ISO 4979:2023
(Main)Water quality — Aquatic toxicity test based on root re-growth in Lemna minor
Water quality — Aquatic toxicity test based on root re-growth in Lemna minor
This document specifies a method for the determination of the inhibition of root re-growth in duckweeds (Lemna minor) by substances and mixtures contained in water or waste water. This method applies to environmental water samples including treated municipal wastewater and industrial effluents.
Qualité de l'eau — Essai de toxicité aquatique basé sur la repousse des racines chez Lemna minor
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INTERNATIONAL ISO
STANDARD 4979
First edition
2023-06
Water quality — Aquatic toxicity test
based on root re-growth in Lemna
minor
Qualité de l'eau — Essai de toxicité aquatique basé sur la repousse
des racines chez Lemna minor
Reference number
ISO 4979:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO 4979:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 4979:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Test organisms . 3
6 Growth medium . 3
6.1 Preparation of stock solution . 3
6.2 Storage and cultivation . 4
7 Apparatus . 4
8 Experimental methods .5
8.1 Preparation of medium . 5
8.2 Preparation of toxicant solution and test dilutions . 6
8.2.1 Test dilutions . 6
8.2.2 Selection of test concentrations . 6
8.3 Control . 6
8.4 Transfer of test organisms . 6
8.5 Culture . 6
8.6 Method of measurements . 7
9 Tests on effects .7
9.1 Reference chemicals . 7
9.2 Statistics . 7
9.3 Validity of test . 7
9.4 Precision . . . 8
10 Expression of results . 8
10.1 Test results . 8
10.2 Expression of results . . 8
11 Test report . 8
Annex A (informative) Root excision and re-growth length measurement .10
Annex B (informative) Interlaboratory precision of control values and EC from the
50
Lemna toxicity test .12
Bibliography .14
iii
© ISO 2023 – All rights reserved
---------------------- Page: 3 ----------------------
ISO 4979:2023(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 of 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2023 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 4979:2023(E)
Introduction
Lemna gibba and L. minor have been most extensively used in phytotoxicity testing and there are
several standard methods which have been adopted by major international standardization agencies,
[1]
for example, ISO 20079:2005, U.S. Environmental Protection Agency , Organization for Economic
[2]
Cooperation and Development . Tests with duckweed have typically favoured measurements of
[3],[4]
frond (e.g. their number, biomass, area, carbon uptake, chlorophyll content ) and require standard
exposure durations of at least 7 d to detect toxicity.
On the other hand, tests based on root elongation are some of the most widely used phytotoxicity
methodologies for terrestrial angiosperms because of their simplicity and rapidity. Despite reports that
roots of L. minor are highly sensitive to environmental stressors and that they play important ecological
[5],[6],[7],[8]
roles by providing stability , little attention has been paid to the roots in Lemna since it was
generally considered that root fragility made their handling for measurements difficult and that it was
impractical to obtain sufficient numbers of individual plants with identical root lengths to initiate tests.
However, the ecotoxicological significance of the root endpoint has been re-evaluated and root length
was shown to be a sensitive, precise and ecologically significant endpoint in comparison with more
[7]
traditional frond growth or biomass endpoints .
The proposed root re-growth bioassay differs in several key aspects from three internationally
standardized methods (ISO, OECD and US EPA):
a) the test can be completed within 72 h;
b) the test vessel is a 24-well cell plate;
c) the required volume of test water samples is 3 ml;
d) roots were excised prior to exposure with subsequent measurements on newly developed roots.
The technique of excising roots prior to exposure means that there is no requirement to pre-select
roots of uniform length, which reduces the handling of these fragile roots.
Artificial severance of roots will possibly never happen in natural settings since root abscission in Lemna
has not been reported previously. However, according to recent studies, the tiny globally distributed
[9]
water ferns of the genus Azolla lost their roots under stress conditions , a phenomenon known
as rapid root abscission. Such shedding sets its fronds free from root-entangled mats and facilitates
their dispersion to a potentially better environment. Therefore, rapid root abscission is considered
[9]
an important survival strategy of Azolla . This can indicate that the endpoint root re-growth has its
ecological relevance.
