SIST EN 17136:2019
(Main)Water quality - Guidance on field and laboratory procedures for quantitative analysis and identification of macroinvertebrates from inland surface waters
Water quality - Guidance on field and laboratory procedures for quantitative analysis and identification of macroinvertebrates from inland surface waters
This standard gives guidance on the estimation of abundance and identification of macro-invertebrates 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.
Wasserbeschaffenheit - Anleitung zu Feld- und Laborverfahren zur quantitativen Analyse und Identifizierung von Makroinvertebraten aus Inland-Oberflächengewässern
Dieses Dokument ist eine Anleitung zur quantitativen Beurteilung der Abundanz und Bestimmung von Makroinvertebraten in Proben aus Binnengewässern. Das Verfahren befasst sich mit der Vorbehandlung (Reinigung), Herstellen von Teilproben, Sortierung und endgültigen Bestimmung von Organismen aus konservierten und lebenden Proben, die aus natürlichen Lebensräumen oder künstlichen Substraten stammen, und ihrem Transport zum Labor. Für die DNA-Analyse wird eine spezifische Anleitung zur Konservierung gegeben.
Qualité de l'eau - Guide sur les modes opératoires de terrain et de laboratoire pour l'analyse quantitative et d'identification des macro-invertébrés des eaux de surface intérieures
Le présent document donne des conseils sur l’estimation quantitative de l’abondance et l’identification des macro-invertébrés dans des échantillons provenant de milieux aquatiques continentaux. La procédure s’applique au prétraitement (nettoyage), au sous-échantillonnage, au tri et à l’identification finale des organismes provenant d’échantillons avec ou sans conservateurs, issus d’habitats naturels ou d’échantillonneurs de colonisation, ainsi qu’à leur transport au laboratoire. Des conseils spécifiques sont donnés au sujet de la conservation en vue de l’analyse ADN.
Kakovost vode - Navodilo za terenske in laboratorijske postopke kvantitativne analize in identifikacije velikih nevretenčarjev v celinskih površinskih vodah
Ta standard podaja navodilo za oceno številčnosti in identifikacijo velikih nevretenčarjev v vzorcih iz celinskih voda. Postopek obravnava predobdelavo (čiščenje), podvzorčenje, razvrščanje in dokončno identifikacijo organizmov iz ohranjenih in živih vzorcev, ki izhajajo iz naravnih habitatov ali umetnih substratov.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.YDQWLWDWLYQHWasserbeschaffenheit - Anleitung zu Feld- und Laborverfahren zur quantitativen Analyse und Identifizierung von Makroinvertebraten aus Inland-OberflächengewässernQualité de l'eau - Guide sur les modes opératoires de terrain et de laboratoire pour l'analyse quantitative et d'identification des macro-invertébrés des eaux de surface intérieuresWater quality - Guidance on field and laboratory procedures for quantitative analysis and identification of macroinvertebrates from inland surface waters13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of water13.060.10Voda iz naravnih virovWater of natural resourcesICS:Ta slovenski standard je istoveten z:EN 17136:2019SIST EN 17136:2019en,fr,de01-maj-2019SIST EN 17136:2019SLOVENSKI
STANDARD
SIST EN 17136:2019
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 17136
February
t r s { ICS
s uä r x rä s r English Version
Water quality æ Guidance on field and laboratory procedures for quantitative analysis and identification of macroinvertebrates from inland surface waters Qualité de l 5eau æ Guide sur les procédures de terrain et de laboratoire pour l 5analyse quantitative et l 5identification des macroæinvertébrés des eaux de surface continentales
Wasserbeschaffenheit æ Anleitung zu Feldæ und Laborverfahren zur quantitativen Analyse und Bestimmung von Makroinvertebraten aus Binnenoberflächengewässern This European Standard was approved by CEN on
s v December
t r s zä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Rue de la Science 23,
B-1040 Brussels
9
t r s { CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s y s u xã t r s { ESIST EN 17136:2019
EN 17136:2019 (E) 2 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 Fixatives and preservatives . 6 5.3.1 General . 6 5.3.2 Formaldehyde, 37 % (volume fraction) . 6 5.3.3 Ethanol, C2H5OH . 7 5.4 Reagents for examination using compound microscopes . 7 5.4.1 General . 7 5.4.2 Koenike . 7 5.4.3 Laevulose syrup . 7 6 Pre-laboratory procedure . 7 6.1 General . 7 6.2 Sample processing in the field . 8 6.3 Transport and storing of samples . 8 7 Laboratory procedure . 9 7.1 General . 9 7.2 Pre-treatment prior to sorting . 10 7.2.1 General . 10 7.2.2 Pre-sorting . 11 7.2.3 Rinsing . 11 7.2.4 Decanting . 11 7.2.5 Sub-sampling . 11 7.3 Quantitative sorting . 12 7.3.1 General . 12 7.3.2 Quantitative sorting on a tray . 13 7.3.3 Quantitative sorting using a stereo-zoom microscope . 14 7.3.4 Screening . 14 8 Identification . 14 8.1 General . 14 8.2 Arachnida . 15 8.3 Chironomids . 15 8.4 Oligochaetes . 15 8.5 Platyhelminthes . 15 9 Quality assurance . 15 Annex A (informative)
