1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16161:2012Water quality Guidance onthe use of in vivo absorptiontechniques for the estimationof chlorophyll-a concentrationin marine and fresh watersamplesBS EN 16161:2012
2、BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16161:2012.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.Thi
3、s publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 72637 8ICS 13.060.70Compliance with a British Standard cannot confer imm
4、unity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 July 2012.Amendments issued since publicationDate Text affectedBS EN 16161:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16161 July 2012 ICS 13.060.70 En
5、glish Version Water quality - Guidance on the use of in vivo absorption techniques for the estimation of chlorophyll-a concentration in marine and fresh water samples Qualit de leau - Lignes directrices sur lutilisation des techniques dabsorption in vivo pour lestimation de la concentration de chlor
6、ophylle-a dans les eaux douces et eaux marines Wasserbeschaffenheit - Anleitung fr die Anwendung der in-vivo-Absorption zur Abschtzung der Chlorophyll a-Konzentration in Meer- und Swasser This European Standard was approved by CEN on 17 May 2012. CEN members are bound to comply with the CEN/CENELEC
7、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 bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to
8、 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 responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the officia
9、l versions. CEN members are the national standards bodies of 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, No
10、rway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in
11、 any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16161:2012: EBS EN 16161:2012EN 16161:2012 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Terms and definitions .54 Principle 65 Apparatus .66 Procedure .76.1 Calibration 76.2 Blank M
12、easurement .76.3 Sample measurement 87 Calculation and Expression of Results .87.1 General 87.2 Datum 87.3 Mathematical routine .97.4 Other factors influencing the chlorophyll-a estimation .98 Quality Assurance .98.1 Repeatability .98.2 Uncertainty .99 Test report . 10Annex A (normative) Published i
13、n vivo specific chlorophyll a absorption spectrum 11Annex B (informative) Determination of the appropriate chlorophyll-a specific spectral absorption coefficient for the IVP system . 13Annex C (informative) Factors influencing the chlorophyll-a estimation . 16C.1 Breakdown pigments . 16C.2 Package e
14、ffect . 17C.3 Chlorophyll-b . 18C.4 Phycocyanin 18Annex D (informative) Examples of paired sample method validation Comparison of extraction and in vivo methods under operational conditions 20D.1 General . 20D.2 The IRH Laboratory Data Set . 20D.3 The Adasa Sistemas Laboratory Data Set . 21Annex E (
15、informative) Validation of the spectrometric technique by determining the chlorophyll-a specific absorption of a set of algal samples 22E.1 Laboratory sample data for validation a*chl_systhe appropriate system unit chlorophyll-a response peak height . 22E.2 In vivo photometric chlorophyll-a and the
16、package effect . 24Bibliography . 25BS EN 16161:2012EN 16161:2012 (E) 3 Foreword This document (EN 16161:2012) 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, eithe
17、r by publication of an identical text or by endorsement, at the latest by January 2013, and conflicting national standards shall be withdrawn at the latest by January 2013. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/o
18、r CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
19、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 the United Kingdom.
20、BS EN 16161:2012EN 16161:2012 (E) 4 Introduction Surveys of chlorophyll and nutrient concentration are fundamental descriptors of primary productivity and eutrophic threat in coastal and inland waters. Chlorophyll-a concentration can be determined by sampling and laboratory analysis using the techni
21、ques described in ISO 10260. Achieving consistent results with this technique requires careful attention during the various steps of the process commonly used, such as during sampling, transport, filtering, freezing, storage and extraction and subsequent pigment estimation. The in vivo technique des
22、cribed here can be applied to surveys where a rapid non-destructive and repeatable measurement capability is required. It can be used either in the field or laboratory. No chemicals are required. Utilised in association with other methods of chlorophyll-a determination such as ISO 10260, HPLC pigmen
23、t analysis and chlorophyll fluorescence measurements techniques, it can help identify sources of inconsistency or be used as an alternative technique in its own right. As chlorophyll-a estimates can be achieved in times as short as one minute, the technique can enhance surveying capability considera
24、bly. This standard describes procedures to implement and verify performance. BS EN 16161:2012EN 16161:2012 (E) 5 1 Scope This European Standard provides guidance in the use of in vivo absorption techniques to quantify chlorophyll-a concentration in marine and fresh waters. This European Standard is
25、comprised of the following: definition of the equipment requirement; a priori data and mathematical tools; recommendations for verification of measurement system performance and consideration of factors that can influence measurements; listing of the procedures to be implemented. 2 Normative referen
26、ces The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ENV
27、 13005, Guide to the expression of uncertainty in measurement 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 absorption coefficient a natural logarithm of the ratio between the light intensity entering and corresponding intensity emerging di
28、rectly through a sample of water divided by the sample path length (in metres) in cases where the scattering of light is negligible Note 1 to entry: The unit is m-1. Note 2 to entry: A spectrophotometer often gives the Log10of the ratio in place of the natural logarithm. 3.2 extinction c sum of loss
29、es of directly transmitted light by absorption and scattering Note 1 to entry: The unit is m-1. Note 2 to entry: The extinction c is related to absorption a and scattering b, by bac += . 3.3 extractive photometric EP method of chlorophyll concentration estimation involving extraction and absorption