It is also well known that Lemna will thrive without any roots. Thus, Lemna roots appear nonessential
organs, but are nonetheless important for plant anchorage, nutrient absorption and cytokinin
biosynthesis. Therefore, the manipulation of roots by simple severance can be an unimportant issue
and does not justify the conclusion that the removal of roots prior to ecotoxicological testing is
inappropriate.
The three-day root re-growth test is useful for the rapid screening of either wastewater effluents or
[8]
hazardous contaminants in natural waters as it is easy to perform, quick to run and cost-effective
to operate for wastewater toxicity screening and can have an operational benefit of testing time since
management decision should be made in a timely manner in the case of unexpected pollution events.
The root re-growth endpoint from this 72-hour protocol is not a direct substitute for the seven-day
growth rate/biomass endpoints.
The present protocol provides detailed information on how to set up and conduct the root re-growth test
with Lemna minor as well as how to analyse toxicity data. This protocol is intended for use with Lemna
minor, but it can also be applied to other Lemna species and Spirodela species with some modifications.
v
© ISO 2023 – All rights reserved
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 4979:2023(E)
Water quality — Aquatic toxicity test based on root re-
growth in Lemna minor
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted 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 root re-growth in duckweeds
(Lemna minor) by substances and mixtures contained in water or waste water. This method applies to
environmental water samples including treated municipal wastewater and industrial effluents.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
axenic culture
monocultures of organisms from a single species, free from fungi, algae and other macrophyte species
[SOURCE: ISO 20079:2005, 3.1]
3.2
coefficient of variation
CV
relative standard deviation, expressed as a percentage
3.3
colony
aggregate of mother and daughter fronds, attached to each other, sometimes referred to as a plant
[SOURCE: ISO 20079:2005, 3.4]
3.4
effective concentration
EC
x
concentration of test sample at which an effect of x % is measured, if compared to the control
[SOURCE: ISO 20079:2005, 3.9, modified — Note 1 to entry deleted]
1
© ISO 2023 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 4979:2023(E)
3.5
frond
individual leaf-like structure on a duckweed colony; the smallest unit (i.e. individual), capable to
reproduce
[SOURCE: ISO 20079:2005, 3.10]
3.6
growth
increase in biomass over time as the result of proliferation of new tissues
Note 1 to entry: In this test, it refers to any parameter of observation.
[SOURCE: ISO 20079:2005, 3.13]
3.7
growth medium
pure water to which reagent-grade chemicals (micronutrients) have been added
3.8
non-axenic culture
monoculture of organisms from a single species (i.e. free from other macrophyte species), which has
not been treated with antimycotic or antibiotic solutions to remove naturally associated bacteria and
fungi
3.9
pre-culture
culture of duckweed used for acclimation of test plants to the test conditions and for the growing of the
plants to be used in the inoculum
[SOURCE: ISO 20079:2005, 3.19]
3.10
pure water
deionized or distilled water
[SOURCE: ISO 19827:2016, 3.4]
3.11
root
plant part that normally grows under water and holds the plant in place
[SOURCE: ISO 20079:2005, 3.20]
3.12
stock culture
culture of a single species of duckweed to conserve the original Lemna species in the laboratory and to
provide inoculum for the pre-culture
Note 1 to entry: It is necessary to use defined and verified strains, because of possible insecurities in species
taxonomy.
[SOURCE: ISO 20079:2005, 3.21, modified — "An address list of suppliers is given in Annex C" has been
deleted from Note 1 to entry.]
