ISO 20665:2008
(Main)Water quality — Determination of chronic toxicity to Ceriodaphnia dubia
Water quality — Determination of chronic toxicity to Ceriodaphnia dubia
ISO 20665:2008 specifies a method for the determination of the chronic toxicity to Ceriodaphnia dubia (Cladocera, Crustacea), based on reproduction inhibition after (7 ± 1) d. The method is applicable to: a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions under the conditions of the test; b) industrial or sewage effluents, if appropriate after decantation, filtration or centrifugation; c) fresh waters; d) aqueous extracts (leachates, eluates, and elutriates). ISO 20665:2008 is not applicable to the testing of aquatic samples from the estuarine or marine environment.
Qualité de l'eau — Détermination de la toxicité chronique vis-à-vis de Ceriodaphnia dubia
L'ISO 20665:2008 spécifie une méthode de détermination de la toxicité chronique vis-à-vis de Ceriodaphnia dubia (Cladocera, Crustacea), basée sur une inhibition de la reproduction au bout de (7 ± 1) jours. Cette méthode est applicable: a) aux substances chimiques solubles ou pouvant être maintenues en suspension ou en dispersion stables dans les conditions de l'essai; b) aux effluents industriels ou aux eaux usées, le cas échéant, après décantation, filtration ou centrifugation; c) aux eaux douces; d) aux extraits aqueux. L'ISO 20665:2008 n'est pas applicable à la réalisation d'essais sur des échantillons aqueux provenant d'un milieu marin ou estuarien.
Kakovost vode - Določevanje kronične strupenosti s Ceriodaphnia dubia
Ta mednarodni standard določa metodo določevanja kronične strupenosti s Ceriodaphnia dubia (Cladocera, Crustacea), osnovan na zaviranje reprodukcije po 7 ± 1) d.
Ta metoda se uporablja za:
a) kemične substance, ki so topne ali ki se lahko ohranijo kot stabilne suspenzije ali disperzije pod preskusnimi pogoji;
b) industrijske ali komunalne odplake, obdelane ali neobdelane, po potrebi po pretočitvi, filtraciji ali centrifugiranju;
c) sladke vode;
d) vodne ekstrakte..
Ta mednarodni standard ne velja za preskušanje vodnih vzorcev iz rečnih ustij ali morskega okolja.
General Information
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 20665
First edition
2008-12-15
Water quality — Determination of chronic
toxicity to Ceriodaphnia dubia
Qualité de l'eau — Détermination de la toxicité chronique vis-à-vis de
Ceriodaphnia dubia
Reference number
ISO 20665:2008(E)
©
ISO 2008
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ISO 20665:2008(E)
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ii © ISO 2008 – All rights reserved
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ISO 20665:2008(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle. 2
5 Test environment. 3
6 Reagents, test organisms and media . 3
7 Apparatus . 5
8 Sampling and samples. 6
9 Procedure . 7
10 Expression of results . 9
11 Validity criteria . 11
12 Precision. 11
13 Test report . 12
Annex A (normative) Preparation of the ELENDT M4 medium. 13
Annex B (normative) Preparation of the moderately hard water medium. 15
Annex C (informative) Preparation of the LC OLIGO medium. 16
Annex D (normative) Procedure for preparing YCT . 18
Annex E (informative) Data collection sheet . 20
Bibliography . 21
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ISO 20665:2008(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 20665 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
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ISO 20665:2008(E)
Introduction
The highlighting of harmful effects for water quality has for several years involved the carrying out of biological
tests. The Cladocera, Ceriodaphnia dubia, is recognised as being representative of the zooplankton species
widely used in aquatic toxicity tests.
The shortness of the chronic toxicity test, (7 ± 1) d, and the low volumes used are major assets for obtaining
relevant results on samples that may be subject to changes during the storage period.
The user should be aware that particular problems could require the specifications of additional marginal
conditions.
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INTERNATIONAL STANDARD ISO 20665:2008(E)
Water quality — Determination of chronic toxicity to
Ceriodaphnia dubia
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International 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 chronic toxicity to Ceriodaphnia
dubia (Cladocera, Crustacea), based on reproduction inhibition after (7 ± 1) d.
The method is applicable to:
a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions
under the conditions of the test;
b) industrial or sewage effluents, if appropriate after decantation, filtration or centrifugation;
c) fresh waters;
d) aqueous extracts.
This International Standard is not applicable to the testing of aquatic samples from the estuarine or marine
environment.
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 5667-16:1998, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 6059, Water quality — Determination of the sum of calcium and magnesium — EDTA titrimetric method
ISO 10523, Water quality — Determination of pH
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
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ISO 20665:2008(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
brood
group or cohort of sibling offspring, consisting of two or more neonates in any test container, during any given
day of the test, released from the adult female during an inter-moult period (i.e. before the carapace is shed by
that female during moulting)
3.2
brood organism
healthy adult female daphnid that produces and releases multiple broods of live neonates
3.3
control batch
series of replicates containing control solution (3.4)
NOTE In this International Standard, 10 replicates constitute the control batch.
3.4
control solution
mixture of test medium and of food without sample under test
3.5
effective concentration producing x % reproduction inhibition
EC
x
estimated concentration of a test sample giving rise to x % reproduction inhibition with respect to the
control batch (3.3), which represents a point of the test sample concentration that is estimated to cause a
designated percent impairment in a quantitative biological function
3.6
neonate
newly born or newly hatched individual
NOTE In this International Standard, a neonate is a first-instar daphnid, < 24 h old.
3.7
reproduction inhibition
comparison between the number of living offspring born from all adults at the end of the test between the
control batch (3.3) and the test batch (3.8)
3.8
test batch
series of replicates containing the same test solution (3.9)
NOTE In this International Standard, 10 replicates constitute a test batch.
3.9
test solution
mixture of test medium, of food and of sample under test
4 Principle
Ceriodaphnia dubia, less than 24 h old at the beginning of the test, are exposed individually to a range of
concentrations of the sample under test for a period of (7 ± 1) d. The test typically ends after 7 d when 60 % of
the control organisms have produced their third brood. The mortality of the adult females and their
reproduction are monitored throughout the exposure time. All other relevant biological parameters can also be
studied.
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ISO 20665:2008(E)
The data obtained allow, using a suitable model, the calculation of the concentration which gives rise to x %
reproduction inhibition, EC , e.g. EC , EC or EC .
x 10 20 50
5 Test environment
Carry out the test in a temperature-controlled room or chamber at (25 ± 2) °C in the test containers. Ensure
that, within one test, the temperature does not vary by more than 2 °C.
Adjust the day/night test cycle (photoperiod) to 16 h of daylight and 8 h of darkness. In the test containers
(7.2), a range of lighting intensity at the air/water interface of 100 lx to 600 lx (7.8) is recommended. Do not
shake or aerate the test containers.
Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that
the test is being performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognised analytical grade, unless otherwise specified.
6.1 Test organisms
Ceriodaphnia dubia neonates are obtained by parthenogenesis from adult females for at least three
generations under the conditions of temperature, photoperiod and food identical to those in the test.
The Ceriodaphnia dubia used for the test shall be less than 24 h old and shall have been taken from a brood
comprising at least eight newly born animals.
The day before the test, isolate from the culture a dozen or more adults that are over 6 d and less than 14 d
old. Isolate each one in a separate container containing food (6.4.1 or 6.4.2) and test medium (6.3.2 or 6.3.3).
Before the test, remove the adults from their containers and count the offspring. Discard all vessels containing
less than eight live offspring.
The Ceriodaphnia dubia may also derive from the hatching of ephippia purchased from a specialised
1)
company . These organisms may be directly used as test organisms.
2)
6.2 Pure water, having a conductivity below 10 µS/cm .
6.3 Test media
6.3.1 General
Two test media are recommended: ELENDT M4 (6.3.2) or moderately hard water (6.3.3). Alternative test
media may be used as long as validity criteria (Clause 11) are met.
