EN ISO 10253:1998
(Main)Water quality - Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum (ISO 10253:1995)
Water quality - Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum (ISO 10253:1995)
Migrated from Progress Sheet (TC Comment) (2000-07-10): Parallel voting procedure ISO/CEN
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema costatum und Phaeodactylum tricornutum (ISO 10253: 1995)
Diese Internationale Norm legt ein Verfahren zur Bestimmung toxischer Effekte chemischer Verbindungen auf das Wachstum mariner Algen fest. Das Verfahren kann auf in Wasser gut lösliche Substanzen angewendet werden, die vor der Untersuchung weder signifikant abgebaut noch eliminiert werden. Anmerkung 1: Mit geringfügigen Änderungen kann das Verfahren auch auf zur Bestimmung der Hemmwirkung von Abwässern angewendet werden, siehe jedoch die Anmerkung zur Tabelle 2.
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec Skeletonema costatum et Phaeodactylum tricornutum (ISO 10253:1995)
Kakovost vode – Preskus zaviranja rasti morskih alg s Skeletonema costatum in Phaeodactylum tricornutum (ISO 10253:1995)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10253:2000
01-januar-2000
Kakovost vode – Preskus zaviranja rasti morskih alg s Skeletonema costatum in
Phaeodactylum tricornutum (ISO 10253:1995)
Water quality - Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum (ISO 10253:1995)
Wasserbeschaffenheit - Wachstumshemmtest mit marinen Algen Skeletonema costatum
und Phaeodactylum tricornutum (ISO 10253: 1995)
Qualité de l'eau - Essai d'inhibition de la croissance des algues marines avec
Skeletonema costatum et Phaeodactylum tricornutum (ISO 10253:1995)
Ta slovenski standard je istoveten z: EN ISO 10253:1998
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 10253:2000 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10253:2000
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SIST EN ISO 10253:2000
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SIST EN ISO 10253:2000
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SIST EN ISO 10253:2000
INTERNATIONAL
STANDARD
First edition
1995-10-01
- Marine algal growth
Water quality
inhibition test with Skeletonema costatum
and Phaeodactylum tricornutum
- Essai d’inhibition de Ia croissance des algues marines
Quake de I’eau
avec Skeletonema costatum et Phaeodactylum tricornutum
Reference number
iSQ 10253:1995(E)
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SIST EN ISO 10253:2000
IISO 10253:1995(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. Esch 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
(1 EC) on all matters of electrotechnical standardization.
Draft International Standards adopted by the technical committees are
circulated to the member bodies for voting. Publication as an International
Standard requires approval by at least 75 % of the member bodies casting
a vote.
International Standard ISO 10253 was prepared by Technical Committee
lSO/TC 147, Water quality, Subcommittee SC 5, Biological methods.
Annex A of this International Standard is for information only.
0 ISO 1995
All rights reserved. Unless otherwise specified, no part of this pubkation may be reproduced
or utilized in any form or by any means, electrorxc or mechanlcal, Inciuding photocopying and
mlcrofilm. wlthout Permission in wntlng from the publisher.
Intern ational Organization for Standa rdization
56 0 CH-121
Case Postale 1 Geneve 20 l Switzerland
Printed In Swltzertand
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SIST EN ISO 10253:2000
INTERNATIONAL STANDARD 0 ISO ISO 10253:1995(E)
Water quality - Marine algal growth inhi.bition test
with Skeletonema costatum and Phaeodactylum
tricornutum
3.3 growth rate: Expression of rate of increase in
1 Scope
cell density with respect to time.
This International Standard specifies a method for the
See 8.2.2.
determination of the toxic effects of Chemical com-
pounds on the growth of marine algae.
3.4 test solution: Mixture of seawater, nutrients
and test substance in which algal cells are incubated.
The method tan be used for testing substances which
are readily soluble in water and are not significantly
3.5 control: Mixture of seawater, nutrients and algal
degraded or eliminated from the test.
cells without test substance.
NOTE 1 With minor changes, the method tan also be
used to determine the inhibitory effects of effluents. See
3.6 effective concentration, EC10 or EC50: The
however the note to table 2.
concentration of test substance which results in re-
spectively a 10 % or 50 % reduction in either growth
or growth rate relative to the controls.
