SIST EN ISO 8692:2012

SLOVENSKI STANDARD
SIST EN ISO 8692:2012
01-junij-2012
1DGRPHãþD
SIST EN ISO 8692:2005
.DNRYRVWYRGH3UHVNXV]DYLUDQMDUDVWLVODGNRYRGQLKDOJ]HQRFHOLþQLPL]HOHQLPL
DOJDPL,62
Water quality - Freshwater algal growth inhibition test with unicellular green algae (ISO
8692:2012)
Wasserbeschaffenheit - Süßwasseralgen-Wachstumshemmtest mit einzelligen
Grünalgen (ISO 8692:2012)
Qualité de l'eau - Essai d'inhibition de la croissance des algues d'eau douce avec des
algues vertes unicellulaires (ISO 8692:2012)
Ta slovenski standard je istoveten z: EN ISO 8692:2012
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 8692:2012 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 8692:2012

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SIST EN ISO 8692:2012


EUROPEAN STANDARD
EN ISO 8692

NORME EUROPÉENNE

EUROPÄISCHE NORM
February 2012
ICS 13.060.70 Supersedes EN ISO 8692:2004
English Version
Water quality - Fresh water algal growth inhibition test with
unicellular green algae (ISO 8692:2012)
Qualité de l'eau - Essai d'inhibition de la croissance des Wasserbeschaffenheit - Süßwasseralgen-
algues d'eau douce avec des algues vertes unicellulaires Wachstumshemmtest mit einzelligen Grünalgen (ISO
(ISO 8692:2012) 8692:2012)
This European Standard was approved by CEN on 14 February 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8692:2012: E
worldwide for CEN national Members.

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SIST EN ISO 8692:2012
EN ISO 8692:2012 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 8692:2012
EN ISO 8692:2012 (E)
Foreword
This document (EN ISO 8692:2012) 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 August 2012, and conflicting national standards shall be withdrawn at
the latest by August 2012.
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 8692:2004.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 8692:2012 has been approved by CEN as a EN ISO 8692:2012 without any modification.

3

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SIST EN ISO 8692:2012

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SIST EN ISO 8692:2012
INTERNATIONAL ISO
STANDARD 8692
Third edition
2012-02-15
Water quality — Fresh water algal growth
inhibition test with unicellular green algae
Qualité de l’eau — Essai d’inhibition de la croissance des algues d’eau
douce avec des algues vertes unicellulaires
Reference number
ISO 8692:2012(E)
©
ISO 2012

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and media . 2
6 Apparatus . 4
7 Procedure . 5
7.1 Preparation of growth medium . 5
7.2 Preparation of pre‑culture and inoculum . 5
7.3 Choice of test sample concentrations . 5
7.4 Preparation of test sample and stock solutions . 5
7.5 Preparation of test and control batches . 6
7.6 Incubation . 6
7.7 Measurements . 7
8 Validity criteria . 7
9 Calculation . 7
9.1 Plotting of growth curves . 7
9.2 Calculation of percentage inhibition . 8
9.3 Determination of E C (e.g. E C and E C ) . 8
r x r 10 r 50
10 Expression of results . 8
11 Interpretation of results . 9
12 Precision . 9
13 Test report . 9
Annex A (informative) Rapid screening of waste water algal growth inhibition . 11
Annex B (informative) Test procedure with algae from algal beads, with direct measurement of algal
growth in spectrophotometric cells .14
Annex C (informative) Procedure for immobilization of algae in alginate beads .19
Bibliography .21
© ISO 2012 – All rights reserved iii

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SIST EN ISO 8692:2012
ISO 8692:2012(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. 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.
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.
ISO 8692 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.
This third edition cancels and replaces the second edition (ISO 8692:2004), which has been technically revised.
iv © ISO 2012 – All rights reserved

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SIST EN ISO 8692:2012
INTERNATIONAL STANDARD ISO 8692:2012(E)
Water quality — Fresh water algal growth inhibition test with
unicellular green algae
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International Standard 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 in accordance with this International
Standard be carried out by suitably qualified staff.
1 Scope
This International Standard specifies a method for the determination of the growth inhibition of unicellular
green algae by substances and mixtures contained in water or by waste water. This method is applicable for
substances that are easily soluble in water.
With modifications to this method, as specified in ISO 14442 and ISO 5667-16, the inhibitory effects of poorly
soluble organic and inorganic materials, volatile compounds, heavy metals and waste water can be tested.
A rapid algal growth inhibition screening test for waste water is described in Annex A.
An alternative test procedure with algae from algal beads, with direct measurement of algal growth in
spectrophotometric cells, is described in Annex B.
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/TR 11044, Water quality — Scientific and technical aspects of batch algae growth inhibition tests
ISO 14442, Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile
compounds, metals and waste water
ISO/TS 20281, Water quality — Guidance on statistical interpretation of ecotoxicity data
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
cell density
n
number of cells per volume of medium
NOTE Cell density is expressed in cells per millilitre.
© ISO 2012 – All rights reserved 1