For alternative test media, supply either a reference to a publication, or for natural waters (in case of effluent
testing) the date of collection, details of storage, handling, and additions, as well as physical chemistry data
relating to major ions [Na(I), K(I), Ca(II), Mg(II), carbonates, chloride, sulfate], pH and dissolved organic
carbon.
1) Microbiotest, Deinze, Belgium, is an example of a supplier able to provide suitable ephippia commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.
2) 1 mS/m.
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ISO 20665:2008(E)
6.3.2 ELENDT M4 medium option
Prepare ELENDT M4 test medium in accordance with Annex A. The test medium thus prepared shall have a
pH of 8,0 ± 0,3 (measured as specified in ISO 10523), a total hardness of (250 ± 20) mg/l (expressed as
CaCO and measured as specified in ISO 6059). Aerate the test medium until the dissolved oxygen
3
concentration has reached the air saturation value and until the pH has stabilised. If necessary, adjust the pH
to 8,0 ± 0,3 using a diluted solution of sodium hydroxide or hydrochloric acid.
NOTE On account of the high hardness of the test medium and of the presence of EDTA within this medium, the
bioavailability of the bivalent metal ions can be reduced, thus resulting in a decrease in the apparent toxicity of these ions.
6.3.3 Moderately hard water medium option
Prepare moderately hard water in accordance with Annex B. The test medium thus prepared shall have a pH
of 7,4 to 7,8 (measured as specified in ISO 10523) and a total hardness of (90 ± 10) mg/l (expressed as
CaCO and measured as specified in ISO 6059).
3
6.4 Food
6.4.1 Option 1: Fish food and two algae diet
3)
The food is composed of fish food, Chlorella vulgaris algae and Pseudokirchneriella subcapitata algae
[1]
(formerly known as Selenastrum capricornutum and Raphidocelis subcapitata) (see NF T90-376 and
Reference [2]).
4)
Prepare a 5 g/l suspension of fish food in the test medium (6.3.2), homogenised with a crusher or any other
means allowing particles of a few micrometers to be obtained. Prepare this suspension each day for the daily
feeding of cultures or during testing.
Grow the algae separately in any suitable medium (e.g. LC OLIGO, Annex C). Use them when the culture is in
6
the exponential growth phase and has reached a density greater than 5 × 10 cells per millilitre. These
cultures may be stored at (4 ± 3) °C, in darkness, for a maximum period of 10 d.
The following constituents should be added to each test solution (9.3) before the transfer of organisms:
6
a) 12 × 10 cells per litre of Chlorella vulgaris;
6
b) 6 × 10 cells per litre of Pseudokirchneriella subcapitata;
c) 500 µl per litre of the fish food suspension.
In any case, the quantity of food shall not constitute more than 10 % of the final volume of each container (9.3).
The use of Chlorella vulgaris and/or Pseudokirchneriella subcapitata algae immobilised in an inert matrix
5)
(gelose), in the form of algae beads is possible. In this case, after dissolving the matrix, centrifuge the algae,
discard the supernatant, and resuspend the algae by shaking in the test medium (6.3.2 or 6.3.3). Repeat this
operation a second time. The algal concentration in the test medium shall be the same as described above.
3) The Freshwater Biological Association, Ambleside, UK, is an example of a supplier able to provide these algae
commercially. This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of this supplier.
4) Sera Micron is 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.
5) Microbiotest, Deinze, Belgium, is an example of a supplier able to provide suitable algal beads commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.
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ISO 20665:2008(E)
6)
6.4.2 Option 2: Yeast/Cerophyll /trout chow and one algae diet
A second food combination based on the US EPA, method 1002.0 (Reference [11], p. 141) and Environment
Canada (Reference [5]) test methods is recommended (see also References [3] and [4]). Daily feeding with
Yeast/Cerophyll/trout chow (YCT) and a single algal species is required for culturing and testing of
Ceriodaphnia. The algal species most commonly used is Pseudokirchneriella subcapitata.
The formula for preparing YCT is given in Annex D. If the YCT/one algae diet is used, mass cultures should
be fed at a rate of:
⎯ 7 ml algae concentrate per litre culture;
⎯ 7 ml YCT concentrate per litre culture.
Individual cultures should be fed at the rate of:
⎯ 0,1 ml algae concentrate per 15 ml culture;
⎯ 0,1 ml YCT concentrate per 15 ml culture.
Food should be added to fresh culture medium immediately before or after the transfer of organisms.
Thoroughly mix algal concentrate and YCT by shaking before dispensing. If the YCT is stored frozen, store
thawed aliquots at (4 ± 3) °C. Discard unused portions of unfrozen or thawed YCT after 2 weeks. Store
unused portions of algal concentrate at (4 ± 3) °C, in darkness, and discard after 10 d.
6.5 Reference substance
Sodium pentachlorophenolate (C Cl ONa), copper sulfate pentahydrate (CuSO ·5H O), sodium chloride
6 5 4 2
(NaCl), or zinc sulfate (ZnSO ) are acceptable.
4
CAUTION — If sodium pentachlorophenolate is used as a reference toxicant, the material safety data
sheet should be consulted prior to use by laboratory personnel due to the hazardous nature of this
substance.
7 Apparatus
Usual laboratory apparatus and in particular the following.
7.1 Temperature-controlled room, chamber or water bath.
7.2 Test containers, made from a chemically inert material.
If closed containers are used, make sure that the capacity is sufficient to allow for a gas phase/aqueous phase
volume ratio of 1:1.
Prior to use, rinse the containers with the test medium (6.3.2 or 6.3.3) or with pure water (6.2).
7.3 Device for the measurement of algal concentration, e.g. a microscope equipped with a
haemocytometer or a particle counter. Indirect methods (e.g. spectrophotometer, turbidimeter, fluorimeter) can
be used if an acceptable correlation with the cellular concentration can be established.
6) Cereal Grass Media – Cerophyll is 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.
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ISO 20665:2008(E)
7.4 Pipette for sampling the Ceriodaphnia dubia, with a sufficient diameter for capturing the animals
while allowing sampling of only a small volume of medium.
7.5 Binocular magnifying glass, with a magnification of at least 8 times and, if possible, a continuous
magnification.
7.6 Image analysis system, to count and measure ceriodaphnids.
7.7 Membrane filtration device, with filters, 0,45 µm, 0,22 µm.
7)
7.8 Light source , providing a range of light intensity at the air/water interface in the test containers (7.2)
of 100 lx to 600 lx.
7.9 Sample collecting bottles, in accordance with ISO 5667-16:1998, 3.2.
7.10 Sieve, of nominal size of openings <100 µm.
8 Sampling and samples
Carry out sampling, transportation and storage of samples in accordance with the general procedures
specified in ISO 5667-16.
Collect samples in bottles made from chemically inert materials (7.9).
Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be
met, cool the sample to 0 °C to 4 °C and test it within 24 h. If it is not possible to perform the test within 72 h,
the sample may be frozen and maintained below −18 °C for testing within 2 months of collection, provided that
characteristics are known to be unaffected by freezing. At the time of testing, homogenise the sample to be
analysed by shaking manually, and, if necessary, allow to settle for 2 h in a container, and sample by drawing
off (use a pipette) the required quantity of supernatant, maintaining the end of the pipette in the centre of the
section of the test tube and half way between the surface of the deposited substances and the surface of the
liquid.
If the raw sample or the decanted supernatant is likely to interfere with the test (due to the presence of micro-
crustaceans, residual suspended matter, protozoa, micro-organisms, etc.), filter through a 0,45 µm membrane
filter (7.7) or centrifuge the raw or decanted sample.
The sample obtained by either of these methods is the sample submitted to testing. It is also used for the
renewal of test solutions. Store this sample in full containers at (4 ± 3) °C in the dark throughout the test
period.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in
ISO 5814) and record these values in the test report (Clause 13).
If the aim of the test is to assess the chronic toxicity without considering extreme pH effects, a second test
may also be carried out after adjustment of the pH value to 8,0 ± 0,3 with hydrochloric acid or sodium
hydroxide solutions. Proceed, if appropriate, as indicated above, for the separation of the suspended matter
formed following the adjustment of the pH. Mention any pH adjustment in the test report (Clause 13).
