Water quality - Guidance on analysis of mesozooplankton from marine and brackish waters

This document specifies a procedure for analysing mesozooplankton in marine and brackish waters. The procedure comprises how to identify and enumerate mesozooplankton to estimate quantitative information on diversity, abundance and biomass with regard to spatial distribution and long-term temporal trends for a given body of water.

Wasserbeschaffenheit - Anleitung zur Analyse von Zooplankton aus marinen und brackigen Gewässern

Dieses Dokument legt ein Verfahren für die Analyse von Mesozooplankton in Meer  und Brackwasser fest. Das Verfahren umfasst die Bestimmung und Zählung von Zooplankton zur Beurteilung quantitativer Informationen zu Diversität, Abundanz und Biomasse hinsichtlich der räumlichen Verteilung und langfristiger zeitlicher Trends für einen bestimmten Wasserkörper.

Qualité de l'eau - Document d'orientation sur l'analyse du mésozooplancton dans les eaux marines et saumâtres

La présente Norme européenne spécifie un mode opératoire pour l’analyse du mésozooplancton dans les eaux marines et saumâtres. Ces modes opératoires déterminent comment identifier et dénombrer le zooplancton, afin d’obtenir des estimations quantitatives sur la diversité, l'abondance et la biomasse en lien avec la distribution spatiale et les tendances temporelles à long terme pour une masse d'eau donnée.

Kakovost vode - Navodilo za analizo mezozooplanktona v morskih in brakičnih vodah

Ta evropski standard določa postopek za analizo mezozooplanktona v morskih in brakičnih vodah. Vsebuje postopke, kako identificirati in šteti zooplankton za oceno kvantitativnih informacij o raznolikosti, številčnosti in biomasi glede na prostorsko porazdelitev in dolgoročne časovne trende za določeno vodno telo.

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Publication Date
02-Apr-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
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03-Apr-2019
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03-Apr-2019

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SLOVENSKI STANDARD
SIST EN 17204:2019
01-julij-2019
Kakovost vode - Navodilo za analizo mezozooplanktona v morskih in brakičnih
vodah
Water quality - Guidance on analysis of mesozooplankton from marine and brackish
water
Wasserbeschaffenheit - Anleitung zur Analyse von Zooplankton aus marinen und
brackigen Gewässern

Qualité de l'eau - Document d'orientation sur l'analyse du mésozooplancton dans les

eaux marines et saumâtres
Ta slovenski standard je istoveten z: EN 17204:2019
ICS:
13.060.10 Voda iz naravnih virov Water of natural resources
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN 17204:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17204:2019
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SIST EN 17204:2019
EN 17204
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2019
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Guidance on analysis of mesozooplankton
from marine and brackish waters

Qualité de l'eau - Document d'orientation sur l'analyse Wasserbeschaffenheit - Anleitung zur Analyse von

du mésozooplancton dans les eaux marines et Zooplankton aus marinen und brackigen Gewässern

saumâtres
This European Standard was approved by CEN on 11 February 2019.

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, Serbia, 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: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17204:2019 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 7

5 Equipment ......................................................................................................................................................... 7

6 Preservatives and other chemicals ........................................................................................................... 8

7 Procedure........................................................................................................................................................... 9

7.1 Sample and sub-sample preparation ....................................................................................................... 9

7.2 Species identification and counting ...................................................................................................... 10

7.2.1 General ............................................................................................................................................................. 10

7.2.2 Statistical requirements for counting ................................................................................................... 11

7.2.3 Calculation of abundance .......................................................................................................................... 12

7.3 Biomass determination .............................................................................................................................. 12

7.3.1 General ............................................................................................................................................................. 12

7.3.2 Calibration of the eyepiece micrometre, counting-graticule and image analysis

software ........................................................................................................................................................... 12

7.3.3 Calculation of carbon biomass based on length measurements ................................................. 13

7.3.4 Calculation of biomass based on carbon conversion factors ....................................................... 13

7.3.5 Calculation of biomass based on standard size classes .................................................................. 14

7.3.6 Calculation of biomass based on individual weight factors .......................................................... 14

8 Reporting ........................................................................................................................................................ 14

9 Quality assurance ......................................................................................................................................... 15

