Water quality - Guidance on the estimation of microalgal biovolume

Development of a harmonised protocol for estimation of algal biovolume including a recommended list of geometrical shapes of most common European phytoplankton taxa meeting the requirements set out in the WFD.
The methods should provide
- the microscopic technique for measurement of algae cell dimensions required for the estimation of phytoplankton biovolume of different phytoplankton taxa including single cells, complex cells shapes and colonies;
- calculation procedures to estimate algal biovolume including biomass relations;
- necessary quality assurance procedures;
- guidance on recommended geometrical shapes for different phytoplankton taxa and the corresponding equations for calculation the biovolume.

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
Public Enquiry End Date
31-Jan-2014
Publication Date
05-Oct-2015
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Sep-2015
Due Date
22-Nov-2015
Completion Date
06-Oct-2015

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SLOVENSKI STANDARD
SIST EN 16695:2015
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
SIST EN 16695:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16695:2015

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SIST EN 16695:2015


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.

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SIST EN 16695:2015
EN 16695:2015 (E)
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

2

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SIST EN 16695:2015
EN 16695:2015 (E)
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.
3

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EN 16695:2015 (E)
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.
4

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SIST EN 16695:2015
EN 16695:2015 (E)
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
3
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.
3
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
5

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SIST EN 16695:2015
EN 16695:2015 (E)
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.
6

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EN 16695:2015 (E)
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.
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EN 16695:2015 (E)
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
2
(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
8
=

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SIST EN 16695:2015
EN 16695:2015 (E)
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.
9

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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:
1
2
V= ××π dh× (7)
4
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EN 16695:2015 (E)

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 boundar
...

SLOVENSKI STANDARD
oSIST prEN 16695:2014
01-januar-2014
Kakovost vode - Navodilo za ocenjevanje biovolumna alg
Water quality - Guidance on the estimation of microalgal biovolume
Wasserbeschaffenheit - Anleitung zur Bestimmung des Phytoplankton-Biovolumens
Ta slovenski standard je istoveten z: prEN 16695
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN 16695:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 16695:2014

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oSIST prEN 16695:2014

EUROPEAN STANDARD
DRAFT
prEN 16695
NORME EUROPÉENNE

EUROPÄISCHE NORM

December 2013
ICS 13.060.70
English Version
Water quality - Guidance on the estimation of microalgal
biovolume
Qualité de l'eau - Lignes directrices pour l'estimation du Wasserbeschaffenheit - Anleitung zur Abschätzung des
biovolume des microalgues Phytoplankton-Biovolumens
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, 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.

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: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16695:2013 E
worldwide for CEN national Members.

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Contents
Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Principle .5
5 Procedure .6
5.1 Determination of required dimensions .6
5.2 Calibration of the eyepiece micrometre .7
5.3 Measurement .8
5.4 Calculation of biovolume .9
5.5 Biovolume biomass relations . 10
6 Quality Assurance . 10
Annex A (informative) List of geometrical shapes . 11
Annex B (informative) Carbon content calculation . 26
Annex C (informative) Alphabetical list of recommended geometrical shapes. 28
Bibliography . 237