7) Grolux is 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.
6 © ISO 2008 – All rights reserved
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ISO 20665:2008(E)
9 Procedure
9.1 Preparation of the stock solutions of substances to be tested
Prepare the stock solution of the test substance by dissolving a known quantity of substance in a specified
volume of test medium (6.3.2 or 6.3.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it may be prepared in advance and stored under these conditions.
For poorly soluble substances in the test medium, refer to the specifications of ISO 5667-16.
9.2 Selection of concentrations
The test shall comprise at least five concentrations of the sample to be tested (Clause 8 or 9.1), selected
within a geometric series with a separation factor not exceeding 3,2.
Take the following criterion into account for selecting the range of concentrations to be examined: to obtain an
EC value, it is desirable that at least one concentration higher by x % than this is used and at least one x %
x
lower.
Produce at least 10 replicates for each concentration. These replicates constitute a test batch (3.8).
Include in each test a control batch (3.3) without any sample to be tested. A control batch is also made up of
at least 10 replicates.
When using a solvent in order to dissolve or disperse the substances, the solvent concentration shall be the
same in all containers. Include a second control containing the solvent at the concentration being used in test
concentrations.
In the case of samples of waters, effluents, and aqueous extracts, the highest tested concentration cannot be
equal to a volume fraction of 100 % of the initial sample on account of the food supply corresponding to a
small percent of the volume of sample being used.
For the purpose of toxicant range-finding or single concentration screening purposes, the test may also be
carried out with a lesser number of concentrations, but in this case, an EC cannot always be estimated.
x
9.3 Preparation of the test and control solutions
Prepare the test solutions by mixing the appropriate volumes of the sample to be tested (Clause 8 or 9.1) or of
its initial dilution with test medium (6.3.2 or 6.3.3) and food (6.4.1 or 6.4.2).
If the fish food/two algae diet (6.4.1) is used for feeding during a test, the quantity of food contained in each
test container shall not be greater than one tenth of the total volume (i.e. 50 ml for a 500 ml test or control
solution).
In the case where the YCT/one algae diet (6.4.2) is used for feeding during a test, the quantity of food
contained in each test container shall be equal to 1,5 % of the total volume in the test container (i.e. 0,1 ml of
YCT and 0,1 ml of algae concentrate added per 15 ml volume).
In both cases, the volume of test and control solutions should be at least (15 ± 2) ml and not exceed
(50 ± 2) ml.
Split the test solution for the replicate containers of each concentration into volumes of (15 ± 2) ml to
(50 ± 2) ml. Prepare the control solutions by mixing the food (6.4.1 or 6.4.2) with the test medium (6.3.2 or
6.3.3). Split the control solution into volumes of approximately (15 ± 2) ml to (50 ± 2) ml in each control
container.
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ISO 20665:2008(E)
9.4 Introduction of the organisms
Place the containers in a temperature-controlled room or chamber (7.1) to obtain a test and control solutions
temperature of (25 ± 2) °C.
As soon as this temperature is attained, introduce one Ceriodaphnia dubia aged less than 24 h (6.1) into each
container (7.2). Use a pipette (7.4) for the transfer, and release the crustaceans under the water surface.
9.5 Renewal of the test and control solutions
When the fish food/two algae diet (6.4.1) is used for feeding during a test, carry out renewal of the test and
control solutions (9.3) according to one of the timetables given in Table 1.
Table 1 — Test timetable for solution renewal (fish food/2 algae diet)
Timetable
a b c
Day 0 1 2 3 4 5 6 7 8
Renewal — yes or no no no or yes yes yes yes yes or no no
a
If the test starts on Thursday, renew test and control solutions; if it starts on Friday, do not renew solutions
b
If the test starts on Thursday, do not renew test and control solutions; if it starts on Friday renew test and control solutions
c
If the test is to continue to an 8th day, renew test and control solutions.
On the days when the timetable requires a renewal of the test and control solutions, transfer the adult females
from the old containers into new containers containing test and control solutions freshly prepared according to
9.3 (fresh food and medium) and bearing the same identification.
It is recommended that the transfer of the adult females take place at the beginning of the illuminated
photoperiod, the quantities of food need to be absorbed prior to the reduction of light. If a degradable
substance or sample is tested, the renewal should be daily, as per the timetable outlined in Table 2.
In the case where the YCT/one algae diet (6.4.2) is used for feeding during a test, renewal of the test and
control solutions (9.3) is carried out according to the timetable given in Table 2.
Table 2 — Test timetable for solution renewal (YCT/one algae diet)
Timetable
a
Day 0 1 2 3 4 5 6 7 8
Renewal — yes yes yes yes yes yes yes or no no
a
If the test is to continue to an 8th day, renew test and control solutions.
On days 1 to 6 where a renewal of the test and control solutions is required, transfer the adult females from
the old containers into new containers containing test and control solutions freshly prepared according to 9.3
(fresh food and medium) and bearing the same identification.
It is recommended that the transfer of the adult females take place at the beginning of the illuminated
photoperiod, t
...
SLOVENSKI STANDARD
SIST ISO 20665:2010
01-september-2010
.DNRYRVWYRGH'RORþHYDQMHNURQLþQHVWUXSHQRVWLV&HULRGDSKQLDGXELD
Water quality - Determination of chronic toxicity to Ceriodaphnia dubia
Qualité de l'eau - Détermination de la toxicité chronique vis-à-vis de Ceriodaphnia dubia
Ta slovenski standard je istoveten z: ISO 20665:2008
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 20665:2010 en,fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 20665:2010
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SIST ISO 20665:2010
INTERNATIONAL ISO
STANDARD 20665
First edition
2008-12-15
Water quality — Determination of chronic
toxicity to Ceriodaphnia dubia
Qualité de l'eau — Détermination de la toxicité chronique vis-à-vis de
Ceriodaphnia dubia
Reference number
ISO 20665:2008(E)
©
ISO 2008
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SIST ISO 20665:2010
ISO 20665:2008(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
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© ISO 2008
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ii © ISO 2008 – All rights reserved
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SIST ISO 20665:2010
ISO 20665:2008(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle. 2
5 Test environment. 3
6 Reagents, test organisms and media . 3
7 Apparatus . 5
8 Sampling and samples. 6
9 Procedure . 7
10 Expression of results . 9
11 Validity criteria . 11
12 Precision. 11
13 Test report . 12
Annex A (normative) Preparation of the ELENDT M4 medium. 13
Annex B (normative) Preparation of the moderately hard water medium. 15
Annex C (informative) Preparation of the LC OLIGO medium. 16
Annex D (normative) Procedure for preparing YCT . 18
Annex E (informative) Data collection sheet . 20
Bibliography . 21
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SIST ISO 20665:2010
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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 20665 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
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SIST ISO 20665:2010
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Introduction
The highlighting of harmful effects for water quality has for several years involved the carrying out of biological
tests. The Cladocera, Ceriodaphnia dubia, is recognised as being representative of the zooplankton species
widely used in aquatic toxicity tests.
The shortness of the chronic toxicity test, (7 ± 1) d, and the low volumes used are major assets for obtaining
relevant results on samples that may be subject to changes during the storage period.
The user should be aware that particular problems could require the specifications of additional marginal
conditions.
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SIST ISO 20665:2010
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SIST ISO 20665:2010
INTERNATIONAL STANDARD ISO 20665:2008(E)
Water quality — Determination of chronic toxicity to
Ceriodaphnia dubia
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International 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 chronic toxicity to Ceriodaphnia
dubia (Cladocera, Crustacea), based on reproduction inhibition after (7 ± 1) d.
The method is applicable to:
a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions
under the conditions of the test;
b) industrial or sewage effluents, if appropriate after decantation, filtration or centrifugation;
c) fresh waters;
d) aqueous extracts.
This International Standard is not applicable to the testing of aquatic samples from the estuarine or marine
environment.