30、measurement BS EN 16161:2012EN 16161:2012 (E) 6 3.4 in vivo photometric IVP method of assessing chlorophyll-a concentration through the use of in vivo spectral photometry 3.5 package effect flattening of a spectral absorption feature arising from excessive absorbing molecule concentration within cel
31、ls 3.6 resolution width at half height of the instrument response function 3.7 scattering coefficient b natural logarithm of the ratio between the light intensity entering and corresponding intensity emerging directly through a sample of water divided by the sample path length (in metres) in cases w
32、here the absorption of light is negligible Note 1 to entry: The unit is m-1. 3.8 spectrum set of data of a sample taken over a defined wavelength range and by a defined resolution 3.9 wavelength range range from minimum to maximum wavelength over which a spectrum is described 4 Principle The in vivo
33、 photometric absorption technique (IVP) is based on: a) the additive nature of absorption of individual constituents within a suspension; b) the use of a priori knowledge about the absorption features of chlorophyll-a in the wavelength area of approximately 675 nm; c) the absence of other components
34、 interfering with spectral features of chlorophyll-a in this region; d) the use of a measurement cell (a cuvette or other sample receptacle) of sufficient length and spectrophotometer of sufficient performance to enable the absorption feature of chlorophyll-a to be identified at the concentration le
35、vels required; e) the availability of a suitable algorithm to identify and quantify the distinctive chlorophyll-a absorption feature within a spectral absorption measurement. 5 Apparatus 5.1 Spectrophotometer, or equivalent, with the capability to determine the absorption spectrum of an in situ samp
36、le of water. The spectral measuring instrument shall be capable of measuring the absorption of the sample in a range between 600 nm and 750 nm with a resolution better than 10 nm. The data capture is carried out in measuring intervals of 5 nm or smaller. BS EN 16161:2012EN 16161:2012 (E) 7 NOTE For
37、example, a spectrophotometer with 2 nm resolution requires the ability to sense changes in absorption around 675 nm of 0,020 3 m-1in order to sense 1 g/l of chlorophyll-a. 5.2 Datum, consisting of the unit response spectrum for chlorophyll-a absorption ()sa in the region around 675 nm for the spectr
38、ophotometer in use. See Annex B for suggested methods of determining this. 5.3 Mathematical routine, capable of determining the quantity C of unit chlorophyll-a spectra present in the sample spectrum. 6 Procedure 6.1 Calibration The reference spectrum should be recorded with a sample of water which
39、is free of constituents with absorption effects likely to mimic or interfere with chlorophyll-a absorption spectral features in the 650 nm to 710 nm region. This measurement should be stored as a system reference spectral signal of the water Iw(). The temperature of the reference water sample should
40、 not differ more than around 10 C from that of the samples to avoid affecting chlorophyll estimation results at the 1 g/l level. If the same measurement cell is used for successive samples, the measuring chamber should be flushed to remove influences of previous measurements before the next measurem
41、ent (see 6.3). This is particularly necessary in cases where a flow-through measurement cell is utilised. Persistent positive residual chlorophyll-a measurements on clean water samples indicate window fouling by chlorophyllous material. Either the measurement cell should be cleaned or the effects of
42、 the persistent fouling should be zeroed out by taking a new clean water reference spectrum. Water free of chlorophyll-a can be generated by filtering drinking water through a pore size of 0,1 m. For the measurement of samples with high content of dissolved salts or colour (especially high content o
43、f humic substances), a representative water sample can be filtered for use as reference. When a measurement cell is emptied there is often a residue. In practice, the number of flushes recommended should be sufficient to bring verification of zero measurements to within two standard deviations of ze
44、ro. If a significant positive residue persists, this might indicate window fouling and either a new reference clean water sample should be taken or the measurement cell should be cleaned. EXAMPLE A sample of clean water free of chlorophyll inserted after a measurement of a sample with 100 g/l of chl
45、orophyll-a may record a residual chlorophyll-a concentration of 2 g/l, or a 2 % residual. Two flushes should reduce this to 2 % of 2 % or 0,04 % and three flushes to 0,008 %. The effect of temperature difference on the system algorithm can be checked by repeating measurements on one sample using dif
46、ferent reference water or sample water temperatures. Such effects arise because of small changes in the absorption of pure water with temperature. These changes are well described in the literature 15 and can influence the curvature of an absorption spectrum near 675 nm. The influence of the tempera
47、ture and the salinity differences between the spectrum of the sample and reference spectrum shall be considered if concentrations around or below 1 g/l need to be determined accurately. These changes are well described in the literature 15 and can influence the shape of an absorption spectrum near 6
48、75 nm. 6.2 Blank Measurement For any instrument system, the standard deviation of the determination of C in a series of measurements on a clean water sample should be determined by statistical methods. This will indicate the contribution of instrument and algorithm fitting noise to the determination
49、 of minimum detectable chlorophyll concentration. For each new reference water, a second measurement of that water used as a sample to determine estimated chlorophyll C should be made. This estimate should not exceed two standard deviations of zero for the BS EN 16161:2012EN 16161:2012 (E) 8 instrument. If this is not the case, flushing, calibration (6.1) and blank measurement (6.2) should be repeated until this condition is
