SIST EN 13946:2003
(Main)Water quality - Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers
Water quality - Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers
This guidance standard establishes a method for sampling and laboratory preparation of benthic diatoms for water quality assessments and studies of temporal change in running waters. It is suitable for use with indices based on the relative abundance of taxa. With appropriate modifications the method can be applied to the study of benthic diatoms in lakes.
Wasserbeschaffenheit - Leitfaden zur Probenahme und Probenaufbereitung von benthischen Kieselalgen in Fließgewässern
Qualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des diatomées benthiques de rivieres
La présente Norme européenne est un guide qui établit une méthode pour l'échantillonnage et la préparation en laboratoire de diatomées benthiques en vue d'évaluer la qualité de l'eau. Les données obtenues par cette méthode conviennent pour la production d'indices de la qualité de l'eau basés sur l'abondance relative des taxons. Avec des modifications appropriées, cette méthode peut s'appliquer a l'étude des diatomées benthiques dans les lacs.
Kakovost vode - Navodilo za rutinsko vzorčenje in predpripravo rečnih bentoških kremenastih alg
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Wasserbeschaffenheit - Leitfaden zur Probenahme und Probenaufbereitung von benthischen Kieselalgen in FließgewässernQualité de l'eau - Guide pour l'échantillonnage en routine et le prétraitement des diatomées benthiques de rivieresWater quality - Guidance standard for the routine sampling and pretreatment of benthic diatoms from rivers13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of water13.060.10Voda iz naravnih virovWater of natural resourcesICS:Ta slovenski standard je istoveten z:EN 13946:2003SIST EN 13946:2003en01-september-2003SIST EN 13946:2003SLOVENSKI
STANDARD
SIST EN 13946:2003
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13946May 2003ICS 13.060.70English versionWater quality - Guidance standard for the routine sampling andpretreatment of benthic diatoms from riversQualité de l'eau - Guide pour l'échantillonnage en routine etle prétraitement des diatomées benthiques de rivièresWasserbeschaffenheit - Leitfaden zur Probenahme undProbenaufbereitung von benthischen Kieselalgen inFließgewässernThis European Standard was approved by CEN on 21 February 2003.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the 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 translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13946:2003 ESIST EN 13946:2003
EN 13946:2003 (E)2ContentspageForeword.3Introduction.31Scope.42Principle.43Terms and definitions.44Equipment.55Reagents.56Procedure.6Annex A (informative)
Methods for cleaning diatoms for microscopic examination.10Bibliography.14SIST EN 13946:2003
EN 13946:2003 (E)3ForewordThis document (EN 13946:2003) has been prepared by Technical Committee CEN/TC 230 “Water analysis”, thesecretariat 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 orby endorsement, at the latest by November 2003, and conflicting national standards shall be withdrawn at the latestby November 2003.Annex A is informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,Slovakia, Spain, Sweden, Switzerland and the United Kingdom.IntroductionWARNING — Persons using this European Standard should be familiar with normal laboratory practice.This standard does not purport to address all of the safety problems, if any, associated with its use. It isthe responsibility of the user to establish appropriate health and safety practices and to ensure compliancewith any national regulatory conditions.Diatoms are an important component of aquatic ecosystems and constitute a water quality monitoring tool wherethe primary objective is either a measure of general water quality or of specific components of water quality (e.g.eutrophication, acidification). The requirement for the monitoring of such processes is inherent in the WaterFramework Directive (2000/60/EC) and Urban Waste Water Treatment Directive (91/271/EEC) in addition to otherEU Directives and international agreements. This European Standard covers aspects of sampling and pre-treatment relevant to assessment of water quality using benthic diatoms. Some aspects may also be relevant tomeasures of ecological integrity. These sampling instructions will result in samples suitable for quantifying relativenumbers of benthic diatom taxa present. If it is necessary to quantify absolute numbers of taxa, or fresh weight perunit area, modifications to the method are required, which are not within the scope of this standard.The use of diatoms as indicators of river water quality is widely accepted both in Europe and the USA. Themethodology is based on the fact that all diatom species have tolerance limits and optima with respect to theirpreference for environmental conditions such as nutrients, organic pollution and acidity. Polluted waters will tend tosupport an increased abundance of those species whose optima correspond with the levels of the pollutant inquestion. Conversely, certain species are intolerant of elevated levels of one or more pollutants, whilst others canoccur in a wide range of water qualities.Methods using diatoms to assess water quality have been developed in several European countries (recent work issummarized in the proceedings of three symposia [1 to 3]. The methodologies for evaluating the diatom data varybut the sampling and pre-treatment processes are similar [4].According to the precise usage to which this standard is to be put it is essential for specifiers and users to agree onany necessary variations or optional procedural details prior to use.All numerical values given in this standard are approximate.SIST EN 13946:2003