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Foreword
This document (prEN 16695:2013) has been prepared by Technical Committee CEN/TC 230 “Water analysis”,
the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of the Water Framework Directive
(2000/60/EC) (WFD), and the Directive on Environmental Quality Standards (Directive 2008/105/EC).
IMPORTANT — This draft European Standard contains in Annex C a very extensive list of common
European phytoplankton taxa down to species level and their proposed geometrical shapes to be
used for biovolume estimations. This list will be the central subject to discussion during the further
revision of the draft, to decide if this list can be reduced for certain taxa to the genus level. The result
will be a much shorter list and a more practicable approach for biovolume estimation.
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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. Thus, biomass is much more relevant
for ecological studies, classification schemes and ecosystem modelling than abundance, particularly because
the energy is available in the form of carbon to other trophic levels of food webs. Therefore, it is important to
determine the biomass of phytoplankton taxa. Because it is not possible to directly analyse the carbon content
on the taxonomic level in natural phytoplankton samples, the biovolume of the phytoplankton cells is a suitable
measure to determine the biomass of an ecosystem according to the taxonomic composition. The biovolume
is an accurate basis for assessing hazards from algae and cyanobacteria which contain noxious or toxic
metabolites, and is used in combination with cell numbers or chlorophyll-a within WHO guidelines and a
number of national regulations for risk assessments. An estimation of the carbon content is possible using
conversion factors (see Annex C). Neither particle size analysis using laser analysis, nor flow cytometry, nor
Coulter Counters, nor chemical analyses of chlorophyll-a as well as total carbon do allow statements on the
taxon level.
The organic substance is not equally distributed within the algal cells. Cells have one or more vacuoles which
are filled with liquid. Organic substances are also dissolved in the vacuoles, but by far the largest part is
located in the cytoplasm. Especially diatoms have extensive vacuoles which can form up to 90% of the total
cell volume in larger species. The cytoplasm is then limited to a narrow region along the cell wall and plasma
threads passing through the vacuole. Depending on the aims of the investigation, the determination of
biovolume can be carried out by microscopy quantifying either cell numbers or cell volumes or plasma
volumes (i.e. cell volume minus vacuole volume). The latter is very difficult to measure with conventional
microscopy, so that in the routine monitoring the total cell volume is determined. For the estimation of the
carbon biomass, the different sizes of the vacuoles are taken into account by applying different conversion
factors and regarding the dependency on the cell size (see Annex C).
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]). Main objective of this document is the standardisation of
the procedure for determining the phytoplankton biovolume in order to achieve comparability of data on an
international level. For that purpose, the microscopic determination of the biovolume in phytoplankton samples
in sedimentation chambers (according to Utermöhl) using an inverted microscope will be described in detail.
The use of a standard catalogue containing basic, composed and fractional geometrical shapes will be
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 justifying the expense. But the usage of agreed geometrical shapes and the application of the respective
equations will improve the comparability of phytoplankton data and will be an important step forward to the
implementation of quality assurance measures in phytoplankton analysis.

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1 Scope
This European Standard describes a general procedure for determination or estimation of biovolume of
marine and freshwater phytoplankton taxa using inverted microscopy (Utermöhl technique).
The determination of phytoplankton abundance and composition according to EN 15204 is a precondition for
the calculation of the biovolume of a phytoplankton sample.
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 14996, Water quality — Guidance on assuring the quality of biological and ecological assessments in the
aquatic environment.
EN 15204, Water quality — Guidance standard for the routine analysis of phytoplankton abundance and
composition using inverted microscopy (Utermöhl technique).
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 a single taxon including the cell wall (if existing) exclusive of lorica and/or mucilaginous
envelopes and cell surface structures such as spines, bristles and scales
3.3
cell biovolume
total volume of a single cell including the cell wall (if existing) exclusive of lorica and/or mucilaginous
envelopes and cell surface structures such as spines, bristles and scales
3.4
plasma biovolume
volume of cytoplasm of a single cell (total cell biovolume minus vacuole volume)
4 Principle
For every phytoplankton taxon, a preferably simple (i.e. with as few dimensions as possible) and best fitting
geometrical shape is assigned (for the catalogue of geometrical shapes see Annex A). If it is not possible to
describe the actual shape with a simple basic geometrical shape, then composite shapes (e.g. cone with half
sphere) or fractional shapes (e.g. half sphere) are used. In most cases, the assignment of a geometrical
shape should be based on a single cell, but for some colony-forming species where individual cells are hardly
to be distinguished or have very complex contours it can be expedient to assign a geometrical shape based
on the shape of the whole colony.
Taxon lists describing the preferred geometrical shapes have been published before (see e.g. [1], [3], [5]),
based on specific taxonomical levels or for particular areas. This guidance document will provide harmonised
geometrical shapes for microalgae spread in European marine, brackish, and freshwater systems. Annex C
contains an alphabetical list for genera, species, subspecies, forms, and varieties with the assigned
geometrical shapes.
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The dimensions needed for the biovolume determination are analysed in the sedimentation chamber by
measuring the cells or counting units of the relevant phytoplankton taxa, using inverted microscopy and an
eyepiece micrometre or image analysis software. The results are reported in micrometres (µm). The
biovolume per taxon and sample is calculated by multiplying the average cell volume of the taxon by the
number of individuals (cells/ml or cells/l) or counting units (e.g. number of 100 µm filament pieces/l).
For taxa with a high variability in cell size (e.g. several diatoms, different stages in life cycle) it is advised to
determine reasonable size classes first and then assign the individuals to both the taxon and the size class.
The appropriate number of size classes depends on the size variation of each taxon. A mean cell volume for
each size class is determined. In routine programmes, standard size classes should be used (e.g. [5]). If
regional lists of size classes were available they should be used instead [5].
Different taxa are quite variable in appearance. Thus, few species sometimes can be rather spherical
(sphere), in the other cases rather elliptical (prolate spheroid). Moreover, different stages within the life cycle
(e.g. cysts) may have various forms. In addition, amoeboid taxa are existing. In the list of Annex C, the
assignments of the geometry have been made according to the common vegetative form. If a corresponding
taxon in the sample to be analysed shows a different shape deviating from the list, an appropriate geometrical
shape will have to be selected according to Annex A.
5 Procedure
5.1 Determination of required dimensions
The required dimensions (e.g. diameter, height, length, width etc.) of the relevant geometrical shape shall be
measured for the taxon of interest. At least 20 individuals per taxon should be measured to ensure that the
standard error will be generally < 10 %.
Depending on the cell size variation, the number of measured cells/counting units should be increased up to
50 individuals ([3], [7]).
If the variability of a taxon is negligibly small, the number of cells measured may be reduced to 5 to 10 cells. In
this case, statistical analysis should verify that the standard error is < 10 %. For that purpose, the standard
deviation can be calculated according to Equation (1):
2
(x− x)

