1、BRITISH STANDARDBS EN15196:2006BS 6068-5.43:2006Water quality Guidance on sampling and processing of the pupal exuviae of Chironomidae (Order Diptera) for ecological assessmentThe European Standard EN 15196:2006 has the status of a British StandardICS 13.060.45g49g50g3g38g50g51g60g44g49g42g3g58g44g5
2、5g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 15196:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2006 BS
3、I 2006ISBN 0 580 48792 XNational forewordThis British Standard is the official English language version of EN 15196:2006. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the responsibility to: A l
4、ist of organizations represented on this subcommittee can be obtained on request to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Stan
5、dards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard
6、 does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European devel
7、opments and promulgate them in the UK.Summary of pagesThis document comprises a front cover, an inside front cover, the EN title page, pages 2 to 9 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd.
8、 No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15196August 2006ICS 13.060.45English VersionWater quality - Guidance on sampling and processing of thepupal exuviae of Chironomidae (Order Diptera) for ecologicalassessmentQualit de leau - Guide dchantillonnage et de traitementdexuv
9、ies nymphales de Chironomidae (Ordre desDiptres) pour lvaluation cologiqueWasserbeschaffenheit - Anleitung zur Probenahme undBehandlung von Exuvien von Chironomidae-Larven(Diptera) zur kologischen UntersuchungThis European Standard was approved by CEN on 26 June 2006.CEN members are bound to comply
10、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 Central Secretar
11、iat 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 Central Secretariat has the same status as the officialve
12、rsions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Swede
13、n, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref.
14、No. EN 15196:2006: EEN 15196:2006 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Principle5 5 Sampling equipment .5 6 Sampling procedure 6 7 Quality assurance7 Bibliography 8 EN 15196:2006 (E) 3 Foreword This document (EN 15196:2006) has been prepared b
15、y 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 February 2007, and conflicting national standards shall
16、be withdrawn at the latest by February 2007. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
17、 Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. WARNING Working in or around water is inherently dangerous. Persons using this standard should be familiar with
18、normal laboratory practice. This 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 guidelines. EN 15196:2006
19、 (E) 4 1 Scope This guidance standard specifies equipment and procedures for collecting floating pupal exuviae of Chironomidae from aquatic habitats; rivers from source to estuary, canals, ponds, lakes and sea coasts. Guidance in preparing specimens for subsequent identification is provided. These s
20、amples provide representative data on relative species abundance, suitable for numerical analysis, classification and monitoring of environmental conditions. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only t
21、he edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. No normative references. 3 Terms and definitions For the purpose of this European Standard, the following terms and definitions apply 3.1 adult terrestrial, reproductiv
22、e phase of the life history 3.2 Chironomidae family of true flies (Diptera) known in the adult stage as non-biting midges 3.3 exuviae cast skin of an arthropod developing to a subsequent life stage NOTE From the Greek word meaning “cast clothes”, there is no singular term. 3.4 larva among aquatic sp
23、ecies of Chironomidae this is the aquatic, juvenile phase of their life history 3.5 leeward shore lake edge to which the wind is blowing 3.6 pupa intermediate stage linking the larval and adult phases of a metamorphosing insect EN 15196:2006 (E) 5 4 Principle A collection of floating chironomid pupa
24、l exuviae can be used for assessing and classifying most water bodies, without changing the equipment or procedure used (see Clause 6). The technique was first used by Thienemann in 1910 12. Chironomidae have exceptional species richness and ecological diversity, with an estimated 2 000 to 3 000 spe
25、cies per biogeographical region 3. Pupae of all aquatic species of Chironomidae rise to the water surface for adult emergence 6. Discarded pupal exuviae float on the water surface due to trapped air and a wax-layered cuticle. After about two days, bacterial attack of the wax layer causes the exuviae
26、 to sink 2, 7, 14. Wind and water currents will cause floating exuviae to drift. In canals and rivers up to 10 m wide and with a mean velocity up to 50 cm sec-1, 90 % of pupal exuviae become trapped behind vegetation or artefacts within 100 m of where the adult emerged and none normally drift furthe
27、r than 500 m 7, 8, 14. A sample of floating debris from a single point along a river or canal will therefore contain an integrated collection of pupal exuviae accumulated over the previous couple of days. These exuviae are associated with individuals, which have colonised available habitats a short
28、distance upstream of the sampling point. In ponds and lakes, pupal exuviae are assumed to drift without hindrance until they reach the shore or sink. A collection of chironomid pupal exuviae from standing water will be representative of recent adult emergence over a wide expanse of water. Owing to t
29、he passive collection of discarded pupal exuviae there is little opportunity for the operator to influence the contents of the sample. In a comparison of a number of 10 minute collections of surface-floating chironomid exuviae with handnet samples of benthic (bottom-dwelling) larvae and pupae, twice
30、 as many species were obtained with exuviae, and these could be processed in one quarter to one third of the time needed to process benthic samples 4. The sample will often contain exuviae from other insect groups and it is therefore important to distinguish them from Chironomidae. It may be possibl
