1、BSI Standards PublicationBS EN 13946:2014Water quality Guidancefor the routine sampling andpreparation of benthic diatomsfrom rivers and lakesBS EN 13946:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 13946:2014. Itsupersedes BS EN 13946:2003 which is with
2、drawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users
3、are responsible for its correctapplication. The British Standards Institution 2014. Published by BSI StandardsLimited 2014ISBN 978 0 580 75638 2ICS 13.060.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theS
4、tandards Policy and Strategy Committee on 31 March 2014.Amendments issued since publicationDate Text affectedBS EN 13946:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13946 March 2014 ICS 13.060.70 Supersedes EN 13946:2003English Version Water quality - Guidance for the routine sampling a
5、nd preparation of benthic diatoms from rivers and lakes Qualit de leau - Guide pour lchantillonnage en routine et le prtraitement des diatomes benthiques de rivires et de plans deau Wasserbeschaffenheit - Anleitung zur Probenahme und Probenaufbereitung von benthischen Kieselalgen aus Fliegewssern un
6、d 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 bibliograph
7、ical 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 responsibilit
8、y 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
9、 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 COMIT EUROPEN DE NORMALISATIO
10、N EUROPISCHES KOMITEE FR 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 EBS EN 13946:2014EN 13946:2014 (E) 2 Contents Page Foreword 3 Introdu
11、ction .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 f
12、or 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
13、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
14、 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
15、.2 Reagents 15 A.5.3 Procedure 15 Bibliography . 17 BS EN 13946:2014EN 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
16、 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 pate
17、nt 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 sam
18、pling 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
19、 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. BS EN 13946:2014EN 13946:2014 (E) 4 Introduction Dia
20、toms 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, a
21、cidification). 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
22、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 p
23、er 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 limi
24、ts 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
25、 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 method
26、ologies 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 p
27、rior 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 responsi
28、bility of the user to establish appropriate health and safety practices and to ensure compliance with any national regulatory conditions. BS EN 13946:2014EN 13946:2014 (E) 5 1 Scope This European Standard specifies a method for the sampling and laboratory preparation of benthic diatoms for ecologica
29、l 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 introduce
30、d 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 substra
31、tum 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 valv
32、es 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 whi
33、ch benthic diatoms are sampled BS EN 13946:2014EN 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 littora
34、l 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
35、 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
36、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
37、(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 an
38、alytical grade but should be of a quality appropriate for the digestion process. BS EN 13946:2014EN 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
39、 a few hours of collection, as long as steps are taken to minimize cell division (i.e. by storage in cool, dark place). Lugols 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 rec
40、ommended 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-hydroxyethy
41、l)piperazin-1-ylethanesulfonic 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
42、 of the silica frustules. This is only necessary in alkaline waters, as diatoms dissolve in alkaline water. 5.1.2 Lugols 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
43、 an air-tight and light-proof container to minimize sublimation. Add 1 to 5 drops of Lugols 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 recommend
44、ed 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
45、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
46、 endorsement by CEN of these products. BS EN 13946:2014EN 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
47、 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
48、 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 f
49、our 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. U
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