EN ISO 10253:2024

SLOVENSKI STANDARD
01-december-2024
Nadomešča:
SIST EN ISO 10253:2017
Kakovost vode - Preskus zaviranja rasti morskih alg s Skeletonema sp. in
Phaeodactylum tricornutum (ISO 10253:2024)
Water quality - Marine algal growth inhibition test with Skeletonema sp. and
Phaeodactylum tricornutum (ISO 10253:2024)
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema sp. und
Phaeodactylum tricornutum (ISO 10253:2024)
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec
Skeletonema sp. et Phaeodactylum tricornutum (ISO 10253:2024)
Ta slovenski standard je istoveten z: EN ISO 10253:2024
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 10253
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2024
EUROPÄISCHE NORM
ICS 13.060.70 Supersedes EN ISO 10253:2016
English Version
Water quality - Marine algal growth inhibition test with
Skeletonema sp. and Phaeodactylum tricornutum (ISO
10253:2024)
Qualité de l'eau - Essai d'inhibition de la croissance des Wasserbeschaffenheit - Wachstumshemmtest mit
algues marines avec Skeletonema sp. et Phaeodactylum marinen Algen Skeletonema sp. und Phaeodactylum
tricornutum (ISO 10253:2024) tricornutum (ISO 10253:2024)
This European Standard was approved by CEN on 29 June 2024.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10253:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 10253:2024) 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 European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2025, and conflicting national standards shall
be withdrawn at the latest by January 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 10253:2016.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 10253:2024 has been approved by CEN as EN ISO 10253:2024 without any modification.

International
Standard
ISO 10253
Fourth edition
Water quality — Marine algal
2024-07
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 10253:2024(en) © ISO 2024
ISO 10253:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 10253:2024(en)
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 Reagents .4
5.2.1 Water .4
5.2.2 Hydrochloric acid or sodium hydroxide solution .4
5.2.3 Synthetic sea water .4
5.2.4 Nutrient stock solution .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 EC(r) .9
x
10 Expression of results . 9
11 Interpretation of results . . 9
12 Test report . 10
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
Annex D (informative) Marine algal growth inhibition test with Phaeodactylum tricornutum
applied in 24-well microwell plates . 19
Bibliography .26

iii
ISO 10253:2024(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 230, Water analysis, in accordance with the Agreement on technical cooperation between
ISO and CEN (Vienna Agreement).
This fourth edition cancels and replaces the third edition (ISO 10253:2016), which has been technically
revised.
The main changes are as follows:
— in Table 2, K PO has been substituted with K HPO in stock solution 3;
3 4 2 4
— Annex D has been added to describe the marine algal growth inhibition test with Phaeodactylum
tricornutum applied in 24-well-microwell plates;
— in Table A.1, dilution step 5 was changed to dilution step 6.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
International Standard ISO 10253:2024(en)
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.
IMPORTANT — It is absolutely essential that tests conducted according to this document be carried
out by suitably qualified 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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
cell density
number of cells per unit volume of medium
−1
Note 1 to entry: The cell density is expressed as x cells ml .

ISO 10253:2024(en)
3.2
specific growth rate
µ
proportional rate of increase in cell density (3.1) per unit of time:
1 dx
μ=×
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 and 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 (3.2) relative to the
controls
Note 1 to entry: The EC value is determined based on the specific growth rate (r).
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.
5 Materials
5.1 Test organisms
Use either of the following marine algae:
a) Skeletonema sp. (CCAP 1077/1C, NIVA BAC 1); or
b) Phaeodactylum tricornutum Bohlin (CCAP 1052/1A, SAG 1090-1a, NIVA BAC 2).

ISO 10253:2024(en)
NOTE 1 Editions of this document before the year 2016 suggested the use of two strains of Skeletonema costatum.
