EN 16695:2015

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

• Status: Published
• Publication Date: 01-Sep-2015
• Withdrawal Date: 30-Mar-2016
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 09-Feb-2022
• Completion Date: 14-Apr-2025
• Directive: 2000/60/EC - Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (substitutes Directive Reference 01/991 - Common Position on WFD)
• Mandate: M/424 - Mandate for standardisation addressed to CEN for the development or improvement standards in support of the water framework directive
• Ref Project: SIST EN 16695:2015 - Water quality - Guidance on the estimation of microalgal biovolume

Overview

EN 16695:2015 - "Water quality - Guidance on the estimation of phytoplankton biovolume" (CEN) defines a harmonized procedure to estimate biovolume of marine and freshwater phytoplankton using inverted microscopy (Utermöhl technique, EN 15204). It covers measurement of cell/counting-unit dimensions, assignment of standard geometrical shapes, volume calculation and conversion to biomass (carbon) estimates. The standard also addresses some heterotrophic protists (<100 µm) and benthic microalgae that occur in pelagic samples.

Key topics and technical requirements

• Scope and principle: Biovolume is derived by measuring representative individuals, assigning standard geometrical shapes, and calculating cell or counting-unit volumes to multiply by abundance.
• Measurement approaches: (1) Representative measurements per sample, (2) size classes based on measurements, (3) standard volumes derived from representative data.
• Geometrical shapes & corrections: A harmonized catalogue of basic and composite shapes (Annex A, D) and geometry correction factors are used to improve comparability.
• Equipment: Inverted microscope (Utermöhl), condenser NA ≥ 0.5 and plan objectives NA ≥ 0.9; total magnification typically 63×–400×; 10× or 12.5× eyepieces. Image analysis software may be used.
• Sampling & preservation: Follow EN 16698 (freshwater) and EN 15972 (marine); use sedimentation chambers per EN 15204; preservatives such as acidic or alkaline Lugol’s iodine are specified.
• Calibration & QA: Eyepiece micrometre, counting graticule and image software must be calibrated with a calibrated object micrometre for each objective/magnification. Statistical requirements, reporting and quality assurance provisions are detailed.
• Supporting annexes: Performance data (Annex B), carbon conversion factors (Annex C), genus-specific measurement guidance (Annex E) and a full alphabetical list of recommended shapes (Annex D).

Applications and users

• Environmental monitoring labs estimating phytoplankton biomass for compliance and trend analysis.
• Water quality and public-health agencies performing risk assessment for harmful algal blooms (HABs) where biovolume informs toxicity risk alongside cell counts and chlorophyll-a.
• Research groups and ecological modelers needing taxon-level biomass inputs for food web or ecosystem models.
• Consultants and industry conducting freshwater and marine impact assessments or monitoring mitigation measures.

Practical benefits include improved comparability of phytoplankton biomass data across laboratories, standardized volume-to-carbon conversions (Annex C), and clearer methods for handling complex morphologies and size variability.

Related standards

• EN 15204 - Enumeration of phytoplankton using inverted microscopy (Utermöhl technique)
• EN 15972 - Quantitative and qualitative investigations of marine phytoplankton
• EN 16698 - Sampling of phytoplankton from inland waters

Keywords: EN 16695:2015, phytoplankton biovolume, Utermöhl technique, inverted microscopy, biovolume estimation, water quality, phytoplankton biomass, geometrical shapes, calibration.

