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

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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.

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SIST EN ISO 10253:2017

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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.

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SIST EN ISO 10253:2017
EN ISO 10253:2016 (E)
Contents Page
European foreword . 3

2

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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.
3

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SIST EN ISO 10253:2017

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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

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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

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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

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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

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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

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SIST EN ISO 10253:2017
ISO 10253:2016(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
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

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SIST EN ISO 10253:2017
ISO 10253:2016(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
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

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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
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 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.
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

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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
2
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
12
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
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.
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.
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.
© ISO 2016 – All rights reserved 5

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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 %
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.
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

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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
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 Skeletone
...

SLOVENSKI STANDARD
oSIST prEN ISO 10253:2016
01-februar-2016
Kakovost vode - Preskus zaviranja rasti morskih alg s Skeletonema sp. in
Phaeodactylum tricornutum (ISO/DIS 10253:2015)
Water quality - Marine algal growth inhibition test with Skeletonema sp. and
Phaeodactylum tricornutum (ISO/DIS 10253:2015)
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema costatum
und Phaeodactylum tricornutum (ISO/DIS 10253:2015)
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec
Skeletonema sp. et Phaeodactylum tricornutum (ISO/DIS 10253:2015)
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 10253:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 10253:2016

---------------------- Page: 2 ----------------------
oSIST prEN ISO 10253:2016
DRAFT INTERNATIONAL STANDARD
ISO/DIS 10253
ISO/TC 147/SC 5 Secretariat: DIN
Voting begins on: Voting terminates on:
2015-12-17 2016-03-17
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
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the International Organization for
Standardization (ISO), and processed under the ISO lead mode of collaboration
as defined in the Vienna Agreement.
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Should this draft be accepted, a final draft, established on the basis of comments
received, will be submitted to a parallel two-month approval vote in ISO and
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FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2015

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ISO/DIS 10253:2015(E)

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All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Contents Page
Foreword . iv
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Principle .2
5 Materials .3
6 Apparatus .5
7 Procedure .6
8 Validity criteria .8
9 Interpretation of data .9
10 Expression of results . 10
11 Interpretation of results . 10
12 Precision . 10
13 Test report . 11
Annex A (informative) Preparation of dilution series of mixtures in sea water (effluents or
elutriates) . 13
Annex B (informative) Test procedure starting from stored algal inocula, and with direct
measurement of algal growth in spectrophotometric cells . 14
Bibliography . 21


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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 WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee 5, Biological
methods.
This third edition cancels and replaces the second edition (ISO 10253:2006), which has been technically
revised as follows:
Inclusion of new Annex B (informative) "Test procedure starting from stored algal inocula, and with
direct measurement of algal growth in spectrophotometric cells".



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DRAFT INTERNATIONAL STANDARD ISO/DIS 10253
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 International Standard 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, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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.
3.1
cell density
number of cells per unit volume of medium (x cells/ml)
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3.2
specific growth rate
µ
proportional rate of increase in cell density per unit of time:
1 d𝑥
𝜇 = × (1/day)
𝑥 d𝑡
where
x is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
-1
Note 1 to entry: Specific growth rate is expressed in inverse days (day ).
3.3
growth medium
mixture of sea water and nutrients which is used for pre-cultures and controls
3.4
test medium
mixture of sea water, nutrients (growth medium (3.3)) and test material in which algal cells are
incubated
3.5
test batch
mixture of sea water, nutrients and test material (test medium (3.4)) inoculated with algae
3.6
control
mixture of sea water, nutrients (growth medium (3.3)) without test material, inoculated with algae
3.7
effective concentration
EC(r)
x
concentration of test substance which results in an x % reduction in specific growth rate relative to the
controls
4 Principle
Mono-specific algal strains are cultured for several generations in a defined medium containing a range
of concentrations of the test substance, prepared by mixing appropriate quantities of nutrient
concentrate, sea water, stock solutions of the test substance, and an inoculum of exponentially growing
algal cells. The test solutions are incubated for a period of 72 h ± 2 h, during which the cell density in
each is measured at intervals of at least every 24 h ± 2 h. Inhibition is measured as a reduction in
specific growth rate, relative to control cultures grown under identical conditions.


