EN 17805:2023

SLOVENSKI STANDARD
01-junij-2023
Kakovost vode - Vzorčenje, zbiranje in konzerviranje okoljske DNK iz vode
Water quality - Sampling, capture and preservation of environmental DNA from water
Wasserbeschaffenheit - Probenahme, Erfassung und Konservierung von Umwelt‑DNA
in Wasser
Qualité de l'eau - Échantillonnage, collecte et conservation de l'ADN environnemental
prélevé dans l'eau
Ta slovenski standard je istoveten z: EN 17805:2023
ICS:
13.060.45 Preiskava vode na splošno Examination of water in
general
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17805
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2023
EUROPÄISCHE NORM
ICS 13.060.70
English Version
Water quality - Sampling, capture and preservation of
environmental DNA from water
Qualité de l'eau - Échantillonnage, collecte et Wasserbeschaffenheit - Probenahme, Erfassung und
conservation de l'ADN environnemental prélevé dans Konservierung von Umwelt-DNA in Wasser
l'eau
This European Standard was approved by CEN on 30 January 2023.

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, 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17805:2023 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 . 7
5 Procedure. 8
5.1 General . 8
5.2 Considerations prior to fieldwork . 8
5.3 Equipment preparation prior to fieldwork . 8
5.4 Sampling the eDNA from water . 8
5.5 Preserving the sample . 9
6 Equipment . 10
7 Preservative solutions. 11
7.1 General . 11
7.2 Examples of preservative solutions . 11
8 Sampling report . 12
8.1 General . 12
8.2 Sample identity and characteristics . 12
8.3 Sampling site . 12
8.4 Sampling conditions . 12
8.5 Sampling . 13
9 Avoiding sample contamination . 13
9.1 General . 13
9.2 Contamination that originates from equipment . 13
9.3 Sampling equipment decontamination procedure . 14
Annex A (informative) Filter types . 15
Bibliography . 16