3.13
stock solution
solution with accurately known analyte concentration (s), prepared from chemicals with an appropriate
purity
[SOURCE: ISO 5667-16:2017, 3.21]
2
© ISO 2023 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 4979:2023(E)
3.14
test sample
discrete portion of sample (taken from, for example, receiving water, waste water, dissolved chemical
substances or mixtures, products and compounds) pre-treated according to the needs of the test (e.g.
dissolution, filtering, neutralization)
[SOURCE: ISO 20079:2005, 3.24]
3.15
test medium
aqueous solution that consists of a particular concentration of prepared test sample mixture of test
water and the sample under test
[SOURCE: ISO 21427-1:2006, 3.4]
4 Principle
All roots shall be removed from Lemna minor fronds, grown in axenic or non-axenic cultures, prior to
exposure to test samples and the growth of newly developed roots during the exposure period of 72 h
will be measured.
To quantify substance-related effects, the root length in the test medium is compared with that of the
controls and the concentration resulting in a relative inhibition of root length to be determined and
expressed as the EC .
(r)x
5 Test organisms
The standard test organism of this test is duckweed, Lemna minor, which is a freshwater-floating plant.
6 Growth medium
6.1 Preparation of stock solution
Prepare stock solution by adding the weighed chemicals according to Table 1 to the desired volume of
pure water for the growth medium and test compound solutions. Pure water should be used for the
dilution of liquid media and test substances.
pH of liquid media shall be adjusted to 6,9 ± 0,1 after adding pure water to each stock solution. Nutrients
should be added in the order of I-II-III-IV-V while preparing the solutions to prevent precipitation.
NOTE A pH of 7 is ideal for the re-g
...
DRAFT INTERNATIONAL STANDARD
ISO/DIS 4979
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:
2022-06-10 2022-09-02
Water quality — Aquatic toxicity test based on root re-
growth in Lemna minor
ICS: 13.060.70
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
This document is circulated as received from the committee secretariat.
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
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 4979:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
---------------------- Page: 1 ----------------------
ISO/DIS 4979:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/DIS 4979:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Test organisms . 3
6 Growth medium . 3
6.1 Preparation of stock solution . 3
6.2 Storage and cultivation . 4
7 Apparatus . 4
8 Experimental methods .6
8.1 Preparation of medium . 6
8.2 Preparation of toxicant solution and test dilutions . 6
8.2.1 Test dilutions . 6
8.2.2 Selection of test concentrations . 6
8.3 Control . 6
8.4 Transfer of test organisms . 6
8.5 Culture . 7
8.6 Method of measurements . 7
9 Tests on effects .7
9.1 Reference chemicals . 7
9.2 Statistics . 7
9.3 Validity of test . 8
9.4 Precision . . . 8
10 Expression of results . 8
10.1 Test results . 8
10.2 Expression of results . . 8
11 Test report . 8
Annex A (informative) Root excision and re-growth length measurement .10
Annex B (informative) Sensitivity of the Lemna root re-growth test .12
-1
Annex C (informative) Interlaboratory precision of control values (mm) and EC (mg L )
50
from the Lemna toxicity test . .15
Bibliography .17
iii
© ISO 2022 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/DIS 4979:2022(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 of 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DIS 4979:2022(E)
Introduction
Lemna gibba and L. minor have been most extensively used in phytotoxicity testing and there are
several standard methods which have been adopted by major international standardization agencies
[4]
e.g. ISO 20079:2005, U.S. Environmental Protection Agency, Organization for Economic Cooperation
[5]
and Development. Tests with duckweed have typically favoured measurements of frond (e.g. their
[9]
number, biomass, area, carbon uptake, chlorophyll content ) require standard exposure durations of
at least 7 d to detect toxicity.
On the other hand, tests based on root elongation are some of the most widely used phytotoxicity
methodologies for terrestrial angiosperms because of their simplicity and rapidity. Despite reports that
roots of L. minor are highly sensitive to environmental stressors and that they play important ecological
[6] [7] [8]
roles by providing stability , , little attention has been paid to the roots in Lemna since it was
generally considered that root fragility made their handling for measurements difficult and that it was
impractical to obtain sufficient numbers of individual plants with identical root lengths to initiate tests.
However, the ecotoxicological significance of the root endpoint has been re‑evaluated and root length
was shown to be a sensitive, precise and ecologically significant endpoint in comparison with more
[9]
traditional frond growth or biomass endpoints.