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 measurements in the same manner as described in
(1).
3) Estimation using standard volumes based on representative measurements: A reasonable
general standard cell or counting unit volume is defined for each taxon once. These standard values
are determined by representative measurements and calculated by the formula of the assigned
geometrical shapes as described in (1).
A geometrical shape shall be assigned to each taxon in all approaches to calculate the cell or counting
unit volume. Thus, to harmonize these approaches the geometrical shapes are pre-assigned to all taxa
(see Annex D). These shapes have been chosen to reflect the corresponding taxa shapes as accurately as
possible, and to allow effective taxa measurement with little effort (i.e. with as few dimensions as
possible; usually only two are necessary). Seventeen different geometrical shapes are utilized (for the
catalogue of geometrical shapes see Annex A). If it is impossible to describe the actual shape of a taxon
with a simple basic geometrical shape, composite shapes (e.g. cone with half sphere) are used. If the
actual geometry of taxa does not fit exactly to the assigned shape, a “geometry correction factor” is used
for the final cell or counting unit volume calculation.
Taxon lists describing the preferred geometrical shapes have been published before (see e.g. [1], [3],
[4]), based on specific taxonomical levels or for particular areas. This guidance document provides
harmonized geometrical shapes for phytoplankton organisms spread across European marine,
brackish, and freshwater systems. Annex D contains an alphabetical list of genera with the assigned
geometrical shapes. If there are divergent forms on species, subspecies, form, or variety level within a
genus they are listed as well.
5 Equipment and preservatives
The following equipment is required for biovolume analysis of phytoplankton samples.
5.1 Inverted microscope equipped with a condenser featuring a numeric aperture (NA) of at least
0,5 and plan objectives with a NA of 0,9 or more allowing for total magnification between 63× and 400×
at a minimum. The microscope should have binocular, bright field (additional phase contrast is useful),
10× or 12,5× eyepieces.
Though inverted microscopy is the recommended method for analysing of phytoplankton, conventional
(non-inverted) compound light microscopes may also be used for measuring phytoplankton under
some conditions.
5.2 Calibrated object micrometre.
5.3 Eyepiece (ocular) micrometre.
5.4 Counting-graticule.
5.5 Sedimentation chambers according to EN 15204.
5.6 Image analysis software, if available.
5.7 Sampling bottles according to EN 15204.
5.8 Preservatives, acidic Lugol’s iodine and/or alkaline Lugol’s iodine according to EN 15204.
6 Procedure
6.1 Sampling and sample preparation
The sampling and determination of phytoplankton abundance and composition is a precondition for the
calculation of the biovolume of a phytoplankton sample. Sampling shall be carried out according to
EN 16698 for freshwater samples and EN 15972 for marine samples. For counting and species
determination, see EN 15204.
The dimensions needed for the biovolume estimation of the relevant phytoplankton taxa are analysed
in sedimentation chambers, which are prepared in the same manner as for counting and species
determination (see EN 15204), using an inverted microscope and an eyepiece micrometre or image
analysis software.
For specific scientific purposes, measurements can be carried out also with a conventional (non-
inverted) compound light microscope.
6.2 Calibration of the eyepiece micrometre, counting-graticule and image analysis
software
The required dimensions for estimation of the cell or counting unit volume shall be measured using an
eyepiece (ocular) micrometre or an image analysis software. For the application of size classes a
calibrated counting-graticule can also be used.
Prior to measurement, all systems shall be calibrated with a calibrated object micrometre for every
microscope and all objectives and eyepieces used.
The scale of commercially available calibrated object micrometres has a length of 1 mm (or 2 mm)
where the millimetre is divided into 100 equal parts. The distance between the graduation lines is
10 µm. By aligning the scale of the eyepiece micrometre with the scale of the object micrometre or the
grid boxes of the counting-graticule, the scale value (S) or conversion factor of the eyepiece micrometre
can be determined for each magnification as follows:
n ×10
obj
S = (1)
n
eye
where
S is the scale value (conversion factor) for the eyepiece micrometre in micrometres (µm);
n is the number of graduation lines of the object micrometre;
obj
neye is the number of graduation lines of the eyepiece micrometre or the number of grid boxes of
the counting-graticule.
The conversion factor should be specified with up to two decimal places. The intervals between the
graduation lines of the scale shall be separately determined with the calibrated object micrometre for
every objective used.
If image analysis software is used, this equipment shall be calibrated with the calibrated object
micrometre separately for every level of magnification, following the instructions in the operating
manual of the software.
6.3 Statistical requirements for determination
The required dimensions of the relevant geometrical shape shall be measured for each taxon of interest.
At least 20 individuals per taxon should be measured to ensure that the standard error of cell or
counting unit volume will be generally < 10 %.
For taxa, which are very variable in size, the number of measured cells/counting units should be
increased until the standard error is < 10 %, to a maximum of 50 individuals ([3], [5]).
Where the size variability of a taxon is small, the number of measured cells may be minimized to only 5
to 10 individuals. In all cases, it is advisable to check that the standard error of cell or counting unit
volume is low. The standard deviation and standard error can be calculated according to Formulae (2)
and (3) as follows:
n
(x − x )
∑ i
i=1
s = (2)
n −1
where
s is the taxon volume standard deviation;
x is the volume of a single cell/counting unit of the taxon;
i
x is the mean volume of all measurements of the taxon;
n is the number of the respective measured cells of the taxon.
σ
x
From the standard deviation s, the standard error of the mean can be calculated using Formula (3):
s
σ = (3)
x
n
To obtain the percentage rangeσ of the standard error, divide the standard error of the mean σ by
