oSIST prEN ISO 10253:2023

Water quality - Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum (ISO/DIS 10253:2023)

ISO 10253:2016 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.

Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema sp. und Phaeodactylum tricornutum (ISO/DIS 10253:2023)

Dieses Dokument legt ein Verfahren zur Bestimmung der Wachstumshemmung der einzelligen marinen Algen-Spezies Skeletonema sp. und Phaeodactylum tricornutum durch im Meerwasser enthaltene Substanzen und Substanzgemische oder durch Umweltwasserproben (Abwasser, Eluate usw.) fest.
Das Verfahren kann für die Untersuchung von leicht wasserlöslichen Substanzen angewendet werden, die durch das Testmedium weder signifikant abgebaut noch in irgendeiner anderen Weise aus diesem eliminiert werden.
ANMERKUNG   Mit Änderungen, wie sie in ISO 14442 und ISO 5667 16 beschrieben sind, kann das Verfahren auch zur Bestimmung der Hemmwirkung von schwer wasserlöslichen organischen und anorganischen Stoffen, flüchtigen Verbindungen, Metallverbindungen, Abwässern, Meerwasserproben und Eluaten von Sedimenten.
WARNUNG — Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortung des Arbeitgebers, angemessene Sicherheits- und Schutzmaßnahmen zu treffen und sicherzustellen, dass diese mit nationalen Festlegungen übereinstimmen.
WICHTIG — Es ist erforderlich, bei den Untersuchungen nach diesem Dokument Fachleute oder Facheinrichtungen einzuschalten.

Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec Skeletonema sp. et Phaeodactylum tricornutum (ISO/DIS 10253:2023)

ISO 10253:2016 spécifie une méthode de détermination de l'inhibition de la croissance des algues marines unicellulaires Skeletonema sp. et Phaeodactylum tricornutum provoquée par des substances et des mélanges présents dans l'eau de mer (effluents, élutriats, etc.).
Cette méthode peut être utilisée pour soumettre à l'essai des substances facilement solubles dans l'eau et qui ne sont pas sensiblement dégradées ou éliminées d'autre manière du milieu d'essai.

Kakovost vode - Preskus zaviranja rasti morskih alg s Skeletonema sp. in Phaeodactylum tricornutum (ISO/DIS 10253:2023)

General Information

• Status: Not Published
• Public Enquiry End Date: 02-May-2023
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: KAV - Water quality
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 03-Mar-2023
• Due Date: 21-Jul-2023
• Completion Date: 09-May-2023
• Ref Project: prEN ISO 10253 - Water quality - Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum (ISO/DIS 10253:2023)

Relations

• Revises: SIST EN ISO 10253:2017 - Water quality - Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum (ISO 10253:2016)
• Effective Date: 01-Apr-2023

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 10253:2023
01-april-2023
Kakovost vode - Preskus zaviranja rasti morskih alg s Skeletonema sp. in
Phaeodactylum tricornutum (ISO/DIS 10253:2023)
Water quality - Marine algal growth inhibition test with Skeletonema sp. and
Phaeodactylum tricornutum (ISO/DIS 10253:2023)
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema sp. und
Phaeodactylum tricornutum (ISO/DIS 10253:2023)
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec
Skeletonema sp. et Phaeodactylum tricornutum (ISO/DIS 10253:2023)
Ta slovenski standard je istoveten z: prEN ISO 10253
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 10253:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 10253:2023

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oSIST prEN ISO 10253:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 10253
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:
2023-02-17 2023-05-12
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
ICS: 13.060.70
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
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BEING ACCEPTABLE FOR INDUSTRIAL,
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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
Reference number
NATIONAL REGULATIONS.
ISO/DIS 10253:2023(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023

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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2023(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 10253
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:

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
ICS: 13.060.70
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2023
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
NOT BE REFERRED TO AS AN INTERNATIONAL
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on STANDARD UNTIL PUBLISHED AS SUCH.
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
IN ADDITION TO THEIR EVALUATION AS
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ISO copyright office
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POTENTIAL TO BECOME STANDARDS TO
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NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 10253:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
ii
  © ISO 2023 – All rights reserved
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022