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5 Materials
5.1 Test organisms
Use either of the following marine algae:
1
a) Skeletonema sp. .(CCAP 1077/1C, NIVA BAC 1); or
b) Phaeodactylum tricornutum Bohlin (CCAP 1052/1A, SAG 1090-1a, NIVA BAC 2).
These algae are important and widely distributed phytoplankton species (phylum Bacillariophyta) in
estuarine and coastal areas.
The recommended algae are available in unialgal, non-axenic cultures from the following sources.
NIVA
Norwegian Institute for Water Research
Gaustadaléen 21
N 0349 Oslo
Norway
CCAP
Dunstaffnage Marine Laboratory
P O Box 3 Oban
Argyll PA37 1QA
United Kingdom
SAG
Collection of Algal Cultures
University of Göttingen
Albrecht-von-Haller Institute for Plant Science
Untere Karspüle 2
37073 Göttingen
Germany
Stock cultures may be maintained in the medium described in 7.1. Regular subculturing is necessary.
Weekly intervals may be necessary for Skeletonema sp., every two or three weeks may be sufficient for
Phaeodactylum tricornutum. The stock cultures may also be maintained for extended periods on richer
algal media such as those recommended by the culture collection. It is recommended to keep the 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
2)
concentrated cultures .

1
The previous editions of this International Standard 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 ISO 10253 has changed the reference from Skeletonema costatum to Skeletonema sp. to avoid
non-compliance for labs that may be using different strains.
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5.2 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.
5.3 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
NaCI 22
MgCI⋅6H O 9,7
2 2
Na SO (anhydrous) 3,7
2 4
CaCI (anhydrous) 1,0
2
KCI 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.
5.4 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,
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.
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Table 2 — Nutrient stock solutions
Nutrient Concentration in stock solution Final concentration in test solution
Stock solution 1
FeCI⋅6H O 48 mg/l 149 µg/l (Fe)
3 2
MnCI⋅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
CoCI⋅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
12
0,10 mg/l 0,05 µg/l
(cyanocobalamin)
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
3
Na2SiO3⋅5H2O 14,9 g/l 14,9 mg/l; 1,97 mg/l Si
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.



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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 [1])), 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.
4
Spectrophotometers may be sufficiently sensitive to measure 10 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.
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 (5.3) 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 cells/ml to 10 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 %
x
should be included in order to provide data for regression analysis.
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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
(5.4) and, if appropriate, to avoid growth inhibition due to a too low salinity, with sea water salts (5.3)
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 found 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 64 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 cells/ml.
A lower initial cell density (three to five-fold 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.


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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 in order to allow CO to enter the vessels. Incubate the test
2
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
2 2
be uniform and in 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.
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 1.
NOTE 1 The light intensity specified above could 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
[2]
temperature of 4 300 K) according to IEC 60081 at a distance of approximately 0,35 m from the algal culture
medium.
NOTE 2 For light-measuring instruments calibrated in lx, an equivalent range of 6 000 lx to 10 000 lx is
acceptable for the test.
Continuously and gently shake the cultures in order to keep the cells in free suspension and to facilitate
CO mass transfer from air to water, and in turn, reduce pH shift.
2
7.7 Measurements
Measure the cell density in each test vessel, including the controls, at least every 24 h ± 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 h ± 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 1 The growth rate of the algae under the specified conditions may vary among different strains of
-1
the species. Results from interlaboratory tests indicate that growth rates above 1,0 d are normally 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 however should always be kept as low as possible, for example,
by performing continuous shaking during the test.
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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 Equation (1):
ln𝑁−ln𝑁
L 0
𝜇 = (1)
𝑡−𝑡
L 0
where
t is the time of test start;
0
t is the time of test termination or the time of the last measurement within the exponential
L
growth period in the control (9.1.);
N is the nomi
...

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