The proposed root re‑growth bioassay differs in several key aspects from three internationally
standardized methods (ISO, OECD and US EPA):
a) the test can be completed within 72 h;
b) the test vessel is a 24-well cell plate;
c) the required volume of test water samples is 3 ml;
d) roots were excised prior to exposure with subsequent measurements on newly developed roots.
The technique of excising roots prior to exposure means that there is no requirement to pre‑select
roots of uniform length, which reduces the handling of these fragile roots.
Artificial severance of roots may never happen in natural settings since root abscission in Lemna has
not been reported previously. However, according to recent studies the tiny globally distributed water
[10]
ferns of the genus Azolla lost their roots under stress conditions a phenomenon known as rapid root
abscission. Such shedding sets its fronds free from root-entangled mats and facilitates their dispersion
to a potentially better environment. Therefore, rapid root abscission is considered an important
[10]
survival strategy of Azolla. This may indicate that the endpoint root re‑growth has its ecological
relevance.
It is also well known that Lemna will thrive without any roots. Thus, Lemna roots appear nonessential
organs, but are nonetheless important for plant anchorage, nutrient absorption and cytokinin
biosynthesis. Therefore, the manipulation of roots by simple severance may not be a serious issue and
does not justify the conclusion that removal of roots prior to ecotoxicological testing is inappropriate.
The 3 d root re‑growth test is useful for the rapid screening of either wastewater effluents or hazardous
[11]
contaminants in natural waters as it is easy to perform, quick to run, and cost‑effective to operate for
wastewater toxicity screening and may have an operational benefit of testing time since management
decision should be made in a timely manner in the case of unexpected pollution events.
The root re‑growth endpoint from this 72‑hour protocol is not a direct substitute for the 7‑day growth
rate/biomass endpoints.
The present protocol provides detailed information on how to set up and conduct the root re-growth test
with Lemna minor as well as how to analyse toxicity data. This protocol is intended for use with Lemna
minor, but it can also be applied to other Lemna species and Spirodela species with some modifications.
v
© ISO 2022 – All rights reserved
---------------------- Page: 5 ----------------------
DRAFT INTERNATIONAL STANDARD ISO/DIS 4979:2022(E)
Water quality — Aquatic toxicity test based on root re-
growth in Lemna minor
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted 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 root re‑growth in duckweeds
(Lemna minor) by substances and mixtures contained in water or waste water. This method applies to
environmental water samples including treated municipal wastewater and industrial effluents.
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 20079, Water quality — Determination of the toxic effect of water constituents and waste water on
duckweed (Lemna minor) — Duckweed growth inhibition test
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
axenic culture
monocultures of organisms from a single species, free from fungi, algae and other macrophyte species
[SOURCE: ISO 20079:2005, 3.1]
3.2
coefficient of variation
CV
relative standard deviation, expressed as a percentage
3.3
colony
aggregate of mother and daughter fronds, attached to each other, sometimes referred to as a plant
[SOURCE: ISO 20079:2005, 3.4]
1
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
ISO/DIS 4979:2022(E)
3.4
effective concentration
EC
x
concentration of test sample (EC ) at which an effect of x % is measured, if compared to the control
x
[SOURCE: ISO 20079:2005, 3.9, modified — Note 1 to entry deleted]
3.5
frond
individual leaf‑like structure on a duckweed colony; the smallest unit (i.e. individual), capable to
reproduce
[SOURCE: ISO 20079:2005, 3.10]
3.6
growth
increase in biomass over time as the result of proliferation of new tissues
Note 1 to entry: In this test it refers to any parameter of observation.