9.1 General ............................................................................................................................................................. 15

9.2 Photo documentation ................................................................................................................................. 15

9.3 Reference collections .................................................................................................................................. 15

9.4 Long term storage of samples .................................................................................................................. 16

Annex A (informative) Advise for length measurements of some selected zooplankton taxa

and taxonomic groups L [µm] .................................................................................................................. 17

Annex B (informative) Coefficients a and b for biomass calculations based on length

measurements ............................................................................................................................................... 19

Annex C (informative) Biomass conversion factors [µg C/Ind.] ............................................................... 20

Annex D (informative) Individual wet weights of selected zooplankton taxa for calculations

of biomass ....................................................................................................................................................... 22

Annex E (informative) Examples of sample preparation devices ............................................................ 27

E.1 Counting chambers ...................................................................................................................................... 27

E.1.1 General ............................................................................................................................................................. 27

E.1.2 Counting Chamber according to Bogorov ............................................................................................ 27

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E.1.3 Ward counting wheel .................................................................................................................................. 27

E.1.4 Tubular Plankton Chambers .................................................................................................................... 28

E.1.5 Utermöhl sedimentation and counting chamber .............................................................................. 28

E.1.6 Dolfuss counting chamber ......................................................................................................................... 29

E.2 Sample splitters ............................................................................................................................................. 29

Bibliography ................................................................................................................................................................. 31

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European foreword

This document (EN 17204:2019) 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 October 2019, and conflicting national standards shall

be withdrawn at the latest by October 2019.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
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Introduction

WARNING — Person using this European Standard should be familiar with normal laboratory

practice. This standard does not purport to address all of the safety problems, if any, associated

with its use. It is the responsibility of the user to establish appropriate health and safety

practices and to ensure compliance with any national and international regulatory conditions.

Mesozooplankton constitute an important part of zooplankton in the pelagic food webs, since these are

the organisms representing the link between primary producers and higher trophic levels.

Mesozooplankton community structure and productivity can be affected by changes in phytoplankton

stocks, species/size composition and phenology. Further, alterations in mesozooplankton can influence

prey availability for zooplanktivores and, thus, fish stock recruitment, as well as sedimentation of the

primary production, which, in turn, may affect food supply to benthic animals and oxygen levels in the

bottom water. [11].

Mesozooplankton comprise a large number of species within a range of total lengths of about 0,2 mm to

20 mm. The main groups are rotifers (Rotatoria), crustacean holozooplankton and merozooplanktonic

larvae of other taxa such as echinoderms, bivalves and crustaceans. Small hydromedusae, ctenophores,

chaetognaths, appendicularians, doliolids, fish eggs and larvae are also considered as part of the

mesozooplanktonic fauna in marine waters. As most protozooplankton species are smaller than 0,2 mm

these are not considered part of the mesozooplankton and hence procedures for sampling and

enumeration of these species are not included in this standard.
For sampling, preservation and storage of mesozooplankton see EN 17218:2019
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1 Scope

This document specifies a procedure for analysing mesozooplankton in marine and brackish waters.

The procedure comprises how to identify and enumerate mesozooplankton to estimate quantitative

information on diversity, abundance and biomass with regard to spatial distribution and long-term

temporal trends for a given body of water.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN 17218:2019, Water quality — Guidance for the sampling of mesozooplankton from marine and

brackish waters using mesh
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
biomass
total mass of living organic material in a given body of water
Note 1 to entry: Unit: g m .
[SOURCE: ISO 6107-3:1993, definition 12, modified – Added note1 to entry]
3.2
metazoan
multicellular animal that develops from embryos
3.3
plankton

organisms drifting or suspended in water, consisting chiefly of minute plants or animals, but including

larger forms having only weak powers of locomotion
[SOURCE: ISO 6107-5:2004, definition 41]
3.4
zooplankton
animals present in plankton
[SOURCE: ISO 6107-5:2004, definition 49]
3.5
merozooplankton

zooplankton that occurs in the plankton for only a part of their life cycle, usually the larvae stage

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3.6
mesozooplankton
zooplankton of 0,2 mm to 20 mm size
3.7
holozooplankton
zooplankton spending their whole life in the pelagic realm
3.8
sampling station
precise location where samples are collected