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 5667-16:1998, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 6059, Water quality — Determination of the sum of calcium and magnesium — EDTA titrimetric method
ISO 10523, Water quality — Determination of pH
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
brood
group or cohort of sibling offspring, consisting of two or more neonates in any test container, during any given
day of the test, released from the adult female during an inter-moult period (i.e. before the carapace is shed by
that female during moulting)
3.2
brood organism
healthy adult female daphnid that produces and releases multiple broods of live neonates
3.3
control batch
series of replicates containing control solution (3.4)
NOTE In this International Standard, 10 replicates constitute the control batch.
3.4
control solution
mixture of test medium and of food without sample under test
3.5
effective concentration producing x % reproduction inhibition
EC
x
estimated concentration of a test sample giving rise to x % reproduction inhibition with respect to the
control batch (3.3), which represents a point of the test sample concentration that is estimated to cause a
designated percent impairment in a quantitative biological function
3.6
neonate
newly born or newly hatched individual
NOTE In this International Standard, a neonate is a first-instar daphnid, < 24 h old.
3.7
reproduction inhibition
comparison between the number of living offspring born from all adults at the end of the test between the
control batch (3.3) and the test batch (3.8)
3.8
test batch
series of replicates containing the same test solution (3.9)
NOTE In this International Standard, 10 replicates constitute a test batch.
3.9
test solution
mixture of test medium, of food and of sample under test
4 Principle
Ceriodaphnia dubia, less than 24 h old at the beginning of the test, are exposed individually to a range of
concentrations of the sample under test for a period of (7 ± 1) d. The test typically ends after 7 d when 60 % of
the control organisms have produced their third brood. The mortality of the adult females and their
reproduction are monitored throughout the exposure time. All other relevant biological parameters can also be
studied.
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SIST ISO 20665:2010
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The data obtained allow, using a suitable model, the calculation of the concentration which gives rise to x %
reproduction inhibition, EC , e.g. EC , EC or EC .
x 10 20 50
5 Test environment
Carry out the test in a temperature-controlled room or chamber at (25 ± 2) °C in the test containers. Ensure
that, within one test, the temperature does not vary by more than 2 °C.
Adjust the day/night test cycle (photoperiod) to 16 h of daylight and 8 h of darkness. In the test containers
(7.2), a range of lighting intensity at the air/water interface of 100 lx to 600 lx (7.8) is recommended. Do not
shake or aerate the test containers.
Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that
the test is being performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognised analytical grade, unless otherwise specified.
6.1 Test organisms
Ceriodaphnia dubia neonates are obtained by parthenogenesis from adult females for at least three
generations under the conditions of temperature, photoperiod and food identical to those in the test.
The Ceriodaphnia dubia used for the test shall be less than 24 h old and shall have been taken from a brood
comprising at least eight newly born animals.
The day before the test, isolate from the culture a dozen or more adults that are over 6 d and less than 14 d
old. Isolate each one in a separate container containing food (6.4.1 or 6.4.2) and test medium (6.3.2 or 6.3.3).
Before the test, remove the adults from their containers and count the offspring. Discard all vessels containing
less than eight live offspring.
The Ceriodaphnia dubia may also derive from the hatching of ephippia purchased from a specialised
1)
company . These organisms may be directly used as test organisms.
2)
6.2 Pure water, having a conductivity below 10 µS/cm .
6.3 Test media
6.3.1 General
Two test media are recommended: ELENDT M4 (6.3.2) or moderately hard water (6.3.3). Alternative test
media may be used as long as validity criteria (Clause 11) are met.
For alternative test media, supply either a reference to a publication, or for natural waters (in case of effluent
testing) the date of collection, details of storage, handling, and additions, as well as physical chemistry data
relating to major ions [Na(I), K(I), Ca(II), Mg(II), carbonates, chloride, sulfate], pH and dissolved organic
carbon.
1) Microbiotest, Deinze, Belgium, is an example of a supplier able to provide suitable ephippia commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.
2) 1 mS/m.
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6.3.2 ELENDT M4 medium option
Prepare ELENDT M4 test medium in accordance with Annex A. The test medium thus prepared shall have a
pH of 8,0 ± 0,3 (measured as specified in ISO 10523), a total hardness of (250 ± 20) mg/l (expressed as
CaCO and measured as specified in ISO 6059). Aerate the test medium until the dissolved oxygen
3
concentration has reached the air saturation value and until the pH has stabilised. If necessary, adjust the pH
to 8,0 ± 0,3 using a diluted solution of sodium hydroxide or hydrochloric acid.
NOTE On account of the high hardness of the test medium and of the presence of EDTA within this medium, the
bioavailability of the bivalent metal ions can be reduced, thus resulting in a decrease in the apparent toxicity of these ions.
6.3.3 Moderately hard water medium option
Prepare moderately hard water in accordance with Annex B. The test medium thus prepared shall have a pH
of 7,4 to 7,8 (measured as specified in ISO 10523) and a total hardness of (90 ± 10) mg/l (expressed as
CaCO and measured as specified in ISO 6059).
3
6.4 Food
6.4.1 Option 1: Fish food and two algae diet
3)
The food is composed of fish food, Chlorella vulgaris algae and Pseudokirchneriella subcapitata algae
[1]
(formerly known as Selenastrum capricornutum and Raphidocelis subcapitata) (see NF T90-376 and
Reference [2]).
4)
Prepare a 5 g/l suspension of fish food in the test medium (6.3.2), homogenised with a crusher or any other
means allowing particles of a few micrometers to be obtained. Prepare this suspension each day for the daily
feeding of cultures or during testing.
Grow the algae separately in any suitable medium (e.g. LC OLIGO, Annex C). Use them when the culture is in
6
the exponential growth phase and has reached a density greater than 5 × 10 cells per millilitre. These
cultures may be stored at (4 ± 3) °C, in darkness, for a maximum period of 10 d.
The following constituents should be added to each test solution (9.3) before the transfer of organisms:
6
a) 12 × 10 cells per litre of Chlorella vulgaris;
6
b) 6 × 10 cells per litre of Pseudokirchneriella subcapitata;
c) 500 µl per litre of the fish food suspension.
In any case, the quantity of food shall not constitute more than 10 % of the final volume of each container (9.3).
The use of Chlorella vulgaris and/or Pseudokirchneriella subcapitata algae immobilised in an inert matrix
5)
(gelose), in the form of algae beads is possible. In this case, after dissolving the matrix, centrifuge the algae,
discard the supernatant, and resuspend the algae by shaking in the test medium (6.3.2 or 6.3.3). Repeat this
operation a second time. The algal concentration in the test medium shall be the same as described above.
3) The Freshwater Biological Association, Ambleside, UK, is an example of a supplier able to provide these algae
commercially. This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of this supplier.
4) Sera Micron is 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.
5) Microbiotest, Deinze, Belgium, is an example of a supplier able to provide suitable algal beads commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.
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SIST ISO 20665:2010
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6)
6.4.2 Option 2: Yeast/Cerophyll /trout chow and one algae diet
A second food combination based on the US EPA, method 1002.0 (Reference [11], p. 141) and Environment
Canada (Reference [5]) test methods is recommended (see also References [3] and [4]). Daily feeding with
Yeast/Cerophyll/trout chow (YCT) and a single algal species is required for culturing and testing of
Ceriodaphnia. The algal species most commonly used is Pseudokirchneriella subcapitata.
The formula for preparing YCT is given in Annex D. If the YCT/one algae diet is used, mass cultures should
be fed at a rate of:
⎯ 7 ml algae concentrate per litre culture;
⎯ 7 ml YCT concentrate per litre culture.
Individual cultures should be fed at the rate of:
⎯ 0,1 ml algae concentrate per 15 ml culture;
⎯ 0,1 ml YCT concentrate per 15 ml culture.
Food should be added to fresh culture medium immediately before or after the transfer of organisms.
Thoroughly mix algal concentrate and YCT by shaking before dispensing. If the YCT is stored frozen, store
thawed aliquots at (4 ± 3) °C. Discard unused portions of unfrozen or thawed YCT after 2 weeks. Store
unused portions of algal concentrate at (4 ± 3) °C, in darkness, and discard after 10 d.