Following a taxonomic review of the Skeletonema genus, several strains originally identified as S. costatum can in fact
be other species. In light of this and to enable continuity in the use of previously accepted strains, since the 2016 edition
of this document, the reference from Skeletonema costatum to Skeletonema sp. has changed to avoid nonconformity for
laboratories that possibly use different strains.
These algae are important and widely distributed phytoplankton species (phylum Bacillariophyta) in
estuarine and coastal areas.
1)
Algae in unialgal, non-axenic cultures are available 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
Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG)
Nikolausberger Weg 18
37073 Goettingen
Germany
Stock cultures may be maintained in the medium described in 7.1. Regular subculturing is necessary. Weekly
intervals can be necessary for Skeletonema sp., every two or three weeks can 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 or in (ninefold) concentrated growth medium (Annex D). It is
1) This information is given for the convenience of users of this document and does not constitute an endorsement by
ISO. The stock cultures are not commercial products, but they are provided by the given sources, and they are tested and
validated for the method described in this document. Equivalent products may be used if they can be shown to lead to the
same results.
ISO 10253:2024(en)
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 Concentrated cultures of the diatom Phaeodactylum tricornutum can also be stored for several months
without losing their viability. Stock cultures for the toxicity tests can easily be prepared from the stored concentrated
2)
cultures .
5.2 Reagents
5.2.1 Water
All water used in the preparation of the synthetic sea water, 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.
5.2.2 Hydrochloric acid or sodium hydroxide solution
If pH-adjustment is necessary (7.1, 7.4) use hydrochloric acid or sodium hydroxide solution:
— hydrochloric acid: HCl, e.g. c(HCl) = 1 mol/l; or
— sodium hydroxide: NaOH, c(NaOH) = 1 mol/l.
5.2.3 Synthetic sea water
For culturing and testing Phaeodactylum tricornutum, the growth medium (7.1) is made up by adding
nutrients to either natural [salinity of (30 ± 5) g/kg] or synthetic (approximate salinity of 33 g/kg) sea
water. For Skeletonema sp., the use of natural sea water can be necessary for the long-term maintenance
of cultures and can also be necessary for the test medium, because a synthetic sea water medium doesn't
always support sufficient growth to meet the test quality criteria. If natural sea water is used, care shall be
taken to ensure that it is not polluted.
Prepare synthetic sea water with the composition given in Table 1 (approximate salinity = 33 g/kg). All the
chemicals used shall be of analytical grade.
Table 1 — Synthetic sea water
Salt Concentration of salt in synthetic sea water
g/l
NaCl 22,0
MgCl ⋅6H O 9,7
2 2
Na SO (anhydrous) 3,7
2 4
CaCl (anhydrous) 1,0
KCl 0,65
NaHCO 0,20
H BO 0,023
3 3
Filter the sea water (synthetic as well as natural one) through a 0,45 µm membrane filter to remove particulate
material and algae. Salinity of the (synthetic) sea water can be measured with e.g. a refractometer.
5.2.4 Nutrient stock solution
Prepare three nutrient stock solutions in water, with the compositions given in Table 2.
2) Concentrated Phaeodactylum tricornutum cultures can be supplied by MicroBioTests Inc. Mariakerke-Gent, Belgium.
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 results.

ISO 10253:2024(en)
Table 2 — Nutrient stock solutions
Nutrient Concentration in stock solution Final concentration in test solution
Stock solution 1
FeCl ⋅6H O 48 mg/l 149 µg/l (Fe)
3 2
MnCl ⋅4H O 144 mg/l 605 µg/l (Mn)
2 2
ZnSO ⋅7H O 45 mg/l 150 µg/l (Zn)
4 2
CuSO ⋅5H O 0,157 mg/l 0,6 µg/l (Cu)
4 2
CoCl ⋅6H O 0,404 mg/l 1,5 µg/l (Co)
2 2
H BO 1 140 mg/l 3,0 mg/l (B)
3 3
Na EDTA 1 000 mg/l 15,0 mg/l
Stock solution 2
Thiamin hydrochloride 50 mg/l 25 µg/l
Biotin 0,01 mg/l 0,005 µg/l
Vitamin B (cyanocobalamin) 0,10 mg/l 0,05 µg/l
Stock solution 3
K HPO 2,46 g/l 2,46 mg/l; 0,438 mg/l P
2 4
NaNO 50,0 g/l 50,0 mg/l; 8,24 mg/l N
Na SiO ⋅5H O 14,9 g/l 14,9 mg/l; 1,97 mg/l Si
2 3 2
NOTE During the preparation of stock solution 3, difficulties can occur in the solubilisation of the silicates with K HPO .