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
3.2
effective concentration
concentration of the test sample (EC ) at which an effect of x % is measured, if compared to the control
x
NOTE To unambiguously denote an EC value deriving from growth rate, it is proposed to use the symbol “E C”.
r
3.3
lowest ineffective dilution
LID
dilution level at which no inhibition, or only effects not exceeding the test-specific variability, are observed
[13]
NOTE Adapted from ISO 15088:2007 , 3.5.
3.4
specific growth rate
m
proportional rate of increase in cell density per time:
1dn
μ =
n dt
where
n is the cell density, expressed in cells per millilitre;
t is the time, expressed in days.
−1
NOTE Specific growth rate is expressed in reciprocal days (day ).
[ISO/TR 11044:2008, 3.2]
4 Principle
Monospecies algal strains are cultured for several generations in a defined medium containing a range of
concentrations of the test sample, prepared by mixing appropriate quantities of growth medium, test sample,
and an inoculum of exponentially growing algal cells. The test batches are incubated for a period of (72 ± 2) h
during which the cell density in each test solution is measured at least every 24 h.
Inhibition is measured as a reduction in specific growth rate relative to control cultures grown under
identical conditions.
5 Reagents and media
5.1 Test organism, using either of the following planktonic fresh water algae species:
1) 2)
a) Desmodesmus subspicatus (R. Chodat) E. Hegewald et A. Schmidt (86.81 SAG );
3)
TM 2) 2) 2)
b) Pseudokirchneriella subcapitata (Korshikov) Hindak (ATCC® 22662 , CCAP 278/4 or 61.81 SAG ).
NOTE 1 The two species do not show identical responses to toxic agents.
NOTE 2 Both algae species are planktonic green algae belonging to the order of Sphaeropleales (Chlorophyta,
Chlorophyceae) and are usually unicellular in culture.
1) This species is formerly known as Scenedesmus subspicatus Chodat.
2) Trade names of strains are examples of suitable strains available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of these products.
3) This species is formerly known as Selenastrum capricornutum Prinz. The new name is currently cited by culture
collections.
2 © ISO 2012 – All rights reserved

