EN 17899:2024

SLOVENSKI STANDARD
oSIST prEN 17899:2022
01-oktober-2022
Kakovost vode - Spektrofotometrijsko določevanje klorofila-a po ekstrakciji z
etanolom za rutinski monitoring kakovosti vode
Water quality - Spectrophotometric determination of chlorophyll-a content by ethanol
extraction for the routine monitoring of water quality
Wasserbeschaffenheit - Spektrophotometrische Bestimmung des Chlorophyll-a-Gehalts
durch Ethanolextraktion für das Routinemonitoring der Wasserqualität
Ta slovenski standard je istoveten z: prEN 17899
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 17899:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 17899:2022
oSIST prEN 17899:2022
DRAFT
EUROPEAN STANDARD
prEN 17899
NORME EUROPÉENNE
EUROPÄISCHE NORM
September 2022
ICS 13.060.70; 71.040.50
English Version
Water quality - Spectrophotometric determination of
chlorophyll-a content by ethanol extraction for the routine
monitoring of water quality
Wasserbeschaffenheit - Spektrophotometrische
Bestimmung des Gehalts an Chlorophyll-a
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 230.
If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17899:2022 E
worldwide for CEN national Members.

oSIST prEN 17899:2022
prEN 17899:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Interferences . 6
6 Reagents . 6
7 Apparatus . 7
8 Procedure. 8
8.1 General . 8
8.2 Sample pre-treatment . 8
8.3 Filtration . 8
8.4 Extraction . 8
8.5 Measurement . 9
9 Quality assurance . 9
9.1 Blank values . 9
9.2 pH after acidification . 9
9.3 Wavelength setting . 9
9.4 Ratio E /E . 9
n z
10 Calculations . 10
11 Expression of results . 11
12 Test report . 11
Annex A (informative) Performance characteristics . 12
Annex B (informative) Theoretical backgrounds . 15
Annex C (informative) Control samples . 16
Annex D (informative) SCOR UNESCO 'Determination of photosynthetic pigments in sea-
water' . 17
Bibliography . 19

oSIST prEN 17899:2022
prEN 17899:2022 (E)
European foreword
This document (prEN 17899:2022) has been prepared by the Technical Committee CEN/TC 230 “Water
analysis”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
oSIST prEN 17899:2022
prEN 17899:2022 (E)
Introduction
Chlorophyll-a is the most common essential photosynthetic pigment present in photoautotrophic
plankton organisms. It is the main component of the dynamically regulated photosystem of these
organisms, in which other accessory pigments are involved, some of which are chemically very similar to
chlorophyll-a. Chlorophyll molecules isolated from the photosystem can be broken down by the influence
of light and acids as well as chlorophyllases.
The chlorophyll-a content depends on the species composition of the phytoplankton, the time, the period,
the place and the depth of sampling. It is also suitable for quantifying the change in the algal biomass (cell
proliferation) in biological tests to check the toxicity or productivity of water or substances dissolved in
water.
The chlorophyll concentration of a water sample can provide information about the trophic state of a
water body. It is used as an easily determinable measure of the phytoplankton biomass and is a key
variable in many trophy scoring systems. Even if this value cannot be used as an absolute measure for the
phytoplankton biomass, the determination of the chlorophyll-a concentration together with other
biomass and bioactivity parameters provides information about the quantitative occurrence and the
potential metabolic performance of the phytoplankton in waters.
Due to the sensitivity of chlorophyll to light, acids and enzymes, there is currently no universally
applicable routine analytical method that enables an accurate, artefact-free and at the same time simple
determination of the chlorophyll-a content in water samples containing phytoplankton. The extractive,
spectrophotometric method described in this document therefore provides an operationally defined
value.
WARNING — Persons using this document should be familiar with usual 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.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably qualified staff.
oSIST prEN 17899:2022
prEN 17899:2022 (E)
1 Scope
This document describes a spectrophotometric method for determining the chlorophyll-a content as a
measure of the amount of phytoplankton for all types of water. Assuming a maximum sample volume of
2 l, chlorophyll-a content values of 5 µg/l or more can be determined. The determination limit can be
calculated by each lab individually and it can be as low as 0,5 µg/l using 2 l of sample (or even more) and
a 50 mm cuvette.
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 5667-4, Water quality — Sampling — Part 4: Guidance on sampling from lakes, natural and man-made
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696)
EN ISO 5667-3, Water quality - Sampling - Part 3: Preservation and handling of water samples (ISO 5667-3)
EN ISO 5667-6, Water quality - Sampling - Part 6: Guidance on sampling of rivers and streams (ISO 5667-6)
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:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
chlorophyll-a
natural plant pigment that is present in phytoplankton, most important photosynthetic pigment of
photoautotrophic organisms
Note 1 to entry: For the purposes of this document, the pigment whose concentration can be determined by
measuring the absorption at 665 nm in an ethanolic solution (without phaeopigments).
3.2
phaeopigments
collective term for chlorophyll breakdown products, which do not contain any magnesium
EXAMPLE Phaeophytin is a phaeopigment.
3.3
phytoplankton
community of free-living, suspended, mainly photosynthetic organisms in aquatic systems comprising
cyanobacteria and algae
3.4
accessory pigments
auxiliary pigments that support photosynthesis
EXAMPLES Carotinoids, phycobilins, chlorophyll-b and chlorophyll-c.
oSIST prEN 17899:2022
prEN 17899:2022 (E)
4 Principle
The determination of the chlorophyll-a concentration according to this document is based on the hot
ethanolic extraction of a water sample’s filter residue and the subsequent absorption measurement at
665 nm, whereby phaeopigments are also recorded. After the quantitative conversion of the chlorophyll-
a into phaeopigments by means of acidification and renewed measurement at 665 nm, the original
chlorophyll-a concentration of the water sample is calculated.
5 Interferences
Macroscopic parts of plants in the water sample (e.g. duckweed, drifted benthic filamentous algae, torn
off macrophyte parts) as well as large zooplankton can falsify the measurement result. They shall be
removed from the water sample, if they should not expressly be included.
In the case of water samples with a pH value of < 5, chlorophyll degradation by acid occurs in the filter
residue. To prevent this, the pH value shall be neutralized by rinsing the filter with about 50 ml of a
0,1 molar ammonium acetate solution (see Reference [7]). This should be done shortly before the end of
the filtration, when there are only a few millilitres of sample left in the filter funnel.
Chlorophyll is very sensitive to light, especially in extracted solution. If the extract is exposed to direct
sunlight or bright artificial light, the pigment is destroyed photochemically.
Changes in the concentration of the extractant due to evaporation are to be avoided by working with
firmly sealable extraction vessels.
During the extraction and homogenization of the filters, turbidities occurs which impairs the precision of
the photometric measurement; it shall be removed by filtration (see 8.1) or centrifugation (see 8.1). The
same applies to turbidities in the extract caused by other reaso
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