s= (1)
n−1
where
s is the corrected sample standard deviation;
x is the sample value;
x is the mean values of all samples;
n is the number of the respective counted samples.
From the standard deviation s, the standard error of the mean, σ , can be calculated using Equation (2):
x
s
σ = (2)
x
n
To obtain the percentage range, σ , of the standard error divide the standard error of the mean σ by the
rel x
mean value x of the samples using Equation (3):
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σ
x
σ = ⋅100 (3)
rel
x
From the results, the 95 % confidence limits can be determined using Equation (4) and (5):
x = x+ (σ ⋅1,96) (4)
u x
x = x− (σ ⋅1,96) (5)
l x
where
x is the upper 95 % confidence limit;
u
x is the lower 95 % confidence limit.
l
Since the required number of individuals to be measured is highly dependent on the variability of the size for a
certain taxon, it is recommended to calculate the biovolume after each measurement, in order to allow
continuous statistical analysis and check the precision. The confidence limits should be set to 95 % as a
measure for precision. By this, only as many cells as necessary for achieving that limit must be counted and
thus, the amount of laboratory work will be minimised.
If not enough cells/counting units of a taxon are included in the sample to be analysed in order to achieve the
minimum statistical requirements described, all the (few) cells of this taxon shall be measured or a mean
standard biovolume may be used (see below).
The required dimensions for determination of the biovolume shall be measured using an eyepiece micrometre
or image analysis software. Prior to measurement, both systems shall be calibrated with a standardised object
micrometre for every microscope and all objectives and oculars used.
Measuring a high number of cells for every taxon in every sample is a time consuming procedure. For routine
monitoring programmes mean cell volumes, used for a particular project and area and calculated from own
measurements, may be used. These mean cell volumes shall be checked regularly by measuring actual cell
dimensions (see 6.2) and calculating actual biovolumes (see 6.3). For taxa having a high variability in cell
sizes and representing more than 50 % of total biovolume, these checks are compulsory.
If taxa show high variability of sizes within size classes, determine and use mean cell volumes per size class.
If the cells are counted directly into size classes, all individuals shall be measured while counted. In this case
a separate measurement of dimensions is not necessary.
5.2 Calibration of the eyepiece micrometre
The scale of commercially available standardised object micrometres has a length of 1 mm and is divided into
100 equal parts. The distance between the graduation lines is 10 µm. By putting the scale of the eyepiece
micrometre over the scale of the object micrometre, the scale value (S) = calibration factor of the eyepiece
micrometre can be determined for each objective magnification as follows:
n ⋅10µm
obj
S= (6)
n
eye
Where
S is the scale value (calibration factor) for the eyepiece micrometre in micrometres (µm);
n is the number of graduation lines of the object micrometre
obj
n is the number of graduation lines of the eyepiece micrometre
eye
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The calibration 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 standardised object micrometre for every objective.
If image analysis software is used, this also shall be calibrated with the standardised object micrometre
separately for every level of magnification, following the instructions in the operating manual of the software.
5.3 Measurement
Measurement of the cells/counting units can be carried out parallel to the counting process or in a separate
step. 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 a 100 and a 1 000 times, in order to
obtain corresponding precision.
By rotating the ocular with the eyepiece micrometre and moving the sedimentation chamber with the
microscope stage, the scale of the eyepiece micrometre is put over the required dimension of the cell/counting
unit to be measured. The number of covered graduation lines is read and with the help of the calibration factor
(see 6.1) the length of the measured dimension is calculated by multiplication. 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 image analysis software is used, the corresponding details of the
operating manual shall be followed.
For some particular taxa, it is often not possible to measure all dimensions needed for calculation of the cell
volume (“hidden dimensions“, e.g. height of prismatic cells of different shapes or small diameter of elliptical
cells). Then it is necessary to estimate the length of missing dimensions as a proportion of the visible
dimensions. The size of the relation to be assessed can be obtained from measurements of other samples if
the position of the cells allows this. Alternatively, corresponding nominal values shall be taken from the
literature.
With some filamentous taxa (e.g. cyanobacteria), it is often difficult to distinguish between individual cells,
especially when the cells are directly interconnected without any gaps. In such cases filament pieces of a fixed
length, e.g. 100 µm, shall be counted and measured (100 µm length and diameter). An alternative and more
precise method 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
finishing the transect. Afterwards, the diameter of at least 20 filaments shall be measured and the median
filament diameter is calculated. To obtain the biovolume of the respective taxon, the sum of total filament
lengths shall be multiplied by the median of filament diameter.

