1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58phytoplankton using inverted microscopy (Utermhl technique)The European Standard EN 15204:2006 has
2、the status of a British StandardICS 13.060.70Water quality Guidance standard on the enumeration of BRITISH STANDARDBS EN 15204:2006BS EN 15204:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 September 2006 BSI 2006ISBN 0 580 48934 5Ame
3、ndments issued since publicationAmd. No. Date Commentscontract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard was published by BSI. It is the UK implementation of EN 15204:2006
4、.The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/5, Biological methods.A list of organizations represented on EH/3/5 can be obtained on request to its secretary.This publication does not purport to include all the necessary provi
5、sions of a EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15204August 2006ICS 13.060.70English VersionWater quality - Guidance standard on the enumeration ofphytoplankton using inverted microscopy (Utermhl technique)Qualit de leau - Norme guide pour lanalyse de routine delabondance et de la compo
6、sition du phytoplancton parmicroscopie inverse (mthode dUtermhl)Wasserbeschaffenheit - Anleitung fr die Zhlung vonPhytoplankton mittels der Umkehrmikroskopie (Utermhl-Technik)This European Standard was approved by CEN on 14 July 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regu
7、lations 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 Secretariat or to any CEN member.This Euro
8、pean 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 officialversions.CEN members are the nationa
9、l 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, Sweden, Switzerland and United Kingdom.
10、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. No. EN 15204:2006: EEN 15204:2006
11、(E) 2 Contents Page Foreword3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle7 5 Equipment and preservatives.7 6 Sample processing9 7 Counting procedure.12 8 Quantitative validation 18 9 Measurement uncertainty .19 Annex A (informative) Optical characteristi
12、cs of inverted microscopes.21 Annex B (informative) Sample treatment23 Annex C (informative) Phytoplankton analysis strategies27 Annex D (informative) Identification30 Annex E (informative) Use of conventional compound microscopes .31 Annex F (informative) Statistical procedure.34 Bibliography 40 Fi
13、gures Figure 1 Random distribution of particles (note the open spaces) . 12 Figure 2 Example of rule for counting cells on the edge of the field. Algae objects crossing both the top and left hand side of grid are not counted whilst those crossing the bottom and right hand side of grid are counted. 1
14、4 Figure F.1 Illustration of collecting algal data for a Run-test . 35 Tables Table 1 Settling times for Lugol preserved seawater samples 12. 11 Table F.F1 Maximum allowable variance for Poisson approximation ( = mean, 2= variance). 35 Table F.F2 Multinomial homogeneity test 36 EN 15204:2006 (E) 3 F
15、oreword This document (EN 15204:2006) 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
16、 February 2007, and conflicting national standards shall 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 Rep
17、ublic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 15204:2006 (E) 4 Introduction The European Water Fr
18、amework Directive (2000/60/EC) has created a need for a uniform procedure to assess ecological quality of surface waters using phytoplankton abundance and composition. This European Standard will meet this need and will help laboratories improve the quality of their analytical results. A single stan
19、dard procedure for the assessment of phytoplankton composition and abundance cannot be given as the questions which drive monitoring programmes are diverse in character and therefore require specific protocols. This European Standard, therefore, aims to provide guidance on basic aspects of microscop
20、ic algal analyses and to provide statistical procedures for the design, optimization and validation of methods and protocols. Though mentioned in Annex C, a method for the estimation of biovolume is not included. WARNING Persons using this European Standard should be familiar with normal laboratory
21、practice. Long periods of microscopic phytoplankton analysis can cause physical fatigue and affect eyesight. Attention should be given to the ergonomics of the microscope and advice from a health and safety practitioner should be sought to ensure that risks are minimized. The use of chemical product
22、s mentioned in this European Standard can be hazardous and users should follow guidelines provided by the manufacturers and take necessary specialist advice. This European Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of th
23、e user to establish appropriate health and safety practices and to ensure compliance with any national regulatory guidelines. EN 15204:2006 (E) 5 1 Scope The procedure described in this European Standard is based on the standard settling technique as defined by Utermhl in 1958 31. It describes a gen
24、eral procedure for the estimation of abundance and taxonomic composition of marine and freshwater phytoplankton by using inverted light microscopy and sedimentation chambers, including the preceding steps of preservation and storage. Emphasis is placed on optimizing the procedure for the preparation
25、 of the microscopic sample. Many of the general principles of the approach described may also be applied to other techniques of enumerating algae (or other entities) using a (conventional) microscope, some of which are described in Annex E. This guidance standard does not cover field collection of s
26、amples or the analysis of picoplankton, quantitative analysis of free-floating mats of Cyanobacteria or specific preparation techniques for diatoms. 2 Normative references Not applicable. 3 Terms and definitions For the purpose of this document, the following terms and definitions apply. 3.1 accurac
27、y closeness of agreement between a test result or measurement result and the true value 3.2 algal object unit/cluster of one or more algal cells encountered during the phytoplankton analysis that is discrete from (liable to settle independently of) other particles in the sample 3.3 detection limit m
28、inimum number and/or size of a specific taxon or group of organisms in a sample at which its presence can be detected with a specified probability NOTE This definition is analogous to the definition used in chemistry (smallest true value of the measurand which is detectable by the measuring method).
