ISO 14442:2006
(Main)Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water
Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water
ISO 14442:2006 provides procedures, not covered by the methods described in ISO 8692 and ISO 10253, for testing difficult substances for inhibition of algal growth. The main subjects covered by ISO 14442:2006 are the methods for preparing the test substance for testing and the procedures needed to carry out an appropriate test. The following test substances are covered by this guideline: poorly soluble pure organic compounds; poorly soluble mixtures of organic substances; poorly soluble inorganic materials; volatile substances; waste waters and environmental samples containing water and sediments; coloured and/or turbid samples; compounds of heavy metals. The following methods of addition are covered: direct; dispersion; water-soluble and water-accommodated fractions. Some guidelines related to the analytical procedures and to the interpretation of the results have been included.
Qualité de l'eau — Lignes directrices pour essais d'inhibition de la croissance algale avec des matières peu solubles, des composés volatils, des métaux et des eaux résiduaires
L'ISO 14442:2006 fournit des modes opératoires, non couverts par les méthodes décrites dans l'ISO 8692 et l'ISO 10253, applicables aux essais d'inhibition de la croissance algale impliquant des substances difficiles. Les principaux sujets traités dans l'ISO 14442:2006 sont les méthodes de préparation de la substance d'essai ainsi que les modes opératoires nécessaires pour effectuer ces essais de façon appropriée. Ces lignes directrices sont applicables aux substances d'essai suivantes: les composés organiques purs peu solubles; les mélanges de substances organiques peu solubles; les matériaux inorganiques peu solubles; les substances volatiles; les eaux résiduaires et les échantillons environnementaux contenant de l'eau et des sédiments; les échantillons colorés et/ou turbides; les composés de métaux lourds. Les méthodes d'addition suivantes sont couvertes: directe; dispersion; fractions en suspension dans l'eau et fractions solubles dans l'eau. Certaines lignes directrices traitant des modes opératoires d'analyse et de l'interprétation des résultats sont également incluses.
Kakovost vode - Smernice za preskuse zaviranja rasti alg s slabo topnimi materiali, hlapnimi spojinami, kovinami in odpadno vodo
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INTERNATIONAL ISO
STANDARD 14442
Second edition
2006-04-01
Water quality — Guidelines for algal
growth inhibition tests with poorly
soluble materials, volatile compounds,
metals and waste water
Qualité de l'eau — Lignes directrices pour essais d'inhibition de la
croissance algale avec des matières peu solubles, des composés
volatils, des métaux et des eaux résiduaires
Reference number
ISO 14442:2006(E)
©
ISO 2006
---------------------- Page: 1 ----------------------
ISO 14442:2006(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.
© ISO 2006
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2006 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 14442:2006(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 2
3 Analytical characterization of test materials and confirmation of concentrations and
stability . 2
4 Poorly soluble organic substances . 3
4.1 General. 3
4.2 Preparation of saturated and supersaturated solutions. 3
4.3 Solvent addition. 4
4.4 Dispersion using an emulsifying agent. 5
4.5 Interference with algal growth and its measurement. 5
5 Poorly soluble mixtures of organic substances. 6
5.1 General. 6
5.2 Preparation of test media. 6
5.3 Test performance. 7
6 Poorly soluble inorganic materials. 7
7 Volatile substances . 8
7.1 General. 8
7.2 Test system and growth medium. 8
7.3 Test performance. 9
7.4 Interference with algal growth. 9
8 Waste waters and environmental aqueous samples . 9
9 Coloured and/or turbid samples . 10
10 Metals and metal compounds . 11
10.1 Introduction . 11
10.2 Modification of algal growth inhibition test procedures for testing materials containing
heavy metals . 11
11 pH buffering. 12
12 Interpretation of the results . 13
Bibliography . 14
© ISO 2006 – All rights reserved iii
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ISO 14442:2006(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 14442 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
This second edition cancels and replaces the first edition (ISO 14442:1999), which has been technically
revised.
iv © ISO 2006 – All rights reserved
---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD ISO 14442:2006(E)
Water quality — Guidelines for algal growth inhibition tests with
poorly soluble materials, volatile compounds, metals and waste
water
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 standard be carried
out by suitably trained staff.
1 Scope
This International Standard provides procedures, not covered by the methods described in ISO 8692 and
ISO 10253, for testing difficult substances for inhibition of algal growth.
The main subjects covered by the guideline are the methods for preparing the test substance for testing and
the procedures needed to carry out an appropriate test. The following test substances are covered by this
guideline:
a) poorly soluble pure organic compounds;
b) poorly soluble mixtures of organic substances;
c) poorly soluble inorganic materials;
d) volatile substances;
e) waste waters and environmental samples containing water and sediments;
f) coloured and/or turbid samples;
g) compounds of heavy metals.
The following methods of addition are covered:
⎯ direct;
⎯ dispersion;
⎯ water-soluble and water-accommodated fractions.
Some guidelines related to the analytical procedures and to the interpretation of the results have been
included.
References to documents describing the background for the testing of difficult substances are given in the
Bibliography.
© ISO 2006 – All rights reserved 1
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ISO 14442:2006(E)
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 8692, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
3 Analytical characterization of test materials and confirmation of concentrations
and stability
Analytical characterization of test substances and materials and the confirmation of their concentrations and
stability in the testing environment is of major concern of regulatory authorities. Such activities are usually not
an integral part of this International Standard algal growth inhibition test methods.
However, there may be situations where analysis may assist in defining the appropriate exposure conditions
of test materials and chemicals and/or in the interpretation of the results.
The relevant properties of substances and materials can be assessed from basic properties such as solubility
in water, partition coefficient (lg P ), Henry's constant, photochemical and hydrolytic stability and
ow
biodegradability.
Analytical confirmation is strongly recommended in order to confirm test substance concentrations and is
required for the calculation of effective concentration (EC) values of volatile substances (Clause 7). If losses
due to adsorption on the test vessels or during transfer of test solutions and media occur, then analytical
confirmation are of particular importance. This aspect is also specified in ISO 5667-16.
Due to the batch test system used for algal growth inhibition tests, loss of substances due to biodegradation
(nearly all algal cultures contain bacteria), photodegradation, hydrolysis and/or adsorption cannot always be
avoided. A decrease in measured concentrations is difficult to prevent by technical means, and is therefore
considered acceptable for algal growth inhibition tests.
The following precautions are suggested for maintaining test substance concentrations in algal growth
inhibition tests:
a) sterilization of media and equipment to reduce the effect of bacterial growth;
b) change of the light quality to prevent photodegradation of test substances;
c) avoidance of contact of test substance with water prior to testing to reduce hydrolytic decomposition;
d) treatment of glassware (e.g. silanization); the effectiveness of such a treatment varies from one chemical
to the other;
e) pre-conditioning of the glassware, before addition of the test media, with the test substance at
concentrations to be used in the test.
The effect of such technical measures is, if relevant and if possible, monitored by chemical analysis.
Water, waste water and organic/inorganic solids/liquids may contain components that may modify the
composition of the algal growth medium (by precipitation of a limiting nutrient, complexation of essential
elements, addition of nutrients), and subsequently may cause effects on algal growth not related to toxic
2 © ISO 2006 – All rights reserved
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ISO 14442:2006(E)
components. If such problems occur, it may be advisable to determine the content of key components of the
test material. Some relevant components are: calcium, magnesium, sodium, potassium, sulfate, chloride,
ammonium, nitrate, phosphate, copper, cobalt, nickel, zinc, cadmium, organic matter [i.e. measured as
Chemical Oxygen Demand (COD) and/or Total Organic Carbon (TOC)].
