Water quality - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes

EN 13946 specifies a method for the sampling and laboratory preparation of benthic diatoms for ecological status and water quality assessments. Data produced by this method are suitable for production of water quality indices based on the relative abundance of taxa.

Wasserbeschaffenheit - Anleitung zur Probenahme und Probenaufbereitung von benthischen Kieselalgen aus Fließgewässern und Seen

Diese Europäische Norm legt ein Verfahren zur Untersuchung aquatischer Makrophyten in Fließgewässern mit dem Ziel fest, den ökologischen Zustand von Gewässern mit Hilfe dieser Organismen abzuschätzen, indem diese als Qualitätselemente eingesetzt werden. Die nach diesem Verfahren erhaltenen Informationen berücksichtigen die Zusammensetzung und Häufigkeitsverteilung der aquatischen Makrophytenflora. Die Grundsätze der in der Europäischen Norm beschriebenen Vorgehensweise können auch für die Überwachung von Gewässerbereichen im Umfeld eines Flusslaufs angewendet werden, z. B. in Seitenkanälen und Altwasserbereichen. Es ist bekannt, dass zur vollständigen Bewertung des ökologischen Zustands weitere Aspekte der biologischen Beschaffenheit betrachtet werden sollten.

Qualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des diatomées benthiques de rivières et de plans d'eau

La présente Norme européenne spécifie une méthode d’échantillonnage et de préparation en laboratoire de diatomées benthiques en vue d’évaluer l’état écologique et la qualité de l’eau. Les données obtenues par cette méthode conviennent pour la production d'indices de qualité de l'eau basés sur l’abondance relative des taxons.

Kakovost vode - Navodilo za rutinsko vzorčenje in pripravo vzorcev bentoških kremenastih alg rek in jezer

Standard EN 13946 določa metodo za vzorčenje in laboratorijsko pripravo bentoških kremenastih alg za ocene ekološkega stanja in kakovosti vode. Podatki, pridobljeni s to metodo, so primerni za pripravo indeksov kakovosti vode, ki temeljijo na relativni številčnosti tega reda.

General Information

Status
Published
Public Enquiry End Date
30-Jun-2012
Publication Date
13-Apr-2014
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
31-Mar-2014
Due Date
05-Jun-2014
Completion Date
14-Apr-2014

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Wasserbeschaffenheit - Anleitung zur Probenahme und Probenaufbereitung von benthischen Kieselalgen aus Fließgewässern und SeenQualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des diatomées benthiques de rivières et de plans d'eauWater quality - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of water13.060.10Voda iz naravnih virovWater of natural resourcesICS:Ta slovenski standard je istoveten z:EN 13946:2014SIST EN 13946:2014en,fr,de01-maj-2014SIST EN 13946:2014SLOVENSKI

STANDARDSIST EN 13946:20031DGRPHãþD
SIST EN 13946:2014
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 13946
March 2014 ICS 13.060.70 Supersedes EN 13946:2003English Version

Water quality - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes

Qualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des diatomées benthiques de rivières et de plans d'eau

Wasserbeschaffenheit - Anleitung zur Probenahme und Probenaufbereitung von benthischen Kieselalgen aus Fließgewässern und Seen This European Standard was approved by CEN on 20 December 2013.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13946:2014 ESIST EN 13946:2014

EN 13946:2014 (E) 2 Contents Page

Foreword ..............................................................................................................................................................3 Introduction .........................................................................................................................................................4 1 Scope ......................................................................................................................................................5 2 Terms and definitions ...........................................................................................................................5 3 Principle ..................................................................................................................................................6 4 Equipment ..............................................................................................................................................6 4.1 Equipment for field sampling ...............................................................................................................6 4.2 Laboratory equipment ...........................................................................................................................6 5 Reagents .................................................................................................................................................6 6 Procedure ...............................................................................................................................................8 6.1 Choice of substratum ............................................................................................................................8 6.2 Sample site selection ............................................................................................................................8 6.3 Sampling methods .................................................................................................................................8 6.3.1 Moveable natural hard surfaces ...........................................................................................................8 6.3.2 Method for sampling vertical man-made surfaces in situ .................................................................9 6.3.3 Use of artificial substrata ................................................................................................................... 10 6.3.4 Sample collection from submerged macrophytes and macroalgae .............................................. 10 6.3.5 Sample collection from emergent macrophytes ............................................................................. 10 6.4 Preparation prior to microscopic examination ................................................................................ 11 6.4.1 Preservation and preliminary laboratory treatment ........................................................................ 11 6.4.2 Methods for cleaning diatoms ........................................................................................................... 11 6.4.3 Preparation of permanent slides ....................................................................................................... 11 Annex A (informative)

