EN 16695:2015
(Main)Water quality - Guidance on the estimation of phytoplankton biovolume
Water quality - Guidance on the estimation of phytoplankton biovolume
This European Standard specifies a procedure for the estimation of biovolume of marine and freshwater phytoplankton taxa using inverted microscopy (Utermöhl technique according to EN 15204), in consideration of some heterotrophic protists (< 100 µm) that are not considered in routine zooplankton analysis and benthic microalgae, which can be found in pelagic water samples.
This European Standard describes the necessary methods for measuring cell dimensions and for the calculation of cell or counting unit volumes to estimate the biovolume in phytoplankton samples. This shall be done using harmonized assignments of geometrical shapes to avoid errors.
Wasserbeschaffenheit - Anleitung zur Abschätzung des Phytoplankton-Biovolumens
Diese Europäische Norm legt ein Verfahren zur Abschätzung des Biovolumens von marinen und limnischen Phytoplankton Taxa unter Anwendung der Umkehrmikroskopie (Utermöhl-Technik) nach EN 15204 fest, und berücksichtigt dabei einige heterotrophe Protisten (< 100 µm), die bei Routineauswertungen von Zooplankton nicht mit angerechnet werden und bentische Mikroalgen, die in pelagischen Wasserproben gefunden werden können.
Diese Europäische Norm beschreibt die erforderlichen Verfahren zur Messung der Zelldimensionen und zur Berechnung des Volumens von Zellen oder Zähleinheiten zur Abschätzung des Biovolumens in Phytoplanktonproben. Um Fehler zu vermeiden, werden harmonisierte geometrische Formen verwendet.
Qualité de l'eau - Lignes directrices pour l'estimation du biovolume des microalgues
La présente Norme européenne spécifie une méthode permettant d'estimer le biovolume des taxons de phytoplancton marin et d'eau douce par microscopie inversée (technique d'Utermöhl selon l'EN 15204), en tenant compte du fait que certains protistes hétérotrophes (< 100 µm), qui ne sont pas pris en considération dans l'analyse de routine du zooplancton et des microalgues benthiques, peuvent se trouver dans des échantillons d'eaux pélagiques.
La présente Norme européenne décrit les méthodes nécessaires pour mesurer les dimensions des cellules et pour calculer les volumes de cellules ou d'unités de comptage afin d'estimer le biovolume d'échantillons de phytoplancton. Pour cela, il est nécessaire d'utiliser des attributions harmonisées de formes géométriques afin d'éviter les erreurs.
Kakovost vode - Navodilo za ocenjevanje biovolumna mikroalg
Razvoj usklajenega protokola za oceno biovolumna alg, vključno s priporočenim seznamom geometričnih oblik najpogostejših evropskih taksonov fitoplanktona, ki izpolnjujejo zahteve iz okvirne direktive o vodah.
Metode naj bi zagotovile:
– mikroskopsko tehniko za merjenje dimenzij celic alg, ki so potrebne za oceno biovolumna fitoplanktona različnih taksonov fitoplanktona, vključno s posamičnimi celicami, oblikami kompleksnih celic in kolonijami;
– računske postopke za oceno biovolumna alg, vključno z razmerji biomase;
– potrebne postopke za zagotavljanje kakovosti;
– navodila za priporočene geometrične oblike za različne taksone fitoplanktona in pripadajoče enačbe za izračun biovolumna.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-november-2015
Kakovost vode - Navodilo za ocenjevanje biovolumna mikroalg
Water quality - Guidance on the estimation of microalgal biovolume
Wasserbeschaffenheit - Anleitung zur Abschätzung des Phytoplankton-Biovolumens
Qualité de l'eau - Lignes directrices pour l'estimation du biovolume des microalgues
Ta slovenski standard je istoveten z: EN 16695:2015
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 16695
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2015
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Guidance on the estimation of
phytoplankton biovolume
Qualité de l'eau - Lignes directrices pour l'estimation Wasserbeschaffenheit - Anleitung zur Abschätzung des
du biovolume des microalgues Phytoplankton-Biovolumens
This European Standard was approved by CEN on 10 July 2015.
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
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16695:2015 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Equipment and preservatives . 6
6 Procedure. 7
6.1 Sampling and sample preparation . 7
6.2 Calibration of the eyepiece micrometre, counting-graticule and image analysis
software . 7
6.3 Statistical requirements for determination . 8
6.4 Measurement . 9
6.4.1 General . 9
6.4.2 Using size classes based on representative measurements . 10
6.4.3 How to deal with hidden dimensions . 10
6.4.4 Measurement of filamentous taxa. 10
6.4.5 Measurement and counting of colony- and coenobium-forming taxa . 11
6.4.6 Measurement of complex geometrical shapes . 12
6.5 Calculation of biovolume . 12
6.6 Biovolume biomass relations . 13
6.7 Reporting . 13
7 Quality Assurance . 13
Annex A (informative) List of geometrical shapes . 14
Annex B (informative) Performance data . 23
Annex C (informative) Carbon content calculation . 28
Annex D (informative) Alphabetical list of recommended geometrical shapes and hidden
dimension factors . 30
Annex E (informative) Example: Genus-specific instruction for measurement of dimensions . 96
Bibliography . 99
European foreword
This document (EN 16695:2015) has been prepared by 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 March 2016 and conflicting national standards shall be
withdrawn at the latest by March 2016.