If the material contains a high concentration of readily degradable organic material, the subsequent bacterial
growth may disturb the algal growth measurement. When untreated (not filtered or centrifuged) waste water is
tested, contamination with other algal species may occur.
4 Poorly soluble organic substances
4.1 General
A pure substance is a substance with one major component containing minor components as impurities.
Poorly soluble substances are those with solubility limits below 100 mg/l in water. If, however, growth
inhibition occurs at concentrations much lower than the solubility limits in water or algal growth medium (the
limit in the medium may be different), then the poorly soluble substance can be tested as a water soluble
substance (added via a stock solution in test medium). This approach is usually not applicable to substances
with a water solubility below 1 mg/l to 10 mg/l (substances with a very low solubility).
The methods described in this clause therefore refer to testing of substances causing effects on algal growth
at concentrations at or around the solubility limit in water and to very low solubility substances.
Testing of nominal concentrations markedly above the solubility limit is not recommended. It may, however, be
unavoidable if the solubility limit in water or algal growth medium (which may be different) is not well
established, or if a substance spontaneously forms dispersions in the test medium.
NOTE Terminology according to Reference [3]:
⎯ water solubility below 100 mg/l: “sparingly soluble”;
⎯ water solubility below 1 mg/l: “very low solubility”.
A number of methods which are available for preparing test solutions of pure substances, are described in
ISO 5667-16. Generally, it is preferred to use mechanical means to prepare stock solutions.
4.2 Preparation of saturated and supersaturated solutions
If the solubility of a substance in water is between 1 mg/l and 100 mg/l, saturated solutions can be prepared
by direct addition of the test substance. A saturated solution is usually prepared by stirring (e.g. magnetic
stirrer or shaking, see also 5.1) an excess amount of the test substance in water for a period in test medium. A
period of 20 h is practical for most substances, but a stirring period of up to three days may be considered to
ensure saturation provided the substance is stable. Lengthy stirring should be carried out in the dark and in
the same temperature range as the growth inhibition test is carried out. Preferably, the equilibrium should be
confirmed by chemical analysis. After a phase separation period of varying length, the clear phase is collected
and tested as the highest concentration. Filtration (through a 0,45 µm membrane filter) or centrifugation may
be useful for removing particulate matter.
Certain membrane filters may interfere with the test substance. The type of filter should be chosen according
to the physico-chemical properties of the test substance and the recommendations of the filter supplier.
Further test concentrations can be prepared by dilution of the saturated solution with test medium. A small
volume of a concentrated suspension of algal culture is then added to the test media to start the test.
A disadvantage of preparing saturated solutions in this way is that trace impurities in the test substance may
be preferentially enhanced in the solution, if they are more soluble than the major component. For this reason,
the quantity of test substance should be the minimum required ensuring that a saturated solution of the test
substance can be achieved.
© ISO 2006 – All rights reserved 3
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ISO 14442:2006(E)
Where possible, prepare stable supersaturated stock solutions with a substance (i.e. stock solution
concentrations in the range 2 to 10 times the saturation value) in test medium by high speed mechanical
1)
stirring [e.g. a high speed blender ] or ultrasonic treatment (a recommended frequency of 20 kHz and a
power output of at least 60 W) for a few minutes to several hours. With both methods, a constant temperature
shall be maintained during the treatment by cooling. If phase separation takes place immediately after the
treatment has ended, one may choose to remove (sinking or floating) particles by filtration through a paper
2)
filter [e.g. Schleicher & Schüll 604 ] or by centrifugation. If dissolved substances are removed by filtration, it
is essential to confirm the actual concentrations in the final solution by chemical analysis. The test solutions
can be prepared by dilution of the supersaturated stock solution.
4.3 Solvent addition
The use of a solvent as a carrier to add a substance to a test medium is considered to be a practical and
convenient method for handling organic substances tested at concentrations below 10 mg/l. The
recommended concentration of solvent does not influence the solubility of substances but assists in a rapid
and complete mixing of substances and test medium.
At concentrations of the test substance below 1 mg/l the solvent addition may be combined with the methods
described in 4.2 to prepare saturated solutions (which are further treated as described in 4.2).
In principle, any organic solvent can be used that meets the following criteria:
a) does not inhibit the algal growth at the highest concentration added;
b) is soluble in water at the recommended concentration;
c) does not interact with medium components;
d) does not react with the test substance;
e) does not biodegrade rapidly;
f) does not interfere with the conditions of illumination.
The concentration of a solvent should not exceed 100 µl/l test medium according to ISO 10253 and ISO 8692.
In practice, this solvent concentration for algal tests can be obtained by the addition of 10 µl solvent per
100 ml of test medium, a solvent volume that can be added with precision. Solvents such as acetone and t-
butanol have been demonstrated to meet most of the stated criteria in algal growth inhibition tests. t-Butanol
however is the less biodegradable one. Dimethylsulfoxide (DMSO) is a very efficient solvent, but might in
some cases interact more easily with test substances and the test organism. Tests have shown that none of
the solvents alone has any effect on algal growth up to a concentration of at least 1 ml/l (Reference [3]). In
exceptional cases, higher solvent concentrations can be used to add higher concentrations of the test
substance than possible with 100 µl/l.
It is recommended that a concentration series is prepared in the selected solvent, and that aliquots of the
stock solutions are added to the test flasks, which already contain the algae and the test medium. Controls
with and without the solvent shall be added to the test concentration series. The solvent concentration should
be the same for all test solutions.
The solvent control group is the appropriate control group for comparisons with treated groups. Each group
shall have the same solvent concentration as the control. For a bioassay in which a solvent is used in
conjunction with the test chemical, the assumptions are that the solvent has no effect on the responses of
1) Ultra Turrax is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
2) Schleicher & Schüll 604 is an example of a suitable product available commercially. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
4 © ISO 2006 – All rights reserved
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ISO 14442:2006(E)
interest and there is no interaction between the test chemical and the solvent. With the addition of a negative
control (i.e. without solvent, as is required in all experiments using a solvent), the assumption regarding a
solvent effect can be tested. However, unless the chemical is also tested in absence of a solvent, the
assumption of no interaction between the solvent and the test chemical cannot be evaluated. Further
guidance on the analysis of data from tests including solvent control can be found in Reference [18].
4.4 Dispersion using an emulsifying agent
The use of an emulsifier to prepare stock or test dispersions is generally the least preferable method. The
nominal test concentrations may easily be considerably higher than the solubility limit in water, and the
emulsifying agent may also influence the availability of a substance to the algal cells. However, if the exposure
conditions with an emulsifier reflect the actual environmental exposure (e.g. pesticide formulations), and other
addition methods appear to be impracticable, this method may be used. No dispersant should be added to
formulated products.
Any emulsifier may be used if it meets the following requirements:
a) no inhibiting effects (direct or indirect) on algal growth at a concentration of 100 mg/l;
b) no or only slight biodegradation within a three-day exposure period;
c) no interference with the nutrient balance of the test medium.