2 4
Therefore, a 14,9 g/l Na SiO ⋅5H O solution can be prepared separately as “stock solution 4”. In this case, add 1 ml of this solution
2 3 2
when preparing the growth medium (7.1).
These stock solutions shall be diluted (see 7.1 and Annex A) to obtain the final nutrient concentrations in the
test solutions.
All the chemicals used shall be of reagent grade quality.
Sterilize stock solutions by filtration through a 0,2 µm membrane filter. Stock solutions 1 and 3 may also be
sterilized by autoclaving at 120 °C for at least 15 min.
Store the stock solutions in the dark at (5 ± 3) °C for a maximum of two months.
6 Apparatus
All equipment which comes into contact with the test medium shall be made of glass or a chemically inert
material.
Use usual laboratory equipment and in addition the following.
6.1 Temperature-controlled cabinet or room, with a white, fluorescent light providing continuous even
illumination, suitable for the lighting requirements specified for the test in 7.6.
6.2 Apparatus for measuring algal cell density, preferably a particle counter or a microscope with a
counting chamber (e.g. Neubauer improved chamber).
Alternatively, determine the state of growth of the algal cultures by an indirect procedure using for
instance a fluorimeter (e.g. in vitro fluorescence, Reference [4]), when sufficiently sensitive and if shown
to be sufficiently well correlated with the cell density. The apparatus used shall be capable of accurately
measuring cell densities as low as the inoculum cell density and to distinguish between algal growth and
disturbing effects, for example, the presence of particulate matter and colour of the sample.
Annex D describes a procedure to perform the test in 24-well-microwell plates with in vivo chlorophyll
fluorimetric determination of algal growth of the species Phaeodactylum tricornutum in a microplate reader.

ISO 10253:2024(en)
4 −1
Spectrophotometers can be sufficiently sensitive to measure 10 algal cells ml providing a sufficient path
length (up to 10 cm) can be used. However, this technique is particularly sensitive to interferences from
suspended material and coloured substances at low cell densities.
Annex B describes a procedure to perform the spectrophotometric measurements of the algal cell density.
6.3 Culture flasks, e.g. conical flasks of capacity 250 ml, with air-permeable stoppers.
6.4 Apparatus for membrane filtration, filters of mean pore diameter 0,2 µm and 0,45 µm.
6.5 Autoclave.
6.6 pH meter.
6.7 Apparatus for salinity, e.g. a refractometer.
7 Procedure
7.1 Preparation of growth medium
Add 15 ml of nutrient stock solution 1, 0,5 ml of nutrient stock solution 2 and 1 ml of nutrient stock solution 3
(Table 2) to approximately 900 ml of natural or synthetic sea water (5.2.3) and then make up to 1 l with the
same sea water (5.2.3).
Adjust the pH to 8,0 ± 0,2 by adding diluted hydrochloric acid or sodium hydroxide solution (5.2.2).
NOTE Complexing of heavy metals by the relatively high concentration of EDTA present in the nutrient medium
can preclude the testing of effluents containing heavy metals. For guidance, see ISO 14442.
7.2 Preparation of pre-culture and inoculum
A pre-culture shall be started two to four days before the beginning of the test (see Note 2 in 5.1).
