prEN 13946

SLOVENSKI STANDARD
01-oktober-2024
Kakovost vode - Navodilo za rutinsko vzorčenje in pripravo vzorcev bentoških
kremenastih alg rek in jezer
Water quality - Guidance standard for the routine sampling and preparation of benthic
diatoms from rivers and lakes
Wasserbeschaffenheit - Anleitung zur Probenahme und Probenaufbereitung von
benthischen Kieselalgen aus Fließgewässern und Seen
Qualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des
diatomées benthiques de rivières et de plans d'eau
Ta slovenski standard je istoveten z: prEN 13946
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2024
ICS 13.060.70 Will supersede EN 13946:2014
English Version
Water quality - Guidance standard for the routine
sampling and preparation of benthic diatoms from rivers
and lakes
Qualité de l'eau - Guide pour l'échantillonnage en Wasserbeschaffenheit - Anleitung zur Probenahme und
routine et le prétraitement des diatomées benthiques Probenaufbereitung von benthischen Kieselalgen aus
de rivières et de plans d'eau Fließgewässern und Seen
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
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: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13946:2024 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 . 6
5 Equipment . 6
5.1 Field sampling . 6
5.2 Laboratory equipment . 7
6 Reagents . 7
6.1 General. 7
6.2 Preservatives . 7
6.3 Reagents for cleaning diatoms for light microscopy . 8
6.4 Reagents for preparing permanent slides . 8
7 Sampling procedure . 8
7.1 Choice of substratum . 8
7.2 Sample site selection . 9
7.3 Sampling methods . 9
7.4 Preparation prior to microscopic examination . 11
Annex A (informative) Methods for cleaning diatoms for microscopic examination . 14
A.1 General. 14
A.2 Method 1: Hot hydrogen peroxide . 14
A.3 Method 2: Cold hydrogen peroxide . 15
A.4 Method 3: Hot hydrogen peroxide with potassium dichromate . 15
A.5 Method 4: Cold acid (permanganate) method of cleaning . 16
A.6 Method . 16
Bibliography . 18

European foreword
This document (prEN 13946:2024) 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 will supersede EN 13946:2014.
This document (prEN 13946:2024) includes the following significant technical changes with respect to
EN 13946:2014:
— the method has been adapted to process the samples obtained for subsequent molecular methods
by adding additional solvents and requirements to avoid contamination of samples.
Introduction
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate health and safety practices and to
ensure compliance with any national regulatory conditions.
Diatoms are an important component of aquatic ecosystems and constitute a water quality monitoring
tool where the primary objective is either a measure of ecological status or the impact of specific
components of water quality (e.g. eutrophication, acidification). The method is appropriate for
assessments required by the Water Framework Directive (2000/60/EC) and Urban Waste Water
Treatment Directive (91/217/EEC) in addition to other EU Directives and international agreements.
This document covers aspects of sampling and preparation relevant to assessment of water quality and
ecological status using benthic diatoms. These instructions will result in samples suitable for
quantifying relative numbers of benthic diatom taxa present using either light microscopy or molecular
methods. If it is necessary to quantify absolute numbers of taxa, or fresh weight per unit area,
modifications to the method are required, which are not within the scope of this document.
The use of diatoms as indicators of river and lake quality is widely accepted both in Europe and beyond.
The methodology is based on observations that all diatom species have distinct preferences for
particular environmental conditions such as nutrients, organic pollution and acidity. Polluted waters
will tend to support an increased abundance of those species whose optima correspond with the levels
of the pollutant in question. Conversely, certain species are intolerant of elevated levels of one or more
pollutants, whilst others may occur in a wide range of water qualities.
Methods using diatoms to assess water quality have been developed in several European countries
(recent work is summarized in references [1] to [4]). The methods for evaluating the data vary but the
sampling and preparation processes are similar [5, 6]. In recent years, molecular methods such as
metabarcoding have been developed to the point where they are suitable for routine use [7, 8] and
modifications to procedures appropriate for this approach have been included in this revision of the
standard.
According to the precise usage to which this standard is to be put it is essential for specifiers and users
to mutually agree on any necessary variations or optional procedural details prior to use.
All numerical values given in this document are approximate.
1 Scope
This document specifies a method for the sampling and laboratory preparation of benthic diatoms for
ecological status and water quality assessments. The sampling and preparation procedures described
can be used for later investigations using either light microscopy or molecular methods. Data produced
by this method are suitable for production of indices based on the relative abundance of taxa.
Analysis using molecular methods is not within the scope of the document.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
3.1
benthic diatoms
diatoms living on natural or artificial substrata, rather than suspended in the water column
3.2
boulder
mineral substratum with a diameter > 256 mm
3.3
cobble
mineral substratum with a diameter > 64 mm and ≤ 256 mm
3.4
ecological status
measure of the structure and functioning of aquatic communities
3.5
euphotic zone
part of the water column in which there is sufficient light for photosynthesis
3.6
frustule
cell wall of diatoms, composed of silica and consisting of two valves linked by two or more girdle bands
3.7
habitat
specific environment in which an organism lives
3.8
introduced substrata
artificial substratum
substratum introduced into river or lake by operator specifically for colonisation by diatoms
3.9
metabarcoding
identification (and quantification) of many species from a single sample based on sequences from a
short DNA/RNA region
3.10
molecular method
method that use molecular genetic technologies in order to elucidate the properties of biological
samples
3.11
pebble
mineral substratum with a diameter > 16 mm ≤ 64 mm
3.12
riffle
shallow part of a stream with swift flow, usually with a broken surface
3.13
substratum
natural or non-natural material from which benthic diatoms are sampled
3.14
taxa
taxonomic units, for example families, genera or species
3.15
valve
structural component of the diatom frustule (3.6)
4 Principle
Benthic diatoms from submerged hard surfaces or submerged macrophytes in rivers, streams or littoral
zones of lakes are sampled in order to produce representative collections of the diatom assemblage
indicative of ecological status and water quality. Samples are cleaned using strong oxidizing agents in
order to prepare diatoms for identification and enumeration, or preserved for analysis by molecular
methods.
The data obtained from the microscopic or molecular methods analysis of these samples are suitable for
the production of diatom based water quality indices (see references[1], [2], and [3]).
5 Equipment
5.1 Field sampling
Apparatus and equipment used for removing biofilms from surfaces shall be free from any
contamination that may bring diatoms or DNA traces from other samples. It is not necessary to use a
brand new toothbrush for every site, but a strict cleaning procedure after use is recommended to avoid
cross contamination [9, 10]. Toothbrushes can be cleaned by rinsing and rubbing to remove coarse
materials. When samples are to be used for metabarcoding, stronger cleaning agents (e.g. a 10 % bleach
solution for 10 min) should be used before thoroughly rinsing and drying.
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