rel x
the mean value x of the samples using Formula (4):
σ
x
σ ×100 (4)
rel
x
From the results, the 95 % confidence limits can be determined using Formulae (5) and (6):
(5)
xx=+×(σ 1,96)
ux
(6)
xx=−×(σ 1,96)
lx
where
=
is the upper 95 % confidence limit;
x
u
is the lower 95 % confidence limit.
x
l
Since the required number of individuals to be measured for any taxon is dependent upon the size
variability of that taxon, it is helpful to calculate the cell or counting unit volume after each
measurement. This will allow continuous statistical analysis and check of precision, thus minimizing the
number of measurements required. Ideally, the confidence limits should be set at 95 % as a measure for
precision. By ensuring that only as many cells as necessary for achieving this limit will be counted, the
amount of laboratory work will be minimized.
If not enough cells/counting units of a taxon are present in the sample in order to achieve the minimum
statistical requirements described, all of the (few) cells of this taxon shall be measured or a mean
standard biovolume may be used (see below).
Measuring a high number of cells for every taxon in every sample is a time consuming procedure. For
routine monitoring programmes mean cell or counting unit volumes calculated from own
measurements, for a particular project and area, may be used. These mean volumes shall be checked
regularly by measuring actual cell dimensions (see 6.4) and calculating actual cell and counting unit
volume (see 6.5). For taxa with a high variability in cell size and representing more than 50 % of the
total biovolume, these checks are strongly advised.
6.4 Measurement
6.4.1 General
Measurement of the cells/counting units can be carried out in a separate step or parallel to the counting
process. Depending on the cell size of the taxa, the determination of the required dimensions (e.g.
diameter, height, length, width, etc.) should be carried out at magnifications between 63× and 1 000×, in
order to obtain corresponding precision.
The so-called empty magnification, which does not reveal any new detail, should be avoided. Therefore,
the total magnification of the microscope should preferably be higher than 500× but smaller than
1 000× of the NA of the used objectives to work in the area of useful magnification.
It is important that the cells to be measured are chosen randomly to avoid a discrimination of special
size classes. This can be achieved by selecting the cells from randomly distributed visual boxes all over
the sedimentation chamber.
If single cells can clearly be distinguished in chain-building or filamentous species or other colony
forming taxa, only one cell per chain, filament or colony should be measured. Filamentous algae often
form lumps and are distributed very unevenly in the chamber. Also in these cases, it shall be ensured
that cells from different lumps are measured.
By rotating the eyepiece micrometre and moving the sedimentation chamber with the microscope
stage, the scale of the eyepiece micrometre is placed over the required dimension of the cell/counting
unit to be measured. The number of covered graduation lines is read, and with the application of the
conversion factor (see 6.2), the length of the dimension is calculated by multiplication.
The measurement results should be reported in micrometres, with up to two decimal places. If the end
of the cell dimension to be measured is between two graduation lines of the ocular scale, the share is to
be estimated to a maximum of one decimal place.
When using the size classes approach based on representative measurements, the calibrated counting-
graticule may also be used for the assignment of the individuals to the corresponding size classes.
When image analysis software is used, the corresponding details of the operating manual shall be
followed.
6.4.2 Using size classes based on representative measurements
The appropriate number of size classes depends on the size variation of each taxon. The size classes, for
example, can be derived from cluster analyses applied to a representative dataset of measured cells of
the taxon (see 6.4.1).
For each size class the average cell or counting unit volume is calculated using the formula of the taxon
specific geometrical shape with mean dimension lengths. By multiplication with the respective
abundance and addition of all size classes of a taxon, the total taxon specific biovolume in the sample is
determined. In routine monitoring programmes, standard size classes should be used (e.g. [4]). If
regional lists of size classes are available, they should be used instead [4]. It is recommended to create a
new size class, when the biovolume of individuals significantly exceeds the biovolume of existing size
classes.
6.4.3 How to deal with hidden dimensions
For some particular taxa, it is often not possible to measure all dimensions needed to calculate the cell
volume (“hidden dimensions“, e.g. height of prismatic cells of different shapes or small diameter of
elliptical cells) during routine analysis. Then it is necessary to estimate the length of missing
dimensions as a proportion of one of the visible dimensions using a calculated species-specific factor.
The visible to hidden size relationship can be obtained from measurements of other samples from the
same sample series or from the same area if the position of the cells allows this. Measurements should
be used preferentially over estimates. The taxon list in Annex D gives suggestions for the dimension
relations for most of the taxa, which usually have a “hidden dimension”. For those taxa, where no
suggestion for the “hidden dimension” is given, corresponding nominal values shall be taken from the
literature if available. If the “hidden dimension” shall be determined exactly for a special problem, the
cells can be turned around in paraffin oil. With special microscopes, it is also possible to measure the
distance between focus of the upper and lower end of the “hidden dimension”.
6.4.4 Measurement of filamentous taxa
With some filamentous taxa (e.g. cyanobacteria), it is often difficult to distinguish individual cells within
a filament, especially when the cells are directly connected without any gaps. In such cases, filament
pieces of a fixed length, e.g. 100 µm or 10 µm, can be counted and measured (100 µm or 10 µm length
and diameter), and multiplied by the total enumeration of this counting unit in the sample.
Alternatively, mean dimensions of filaments can be measured to calculate the volume of one filament, a
