EN ISO 20079:2006

SLOVENSKI STANDARD
01-februar-2007
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Water quality - Determination of the toxic effect of water constituents and waste water on
duckweed (Lemna minor) - Duckweed growth inhibition test (ISO 20079:2005)
Wasserbeschaffenheit - Bestimmung der toxischen Wirkung von Wasserinhaltsstoffen
und Abwasser gegenüber Wasserlinsen (Lemna minor) - Wasserlinsen-
Wachstumshemmtest (ISO 20079:2005)
Qualité de l'eau - Détermination de l'effet toxique des constituants de l'eau et des eaux
résiduaires vis-a-vis des lentilles d'eau (Lemna minor) - Essai d'inhibition de la
croissance des lentilles d'eau (ISO 20079:2005)
Ta slovenski standard je istoveten z: EN ISO 20079:2006
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 20079
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2006
ICS 13.060.70
English Version
Water quality - Determination of the toxic effect of water
constituents and waste water on duckweed (Lemna minor) -
Duckweed growth inhibition test (ISO 20079:2005)
Qualité de l'eau - Détermination de l'effet toxique des Wasserbeschaffenheit - Bestimmung der toxischen
constituants de l'eau et des eaux résiduaires vis-à-vis des Wirkung von Wasserinhaltsstoffen und Abwasser
lentilles d'eau (Lemna minor) - Essai d'inhibition de la gegenüber Wasserlinsen (kursiv(Lemna minor)) -
croissance des lentilles d'eau (ISO 20079:2005) Wasserlinsen-Wachstumshemmtest (ISO 20079:2005)
This European Standard was approved by CEN on 17 August 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20079:2006: E
worldwide for CEN national Members.

Foreword
The text of ISO 20079:2005 has been prepared by Technical Committee ISO/TC 147 "Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 20079:2006 by
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 March 2007, and conflicting national standards shall be withdrawn at
the latest by March 2007.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden,
Switzerland and United Kingdom.

Endorsement notice
The text of ISO 20079:2005 has been approved by CEN as EN ISO 20079:2006 without any modifications.

INTERNATIONAL ISO
STANDARD 20079
First edition
2005-11-01
Water quality — Determination of the
toxic effect of water constituents and
waste water on duckweed (Lemna
minor) — Duckweed growth inhibition
test
Qualité de l'eau — Détermination de l'effet toxique des constituants de
l'eau et des eaux résiduaires vis-à-vis des lentilles d'eau (Lemna
minor) — Essai d'inhibition de la croissance des lentilles d'eau

Reference number
ISO 20079:2005(E)
©
ISO 2005
ISO 20079:2005(E)
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ii © ISO 2005 – All rights reserved

ISO 20079:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 3
5 Interferences . 4
6 Apparatus . 4
7 Reagents. 4
8 Test organisms . 7
9 Stock cultures and pre-cultures. 7
10 Procedure . 8
11 Validity criteria . 10
12 Expression of results . 10
13 Estimation of EC(r) values for frond number and the second observation parameter . 12
x
14 Documentation of results . 12
15 Precision. 12
16 Test report . 13
Annex A (informative) Preparation of the nutrient media . 14
Annex B (informative) Measurement of the lowest ineffective dilution (LID) of a waste water —
A simplified evaluation for testing of waste water. 19
Annex C (informative) Suppliers of Lemna species . 22
Bibliography . 23

ISO 20079:2005(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 20079 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2005 – All rights reserved

ISO 20079:2005(E)
Introduction
The duckweed species Lemna minor is used as model organism for higher water plants. Duckweeds are
monocotyledonous, free-floating angiosperms and belong to the Arales within the subclass of Aridae.
Duckweeds are fast growing higher plants, spreading from the tropic to the arctic zone. As primary producers
they are a food source for waterfowl, fish and small animals and serve as physical support for a variety of
small invertebrates.
Duckweed can be damaged by water constituents and effluents (see Annex B). The subsequent inhibition of
growth is calculated from the observation parameters (frond number, frond area, chlorophyll, dry weight) by a
number of defined calculation methods.
EC values are determined to allow for an assessment of toxic effects of water constituents (e.g. chemicals,
plant protection products). The evaluation for at least two observation parameters is based on the average
specific growth-rates.
The test is designed for measurement of response of substances dissolved in water. This includes the
definition of a fixed dilution step, or a concentration of the test sample at which a parameter of observation
(endpoint) is inhibited relative to a control for a defined percentage.
INTERNATIONAL STANDARD ISO 20079:2005(E)

