1、BSI Standards PublicationBS EN 16695:2015Water quality Guidanceon the estimation ofphytoplankton biovolumeBS EN 16695:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16695:2015.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3
2、/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 are responsible for its correctapplication. The British Standards Institution 2015. Pu
3、blished by BSI StandardsLimited 2015ISBN 978 0 580 84590 1ICS 13.060.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 September 2015.Amendments issued since pu
4、blicationDate Text affectedBS EN 16695:2015EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16695 September 2015 ICS 13.060.70 English Version Water quality - Guidance on the estimation of phytoplankton biovolume Qualit de leau - Lignes directrices pour lestimation du biovolume des microalgues W
5、asserbeschaffenheit - Anleitung zur Abschtzung des Phytoplankton-Biovolumens This European Standard was approved by CEN on 10 July 2015. 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
6、 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
7、 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, Czec
8、h 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. EU
9、ROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16695:2015 EBS
10、 EN 16695:2015EN 16695:2015 (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 and preservatives 6 6 Procedure. 7 6.1 Sampling and sample preparation 7 6.2 Calibration of the eyepiece micrometre, countin
11、g-graticule and image analysis software 7 6.3 Statistical requirements for determination 8 6.4 Measurement 9 6.4.1 General 9 6.4.2 Using size classes based on representative measurements . 10 6.4.3 How to deal with hidden dimensions . 10 6.4.4 Measurement of filamentous taxa 10 6.4.5 Measurement and
12、 counting of colony- and coenobium-forming taxa 11 6.4.6 Measurement of complex geometrical shapes 12 6.5 Calculation of biovolume . 12 6.6 Biovolume biomass relations 13 6.7 Reporting 13 7 Quality Assurance 13 Annex A (informative) List of geometrical shapes 14 Annex B (informative) Performance dat
13、a 23 Annex C (informative) Carbon content calculation . 28 Annex D (informative) Alphabetical list of recommended geometrical shapes and hidden dimension factors 30 Annex E (informative) Example: Genus-specific instruction for measurement of dimensions 96 Bibliography . 99 BS EN 16695:2015EN 16695:2
14、015 (E) 3 European foreword This document (EN 16695:2015) 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 endorseme
15、nt, at the latest by March 2016 and conflicting national standards shall be withdrawn at the latest by March 2016. 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 an
16、y or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this
17、 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
18、, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16695:2015EN 16695:2015 (E) 4 Introduction The abundance or number of counting units of individual phytoplankton taxa does not necessarily reflect the real ratio of single taxa to the complete biomass of a phytoplankton com
19、munity. Few big cells/counting units can contribute far more biomass to the system than many small ones. Hence, abundance data alone is often not an ideal measurement of population size. Biomass estimations give very important information for ecological studies, classification schemes and ecosystem
20、modelling. Therefore, it is necessary to determine the biomass of phytoplankton taxa, particularly because phytoplankton delivers energy in the form of carbon, to other trophic levels of food webs. It is not possible to directly analyse the carbon content on the taxonomic level in natural phytoplank
21、ton samples, therefore the biovolume of the phytoplankton taxa is a suitable measure to determine the biomass of an ecosystem according to the taxonomic composition. Neither particle size analysis using laser analysis, nor flow cytometry, nor Coulter Counters, nor chemical analyses of chlorophyll-a
22、concentration as well as total carbon allow statements on the taxon level. An estimation of the carbon content is possible using conversion factors (see Annex C). Further, the biovolume is a quantitative basis for assessing hazards from those algae and cyanobacteria, which (can) contain noxious or t
23、oxic metabolites, and is used in combination with cell numbers or chlorophyll-a concentration within WHO guidelines and national regulations for risk assessments. Up to now, various guidelines for estimating the biovolume of microalgae have been used in different national and international monitorin
24、g programs (e.g. 1, 2, 3, 4). The main objective of this document is the standardization of the procedure for determining the phytoplankton biovolume in order to achieve comparability of data. For this reason, the estimation of the biovolume in phytoplankton samples in sedimentation chambers (accord
25、ing to Utermhl) using an inverted microscope will be described in detail. This European Standard is also applicable for image analysis of pictures derived from microscope and flow cytometry cameras. The use of a standard catalogue containing basic and some composed geometrical shapes is recommended.
