqueous sample, in %) for which an effect of
x% is expected compared to the control
3.2
frond
leaf-like structure which develops from a germinated turion
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3.3
growth
increase in size of the first frond developing from a germinated turion
3.4
growth medium
nutrient medium used for the germination of the turions and the growth of the fronds
3.5
inoculation
transfer of a germinated turion with its small frond in all the test wells at the start of the toxicity test
3.6
pure water
deionized or distilled water with a conductivity below 10 µS/cm
3.7
root
part of the plant which develops underneath a frond
3.8
stock culture
laboratory culture of the duckweed for the production of the turions
3.9
test dilution medium
growth medium which is also used as test dilution medium
3.10
test sample
portion of the collected material or the dissolved chemical to be used for the preparation of the dilution
series
3.11
turion
small vegetative bud which develops from a colony of the duckweed under specific environmental
conditions
4 Principle
Turions produced by culturing Spirodela polyrhiza, or taken from test tubes in which they are stored
(see Annex A) are transferred to a Petri dish containing growth medium, and incubated for 3 d at 25 °C
-2 -1
s ).
with continuous illumination of at least 6 000 lx (corresponding approximately to 85 µE m
During this time the turions germinate and produce a small (first) frond (see Figure 1).
One germinated turion with its first frond is then taken from the Petri dish and inoculated into each cup
of a 6 × 8 multiwell test plate which contains the toxicant dilutions and the negative control (each of
which is prepared in growth medium).
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Key
1 turion
2 first frond
Figure 1 — Enlargement of a germinated turion with its first frond, in a cup of the test plate
On completion of the inoculations, a photo of the multiwell is taken (= at t0h) with a digital camera and
transferred to a computer file.
The multiwell is subsequently incubated for 3 d at (25 ± 1) °C with continuous illumination of minimum
6 000 lx, after which a photo is again taken (= at t72h) and transferred to a computer file.
The area of the first frond in each test cup is measured with the aid of an image analysis programme, on
the 2 photos of the multiwell (i.e. taken at t0h and at t72h).
The growth of the first fronds in the controls and in the test concentrations or dilutions is calculated as
the difference between the t72h areas and the t0h areas, after which the growth inhibition and the 72 h
EC50 or ECx values are determined.
5 Test organisms
The test species used in this International Standard is the duckweed Spirodela polyrhiza (L.) Schleid.
The test organisms are obtained by germination of (stored) turions.
Turions can be produced in the laboratory according to the procedure described in Annex A.
1
They can also be purchased from a commercial source
6 Growth medium
The growth medium (3.4) used for the germination of the turions and the growth of the duckweeds
during the toxicity test is the "modified STEINBERG medium" which is described and used in ISO 20079
and OECD guideline for testing chemicals (see Reference [1]).
This medium will also be used to prepare the toxicant dilutions.