Add sufficient cells from the algal stock culture to the growth medium (7.1) to obtain a sufficiently low cell
3 −1 4 −1
density of, e.g. 2 × 10 algal cells ml to 10 algal cells ml for three days pre-culturing, in order to maintain
exponential growth until the start of the test. The pre-culture shall be incubated under the same conditions
as those in the test. Measure the cell density in the pre-culture immediately before use, to calculate the
required inoculum volume.
7.3 Choice of test concentrations
Algae should be exposed to concentrations of the test substance in a geometric series with a ratio not
exceeding 3,2 (e.g. ratio of 1,8: 1,0 mg/l, 1,8 mg/l, 3,2 mg/l, 5,8 mg/l and 10,4 mg/l).
The concentrations should be chosen to obtain at least one inhibition below and one inhibition above the
intended EC(r) parameter. Additionally, at least two levels of inhibition between 10 % and 90 % should be
x
included to provide data for regression analysis.
NOTE A suitable concentration range is best determined by carrying out a preliminary range-finding test
covering several orders of magnitude of difference between test concentrations. Replication of test concentrations is
not a requirement in the preliminary test.
7.4 Preparation of test substance stock solutions
Prepare stock solutions by dissolving the test substance in growth medium (7.1). Modifications are required
when the test substance does not readily dissolve in the test medium, as described in ISO 14442 and
ISO 5667-16.
ISO 10253:2024(en)
Use a concentrated growth medium as specified in Annex A and, if necessary, when testing water samples
(effluents, elutriates, etc.), to avoid growth inhibition due to low salinity. Use seawater salts (5.2.3) to adjust
the salinity of the sample to the salinity of the growth medium. An example of a dilution scheme for sea
water samples is given in Annex A.
Carry out the test without adjusting the pH after addition of the test substance. However, some substances
can exert a toxic effect due to extreme acidity or alkalinity. To determine the toxicity of a substance
independent of pH, adjust the pH of the stock solution (before the dilution in series) to 8,0 ± 0,2, using either
hydrochloric acid or sodium hydroxide solution (5.2.2). The concentration of acid or base should be such that
the volume change is as small as possible.
7.5 Preparation of test and control batches
Prepare the test batches by mixing the appropriate volumes of test substance stock solutions (7.4), growth
medium (7.1) and inoculum (7.2) in the test vessels. The total volume, the concentration of added growth
medium nutrients and the cell density shall be the same in all test batches.
The initial cell density shall be sufficiently low to allow for exponential growth in the control culture
throughout the test duration, or for at least the time required to achieve a factor 16 increase of cell density,
without a pH drift of more than 1,0 pH units (see Clause 8). Therefore, the initial cell densities shall not
4 −1
exceed 10 algal cells ml .
A lower initial cell density (three to fivefold lower) is recommended for Skeletonema sp. due to its higher cell
volume and growth rate. Take into account the chain-formation of Skeletonema sp. when determining the
initial cell density.
Prepare at least three replicates for each test substance concentration. To a further six vessels, add only
growth medium and inoculum with no test substance. These vessels serve as controls.
If appropriate (e.g. environmental, coloured or turbid samples), prepare a concentration series, single vessels
only, of the test substance without algae to serve as a background for the cell density determinations.
The test design may be altered, based on statistical consideration, to increase the number of concentrations
and reduce the number of replicates per concentration.
Measure the pH of samples of each concentration of the test solution and of the controls.
7.6 Incubation
The test vessels shall be sufficiently covered to avoid airborne contamination and to reduce water
evaporation, but they shall not be airtight to allow CO to enter the vessels. Incubate the test vessels at a
nominal temperature of 20 °C, under continuous white light. The temperature shall not vary by more than
2 °C during the test. The photon fluence rate at the average level of the test solutions shall be uniform and in
2 2
the range 60 µmol/m s to 120 µmol/m s, when measured in the photosynthetically effective wavelength
range of 400 nm to 700 nm using an appropriate receptor.