European foreword
This document (EN 17805:2023) 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 September 2023, and conflicting national standards
shall be withdrawn at the latest by September 2023.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, 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 the
United Kingdom.
Introduction
WARNING — Persons using this document should be familiar with water sampling protocols to
assess biological diversity. 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.
Moreover, the need of notification, obtaining certificates or permits prior to sampling,
depending on national or international laws and regulations such as the Nagoya Protocol on
Access to Genetic Resources (https://www.cbd.int/abs/), needs to be considered.
The monitoring of organisms is key to the assessment of the status of aquatic ecosystems and is
required by national and international legislation such as the European Union Water Framework
Directive (2000/60/EC). A range of methods have been described how to monitor organisms in aquatic
environments, leading to a wide range of European standards (e.g. EN 14011:2003, EN 14757:2015,
EN 15460:2007). These approaches, however, necessitate the capture and/or collection of the
organisms of interest, which can be a laborious and time-consuming process.
The possibility to detect the presence of organisms and/or quantify relative abundance (e.g. [6]) in
aquatic environments via the analysis of environmental DNA (eDNA) provides a novel means to
monitor biodiversity across a wide range of taxonomic groups, including microorganisms, plants and
animals ([7][8][9]). This approach allows to examine organismic diversity without the need to directly
isolate and capture organisms and it is expected to play a key role for future biomonitoring aiming at
temporally and spatially highly resolved species inventories [10]. Albeit the power of the eDNA
approach has been repeatedly reported [11], there is a great need for standardizing the application of
eDNA-based assessment of aquatic biodiversity ([12], [13]). Note, however, that eDNA-based
biomonitoring currently does not allow to obtain certain population parameters (e.g. individual size,
sex) which can be obtained by traditional sampling techniques.
This document provides guidance how to sample and preserve eDNA from water samples, addressing
the first and crucial step for any further downstream eDNA-based analyses of biodiversity. A specific
technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for
metabarcoding analyses is CEN/TR 17245:2018.
1 Scope
This document specifies procedures for sampling, capture and preservation of environmental DNA
(eDNA) in aquatic environments, stemming from organisms that are or have recently been present in a
waterbody, have visited it or whose DNA has been introduced to the waterbody through some
mechanism. This document also covers procedures for avoiding sample contamination and ensuring
DNA quality, key properties of the filtering procedure and equipment and reporting standards.
This document does not include the collection of eDNA from biofilms, sediments or similar sample types
and does not cover sampling designs.
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:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
cross-contamination
unintended transfer of any source of and/or DNA from one sample to another sample
3.2
decontamination
procedure to remove any source and/or trace of DNA from material that might come into contact with
the sample
3.3
enclosed filter
filtering device where the filter membrane is encapsulated and where the inflow and outflow can be
closed for transport and storage
Note 1 to entry: The eDNA contained on the filter is typically extracted without removing the membrane from the
filter capsule greatly reducing the risk of contamination of samples. See Figure A.1 C. in Annex A.
3.4
environmental DNA
eDNA
material stemming e.g. from dead or from living organisms and include single-stranded (ss) and double-
stranded (ds) DNA fragments from nuclear and mitochondrial/plastid DNA of eukaryotes as well as
plasmid DNA of prokaryotes
Note 1 to entry: Subsuming DNA from various sources such as unicellular or small multicellular organisms or
tissue particles (e.g. shed cells, faeces) and gametes of multicellular organisms.
3.5
field equipment blank
sample obtained from processing target DNA-free water (e.g. distilled water) through all the equipment
used and covering all procedures involved in the eDNA sampling process to allow checking that the
equipment and procedures do not introduce DNA contamination
3.6
housed filter
systems in which a filter membrane is protected within a solid housing during the filtration process
Note 1 to entry: The filters are removed from the housing for eDNA extraction. The housing can be opened and
the filter removed for preservation and later processing. See Figure A.1 B. in Annex A.
3.7
lysis buffer
buffer solution to preserve DNA present in the sample and to lyse/open cells as a first step of the DNA
extraction
3.8
internal positive control
IPC
known fragment of synthetic or natural DNA containing an amplifiable and quantifiable sequence that
will not naturally occur in the sample
Note 1 to entry: The IPC can be added to the sample or the preservation/lysis buffer at a known concentration to
verify the efficiency of DNA preservation, DNA extraction, DNA amplification and DNA identification.
3.9
open filter
filtering device including filtration towers (laboratory) and filtration backpacks from which the filter
membrane has to be removed by hand for further processing
Note 1 to entry: See Figure A.1 A. in Annex A.
3.10
pre-filter
filter membrane, mesh or hose strainer with a larger pore-size than the main filter membrane (for
capturing the eDNA) through which water is passed first to remove larger particles of sediment, plant
material or algae to increase the volume of water that can be filtered before saturation of the main filter
3.11
sample contamination
process by which exogenous DNA is unintentionally introduced to the sample during the sampling
process
Note 1 to entry: DNA that is already present in the water before the eDNA sampling was undertaken is not
considered as contamination.
3.12
target DNA
any source and/or trace of DNA from the surveyed species/taxa
4 Principle
A representative water sample from the surveyed water body is sampled according to an appropriate
sampling design to capture and separate eDNA from the water sample. During the whole procedure
(cross-)sample contamination with target DNA is avoided and eDNA integrity is guaranteed.
An overview on the key steps and considerations for the eDNA water sampling process is provided in
Figure 1.
NOTE Numbers in parentheses refer to the respective clause/subclause.
Figure 1 — Key steps and considerations for the eDNA water sampling process
5 Procedure
5.1 General
Water shall be sampled to capture and separate eDNA via filtration or other processes. The probability
of obtaining eDNA from the targeted organism(s) is positively correlated with:
— the number of samples per waterbody;
— the spatial representativeness of the samples;
— the volume of water filtered;
— the optimum sampling time point/season with regard to the organism(s) eDNA shedding rates,
abundances, metabolic activity and locomotion.
5.2 Considerations prior to fieldwork
Depending on the different applications/goals of each eDNA survey, the most appropriate sampling
conditions and design shall be assessed based on case-by-case evidence to obtain water samples
representative of the water body and the organisms which shall be monitored. These might include
hydrological, meteorological, seasonal/temporal and biological/ecological variation.
This is particularly important in lentic (non-flowing) water bodies since eDNA is often unevenly
distributed when the water is not well mixed. Representative sampling can be achieved by merging
subsamples collected at different points in the water body, or alternatively by continuous sampling
systems that move across the water body while drawing up water. When surveying deep water bodies
and targeting deep water dwelling organisms, it may be necessary to sample water from depth.
To maximize the probability of capturing target DNA, the following shall be considered when planning
where and when to collect samples and subsamples:
1) Features of the water body, including its size, depth, flow and the distribution of microhabitats as
well as inlets/outlets of the waterbody.
2) Biology of all target taxa, including habitat preferences and lifecycle. Detection probability for
individual species can be increased by timing sampling to coincide with times of intense activity
(e.g. spawning). Temporal variations in the amounts of released eDNA by the target species needs
to be considered. It is also important to consider whether target taxa are likely to be present in the
water body at the time of sampling, especially in the case of amphibious or migratory species.
5.3 Equipment preparation prior to fieldwork
Prior to fieldwork the collecting vessels and equipment need to be cleaned to avoid contamination (for
detailed instructions, see Clause 9).
5.4 Sampling the eDNA from water
Various systems are used for sampling and filtering water. Some involve initially gathering water into a
collecting vessel where it is mixed and then filtered subsequently; other systems filter the water
directly as it is drawn up from the water body. When the water is not filtered directly in the water body,
the filtration can be carried out on the shore or in the laboratory.
Water shall be sampled and/or filtered to capture tissue fragments, cells and DNA. This may be
achieved manually with syringes or using a hand or powered pump. If a pump is used and water passes
through a pump tubing before reaching a filter then a new or decontaminated pump tubing shall be
used for each sample.
If you are merging subsamples into a pooled sample, ensure that they are well mixed before starting to
filter.
5.5 Preserving the sample
5.5.1 General
The eDNA contained in the coll
...