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
The strains recommended are available in unialgal, non-axenic cultures from the following collections:
— SAG — Sammlung von Algenkulturen Göttingen [Göttingen Algal Culture Collection], Germany
www.epsag.uni-goettingen.de (viewed 2012-01-30);
— ATCC — American Type Culture Collection, USA
www.atcc.org (viewed 2012-01-30);
— CCAP — Culture Collection of Algae and Protozoa, UK
www.ccap.ac.uk (viewed 2012-01-30);
— ALCP — Algothèque du Laboratoire de Cryptogamie, France
www.mnhn.fr (viewed 2012-01-30).
Stock cultures can be maintained in the medium specified in 5.3. and 7.1. However, frequent subculturing is
necessary (once a week) to prevent failure of growth. The stock culture can be maintained for extended periods
on richer algal media such as those recommended by the culture collection.
4)
Alternatively, algae can be stored for several months on agar plates or in alginate beads without losing their
[1]
viability . The algae can be easily recovered from the agar or liberated from the algal beads (see Annex C)
when needed to perform the toxicity tests.
The appearance of the cells and the identity of the test organisms should be confirmed by microscopy.
5.2 Water, deionized or of equivalent purity (conductivity <10 µS/cm), for use in the preparation of the growth
medium and test substance solutions.
Take special care to avoid contamination of the water by inorganic or organic substances during preparation
and storage. Do not use equipment made of copper.
5.3 Nutrients
Prepare four nutrient stock solutions in water, according to the compositions given in Table 1.
These solutions are eventually diluted (see 7.1 and 7.4) to achieve the final nutrient concentrations in the test
solutions. However, the macronutrients may instead be added directly to the water.
All chemicals used shall be of reagent-grade quality.
Sterilize the stock solutions by membrane filtration (mean pore diameter 0,2 µm) or by autoclaving [(120 ± 2) °C,
15 min]. Store the solutions in the dark at 4 °C.
Do not autoclave stock solution 4 in order to avoid evaporative loss of NaHCO , but sterilize it by membrane filtration.
3
4) The algal beads supplied by MicroBioTests Inc., Mariakerke-Gent, Belgium are an example of a suitable product
available commercially. 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 the validity criteria specified in this document are
fulfilled.
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SIST EN ISO 8692:2012
ISO 8692:2012(E)
Table 1 — Mass concentrations of nutrients in the test solution
Final mass
Mass concentration
Stock solution Nutrient concentration
in stock solution
in test solution
NH Cl 1,5 g/l 15 mg/l (N: 3,9 mg/l)
4
MgCl⋅6H O 1,2 g/l 12 mg/l (Mg: 2,9 mg/l)
2 2
1: Macronutrients CaCl⋅2H O 1,8 g/l 18 mg/l (Ca: 4,9 mg/l
2 2
MgSO⋅7H O 1,5 g/l 15 mg/l (S: 1,95 mg/l)
4 2
KH PO 0,16 g/l 1,6 mg/l (P: 0,36 mg/l)
2 4
FeCl⋅6H O 64 mg/l 64 µg/l (Fe: 13 µg/l)
3 2
2: Fe-EDTA
Na EDTA⋅2H O 100 mg/l 100 µg/l
2 2
a
H BO 185 mg/l 185 µg/l (B: 32 µg/l)
3 3
MnCl⋅4H O 415 mg/l 415 µg/l (Mn: 115 µg/l)
2 2
ZnCl 3 mg/l 3 µg/l (Zn: 1,4 µg/l)
2
3: Trace elements
CoCl⋅6H O 1,5 mg/l 1,5 µg/l (Co: 0,37 µg/l)
2 2
CuCl⋅2H O 0,01 mg/l 0,01 µg/l (Cu: 3,7 ng/l)
2 2
Na MoO⋅2H O 7 mg/l 7 µg/l (Mo: 2,8 µg/l)
2 4 2
4: NaHCO NaHCO 50 g/l 50 mg/l (C: 7,14 mg/l)
3 3
a
H BO can be dissolved by the addition of 0,1 mol/l NaOH.
3 3
6 Apparatus
All equipment that comes in contact with the test medium shall be made of glass or other chemically inert material.
Usual laboratory apparatus and, in particular, the following.
6.1 Temperature‑controlled cabinet or room, with white fluorescent light, providing continuous, uniform
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 capable of counting particles
in the size range 2,5 µm to 25 µm (spherical diameters), or a microscope and a counting chamber.
Alternatively, the algal densities may be determined by an indirect procedure using for instance a fluorimeter
[2] 5) [3]
(e.g. in vitro fluorescence or DCMU -enhanced in vivo fluorescence ), when sufficiently sensitive and if
shown to be sufficiently well correlated with cell density. The apparatus used shall be capable of measuring
4
cell densities as low as 10 cells/ml and of distinguishing between algal growth and disturbing effects, e.g. the
presence of particulate matter and the colour of the sample. Spectrophotometers may be sufficiently sensitive
4
to measure 10 cells/ml, providing a sufficient pathlength (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
(see ISO/TR 11044).
6.3 Culture vessels (glass), e.g. 250 ml conical flasks with air-permeable stoppers.
6.4 Apparatus for membrane filtration, using filters of mean pore diameter 0,2 µm.
6.5 Autoclave.
6.6 pH meter.
5) DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea (CAS No. 330-54-1).
4 © ISO 2012 – All rights reserved

