SIST EN ISO 10253:2006

SLOVENSKI OSIST prEN ISO 10253:2004

PREDSTANDARD
december 2004
Kakovost vode – Preskus zaviranja rasti morskih alg s Skeletonema costatum
in Phaeodactylum tricomutum
Water Quality - Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricomutum
ICS 13.060.70 Referenčna številka
OSIST prEN ISO 10253:2004(en;fr;de)
©  Standard je založil in izdal Slovenski inštitut za standardizacijo. Razmnoževanje ali kopiranje celote ali delov tega dokumenta ni dovoljeno

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EUROPEAN STANDARD
DRAFT
prEN ISO 10253
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2004
ICS Will supersede EN ISO 10253:1998
English version
Water Quality - Marine algal growth inhibition test with
Skeletonema costatum and Phaeodactylum tricomutum
Qualité de l'eau - Essai d'inhibition de la croissance des
algues marines avec Skeletonema costatum et
Phaeodactylum tricomutum
This draft European Standard is submitted to CEN members for parallel enquiry. It has been drawn up by the Technical Committee
CEN/TC 230.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which
stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language
made by translation under the responsibility of a CEN member into its own language and notified to the Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN ISO 10253:2004: E
worldwide for CEN national Members.

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prEN ISO 10253:2004 (E)


Foreword

This document (prEN ISO 10253:2004) has been prepared by Technical Committee ISO/TC 147
"Water quality" in collaboration with Technical Committee CEN/TC 230 "Water analysis", the
secretariat of which is held by DIN.

This document is currently submitted to the parallel Enquiry.

This document will supersede EN ISO 10253:1998.

Endorsement notice

The text of ISO/DIS 10253:2004 has been approved by CEN as prEN ISO 10253:2004 without
any modifications.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 10253
ISO/TC 147/SC 5 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2004-09-02 2005-02-02
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION • МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ • ORGANISATION INTERNATIONALE DE NORMALISATION
Water quality — Marine algal growth inhibition test with
Skeletonema costatum and Phaeodactylum tricornutum
Qualité de l'eau — Essai d'inhibition de la croissance des algues marines avec Skeletonema costatum et
Phaeodactylum tricornutum
[Revision of first edition (ISO 10253:1995)]
ICS 13.060.70

ISO/CEN PARALLEL ENQUIRY
The CEN Secretary-General has advised the ISO Secretary-General that this ISO/DIS covers a subject
of interest to European standardization. In accordance with the ISO-lead mode of collaboration as
defined in the Vienna Agreement, consultation on this ISO/DIS has the same effect for CEN
members as would a CEN enquiry on a draft European Standard. Should this draft be accepted, a
final draft, established on the basis of comments received, will be submitted to a parallel two-month FDIS
vote in ISO and formal vote in CEN.
To expedite distribution, this document is circulated as received from the committee secretariat.
ISO Central Secretariat work of editing and text composition will be undertaken at publication
stage.
Pour accélérer la distribution, le présent document est distribué tel qu'il est parvenu du
secrétariat du comité. Le travail de rédaction et de composition de texte sera effectué au
Secrétariat central de l'ISO au stade de publication.
THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE
REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.
© International Organization for Standardization, 2004

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ISO/DIS 10253
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©
ii ISO 2004 – All rights reserved

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ISO/DIS 10253
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Principle. 2
5 Materials . 2
5.1 Test organisms. 2
5.2 Water. 3
5.3 Seawater. 3
5.4 Nutrients . 4
6 Apparatus . 5
7 Procedure . 6
7.1 Preparation of growth medium. 6
7.2 Preparation of pre-culture and inoculum . 6
7.3 Choice of test concentrations. 6
7.4 Preparation of test substance stock solutions. 6
7.5 Preparation of test and control batches. 7
7.6 Incubation . 7
7.7 Measurements. 8
8 Validity criteria. 8
9 Interpretation of data. 8
9.1 Plotting growth curves. 8
9.2 Calculation of percentage inhibition . 8
9.3 Determination of E C . 9
r x
10 Expression of results. 9
11 Interpretation of results . 9
12 Reproducibility. 10
13 Test report . 10
Annex A (informative) Preparation of dilution series of mixtures in seawater (effluents or
elutriates) . 12
Bibliography . 14

© ISO 2004 – All rights reserved iii

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ISO/DIS 10253
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 10253 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2004 – All rights reserved

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DRAFT INTERNATIONAL STANDARD ISO/DIS 10253