NOTE The lengths of all fractions within the counting grid (black) are measured, excluding fractions outside of the
counting grid boundaries [6].
Figure 1 — Determining biovolume for filaments without distinct cell boundaries using a counting grid
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There are also colony-forming species existing, in which single cells can be distinguished. However, due to
uneven and complex geometries these are often difficult to be classified and measured. In such cases, it may
be easier to classify and measure the entire colony with its geometry. In Annex B, this is specified for the
respective taxa.
Some taxa show very complex cell outlines requiring a composition of multiple geometrical shapes and thus,
the application of complicated equations for the 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 efforts for the determination. However, depending on the required precision, more complex and thus
more precise geometric subdivisions shall be applied to those taxa. Another possibility is the resorting to
already determined cell volumes from literature (mean standard biovolume), bearing in mind that the size of
the cells depends on a number of environmental factors and thus, may vary widely. In any case, everything
shall be documented in the protocol.
5.4 Calculation of biovolume
The cell 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.). 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, but the apical ends of the cell are rounded. For such cases, the cell volume shall be
multiplied by a correction factor, which is given in Annex C for relevant taxa.
The average cell volumes of the various taxa shall be calculated as the median of all individual cell volumes.
The median is used, because it is more robust to extreme values compared to the arithmetic mean. These
calculations shall be carried out for every phytoplankton taxon identified.
The biovolume per taxon is calculated by multiplying the number of cells/l (or cells/ml) or counting units (e.g.
3
number of 100 µm filament pieces/l) with the median of the determined taxon-specific cell volumes (µm ):
~
n ⋅V
i i
V =
bio, i
9
10
where
3
V is the biovolume of taxon i in cubic millimetres per litre (mm /l);
bio,i
-1
n is the number of cells (or number of counting units) of taxon i per litre (l );
i
~
3
V is the median of the cell volumes of taxon i in cubic micrometres (µm );
i
The total biovolume to be determined for each sample results from the sum of the biovolumes determined for
each phytoplankton taxon.
3
The specific cell volumes are expressed in cubic micrometres (µm ) without decimal places or for
picoplankton with two significant places. The biovolume of a single taxon and the complete sample is given in
3
cubic millimetres per litre (mm /l) with three significant places.
NOTE 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 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 correction factors has not the highest priority. In other cases
where it might be mandatory to determine the exact biomass, correction factors should be derived from comparisons
between fixated and living organisms. This has to be noted down in the protocol.
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5.5 Biovolume biomass relations
3
Assuming a density of organisms being equal to water (1,0 g/cm , [4]), the biomass (wet weight) may be
estimated as follows:
3 3 3
1 mm /l = 1 cm /m = 1 mg/l;
3 3 6 3
1 mm /m = 10 µm /l = 1 µg/l.
NOTE For the estimation of the carbon content, see Annex B.
6 Quality Assurance
The quality assurance associated with this European Standard should be in accordance with EN 14996.
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Annex A
(informative)