The following emulsifying agents have been demonstrated to meet the stated criteria, but others may be used
if required by the properties of the test substance:
3)
⎯ polyoxyethylene ethers ;
4)
⎯ alkyl polyoxyethylene sorbitan ;
5)
⎯ alkyl sorbitan .
A dispersion may be prepared by mixing appropriate amounts of the test substance and the chosen emulsifier
by one of the methods described in 4.2. The concentration of the emulsifier should not exceed 100 mg/l. The
selection of the best emulsifier is made by visually assessing the homogeneity of the stock dispersion.
Additional controls shall be added containing the same emulsifier concentration as in the test media. The use
of the emulsifier controls in the data analysis is as described for solvent controls in 4.3.
4.5 Interference with algal growth and its measurement
If nominal test substance concentrations above the solubility limit or dispersions are tested, relatively high
particle densities may occur in the test medium. High background particle numbers may disturb the growth
measurements when using a particle counter or a spectrophotometer. For this reason, a background test
substance concentration series without algae shall be included as a background correction of the
measurements.
Usually quite high particle densities (i.e. at the same density level as the inoculum) are acceptable at the start
of the test, as their influence on the subsequent measurements is progressively less due to the algal growth.
3) Brij 56 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
4) Tween 80 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
5) Span 20 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
© ISO 2006 – All rights reserved 5
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ISO 14442:2006(E)
If treatments to reduce the particle densities (i.e. by filtration or centrifugation) should lead to a considerable
loss of soluble substance, testing with particles present is preferred. In extreme cases, the growth can be
determined by other methods or validated by counting of algal cells with a microscope.
Fluorimetric measurement of solvent extracted pigments (Reference [5]) may be an attractive indirect method
of estimating the algal biomass, which eliminates interferences from particles. The method is however indirect
as pigment content may vary with growth conditions.
Bacterial growth on biodegradable test substances or auxiliary substances (i.e. solvents or emulsifiers) cannot
be prevented, as the algal cultures nearly always contain bacteria. The growth can be delayed however by
working under aseptic conditions as much as possible and using sterilised equipment and media. A significant
interference is expected only at the highest concentrations tested (i.e. in the range of 10 mg/l to 100 mg/l) of
6)
highly degradable substances [e.g. with a BOD /COD ratio of 0,5 or higher].
5
Solvents and in particular emulsifiers may inhibit or stimulate the algal growth. A stimulating effect is probably
due to carbon dioxide or other nutrients released by degradation (at high cell densities the growth of the algal
culture is often carbon limited). Stimulating effects may complicate the calculation of the EC values (see
Clause 12).
5 Poorly soluble mixtures of organic substances
5.1 General
Mixtures of organic substances refer to both homogenous aggregates of a number of compounds with
different physico-chemical and/or chemical properties, which cannot be easily separated into their component
parts by physical means (e.g. oil products, mixtures of isomers), and formulated products (preparations such
[3]
as formulated pesticides and oil based drilling fluids ).
The method of choice for testing mixtures containing poorly soluble substances and/or volatile substances is
[2]
the preparation of Water-accommodated fractions (WAFs) by stirring and phase separation . A WAF is an
aqueous medium containing only that fraction of a substance which remains in the aqueous phase after the
preparation procedure is terminated. Components of the test substance may be present either in true solution
or as a stable emulsion. When filtered through suitable filters, Water-soluble fractions (WSFs) are obtained.
As the (assumed) equilibrium between the test substance and the aqueous phase depends on the test
substance to liquid ratio, a WAF is prepared for each test concentration separately and should not be diluted.
If however, stable dispersions are formed by the WAF preparation, these can further be treated according
to 4.4.
In 5.2, the general preparation procedure for a WAF is described.
In testing WAFs, the results are expressed in terms of loading rates instead of the usual concentration term.
Loading rate is the amount of test substance from which a WAF is prepared and is equivalent to the nominal
concentration. The final results also shall be expressed as EL , and EL values, where L represents the
50 10
loading rate.
5.2 Preparation of test media
Water-accommodated fractions (WAFs) are prepared by mixing the test substance with the algal growth
medium at a range of loading rates in clean mixing vessels, using a suitable mixing apparatus. The mixing
vessels shall be cylindrical and fitted with a drain port near the bottom for drawing off the WAF (commercially
available aspirator bottles are quite acceptable). The mixing vessel volume shall be large enough to prepare
the volume of WAF required for the exposure (and for sampling for analysis if relevant).
6) BOD = biochemical oxygen demand; COD = chemical oxygen demand.
6 © ISO 2006 – All rights reserved
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ISO 14442:2006(E)
The vessel volume has also to be small enough to minimize headspace whilst maintaining optimum surface
contact between test material and the growth medium. The containers should preferably be sealed with
ground glass stoppers, although PTFE-lined screw caps or tightly fitted, aluminium foil-covered neoprene
stoppers may be acceptable. The loss of volatiles is prevented by tightly sealing the vessels, which should be
incubated in the dark to prevent photochemical degradation of dissolved components.
A magnetic stirring bar (or
...
SLOVENSKI STANDARD
SIST ISO 14442:2007
01-februar-2007
Kakovost vode - Smernice za preskuse zaviranja rasti alg s slabo topnimi
materiali, hlapnimi spojinami, kovinami in odpadno vodo
Water quality -- Guidelines for algal growth inhibition tests with poorly soluble materials,
volatile compounds, metals and waste water
Qualité de l'eau -- Lignes directrices pour essais d'inhibition de la croissance algale avec
des matières peu solubles, des composés volatils, des métaux et des eaux résiduaires
Ta slovenski standard je istoveten z: ISO 14442:2006
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 14442:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST ISO 14442:2007
---------------------- Page: 2 ----------------------
SIST ISO 14442:2007
INTERNATIONAL ISO
STANDARD 14442
Second edition
2006-04-01
Water quality — Guidelines for algal
growth inhibition tests with poorly
soluble materials, volatile compounds,
metals and waste water
Qualité de l'eau — Lignes directrices pour essais d'inhibition de la
croissance algale avec des matières peu solubles, des composés
volatils, des métaux et des eaux résiduaires
Reference number
ISO 14442:2006(E)
©
ISO 2006
---------------------- Page: 3 ----------------------
SIST ISO 14442:2007
ISO 14442:2006(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.
© ISO 2006
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2006 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 14442:2007
ISO 14442:2006(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 2
3 Analytical characterization of test materials and confirmation of concentrations and
stability . 2
4 Poorly soluble organic substances . 3
4.1 General. 3
4.2 Preparation of saturated and supersaturated solutions. 3
4.3 Solvent addition. 4
4.4 Dispersion using an emulsifying agent. 5
4.5 Interference with algal growth and its measurement. 5
5 Poorly soluble mixtures of organic substances. 6
5.1 General. 6
5.2 Preparation of test media. 6
5.3 Test performance. 7
6 Poorly soluble inorganic materials. 7
7 Volatile substances . 8
7.1 General. 8
7.2 Test system and growth medium. 8
7.3 Test performance. 9
7.4 Interference with algal growth. 9
8 Waste waters and environmental aqueous samples . 9
9 Coloured and/or turbid samples . 10
10 Metals and metal compounds . 11
10.1 Introduction . 11
10.2 Modification of algal growth inhibition test procedures for testing materials containing
heavy metals . 11
11 pH buffering. 12
12 Interpretation of the results . 13
Bibliography . 14
© ISO 2006 – All rights reserved iii
---------------------- Page: 5 ----------------------
SIST ISO 14442:2007
ISO 14442:2006(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 14442 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
This second edition cancels and replaces the first edition (ISO 14442:1999), which has been technically
revised.
iv © ISO 2006 – All rights reserved
---------------------- Page: 6 ----------------------
SIST ISO 14442:2007
INTERNATIONAL STANDARD ISO 14442:2006(E)
Water quality — Guidelines for algal growth inhibition tests with
poorly soluble materials, volatile compounds, metals and waste
water
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 standard be carried
out by suitably trained staff.