29、 3.4 error difference between an individual result and the true value 3.5 fixation protection from disintegration of the morphological structure of organisms 3.6 microscope counting field delimited area (e.g. a square or grid) in the microscope field of view, used for enumeration 3.7 nanoplankton sm
30、all algae between 2 m and 20 m in size EN 15204:2006 (E) 6 3.8 numeric aperture (NA) difference in refraction index of the medium between objective and object multiplied by the sine of half the angle of incident light 3.9 performance characteristic characteristics of a specific analysis protocol whi
31、ch encompass qualitative and quantitative aspects for data precision, bias, method sensitivity and range of conditions over which a method yields satisfactory data 3.10 phytoplankton community of free-living, suspended, mainly photosynthetic organisms in aquatic systems comprising Cyanobacteria and
32、algae 3.11 picoplankton very small algae between 0,2 m and 2 m in size 3.12 precision closeness of agreement between independent test/measurement results obtained under stipulated conditions 3.13 preservation process that protects organic substances from decay 3.14 (analysis) protocol specific analy
33、tical procedure concerning (sub)sample volume, magnification, number of cells to count, taxonomic level of identification etc. 3.15 repeatability precision under repeatability conditions 3.16 repeatability conditions conditions where independent test/measurement results are obtained with the same me
34、thod on identical test/measurement items in the same test or measuring facility by the same operator using the same equipment within short intervals of time NOTE This definition should be interpreted as the error occurring between replicate sub-samples from the same sample, counted using the same co
35、unting chamber, performed by one analyst using one microscope in a continuous run on one day. 3.17 reproducibility precision under reproducibility conditions 3.18 reproducibility conditions conditions where independent test/measurement results are obtained with the same method on identical test/meas
36、urement items in different test or measurement facilities with different operators using different equipment EN 15204:2006 (E) 7 3.19 uncertainty parameter associated with the result of a counting that characterizes the dispersion of values that could reasonably be attributed to the measurand 3.20 v
37、alidation confirmation by examination and the provision of effective evidence that the particular requirements for a specific intended use are fulfilled 4 Principle After preservation and storage, if applicable, the sample is homogenized and a sub sample is placed in a sedimentation chamber. When th
38、e algae have settled to the bottom of the chamber, they are identified and counted using an inverted microscope. 5 Equipment and preservatives 5.1 Sampling bottles A sampling bottle should meet the following requirements (the relevance of some of these may depend on the duration of storage of the sa
39、mple): the bottle should be clean and easily be cleaned. It should not be permeable to, or react with, the preservative used; the bottle should be transparent (so that the state of preservation and the presence of aggregates can be examined easily), but stored in the dark.; the combination of bottle
40、 and screw cap should ensure a closure that is watertight (to facilitate homogenisation) and almost gastight (to minimize evaporation) to allow long periods of storage; the neck of the bottle should be wide enough for filling the counting chamber. The bottle should not be too large for easy handling
41、 and filling of the counting chamber: generally, a volume of some 100 ml to 200 ml is satisfactory; to facilitate homogenisation, bottles should not be filled completely with sampling water (preferably fill to around 80 %). 5.2 Sedimentation chamber Sedimentation chambers consist of a vertical colum
42、n, with a base through which the contents can be observed with an inverted microscope. The column is filled with a sample and the particles in the sample are allowed to settle on the bottom of the chamber. By using a relatively small cross-sectional area in comparison with column height, the sample
43、can be concentrated effectively. A common type of chamber has 2 pieces: a top-piece column that is placed above a well in a base-piece, the top-piece being slid aside and replaced with a cover glass once the algae have settled on the bottom. Sedimentation chambers may be square or circular. The thic
44、kness of the base plate should not exceed 0,17 mm as this directly affects image quality. Counting chambers should be calibrated so that the volume of sub-sample contained can be determined. Counting chambers should be cleaned and dried between uses. For best results, cleaning should include washing
45、 with detergent using a soft paintbrush or small scrubbing brush; afterwards, the chamber should be rinsed in distilled water. Other agents that can be used, depending on the chamber material, are methanol, ethanol (90 %), commercial denatured alcohol or isopropanol. EN 15204:2006 (E) 8 5.3 Inverted
46、 microscope The use of an inverted microscope allows the algae, settled on the bottom of the chamber, to be brought into clear focus (see Annex A). The optical properties of the microscope determine the discriminating potential and hence the identification possibilities. For phytoplankton counting,
47、an inverted microscope should be equipped with a condenser with a NA of at least 0,5 and plan objectives with a NA of 0,9 or more (see Annex A). Phase-contrast and/or Normarski interference-contrast is usually used in marine phytoplankton analysis. It can assist greatly in the identification of cert
48、ain taxa, including flagellates, diatoms and delicate forms such as chrysophytes. Ideally, the microscope should be equipped with a (digital) camera. The microscope should have binocular, wide-field 10 or 12,5 eyepieces. One eyepiece should be equipped with a calibrated ocular micrometer. The other
49、eyepiece should be equipped for counting by use of an appropriate calibrated counting-graticule: a) for counting of randomly-selected microscope fields, the graticule should have a square field or grid (available commercially, e.g. a Whipple disc), or the equivalent using 4 crossing threads, or b) for counting transects or the whole chamber, 2 parallel threads within the eyepiece forming a transect, preferably with a third vertical thread crossing the other two in the centre. The ocular micr
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