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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
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
5.2.1 Water . 4
5.2.2 Synthetic Sea water . 4
5.2.3 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 . 7
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 . 9
9.3 Determination of EC(r) . 9
x
10 Expression of results . 9
11 Interpretation of results . .10
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 .27
iii
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(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.
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 documents 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).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
This fourth edition cancels and replaces the second edition (ISO 10253:2016), which has been
technically revised.
The main changes compared to the previous edition are as follows:
— in 5.2 Table 2, substitution of K PO 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.
iv
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oSIST prEN ISO 10253:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 10253:2022(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 https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// 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.
1
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
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
Note 1 to entry: The EC value is determined on the basis of 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.
2
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
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

 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 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 or in (ninefold) concentrated growth
medium (Annex D). It is recommended to keep the stock culture in the medium described in 7.1 and in
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. costatum may in fact be other
species. In light of this and to enable continuity in the use of previously accepted strains, the present revision of this
document has changed the reference from Skeletonema costatum to Skeletonema sp. to avoid non-compliance for
labs that may be using different strains.
3
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
an exponential growth phase immediately before preparing the pre-culture for testing as described in
7.2.
NOTE 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
2)
concentrated 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 Synthetic Sea water
For culturing and testing Phaeodactylum tricornutum, the growth medium (7.1) is made up by adding
nutrients to either natural [salinity = (30 ± 5) g/kg] or synthetic sea water (approximate salinity = 33 g/
kg). For Skeletonema sp., the use of natural sea water may be necessary for the long-term maintenance
of cultures and may also be necessary for the test medium, because a synthetic sea water medium may
not 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
Concentration of salt in synthetic sea water
Salt
g/l
NaCl 22
MgCl ⋅6H O 9,7
2 2
Na SO (anhydrous) 3,7
2 4
CaCl (anhydrous) 1,0
2
KCl 0,65
NaHCO 0,20
3
H BO 0,023
3 3
Filter the sea water (synthetic as well as natural one) through a 0,45 µm membrane filter in order to
remove particulate material and algae. Salinity of the (synthetic) sea water can be measured with e.g. a
refractometer.
5.2.3 Nutrients
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.
4
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
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
2
Stock solution 2
Thiamin hydrochloride 50 mg/l 25 µg/l
Biotin 0,01 mg/l 0,005 µg/l
Vitamin B (cyanocobala-
12
0,10 mg/l 0,05 µg/l
min)
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
3
Na SiO ⋅5H O 14,9 g/l 14,9 mg/l; 1,97 mg/l Si
2 3 2
These stock solutions have to 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 4 °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 normal laboratory apparatus 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.
5
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
4
Spectrophotometers may 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 (see Table 2) to approximately 900 ml of natural or synthetic sea water (5.2.2) and then make
up to 1 l with the same sea water.
Adjust the pH to 8,0 ± 0,2 by adding dilute hydrochloric acid or sodium hydroxide solution.
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 in 5.1).
Add sufficient cells from the algal stock culture to the growth medium (7.1) to obtain a sufficiently low
3 4
cell 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, in order 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 %
x
should be included in order 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.
6
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oSIST prEN ISO 10253:2023
ISO/DIS 10253:2022(E)
7.4 Preparation of test substance stock solutions
Prepare stock solutions by dissolving the test substance in growth medium (7.1). Modifications
are necessary when the test substance does not readily dissolve in the test medium, as described in
ISO 14442 and ISO 5667-16.
When testing water samples (effluent, elutriates, etc.), use concentrated growth medium according to
Annex A and, if appropriate, to avoid growth inhibition due to a too low salinity, with sea water salts
(5.2.2) to bring the salinity of the sample up to the salinity of the growth medium. An example of a
dilution scheme for sea water samples is given in Annex A.
Normally, carry out the test without adjusting the pH after addition of the test substance. However,
some substances may exert a toxic effect due to extreme acidity or alkalinity. In order to determine the
toxicity of a substance independent of pH, adjust the pH of the master stock solution (before the dilution
in series) to 8,0 ± 0,2, using either hydrochloric acid or sodium hydroxide solution. The concentration of
acid or base should be such as 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, concentration of added
growth medium nutrients and cell density shall be the same in all test batches.
The initial cell density shall be sufficiently low to allow 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
4
shall not 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 an
...