value that is then multiplied by the number of filaments in the sample.
A third method, which is more precise for filamentous forms, particularly those, which have no distinct
boundaries between cells, is as follows: Instead of counting individual filaments, the total length of the
fraction of each filament that is within the boundaries of a counting grid shall be measured, ignoring the
fraction outside of the grid boundaries (see Figure 1). The sum of the total length of all fractions of
filaments within the grid shall be calculated after counting of the transect is completed. Afterwards, the
diameter of at least 20 filaments shall be measured and the mean filament diameter is calculated. To
obtain the biovolume of the respective taxon, the sum of total filament lengths (h) shall be multiplied
with the square of the median filament diameter and the factor of π/4, because the filament is a cylinder
with the volume:
V= ××π dh× (7)
Figure 1 — Determining biovolume for filaments without distinct cell boundaries using a
counting grid
NOTE The lengths of all fractions within the counting grid (black rectangle) are measured, excluding fractions
outside of the counting grid boundaries [6].
6.4.5 Measurement and counting of colony- and coenobium-forming taxa
In most cases, the assignment of a geometrical shape should be based on the shape of an individual cell.
For some colony- and coenobium-forming species where individual cells are difficult to distinguish or
have very complex contours, it can be expedient to assign a geometrical shape based on the shape of the
whole colony or coenobium or to use ultrasonic treated samples (where the association of cells have
become disintegrated into individual cells) for counting and measuring the cells.
For example in some species, small individual cells are aggregated into compact spatial colonies. The
individual cells are usually indistinguishable, and the number may be very difficult to assess in these
colonies or coenobia. In such cases, the estimation of the volume can be based on the geometry of the
entire colony or coenobium, in particular during the analysis of routine monitoring samples. For some
taxa an additional geometrical shape correction factor shall be taken into account for the volume
calculation. The same applies for colony- or coenobium-forming taxa with very complex contours of
individual cells. Table 1 lists examples. In Annex D, it is specified for the respective taxa if the entire
colony or coenobium with its geometry should be measured.
Table 1 — Geometrical shapes and correction factors for some colony- and coenobium-forming
taxa.
Taxon name Geometry based on Geometrical shape Hidden dimension factor
colony correction factor
Woronichinia sphere 0,2 -
Coelosphaerium sphere 0,2 -
Snowella sphere 0,75 -
Botryococcus spheroid - -
Eudorina sphere 0,25 -
Pandorina sphere - -
Coelastrum sphere - -
Pediastrum cylinder species dependend height of colony = height of
single cell (factor see Annex D)
Crucigenia cuboid species dependend third edge length (height) =
0,5×second edge length
(width)
A more precise method to achieve the biovolume of such colonial forms, especially the cyanobacterial
genera Microcystis, Aphanothece, Aphanocapsa, and the Dolichospermum species forming “ball of yarn”
colonies, is to measure cell dimensions and the greatest axial linear dimension of the colony in the
sedimented sample, and then separate the colonies by ultrasonic treatment, and count the individual
cells in the treated sample [7].
6.4.6 Measurement of complex geometrical shapes
Some taxa show very complex cell outlines requiring a composition of multiple geometrical shapes and
thus, the application of complicated formulae for biovolume calculations. In order to ease that work,
some simplified combined forms have been assigned to these taxa that can be easily measured and will
require only minimum effort for the estimation. However, depending on the required precision, more
complex and thus more precise geometrical subdivisions shall be applied to those taxa. Another
possibility is to resort to pre-determined cell volumes from literature (mean standard cell or counting
unit volume), bearing in mind that the size of the cells depends on a number of environmental factors
and thus, may vary widely. On the other hand, cell volume estimation for some species is easier on half-
cell basis (for example in some desmid genera like Cosmarium and Staurastrum).
In any case, everything shall be documented in the protocol. As an example, Annex E shows how to
measure the needed dimensions for four taxa from the conducted interlaboratory comparison as well as
for two additional taxa, which are not easy to measure.
6.5 Calculation of biovolume
The cell or counting unit volume is calculated on the basis of the taxon-specific geometrical shape and
the linear dimensions determined for the individual cell or counting unit (e.g. diameter, height, length,
width, etc.; see Annex A). For some taxa, the associated geometrical form will not fit exactly to the actual
cell shape, for example, if a cuboid is assigned to a pennate diatom with rounded apical cell ends. For
such cases, the cell volume shall be multiplied by a correction factor, which is given in Annex D for
relevant taxa.
The average cell or counting unit volumes of the various taxa shall be generally calculated as the median
of all individual cell or counting unit volumes.
NOTE If the individual volumes per taxon are normally distributed (according to Chi-squared test or
Kolmogorov-Smirnov test), the arithmetic mean can be used for the calculation of the average volume instead of
the median. For answering specific questions in ecology, it can be necessary to use also the arithmetic mean for
non-normally distributed values.
These calculations shall be carried out for every phytoplankton taxon recorded in all or selected
samples using the “representative measurement” approach (see Clause 4) or basically once for
characteristic samples using the “standard factor” approach (see Clause 4). If applying the “size class”
approach (see Clause 4 and 6.4.2) the average cell or counting unit volume for each size class is
calculated using the mean of the upper and lower dimensions of size class borders.
The biovolume per taxon in a sample is calculated by multiplying the number of cells/l (or cells/ml) or
counting units (e.g. number of 100 µm filament pieces per liter) with the median (or mean) of the
determined taxon-specific cell or counting unit volumes (µm ) as measured by one of the three methods
listed above:

nV×
ii
V = (8)
bio, i
where
Vbio,
i is the biovolume of taxon or size class i in cubic millimetres per litre (mm /l);
-1
n
i is the number of cells (or number of counting units) of taxon or size class i per litre (l );
is the median (or mean) of the cell or counting unit volumes of taxon or size class i in cubic

V
i
micrometres (µm ).
The total biovolume to be determined for each sample results from the sum of the biovolume
determined for each phytoplankton taxon or size class.
Statistical performance data from the conducted European wide interlaboratory comparison for
validation are listed in Annex B.
6.6 Biovolume biomass relations
In phytoplankton ecology, the biomass is usually expressed as chlorophyll-a concentration (µg/l),
biovolume (mm /l) or carbon content (µg/l). Assuming the density of organisms being equal to the
density of water (1,0 g/cm , [8]), the biomass as wet weight may be estimated as follows:
3 3 3
1 mm /l (biovolume) = 1 cm /m (biovolume) = 1 mg/l (wet weight);
3 3 6 3
1 mm /m (biovolume) = 10 µm /l (biovolume) = 1 µg/l (wet weight).
NOTE For the estimation of the carbon content, see Annex C.
6.7 Reporting
The specific cell or counting unit volumes are expressed in cubic micrometres (µm ) without decimal
places or for picoplankton with two significant places (e.g. 4,2 µm ). The biovolume of a single taxon
3 3
and the complete sample is given in cubic millimetres per litre (mm /l or mm /ml) with three
3 3 3 3
significant digits or places (e.g. 12,5 mm /l, 3,75 mm /l, 0,138 mm /l, 0,004 mm /l).
As an example, the taxon specific part of a sample report can contain information as shown in Table 2.
Table 2 — Example report for taxon specific sample part.
Taxon name Abundance Geometry Average cell Total Carbon content
volume biovolume
(according to
…)
3 3
n/l µm mm /l µg/l
Thalassiosira 12 109 cylinder 9 817 0,119 6,53
nordenskioeldii
Odontella aurita 265 725 elliptic cylinder 14 091 3,74 188,5
….
Sum of sample 5 765 180  4,43 231,2
Some taxa (e.g. flagellates without solid cell wall) are inclined to shrink during the fixation process.
Shrinkage depends on many influencing factors (preservative, life stage, physiological status, species,
etc., even diatoms can shrink). Often it is difficult to find good correction factors for this process in the
literature. As a consequence, applying a general correction factor to preserved material can lead either
to overestimation or to underestimation of the biovolume. For monitoring activities with the objective
of detecting trends, the exact compensation by preservation correction factors is not of the highest
priority. In other cases where it might be mandatory to determine the exact biomass, correction factors
for influence of fixation should be derived from comparisons between fixated and living organisms. This
shall be noted in the protocol.
7 Quality Assurance
The quality assurance associated with this European Standard should be in accordance with EN 14996.
Annex A
(informative)
List of geometrical shapes
Table A.1 provides information about 17 different geometrical shapes used for the biovolume
estimation of phytoplankton. The first column simply contains a consecutive number (ID-number) for
each shape. Listed in the second column is the official geometrical name of the form alongside any other
synonymous names. The images of each shape show a 3D-view, a cross-section, and a longitudinal-
section in which the dimensions are designated to be used for biovolume calculation. The third column
shows some general information about each shape, a description about the needed dimensions as well
as some taxon examples. Firstly, it is listed if it is a basic shape or a composed form. If the latter is the
case it is also indicated from which basic shapes the form is composed. The volume calculation
precision distinguishes between exact calculation, if there is a clearly defined formula for volume, and
approximate calculation, if the formula is derived by approximate integration. The dimensions
necessary for calculation of biovolume are listed with full name and abbreviation as well as with
information about measurability of these dimensions in phytoplankton organisms using an inverted
microscope. “m” means that this dimension is measurable, “hd” means that this is a hidden dimension
that cannot be measured in a sedimentation chamber. If both specifications are listed (m; hd), this
indicates that cells can have different positions, the orientation of which can affect if the dimension is
measurable or not. Finally, some typical taxon examples for each shape are listed. In the last column, the
formulae needed for biovolume calculation are specified, with the deduced form based on diameter that
can clearly be measured in the cells, and with the original geometrical form based on radius.
Table A.1 — Geometrical shapes
ID Geometrical shape (Synonyms) Information Volume formula
1 Sphere basic shape
Vd= ××π
Volume calculation precision:
d
For radius r = :
exact
Dimensions:
diameter (d) - m
Vr= ××π
Examples:
Chroococcus, Coelastrum,
Gloeocapsa, Microcystis, Oblea,
Pterosperma
cross section
ID Geometrical shape (Synonyms) Information Volume formula
2 Spheroid basic shape
V= ××π dh×
rotational ellipsoid
ellipsoid of revolution
Volume calculation precision:
d
For radius r = :
exact
Dimensions:
diameter (d) - m
V= ××π rh× ×
3 2
height (h) - m
Examples:
Aphanothece, Chlamydomonas,
Desmodesmus, Trachelomonas,
Woronichinia
cross section  longitudinal section