6.5 Reference substance
Sodium pentachlorophenolate (C Cl ONa), copper sulfate pentahydrate (CuSO ·5H O), sodium chloride
6 5 4 2
(NaCl), or zinc sulfate (ZnSO ) are acceptable.
4
CAUTION — If sodium pentachlorophenolate is used as a reference toxicant, the material safety data
sheet should be consulted prior to use by laboratory personnel due to the hazardous nature of this
substance.
7 Apparatus
Usual laboratory apparatus and in particular the following.
7.1 Temperature-controlled room, chamber or water bath.
7.2 Test containers, made from a chemically inert material.
If closed containers are used, make sure that the capacity is sufficient to allow for a gas phase/aqueous phase
volume ratio of 1:1.
Prior to use, rinse the containers with the test medium (6.3.2 or 6.3.3) or with pure water (6.2).
7.3 Device for the measurement of algal concentration, e.g. a microscope equipped with a
haemocytometer or a particle counter. Indirect methods (e.g. spectrophotometer, turbidimeter, fluorimeter) can
be used if an acceptable correlation with the cellular concentration can be established.
6) Cereal Grass Media – Cerophyll is 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.
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SIST ISO 20665:2010
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7.4 Pipette for sampling the Ceriodaphnia dubia, with a sufficient diameter for capturing the animals
while allowing sampling of only a small volume of medium.
7.5 Binocular magnifying glass, with a magnification of at least 8 times and, if possible, a continuous
magnification.
7.6 Image analysis system, to count and measure ceriodaphnids.
7.7 Membrane filtration device, with filters, 0,45 µm, 0,22 µm.
7)
7.8 Light source , providing a range of light intensity at the air/water interface in the test containers (7.2)
of 100 lx to 600 lx.
7.9 Sample collecting bottles, in accordance with ISO 5667-16:1998, 3.2.
7.10 Sieve, of nominal size of openings <100 µm.
8 Sampling and samples
Carry out sampling, transportation and storage of samples in accordance with the general procedures
specified in ISO 5667-16.
Collect samples in bottles made from chemically inert materials (7.9).
Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be
met, cool the sample to 0 °C to 4 °C and test it within 24 h. If it is not possible to perform the test within 72 h,
the sample may be frozen and maintained below −18 °C for testing within 2 months of collection, provided that
characteristics are known to be unaffected by freezing. At the time of testing, homogenise the sample to be
analysed by shaking manually, and, if necessary, allow to settle for 2 h in a container, and sample by drawing
off (use a pipette) the required quantity of supernatant, maintaining the end of the pipette in the centre of the
section of the test tube and half way between the surface of the deposited substances and the surface of the
liquid.
If the raw sample or the decanted supernatant is likely to interfere with the test (due to the presence of micro-
crustaceans, residual suspended matter, protozoa, micro-organisms, etc.), filter through a 0,45 µm membrane
filter (7.7) or centrifuge the raw or decanted sample.
The sample obtained by either of these methods is the sample submitted to testing. It is also used for the
renewal of test solutions. Store this sample in full containers at (4 ± 3) °C in the dark throughout the test
period.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in
ISO 5814) and record these values in the test report (Clause 13).
If the aim of the test is to assess the chronic toxicity without considering extreme pH effects, a second test
may also be carried out after adjustment of the pH value to 8,0 ± 0,3 with hydrochloric acid or sodium
hydroxide solutions. Proceed, if appropriate, as indicated above, for the separation of the suspended matter
formed following the adjustment of the pH. Mention any pH adjustment in the test report (Clause 13).
7) Grolux is 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.
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SIST ISO 20665:2010
ISO 20665:2008(E)
9 Procedure
9.1 Preparation of the stock solutions of substances to be tested
Prepare the stock solution of the test substance by dissolving a known quantity of substance in a specified
volume of test medium (6.3.2 or 6.3.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it may be prepared in advance and stored under these conditions.
For poorly soluble substances in the test medium, refer to the specifications of ISO 5667-16.
9.2 Selection of concentrations
The test shall comprise at least five concentrations of the sample to be tested (Clause 8 or 9.1), selected
within a geometric series with a separation factor not exceeding 3,2.
Take the following criterion into account for selecting the range of concentrations to be examined: to obtain an
EC value, it is desirable that at least one concentration higher by x % than this is used and at least one x %
x
lower.
Produce at least 10 replicates for each concentration. These replicates constitute a test batch (3.8).
Include in each test a control batch (3.3) without any sample to be tested. A control batch is also made up of
at least 10 replicates.
When using a solvent in order to dissolve or disperse the substances, the solvent concentration shall be the
same in all containers. Include a second control containing the solvent at the concentration being used in test
concentrations.
In the case of samples of waters, effluents, and aqueous extracts, the highest tested concentration cannot be
equal to a volume fraction of 100 % of the initial sample on account of the food supply corresponding to a
small percent of the volume of sample being used.
For the purpose of toxicant range-finding or single concentration screening purposes, the test may also be
carried out with a lesser number of concentrations, but in this case, an EC cannot always be estimated.
x
9.3 Preparation of the test and control solutions
Prepare the test solutions by mixing the appropriate volumes of the sample to be tested (Clause 8 or 9.1) or of
its initial dilution with test medium (6.3.2 or 6.3.3) and food (6.4.1 or 6.4.2).
If the fish food/two algae diet (6.4.1) is used for feeding during a test, the quantity of food contained in each
test container shall not be greater than one tenth of the total volume (i.e. 50 ml for a 500 ml test or control
solution).
In the case where the YCT/one algae diet (6.4.2) is used for feeding during a test, the quantity of food
contained in each test container shall be equal to 1,5 % of the total volume in the test container (i.e. 0,1 ml of
YCT and 0,1 ml of algae concentrate added per 15 ml volume).
In both cases, the volume of test and control solutions should be at least (15 ± 2) ml and not exceed
(50 ± 2) ml.
Split the test solution for the replicate containers of each concentration into volumes of (15 ± 2) ml to
(50 ± 2) ml. Prepare the control solutions by mixing the food (6.4.1 or 6.4.2) with the test medium (6.3.2 or
6.3.3). Split the control solution into volumes of approximately (15 ± 2) ml to (50 ± 2) ml in each control
container.
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9.4 Introduction of the organisms
Place the containers in a temperature-controlled room or chamber (7.1) to obtain a test and control solutions
temperature of (25 ± 2) °C.
As soon as this temperature is attained, introduce one Ceriodaphnia dubia aged less than 24 h (6.1) into each
container (7.2). Use a pipette (7.4) for the transfer, and release the crustaceans under the water surface.
9.5 Renewal of the test and control solutions
When the fish food/two algae diet (6.4.1) is used for feeding during a test, carry out renewal of the test and
control solutions (9.3) according to one of the timetables given in Table 1.
Table 1 — Test timetable for solution renewal (fish food/2 algae diet)
Timetable
a b c
Day 0 1 2 3 4 5 6 7 8
Renewal — yes or no no no or yes yes yes yes yes or no no
a
If the test starts on Thursday, renew test and control solutions; if it starts on Friday, do not renew solutions
b
If the test starts on Thursday, do not renew test and control solutions; if it starts on Friday renew test and control solutions
c
If the test is to continue to an 8th day, renew test and control solutions.
On the days when the timetable requires a renewal of the test and control solutions, transfer the adult females
from the old containers into new containers containing test and control solutions freshly prepared according to
9.3 (fresh food and medium) and bearing the same identification.
It is recommended that the transfer of the adult females take pla
...