NOTE 1 It is important to note that the method of measurement, and in particular the type of receptor (collector),
affects the measured value. Spherical receptors (which respond to direct and reflected light from all angles above
and below the plane of measurement) and “cosine” receptors (which respond to light from all angles above the
measurement plane) are preferred to unidirectional receptors and give higher readings for a multi-point light source
of the type described in Note 2.
NOTE 2 The light intensity specified above can be obtained using between four to seven fluorescent lamps (power
rating 30 W) of the universal white, natural type, i.e. a rated colour of standard colour 2 (a colour temperature of
4 300 K) according to IEC 60081 at a distance of approximately 0,35 m from the algal culture medium.
NOTE 3 For light-measuring instruments calibrated in lx, an equivalent range of 3 000 lx to 6 000 lx is acceptable
for the test.
Continuously and gently shake the cultures to keep the cells in free suspension and to facilitate CO mass
transfer from air to water, and in turn, reduce pH shift.

ISO 10253:2024(en)
7.7 Measurements
Measure the cell density in each test vessel, including the controls, at least every (24 ± 2) h. These
measurements are usually made on small volumes which are removed from the test solution and not
replaced. Before measurement, the test batches should be mixed thoroughly.
The test shall last for (72 ± 2) h. At the end of the test, measure the pH of each test batch (7.5) and of the
controls (7.5). Confirm the appearance of the cells and the identity of the test organism by microscopy.
8 Validity criteria
Consider the test valid if the following conditions are met.
a) The control cell density shall have increased by a factor of more than 16 in 72 h. This increase
−1
corresponds to a specific growth rate (9.2) of 0,9 d .
NOTE The growth rate of the algae under the specified conditions can vary among different strains of
−1
the species. Results from validation interlaboratory trials indicate that growth rates above 1,0 d are usually
obtained with both species.
b) The variation coefficient of the control specific growth rates should not exceed 7 %.
c) The control pH shall not have increased by more than 1,0 during the test.
Variations in pH during the test can have a significant influence on the results and therefore a limit
of ± 1,0 unit is set. These variations should always be kept as low as possible, for example, by continuous
shaking during the test.
9 Interpretation of data
9.1 Plotting growth curves
Tabulate the cell density measurements, or other parameters correlated with cell density in the test media,
according to the concentration of test sample and the time of measurement.
Plot a growth curve for each test concentration and control, as a graph of the logarithm of the mean cell
density against time. A linear growth curve indicates exponential growth, whereas a levelling off indicates
that cultures have entered the stationary phase.
If the control cultures show declining growth rate towards the end of the exposure period, inhibited cultures
may tend to catch up with the controls, falsely indicating a decreased growth inhibiting effect. In this case,
perform the calculations of growth rate and growth inhibition based on the last measurement within the
exponential growth period in the control cultures.
9.2 Calculation of percentage inhibition
Calculate first the average specific growth rate, μ, for each test culture, using Formula (1):
lnNN−ln
L 0
μ= (1)
tt−
L 0
ISO 10253:2024(en)
where
t is the time of test start;
t is the time of test termination or the time of the last measurement within the exponential growth
L
period in the control (9.1);
N is the nominal initial cell density;
N is the measured cell density at time t .
L L
Alternatively, determine the growth rate from the slope of the regression line in a plot of the logarithm of
the mean cell density against time (9.1).
Calculate mean values of μ for the control. Calculate the percentage inhibition for each individual test flask
using Formula (2):
μμ−
c i
I =×100 (2)
μi
μ
c
where
I is the percentage inhibition (growth rate) for test flask i;
μi
μ is the growth rate for test flask i;
i
μ
is the mean growth rate for the control.
c
9.3 Determination of EC(r)
x
Tabulate and plot for each individual flask the percentage inhibition (I ) against the test concentration on a
μi
logarithmic scale. If the scatter of data points is large, plot means of replicates with corresponding standard
deviations.