Note 1 to entry: A sampling station is defined by its geographical position (latitude, longitude), its depth

(relative to chart datum and normalized to mean low water as given in tide tables), the sampling depth and any

other invariant or physical condition. The station is delineated using the given level of precision. In case of doubts

when sampling stations have to be re-identified, most weight should be placed on depth and type of bottom, if

known.
[SOURCE: EN ISO 16665:2013, definition 2.2.5, modified - Added note 1 to entry]
3.9
sub-sample
portion removed from a sample and intended to be representative of that sample
[SOURCE: EN ISO 5667-6:2016, definition 3.13]
3.10
semi-quantitative analysis
analysis of relative abundance of a taxon or group of organisms in a sample
3.11
quantitative analysis

analysis of absolute number per volume of a taxon or group of organisms in a sample

4 Principle

The determination of the abundance and biomass of zooplankton in samples of natural communities is

based on microscopic counting and measuring of individuals of a representative sample, see Annex A

for examples of length measurements of some selected zooplankton taxa. The total biomass of each

taxon in the sample is determined by multiplying abundance with established length-weight conversion

factors (see Annex B), with species-specific carbon conversion factors (see Annex C) or species-specific

weight conversion factors.
5 Equipment
The following equipment is required for the analysis of zooplankton samples.

5.1 Stereoscopic microscope, objective and oculars with sufficient resolving power and

magnification for the purpose of the analysis, lighting: incident and transmitted light, preferably cold-

light source.

In some cases, the use of a bright-field or inverted compound 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

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for total magnification to 125 × at a minimum is recommended. The microscopes should have binocular,

bright field (additional phase contrast is helpful), 10 × or 12,5 × eyepieces.
5.2 Calibrated object micrometre.
5.3 Eyepiece (ocular) micrometre.

5.4 Counting chambers, for example Dolfuss chambers, Bogorov-chambers or Mini-Bogorov-

chambers and when using an inverted microscope: Utermöhl chambers, see E.1 or Petri dishes with

marked fields.

5.5 Sub-sample equipment, sample splitter for example Plunger sampling pipette according to

Hensen, Folsom plankton sample divider, Motoda box splitter or Kott splitter, see E.2.

5.6 Sieves, of a mesh size approximately half of the mesh size of the sampling net.

5.7 Microprobes, microforceps.
5.8 Dispenser.
5.9 Refrigerator.
5.10 Counting devices and/or image analysis software.
5.11 Fume hood, for the work with formaldehyde.
6 Preservatives and other chemicals
The following preservatives are required for analysis of zooplankton samples:

6.1 4 % formaldehyde (CH O) solution. 1 part 40 % formaldehyde solution and 9 parts water. The

formaldehyde solution has to be buffered to pH 8 to pH 8,2 with disodiumtetraborate (borax,

Na B O ·10H O).
2 4 3 2

NOTE Formaldehyde is an organic compound which is available in liquid form. Formalin is a commercially

sold aqueous saturated solution of formaldehyde at ∽40 % volume fraction or ∽37 % mass fraction. However, the

precise contents may vary slightly between producers. A small amount of stabilizer, such as methanol, is usually

added to limit oxidation and polymerization. A typical commercial-grade formaldehyde solution may contain 10 %

to 12 % of methanol by volume.

WARNING — Beware of formaldehyde vapours. Do not store large numbers of samples in small work

areas.
6.2 Ethanol. 96 % or 99 % C H OH.
2 5

6.3 Lugol’s Iodine. Acidified Lugol’s Iodine: Dissolve 100 g KI (potassium iodide) in 1 l of

demineralized water; then add 50 g iodine (crystalline), shake until it is dissolved and add 100 ml of

anhydrous acetic acid. As this solution is close to saturation, any precipitate should be removed by

decanting the solution before use.