Methods for cleaning diatoms for microscopic examination ................................ 13 A.1 General ................................................................................................................................................. 13 A.2 Method 1: Hot hydrogen peroxide .................................................................................................... 13 A.2.1 Apparatus ............................................................................................................................................ 13 A.2.2 Reagents .............................................................................................................................................. 13 A.2.3 Procedure ............................................................................................................................................ 13 A.3 Method 2: Cold hydrogen peroxide .................................................................................................. 14 A.3.1 Apparatus and reagents ..................................................................................................................... 14 A.3.2 Procedure ............................................................................................................................................ 14 A.4 Method 3: Hot hydrogen peroxide with potassium dichromate .................................................... 14 A.4.1 Apparatus ............................................................................................................................................ 14 A.4.2 Reagents .............................................................................................................................................. 14 A.4.3 Procedure ............................................................................................................................................ 14 A.5 Method 4: Cold acid (permanganate) method of cleaning ............................................................. 15 A.5.1 Apparatus ............................................................................................................................................ 15 A.5.2 Reagents .............................................................................................................................................. 15 A.5.3 Procedure ............................................................................................................................................ 15 Bibliography ..................................................................................................................................................... 17

SIST EN 13946:2014

EN 13946:2014 (E) 3 Foreword This document (EN 13946:2014) 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 2014, and conflicting national standards shall be withdrawn at the latest by September 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 13946:2003. This document contains the following technical changes in comparison with the previous edition: — this European Standard is now also applicable for the sampling of benthic diatoms in lakes. According to the CEN-CENELEC Internal Regulations, the national standards organizations 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 13946:2014

EN 13946:2014 (E) 4 Introduction 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 based on diatoms as one compartment of the ecosystem or the impact of specific components of water quality (e.g. eutrophication, acidification). The requirement for the monitoring of such processes is inherent in the Water Framework Directive (2000/60/EC) [7] and Urban Waste Water Treatment Directive (91/271/EEC) [8] in addition to other EU Directives and international agreements. This European Standard covers aspects of sampling and preparation relevant to assessment of water quality and ecological status using benthic diatoms. These sampling instructions will result in samples suitable for quantifying relative numbers of benthic diatom taxa present. 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 European Standard. The use of diatoms as indicators of river and lake quality is widely accepted both in Europe and the USA. The methodology is based on the fact that all diatom species have tolerance limits and optima with respect to their preference for 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 the proceedings of three symposia [1] to [3]. The methodologies for evaluating the diatom data vary but the sampling and preparation processes are similar [5, 6]. According to the precise usage to which this European 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 standard are approximate. WARNING — Persons using this European Standard should be familiar with usual laboratory practice. This European Standard 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. SIST EN 13946:2014

EN 13946:2014 (E) 5 1 Scope This European Standard specifies a method for the sampling and laboratory preparation of benthic diatoms for ecological status and water quality assessments. Data produced by this method are suitable for production of water quality indices based on the relative abundance of taxa. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 artificial substratum introduced substratum substratum introduced into river or lake by operator specifically for colonization by diatoms 2.2 benthic diatoms diatoms living on natural or artificial substrata, rather than suspended in the water column 2.3 boulder mineral substratum with a diameter > 256 mm 2.4 cobble mineral substratum with a diameter > 64 mm and ≤ 256 mm 2.5 ecological status measure of the structure and functioning of aquatic communities 2.6 euphotic zone part of the water column in which there is sufficient light for photosynthesis 2.7 frustule cell wall of diatoms, composed of silica and consisting of two valves linked by two or more girdle bands 2.8 habitat specific environment in which an organism lives 2.9 pebble mineral substratum with a diameter > 16 mm and ≤ 64 mm 2.10 riffle shallow part of a stream with swift flow, usually with a broken surface 2.11 substratum natural or non-natural material from which benthic diatoms are sampled SIST EN 13946:2014

EN 13946:2014 (E) 6 2.12 taxa taxonomic units, for example families, genera or species 2.13 valve structural component of the diatom frustule 3 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 water quality. Samples are cleaned using strong oxidizing agents in order to prepare diatoms for identification and enumeration. The data obtained from the microscopic analysis of these samples are suitable for the production of diatom based water quality indices (see [1], [2], and [3]). 4 Equipment 4.1 Equipment for field sampling 4.1.1 Appropriate water safety equipment 4.1.2 Waders 4.1.3 Stiff toothbrush (or other similar instrument) or knife (or other suitable blade). 4.1.4 Plastic tray (approximately 30 cm × 20 cm or larger). 4.1.5 Sample bottles with a tight fitting lid. 4.1.6 Indelible marker pen (or other means of labelling samples). If labels are used, these shall be capable of withstanding wet conditions. 4.1.7 Hoe, with a fine-meshed net attached, attached to long handle (if vertical hard surfaces are to be sampled). 4.1.8 Glass-bottomed box or bucket (“Aquascope”) used for finding suitable substrata under some circumstances. 4.1.9 Plastic bags 4.2 Laboratory equipment See Annex A. 5 Reagents Reagents used in the preparation of the diatom frustules need not be of analytical grade but should be of a quality appropriate for the digestion process. SIST EN 13946:2014