Water quality — Determination of the toxic effect of water
constituents and waste water on duckweed (Lemna minor) —
Duckweed growth inhibition test
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard 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 International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination of the growth-inhibiting response of
duckweed (Lemna minor) to substances and mixtures contained in water, treated municipal wastewater and
industrial effluents.
2 Normative references
The following referenced documents are indispensable for the application 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
ISO 10260, Water quality — Measurement of biochemical parameters — Spectrometric determination of the
chlorophyll-a concentration
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
axenic cultures
monocultures of organisms from a single species, free from fungi, algae and other macrophyte species
3.2
calculation parameters
parameters for the estimation of toxicity derived from any parameters of observation by different methods of
calculation
EXAMPLE Growth-rates derived from frond number, frond area, chlorophyll and dry weight are calculation
parameters in this International Standard.
ISO 20079:2005(E)
3.3
chlorosis
loss of pigment (yellowing of frond tissue)
3.4
colony
aggregate of mother and daughter fronds, attached to each other, sometimes referred to as a plant
3.5
control batch
control medium, including organisms used for testing
3.6
control medium
combination of dilution water and/or nutrient medium used in the test
3.7
dilution water
water added to the test sample to prepare a series of defined dilutions
3.8
doubling time
quotient of natural logarithm of 2 (ln 2) divided by average specific growth-rate
3.9
effective concentration
concentration of the test sample (EC ) at which an effect of x % is measured, if compared to the control
x
NOTE To unambiguously denote an EC value deriving from growth-rate, it is proposed to use the symbol “EC(r)”,
followed by the observation parameter used, e.g. EC(r) (frond number).
3.10
frond
individual leaf-like structure on a duckweed colony; the smallest unit (i.e. individual), capable of reproducing
3.11
frond area
total area of all fronds visible from vertically above
3.12
frond number
all fronds protruding from a mother frond which are directly visible from above without magnification
3.13
growth
increase in biomass over time as the result of proliferation of new tissues
NOTE In this test it refers to any parameter of observation.
3.14
growth-rate
calculation parameter defined as quotient of the difference of the natural logarithms of a parameter of
observation and the respective time period
NOTE If the time period comprises the total duration of the test, the term is referred to as average specific
growth-rate. If the period between two measurements within the test period is used, the term is named segmented
growth-rate (see 12.1.2).
2 © ISO 2005 – All rights reserved

ISO 20079:2005(E)
3.15
inoculum
number of fronds (colonies) added to the test batch at the beginning of the test
3.16
necrosis
localised dead frond tissue (i.e. brown or white)
3.17
nutrient medium
solution of nutrients and micronutrients in water which are essential for the growth of duckweed
3.18
observation parameters
observed or measured biomass parameters like frond number, frond area, chlorophyll, dry weight, which are
measured or counted once or repeatedly by observation or measurement
NOTE These parameters are relevant for the assessment of growth and vitality of the test organisms (e.g. frond
number, frond area or chlorophyll content, dry weight).
3.19
pre-culture
culture of duckweed used for acclimatisation of test plants to the test conditions and for the growing of the
plants to be used in the inoculum
3.20
root
that part of the Lemna plant that assumes a root-like structure
3.21
stock culture
culture of a single species of duckweed to conserve the original defined Lemna species in the laboratory and
to provide inoculum for the pre-culture
NOTE It is necessary to use defined and verified strains, because of possible insecurities in species taxonomy. An
address list of suppliers is given in Annex C.
3.22
test batch
test medium including organisms used for testing
3.23
test medium
combination of test sample, dilution water and/or nutrient medium used in the test
3.24
test sample
discrete portion of a sample (taken from i.e. receiving water, waste water, dissolved chemical substances or
mixtures, products and compounds) pretreated according to the needs of this test (e.g. dissolution, filtering,
neutralisation)
4 Principle
Plants of the species Lemna minor are allowed to grow as monocultures in different concentrations of the test
sample over a period of seven days. The objective of the test is to quantify substance-related effects on
vegetative growth over this period based on assessments of frond number, and also on assessments on
biomass (total frond area, dry weight or chlorophyll). To quantify substance-related effects, the growth-rate in
the test solutions is compared with that of the controls and the concentration resulting in a specified x %
inhibition of growth-rate is determined and expressed as the EC(r) .
x
ISO 20079:2005(E)
5 Interferences
Non-soluble, poorly soluble, volatile, bio- or photodegradable substances or substances reacting with the
dilution water or the nutrient medium or changing their state during the test, may falsify or reduce the

