kSIST FprEN 17805:2022

Water sampling for capture of macrobial environmental DNA in aquatic environments

Water sampling for capture of environmental DNA (eDNA) in aquatic environments. eDNA stems from organisms which are or have recently been living in the water body and does not include eDNA found in sediments or similar sample types. Covers procedures for avoiding sample contamination and ensuring DNA quality, key properties of the filtering procedure and equipment, and reporting standards.

Wasserprobenahme zum Nachweis aquatischer Umwelt-DNA

Dieses Dokument legt Verfahren für die Probenahme, den Fang und die Konservierung von Umwelt DNA (eDNA) in Gewässern fest, die von Organismen stammen, die sich in einem Gewässer aufhalten oder aufgehalten haben, das Gewässer besucht haben oder deren DNA durch irgendeinen Mechanismus in das Gewässer gelangt ist. Dieses Dokument behandelt auch Verfahren zur Vermeidung von Proben¬kontaminationen und zur Sicherstellung der DNA Qualität, Schlüsseleigenschaften des Filtrationsverfahrens und der Ausrüstung sowie Berichtsstandards.
Dieses Dokument befasst sich nicht mit der Gewinnung von eDNA aus Biofilmen, Sedimenten oder ähnlichen Probenarten und geht nicht auf die Gestaltung der Probenahme ein.

Techniques de prélèvement d’eau en vue de l’analyse de l’ADN environnemental dans les milieux aquatiques

Le présent document spécifie des modes opératoires d'échantillonnage, de capture et de conservation de l’ADN environnemental (ADNe) dans des milieux aquatiques, provenant d’organismes qui sont ou qui ont été récemment présents dans une masse d’eau, qui l’ont parcouru ou dont l’ADN a été introduit dans la masse d’eau par un mécanisme précis. Le présent document porte également sur les modes opératoires permettant d'éviter la contamination des échantillons et de contrôler la qualité de l’ADN, les principales propriétés du mode opératoire et de l'équipement de filtration et les normes relatives au compte-rendu.
Le présent document n’inclut pas la collecte d’ADNe de biofilms, sédiments ou autres types d'échantillons similaires et n’aborde pas la question des plans d'échantillonnage.

[Not translated]

General Information

• Status: Not Published
• Public Enquiry End Date: 02-Feb-2022
• ICS: 13.060.45 - Examination of water in general
• Technical Committee: KAV - Water quality
• Current Stage: 5020 - Formal vote (FV) (Adopted Project)
• Start Date: 14-Nov-2022
• Due Date: 02-Jan-2023
• Completion Date: 13-Dec-2022
• Directive: 2000/60/EC - Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (substitutes Directive Reference 01/991 - Common Position on WFD)
• Ref Project: FprEN 17805 - Water quality - Sampling, capture and preservation of environmental DNA from water

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN 17805:2022
01-januar-2022
[Not translated]

Water sampling for capture of macrobial environmental DNA in aquatic environments

Wasserprobenahme zum Nachweis aquatischer Umwelt-DNA

Techniques de prélèvement deau en vue de lanalyse de lADN environnemental dans les

milieux aquatiques
Ta slovenski standard je istoveten z: prEN 17805
ICS:
13.060.45 Preiskava vode na splošno Examination of water in
general
oSIST prEN 17805:2022 en,fr,de

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

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oSIST prEN 17805:2022
DRAFT
EUROPEAN STANDARD
prEN 17805
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2021
ICS
English Version
Water sampling for capture of macrobial environmental
DNA in aquatic environments

Techniques de prélèvement d¿eau en vue de l¿analyse Wasserprobenahme zum Nachweis aquatischer

de l¿ADN environnemental dans les milieux aquatiques Umwelt-DNA

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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17805:2021 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

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

5 General Procedure .......................................................................................................................................... 8

5.1 General ................................................................................................................................................................ 8

5.2 Sampling ............................................................................................................................................................. 8

5.3 Equipment preparation prior to fieldwork ........................................................................................... 8

5.4 Collecting the water and capturing the eDNA ....................................................................................... 8

5.5 Preserving the sample ................................................................................................................................... 9

6 Equipment ......................................................................................................................................................... 9

7 Preservative solutions................................................................................................................................ 11

7.1 General ............................................................................................................................................................. 11

7.2 Examples of preservative solutions ...................................................................................................... 11

8 Recommended key information to record pre-planning............................................................... 11

8.1 General ............................................................................................................................................................. 11

8.2 Sample identity and characteristics ...................................................................................................... 12

8.3 Sampling site .................................................................................................................................................. 12

8.4 Sampling conditions .................................................................................................................................... 12

8.5 Sampling .......................................................................................................................................................... 12

9 Avoiding sample contamination ............................................................................................................. 13

9.1 General ............................................................................................................................................................. 13

9.2 Contamination that originates from equipment ............................................................................... 13

9.3 Contamination that originates from the person taking the samples ........................................ 13

9.4 Sampling equipment decontamination procedure .......................................................................... 13

9.5 Materials and equipment in direct contact with the water sample ........................................... 14

9.6 Materials and equipment not in direct contact with the water sample ................................... 14

Bibliography ................................................................................................................................................................. 15

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European foreword

This document (prEN 17805:2021) 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.
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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.

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 14757:2005,

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. [1]) 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

([2][3]). 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 [4]. Albeit the power of the eDNA approach has been

repeatedly reported [5], there is a great need for standardizing the application of eDNA-based assessment

of aquatic biodiversity ([6][7]). 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 collect 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.
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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 terminological 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/

Note 1 to entry: Not all definitions listed below are necessarily applicable to all studies. Only those which are

relevant to the aims and objectives of the study in question are required.
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 unit

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 filter from the filter

capsule greatly reducing the risk of contamination of samples
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 gamets of multicellular
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3.5
field equipment blank

sample obtained from processing target-DNA-free water through all the equipment used and covering all

procedures involved in the eDNA sampling process to ensure that the equipment and procedures do not

introduce contamination
3.6
housed filter unit

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
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 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 unit

filtering device from which the filter membrane has to be removed by hand for further processing

including filtration towers (laboratory) and filtration backpacks
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
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4 Principle

A representative water sample from the surveyed water body is collected 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 respective clause/subclause.
Figure 1 —Key steps and considerations for the eDNA water sampling process
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5 General Procedure
5.1 General

Water shall be collected to capture and separate eDNA via filtration or other processes. The probability

of collecting 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 Sampling

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 collect 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). 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 instruction see 8.4).
5.4 Collecting the water and capturing the eDNA

Various systems are used for collecting and filtering water. Some involve initially gathering water into a

collecting vessel where it is mixed and then filtered at the shore; 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 collected 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.
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If you are merging subsamples into a pooled sample, ensure that they are well mixed before starting to

filter.
5.5 Preserving the sample

The eDNA contained in the collected water shall ideally be separated from the water immediately in the

field, and the obtained eDNA sample (e.g. filter, precipitate) shall be immediately preserved in the field

for transportation to the laboratory and storag
...