Calcareous structures of organisms are damaged by acidified Lugol’s Iodine; therefore these organisms

should not be preserved with acidified Lugol’s Iodine. Acidified Lugol’s Iodine is mostly used for

protozooplankton and microzooplankton, for mesozooplankton samples are usually fixed with

formaldehyde or ethanol.
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6.4 Mastail and Battaglia solution. Prepare separate solutions by dissolving 8 g
buthylhydroxyanisol (BHA, C H O) in 500 ml propane-1-2-diol (C H O) and 20 g
22 32 4 3 8 2

ethylenediaminetetraacetic acid (EDTA, C H N O Na ·2H O) in 500 ml demineralized water. Add

10 14 2 8 2 2

both solutions to 2 l of 40 % formaldehyde solution while stirring and buffer to pH 8 with sodium

glycerophosphate (C H Na O P·H O). After buffering add 2 g ascorbic acid (C H O ) and

3 7 2 6 2 6 8 6

demineralized water up to 5 l. Samples are preserved by adding 6 ml of the stock solution per 100 ml of

sample in sea water.

NOTE This solution improves the preservation of chromatophores, which are key features for identification

of decapod larvae or fish eggs and larvae.
The following chemicals may be useful for analysis of zooplankton samples:

6.5 1-Hexadecanol (Cetyl alcohol, CH (CH ) CH OH) to reduce the surface tension (few drops per

3 2 14 2
100 ml).

6.6 Eosin Y, for staining of animals in phytoplankton rich samples (few drops per 100 ml).

6.7 Observation fluid, for example Steedmans observation fluid; 5 ml l 1-phenoxypropan-2-ol

(C H O ) and 45 ml l-1 propane-1-2-diol (C H O ) in demineralized water.
9 12 2 3 8 2
7 Procedure
7.1 Sample and sub-sample preparation

For sampling and storage of zooplankton samples from marine waters, see EN 17218:2019. All samples

should be retained in storage until the subsequent investigation is completed.

To remove the formaldehyde from the sample before the microscopic investigation the sample should

be filtered through a sieve and rinsed with filtered or sieved tap water. The mesh size of the sieves shall

be considerably smaller than the mesh size of the plankton net used for sampling. All activities should

occur under a fume hood to deflect the formaldehyde fume.

The formaldehyde should be collected in the sample vessel and reused to preserve the zooplankton

organisms after completion of the microscopic analysis.

The zooplankton should be transferred under careful rinsing from the sieve with filtered tap water into

a glass vessel and refilled to a certain volume depending of the density of organisms. The whole

zooplankton sample should be filled in a counting chamber or divided in case organisms are too densely

concentrated. Depending on the splitting device, the sample should be concentrated by sieving or

diluted with tap water as necessary.

Before filling the splitter the volume of the total sample shall be measured in a graduated glass or

plastic cylinder. The volume shall be noted in the protocol.

If the sample contains large clumps of plankton (e.g. by gelatinous organisms or Cercopagis) or

macroalgae, these shall be carefully removed and placed on a very large mesh. The clump should be

gently rinsed with water or seawater while pulling at the clumps with forceps to free trapped

organisms, which are returned to the sample before splitting or analysis. All the organisms still attached

to the clump shall be identified, counted and recorded.

To divide the sample into defined subsamples a calibrated Plunger sampling pipette according to

Hensen, a Folsom plankton sample divider (splits into two subsamples), a Motoda box splitter or a Kott

splitter (splits into eight subsamples) is recommended, see E.2.
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The splitter shall be placed on a level surface and operated in a way that ensures a homogenous split of

the sample.

After that, the sample shall be mixed intensively until all organisms are distributed homogenous in the

sample volume. A few drops of a detergent should be added to allow the cladocerans to mix in the

sample. The mixed sample should be splitted. The procedure can be repeated with an aliquot in case the

sample is still too dense. A known aliquot or the whole sub sample shall be filled in the counting

chambers.

For the work with Stempel-pipette mix the sample thoroughly and remove a 1 ml subsample

immediately. Make sure no air bubbles or large lumps of detritus are in the sample chamber. Rinse the

content of the pipette into a counting chamber.

The sub sample volumes have also to be recorded for calculating the abundance per sample. The count

multiplied by the inverse of the split is the estimated number of organism in the sample.

NOTE 1 Non-random distribution in the sample during sub-sampling is the most important source of error.

NOTE 2 The Kott Splitter, which produces eight sub samples, is somewhat better in precision, but is more time-

consuming to handle, while the Folsom sample divider splits samples into halves.

The counting chambers should be cleaned with tap water immediately after finishing the analysis to

avoid adhesion of dried organisms.