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 has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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.
Introduction
The abundance or number of counting units of individual phytoplankton taxa does not necessarily
reflect the real ratio of single taxa to the complete biomass of a phytoplankton community. Few big
cells/counting units can contribute far more biomass to the system than many small ones. Hence,
abundance data alone is often not an ideal measurement of population size. Biomass estimations give
very important information for ecological studies, classification schemes and ecosystem modelling.
Therefore, it is necessary to determine the biomass of phytoplankton taxa, particularly because
phytoplankton delivers energy in the form of carbon, to other trophic levels of food webs. It is not
possible to directly analyse the carbon content on the taxonomic level in natural phytoplankton
samples, therefore the biovolume of the phytoplankton taxa is a suitable measure to determine the
biomass of an ecosystem according to the taxonomic composition. Neither particle size analysis using
laser analysis, nor flow cytometry, nor Coulter Counters, nor chemical analyses of chlorophyll-a
concentration as well as total carbon allow statements on the taxon level. An estimation of the carbon
content is possible using conversion factors (see Annex C).
Further, the biovolume is a quantitative basis for assessing hazards from those algae and cyanobacteria,
which (can) contain noxious or toxic metabolites, and is used in combination with cell numbers or
chlorophyll-a concentration within WHO guidelines and national regulations for risk assessments.
Up to now, various guidelines for estimating the biovolume of microalgae have been used in different
national and international monitoring programs (e.g. [1], [2], [3], [4]). The main objective of this
document is the standardization of the procedure for determining the phytoplankton biovolume in
order to achieve comparability of data. For this reason, the estimation of the biovolume in
phytoplankton samples in sedimentation chambers (according to Utermöhl) using an inverted
microscope will be described in detail.
This European Standard is also applicable for image analysis of pictures derived from microscope and
flow cytometry cameras. The use of a standard catalogue containing basic and some composed
geometrical shapes is recommended. Of course, such a standard list will not reflect the variety of all
naturally existing shapes and will not match the exact biovolume values of each taxon. It will always be
a compromise between accuracy and efficiency. However, the usage of agreed geometrical shapes and
the application of the relevant formulae will improve the comparability of phytoplankton data and will
be an important step forward for the implementation of quality assurance measures in phytoplankton
analysis.
1 Scope
This European Standard specifies a procedure for the estimation of biovolume of marine and
freshwater phytoplankton taxa using inverted microscopy (Utermöhl technique according to
EN 15204), in consideration of some heterotrophic protists (< 100 µm) that are not considered in
routine zooplankton analysis and benthic microalgae, which can be found in pelagic water samples.
This European Standard describes the necessary methods for measuring cell dimensions and for the
calculation of cell or counting unit volumes to estimate the biovolume in phytoplankton samples. This
shall be done using harmonized assignments of geometrical shapes to avoid errors.
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.
EN 15204, Water quality - Guidance standard on the enumeration of phytoplankton using inverted
microscopy (Utermöhl technique)
EN 15972, Water quality - Guidance on quantitative and qualitative investigations of marine
phytoplankton
EN 16698, Water quality - Guidance on quantitative and qualitative sampling of phytoplankton from
inland waters
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
biomass
total mass of living organic matter within a system or taxon
3.2
biovolume
total volume of (living) organisms within a system or taxon
Note 1 to entry: The biovolume is usually expressed in cubic millimetres per litre (mm /l).
3.3
cell volume
counting unit volume
total volume of a single cell or one counting unit
Note 1 to entry: The cell volume or counting unit volume includes the cell wall (if existing) but excludes lorica
and/or mucilaginous envelopes and cell surface structures such as spines, bristles and scales.
Note 2 to entry: The cell volume or counting unit volume is usually expressed in cubic micrometres (µm ).
4 Principle
Generally, the estimation of the total or taxon specific biovolume in phytoplankton samples of natural
communities or cultures is based on measurements of a representative number of individuals. By
multiplying the average or median cell or counting unit volume with the abundance, the total biovolume
of each taxon in the sample is determined.
Three approaches are feasible:
1) Estimation by representative measurement: A representative number of individuals (in most
cases single cells) or counting units of all recorded or dominating taxa is measured in each sample
or a specified number of samples within a comparable series. These data are used to calculate the
average or median cell or counting unit volume of each taxon using the applied geometrical
formulae.
2) Estimation using size classes based on representative measurements: For taxa with a high
variability in cell size (e.g. several diatoms, different stages in life cycle) reasonable size classes can
be determined first, and then the individuals are assigned to both the relevant taxon and size class.
Basis for the definition of the size classes are measureme
...
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