1 Scope
This International Standard provides procedures, not covered by the methods described in ISO 8692 and
ISO 10253, for testing difficult substances for inhibition of algal growth.
The main subjects covered by the guideline are the methods for preparing the test substance for testing and
the procedures needed to carry out an appropriate test. The following test substances are covered by this
guideline:
a) poorly soluble pure organic compounds;
b) poorly soluble mixtures of organic substances;
c) poorly soluble inorganic materials;
d) volatile substances;
e) waste waters and environmental samples containing water and sediments;
f) coloured and/or turbid samples;
g) compounds of heavy metals.
The following methods of addition are covered:
⎯ direct;
⎯ dispersion;
⎯ water-soluble and water-accommodated fractions.
Some guidelines related to the analytical procedures and to the interpretation of the results have been
included.
References to documents describing the background for the testing of difficult substances are given in the
Bibliography.
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ISO 14442:2006(E)
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 8692, Water quality — Freshwater algal growth inhibition test with unicellular green algae
ISO 10253, Water quality — Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
3 Analytical characterization of test materials and confirmation of concentrations
and stability
Analytical characterization of test substances and materials and the confirmation of their concentrations and
stability in the testing environment is of major concern of regulatory authorities. Such activities are usually not
an integral part of this International Standard algal growth inhibition test methods.
However, there may be situations where analysis may assist in defining the appropriate exposure conditions
of test materials and chemicals and/or in the interpretation of the results.
The relevant properties of substances and materials can be assessed from basic properties such as solubility
in water, partition coefficient (lg P ), Henry's constant, photochemical and hydrolytic stability and
ow
biodegradability.
Analytical confirmation is strongly recommended in order to confirm test substance concentrations and is
required for the calculation of effective concentration (EC) values of volatile substances (Clause 7). If losses
due to adsorption on the test vessels or during transfer of test solutions and media occur, then analytical
confirmation are of particular importance. This aspect is also specified in ISO 5667-16.
Due to the batch test system used for algal growth inhibition tests, loss of substances due to biodegradation
(nearly all algal cultures contain bacteria), photodegradation, hydrolysis and/or adsorption cannot always be
avoided. A decrease in measured concentrations is difficult to prevent by technical means, and is therefore
considered acceptable for algal growth inhibition tests.
The following precautions are suggested for maintaining test substance concentrations in algal growth
inhibition tests:
a) sterilization of media and equipment to reduce the effect of bacterial growth;
b) change of the light quality to prevent photodegradation of test substances;
c) avoidance of contact of test substance with water prior to testing to reduce hydrolytic decomposition;
d) treatment of glassware (e.g. silanization); the effectiveness of such a treatment varies from one chemical
to the other;
e) pre-conditioning of the glassware, before addition of the test media, with the test substance at
concentrations to be used in the test.
The effect of such technical measures is, if relevant and if possible, monitored by chemical analysis.
Water, waste water and organic/inorganic solids/liquids may contain components that may modify the
composition of the algal growth medium (by precipitation of a limiting nutrient, complexation of essential
elements, addition of nutrients), and subsequently may cause effects on algal growth not related to toxic
2 © ISO 2006 – All rights reserved
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components. If such problems occur, it may be advisable to determine the content of key components of the
test material. Some relevant components are: calcium, magnesium, sodium, potassium, sulfate, chloride,
ammonium, nitrate, phosphate, copper, cobalt, nickel, zinc, cadmium, organic matter [i.e. measured as
Chemical Oxygen Demand (COD) and/or Total Organic Carbon (TOC)].
If the material contains a high concentration of readily degradable organic material, the subsequent bacterial
growth may disturb the algal growth measurement. When untreated (not filtered or centrifuged) waste water is
tested, contamination with other algal species may occur.
4 Poorly soluble organic substances
4.1 General
A pure substance is a substance with one major component containing minor components as impurities.
Poorly soluble substances are those with solubility limits below 100 mg/l in water. If, however, growth
inhibition occurs at concentrations much lower than the solubility limits in water or algal growth medium (the
limit in the medium may be different), then the poorly soluble substance can be tested as a water soluble
substance (added via a stock solution in test medium). This approach is usually not applicable to substances
with a water solubility below 1 mg/l to 10 mg/l (substances with a very low solubility).
The methods described in this clause therefore refer to testing of substances causing effects on algal growth
at concentrations at or around the solubility limit in water and to very low solubility substances.
Testing of nominal concentrations markedly above the solubility limit is not recommended. It may, however, be
unavoidable if the solubility limit in water or algal growth medium (which may be different) is not well
established, or if a substance spontaneously forms dispersions in the test medium.
NOTE Terminology according to Reference [3]:
⎯ water solubility below 100 mg/l: “sparingly soluble”;
⎯ water solubility below 1 mg/l: “very low solubility”.
A number of methods which are available for preparing test solutions of pure substances, are described in
ISO 5667-16. Generally, it is preferred to use mechanical means to prepare stock solutions.
4.2 Preparation of saturated and supersaturated solutions
If the solubility of a substance in water is between 1 mg/l and 100 mg/l, saturated solutions can be prepared
by direct addition of the test substance. A saturated solution is usually prepared by stirring (e.g. magnetic
stirrer or shaking, see also 5.1) an excess amount of the test substance in water for a period in test medium. A
period of 20 h is practical for most substances, but a stirring period of up to three days may be considered to
ensure saturation provided the substance is stable. Lengthy stirring should be carried out in the dark and in
the same temperature range as the growth inhibition test is carried out. Preferably, the equilibrium should be
confirmed by chemical analysis. After a phase separation period of varying length, the clear phase is collected
and tested as the highest concentration. Filtration (through a 0,45 µm membrane filter) or centrifugation may
be useful for removing particulate matter.
Certain membrane filters may interfere with the test substance. The type of filter should be chosen according
to the physico-chemical properties of the test substance and the recommendations of the filter supplier.
Further test concentrations can be prepared by dilution of the saturated solution with test medium. A small
volume of a concentrated suspension of algal culture is then added to the test media to start the test.
A disadvantage of preparing saturated solutions in this way is that trace impurities in the test substance may
be preferentially enhanced in the solution, if they are more soluble than the major component. For this reason,
the quantity of test substance should be the minimum required ensuring that a saturated solution of the test
substance can be achieved.
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Where possible, prepare stable supersaturated stock solutions with a substance (i.e. stock solution
concentrations in the range 2 to 10 times the saturation value) in test medium by high speed mechanical
1)
stirring [e.g. a high speed blender ] or ultrasonic treatment (a recommended frequency of 20 kHz and a
power output of at least 60 W) for a few minutes to several hours. With both methods, a constant temperature
shall be maintained during the treatment by cooling. If phase separation takes place immediately after the
treatment has ended, one may choose to remove (sinking or floating) particles by filtration through a paper
2)
filter [e.g. Schleicher & Schüll 604 ] or by centrifugation. If dissolved substances are removed by filtration, it
is essential to confirm the actual concentrations in the final solution by chemical analysis. The test solutions
can be prepared by dilution of the supersaturated stock solution.