26、 Of course, such a standard list will not reflect the variety of all naturally existing shapes and will not match the exact biovolume values of each taxon. It will always be a compromise between accuracy and efficiency. However, the usage of agreed geometrical shapes and the application of the relev
27、ant formulae will improve the comparability of phytoplankton data and will be an important step forward for the implementation of quality assurance measures in phytoplankton analysis. BS EN 16695:2015EN 16695:2015 (E) 5 1 Scope This European Standard specifies a procedure for the estimation of biovo
28、lume of marine and freshwater phytoplankton taxa using inverted microscopy (Utermhl technique according to EN 15204), in consideration of some heterotrophic protists (h thread: d h basic shape Volume calculation precision: exact Dimensions: diameter (d) - m height (h) - m; hd Examples: Aphanizomenon
29、, Aulacoseira, Coscinodiscus, Phormidium, Rhizosolenia, Thalassiosira = 214V dh For radius =2dr : =2V rh 5 Elliptic cylinder prism on elliptic base oval cylinder cross section longitudinal sections basic shape Volume calculation precision: exact Dimensions: large diameter (d1) - m small diameter (d2
30、) - m; hd height of cylinder (h) - hd; m Examples: Achnanthes, Chaetoceros, Fragilaria, Odontella, Surirella = 1214V ddh For radius r: =12V rrh with =112dr ; =222dr BS EN 16695:2015EN 16695:2015 (E) 17 ID Geometrical shape (Synonyms) Information Volume formula 6 Lanceolate cylinder naviculoid cross
31、section longitudinal sections basic shape Volume calculation precision: approximate Dimensions: large diameter (d1) - m small diameter (d2) - m; hd height of cylinder (h) - hd; m Examples: Haslea, Navicula =122V ddh For radius r: =128V rrh with =112dr ; =222dr 7 Cone cross section longitudinal secti
32、on basic shape Volume calculation precision: exact Dimensions: diameter (d) - m slant height (s) - m height of cone (z) - hd NOTE With an inverted microscope, the slant height can exactly be measured. The cone height is a hidden dimension because of the oblique projection. Examples: Calciopappus, Mi
33、nuscula, Pyramimonas longicauda = 22 21112 4V ds d For radius =2dr and height = 22z sr: = 213V rz BS EN 16695:2015EN 16695:2015 (E) 18 ID Geometrical shape (Synonyms) Information Volume formula 8 Double cone two cones cross section longitudinal section composed shape basic shape: cone Volume calcula
34、tion precision: exact Dimensions: diameter (d) - m height of double cone (z) - m NOTE Due to the position in the sedimentation chamber, the total height can only be measured approximately because of the oblique projection. Examples: Gyrodinium spirale, Heterocapsa triquetra, Protoperidinium crassipe
35、s = 2112V dz For radius =2dr : = 211232V rz 9 Cuboid rectangular box rectangular parallelepiped cross section longitudinal sections basic shape Volume calculation precision: exact Dimensions: first edge length (a) - m second edge length (b) - m; hd third edge length (c) - hd; m Examples: Bacillaria,
36、 Hantzschia, Pinnularia, Tabellaria =V abc BS EN 16695:2015EN 16695:2015 (E) 19 ID Geometrical shape (Synonyms) Information Volume formula 10 Rhombic prism parallelepiped prism on parallelogram base cross section longitudinal sections basic shape Volume calculation precision: exact Dimensions: large
37、 diagonal (d1) - m small diagonal (d2) - m; hd height of prism (h) - hd; m Examples: Brachysira, Nitzschia, Pseudo-nitzschia =1212V ddh 11 Trapezoid prism cross section longitudinal section basic shape Volume calculation precision: exact Dimensions: length of large trapezoid side (l1) - m length of