For routine counting of larger zooplankton taxa a stereomicroscope should be used, which allows

manipulation of the specimens during identification. For smaller organisms, an inverted microscope

should be used. For special investigations during identification, a compound microscope should be

used.
7.2 Species identification and counting
7.2.1 General

Species identification and counting are the basis of all zooplankton community analysis. Depending on

the aim of the investigation all taxa appearing in the sample are to be determined or only dominant

organisms and groups.

If possible, the complete sample shall be analysed. At least 100 individuals shall be counted. With higher

abundance, representative subsamples shall be analysed.

A hierarchical counting technique should be used to obtain density estimates for all taxa. This

procedure consists of first identifying all specimens (adults and development stages) and counting at

least 100 individuals of the occurring dominating taxonomic groups, excluding nauplii, rotifers and

tintinnids. If these minimum counts are not achieved in one subsample, additional subsamples shall be

counted. The taxonomic group(s) that reached 100 individuals in the previous subsample(s), do not

need to be counted in the next subsample(s). The precision of calculated abundance for organisms of

the first three groups, that will be counted up to 100 specimens, amounts to 20 % [11]. The estimation

of abundance for other groups (“tail”) will be less precise [10].

All individuals should be counted to avoid heterogeneities due to splitting. While counting the

settlement of all organisms to the bottom shall be ensured. It is possible to sink floating Microcrustacea

by gently pressing them down using the microprobe or by adding a drop of dilute laboratory detergent

(e.g. Cetyl alcohol). If a sample cannot be completely analysed and archived within 2 days, the sample

should be kept in the refrigerator and preservative added to prevent the degradation of the sample.

For the quantitative analysis of microzooplankton organisms of particular interest (e.g. tintinnids or

naupliar stages) abundance can be estimated semiquantitatively from the first subsample. For the

quantitative analysis of macrozooplankton organisms and rare species of particular interest (e.g. non-

indigenous species) the whole sample shall be scanned through.
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NOTE 1 Although macrozooplankton, nauplii, rotifers and tintinnids may fall outside the size range of

mesozooplankton, as do many of the merozooplankton, there is a considerable amount of historic data on these

groups. Thus, their presence or absence in the sample might be reported.

The fauna shall be identified to the lowest taxonomic level possible or that appropriate to the aim of the

survey.

Copepods shall be at least classified according to species, developmental stage (copepodites CI-III and

CIV-V classified as younger and older copepodites, respectively), and sex (adults); naupliar stages

should not be separated. Rotifers and cladocerans should be identified to the lowest possible taxonomic

level; moreover, the latter should be classified according to sex, and females as ovigerous or non-

ovigerous.

NOTE 2 The abundance of younger copepodite stages (CI-CIII) of copepods are generally underestimated by

the nets used for collecting mesozooplankton samples.

The nomenclature used shall be in accordance with recent editions of general faunal works and an

agreed regularly updated literature checklist or relevant catalogues of zooplankton fauna. For using the

correct names, the rules of the International Code of Zoological Nomenclature [19] shall be used. For

information on the validity of used taxonomic designations international databases, e.g. World Register

of Marine Species (WoRMS) [18] or the database of Marine Planktonic Copepods [20] should be

consulted.

Reference shall be made to keys and guides relating to the relevant geographical area and preferably in

the working language of the staff. Care should be taken to check descriptions of species and not just to

match illustrations.

The list of all taxa identified by analysts within the laboratory should include identification

characteristics and relevant references in the literature for each taxon. Drawings or digital photographs

of taxa observed should be made, see 9.2. A reference collection should be confirmed by specialists, see

9.3. Taxa shall never be identified beyond the level at which the analyst is confident.

7.2.2 Statistical requirements for counting

At least 100 individuals per taxon of interest shall be counted to ensure that the standard error of

counting would be < 20 %.

If not a sufficient number of individuals of a taxon are present in the sample to achieve the minimum

statistical requirements described, all the occurrences of this taxon shall be counted.

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7.2.3 Calculation of abundance

The abundance (number of individuals per volume unit) of the taxa or taxonomical groups per m shall

be calculated as follows:
Ind total sample
= NV/ (1)
Net
sub sample
where
Ind are the Individuals;
N is t
...

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