EN 17899:2024

SLOVENSKI STANDARD
01-december-2024
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
Qualité de l'eau - Détermination spectrophotométrique de la teneur en chlorophylle a par
extraction à l'éthanol pour la surveillance de routine de la qualité de l'eau
Ta slovenski standard je istoveten z: EN 17899:2024
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17899
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2024
EUROPÄISCHE NORM
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
Qualité de l'eau - Détermination spectrophotométrique Wasserbeschaffenheit - Bestimmung des Chlorophyll-
de la teneur en chlorophylle a par extraction à l'éthanol a-Gehalts durch Ethanolextraktion für die
pour la surveillance de routine de la qualité de l'eau routinemäßige Überwachung der Wasserqualität
This European Standard was approved by CEN on 10 June 2024.

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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17899:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Interferences . 7
6 Reagents . 7
7 Apparatus . 8
8 Procedure. 9
8.1 General . 9
8.2 Sample pre-treatment . 9
8.3 Filtration . 9
8.4 Extraction . 9
8.5 Measurement . 10
9 Quality assurance . 10
9.1 Blank values . 10
9.2 pH after acidification . 10
9.3 Wavelength setting . 10
9.4 Ratio A/A' . 11
10 Calculations . 11
11 Expression of results . 12
12 Test report . 12
Annex A (informative) Performance data . 13
A.1 Interlaboratory trial study in the Netherlands: . 13
A.2 Validation interlaboratory trial in Germany: . 13
Annex B (informative) Theoretical backgrounds . 16
B.1 Meaning of the determination of the chlorophyll-a content . 16
B.2 Acidifying extracts . 16
B.3 Calculation of Phaeopigment concentration . 16
Annex C (informative) Control samples . 17
C.1 Introduction . 17
C.2 Surface water . 17
C.3 Frozen spinach extracts . 17
C.4 Freeze-dried kale . 17
C.5 Algae culture. 18
Annex D (informative) SCOR-Unesco method for determination of chlorophyll in sea-water . 19
D.1 Measurement of chlorophyll a, b, c and d with SCOR-Unesco method using chromatic
equations . 19
D.2 Performance characteristics and comparison of methods . 20
Bibliography . 21
European foreword
This document (EN 17899:2024) has been prepared by the 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 January 2025, and conflicting national standards shall
be withdrawn at the latest by January 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
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.
The chlorophyll-a content depends on the species composition of the phytoplankton, the time of day and
season, 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 of 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. As extracting agent hot ethanol is used. Various other extractants (e.g. acetone or methanol) are
described in the literature, but these may have lower extraction efficiency or are toxicologically
problematic.
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.
Annexes A, B, C and D of this document are for information only.
1 Scope
This document describes a spectrophotometric method for determining the chlorophyll-a content
corrected for phaeopigments as a measure of the amount of phytoplankton for all types of surface water
including marine water. The determination limit is usually 2 µg/l to 5 µg/l and is calculated by each
laboratory individually. It can be as low as 0,5 µg/l using 2 l of sample (or even more) and a 50 mm
cuvette.
NOTE In some measurement programs like marine studies on time series data and ecological
status/classification no correction for phaeopigments is used and acidification is omitted, e.g. as recommended by
OSPAR.
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.
EN ISO 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples
(ISO 5667-3)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://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 absorbance at 665 nm in an ethanolic solution corrected for 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
EXAMPLE Carotinoids, phycobilins, chlorophyll-b and chlorophyll-c.
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 absorbance 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 chlorophyll-a
concentration of the water sample corrected for phaeopigments is calculated with the formula defined
by Lorenzen (see Reference [12]).
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 [8]). 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, turbidity may occur which impairs the precision
of the photometric measurement; it shall be removed by filtration or centrifugation (see 8.4). The same
applies to turbidity in the extract caused by other reasons (e.g. after acidification).
Pigments of (rarely occurring) autotrophic bacteria (e.g. chlorobium-chlorophyll, “bacterioviridin”) may
influence the determination of chlorophyll-a.
6 Reagents
Use reagents of an analytical pure quality and deionized or destilled water.
6.1 Ethanol, C H OH, volume fraction 96 %.
2 5
Pure ethanol and ethanol denatured with methanol or methyl ethyl keton (MEK) do not interfere with
the determination of chlorophyll-a and can also be used.