EN 13946:2003 (E)41 ScopeThis guidance European Standard establishes a method for the sampling and laboratory preparation of benthicdiatoms for water quality assessments. Data produced by this method are suitable for production of water qualityindices based on the relative abundance of taxa. With appropriate modifications the method can be applied to thestudy of benthic diatoms in lakes.2 PrincipleBenthic diatoms from submerged hard surfaces or submerged macrophytes in rivers or streams are sampled inorder to produce representative collections of the diatom assemblage indicative of water quality. Samples arecleaned 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-basedwater quality indices (see references 1, 2, and 3).3 Terms and definitionsFor the purposes of this European Standard, the following terms and definitions apply:3.1artificial substratumsubstratum introduced into river by operator specifically for colonisation by diatoms3.2benthic diatomsdiatoms living on substrata, rather than suspended in the water column3.3bouldermineral substratum with a diameter > 256 mm3.4cobblemineral substratum with a diameter > 64 mm and £ 256 mm3.5euphotic zonethe part of the water column in which there is sufficient light for photosynthesis3.6frustulecell wall of diatoms, composed of silica and
consisting of two valves linked by two or more girdle bands3.7habitatthe specific environment in which an organism lives3.8pebblemineral substratum with a diameter > 16, £ 64 mm3.9riffleshallow part of a stream with swift flow, usually with a broken surface3.10substratumnatural or non-natural material from which benthic diatoms are sampledSIST EN 13946:2003
EN 13946:2003 (E)53.11taxataxonomic units, for example families, genera or species3.12valvestructural component of the diatom frustule (3.6)4 Equipment4.1 Field sampling¾ Appropriate water safety equipment;¾ Waders;¾ Stiff toothbrush (or other similar instrument) or knife (or other suitable blade);¾ Plastic tray (approximately 30 cm ´ 20 cm or larger);¾ Sample bottle with a tightly fitting lid;¾ Indelible marker pen (or other means of labelling samples). If labels are used, these shall be capable ofsurviving wet conditions;¾ Hoe, with a fine-meshed net attached and a long handle(if vertical hard surfaces are to be sampled);¾ A glass-bottomed box or bucket (“Aquascope”) is useful for finding suitable substrata under somecircumstances.4.2 LaboratorySee annex A.5 Reagents5.1 GeneralReagents used in the preparation of the diatom frustules need not be of analytical grade but should be of a qualityappropriate for the digestion process.5.2 PreservativesThese are required to stop cell division of diatoms and decomposition of organic matter. No preservative isnecessary if the sample is to be processed within a few hours of collection, as long as steps are taken to minimizecell division (i.e. by storage in cool, dark place). Lugol’s iodine can be used for short-term storage; however, it isnot suitable for long-term storage, due to problems caused by sublimation. Buffered formaldehyde or ethanol arerecommended for long-term storage of samples. Samples can also be deep-frozen.5.2.1 General5.2.2 Buffered 4% v/v (minimum) formaldehyde (HCHO) solutionDilute a stock solution of formaldehyde to 4 % in a solution buffered to pH 7. Suitable buffers include HEPES (N-2-hydroxymethylpiperazine-N-2’-sulfonic acid), borate and hexamethylenetetramine.SIST EN 13946:2003
EN 13946:2003 (E)6A final solution of 1 % to 4 % (v/v) is recommended (the quantity required will depend upon the amount of organicmatter present in the sample).NOTEThe buffer is necessary to prevent dissolution of the silica frustules.5.2.3 Lugol’s iodineDissolve 2 g potassium iodide and 1 g iodine crystals in 300 ml distilled or demineralised water. The resultant liquidshould be straw coloured. It should be stored in an air-tight and light-proof container to minimise sublimation.Add 1 to 5 drops of Lugol’s iodine per 100 ml sample to give a final “straw” colour. More may be necessary ifsamples are rich in organic matter.NOTESome recipes for Lugol’s iodine include acetic acid or glutaraldehyde to prevent loss of flagella. These reagentsshould be omitted when the solution is to be used for diatoms, as they can lead to the dissolution of silica.70% Ethanol (C2H5OH) can also be used for this purpose.5.3 Reagents for cleaning diatomsSee annex A.5.4 Reagents for preparing permanent slidesA diatom mountant with a refractive index > 1,6 is required. Proprietary brands include Naphrax and Hyrax.6 Procedure6.1 Choice of substratumDiatoms can be found growing on most submerged surfaces; however, the composition of the community variesdepending upon the substratum chosen. Ideally, a single substratum should be used at all sites included in asurvey.Areas of the river bed with naturally occurring moveable hard surfaces (large pebbles, cobbles and boulders) arerecommended wherever possible. If such hard surfaces do not occur naturally, then it