1
The turions supplied by MicroBioTests Inc. Mariakerke-Gent, Belgium are an example of a suitable product
available commercially. This information is given for the convenience of users of this International Standard and
does not constitute an endorsement by ISO of this product.
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The growth medium is composed of "macroelements" and "microelements" of which stock solutions are
prepared according to Table 1 and Table 2 respectively.
6.1 Preparation of stock solutions
Prepare the 8 stock solutions by adding the prescribed weight of the chemicals to 1 l of pure water
(3.6).
Table 1 — Macroelements stock solutions
Macroelements (50-fold concentrated) g/l
KNO 17,50
3
Stock solution 1 KH PO 4,5
2 4
K HPO 0,63
2 4
Stock solution 2 MgSO ·7H O 5,00
4 2
Stock solution 3 Ca(NO3)2·4H2O 14,75
Table 2 — Microelements stock solutions
Microelements (1 000-fold concentrated) mg/l
Stock solution 4 H BO 120,00
3 3
Stock solution 5 ZnSO ·7H O 180,00
4 2
Stock solution 6 Na2MoO4·2H2O 44,0
Stock solution 7 MnCl ·4H O 180,00
2 2
Stock solution 8 FeCl ·6H O 760,00
3 2
EDTA disodium-dihydrate 1 500,00
Stock solutions 2 and 3, and 4 to 7 may be pooled (taking into account the required concentrations).
6.2 Preparation of the final concentration of modified STEINBERG medium
Add 20 ml each of stock solutions 1, 2 and 3 to about 900 ml pure water (3.6) in a 1 l volumetric flask.
Then add 1,0 ml each of stock solutions 4, 5, 6, 7 and 8.
Fill the volumetric flask to 1 000 ml with pure water.
The pH of the growth medium should be 5,5 ± 0,2 and can be adjusted with either HCl or NaOH.
Once prepared, the growth medium has a relatively short shelf life and should be used within 2 weeks
after preparation.
7 Apparatus
Usual laboratory equipment and in particular the following.
7.1 Temperature-controlled cabinet or room, or incubator, with white fluorescent light providing
continuous uniform illumination of at least 6 000 lx at the surface of the turion germination Petri dish
and the multiwell test plate.
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7.2 Lux meter, for the measurement of the light intensity at the surface of the turion germination
Petri dish and the multiwell test plate.
7.3 pH meter, for checking and/or adjustment of the pH of the growth medium.
7.4 Laboratory glassware, for the preparation of the test concentrations (volumetric flasks,
graduated cylinders, pipettes, test tubes).
7.5 Petri dishes, diameter 9 cm, with lid, for the germination of the turions.
7.6 Microsieve, 100 µm mesh, for rinsing the stored turions.
7.7 Multiwells, 6 × 8 cups, as test plates.
7.8 Plastic spatula, for the transfer of the germinated turions in the multiwell cups.
7.9 Digital camera, to take a picture of the multiwell with the growing duckweeds.
7.10 Image analysis system, for the measurement of the area of the first fronds.
8 Reference chemicals
8.1 3,5-dichlorophenol, analytical grade >99 % purity.
8.2 Potassium chloride, KCl, analytical grade >99 % purity.
9 Procedure
9.1 Germination of the Spirodela polyrhiza turions
When using turions from a culture of Spirodela polyrhiza, place the turions in a Petri dish (7.5) and pour
30 ml growth medium (3.4).
Starting from stored turions, take a tube with stored turions and shake it slightly to re-suspend the
turions.
Pour the contents of the tube in the microsieve (7.6) and rinse with pure water (3.6) to remove the
storage medium.
Put 10 ml growth medium (3.4) in the Petri dish (7.5).
Turn the microsieve upside down and flush all the turions in the Petri dish by pouring 10 ml growth
medium over the surface of the microsieve.
Fill the Petri dish further by adding 10 ml growth medium.
Cover the Petri dish with the transparent lid and place it in the incubator or in the temperature
conditioned room (7.1).
Incubate the Petri dish for 3 d (72 h ± 1 h) at (25 ± 1) °C, with continuous illumination (at least 6 000 lx
at the surface of the Petri dish).
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NOTE Both germination of the turions and the growth of the first fronds are "very substantially" dependent
on temperature and illumination conditions. It is therefore important that the prescribed values of temperature
and illumination be respected as closely as possible.
9.2 Tests on effluents (and waste waters)
9.2.1 Addition of concentrated growth medium to the effluent sample
Transfer about 80 ml effluent in a 100 ml calibrated flask.
Add 2 ml of each of the stock solutions 1, 2 and 3, and 100 µl of each of the stock solutions 4, 5, 6, 7 and
8 to the calibrated flask.
Fill the flask to the 100 ml mark with the effluent, stopper the flask and shake thoroughly to
homogenize the contents.
NOTE The addition of 6,5 ml growth medium (3.4) to 93,5 ml effluent, dilutes the effluent sample by about
6 %. This means that the highest effluent concentration which will be tested is about 94 % of the original effluent
sample.
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9.2.2 Preparation of the effluent dilutions
A 1:1 dilution series is prepared from the 94 % effluent (see Figure 2).

Key
1 effluent with growth medium
2 growth medium
a effluent
Figure 2 — Preparation of the 1:1 effluent dilution series
9.2.3 Procedure
Take 5 test tubes of 20 ml contents and label them C1 ,C2, C3, C4 and C5.
Add 20 ml effluent (containing growth medium) to test tube C1.
Add 10 ml growth medium (as dilution medium) to the tubes C2, C3, C4 and C5.
Transfer 10 ml effluent from tube C1 to tube C2, and cap and shake the test tube.
Transfer 10 ml test dilution from tube C2 to tube C3, and cap and shake the test tube.
Repeat this procedure for the next dilutions.
The concentrations of the effluent that will be tested are shown in Table 3.
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Table 3 — Dilution series of the effluent
Test tube Effluent concentration
(in %)
C1 93,50
C2 46,75
C3 23,37
C4 11,68
C5 5,84
9.3 Tests on chemical compounds
If the "approximate toxicity" (= the order of magnitude) of the chemical compound to be tested is not
known, a "Range finding test" has to be performed first to determine the 0 % to 100 % tolerance range
of the duckweeds to the toxicant.
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9.3.1 Range finding test
A dilution series 1:10 is prepared in test tubes of 10 ml, in growth medium (as dilution medium).
Figure 3 shows an example for preparation of a concentration range of a chemical from 100 mg/l down
to 0,01 mg/l.