4.3 Solvent addition
The use of a solvent as a carrier to add a substance to a test medium is considered to be a practical and
convenient method for handling organic substances tested at concentrations below 10 mg/l. The
recommended concentration of solvent does not influence the solubility of substances but assists in a rapid
and complete mixing of substances and test medium.
At concentrations of the test substance below 1 mg/l the solvent addition may be combined with the methods
described in 4.2 to prepare saturated solutions (which are further treated as described in 4.2).
In principle, any organic solvent can be used that meets the following criteria:
a) does not inhibit the algal growth at the highest concentration added;
b) is soluble in water at the recommended concentration;
c) does not interact with medium components;
d) does not react with the test substance;
e) does not biodegrade rapidly;
f) does not interfere with the conditions of illumination.
The concentration of a solvent should not exceed 100 µl/l test medium according to ISO 10253 and ISO 8692.
In practice, this solvent concentration for algal tests can be obtained by the addition of 10 µl solvent per
100 ml of test medium, a solvent volume that can be added with precision. Solvents such as acetone and t-
butanol have been demonstrated to meet most of the stated criteria in algal growth inhibition tests. t-Butanol
however is the less biodegradable one. Dimethylsulfoxide (DMSO) is a very efficient solvent, but might in
some cases interact more easily with test substances and the test organism. Tests have shown that none of
the solvents alone has any effect on algal growth up to a concentration of at least 1 ml/l (Reference [3]). In
exceptional cases, higher solvent concentrations can be used to add higher concentrations of the test
substance than possible with 100 µl/l.
It is recommended that a concentration series is prepared in the selected solvent, and that aliquots of the
stock solutions are added to the test flasks, which already contain the algae and the test medium. Controls
with and without the solvent shall be added to the test concentration series. The solvent concentration should
be the same for all test solutions.
The solvent control group is the appropriate control group for comparisons with treated groups. Each group
shall have the same solvent concentration as the control. For a bioassay in which a solvent is used in
conjunction with the test chemical, the assumptions are that the solvent has no effect on the responses of
1) Ultra Turrax is an example of a suitable product available commercially. This information is given for the convenience
of users of this International Standard and does not constitute an endorsement by ISO of this product.
2) Schleicher & Schüll 604 is an example of a suitable product available commercially. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.
4 © ISO 2006 – All rights reserved
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ISO 14442:2006(E)
interest and there is no interaction between the test chemical and the solvent. With the addition of a negative
control (i.e. without solvent, as is required in all experiments using a solvent), the assumption regarding a
solvent effect can be tested. However, unless the chemical is also tested in absence of a solvent, the
assumption of no interaction between the solvent and the test chemical cannot be evaluated. Further
guidance on the analysis of data from tests including solvent control can be found in Reference [18].
4.4 Dispersion using an emulsifying agent
The use of an emulsifier to prepare stock or test dispersions is generally the least preferable method. The
nominal test concentrations may easily be considerably higher than the solubility limit in water, and the
emulsifying agent may also influence the availability of a substance to the algal cells. However, if the exposure
conditions with an emulsifier reflect the actual environmental exposure (e.g. pesticide formulations), and other
addition methods appear to be impracticable, this method may be used. No dispersant should be added to
formulated products.
Any emulsifier may be used if it meets the following requirements:
a) no inhibiting effects (direct or indirect) on algal growth at a concentration of 100 mg/l;
b) no or only slight biodegradation within a three-day exposure period;
c) no interference with the nutrient balance of the test medium.
The following emulsifying agents have been demonstrated to meet the stated criteria, but others may be used
if required by the properties of the test substance:
3)
⎯ polyoxyethylene ethers ;
4)
⎯ alkyl polyoxyethylene sorbitan ;
5)
⎯ alkyl sorbitan .
A dispersion may be prepared by mixing appropriate amounts of the test substance and the chosen emulsifier
by one of the methods described in 4.2. The concentration of the emulsifier should not exceed 100 mg/l. The
selection of the best emulsifier is made by visually assessing the homogeneity of the stock dispersion.
Additional controls shall be added containing the same emulsifier concentration as in the test media. The use
of the emulsifier controls in the data analysis is as described for solvent controls in 4.3.
4.5 Interference with algal growth and its measurement
If nominal test substance concentrations above the solubility limit or dispersions are tested, relatively high
particle densities may occur in the test medium. High background particle numbers may disturb the growth
measurements when using a particle counter or a spectrophotometer. For this reason, a background test
substance concentration series without algae shall be included as a background correction of the
measurements.
Usually quite high particle densities (i.e. at the same density level as the inoculum) are acceptable at the start
of the test, as their influence on the subsequent measurements is progressively less due to the algal growth.
3) Brij 56 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
4) Tween 80 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
5) Span 20 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
© ISO 2006 – All rights reserved 5
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If treatments to reduce the particle densities (i.e. by filtration or centrifugation) should lead to a considerable
loss of soluble substance, testing with particles present is preferred. In extreme cases, the growth can be
determined by other methods or validated by counting of algal cells with a microscope.
Fluorimetric measurement of solvent extracted pigments (Reference [5]) may be an attractive indirect method
of estimating the algal biomass, which eliminates interferences from particles. The method is however indirect
as pigment content may vary with growth conditions.
Bacterial growth on biodegradable test substances or auxiliary substances (i.e. solvents or emulsifiers) cannot
be prevented, as the algal cultures nearly always contain bacteria. The growth can be delayed however by
working under aseptic conditions as much as possible and using sterilised equipment and media. A significant
interference is expected only at the highest concentrations tested (i.e. in the range of 10 mg/l to 100 mg/l) of
6)
highly degradable substances [e.g. with a BOD /COD ratio of 0,5 or higher].
5
Solvents and in particular emulsifiers may inhibit or stimulate the algal growth. A stimulating effect is probably
due to carbon dioxide or other nutrients released by degradation (at high cell densities the growth of the algal
culture is often carbon limited). Stimulating effects may complicate the calculation of the EC values (see
Clause 12).
5 Poorly soluble mixtures of organic substances
5.1 General
Mixtures of organic substances refer to both homogenous aggregates of a number of compounds with
different physico-chemical and/or chemical properties, which cannot be easily separated into their component
parts by physical means (e.g. oil products, mixtures of isomers), and formulated products (preparations such
[3]
as formulated pesticides and oil based drilling fluids ).
The method of choice for testing mixtures containing poorly soluble substances and/or volatile substances is
[2]
the preparation of Water-accommodated fractions (WAFs) by stirring and phase separation . A WAF is an
aqueous medium containing only that fraction of a substance which remains in the aqueous phase after the
preparation procedure is terminated. Components of the test substance may be present either in true solution
or as a stable emulsion. When filtered through suitable filters, Water-soluble fractions (WSFs) are obtained.
As the (assumed) equilibrium between the test substance and the aqueous phase depends on the test
substance to liquid ratio, a WAF is prepared for each test concentration separately and should not be diluted.
If however, stable dispersions are formed by the WAF preparation, these can further be treated according
to 4.4.