NORME ISO
INTERNATIONALE 20665
Première édition
2008-12-15
Qualité de l'eau — Détermination de la
toxicité chronique vis-à-vis de
Ceriodaphnia dubia
Water quality — Determination of chronic toxicity to Ceriodaphnia dubia
Numéro de référence
ISO 20665:2008(F)
©
ISO 2008
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ISO 20665:2008(F)
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© ISO 2008
Droits de reproduction réservés. Sauf prescription différente, aucune partie de cette publication ne peut être reproduite ni utilisée sous
quelque forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie et les microfilms, sans l'accord écrit
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Publié en Suisse
ii © ISO 2008 – Tous droits réservés
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ISO 20665:2008(F)
Sommaire Page
Avant-propos. iv
Introduction . v
1 Domaine d'application. 1
2 Références normatives . 1
3 Termes et définitions. 2
4 Principe. 2
5 Environnement de l'essai. 3
6 Réactifs . 3
7 Appareillage . 6
8 Échantillonnage et échantillons. 6
9 Mode opératoire . 7
10 Expression des résultats . 10
11 Critères de validité. 11
12 Fidélité . 12
13 Rapport d'essai . 12
Annexe A (normative) Préparation du milieu ELENDT M4. 14
Annexe B (normative) Préparation du milieu «eau de dureté moyenne». 16
Annexe C (informative) Préparation du milieu de LC OLIGO. 17
Annexe D (normative) Mode opératoire pour la préparation à base de levures/Cerophyll/granulés
pour truite . 19
Annexe E (informative) Feuille de collecte de données . 21
Bibliographie . 22
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ISO 20665:2008(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée
aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du
comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,
Partie 2.
La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur
publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne
pas avoir identifié de tels droits de propriété et averti de leur existence.
L'ISO 20665 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité SC 5,
Méthodes biologiques.
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ISO 20665:2008(F)
Introduction
La mise en évidence d'effets néfastes pour la qualité de l'eau passe, depuis plusieurs années, par la
réalisation d'essais biologiques. Les cladocères, Ceriodaphnia dubia, sont reconnus comme étant
représentatifs du zooplancton largement utilisé lors des essais de toxicité aquatique.
La brièveté de l'essai de toxicité chronique, (7 ± 1) jours, et les faibles volumes utilisés sont des atouts
majeurs pour l'obtention de résultats appropriés sur des échantillons qui sont susceptibles de subir des
modifications pendant la période de conservation.
Il convient que l'utilisateur soit conscient du fait que des problèmes particuliers peuvent nécessiter la
spécification de conditions marginales supplémentaires.
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NORME INTERNATIONALE ISO 20665:2008(F)
Qualité de l'eau — Détermination de la toxicité chronique vis-à-
vis de Ceriodaphnia dubia
AVERTISSEMENT — Il convient que l'utilisateur de la présente Norme internationale connaisse bien
les pratiques courantes de laboratoire. La présente Norme internationale n'a pas pour but de traiter
tous les problèmes de sécurité qui sont, le cas échéant, liés à son utilisation. Il incombe à l'utilisateur
d'établir des pratiques appropriées en matière d'hygiène et de sécurité et de s'assurer de la
conformité à la réglementation nationale en vigueur.
IMPORTANT — Il est absolument essentiel que les essais conduits conformément à la présente
Norme internationale soient effectués par du personnel ayant reçu une formation adéquate.
1 Domaine d'application
La présente Norme internationale spécifie une méthode de détermination de la toxicité chronique vis-à-vis de
Ceriodaphnia dubia (Cladocera, Crustacea), basée sur une inhibition de la reproduction après (7 ± 1) jours.
Cette méthode est applicable:
a) aux substances chimiques solubles ou pouvant être maintenues en suspension ou en dispersion stables
dans les conditions de l'essai;
b) aux effluents industriels ou aux eaux usées, le cas échéant, après décantation, filtration ou centrifugation;
c) aux eaux douces;
d) aux extraits aqueux.
La présente Norme internationale n'est pas applicable à la réalisation d'essais sur des échantillons aqueux
provenant d'un milieu marin ou estuarien.
2 Références normatives
Les documents de référence suivants sont indispensables pour l'application du présent document. Pour les
références datées, seule l'édition citée s'applique. Pour les références non datées, la dernière édition du
document de référence s'applique (y compris les éventuels amendements).
ISO 5667-16:1998, Qualité de l'eau — Échantillonnage — Partie 16: Lignes directrices pour les essais
biologiques des échantillons
ISO 5814, Qualité de l'eau — Dosage de l'oxygène dissous — Méthode électrochimique à la sonde
ISO 6059, Qualité de l'eau — Dosage de la somme du calcium et du magnésium — Méthode titrimétrique à
l'EDTA
ISO 10523, Qualité de l'eau — Détermination du pH
ISO/TS 20281, Qualité de l'eau — Lignes directrices relatives à l'interprétation statistique de données
écotoxicologiques
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ISO 20665:2008(F)
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
3.1
portée
groupe ou cohorte de nouveau-nés, composé de deux nouveau-nés (ou plus) dans un récipient d'essai
pendant un jour donné de la période d'essai engendrés par la femelle adulte entre deux périodes de mue
(c'est-à-dire avant que la carapace ne soit rejetée par ladite femelle pendant la mue)
3.2
organisme mature
daphnie femelle adulte, saine qui produit et engendre de multiples portées de nouveau-nés vivants
3.3
lot témoin
série de répliques contenant la solution témoin (3.4)
NOTE Dans la présente Norme internationale, 10 répliques constituent le lot témoin.
3.4
solution témoin
mélange de milieu d'essai et de nourriture sans l'échantillon soumis à essai
3.5
concentration effective produisant x % d'inhibition de la reproduction
CE
x
concentration estimée d'un échantillon pour essai produisant x % d'inhibition de la reproduction (3.7) par
rapport au lot témoin (3.3), ce qui représente un point de la concentration de l'échantillon pour essai qui
selon les estimations provoque un pourcentage donné de réduction d'une fonction biologique de caractère
quantitatif
3.6
nouveau-né
nouveau-né ou individu récemment éclos
NOTE Dans la présente Norme internationale, un nouveau-né est une daphnie de premier stade, âgée de moins de
24 h.
3.7
inhibition de la reproduction
comparaison portant sur le nombre de jeunes vivants engendrés par l'ensemble des adultes à la fin de l'essai
entre le lot d'essai (3.8) et le lot témoin (3.3)
3.8
lot d'essai
série de répliques contenant la même solution d'essai (3.9)
NOTE Dans la présente Norme internationale, 10 répliques constituent un lot d'essai.
3.9
solution d'essai
mélange constitué du milieu d'essai, de nourriture et de l'échantillon soumis à essai
4 Principe
De jeunes Ceriodaphnia dubia, âgées de moins de 24 h au début de l'essai sont exposées individuellement, à
une gamme de concentrations de l'échantillon soumis à essai pendant une période de (7 ± 1) jours. En
général, l'essai se termine après 7 jours une fois que 60 % des organismes témoins ont engendré leur
2 © ISO 2008 – Tous droits réservés
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ISO 20665:2008(F)
troisième portée. La mortalité des femelles adultes et leur reproduction sont contrôlées pendant toute la durée
d'exposition. Tout autre paramètre biologique pertinent peut également être étudié.
Les données obtenues permettent, en utilisant un modèle approprié, de calculer la concentration qui provoque
x % d'inhibition de la reproduction (CE ), par exemple CE , CE ou CE .
x 10 20 50
5 Environnement de l'essai
Réaliser l'essai dans une enceinte ou un local thermostaté permettant d'obtenir (25 ± 2) °C dans les récipients
d'essai. S'assurer qu'au cours d'un même essai la température ne varie pas plus de 2 °C.
Régler le cycle jour/nuit (photopériode) sur 16 h de jour et 8 h d'obscurité. Il est recommandé d'avoir un
éclairage (7.8) d'une intensité comprise dans une gamme de 100 lx à 600 lx au niveau de l'interface air/eau
dans les récipients d'essai (7.2). Ne pas agiter ni aérer les récipients d'essai.
Maintenir l'atmosphère exempte de vapeurs ou poussières toxiques. L'emploi de solutions témoins permet de
confirmer que l'essai se déroule bien dans une atmosphère exempte de vapeurs ou poussières toxiques.
6 Réactifs
Utiliser uniquement des réactifs de qualité analytique reconnue, sauf spécification contraire.
6.1 Organismes pour essai
Les Ceriodaphnia dubia nouveau-nées sont obtenues par parthénogenèse à partir de femelles adultes, sur au
moins trois générations dans les conditions de température, de photopériode et de nourriture identiques à
celles de l'essai.
