Water quality - Marine algal growth inhibition test with Skeletonema sp. and Phaeodactylum tricornutum (ISO 10253:2016)

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.

Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema costatum und Phaeodactylum tricornutum (ISO 10253:2016)

Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec Skeletonema sp. et Phaeodactylum tricornutum (ISO 10253:2016)

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 10253:2016)

Ta dokument določa metodo za določevanje zaviranja rasti enoceličnih morskih alg Skeletonema sp. in Phaeodactylum tricornutum s snovmi in zmesmi, ki jih vsebuje morska voda, ali z vzorci okoljske vode (odplake, elutriati itd.).
To metodo je mogoče uporabiti za preskušanje snovi, ki so zlahka topne v vodi in ne razpadejo občutno ali se izločijo na kakršen koli način iz preskusnega medija.
OPOMBA: s spremembami, navedenimi v standardih ISO 14442 in ISO 5667–16, se lahko uporabi za preskušanje zaviralnih učinkov slabo
topnih in anorganskih snovi, hlapnih spojin, kovinskih spojin, odplak, vzorcev morske vode in elutriatov usedlin.

General Information

Status
Published
Public Enquiry End Date
01-Mar-2016
Publication Date
17-May-2017
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Feb-2017
Due Date
09-Apr-2017
Completion Date
18-May-2017

RELATIONS

Buy Standard

Standard
SIST EN ISO 10253:2017
English language
26 pages
sale 10% off
Preview
sale 10% off
Preview

e-Library read for
1 day

Standards Content (sample)

SLOVENSKI STANDARD
SIST EN ISO 10253:2017
01-junij-2017
1DGRPHãþD
SIST EN ISO 10253:2006
Kakovost vode - Preskus zaviranja rasti morskih alg s Skeletonema sp. in
Phaeodactylum tricornutum (ISO 10253:2016)
Water quality - Marine algal growth inhibition test with Skeletonema sp. and
Phaeodactylum tricornutum (ISO 10253:2016)
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema costatum
und Phaeodactylum tricornutum (ISO 10253:2016)
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec
Skeletonema sp. et Phaeodactylum tricornutum (ISO 10253:2016)
Ta slovenski standard je istoveten z: EN ISO 10253:2016
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 10253:2017 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 10253:2017
---------------------- Page: 2 ----------------------
SIST EN ISO 10253:2017
EN ISO 10253
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2016
EUROPÄISCHE NORM
ICS 13.060.70 Supersedes EN ISO 10253:2006
English Version
Water quality - Marine algal growth inhibition test with
Skeletonema sp. and Phaeodactylum tricornutum (ISO
10253:2016)

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 10253:2016) tricornutum (ISO 10253:2016)
This European Standard was approved by CEN on 22 October 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10253:2016 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 10253:2017
EN ISO 10253:2016 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 10253:2017
EN ISO 10253:2016 (E)
European foreword

This document (EN ISO 10253:2016) 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 May 2017, and conflicting national standards shall be

withdrawn at the latest by May 2017.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent

rights.
This document supersedes EN ISO 10253:2006.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 10253:2016 has been approved by CEN as EN ISO 10253:2016 without any modification.

---------------------- Page: 5 ----------------------
SIST EN ISO 10253:2017
---------------------- Page: 6 ----------------------
SIST EN ISO 10253:2017
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 10253:2016(E)
ISO 2016
---------------------- Page: 7 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(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

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

© ISO 2016 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(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 third edition cancels and replaces the second edition (ISO 10253:2006), which has been technically

revised.
iv © ISO 2016 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 10253:2017
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.
© ISO 2016 – All rights reserved 1
---------------------- Page: 11 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)
3.2
specific growth rate
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.
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)

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
---------------------- Page: 12 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)
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).

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. 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.
© ISO 2016 – All rights reserved 3
---------------------- Page: 13 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)

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 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 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 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
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 in order to

remove particulate material and algae.
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 © ISO 2016 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(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
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 PO 3,0 g/l 3,0 mg/l; 0,438 mg/l P
3 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

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.

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.

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.
© ISO 2016 – All rights reserved 5
---------------------- Page: 15 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)

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.
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. 1,0 mg/l, 1,8 mg/l, 3,2 mg/l, 5,6 mg/l and 10 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 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.
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.), spike them with the nutrient stock solutions

(5.2.3) 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

6 © ISO 2016 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 10253:2017
ISO 10253:2016(E)

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

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 Skeletone

...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.