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
7 Procedure
7.1 Preparation of growth medium
Prepare a growth medium by adding an appropriate volume of the nutrient stock solutions (5.3) to water (5.2).
Add to approximately 500 ml of water (5.2):
— 10 ml of stock solution 1 (5.3);
— 1 ml of stock solution 2 (5.3);
— 1 ml of stock solution 3 (5.3);
— 1 ml of stock solution 4 (5.3).
Make up to 1 000 ml with water.
Before use, equilibrate the medium by leaving overnight in contact with air, or by bubbling filtered air through it
for 30 min. After equilibration, adjust the pH if necessary to 8,1 ± 0,2, using either 1 mol/l hydrochloric acid or
1 mol/l sodium hydroxide solution.
This growth medium is buffered by hydrogencarbonate and atmospheric CO . Different pH values may be
2
−
obtained by modifying the concentration of HCO and/or the atmospheric CO concentration (requires closed
2
3
vessels) as specified in ISO 14442. Should such modifications be required in order to perform a test at a
different, specific pH value, these should be clearly motivated and reported.
7.2 Preparation of pre‑culture and inoculum
A pre-culture shall be started 2 d to 4 d before the beginning of the test. Growth medium (7.1) is inoculated
3 4
at a sufficiently low cell density (e.g. 5 × 10 cells/ml to 10 cells/ml for 3 d pre-culturing) in order to maintain
exponential growth until test start. The pre-culture shall be incubated under the same conditions as those in
the test (7.6).
This exponentially growing pre-culture is used as an inoculum for 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 sample concentrations
Algae should be exposed to concentrations of the test sample 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 E C parameter. Additionally, at least two levels of inhibition between 10 % and 90 % should be
r x
included in order to provide data for regression analysis.
A limit test with only one concentration can be conducted to demonstrate absence of toxicity. The number of
replicates for this one concentration should be at least six.
In case the “lowest ineffective dilution” (LID) of a waste water is to be determined, the following dilution series
shall be used: 1:1,25, 1:2, 1:3, 1:4, 1:6, 1:8, 1:12.
NOTE A suitable concentration range is best determined by carrying out a preliminary range-finding test covering
several orders of magnitude of difference in test concentration. Replication of test concentrations is not a requirement in
the preliminary test.
7.4 Preparation of test sample and stock solutions
Test sample may be aqueous (e.g. waste water) or non-aqueous (e.g. chemical substance or mixture of
chemicals) for which the inhibitory effects on the growth of algae shall be determined.
© ISO 2012 – All rights reserved 5

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
If the test sample is aqueous (e.g. waste water), pre-treatment (e.g. filtration, neutralization) should be considered
depending on the nature of the sample and the purpose of the test. Add nutrient stock solutions (5.3), prepared
in accordance with 7.1, to the sample.
For non-aqueous test samples, preparation of stock solutions is generally necessary. The method for preparation
of the stock solutions should be carefully chosen based on the properties of the sample. Stock solutions are
usually prepared by dissolving the test sample in growth medium. Modifications are necessary when the test
sample does not readily dissolve in the growth medium as specified in ISO 14442 and ISO 5667-16.
Usually, the test shall be carried out without adjustment of the pH of the medium after addition of the test
sample. However, some substances may exert a toxic effect due to extreme acidity or alkalinity. In order to
determine the toxicity of a sample independent of pH, adjust the pH of the aqueous sample or stock solution
(before the dilution in series) to that of the culture medium using either 1 mol/l hydrochloric acid or 1 mol/l
sodium hydroxide solution (see ISO 5667-16).
7.5 Preparation of test and control batches
Prepare the test batches by mixing the appropriate volumes of test sample or test sample stock solutions (7.4),
growth medium (7.1) and inoculum (7.2) in the test vessels. The total volume, concentrations of added growth
medium nutrients and cell density shall be the same in all vessels. Prepare at least three replicate batches for
each test sample concentration.
The initial cell density shall be sufficiently low to allow exponential growth in the control culture throughout the
test duration without a pH drift of more than 1,5 pH units (see Clause 8). Therefore, the initial cell densities shall
4
not exceed 10 cells/ml.
Prepare six replicate control batches by adding the appropriate volume of inoculum to growth medium.
Measure the pH of a replicate batch at each test concentration and in one control replicate.
If appropriate, prepare a single concentration series of the test sample without algae to serve as background
for the cell density determinations.
The number of replicates per concentration can be reduced based on statistical considerations (see
ISO/TS 20281), if increasing the number of concentrations and reducing the concentration spacing.
If chemicals are tested for registration purposes, the exposure concentration at the start, during, and at the end
of exposure shall be verified by specific chemical analysis. This can require preparation of additional batches
[4]
for analysis. Further information can be found in OECD 201 .
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 (a small hole is sufficient). Incubate the
2
test vessels at (23 ± 2) °C, under continuous white light. The light intensity at the average level of the test media
2 2
shall be homogeneous within ±10 % 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, in particular the type of receptor (collector), affects
the measured value. Spherical receptors (which respond to 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. They give higher readings for a multipoint light source of the type
described in the Note.
NOTE The light intensity specified in the first paragraph of this subclause can be obtained using four to six fluorescent
lamps of the universal white (natural) type, i.e. a rated colour of standard colour 2 (colour temperature of 4 300 K). The
optimum distance of the lamps is approximately 0,35 m from the algal culture medium.
For light-measuring instruments calibrated in lux, an equivalent range of 6 000 lx to 10 000 lx is acceptable
for the test.
6 © ISO 2012 – All rights reserved

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SIST EN ISO 8692:2012
ISO 8692:2012(E)
Testing of coloured test solutions requires specific modifications as specified in ISO 14442.
Continuously
...