List of geometrical shapes
Table A.1 — Geometrical shapes (1 of 15)
ID Geometrical shape (Synonyms) Information Volume equation
Sphere basic shape
1
1
3
V= ⋅π⋅ d
6

Volume calculation precision: d
For radius r= :
exact
2
Dimensions:
4
3
V= ⋅π⋅ r
A: diameter (d)
3


2 Half sphere fraction shape
1
3
V= ⋅π⋅ d
hemisphere
12
basic shape: sphere
Volume calculation precision: d
For radius r= :
exact
2
Dimensions:
1 4
3
V= ⋅ ⋅π⋅ r
A: diameter (d)
2 3

3 Prolate spheroid basic shape
1
2
V= π⋅ d ⋅ h
rotational ellipsoid
6

ellipsoid of revolution
Volume calculation precision: d
For radius r= :
exact
2
Dimensions:
4 1
2
V= ⋅π⋅ r ⋅ ⋅ h
A: diameter (d)
3 2
B: height (h)

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Table A.1 (2 of 15)
ID Geometrical shape (Synonyms) Information Volume equation
Cymbelloid fraction shape
4
2 β β c
2
V= ⋅π⋅ r ⋅ h⋅ ;sin =
Cymbella-shape
3 360 2 2⋅ r
basic shape: prolate spheroid
segment of prolate spheroid
Volume calculation precision:
2 1  c 
2
exact V= ⋅π⋅ r ⋅ ⋅ arcsin
 
3 180 2⋅ r
 
Dimensions:
A: height (h)
B: radius (r)
C: width of “wedge” (c)
D: angle at "wedge" end (β)

5 Double prolate spheroid composed shape
1
2
V= ⋅π⋅ d ⋅ h
12
basic shape: prolate spheroid
Volume calculation precision:
d
exact For radius r= :
2
Dimensions:
4 1
2

V= 2⋅ ⋅π⋅ r ⋅ ⋅ h
A: total diameter (d)
3 2
B: height (h)

6 Oblate spheroid basic shape
1
2
V= ⋅π⋅ d ⋅ h
rotational ellipsoid
6

ellipsoid of revolution
Volume calculation precision:
4 1
2
V= ⋅π⋅ r ⋅ ⋅ h
exact
3 2
Dimensions:
A: diameter (d)
B: height (h)

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Table A.1 (3 of 15)
ID Geometrical shape (Synonyms) Information Volume equation
Ellipsoid basic shape
7
1
V= ⋅π⋅ d ⋅ d ⋅ h
1 2
tri-axial ellipsoid,
6

flattened ellipsoid
Volume calculation precision:
For radius r:
exact
4
Dimensions: V= ⋅π⋅ r⋅ r ⋅ r
1 2 3
3
A: large diameter (d )
1
with
B: small diameter (d )
2
d d h
1 2
r = ;  r = ;  r =
1 2 3
C: height (h) 2 2 2

8 Cylinder basic shape
1
2
V= ⋅π⋅ d ⋅ h
circle based cylinder
4

Volume calculation precision:
d
For radius r= :
exact
2
Dimensions:
2
V=π⋅ r ⋅ h
A: diameter (d)
B: height (h)

9 Elliptic cylinder basic shape
1
V= ⋅π⋅ d ⋅ d ⋅ h
1 2
prism on elliptic base
4

oval cylinder
Volume calculation precision:
For radius r:
exact
V=π⋅ r⋅ r ⋅ h
1 2
Dimensions:
A: large diameter (d ) with
1
d d
B: small diameter (d )
1 2
2
r = ;  r =
1 2
2 2
C: height of cylinder (h)

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Table A.1
...

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