3 Ellipsoid basic shape
V= ××π d ×d ×h
tri-axial ellipsoid,
flattened ellipsoid
Volume calculation precision:
For radius r:
exact
V= ××π r×r×r
Dimensions:
1 23
large diameter (d ) - m
with
small diameter (d ) - hd
d d h
1 2
r = ;  r = ;  r =
height (h) - m 1 2 3
2 2 2
Examples:
Amphidinium, Cosmarium,
Dinophysis, Gymnodinium,
Mallomonas
cross section  longitudinal section

ID Geometrical shape (Synonyms) Information Volume formula
4 Cylinder basic shape
V= ××π dh×
circle based cylinder
Volume calculation precision:
d
For radius r = :
exact
Dimensions:
V=π××rh
diameter (d) - m
height (h) - m; hd
Examples:
Aphanizomenon, Aulacoseira,
Coscinodiscus, Phormidium,
Rhizosolenia, Thalassiosira
cross section  longitudinal section
disk: d >>h
thread: d << h
5 Elliptic cylinder basic shape
V= ××π d ×d ×h
prism on elliptic base 12
oval cylinder
Volume calculation precision:
For radius r:
exact
V=π×rr××h
Dimensions:
with
large diameter (d ) - m
d d
1 2
small diameter (d ) - m; hd
r = ;  r =
1 2
2 2
height of cylinder (h) - hd; m
Examples:
Achnanthes, Chaetoceros,
Fragilaria, Odontella, Surirella