2 Principle
3.7 no observed effect concentration, NOEC: The
highest concentration tested at which there is no
Monospecific algal cells are cultured for several gen-
statistically significant reduction of growth or growth
erations in a defined medium containing a range of
rate relative to the controls.
concentrations of the test substance, prepared by
mixing appropriate quantities of nutrient concentrate,
seawater, stock solutions of the test substance, and
4 Materials
an inoculum of exponentially growing algal cells. The
test solutions are incubated for a minimum period of
72 h, during which the cell density in each is meas-
4.1 Test organisms
ured at intervals of at least every 24 h. Inhibition is
measured as a reduction in growth, or growth rate,
Use either of the following marine algae.
relative to control cultures grown under identical con-
ditions.
a) Skeletonema costatum (Greville) Cleve (CCAP
1077/lC, NIVA BAC 1, ISTPM P4 - Bouin).
or
3 Definitions
b) Phaeodactylum tricornutum Bohlin
(CCAP
For the purposes of this International Standard, the
1052/1 A - Oban, 1090/1A Göttingen, NIVA BAC
following definitions apply.
2, ISTPM Pl).
3.1 ceIl density: Number of cells per unit volume.
These algae are important and widely distributed
pianktonic phytoplankton species (Phylum
3.2 growth: Increase in cell density. Bacillariophyta) in estuarine and coastai areas.
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SIST EN ISO 10253:2000
ISO 10253:1995(E)
The strains recommended are available in unialgal,
non-axenic cultures from the following sources: Table 1 - Synthetic seawater
Concentration of salt in
N IVA:
Norwegian Institute for Water Research
Salt
synthetic seawater
P.O. Box 173 Kjelsas
sll
N-O41 1 Oslo
Norway
22
NaCI
ISTPM Pl
MgCI,GH,O
9,7
ISTPM P4 - I I
Bouin: INERIS
Na,SO, (anhydrous)
3,7
r---- 1
9, rue de Rocroy
CaCI, (anhydrous)
w
l- 1
75010 Paris
France
KCI 0,65
l-
1
CCAP: Dunstaffnage Marine Laboratory
NaHCO, 0,20
r- 1
P.0 Box 3 Oban
Salts of H,BO, 0,023
r- 1
Argyll PA34 4AD
United Kingdom
Sterilize the seawater by membrane filtration (5.4).
Göttingen: Collection of Algal Cultures
Institute of Plant Physiology
University of Göttingen
Nikolausberger Weg 18
4.4 Nutrients
D-3400 Göttingen
Germany
Prepare three nutrient stock solutions in water, with
the compositions given in table 2.
NOTE 2 Stock cultures may be maintained in the medium
(see 4.3 and 6.1). Regular subculturing is necessary. Weekly
intervals may be necessary for Skeletonema; every two or
Table 2 - Nutrient stock solutions
three weeks may be sufficient for Phaeodactylum.
Final
Nutrient Concentration
in stock concentration
solution in test
4.2 Water
Solution
Stock Solution 1
All water used in the preparation of the synthetic
FeCI,.GH,O 48 mg/1
149 pg/l Fe)
seawater, nutrient medium and test substance sol-
MnCl,.4H,O 144 mg/1 605 pg/l (Mn)
utions shall be deionized or of equivalent purity. Take
ZnS0,.7H,O 45 mg/1
150 cig/l (Zn)
special care to avoid contamination of the water by
CuS0,5H,O 0,157 mg/1
08 pg/l Ku)
inorganic or organic substances during preparation
C0CI,~6H,0 0,404 mg/1
1,5 pg/l (Co)
and storage. Equipment made of topper shall not be
1 140 mg/1
17J cig/l
HW3
used.
