ISO 10253:2016

INTERNATIONAL ISO
STANDARD 10253
Third edition
2016-11-15
Water quality — Marine algal
growth inhibition test with
Skeletonema sp. and Phaeodactylum
tricornutum
Qualité de l’eau — Essai d’inhibition de la croissance des algues
marines avec Skeletonema sp. et Phaeodactylum tricornutum
Reference number
©
ISO 2016
© ISO 2016, Published in Switzerland
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ii © ISO 2016 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Materials . 3
5.1 Test organisms . 3
5.2 Reagents. 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 . 7
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 EC(r) .
x 9
10 Expression of results . 9
11 Interpretation of results . 9
12 Test report . 9
Annex A (informative) Preparation of dilution series of mixtures in sea water(effluents
or elutriates) .11
Annex B (informative) Test procedure starting from stored algal inocula, and with direct
measurement of algal growth in spectrophotometric cells .12
Annex C (informative) Performance data .18
Bibliography .19
Foreword
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The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
This third edition cancels and replaces the second edition (ISO 10253:2006), which has been technically
revised.
iv © ISO 2016 – All rights reserved

INTERNATIONAL STANDARD ISO 10253:2016(E)
Water quality — Marine algal growth inhibition test with
Skeletonema sp. and Phaeodactylum tricornutum
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this document be
carried out by suitably trained staff.
1 Scope
This document specifies a method for the determination of the inhibition of growth of the unicellular
marine algae Skeletonema sp. and Phaeodactylum tricornutum by substances and mixtures contained in
sea water or by environmental water samples (effluents, elutriates, etc.).
The method can be used for testing substances that are readily soluble in water and are not significantly
degraded or eliminated in any other way from the test medium.
NOTE With modifications, as described in ISO 14442 and ISO 5667–16, the inhibitory effects of poorly
soluble organic and inorganic materials, volatile compounds, metal compounds, effluents, marine water samples
and elutriates of sediments can be tested.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials,
volatile compounds, metals and waste water
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
cell density
number of cells per unit volume of medium
Note 1 to entry: The cell density is expressed as x cells/ml.
3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1 dx
μ =× (/1 day)
x dt
where
x is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
−1
Note 1 to entry: Specific growth rate is expressed in inverse days (day ).
3.3
growth medium
mixture of sea water and nutrients which is used for pre-cultures and controls
3.4
test medium
mixture of sea water, nutrients [growth medium (3.3)] and test material in which algal cells are
incubated
3.5
test batch
mixture of sea water, nutrients and test material [test medium (3.4)] inoculated with algae
3.6
control
mixture of sea water, nutrients [growth medium (3.3)] without test material, inoculated with algae
3.7
effective concentration
EC(r)
x
concentration of test substance which results in an 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, sea water, stock solutions of the test substance, and an inoculum of exponentially growing
algal cells. The test solutions are incubated for a period of (72 ± 2) h, during which the cell density
in each is measured at intervals of at least every (24 ± 2) h. Inhibition is measured as a reduction in
specific growth rate, relative to control cultures grown under identical conditions.
2 © ISO 2016 – All rights reserved

5 Materials
5.1 Test organisms
Use either of the following marine algae:
1)
a) Skeletonema sp. (CCAP 1077/1C, NIVA BAC 1); or
b) Phaeodactylum tricornutum Bohlin (CCAP 1052/1A, SAG 1090-1a, NIVA BAC 2).
These algae are important and widely distributed phytoplankton species (phylum Bacillariophyta) in
estuarine and coastal areas.
The recommended algae are available in unialgal, non-axenic cultures from the following sources.
NIVA
Norwegian Institute for Water Research
Gaustadaléen 21
N 0349 Oslo
Norway
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
Stock cultures may be maintained in the medium described in 7.1. Regular subculturing is necessary.
Weekly intervals may be necessary for Skeletonema sp., every two or three weeks may be sufficient
for Phaeodactylum tricornutum. 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
1) The previous editions of this document suggested the use of two strains of Skeletonema costatum. Following a
taxonomic review of the Skeletonema genus, several strains originally identified as S. costa
...