cross section  longitudinal sections

ID Geometrical shape (Synonyms) Information Volume formula
6 Lanceolate cylinder basic shape
V= ×d××d h
naviculoid
π
Volume calculation precision:
For radius r:
approximate
Dimensions: V= ×rr××h
π
large diameter (d ) - m
with
small diameter (d ) - m; hd
d d
1 2
r = ;  r =
height of cylinder (h) - hd; m 1 2
2 2
Examples:
Haslea, Navicula
cross section  longitudinal sections
7 Cone basic shape
2 2 2
V ××π d× s− ×d
12 4
Volume calculation precision:
d
exact
For radius r = and
Dimensions:
diameter (d) - m
height z s− r :
slant height (s) - m
V= ××π rz×
height of cone (z) - hd
NOTE With an inverted
microscope, the slant height
can exactly be measured. The
cone height is a hidden
dimension because of the
oblique projection.
Examples:
Calciopappus, Minuscula,
Pyramimonas longicauda
cross section  longitudinal section
=
=
ID Geometrical shape (Synonyms) Information Volume formula
8 Double cone composed shape
V ××π d× z
two cones
basic shape: cone
Volume calculation precision:
d
For radius r = :
exact
Dimensions:
1 1
diameter (d) - m
V=2× ×π×rz× ×
height of double cone (z) - m
NOTE Due to the position
in the sedimentation chamber,
the total height can only be
measured approximately
because of the oblique
projection.
Examples:
Gyrodinium spirale,
cross section  longitudinal section
Heterocapsa triquetra,
Protoperidinium crassipes
9 Cuboid basic shape
V= a××bc
rectangular box
rectangular parallelepiped
Volume calculation precision:
exact
Dimensions:
first edge length (a) - m
second edge length (b) - m; hd
third edge length (c) - hd; m
Examples:
Bacillaria, Hantzschia,
Pinnularia, Tabellaria
cross section  longitudinal sections
=
...