Na,EDTA 1) 1 000 mg/1
15,O l-g/1
Stock Solution 2
Thiamin hydrochlo-
4.3 Seawater
50 mg/1
25 l-4
ride
Biotin 0,Ol mg/1
0,005 cigll
For culturing and testing Phaeodactylum, the medium
Vitamin B,,
0,lO mg/1
0,05 lall
(6.1) is made up by adding nutrients to either natura1
(cyanocobalamin)
or synthetic seawater. For Skeletonema, the use of
Stock Solution 3
natura1 seawater is necessaty for the long-term main-
tenance of cultures, and may also be necessary for
3,O g/l 3,O mg/1
Km4
50,O mg/1
NaNO,
the test medium because a synthetic seawater me- 50,O 911
Na,SiO,-5H,O 14,9 mg/1
149 g/l
dium may not always support sufficient growth to
meet the test quality criteria. If natura/ seawater [of
1) Complexing of heavy metals by the relatively high
salinity 30 %O @/VI) + 5 %O (m/m)] is used, care shall
concentration of EDTA present in the nutrient medium
be taken to ensure that it is not polluted.
may preclude the testing of effluents containing heavy
metals.
Prepare synthetic seawater with the composition
given in table 1.
NOTE 3 These stock solutions will eventually be diluted
(see 6.1) to obtain the final nutrient concentrations In the
All the chemicals used shall be of analytical grade.
test solutions.
2
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SIST EN ISO 10253:2000
cJ ISO
ISO ~o~53:1995(E)
All the chemicals used shall be of analytical grade.
6.2 Preparation of inoculum
Sterilize stock solutions 1 and 3 by autoclaving at
The algal inoculum for the test shall be taken from an
120 “C for at least 15 min, and stock Solution 2 by
exponentially growing pre-culture. The pre-culture
membrane filtration (5.4).
shall be set up 3 d k 1 d before the Start of the test,
as follows.
Store the solutions in the dark at 4 “C.
Add sufficient cells from the algal stock culture to the
culture medium (6.1) to obtain an initial cell density
5 Apparatus
of approximately 2 x IO3 to IO4 cells per millilitre.
Maintain the pre-culture under the same conditions
All equipment which will come into contact with the
as those in the test (see 6.6) for 3 d + 1 d. After this,
test medium shall be made of glass or a chemically
the pre-culture should be in exponential growth and
inert material.
of sufficient cell density to be used as an inoculum for
Normal laboratory apparatus and
the test. Measure the cell density in the pre-culture
immediately before use (see 6.7) in Order to calculate
the required inoculum volume.
5.1 Temperature-controlled cabinet or room, with
a white fluorescent light providing continuous even il-
lumination, suitable for the lighting requirements
6.3 Choice of test concentrations
specified for the test in 6.6.
The concentrations of substance to be tested shall
normally follow a geometric Progression, for example
5.2 Apparatus for measuring algal cell density,
10 mg/l; 3,2 mg/l; 1 ,0 mg/l; 0,32 mg/l; . . . . 0,Ol mg/l.
preferably a particle counter, or a microscope with a
counting chamber. Alternatively, determine the state
If possible, the concentrations shall be Chosen to ob-
of growth of the algal cultures by an indirect pro-
tain several (i.e. 4 or 5) levels of inhibition of growth
cedure using a spectrometer, turbidimeter or
ranging from less than 10 % to greater than 90 %.
fluorimeter, when sufficiently sensitive and if shown
to be sufficiently well correlated with the cell density.
NOTE 4 A suitable concentration range is best deter-
The apparatus used shall be capable of accurately
mined by carrying out a preliminary range-finding test
measunng cell densities as low as IO4 cells per milli-
covering several orders of magnitude of differente between
litre and to distinguish between algal growth and dis-
test concentrations. Replication of test concentrations is
turbing effects, for example the presence of unnecessary during this preliminary test.
particulate matter and colour of the Sample.
6.4 Preparation of test substance stock
5.3 Culture flasks, for example conical flasks of ca-
Solution
pacity 250 ml, with air-permeable Stoppers.
Prepare stock solutions of the test substance, where
necessary, in the algal growth medium by dilution.
5.4 Apparatus for membrane filtration, with filters
The concentration of test substance in the stock sol-
of mean pore diameter 0,2 Pm.
utions shall be such that, when added to the test
vessels containing growth medium inoculated with
5.5 Autoclave.
the algae, the intended range of test concentrations
is obtained.
5.6 pH-meter.
Normally, the test shall be carried out without adjust-
ing the pH. However, some substances may exert a
6 Procedure
toxic effect due to extreme acidity or alkalinity. In or-
der to determine the toxicity of a substance inde-
6.1 Preparation of culture medium
pendent of pH, adjust the pH of the master stock
Solution (before the dilution in series) to that of the
Add 15
...
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