In 5.2, the general preparation procedure for a WAF is described.
In testing WAFs, the results are expressed in terms of loading rates instead of the usual concentration term.
Loading rate is the amount of test substance from which a WAF is prepared and is equivalent to the nominal
concentration. The final results also shall be expressed as EL , and EL values, where L represents the
50 10
loading rate.
5.2 Preparation of test media
Water-accommodated fractions (WAFs) are prepared by mixing the test substance with the algal growth
medium at a range of loading rates in clean mixi
...
NORME ISO
INTERNATIONALE 14442
Deuxième édition
2006-04-01
Qualité de l'eau — Lignes directrices
pour essais d'inhibition de la croissance
algale avec des matières peu solubles,
des composés volatils, des métaux et des
eaux résiduaires
Water quality — Guidelines for algal growth inhibition tests with poorly
soluble materials, volatile compounds, metals and waste water
Numéro de référence
ISO 14442:2006(F)
©
ISO 2006
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ISO 14442:2006(F)
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ii © ISO 2006 – Tous droits réservés
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ISO 14442:2006(F)
Sommaire Page
Avant-propos. iv
1 Domaine d'application. 1
2 Références normatives . 2
3 Caractérisation analytique des matériaux d'essai et confirmation des concentrations et
de la stabilité . 2
4 Substances organiques peu solubles . 3
4.1 Généralités . 3
4.2 Préparation de solutions saturées et sursaturées . 4
4.3 Addition de solvants. 4
4.4 Dispersion à l'aide d'un agent émulsifiant . 5
4.5 Interférence avec la croissance algale et son mesurage . 6
5 Mélanges peu solubles de substances organiques. 7
5.1 Généralités . 7
5.2 Préparation des milieux d'essai . 7
5.3 Réalisation de l'essai. 8
6 Matériaux solides inorganiques peu solubles. 8
7 Substances volatiles . 9
7.1 Généralités . 9
7.2 Système d'essai et milieu de culture . 10
7.3 Rendement de l'essai . 10
7.4 Interférences avec la croissance algale . 10
8 Eaux résiduaires et échantillons environnementaux aqueux. 11
9 Échantillons colorés et/ou turbides. 12
10 Métaux et composés métalliques. 12
10.1 Introduction . 12
10.2 Modification des modes opératoires d'essai d'inhibition de la croissance algale pour
l'essai de matériaux contenant des métaux lourds. 13
11 Tamponnage du pH . 14
12 Interprétation des résultats . 15
Bibliographie . 16
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ISO 14442:2006(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 14442 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité SC 5,
Méthodes biologiques.
Cette deuxième édition annule et remplace la première édition (ISO 14442:1999), qui a fait l'objet d'une
révision technique.
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NORME INTERNATIONALE ISO 14442:2006(F)
Qualité de l'eau — Lignes directrices pour essais d'inhibition de
la croissance algale avec des matières peu solubles, des
composés volatils, des métaux et des eaux résiduaires
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
de mettre en place 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
soient effectués par un personnel ayant reçu une formation adéquate.
1 Domaine d'application
La présente Norme internationale a pour objectif de fournir des modes opératoires, non couverts par les
méthodes décrites dans l'ISO 8692 et l'ISO 10253, applicables aux essais d'inhibition de la croissance algale
impliquant des substances difficiles.
Les principaux sujets traités dans ces lignes directrices sont les méthodes de préparation de la substance
d'essai ainsi que les modes opératoires nécessaires pour effectuer ces essais de façon appropriée. Ces
lignes directrices sont applicables aux substances d'essai suivantes:
a) composés organiques purs peu solubles;
b) mélanges de substances organiques peu solubles;
c) matériaux inorganiques peu solubles;
d) substances volatiles;
e) eaux résiduaires et échantillons environnementaux contenant de l'eau et des sédiments;
f) échantillons colorés et/ou turbides;
g) composés de métaux lourds.
Les méthodes d'addition suivantes sont couvertes:
⎯ directe;
⎯ dispersion;
⎯ fractions en suspension dans l'eau et fractions solubles dans l'eau.
Certaines lignes directrices traitant des modes opératoires d'analyse et de l'interprétation des résultats sont
également incluses.
Les références faites à des documents décrivant le contexte relatif aux essais sur des substances difficiles
sont récapitulées dans la Bibliographie.
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ISO 14442:2006(F)
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, Qualité de l'eau — Échantillonnage — Partie 16: Lignes directrices pour les essais biologiques
des échantillons
ISO 8692, Qualité de l'eau — Essai d'inhibition de la croissance des algues d'eau douce avec des algues
vertes unicellulaires
ISO 10253, Qualité de l'eau — Essai d'inhibition de la croissance des algues marines avec Skeletonema
costatum et Phaeodactylum tricornutum
3 Caractérisation analytique des matériaux d'essai et confirmation des
concentrations et de la stabilité
La caractérisation analytique des substances et des matériaux soumis à essai ainsi que la confirmation de
leur concentration et de leur stabilité dans l'environnement d'essai est une préoccupation majeure pour les
autorités réglementaires. Ces activités ne font normalement pas partie intégrante des méthodes d'essai
d'inhibition de la croissance algale développées dans la présente Norme internationale.
Cependant, il peut exister des situations dans lesquelles l'analyse peut contribuer à mieux définir les
conditions d'exposition des matériaux et des substances chimiques d'essai et/ou à faciliter l'interprétation des
résultats.
Les caractéristiques importantes des substances et matériaux peuvent être évaluées à partir des
caractéristiques de base telles que la solubilité dans l'eau, le coefficient de partage (lg P ), la constante de
ow
Henry, la stabilité photochimique et hydrolytique ainsi que la biodégradabilité.
Il est fortement conseillé de procéder à une analyse des concentrations de la substance chimique d'essai,
concentrations qui sont exigées pour le calcul des valeurs de concentration effective (CE) des substances
volatiles (Article 7). Si des pertes de substance ont lieu par adsorption sur les parois des récipients d'essai ou
pendant le transfert des solutions et du milieu, cette confirmation analytique sera essentielle. Cet aspect est
également spécifié dans l'ISO 5667-16.
En raison du système d'essai sous forme de lots utilisé pour les essais d'inhibition de la croissance algale, il
n'est pas toujours possible d'éviter les pertes de substance dues à la biodégradation (la plupart des cultures
algales contiennent des bactéries), la photodégradation, l'hydrolyse et/ou l'adsorption. La diminution des
concentrations mesurées étant difficile à empêcher par des moyens techniques, elle est considérée comme
acceptable dans le cadre des essais d'inhibition de la croissance algale.
Dans le cadre d'essais d'inhibition de la croissance algale, il est suggéré de prendre les précautions suivantes
pour maintenir à un niveau constant les concentrations de la substance d'essai:
a) stérilisation des milieux de culture et des matériels utilisés pour réduire les effets de la croissance
bactérienne;
b) changement de la qualité de la lumière pour empêcher une photodégradation des substances d'essai;
c) absence de tout contact de la substance d'essai avec l'eau avant l'essai pour réduire les risques liés à la
décomposition par hydrolyse;
d) traitement de la verrerie (par exemple par silanisation); ce type de traitement est plus ou moins efficace
selon la substance chimique concernée;
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ISO 14442:2006(F)
e) préconditionnement de la verrerie avec la substance d'essai aux concentrations utilisées au cours de
l'essai avant l'ajout du milieu d'essai.