Les Ceriodaphnia dubia utilisées pour l'essai doivent être âgées de moins de 24 h et doivent être issues d'une
portée comprenant au moins huit animaux nouvellement nés.
La veille de l'essai, isoler à partir de l'élevage une douzaine d'adultes (ou plus) âgés de plus de 6 jours et de
moins de 14 jours. Isoler chacun dans un récipient distinct contenant de la nourriture (6.4.1 ou 6.4.2) et du
milieu d'essai (6.3.2 ou 6.3.3). Avant l'essai, retirer les adultes des récipients et dénombrer la progéniture.
Éliminer tous les récipients contenant moins de huit jeunes vivants.
Les Ceriodaphnia dubia peuvent aussi provenir de l'éclosion d'éphippies achetées auprès d'une société
1)
spécialisée . Ces organismes peuvent être utilisés directement comme organismes pour essai.
2)
6.2 Eau pure, eau de conductivité inférieure à 10 µS/cm .
6.3 Milieu d'essai
6.3.1 Généralités
Deux milieux d'essai sont recommandés: ELENDT M4 (6.3.2) ou une eau de dureté moyenne (6.3.3). Il est
possible d'utiliser d'autres milieux d'essai à condition que les critères de validité (Article 11) soient satisfaits.
1) Les éphippies fournies par MicroBiotests, Mariakerke (Gent), Belgique sont un exemple de produit approprié
disponible sur le marché. Cette information est donnée à l'intention des utilisateurs de la présente Norme internationale et
ne signifie nullement que l'ISO approuve ou recommande l'emploi exclusif des produits de ce fournisseur.
2) 1 mS/m.
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ISO 20665:2008(F)
Dans ce dernier cas, fournir la référence d'une publication ou, pour des eaux naturelles (dans le cas d'essai
sur effluents), la date de prélèvement, le type de conservation, le mode de manipulation, les ajouts effectués
et les caractéristiques physico-chimiques concernant les principaux ions [Na(I), K(I), Ca(II), Mg(II), carbonates,
chlorure, sulfate) ainsi que le pH et le carbone organique dissous (COD).
6.3.2 Option: Milieu ELENDT M4
Préparer le milieu d'essai ELENDT M4 conformément à l'Annexe A. Le milieu d'essai, ainsi préparé, doit avoir
un pH de 8,0 ± 0,3 (mesuré comme spécifié dans l'ISO 10523) et une dureté totale de (250 ± 20) mg/l
(exprimée en CaCO et mesurée comme spécifié dans l'ISO 6059). Aérer le milieu d'essai jusqu'à ce que la
3
concentration en oxygène dissous ait atteint la valeur de saturation dans l'air et jusqu'à stabilisation du pH. Si
nécessaire, ajuster le pH à 8,0 ± 0,3 à l'aide d'une solution diluée d'hydroxyde de sodium ou d'acide
chlorhydrique.
NOTE Étant donné la valeur élevée de la dureté du milieu d'essai et la présence d'EDTA dans ce milieu, la
biodisponibilité des ions métalliques bivalents peut être réduite, entraînant de ce fait une diminution de la toxicité
apparente de ces ions.
6.3.3 Option: Milieu «eau de dureté moyenne»
Préparer l'eau de dureté moyenne conformément à l'Annexe B. Le milieu d'essai, ainsi préparé, doit avoir un
pH compris entre 7,4 et 7,8 (mesuré comme spécifié dans l'ISO 10523) et une dureté totale de (90 ± 10) mg/l
et mesurée comme spécifié dans l'ISO 6059).
(exprimée en CaCO
3
6.4 Nourriture
6.4.1 Option 1: Alimentation à base de nourriture pour poissons et de deux espèces d'algues
La nourriture est composée de nourriture pour poissons et des algues Chlorella vulgaris et Pseudo-
3)
kirchneriella subcapitata (dénommées auparavant Selenastrum capricornutum et Raphidocelis subcapitata)
[1]
(voir NF T90-376 et Référence [2]).
4)
Préparer une suspension de nourriture pour poissons à 5 g/l dans le milieu d'essai (6.3.2), homogénéisée au
broyeur ou avec tout autre moyen permettant l'obtention de particules de quelques micromètres. Préparer
cette suspension chaque jour pour nourrir les élevages, ou durant les essais.
Cultiver les algues séparément dans tout milieu approprié (par exemple LC OLIGO, Annexe C). Les utiliser
lorsque la culture est en phase exponentielle de croissance et qu'elle a atteint une densité supérieure à
6
5 × 10 cellules par millilitre. Ces cultures peuvent être conservées à (4 ± 3) °C à l'abri de la lumière, pendant
une durée maximale de 10 jours.
Il convient d'ajouter les constituants suivants à chaque solution d'essai (9.3) avant l'introduction des
organismes:
6
a) 12 × 10 cellules par litre de Chlorella vulgaris;
6
b) 6 × 10 cellules par litre de Pseudokirchneriella subcapitata;
c) 500 µl par litre de la suspension de nourriture pour poissons.
3) Les algues fournies par Freshwater Biological Association, Ambleside, Royaume-Uni, sont un exemple de produit
approprié disponible sur le marché. Cette information est donnée à l'intention des utilisateurs de la présente Norme
internationale et ne signifie nullement que l'ISO approuve ou recommande l'emploi exclusif des produits de ce fournisseur.
4) «Sera micron» est un exemple de produit approprié disponible sur le marché. Cette information est donnée à
l'intention des utilisateurs de la présente Norme internationale et ne signifie nullement que l'ISO approuve ou recommande
l'emploi exclusif du produit ainsi désigné.
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ISO 20665:2008(F)
Dans tous les cas, la quantité de nourriture ne doit pas constituer plus de 10 % du volume final de chaque
récipient (9.3).
L'utilisation des algues Chlorella vulgaris et/ou Pseudokirchneriella subcapitata immobilisées dans une
5)
matrice inerte (gélose), sous la forme de billes d'algues est possible. Dans ce cas, après dissolution de la
matrice, centrifuger les algues, éliminer le surnageant et remettre les algues en suspension par agitation dans
du milieu d'essai (6.3.2 ou 6.3.3). Recommencer cette opération une seconde fois. La concentration algale
dans le milieu d'essai doit être la même que celle décrite ci-dessus.
6)
6.4.2 Option 2: Alimentation à base de levures/Cerophyll /granulés pour truite et d'une seule algue
Une seconde combinaison d'aliments basée sur la méthode d'essai US EPA méthode 1002.0 (Référence [11],
p. 141) et les méthodes d'essai Environment Canada, (Référence [5]) est recommandée (voir aussi
Références [3] et [4]). Une alimentation quotidienne à base de levures/Cerophyll/granulés pour truite et une
seule espèce d'algue est nécessaire pour l'élevage de Ceriodaphnia et les essais auxquels celles-ci sont
soumises. L'espèce algale la plus communément utilisée est Pseudokirchneriella subcapitata.
La formule utilisée pour la préparation à base de levures/Cerophyll/granulés pour truite est donnée dans
l'Annexe D. Si l'on choisit l'alimentation levures/Cerophyll/granulés pour truite et une seule algue, il convient
de fournir la nourriture aux élevages de masse selon les quantités suivantes:
⎯ 7 ml d'algues concentrées par litre de culture;
⎯ 7 ml de levures/Cerophyll/granulés pour truite concentrés par litre de culture.
Il convient d'alimenter les cultures individuelles selon les quantités suivantes:
⎯ 0,1 ml d'algues concentrées pour 15 ml de culture;
⎯ 0,1 ml de levures/Cerophyll/granulés pour truite concentrés pour 15 ml de culture.
Il convient d'ajouter la nourriture au milieu de culture frais immédiatement avant ou après l'introduction des
organismes.