EN 13946:2014 (E) 7 5.1 Preservatives Preservatives are required to stop cell division of diatoms and decomposition of organic matter. No preservative is necessary if the sample is to be processed within a few hours of collection, as long as steps are taken to minimize cell division (i.e. by storage in cool, dark place). Lugol’s iodine can be used for short-term storage; however, it is not suitable for long-term storage, due to problems caused by sublimation. Buffered formaldehyde or ethanol are recommended for long-term storage of samples. Samples may also be deep-frozen. 5.1.1 Buffered formaldehyde solution, HCHO, volume fraction at least 4 %. Dilute a stock solution of formaldehyde to a volume fraction of 4 % in a solution buffered to pH 7. Suitable buffers include HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), borate and hexamethylene-tetramine. A final solution with a volume fraction of 1 % to 4 % in the sample is recommended. The quantity required will depend upon the amount of organic matter present in the sample. NOTE The buffer is necessary to prevent dissolution of the silica frustules. This is only necessary in alkaline waters, as diatoms dissolve in alkaline water. 5.1.2 Lugol’s iodine Dissolve 2 g potassium iodide and 1 g iodine crystals in 300 ml distilled or demineralized water. The resultant liquid should be dark brown coloured. It should be stored in an air-tight and light-proof container to minimize sublimation. Add 1 to 5 drops of Lugol’s iodine per 100 ml sample to give a final dark brown colour. More may be necessary if samples are rich in organic matter. 5.1.3 Ethanol, C2H5OH, volume fraction 70 %. A final concentration of 20 % is recommended for medium-term storage. 5.1.4 Diluted hydrochloric acid, HCl, volume fraction 10 %. 10 % hydrochloric acid can also be used for medium-term storage. By adding acid, the diatoms detach from their substrata and iron- and calcium-complexes will dissolve. 5.2 Reagents for cleaning diatoms See Annex A. 5.3 Reagents for preparing permanent slides A diatom mountant with a refractive index > 1,6 is required (e.g. Naphrax1)).

1) Naphrax is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. SIST EN 13946:2014

EN 13946:2014 (E) 8 6 Procedure 6.1 Choice of substratum Diatoms can be found growing on most submerged surfaces. However, the composition of the assemblage varies depending upon the substratum chosen. Ideally, a single substratum should be used at all sites included in a survey. Areas of the river bed or lake littoral zone with naturally occurring moveable hard surfaces (large pebbles, cobbles and boulders) are recommended wherever possible. If such hard surfaces do not occur naturally, then it is also possible to sample vertical faces of man-made structures such as quays and bridge supports (as long as these are not made from wood). Other man-made hard surfaces, such as bricks may also be sampled, if these have been in the river or lake for long enough to ensure that assemblages are in equilibrium with their environment. At least four weeks is recommended but the period depends upon environmental conditions. See also 6.3.3. Samples of diatoms may also be collected from submerged macrophytes. Where possible, comparative studies in rivers or lakes should be based on samples collected from the same macrophyte species (or group of morphologically similar species). There will also be situations where other substrata (e.g. sand, finer silts) are characteristic of the water body. These can also be sampled; however, there is no widespread agreement on appropriate methods. Users should consult the technical literature and conduct preliminary experiments before deciding on their approach. Consideration should also be given to the introduction of artificial substrata within the euphotic zone. 6.2 Sample site selection A segment of river or lake shore that has substrata suitable for sampling should be selected. As a general rule, this should be about 10 m in length, but longer segments can be appropriate, depending upon the physical uniformity of the sampling site and the availability of substrata. In rivers “riffles” are preferred, as these tend to have a good variety of natural hard surfaces (see 6.1) but “runs” and “glides” are also suitable. A detailed description of the site (location, width, depth, substratum type, percent cover of macrophytes, shade, etc.) is required on the first occasion that a sample is collected. A photographic record is also recommended. This information serves as an aid to data interpretation and to help future samplers locate the site. On subsequent visits, notes may be limited to major changes that have occurred since the previous visit, and any variations in sampling protocol employed. 6.3 Sampling methods 6.3.1 Moveable natural hard surfaces In general, cobbles are the preferred substratum for sampling, as these balance substratum stability (allowing diatom communities to develop) with manoeuvrability. Pebbles and boulders may also be used. At least five cobbles should be sampled. However, if cobbles are unavailable, then either five small boulders or ten pebbles should be sampled. An area of approximately 10 cm2 or more should be scraped or brushed. If fewer suitable substrata are available, then a note of this should be made. The following microhabitat conditions should be fulfilled: a) Areas of heavy shade should be avoided. If it may not be avoided, then a note should be made to this effect. Areas very close to the bank should also be avoided. b) The substrata shall be submerged for long enough to ensure that assemblages are in equilibrium with their environment. At least four weeks is recommended but the period depends upon environmental conditions. It shall be ensured that the surfaces are submersed in water at all times and exposure to air is SIST EN 13946:2014

EN 13946:2014 (E) 9 avoided. All depths that may be easily sampled wearing waders are usually suitable, as long as these remain in the euphotic zone. c) In general, samples should be collected from within the main flow of the river at the sample site. Zones of ve

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