6.1.1 Ethanol, C H OH, volume fraction 90 %.
2 5
Add 60 ml of water to 900 ml of ethanol 96 % (6.1).
6.2 Hydrochloric acid, HCl, 12 mol/l, ρ = 1,19 g/ml.
6.2.1 Hydrochloric acid, HCl, 0,4 mol/l.
Add 10 ml of concentrated hydrochloric acid (6.2) to 290 ml of water.
6.3 Ammoniumacetate solution, C H NO , 0,1 mol/l.
2 7 2
7 Apparatus
Special attention using laboratory equipment and devices is needed to minimize the influence of UV light.
7.1 Vacuum pump, preferably diaphragm vacuum pump with barometer, with suction bottle with a
volume of 2 l to 5 l, with silicone or rubber stopper.
7.2 Filtration apparatus for water samples with tightly closing funnel, preferably for filter diameters
of 45 mm to 55 mm.
7.3 Filtration apparatus for extracts, e.g. Witt’s pot to accommodate a volumetric flask, 200 mm high
and internal width 150 mm, with side tube for hose attachment, flat ground lid with central tube, rough
ground for rubber stopper connection, and filter attachment for round filters with low dead volume (e.g.
perforated plate as filter support).
NOTE The filtration apparatus can be protected from excessive incidence of light by means of an opaque film
or by painting it - with the exception of a viewing window.
7.4 Glass fibre filter, made of borosilicate glass, without binding agent, separation efficiency > 98 %
for particles > 0,7 µm, recommended diameter 45 mm to 55 mm.
7.5 Filters for clearing of extracts, ash-free, slow-filtering paper filters with high separation
efficiency or cellulose acetate membrane filters with a pore size of 0,45 µm.
Cellulose nitrate filters cannot be used because of losses of chlorophyll-a.
7.6 Extraction vessel, protected from light, tightly closable, e.g. wide neck bottles or vials made of
(amber) glass with screw cap, nominal volume approx. 30 ml to 100 ml. If clarification of the extract is
done by centrifugation, glass vials or centrifuge tubes should be used.
7.7 Centrifuge, with an acceleration of at least 3000 g, suitable for glass vials or centrifuge tubes with
tightly fitting screw caps, nominal volume 15 ml to 50 ml; light protected (e.g. made of brown
polypropylene), also transparent when working in darkened rooms.
7.8 Shaking water bath, set at (75 ± 1) °C.
7.9 Spectrophotometer, suitable for absorbance measurements at 665 nm and 750 nm, with the
following recommended performance characteristics: spectral bandwidth ≤ 2 nm, photometric
accuracy ≤ ± 0,005 at 1 abs. (decadic absorbance), wavelength accuracy ≤ ± 1 nm.
NOTE Suitable devices are in particular split-beam, dual-beam or reference beam path spectrophotometers.
Devices with an integrated self-test to check for their correct function are recommended.
7.10 Rectangular cuvette, with a path length of 10 mm to 50 mm (or even 100 mm can be used).
NOTE In most cases, the use of 50 mm cuvettes will be suitable.
7.11 Freezer, with a temperature lower than −18 °C.
7.12 Freezer, with a temperature lower than −80 °C.
7.13 pH meter with electrode, suitable for pH measurements in alcohol.
8 Procedure
8.1 General
Direct sunlight or bright artificial light shall be avoided in all steps of the procedure. The extraction and
the analysis shall take place in subdued light in the laboratory. Execution in a darkened room with low
light is recommended. The illuminance should not exceed the level necessary for safe working with dark-
adapted eyes. Execution in a room with UV free lamps is recommended.
8.2 Sample pre-treatment
The water samples are preserved according to EN ISO 5667-3 and transported at (5 ± 3) °C in the dark.
Macroscopic plant material and/or water fleas can contribute to the chlorophyll-a content. If present,
pre-filtering shall be carried out over plankton gauze of a mesh width of 1 mm. The water samples should
be filtered as quickly as possible, at the latest 24 h after the sample was taken. Freezing of the water
samples is not permitted.
8.3 Filtration
The filtration system shall be clean before use and rinsed with water. After the water sample has been
sufficiently homogenized, a volume (V ) appropriate to the required absorbance range is filtered through
a glass fibre filter (7.4) preferably in subdued light. Algae deposited on the wall of the filter bowl shall be
rinsed with water to be collected on the filter. With high concentrations chlorophyll-a, 100 m
...