Si nécessaire et si possible, contrôler par des analyses chimiques l'effet de ces mesures techniques.
L'eau, les eaux résiduaires, les liquides/solides organiques/inorganiques peuvent contenir des composés
susceptibles de modifier la composition du milieu de culture des algues (par précipitation d'un nutriment
limitant, complexation d'éléments essentiels, addition de nutriments) et par voie de conséquence, d'affecter de
façon non négligeable la croissance algale sans implication de composés toxiques. En présence de ce type
de problèmes, il peut être recommandé de déterminer la teneur de ces composés clés du matériau d'essai.
Parmi ces composés, on compte: le calcium, le magnésium, le sodium, le potassium, les sulfates, les
chlorures, l'ammonium, les nitrates, les phosphates, le cuivre, le cobalt, le nickel, le zinc, le cadmium, les
matières organiques [mesurées en termes de demande chimique en oxygène (DCO) et/ou de carbone
organique total (COT)].
Si le matériau a une forte teneur en substances organiques facilement dégradables, la croissance bactérienne
qui en résulte peut perturber les mesurages de la croissance algale. Les essais portant sur des eaux
résiduaires non traitées (non filtrées ou non centrifugées) peuvent entraîner une contamination par d'autres
espèces algales.
4 Substances organiques peu solubles
4.1 Généralités
Une substance pure est une substance constituée d'un composé principal et de composés secondaires à
l'état d'impuretés. On entend par substances peu solubles les substances dont la limite de solubilité dans
l'eau est inférieure à 100 mg/l. Cependant, si le phénomène d'inhibition de la croissance se produit à des
concentrations bien inférieures à la limite de solubilité dans l'eau ou dans le milieu de croissance des algues
(la limite de solubilité dans le milieu d'essai peut être différente de celle observée dans l'eau), les substances
peu solubles peuvent alors être soumises à l'essai comme des substances solubles dans l'eau (ajoutées via
une solution mère dans le milieu d'essai). Cette approche ne s'applique généralement pas aux substances
dont la solubilité dans l'eau est inférieure à 1 mg/l jusqu'à 10 mg/l (substances très peu solubles).
Les méthodes décrites dans le présent article font donc référence à des essais sur des substances affectant
la croissance algale à des concentrations qui correspondent à la limite de solubilité dans l'eau ou qui sont
proches de celle-ci, ainsi qu'à des substances très peu solubles.
Il n'est pas conseillé de procéder à des essais avec des concentrations nominales nettement supérieures à la
limite de solubilité. Cependant, cela peut se révéler inévitable si les limites de solubilité dans l'eau ou dans le
milieu de culture des algues (qui peuvent être différentes) ne sont pas connues avec exactitude ou si une
substance se disperse spontanément dans le milieu d'essai.
NOTE Terminologie selon Référence [3]:
⎯ solubilité dans l'eau inférieure à 100 mg/l: «peu soluble»;
⎯ solubilité dans l'eau inférieure à 1 mg/l: «très peu soluble».
L'ISO 5667-16 décrit un certain nombre de méthodes permettant de préparer les solutions pour soumettre à
essai des substances pures. Pour préparer les solutions mères, il est conseillé, en général, d'utiliser des
moyens mécaniques.
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ISO 14442:2006(F)
4.2 Préparation de solutions saturées et sursaturées
Si la solubilité dans l'eau d'une substance est comprise entre 1 mg/l et 100 mg/l, il est possible de préparer
des solutions saturées par addition directe de la substance d'essai. Une solution saturée est en général
préparée en agitant (par exemple à l'aide d'un agitateur magnétique ou en secouant le récipient; voir
également 5.1) une quantité en excès de la substance d'essai dans l'eau pendant un certain temps dans le
milieu d'essai. Une période de 20 h suffit dans la plupart des cas, mais on peut envisager de la prolonger à
trois jours pour obtenir une saturation, à condition que la substance soit stable. Il convient que les agitations
prolongées soient faites à l'abri de la lumière et à une température équivalente à celle de l'essai d'inhibition de
la croissance. Il est conseillé de confirmer l'état d'équilibre en procédant à une analyse chimique. Après une
période plus ou moins longue pendant laquelle s'opère la séparation des phases, recueillir la phase limpide et
l'utiliser pour l'essai en tant que concentration la plus élevée. Pour éliminer les particules en suspension, il
peut s'avérer utile d'avoir recours à une filtration (sur une membrane de 0,45 µm) ou à une centrifugation.
Certains types de membranes peuvent créer des interférences avec la substance soumise à l'essai. Il
convient de la choisir en tenant compte des propriétés physico-chimiques de la substance d'essai et des
recommandations du fabricant.
D'autres concentrations d'essai peuvent être préparées en diluant la solution saturée avec le milieu d'essai.
Ensuite, pour commencer l'essai, un petit volume de la suspension algale concentrée mise en culture est
ajouté aux milieux d'essai.
L'inconvénient qu'il y a à préparer les solutions saturées de cette manière réside dans le fait que les
impuretés se trouvant à l'état de traces dans la substance d'essai peuvent être présentes en plus grande
proportion dans la solution si elles sont plus solubles que le composé principal. En conséquence, il convient
que la quantité de substance soumise à l'essai corresponde au minimum requis pour obtenir une solution
saturée de la substance d'essai.
Si possible, préparer des solutions mères sursaturées stables à partir de la substance dans le milieu d'essai
(c'est-à-dire des solutions mères à des concentrations de l'ordre de 2 à 10 fois la valeur de saturation), à l'aide
1)
d'un dispositif mécanique d'agitation à grande vitesse [par exemple un mélangeur grande vitesse ] ou en
leur appliquant un traitement aux ultrasons (la fréquence recommandée étant de 20 kHz pour une puissance
d'au moins 60 W en sortie) pendant une période pouvant varier de quelques minutes à plusieurs heures.
Quelle que soit la méthode, une température constante doit être maintenue par refroidissement pendant le
traitement. Si la séparation des phases se produit immédiatement après la fin du traitement, il est possible
d'éliminer les particules (submergées ou flottantes) par filtration sur un papier-filtre [par exemple,
2)
Schleicher & Schüll 604 ] ou par centrifugation. Si cette filtration élimine certaines substances dissoutes, il
est essentiel de confirmer les concentrations réelles de la solution finale en procédant à une analyse chimique.
Les solutions d'essai peuvent être préparées en diluant la solution mère sursaturée.
4.3 Addition de solvants
L'utilisation d'un solvant comme vecteur de transfert de la substance dans un milieu d'essai est un moyen
pratique, approprié à la manipulation des substances organiques soumises à essai à des concentrations
inférieures à 10 mg/l. La concentration recommandée de solvant est sans effet sur la solubilité des
substances mais facilite un mélange rapide et complet des substances et du milieu d'essai.
Lorsque la concentration de la substance d'essai est inférieure à 1 mg/l, il est possible d'avoir recours, en plus
du solvant, aux méthodes décrites en 4.2 pour préparer des solutions saturées (qui sont traitées
ultérieurement comme indiqué en 4.2).
1) Le mélangeur grande vitesse «Ultra Turrax» est un exemple de produit approprié disponible dans le commerce. Cette
information est donnée par souci de commodité à l'intention des utilisateurs de la présente Norme internationale et ne
saurait constituer un engagement de l'ISO à l'égard de ce produit.