38、small trapezoid side (l2) - m height of trapezoid (z) - m; hd height of prism (h) - hd; m Examples: Crucigenia fenestrata ( )= + 1212V l l zh BS EN 16695:2015EN 16695:2015 (E) 20 ID Geometrical shape (Synonyms) Information Volume formula 12 Triangular prism prism on triangle base cross section longi
39、tudinal section basic shape Volume calculation precision: exact Dimensions: length of triangle side (l) - m height of triangle (m) - m; hd height of prism (h) - hd; m NOTE For a prism on basis of an equilateral triangle, only the triangle side is needed. The triangle height can be expressed by this
40、side. Examples: Bellerochea, Ditylum, Goniochloris, Lithodesmium, Triceratium = 12V lmh or = 2134V lh for a prism on basis of an equilateral triangle with =132ml 13 Pyramid Cross sections Top view basic shape Volume calculation precision: exact Dimensions: length of first basic side (l1) - m length
41、of second basic side (l2) - m; hd height of pyramid (h) - hd; m Examples: Gomphonema, Licmophora =1213V llh BS EN 16695:2015EN 16695:2015 (E) 21 ID Geometrical shape (Synonyms) Information Volume formula 14 Tetrahedron lateral view top view basic shape Volume calculation precision: exact Dimensions:
42、 edge length (a) - m height of tetrahedron (h) hd Examples: Staurastrum, Staurodesmus, Tetraedriella, Tetraedron =21312V ah or =31212Va for a regular tetrahedron with =163ha; 15 Spindle cross section longitudinal section basic shape Volume calculation precision: approximate Dimensions: diameter (d)
43、- m height (h) - m Examples: Ankistrodesmus, Closterium, Monoraphidium = 2215V dh For radius =2dr : = 2815V rh BS EN 16695:2015EN 16695:2015 (E) 22 ID Geometrical shape (Synonyms) Information Volume formula 16 Cone with half sphere cone with hemisphere cross section longitudinal section composed sha
44、pe basic shapes: cone, sphere Volume calculation precision: exact Dimensions: diameter (d) - m total height (h) - m NOTE Due to the position in the sedimentation chamber, the total height can only be measured approximately because of the oblique projection. Examples: Gonyaulax, Hemiselmis, Katodiniu
45、m, Teleaulax = +21112 2V dh d For radius =2dr : ( )= +231 143 23V r hr r17 Ceratium-shape lateral view Complicated composed shape Volume calculation precision: approximate Dimensions: girdle diameter (d) - m Examples: Ceratium = yV xd x and y are factors derived from measurements 4, 9 Ceratium x: 0,
46、32359 y: 2,9953 C, furca x: 2,3038 y: 2,532 C, fusus x: 35,198 y: 1,9156 C, hirundinella x: 2,3038 y: 2,532 C, horridum x: 0,32437 y: 3,0474 C, lineatum x: 1,2375 y: 2,5989 C, longipes x: 0,32437 y: 3,0474 C, macroceros x: 0,32437 y: 3,0474 C, tripos x: 0,32359 y: 2,9953 BS EN 16695:2015EN 16695:201
47、5 (E) 23 Annex B (informative) Performance data The performance data given in Table B.1 to Table B.4 were determined in a European interlaboratory comparison for validation carried out in March 2014 on two mixed marine and two freshwater samples taken from the North Sea (Helgoland, Germany), the Bal
48、tic Sea (Stralsund, Germany) and different lakes around Oldenburg (Germany) and Bad Saarow (Germany). 52 participants from the following European countries have provided results: Denmark (4), Finland (3), France (1), Germany (20), Ireland (1), Italy (3), Lithuania (3), Norway (1), Poland (2), Portug
49、al (3), Spain (2), Sweden (3) and United Kingdom (6). All results have been calculated according to the German standard document DIN 38402-45:2014-06. BS EN 16695:2015EN 16695:2015 (E) 24 Table B.1 Performance data for measurements of first dimension in biovolume analysis of phytoplankton species in natural samples - Abbreviations for dimensions according to Annex A. Taxon name l n X xmedxmeanxHSsRCV,RsrCV,rm m m m m % m % Dactyliosol