reproducibility of the results (see ISO 5667-16). Special consideration is necessary in the case of substances
accumulated at the water surface as this may increase the effects on duckweed.
6 Apparatus
The test design determines the requirements for the apparatus.
6.1 Cylindrical vessels, (glass beakers, crystallising dishes, Petri dishes).
Minimum volume of 150 ml (for 2/3 of total volume, i.e. 100 ml of test solution).
6.2 Uniform glass coverings.
Covers may be provided to minimize evaporation and accidental contamination.
6.3 Facilities with constant temperature and illumination, temperature controlled room or water bath,
incubator or environmental chamber.
6.4 Spectrometer to monitor chlorophyll, 665 nm and 750 nm.
6.5 Lumino-meter, to be used to measure light intensity.
6.6 pH-meter.
6.7 Tweezers.
6.8 Glassware, for the preparation of different concentration series and nutrient medium (volumetric flasks,
graduated cylinders, pipettes, Petri dishes).
6.9 Image analysis system, to measure frond number and frond area.
6.10 Autoclave.
6.11 Filtration device, for sterile filtration.
7 Reagents
Use only reagents of recognised analytical grade.
7.1 Dilution water, distilled or deionised water or water of equivalent purity, conductivity u 10 µS/cm.
7.2 Hydrochloric acid, for example c(HCl) = 0,1 mol/l.
7.3 Sodium hydroxide solution, for example c(NaOH) = 0,1 mol/l.
7.4 Glucose, C H O .
6 12 6
7.5 Agar medium.
See Annex A.
4 © ISO 2005 – All rights reserved

ISO 20079:2005(E)
7.6 Nutrient media, modified STEINBERG medium (see Table 1).
Generally the modified STEINBERG medium shall be used for all applications within the scope of the guideline,
i.e. water constituents and wastewaters. In some cases, use of the media described in Annex A may also be
suitable as long as all validity criteria are fulfilled.
Table 1 — pH-stabilised STEINBERG medium (modified by Altenburger)
Substance Nutrient medium
Macroelements Molecular mass mg/l mmol/l
KNO 101,12 350,00 3,46
Ca(NO )⋅4HO 236,15 295,00 1,25
3 2 2
KH PO 136,09 90,00 0,66
2 4
K HPO 174,18 12,60 0,072
2 4
MgSO⋅7HO 246,37 100,00 0,41
4 2
Microelements Molecular mass µg/l µmol/l
H BO 61,83 120,00 1,94
3 3
ZnSO⋅7HO 287,43 180,00 0,63
4 2
Na MoO⋅2HO 241,92 44,00 0,18
2 4 2
MnCl ·4HO 197,84 180,00 0,91
2 2
FeCl⋅6HO 270,21 760,00 2,81
3 2
EDTA Disodium-dihydrate 372,24 1 500,00 4,03
7.6.1 Concentrations and stock solutions (see Tables 2 and 3).
Prepare the nutrient medium from single solutions. The required concentrations of pre-culture and test
medium are obtained by dilution if 10-fold concentrated medium is prepared.
Table 2 — Stock solutions (macroelements)
Macroelements (50-fold concentrated) g/l
Stock solution 1:
KNO 17,50
KH PO 4,5
2 4
K HPO 0,63
2 4
Stock solution 2:
MgSO⋅7HO 5,00
4 2
Stock solution 3:
Ca(NO )⋅4HO 14,75
3 2 2
ISO 20079:2005(E)
Table 3 — Stock solutions (microelements)
Microelements (1 000-fold concentrated) mg/l
Stock solution 4:
H BO 120,0
3 3
Stock solution 5:
ZnSO⋅7HO 180,0
4 2
Stock solution 6:
Na MoO⋅2HO 44,0
2 4 2
Stock solution 7:
MnCl⋅4HO 180,0
2 2
Stock solution 8:
FeCl⋅6HO 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).
For longer shelf life, treat stock solutions in an autoclave at 121 °C for 20 min or alternatively carry out a
sterile filtration (0,2 µm). For stock solution 8, sterile filtration (0,2 µm) is strongly recommended.
7.6.2 Preparation of the final concentration of modified STEINBERG medium
Add 20 ml each of stock solutions 1, 2 and 3 (see Table 2) to about 900 ml water (7.1).
Then add 1,0 ml each of stock solutions 4, 5, 6, 7 and 8 (see Table 3) to avoid precipitation.
The pH should be 5,5 ± 0,2 [adjust by addition of a minimised volume of NaOH solution (7.3) or HCl (7.2)].
Adjust with water (7.1) to 1 000 ml.
If stock solutions are sterilized and appropriate water is used, no further sterilisation is necessary. If
sterilisation is done with the final medium, stock solution 8 should be added after autoclaving (at 121 °C for
20 min).
7.6.3 Preparation of 10-fold-concentrated modified STEINBERG medium
Add 20 ml each of stock solutions 1, 2 and 3 (see Table 2) to about 30 ml water (7.1).
Then add 1,0 ml each of stock solutions 4, 5, 6, 7 and 8 (see Table 3) to avoid precipitation. Adjust with water
(7.1) to 100 ml.
If stock solutions are sterilized and appropriate water is used, no further sterilisation is necessary. If
sterilisation is done with the final medium, stock solution 8 should be added after autoclaving (at 121 °C for
20 min).
The pH of the medium (final concentration) should be 5,5 ± 0,2.
For the assessment of mining effluents, metal substances added to water or other samples which may contain
pre-dominantly metals, it may be appropriate to use a modified APHA test medium. Using modified APHA (i.e.
without EDTA, see Annex A) would make it necessary to change medium from modified STEINBERG medium to
[12]
modified APHA between pre-culture and an acclimatization phase before the test . This change does not
conform with 9.2, but this is to be accepted in this case. All details on handling and use of APHA are included
in Annex A.
6 © ISO 2005 – All rights reserved