Examples of sample splitters and sorting tray . 16 Bibliography . 18
SIST EN 17136:2019
EN 17136:2019 (E) 3 European foreword This document (EN 17136:2019) 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 August 2019, and conflicting national standards shall be withdrawn at the latest by August 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. SIST EN 17136:2019
EN 17136:2019 (E) 4 Introduction 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. Macroinvertebrates are important in water quality assessment and form one of the biological quality elements 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 standard aims to provide a general procedure for the quantitative analysis of macroinvertebrate samples for the WFD in particular and scientific studies in general. It is not intended to give a full and exact overview of all possible methods. Although specific situations and investigation objectives may require adjustments to the outlined procedure the general principles given in this guideline should still be valid. SIST EN 17136:2019
EN 17136:2019 (E) 5 1 Scope This document gives guidance on the quantitative 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 unpreserved samples originating from natural habitats or artificial substrates and their transport to the laboratory. Specific guidance is given for preservation for DNA-analysis. 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: — IEC Electropedia: available at http://www.electropedia.org/ — ISO Online browsing platform: available at http://www.iso.org/obp
3.1 fixation protection from disintegration of the morphological structure of organisms 3.2 macroinvertebrate 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 3.3 matrix components of a sample other than the macroinvertebrates 3.4 preservation process that protects organic substances from decay 4 Principle The identification and quantification of macroinvertebrates comprises both field and laboratory procedures. After an optional pre-treatment of rinsing and sieving in the field, macroinvertebrate samples are transported to the laboratory where the samples are further processed. Macroinvertebrates are taken out of the matrix material by a sample dependent technique, quantified and sorted in functional groups for identification using a microscope or DNA-analysis. Several techniques exist to clean (remove unwanted matrix material) the sample and sort the organisms. The most suitable technique should be selected by visual inspection of the individual sample. Dependent on the objective of the analysis samples can be preserved in the field or cooled and processed unpreserved. SIST EN 17136:2019
EN 17136:2019 (E) 6 5 Equipment, preservatives and reagents 5.1 Sieves Each new sieve should be checked for the right mesh size before first use. The smallest (typically 500 µm) sieve (having a significant effect on the validity of the analysis) is critical, its mesh should be guaranteed by the manufacturer attestation or control, providing metrological traceability. It should regularly visually be inspected for visible damage before every use 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 A low-power stereo-zoom microscope (typical magnification ~10x to 60x) can be used for accurate quantitative sorting and most identifications. For the species identification of specific groups like Chironomidae and Oligochaeta a compound microscope (typical magnification 40x to 400x) should be used. 5.3 Fixatives and preservatives 5.3.1 General There is a distinct difference between fixation and preservation. In general, fixation is preferred for morphological analysis. Within the context of freshwater macroinvertebrate analysis ethanol as a preservative is the most often used chemical for preserving samples for quantification and identification. When ethanol is used as a preservative formaldehyde fixation can be left out for macroinvertebrate analysis. WARNING — Formaldehyde is hazardous to humans (CMR substance, see ECHA) even at very low concentrations in air. Formaldehyde fixation should be avoided as much as possible. Preservation by ethanol and formaldehyde fixation affects the estimation of biomass. Although weight loss by ethanol is considered to be more significant than by formaldehyde 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. After the fixative or preservative has been added the sample should be gently turned to disperse the preservative. 