is possible to sample verticalfaces of man-made structures such as quays and bridge supports (so long as these are not made from wood).Other man-made hard surfaces, such as bricks can also be sampled, if these have been in the river for longenough to ensure that assemblages are in equilibrium with their environment. At least four weeks is recommendedbut the period depends upon environmental conditions. See also comments in 6.3.3.In deeper rivers where the underlying substrata are finer silts and sands (and when no hard substrata areavailable) consideration should be given to the introduction of artificial substrata within the euphotic zone.Samples of diatoms can also be collected from submerged macrophytes. Where possible, comparative studies inrivers should be based on samples collected from the same macrophyte species (or group of morphologicallysimilar species).6.2 Sample site selectionA segment of river that has substrata suitable for sampling should be selected. As a general rule, this should beabout 10 m in length, but longer lengths may be appropriate, depending upon the physical uniformity of the riverand the availability of substrata. “Riffles” are preferred, as these tend to have a good variety of natural hardsurfaces (6.1).A detailed description of the site (location, width, depth, substratum type, percent cover of macrophytes, shadeetc.) is required on the first occasion that a sample is collected. A photographic record is also recommended. Thisinformation serves as an aid the interpretation of data and to help future samplers locate the site. On subsequentvisits, notes may be limited to major changes that have occurred since the previous visit, and any variations insampling protocol employed.SIST EN 13946:2003
EN 13946:2003 (E)76.3 Sampling methods6.3.1 Moveable natural hard surfacesIn general, cobbles are the preferred substratum for sampling, as these balance substratum stability (allowingdiatom communities to develop) with manoeuvrability. Pebbles and boulders can also be used. At least five cobblesshould be sampled. However, if cobbles are unavailable, then either 5 small boulders or 10 pebbles should besampled. An area of approximately 10 cm2 or more should be scraped. If fewer suitable substrata are available,then a note should be made to this effect.The following microhabitat conditions should be fulfilled:1) areas of heavy shade should be avoided (if it cannot be avoided, then a note should be made to thiseffect). Areas very close to the bank should also be avoided;2) the substrata shall be submerged for long enough to ensure that assemblages are in equilibrium with theirenvironment. At least four weeks is recommended but the period depends upon environmental conditions.The precise depth is unimportant so long as the surfaces have not been exposed to air. All depths thatcan be easily sampled wearing waders are usually suitable, so long as these remain in the euphotic zone;3) in general, samples should be collected from within the main flow of the river at the sample site. Zones ofvery slow current (approx. £ 20 cm s-1) should be avoided as these allow the build-up of loosely attacheddiatoms, silt and other debris.Collect a selection of substrata from a variety of locations within the sample site, which fulfil the microhabitatrequirements listed above. Where suitable substrata are very abundant, random or stratified sampling strategiesmay be appropriate within a defined sample site.Remove any loosely attached surface contamination (e.g. organic debris) by washing the substratum briefly in thestream water. Place the substrata in a tray, along with approximately 50 ml of river water.Wash a stiff toothbrush in clean river water and rub it on a clean surface in order to minimise any diatomcontamination from previous samples. Brush the upper surface of the substratum vigorously to remove the diatomfilm, rinsing the toothbrush periodically in the water in order to transfer the diatoms.A knife or other sharp instrument can also be used to remove the diatom film. This will be more effective atremoving firmly-attached diatoms, but will be less efficient at penetrating crevices on rough surfaces, may causemore damage to frustules and may lead to more rock particles being transferred to the sample. However, it isunlikely that there will be any quantitative difference in results. The knife should also be rinsed in river water andcleaned before use.Alternatively, the diatom film can be removed using a toothbrush or knife and washed directly from the surface ofthe substratum into a sample bottle. The toothbrush or knife can also be used to remove diatoms from thesubstratum and then rinsed in some stream water collected directly into a sample bottle, if this is preferred.If > 75 % of substrata are smothered with filamentous algae, these should be sampled in preference to substratalacking such growths. Remove as many of the filaments as possible prior to brushing or scraping, as above.Replace the substratum in the stream, and repeat the process for the other replicate substrata. Transfer the water,which should now be brown and turbid due to the presence of diatoms, from the tray into the sample jar.Label the sample bottle with details relevant to the sample. Transfer the sample to the laboratory in a cool, darkplace. If samples are brought to the laboratory with
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