[SOURCE: ISO 20079:2005, 3.13]
3.7
growth medium
pure water to which reagent-grade chemicals (micronutrients) have been added
3.8
non-axenic culture
monoculture of organisms from a single species (i.e. free from other macrophyte species), which has
not been treated with antimycotic or antibiotic solutions to remove naturally associated bacteria and
fungi
3.9
pre-culture
culture of duckweed used for acclimation of test plants to the test conditions and for the growing of the
plants to be used in the inoculum
[SOURCE: ISO 20079:2005, 3.19]
3.10
pure water
deionized or distilled water
[SOURCE: ISO 19827:2016, 3.4]
3.11
root
the part of the Lemna plant that assumes a root-like structure
[SOURCE: ISO 20079:2005, 3.20]
3.12
stock culture
culture of a single species of duckweed to conserve the original Lemna species in the laboratory and to
provide inoculum for the pre-culture
Note 1 to entry: It is necessary to use defined and verified strains, because of possible insecurities in species
taxonomy.
[SOURCE: ISO 20079:2005, 3.21, modified — "An address list of suppliers is given in Annex C" deleted in
the note]
2
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/DIS 4979:2022(E)
3.13
stock solution
solution with accurately known analyte concentration (s), prepared from chemicals with an appropriate
purity
[SOURCE: ISO 5667-16:2017, 3.21]
3.14
test sample
discrete portion of sample (taken from i.e. receiving water, waste water, dissolved chemical substances
or mixtures, products and compounds) pre‑treated according the to the needs of this test (e.g.
dissolution, filtering, neutralization)
[SOURCE: ISO 20079:2005, 3.24]
3.15
test medium
aqueous solution that consists of a particular concentration of prepared test sample mixture of test
water and the sample under test
[SOURCE: ISO 21427-1:2006, 3.4]
4 Principle
All roots (3.11) shall be removed from Lemna minor fronds (3.4), grown in axenic (3.7) or non‑axenic
cultures (3.8), prior to exposure to test sample and the growth of newly developed roots during the
exposure period of 72 h will be measured.
To quantify substance‑related effects, the root length in the test medium (3.15) is compared with that
of the controls and the concentration resulting in a relative inhibition of root length to be determined
and expressed as the EC(r)x.
5 Test organisms
The standard test organism of this test is duckweed, Lemna minor, which is a freshwater‑floating plant.
6 Growth medium
6.1 Preparation of stock solution
Prepare stock solution (3.13) by adding the weighed chemicals according to Table 1 to the desired
volume of pure water (3.10) for the growth medium and test compound solutions. Pure water should be
used for the dilution of liquid media and test substances.
pH of liquid media shall be adjusted to 6,9 ± 0,1 after adding pure water to each stock solution. Nutrients
should be added in the order of I-II-III-IV-V while preparing the solutions to prevent precipitation.
NOTE A pH of 7 is ideal for the re-growth of Lemna roots and pH 6,9 ± 0,1 is proposed to be the appropriate
pH for the toxicity tests, as this is also the same pH range that is measured in the Steinberg medium.
Liquid media can be stored for up to one month at room temperature in the dark.
Table 1 — Main ingredients of STEINBERG medium
Stock solution Chemicals Stock concentration Final concentration
-1 -1
g L ml L
Macro-elements
3
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/DIS 4979:2022(E)
Table 1 (continued)
Stock solution Chemicals Stock concentration Final concentration
-1 -1
g L ml L
I KNO 17,5 20
3
K HPO 4,5
2 4
KH PO 0,63
2 4
II MgSO ·7H O 5 20
4 2
III Ca(NO ) ·4H O 14,75 20
3 2 2
Micro-elements
IV H BO 0,12 1
3 3
ZnSO ·7H O 0,18
4 2
Na MoO ·2H O 0,044
2 4 2
MnCl ·4H O 0,18
2 2
V FeCl ·6H O 0,76 1
3 2
Na -EDTA·2H O 1,5
2 2
6.2 Storage and cultivation
Culture fronds of Lemna minor in growth medium (3.6) as shown in Table 1. The fronds (3.4) shall
-2 -1
be cultured at (25 ± 1) °C, given 24 h continuous white light at an intensity of 30 μmol m s to
-2 -1
40 μmol m s (1,500 lx to 2,000 lx). The medium shall be replaced at an interval of 7 d and the storage
culture can be kept continually unless uncontrolled contamination occurs. Clo
...
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