ISO 20079:2005(E)
For the assessment of water constituents (chemicals), the OECD medium (modified SIS) could also be used
(see Annex A). The concentration of the respective nutrient medium shall be kept constant in all treatments
and controls.
7.7 Reference substances
7.7.1 3,5-dichlorophenol
3,5-dichlorophenol analytical grade > 99 % purity.
7.7.2 Potassium chloride, KCl.
8 Test organisms
For use in this International Standard, Lemna minor (with a documentation of origin) is the recommended
species. Plants obtained from a wild population need a confirmation of their taxonomy.
9 Stock cultures and pre-cultures
9.1 Stock cultures
Stock cultures are kept axenically. Addition of glucose (1 %) enables the recognition of microbial infections.
For solid medium, about 1 % of agar may be added (approved media are listed in Annex A). Stock cultures
may be maintained at low light conditions and ambient temperature for several months without re-inoculation if
evaporation is minimized by covering the Erlenmeyer flask with a plug or cap and aluminium foil (see also
Annex A).
9.2 Pre-cultures
Initiate cultures used for toxicity tests at 7 d to 10 d prior to the test, using test medium and test conditions.
NOTE 1 A longer adaptation-time may be required in the case of change of the nutrient medium between stock culture
and pre-culture.
NOTE 2 Where the modified APHA-medium is to be used as testing medium, specific treatment will be necessary (see
Annex A).
Pre-cultured duckweed to be used in toxicity tests should meet the following health criteria:
⎯ exponential growth;
⎯ the number of fronds in the pre-culture should have a seven-fold increase by the end of 7 d (i.e.
r W 0,275 per d or doubling time u 2,5 d);
⎯ the culture should consist of young, rapidly growing colonies with bright green colour without visible
lesions, chlorosis or necrosis;
⎯ a large number of single fronds or small colonies is an indication of environmental stress and these plants
should not be used in testing;
⎯ the depth of the medium should be at least 3 cm.
To minimise lag-phases caused by interactions between colonies, it should be assured that the covering is
less than 50 % of the total available surface (no crowding). All colonies used should originate from the same
pre-culture.
ISO 20079:2005(E)
10 Procedure
10.1 General
The general recommendations as specified in ISO 5667-16 are to be taken into account. Adjust the pH value
of the test sample to ± 0,2 of the pH value of the nutrient medium in the control by adding hydrochloric acid
(7.2) or sodium hydroxide solution (7.3). Dilution should be kept at a minimum. No later adjustment should be
made.
NOTE Duckweed generally has no growth problems between pH 5 and pH 9. Therefore adjustment of pH is usually
not necessary as long as the pH of the sample is between 5 and 8, depending on buffer capacity.
Neutralisation is not allowed if the effect of pH is to be reflected in the test results or if physical modification or chemical
reaction is observed due to pH adjustment.
10.2 Preparation of concentration series for EC(r) value assessment
x
If the EC(r) is to be estimated, a sufficient number of concentrations is used to define the EC(r) at an
x x
appropriate confidence level. An appropriate test design consists of a geometric series of at least five
concentrations. At least one measured inhibition value for the intended EC(r) should be below and one above
x
the EC(r) to be estimated, and three or more values should be other than 0 % or 100 % inhibition. Otherwise,
x
confidence limits might be too large.
10.3 Test
10.3.1 Test for EC(r)
x
For EC(r) assessment (see also ISO 5667-16), use at least 3 re
...