Fit a suited nonlinear model to the experimental data points by regression analysis (for example, see
References [2], [5] and [6]) to determine EC(r) values, preferably with their confidence intervals.
x
If data are too few or uncertain for regression analysis, or if inhibitions appear not to follow a regular
concentration response relation (e.g. stimulation occurs), then a graphical method may be applied. In this
case, draw a smooth eye-fitted curve of the concentration response relationship and read EC(r) values from
x
this graph.
10 Expression of results
Denote EC and EC values based on growth rate as EC(r) and EC(r) . Also, clearly indicate the time span
10 50 10 50
used for the determination, for example, EC(r) (0 h to 72 h). Quote EC(r) and EC(r) values, usually in
50 10 50
milligrams per litre (mg/l), millilitres per litre (ml/l), or %.
11 Interpretation of results
EC and EC values are toxicological data derived from a laboratory experiment carried out under defined
10 50
standard conditions. They give an indication of potential hazards but cannot be used directly to predict
effects in the natural environment. When interpreting EC and EC values, take into account the shape
10 50
of the growth curves. Certain features of these curves (for example, delayed onset of growth, good initial
growth that is not sustained) can help to indicate the mode of action of the toxic substance concerned.

ISO 10253:2024(en)
12 Test report
This test report shall contain at least the following information:
a) the test method used, together with a reference to this document, i.e. ISO 10253:2024;
b) all data required for identification of the test sample, e.g. test substance chemical identification data;
c) test organism: species, origin, strain number, method of cultivation;
d) test details:
— start date and duration;
— method of preparations;
— nominal and measured concentrations tested;
— composition of medium;
— source and salinity of sea water;
— culturing apparatus and incubation procedure;
— light intensity and quality;
— temperature in temperature-controlled cabinet or incubator;
— pH of test solutions at the start and end of the test;
— method for measuring cell density, and, if appropriate, method to correct for background values;
e) results:
— cell density in each test vessel at each measuring point;
— mean cell density for each test concentration (and control) at each measuring point;
— growth curves (logarithm of cell density against time);
— relationship between concentration and effect (percentage inhibition values against concentration)
in table or graphical representation: for example, percentage inhibition on probit-scaled ordinate
against concentration on logarithmic-scaled abscissa;
— EC(r) value and method of determination;
— EC(r) value and method of determination;
— other observed effects;
— if appropriate, results of positive controls, control chart.

ISO 10253:2024(en)
Annex A
(informative)
Preparation of dilution series of mixtures in sea water(effluents or
elutriates)
A.1 General
When testing mixtures in sea water (wastewater or elutriates) in dilution series, the natural or synthetic
sea water (see 5.2.3) should be used as dilution water and added nutrient concentrations should be the
same in all dilutions and equal to the final test concentration stated in 5.2.3. For that reason, the use of a
concentrated growth medium is recommended.
A.2 Preparation of concentrated growth medium
Add 135 ml of stock solution 1, 4,5 ml of stock solution 2 and 9 ml of stock solution 3 to approximately
700 ml natural or synthetic sea water (5.2.3) and then make up to 1 l with the same sea water.
Adjust the pH to 8,0 ± 0,2 by adding diluted hydrochloric acid or sodium hydroxide solution (5.2.2).
A.3 Preparation of the dilution series of test media
Prepare the dilution series of test media by mixing the volumes of concentrated growth medium (A.2),
inoculum (7.2), sample (effluent or elutriates) and dilution water (natural or synthetic sea water 5.2.3) in the
test vessels following the scheme of Table A.1. The total volume should be the same in all the vessels.
For further instruction concerning initial cell density and test design, follow 7.5.
Table A.1 — Preparation of dilution series — Concentration of test and control batch
Dilution Dilution step Inoculum Sample Sea water Concentrated End volume
D (7.2) (7.4) (5.2.3) growth medium
(A.2)
ml ml ml ml ml
1 in 1,25 1 10 80 ‒ 10 100
1 in 2 2 10 50 30 10 100
1 in 3 3 10 33,33 46,67 10 100
1 in 4 4 10 25 55 10 100
1 in 6 6 10 16,67 63,33 10 100
1 in
...