ISO 4979:2023

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 2023
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ii
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
Lemna toxicity test .12
Bibliography .14
iii
Foreword
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This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
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iv
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
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
environme
...