Key
1 25 ml volumetric flask
2 growth medium
a) stock solution
b) test concentrations
a fill to the mark
Figure 3 — Preparation of a 100 mg/l to 0,01 mg/l dilution series for a range finding test on a
pure chemical
9.3.2 Definitive test
The dilution series to be prepared span the range of the lowest concentration producing 100 % effect,
to the highest one producing less than 10 % effect in the range finding test.
A dilution series is prepared, starting with a test concentration of the toxicant which was the lowest one
giving 100 % growth inhibition in the range finding test.
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A logarithmic dilution series (10 - 5,6 - 3,2 - 1,8 - 1 + the negative control) is then prepared in 10 ml test
tubes, with the volumes of growth medium and toxicant indicated in Figure 4.
The actual concentrations of the toxicant in the tubes (which will be needed for the EC50 or ECx
determination) are calculated as follows:
C1 = …. mg/l
C2 = 0,56 × C1 = … mg/l
C3 = 0,32 × C1 = … mg/l
C4 = 0,18 × C1 = … mg/l
C5 = 0,10 × C1 = … mg/l

Key
1 25 ml toxicant solution (in growth medium) = C1
2 growth medium
3 control
Figure 4 — Preparation of a 10-1 logarithmic dilution series for a definitive test on a pure
chemical
9.4 Filling of the test plate with the toxicant dilutions
The test is performed in a 6 × 8 cups multiwell (7.7), with 8 replicates per test concentration and the
negative control.
9.4.1 Procedure (see Figure 5)
Put 1 ml growth medium (3.4) in the 8 cups of row A (= the control row).
Put 1 ml of each toxicant concentration in the rows B to F.
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The distribution of the test solutions shall always be carried out starting with the control row on top of
the multiwell (row A), followed in sequence by the rows receiving increasing toxicant concentrations
(rows B to F)

Key
1 control medium
Figure 5 — Filling of the multiwell test plate with the toxicant dilutions (C1 to C5)
9.5 Transfer of the germinated turions in the test cups
Take the Petri dish (7.5) with the turions out of the incubator and transfer, with the aid of a spatula, one
germinated turion into each cup of the multiwell test plate.
NOTE Germinated turions can easily be distinguished from turions which have not germinated, by the
presence of a (small) first frond on one side of the turion, and by small roots underneath the frond.
The transfer of the germinated turions in the test cups shall be started with the control row (row A on
top of the multiwell) and continued in the next rows, in sequence of the increasing test concentrations.
The transfers must be carried out "randomly", i.e. one has to avoid picking up and transferring the
turions with "the largest" fronds first!
9.6 Photo of the multiwell at the start of the toxicity test
On completion of the transfers of the germinated turions (with their small first fronds), take a digital
photo (= at t0h) and transfer the picture to a computer file.
The digital camera (7.9) shall not be held "too close" to the multiwell since this will lead to a
"distortion" of the view of the cups in the columns on the right and on the left side. The latter cups must
also have a round (and not an oval) look.
To increase the contrast between the turions and the first small fronds in the photo, it is advised to
place the multiwell on a light table, or on a white background.


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9.7 Incubation of the multiwell
Put the cover on the multiwell and put the test plate in the incubator (7.1).
Incubate the multiwell at (25 ± 1) °C for 3 d (72 ± 1) h with a continuous illumination of 6 000 lx
(measured at the top of the test plate).
Similarly as mentioned above for the germination of the turions, the prescribed temperature and
illumination must be respected as closely as possible.
9.8 Photo of the multiwell at the end of the toxicity test
At the end of the 3 d incubation (= at t72h), take the multiwell out of the incubator, remove the lid, and
take again a digital photo (preferably on a light table or a white background), with subsequent transfer
of the picture to a computer file.
9.9 Measurement of the area of the first fronds
Area measurements of the first fronds have to be made in all the test cups, on the (computer stored)
photos of the multiwell test plate taken at t0h and at t72h.
The area measurements are made with the aid of an appropriate image analysis (7.10) program (such
as e.g. image J which is an open access imag
...

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