Water quality — Marine algal growth inhibition test with
Skeletonema costatum and Phaeodactylum tricornutum
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.
1 Scope
This International Standard specifies a method for the determination of the inhibition of growth of unicellular
marine algae by substances and mixtures contained in seawater.
The method can be used for testing substances that are readily soluble in water and are not significantly
degraded or eliminated from the test.
NOTE With modifications as described in ISO/DIS 14442 and ISO 5667-16, the inhibitory effects of poorly soluble
organic and inorganic materials, volatile compounds, metals, effluents, marine water samples and elutriates of sediments
can be tested.
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
1)
ISO/DIS 14442 , Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials,
volatile compounds, metals and waste water
1)
ISO/TS 20281 , Water quality — Guidance on statistical interpretation of ecotoxicity data
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
cell density
number of cells per unit volume of medium (x cells/ml)

1) Under preparation.
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ISO/DIS 10253
3.2
specific growth rate
1 dx
proportional rate of increase in cell density per unit of time : ´ (1/day)
x dt
3.3
growth medium
mixture of seawater and nutrients which is used for pre-cultures and controls
3.4
test medium
mixture of seawater, nutrients (growth medium 3.3) and test material in which algal cells are incubated
3.5
test batch
mixture of seawater, nutrients and test material (test medium 3.4) inoculated with algae
3.6
control
mixture of seawater, nutrients (growth medium 3.3) without test material, inoculated with algae
3.7
effective concentration, E C
r x
concentration of test substance which results in a x % reduction in specific growth rate relative to the controls
4 Principle
Mono-specific algal strains are cultured for several generations in a defined medium containing a range of
concentrations of the test substance, prepared by mixing appropriate quantities of nutrient concentrate,
seawater, stock solutions of the test substance, and an inoculum of exponentially growing algal cells. The test
solutions are incubated for a period of 72 h ± 2 h, during which the cell density in each is measured at
intervals of at least every 24 h ± 2 h. Inhibition is measured as a reduction in specific growth rate, relative to
control cultures grown under identical conditions.
5 Materials
5.1 Test organisms
Use either of the following marine algae :
a) Skeletonema costatum (Greville) Cleve (CCAP 1077/1C, NIVA BAC 1, ISTPM P4) ; or
b) Phaeodactylum tricornutum Bohlin (CCAP 1052/1A, SAG 1090-1a , NIVA BAC 2, ISTPM P1).
These algae are important and widely distributed phytoplankton species (phylum Bacillariophyta) in estuarine
and coastal areas.
The strains recommended are available in unialgal, non-axenic cultures from the following sources.
NIVA Norwegian Institute for Water Research
P.O Box 173 Kjelsås
N-0411 0slo
Norway
2 © ISO 2004 – All rights reserved

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ISO/DIS 10253
ISTPM INERIS
9, rue de Rocroy
75010 Paris
France
CCAP Dunstaffnage Marine Laboratory
P O Box 3 Oban
Argyll PA37 1QA
United-Kingdom
SAG Collection of Algal Cultures
University of Göttingen
Albrecht-von-Haller Institute for Plant Science
Untere Karspüle 2
37073 Göttingen
Germany
NOTE 1 Stock cultures may be maintained in the medium described in 7.1. Regular subculturing is necessary. Weekly
intervals may be necessary for Skeletonema, every two or three weeks may be sufficient for Phaeodactylum. The stock
cultures may also be maintained for extended periods on richer algal media such as those recommended by the culture
collection. It is recommended to keep the stock culture in the medium described in 7.1 and in an exponential growth phase
immediately before preparing the pre-culture for testing as described in 7.2.
NOTE 2 Like many freshwater algae, the diatom Phaeodactylum tricornutum can also be stored for several months in
alginate beads, without losing its viability [1]. The algae can be liberated from the algal beads when needed to perform the
2)
toxicity tests .
5.2 Water
All water used in the preparation of the synthetic seawater, growth medium and test substance solutions shall
be deionized or of equivalent purity. Take special care to avoid contamination of the water by inorganic or
organic substances during preparation and storage. Equipment made of copper shall not be used.
5.3 Seawater
For culturing and testing Phaeodactylum, the growth medium (7.1) is made up by adding nutrients to either
natural or synthetic seawater of salinity 30 ± 5 g/kg. For Skeletonema, the use of natural seawater may be
necessary for the long-term maintenance of cultures, and may also be necessary for the test medium because
a synthetic seawater medium may not always support sufficient growth to meet the test quality criteria. If
natural seawater is used, care shall be taken to ensure that it is not polluted.
Prepare synthetic seawater with the composition given in Table 1 (approximate salinity = 33 g/kg). All the
chemicals used shall be of analytical grade.

2) The algae beads supplied by MICROBIOTESTS Inc., Venecoweg 19, 9810 Nazareth, Belgium, Tel (32) 9 380 8545,
Fax (32) 9 380 8546, Email microbiotests@skynet.be, are an example of a suitable commercially available product. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
product. Equivalent products may be used if they can be shown to lead to the same res ults.
© ISO 2004 – All rights reserved 3

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ISO/DIS 10253
Tabl
...