Bien mélanger le concentré algal et la préparation de levures/Cerophyll/granulés pour truite en l'agitant avant
la distribution. Si la préparation de levures/Cerophyll/granulés pour truite est conservée à l'état congelé,
conserver des aliquotes décongelées à (4 ± 3) °C. Éliminer les portions inutilisées de la préparation de
levures/Cerophyll/granulés pour truite non congelées ou décongelées après deux semaines. Conserver les
volumes inutilisés de concentré algal à (4 ± 3) °C à l'abri de la lumière, puis les éliminer au bout de 10 jours.
6.5 Substance de référence
Le pentachlorophénate de sodium (C Cl ONa), le sulfate de cuivre pentahydraté (CuSO , 5H O), le chlorure
6 5 4 2
de sodium (NaCl) ou le sulfate de zinc (ZnSO ) sont acceptables.
4
ATTENTION — En cas d'utilisation du pentachlorophénate de sodium en tant que toxique de référence,
il convient de consulter sa fiche de données de sécurité avant tout usage par le personnel du
laboratoire, en raison de la nature dangereuse de cette substance.
5) Les billes d'algues fournies par Microbiotest, Deinze, Belgique sont un exemple de produit approprié disponible sur le
marché. Cette information est donnée à l'intention des utilisateurs de la présente Norme internationale et ne signifie
nullement que l'ISO approuve ou recommande l'emploi exclusif des produits de ce fournisseur.
6) Cereal Grass Media — Cerophyll est un exemple de produit approprié disponible sur le marché. Cette information est
donnée à l'intention des utilisateurs de la présente Norme internationale et ne signifie nullement que l'ISO approuve ou
recommande l'emploi exclusif du produit ainsi désigné.
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ISO 20665:2008(F)
7 Appareillage
Matériel courant de laboratoire et en particulier ce qui suit:
7.1 Local, enceinte ou bain-marie thermostaté.
7.2 Récipients d'essai, en matériau chimiquement inerte.
En cas d'utilisation de récipients fermés, s'assurer que la capacité est suffisante pour que le rapport phase
gazeuse/phase aqueuse soit de 1:1.
Avant utilisation, rincer les récipients avec le milieu d'essai (6.3.2 ou 6.3.3) ou avec de l'eau pure (6.2).
7.3 Dispositif permettant de mesurer la concentration algale, par exemple microscope équipé d'un
hématimètre ou d'un compteur de particules. Des méthodes indirectes (par exemple spectrophotomètre,
turbidimètre, fluorimètre) peuvent être utilisées si une corrélation acceptable avec la concentration cellulaire
peut être établie.
7.4 Pipette de prélèvement de Ceriodaphnia dubia, de diamètre suffisant pour capturer les animaux tout
en permettant de ne prélever qu'un faible volume de milieu.
7.5 Loupe binoculaire, grossissant 8 fois (ou plus) et, si possible, permettant d'obtenir un grossissement
continu.
7.6 Système d'analyse d'image, pour compter et mesurer les cériodaphnies.
7.7 Dispositif de filtration sur membranes, avec filtres de 0,45 µm, 0,22 µm.
7)
7.8 Éclairage , capable de fournir une intensité lumineuse comprise dans une gamme de 100 lx à 600 lx
au niveau de l'interface air/eau dans les récipients d'essai (7.2).
7.9 Flacons de collecte d'échantillons, conformes à l'ISO 5667-16:1998, 3.2.
7.10 Tamis, de dimension nominale d'ouverture <100 µm.
8 Échantillonnage et échantillons
Procéder à l'échantillonnage, au transport et à la conservation des échantillons conformément aux modes
opératoires généraux spécifiés dans l'ISO 5667-16.
Recueillir les échantillons dans des flacons en matériaux chimiquement inertes (7.9).
Procéder à l'essai de toxicité dès que possible, idéalement dans les 12 h suivant la collecte des échantillons.
Si cet intervalle de temps ne peut pas être respecté, refroidir l'échantillon entre 0 °C et 4 °C et soumettre
l'échantillon à l'essai dans les 24 h. S'il n'est pas possible de réaliser l'essai dans les 72 h, l'échantillon peut
être congelé et maintenu à une température inférieure à −18 °C, en vue d'effectuer l'essai dans les 2 mois
après le prélèvement à condition de s'assurer que ses caractéristiques ne sont pas altérées par la congélation.
Au moment de l'essai, homogénéiser l'échantillon à analyser par agitation manuelle et, si nécessaire, laisser
décanter pendant 2 h dans un récipient; prélever (à la pipette) la quantité requise de surnageant en
maintenant l'extrémité de la pipette au centre de la section de l'éprouvette et à mi-distance entre la surface du
précipité et la surface du liquide.
7) Grolux est un exemple de produit approprié disponible sur le marché. Cette information est donnée à l'intention des
utilisateurs de la présente Norme internationale et ne signifie nullement que l'ISO approuve ou recommande l'emploi
exclusif du produit ainsi désigné.
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ISO 20665:2008(F)
Dans le cas où l'échantillon brut ou le surnageant de décantation est susceptible de perturber l'essai (du fait
de la présence de microcrustacés, de matières en suspension résiduelles, de protozoaires, de
micro-organismes, etc.), filtrer sur membrane filtrante de 0,45 µm (7.7) ou centrifuger l'échantillon brut ou
décanté.
L'échantillon obtenu par l'une ou l'autre de ces méthodes est l'échantillon soumis à essai. Il est également
utilisé pour le renouvellement des solutions d'essai. Conserver cet échantillon dans des récipients
complètement remplis à (4 ± 3) °C dans l'obscurité pendant toute la période d'essai.
Mesurer le pH (comme spécifié dans l'ISO 10523) et de la concentration en oxygène dissous (comme spécifié
dans l'ISO 5814) et enregistrer ces valeurs dans le rapport d'essai (Article 13).
Si l'objectif visé consiste à évaluer la toxicité chronique sans tenir compte des effets de pH extrêmes, il est
également possible d'effectuer un second essai après ajustement du pH à 8,0 ± 0,3 avec des solutions
d'acide chlorhydrique ou d'hydroxyde de sodium. Procéder s'il y a lieu, comme indiqué ci-dessus pour la
séparation des matières en suspension formées à la suite de l'ajustement du pH. Mentionner tout ajustement
du pH dans le rapport d'essai (Article 13).
9 Mode opératoire
9.1 Préparation des solutions mères des substances soumises à essai
Préparer la solution mère de la substance soumise à essai par dissolution d'une quantité connue de
substance dans un volume spécifié de milieu d'essai (6.3.2 ou 6.3.3) au moment de l'emploi. Toutefois, si la
solution mère de la substance est stable dans certaines conditions, elle peut être préparée à l'avance et
conservée dans lesdites conditions.
Pour les substances faiblement solubles dans le milieu d'essai, se reporter aux spécifications de
l'ISO 5667-16.
9.2 Choix des concentrations
L'essai doit comprendre au moins cinq concentrations de l'échantillon soumis à essai (Article 8 ou 9.1), avec
un pas de raison géométrique inférieur ou égal à 3,2 entre chaque concentration.
Tenir compte du critère suivant pour choisir les séries de concentrations à examiner: pour obtenir une valeur
de CE , il est souhaitable que cette valeur soit encadrée par au moins deux concentrations, l'une induisant un
x
effet supérieur à x % et l'autre un effet inférieur à x %.
Réaliser au moins dix répliques pour chaque concentration. Ces répliques constituent un lot d'essai (3.8).
Inclure dans chaque essai un lot témoin (3.3) sans échantillon pour essai. Un lot témoin est également
constitué d'au moins dix répliques.
Lorsqu'un solvant est utilisé pour mettre en solution ou disperser les substances, la concentration en solvant
doit être la même dans tous les récipients. Inclure un second lot témoin contenant le solvant à la
concentration utilisée dans les concentrations d'essai.
S'il s'agit d'échantillons d'eaux, d'effluents, et d'extraits aqueux, la plus forte concentration soumise à essai ne
peut pas être égale à une fraction volumique de 100 % de l'échantillon initial du fait que l'apport de nourriture
correspond à un faible pourcentage du volume d'échantillon utilisé.
Pour l'établissement de la gamme de toxicité ou dans un objectif de criblage à une concentration unique,
l'essai peut également être réalisé avec un nom
...
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