2) Le papier-filtre «Schleicher & Schüll 604» est un exemple de produit approprié disponible dans le commerce. Cette
information est donnée par souci de commodité à l'intention des utilisateurs de la présente Norme internationale et ne
saurait constituer un engagement de l'ISO à l'égard de ce produit.
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ISO 14442:2006(F)
En principe, n'importe quel solvant organique peut être utilisé à condition qu'il satisfasse aux critères suivants:
a) ne pas inhiber la croissance algale à la concentration la plus élevée;
b) être soluble dans l'eau à la concentration recommandée;
c) ne pas interagir avec les composés du milieu;
d) ne pas réagir avec la substance d'essai;
e) ne pas être rapidement biodégradable;
f) ne pas interférer avec les conditions d'éclairage.
Il convient que la concentration d'un solvant ne soit pas supérieure à 100 µl/l de milieu d'essai conformément
à l'ISO 10253 et à l'ISO 8692. En pratique, pour les essais d'inhibition de la croissance algale, cette
concentration en solvant peut être obtenue en ajoutant 10 µl de solvant pour 100 ml de milieu d'essai, ce
volume de solvant pouvant être ajouté avec précision. Utilisés dans les essais d'inhibition de la croissance
algale, les solvants tels que l'acétone et le t-butanol ont démontré qu'ils remplissaient la plupart des critères
indiqués. Le t-butanol est, cependant, le moins biodégradable des deux. Le diméthylsulfoxyde (DMSO) est un
solvant très efficace mais susceptible, dans certains cas, d'interagir plus facilement avec les substances et
l'organisme d'essai. Des essais ont permis de démontrer qu'aucun des solvants utilisés seuls n'a d'effet sur la
croissance algale jusqu'à une concentration de 1 ml/l (Référence [3]). Dans certains cas exceptionnels, il est
possible d'utiliser des concentrations de solvants supérieures à 100 µl/l, pour pouvoir ajouter des
concentrations plus élevées de la substance d'essai.
Il est recommandé de préparer une gamme de concentrations dans le solvant choisi et d'ajouter des parties
aliquotes des solutions mères dans les flacons contenant déjà le milieu d'essai et les algues. Des témoins
avec et sans solvant devront être ajoutés à cette gamme de concentrations d'essai. Il convient que la
concentration en solvant soit la même pour toutes les solutions d'essai.
Le groupe témoin contenant le solvant est approprié pour la comparaison avec les groupes traités. Chaque
groupe doit avoir la même concentration de solvant que le témoin. Dans le cas d'un bioessai dans lequel le
solvant est utilisé conjointement avec le produit chimique d'essai, il est supposé que le solvant n'a aucun effet
sur les réponses recherchées et qu'aucune interaction ne se produit entre le produit chimique d'essai et le
solvant. Il est possible de vérifier le postulat concernant l'effet du solvant en ajoutant un témoin négatif (c'est-
à-dire sans solvant, comme exigé pour toutes les expériences utilisant un solvant). Cependant, à moins que le
produit chimique ne soit également soumis à l'essai en l'absence de solvant, il n'est pas possible de vérifier le
postulat concernant l'absence d'interaction entre le solvant et le produit chimique. Des lignes directrices
supplémentaires sur l'analyse des données issues d'essais incluant un témoin du solvant sont données
dans la Référence [18].
4.4 Dispersion à l'aide d'un agent émulsifiant
Il est généralement déconseillé d'utiliser un agent émulsifiant pour préparer des dispersions mères ou d'essai.
Les concentrations nominales d'essai peuvent facilement être bien supérieures à la limite de solubilité dans
l'eau et l'agent émulsifiant peut également influer sur la disponibilité d'une substance vis-à-vis des cellules
algales. Toutefois, si les conditions d'exposition avec un émulsifiant sont le reflet des conditions
environnementales réelles d'exposition (par exemple formulations des pesticides) et que les autres méthodes
d'addition se révèlent irréalisables, il est possible d'avoir recours à cette méthode. Il convient de ne pas
ajouter de dispersant aux formulations.
Un émulsifiant, quel qu'il soit, peut être utilisé à condition qu'il réponde aux exigences suivantes:
a) pas d'effet inhibiteur (direct ou indirect) sur la croissance algale à une concentration de 100 mg/l;
b) pas ou peu de biodégradation observable sur une période d'exposition de trois jours;
c) pas d'interférence avec l'équilibre nutritionnel du milieu d'essai.
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ISO 14442:2006(F)
Il a été démontré que les agents émulsifiants suivants remplissaient les critères énoncés mais d'autres agents
peuvent être utilisés si les propriétés de la substance d'essai le nécessitent.
3)
⎯ éthers de polyoxyéthylène ;
4)
⎯ alkyl polyoxyéthylène sorbitan ;
5)
⎯ alkyl sorbitan
Une dispersion peut être préparée en mélangeant, en quantités appropriées, la substance d'essai et
l'émulsifiant choisi, selon l'une des méthodes décrites en 4.2. Il convient que la concentration de l'émulsifiant
ne dépasse pas 100 mg/l. Le choix de l'émulsifiant le mieux adapté s'effectue en évaluant visuellement
l'homogénéité de la suspension mère.
D'autres témoins doivent être ajoutés contenant la même concentration d'émulsifiant que dans le milieu
d'essai. L'utilisation des témoins émulsifiants pour l'analyse des données est identique à celle décrite pour les
témoins de solvant en 4.3.
4.5 Interférence avec la croissance algale et son mesurage
Si les essais sont réalisés sur des concentrations nominales de la substance d'essai supérieures à sa limite
de solubilité ou sur des suspensions, la concentration de particules dans le milieu d'essai peut s'avérer
relativement élevée. Un nombre élevé de particules peut perturber les mesurages de la croissance effectués
à l'aide d'un compteur de particules ou d'un spectrophotomètre. Pour cette raison, une gamme de
concentrations de la substance d'essai ne contenant pas d'algues doit être incluse pour corriger le bruit de
fond lors des mesures.
En général, des concentrations de particules relativement élevées (c'est-à-dire du même niveau de
concentration que celle de l'inoculum) sont acceptables au début de l'essai car leur influence sur les
mesurages ultérieurs diminue progressivement, en raison de la croissance algale.
Si des traitements pour réduire les concentrations de particules (par filtration ou centrifugation) conduisent à
une perte importante de substance soluble, il est préférable d'effectuer les essais sans éliminer les particules.
Dans certains cas extrêmes, la croissance peut être déterminée par d'autres méthodes ou être validée par un
comptage au microscope des cellules algales.
La mesure fluorimétrique de pigments extraits à l'aide de solvants (Référence [5]) peut être une méthode
indirecte intéressante pour estimer la biomasse algale, ce qui élimine les interférences dues aux particules.
Cette méthode est cependant indirecte dans la mesure où la teneur en pigments peut varier selon les
conditions de croissance.
La croissance bactérienne sur des substances d'essai biodégradables ou des substances auxiliaires (telles
que les solvants ou émulsifiants) ne peut être évitée, puisque les cultures d'algues contiennent pratiquement
toujours des bactéries. Cette croissance peut cependant être retardée si l'on travaille le plus possible dans
des conditions d'asepsie et en utilisant du matériel et des milieux stériles. Une interférence significative n'est à
...
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