31、e to use these non-chironomid exuviae for water quality assessment but little is known on their reliability in representing species inhabiting the waterbody sampled. Mayfly exuviae break up rapidly in turbulent water while some caddisfly species leave their pupal exuviae in the larval case and this
32、would bias any sample of floating exuviae. 5 Sampling equipment 5.1 Handnet, consisting of an extendable handle and frame supporting a net of mesh, sized no greater than 250 m. Although an extendable handle and frame is not essential, it enables the operator to collect a sample without entering the
33、water body. 5.2 Coarse mesh sieve, 30 cm diameter, 4 mm mesh or greater. 5.3 Fine mesh sieve, 30 cm diameter, 250 m mesh or smaller. 5.4 Bucket and/or bowl, 30 cm diameter mouth, approx 10 l. 5.5 Pot with water-tight lid, 100 ml to 250 ml. 5.6 Low-power microscope, x15 to x100. 5.7 High power micros
34、cope, x100 to x400. 5.8 Fine mesh net or sieve, for sub-sampling. EN 15196:2006 (E) 6 6 Sampling procedure 6.1 Sampling season and frequency In northern temperate regions, adult emergence largely occurs during the warmest 5 to 10 months of the year 1. Any combination of three monthly river samples b
35、etween April and September has been found to yield at least 80 % of the available genera of chironomids found from 12 consecutive monthly samples 11. For lakes, 80 % to 90 % of species obtained from monthly samples during April to October have been obtained by combining data from any four of the sam
36、ples, the lower figure of 80 % being obtained by permutations of four consecutive months 10. To verify these data for other regions it will be necessary to collect pupal skin samples monthly, as detailed in paragraph 6.2, and subsample according to 6.3. The period of emergence for most species can b
37、e determined from a histogram of species richness for each month. The minimum number of samples to obtain 80 % to 90 % of species during the emergence season can then be determined by calculating the percentage of species obtained for all permutations of 1, 2, 3, 4 or more samples during that period
38、. 6.2 Sample collection The same procedure is applicable to all types of water body that are sufficiently large to insert a net. With an extendable handnet the operator can skim the surface of the water from the bank or shore without entering the water. In flowing water, the number of floating pupal
39、 exuviae will be greater upstream of obstructions at the water surface; overhanging bankside vegetation, emergent vegetation, weirs, navigation locks, moored boats etc. In standing water, the greatest collection of pupal exuviae will be found on the leeward shore. The net will fill with debris, incl
40、uding exuviae, as it is dragged through the water surface. If necessary, the debris can be regularly emptied into a bucket containing water from the same site. In practice, sufficient pupal exuviae for the recommended method (see 6.3) are likely to be obtained before the net is full of debris. There
41、 is no need to time the sample, it is only necessary to obtain a sufficient number of exuviae from which a random sub-sample will be extracted. After sampling has finished, the contents of the bucket can be poured through the coarse sieve stacked on top of the fine sieve (Figure 1). The debris retai
42、ned within the coarse sieve is emptied back into the bucket containing a fresh supply of source water. The coarse sieve is restacked above the fine sieve and the contents of the bucket poured through the sieve stack a second time. This procedure is repeated at least once more. The contents of the co
43、arse sieve are now discarded and the contents of the fine sieve gathered up and transferred to the pot. Sufficient absolute alcohol or industrial methylated spirit is added to the pot to cover the contents. If desired, the sieving may be performed in the laboratory. In this case the contents of the
44、bucket can be strained back through the handnet, emptied into a plastic bag and sealed without the addition of any liquid. Figure 1 EN 15196:2006 (E) 7 6.3 Sample processing 6.3.1 Quantitative In the laboratory the sample should be floated in a bucket or bowl of water. After stirring the contents of
45、 the bowl, an aliquot of sample can be sub-sampled with a tea strainer or small fine mesh sieve. The sub-sample is emptied onto a petri dish (approximately 9 cm diameter) and sufficient water added to wet the whole inside base of the dish and float the exuviae. While viewing the dish contents throug
46、h a low-power binocular microscope all chironomid pupal exuviae should be picked out from the petri dish with fine forceps and placed in 70 % alcohol. It should be ensured to pick out all chironomid pupal exuviae from the petri dish since the larger, darker species are likely to be selected first. A
47、 sub-sample of 200 exuviae using this technique provides an unbiased representation of the species present in the original sample 8, 15. Consequently, the sub-sample should be checked for exuviae abundance before any are picked-out. If there are clearly too many exuviae a smaller sub-sample should b
48、e taken. 6.3.2 Qualitative If it is only possible to collect a single sample from a site, rather than the recommended three (rivers) or four (lakes) monthly visits, then it is recommended that at least 500 exuviae are sub-sampled. Owing to the broad adult emergence periods of northern temperate chir
49、onomids 9, 10, 11 many species will be represented in this sub-sample, despite the sample occurring outside of their peak emergence period. For this reason, data for a single sample should be used only for qualitative analysis (presence/absence). 6.3.3 Slide preparation Many genera can be identified while observed under a low-power microscope. To identify a species, a temporary mount of sub-sampled exuviae placed in 70 % alcohol on a glass slide under a cover slip will allow subsequent manipulation of the specimen if any morpholog
