SIST EN 17136:2019

SLOVENSKI STANDARD
oSIST prEN 17136:2017
01-julij-2017
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Water quality - Guidance on field and laboratory procedures for quantitative analysis and
identification of macro-invertebrates from inland surface waters
Wasserbeschaffenheit - Anleitung zu Feld- und Laborverfahren zur quantitativen Analyse
und Identifizierung von Makroinvertebraten aus Inland-Oberflächengewässern
Ta slovenski standard je istoveten z: prEN 17136
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
oSIST prEN 17136:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17136:2017


DRAFT
EUROPEAN STANDARD
prEN 17136
NORME EUROPÉENNE

EUROPÄISCHE NORM

June 2017
ICS 13.060.10
English Version

Water quality - Guidance on field and laboratory
procedures for quantitative analysis and identification of
macro-invertebrates from inland surface waters
 Wasserbeschaffenheit - Anleitung zu Feld- und
Laborverfahren zur quantitativen Analyse und
Identifizierung von Makroinvertebraten aus Inland-
Oberflächengewässern
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, 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: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17136:2017 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 . 5
5 Equipment, preservatives and reagents . 6
5.1 Sieves . 6
5.2 Microscope . 6
5.3 Preservatives and fixatives . 7
5.4 Reagents for examination using compound microscopes . 7
6 Procedure . 8
6.1 General . 8
6.2 Selection of preservatives . 8
6.3 Sample processing in the field . 8
6.4 Transport and storing of samples . 9
7 Laboratory procedure . 9
7.1 General . 9
7.2 Pre-treatment prior to sorting . 10
7.3 Quantitative sorting . 12
8 Identification . 13
8.1 General . 13
8.2 Acari . 14
8.3 Chironomids . 14
8.4 Oligochaetes . 14
8.5 Platyhelminthes . 14
9 Quality assurance . 14
Annex A (informative) Examples of sample splitters and sorting tray . 15
Bibliography . 17


