SIST EN ISO 20227:2017

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)

This International Standard specifies a method for the determination of the inhibition of the growth of the first fronds of Spirodela polyrhiza germinated from dormant 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
• Public Enquiry End Date: 24-Jul-2016
• Publication Date: 19-Nov-2017
• ICS: 07.100.20 - Microbiology of water
• 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: 20-Oct-2017
• Due Date: 25-Dec-2017
• Completion Date: 20-Nov-2017
• Ref Project: EN ISO 20227:2017 - 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)

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

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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.

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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. [phone]
Fax [phone]
[e-mail]
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

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.

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
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.
...