SIST EN ISO 20227:2017
(Main)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)
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
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 stockculture 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
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 unabhängigemmikrobiologischem 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.---------------------- Page: 3 ----------------------
SIST EN ISO 20227:2017
EN ISO 20227:2017 (E)
Contents Page
European foreword ....................................................................................................................................................... 3
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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
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
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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
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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
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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
[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 referencesThe 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 definitionsFor 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
3.2frond
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.
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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
3.6pure 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
3.8stock 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
conditions4 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 −1with 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 mediumAdd 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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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.
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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....
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