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European foreword
This document (prEN 17136:2017) 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.
WARNING — Working in or around water is inherently dangerous. Care should be taken
when working with different and generally toxic preservatives. This standard does not
purport to address the safety problems associated with its use. It is the responsibility of
the user to adopt appropriate health and safety practices in compliance with national
regulatory conditions.
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Introduction
Macroinvertebrates are important in water quality assessment and form one of the biological
quality element in the EC Water Framework Directive (WFD 2000/60/EC). Macroinvertebrates
are a very diverse group of organisms for which an efficient and effective procedure for analysis
is not always obvious. Their large size range, very different appearances and the diverse range of
habitats they live in produce specific problems for collecting and analysis. This guidance aims to
provide general guidance for the quantitative processing of macroinvertebrate samples to
reduce sample processing related variation for WFD in particular and scientific studies in
general. It is not intended to give a full and exact overview of all possible methods. Specific
situations and investigation purposes may require expert judgement based deviations from the
outlined procedure but the general principles given in this guideline should always apply.
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1 Scope
This European Standard gives guidance on the estimation of abundance and identification of
macroinvertebrates in samples taken from inland waters. The procedure deals with pre-
treatment (cleaning), sub-sampling, sorting, and final identification of organisms from preserved
and live samples originating from natural habitats or artificial substrates. Specific guidance is
given for preservation for DNA-analysis.
2 Normative references
Not applicable.
3 Terms and definitions
For the purpose of this document, the following terms and definitions apply.
3.1
macro invertebrate
Invertebrate that is easily visible without magnification (> 0,5 mm) and generally belongs to the
group of organisms that live for at least one life stage on or in the bottom substrate or
vegetation of inland surface waters
3.2
matrix
components of a sample other than the macroinvertebrates
4 Principle
The process for the identification and estimation of the abundance of macroinvertebrates
comprises both field and laboratory procedures. A pre-treatment of a fresh sample should be
performed in the field or upon arrival in the laboratory to allow for efficient analysis. Pre-
treatment consists of rinsing and sieving to remove as much matrix as possible. If needed,
specific groups (Table 1) can directly be sorted, removed and identified. After pre-treatment the
sample should be immediately cooled and/or preserved by addition of preservative. As
alternative to cooling or preservation samples can also be frozen at ≤ −18°C. Freezing is not
suitable for Platyhelminthes and Annelida.
In the laboratory the need and selection of a subsequent sorting step should be determined by
visual inspection of the composition of matrix material (silt, clay, sand, macrophytes) and
number of organisms. Such a procedure may comprise further rinsing and cleaning and/or sub-
dividing of the sample. In case of preserved samples small trays are used in combination with a
stereo-zoom microscope. For unpreserved samples macroinvertebrates are sorted out visually
(naked eye or magnifying glass) on a tray. Organisms are immediately identified to the main
taxonomic groups. Then, individuals from each group are identified to the desired taxonomic
level.
Generally the smallest mesh size for collecting the macroinvertebrates during the analysis is
500 µm (e.g. sampling EN ISO 10870) as for most waters it is the best combination for trapping
the macroinvertebrates and at the same time allowing effective rinsing. In specific situations
(e.g. clear stony rivers) a smaller mesh size for maximum trapping of small macroinvertebrates
can be selected.
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For samples intended for DNA analysis, special emphasis should be put on working with clean
equipment during the whole process to minimize the risk of cross-contamination during both
field and laboratory procedures.
Table 1 — Typical taxonomic groups of macroinvertebrates
Bryozoa, Cnidaria, PoriferaF
Platyhelminthes
Annelida (Hirudinea, Oligochaeta, Polychaeta)
Crustacea-(Decapoda, Isopoda, Amphipoda, Mysida, remaining)
Diptera- (Chironomidae, remaining)
Arachnida
Coleoptera
Ephemeroptera
Heteroptera
Lepidoptera
Odonata
Trichoptera
Insecta-remaining
Bivalvia
Gastropoda
5 Equipment, preservatives and reagents
5.1 Sieves
Each new sieve shall be carefully checked for the exact mesh size before being put into service. If
the mesh size does not fulfil the desired quality grade the sieve shall be excluded from use.
Subsequently, before every use a sieve should be inspected for damage. The critical and smallest
(typically 500 µm) sieves (having a significant effect on the validity of the analysis) should be
fully traceable calibrated.
For DNA-based studies the removal of larger, visible contaminants by washing the equipment in
water only is not sufficient. Instead sieves should be cleaned preferably with an appropriate
bleach solution for each consecutive sample location.
5.2 Microscope
For accurate quantitative sorting and most identifications a low-power stereo-zoom microscope
(typical magnification ~10x-60x) is needed. For the species identification of specific groups like
Chironomidae and Oligochaeta a compound microscope (typical magnification 40x-400x) should
be used.
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5.3 Preservatives and fixatives
5.3.1 General
There is a distinct difference between fixation and preservation. In general fixation is the most
desirable for morphological analysis but specimens should never be stored in any fixative for
long or even short periods of time. Within the context of macro-invertebrate analysis ethanol as
a preservative and formaldehyde as a fixative are by tradition the most often used chemicals.
5.3.2 Ethanol, C H OH
2 5
The final ethanol concentration in the sample should be about 70 % (≥80 % for samples
intended for DNA extraction) to ensure preservation. Therefore ethanol 96 % should be added
to the sample in most cases to compensate for dilution by sample water. Ethanol concentrations
well above 70 % will harden many organisms rendering them difficult to manipulate and
identify. Ethanol concentrations below 70 % will lead to tissue degradation.
Macroinvertebrate samples used for DNA studies or DNA-based identification that need to be
stored for extended times before analysis (e.g. DNA barcoding) should be placed within two days
of sampling in ≥ 90 % ethanol and preferably stored in a cool and dark place. At concentrations
below 80 % DNA will degenerate.
5.3.3 Formaldehyde, 37 % (volume fraction)
Formaldehyde should be added to the sample to give a final concentration of 4 %–6 %. It is most
suitable for fixation of organisms (avoiding damaging and shrinking). Formaldehyde is not
suitable for DNA analysis and should be avoided as much as possible at it is hazardous to
humans. Formaldehyde fixation should be limited to 24 h after which the organisms should be
rinsed thoroughly and transferred to 70 % ethanol for (long-term) storage.
Preservation by ethanol and formalin fixation affects the estimation of biomass. Although weight
loss by ethanol is considered to be more significant than by formalin no unambiguous scientific
data are available on the exact effect of either preservative. Therefore, prior to estimating
biomass on the basis of preserved organisms the bias by using preserved material should be
assessed for the specific taxonomic groups under investigation and the relevant conditions of
sampling and preservation.
5.4 Reagents for examination using compound microscopes
5.4.1 General
Many chemicals are known to enhance the microscope image quality in order to be able to
accurately view diagnostic features. Koenike and Laevulose syrup are the most often used
reagents. Both solution can be stored at room temperature for 1 year.
5.4.2 Koenike
Mix 50 ml of glycerol with 30 ml of water and add 20 ml of acetic acid. This solution can be
stored at room temperature for an unlimited period of time. “Koenike’s Gemisch” is used for
identification and long-term storage of Acari.
5.4.3 Laevulose syrup
Dilute 25 g D(-)fructose in 25 ml water by proper stirring (magnetic stirring bar). The solution
can be slightly heated. Thereafter add 25 mL of lactic acid and stir again. Laevulose is used for
the identification of Oligochaeta and Chironomidae.
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6 Procedure
6.1 General
If live sample sorting is used samples should be sorted preferably within 24 h (max 48 h) and
measures should be taken to prevent predation [1] and dying of organisms by oxygen depletion.
Generally logistic considerations and negative effects of predation necessitate the preservation
of samples before sorting. In case of preserved samples it can be advantageous to select specific
groups (e.g. Platyhelminthes) from the live sample in the field for identification to anticipate the
negative effects of nonspecific preservation for some organism groups.
6.2 Selection of preservatives
Ethanol is the best general preservative. Before adding ethanol the sample should be drained to
minimize the dilution with excess sample water. In samples with absorbing material and/or
relatively large organisms (e.g. numerous Molluscs) the initial 96 % ethanol should be replaced
once to ensure a final concentration (approximately 70 %) in the sample. The sample container
should be filled not more than one third with material. Ethanol should be added until the sample
is completely immersed and the container is filled to achieve a concentration of 75 %. Ethanol
concentration should be checked on a regular basis with an ethanol meter. This check can best
be carried out in a graduated cylinder.
If formaldehyde is used for preservation (not recommended) samples should not be drained.
Usually 37 % formaldehyde is used to obtain a final concentration of 4 %–6 %.
After the preservative has been added the sample should be gently turned to disperse the
preservative.
Ethanol preserved samples can be stored for a long time (several months). Formaldehyde fixed
samples should be transferred to ethanol if they are kept for more than a few months before
sort
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