5.3.2 Formaldehyde, 37 % (volume fraction) Formaldehyde 37 % should be added to an undrained sample with a final concentration of 4 % to 6 %. This concentration provides a good fixation of organisms (avoiding damaging and shrinking). Formaldehyde is not a suitable fixative for DNA analysis. Formaldehyde samples should always be thoroughly rinsed with water on a sieve prior to processing. Long-term storage in formaldehyde can seriously affect the colour of specimens and dissolves the shells of Mollusca (if not buffered). To prevent this, formaldehyde fixed samples can be transferred to 70 % ethanol. Transferring the sample to ethanol can already be done after 24 h of fixation. The sample should be thoroughly rinsed with water to remove the free formaldehyde before adding the ethanol. SIST EN 17136:2019
EN 17136:2019 (E) 7 5.3.3 Ethanol, C2H5OH Before addition of ethanol the sample should be drained to minimize the dilution with excess sample water. In most cases ethanol 96 % should be added to compensate for dilution by the residual sample water. In samples with absorbing material and/or relatively large organisms (e.g. numerous molluscs) the initial 96 % ethanol should be replaced after a few days to ensure a permanent concentration of approximately 70 % in the sample. The sample container should not be filled for 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 concentrations well above 70 % will harden many organisms rendering them difficult to manipulate and identify. Ethanol concentrations below 70 % will lead to tissue degradation. Initial ethanol concentrations should be checked sample-wise with an alcohol meter. Unfixed macroinvertebrate samples preserved with ethanol can be stored for several years. 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. 5.4 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 describe diagnostic features. The most relevant chemicals including mounting media can be found in group specific identification literature. Koenike and Laevulose syrup, presented below, are the most often used reagents. Koenike can also be used as a preservative for Arachnida (mites). 5.4.2 Koenike Mix 50 ml of glycerol with 30 ml of water and add 20 ml of acetic acid. This clear solution can be stored at room temperature for an unlimited period of time. 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. This clear solution can be stored at room temperature for an unlimited period of time. 6 Pre-laboratory procedure 6.1 General The pre-laboratory procedure starts with a visual inspection of the sample to assess the amount of matrix material (silt, clay, sand, macrophytes) and the number of organisms. With this information the right procedure for pre-treatment and sorting can be selected. It can be done in the field directly after sampling or in the laboratory. The general objective is to select the organisms in the most effective way (least amount of time). If sub-sampling is necessary it should be done in a random way. When not all organisms have to be identified selectivity in sorting of taxa should be avoided. Before collecting the organisms the sample should be rinsed and cleaned and/or sub-divided in smaller portions. In case of unpreserved samples macroinvertebrates are collected alive in larger trays with the naked eye or with the use of a magnifying glass. SIST EN 17136:2019
EN 17136:2019 (E) 8 In case of preserved sampl
...
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
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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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oSIST prEN 17136:2017
prEN 17136:2017 (E)
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
2
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oSIST prEN 17136:2017
prEN 17136:2017 (E)
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.
3
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oSIST prEN 17136:2017
prEN 17136:2017 (E)
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.
4
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prEN 17136:2017 (E)
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.
5
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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.
6
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prEN 17136:2017 (E)
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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