1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 23753-1:2011Soil Quality Determinationof dehydrogenase activity insoilPart 1: Method using triphenyltetrazoliumchloride (TTC) (ISO 23753-1:2005)BS EN ISO 23753-1:2011 B
2、RITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 23753-1:2011. It is identical to ISO 23753-1:2005.The UK participation in its preparation was entrusted to Technical Committee EH/4, Soil quality.A list of organizations represented on this committee can be obta
3、ined on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. BSI 2011 ISBN 978 0 580 73166 2 ICS 13.080.30 Compliance with a British Standard cannot confer immunity from legal obligations.
4、This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2011.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 23753-1 July 2011 ICS 13.080.30 English Version Soil Quality - Determin
5、ation of dehydrogenase activity in soil - Part 1: Method using triphenyltetrazolium chloride (TTC) (ISO 23753-1:2005) Qualit du sol - Dtermination de lactivit des dshydrognases dans les sols - Partie 1: Mthode au chlorure de triphnylttrazolium (CTT) (ISO 23753-1:2005)Bodenbeschaffenheit - Bestimmung
6、 der Dehydrogenaseaktivitt in Bden - Teil 1: Verfahren mit Triphenyltetrazoliumchlorid (TTC) (ISO 23753-1:2005) This European Standard was approved by CEN on 17 June 2011. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this Europea
7、n 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 any CEN member. This European Standard exists in three official versions (
8、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 official versions. CEN members are the national standards bodies of Austria, Belgi
9、um, Bulgaria, Croatia, 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. EUROPEAN COMMI
10、TTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 23753-1:2011: EBS EN ISO 23753-1:2
11、011EN ISO 23753-1:2011 (E) 3 Foreword The text of ISO 23753-1:2005 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 23753-1:2011 by Technical Committee CEN/TC 345 “Characterization of soil
12、s” the secretariat of which is held by NEN. 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 January 2012, and conflicting national standards shall be withdrawn at the latest by January 2012. Atten
13、tion 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 any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of
14、the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Roman
15、ia, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 23753-1:2005 has been approved by CEN as a EN ISO 23753-1:2011 without any modification. BS EN ISO 23753-1:2011ISO 23753-1:2005(E) ISO 2005 All rights reserved iiiContents Page Foreword iv I
16、ntroduction v 1 Scope . 1 2 Normative references . 1 3 Principle. 1 4 Reagents and materials . 2 5 Apparatus 3 6 Procedure 3 7 Calculation. 3 8 Test report . 4 Bibliography . 5 BS EN ISO 23753-1:2011ISO 23753-1:2005(E) iv ISO 2005 All rights reservedForeword ISO (the International Organization for S
17、tandardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the ri
18、ght to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Internationa
19、l Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as
20、an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO
21、 23753-1 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. ISO 23753 consists of the following parts, under the general title Soil quality Determination of dehydrogenase activity in soils: Part 1: Method using triphenyltetrazolium chloride (TTC) Par
22、t 2: Method using iodotetrazolium chloride (INT) BS EN ISO 23753-1:2011ISO 23753-1:2005(E) ISO 2005 All rights reserved vIntroduction The soil microflora is responsible for the decomposition and conversion of organic substances, aggregation stability and the carbon, nitrogen, sulfur and phosphorus c
23、ycles. Dehydrogenases, as respiratory chain enzymes, play a major role in the energy production by organisms. They oxidize organic compounds by transferring two hydrogen atoms. Dehydrogenases are essential components of the enzyme system of microorganisms. Dehydrogenase activity can therefore be use
24、d as an indicator of biological redox systems and as a measure of microbial activity in the soil. BS EN ISO 23753-1:2011BS EN ISO 23753-1:2011INTERNATIONAL STANDARD ISO 23753-1:2005(E) ISO 2005 All rights reserved 1Soil quality Determination of dehydrogenase activity in soils Part 1: Method using tr
25、iphenyltetrazolium chloride (TTC) 1 Scope This part of ISO 23753 specifies a method for determining the dehydrogenase activity in soil using 2,3,5-triphenyltetrazolium chloride (TTC). It is not applicable for determining the dehydrogenase activity in the upper layers (L, F, H horizons) of forest hum
26、us forms with low microbial activity (e.g. mor), or in soils showing reducing properties (e.g. waterlogged soils). 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated refer
27、ences, the latest edition of the referenced document (including any amendments) applies. ISO 10381-6, Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory ISO 10390, Soil quality Determination of pH
28、 ISO 11259, Soil quality Simplified soil description ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method 3 Principle TTC solution is added to a soil sample and the mixture is incubated at 25 C for 16 h. The triphenylformazan (TPF) released is extr
29、acted with acetone and determined by photometry at a wavelength of 485 nm. NOTE 1 The method is based on a modified version of the method reported in Reference 3. NOTE 2 Other extraction liquids than acetone may be used (e.g. ethanol, acetone-CCl4mixture). According to Reference 1, methanol is not s
30、uitable because the extraction efficiency is influenced by the soil and the soil water content. In the case of soil having reducing characteristics (e.g. waterlogged soil), dehydrogenase activity should not be used as a measure of the biological activity in the soil 2. Abiotic components, such as ir
31、on(II) compounds or sulfides, can reduce TTC. NOTE 3 Organic solvents can extract excessive amounts of humic substances from humus rich soils (e.g. L, F, H horizons, mouldy peat), giving blank values (XLP) which do not differ from measured values (XVP) in soils with low microbial activity. BS EN ISO
32、 23753-1:2011ISO 23753-1:2005(E) 2 ISO 2005 All rights reserved4 Reagents and materials 4.1 Soil Take and prepare soil samples as specified in ISO 10381-6. If samples which have been sieved in the fresh state cannot be analysed immediately, they may be kept for up to three months at 4 C. Determine t
33、he dry matter content of the sample in accordance with ISO 11465. The storage can affect the enzyme activity and hence dehydrogenase activity of samples with different storage times should not be compared. 4.2 Hydrochloric acid, c(HCl) = 1 mol/l. 4.3 Tris buffer solution, c = 0,1 mol/l. Dissolve 12,
34、11 g of tris(hydroxymethyl)aminomethane in 600 ml of distilled water and adjust the pH value to a) 7,8 for acidic soil (pH less than 6); b) 7,6 for neutral soil (pH 6 to 7); c) 7,4 for carbonate-rich soil (pH greater than 7); using 1 mol/l hydrochloric acid and make up to 1 000 ml with distilled wat
35、er. Determine pH value in accordance with ISO 10390. 4.4 Substrate solution (TTC) The optimum substrate concentration is generally between 0,1 % and 2 % TTC, depending on soil textural class and humus content. Determine the soil texture in accordance with ISO 11259. Dissolve 2,3,5-triphenyltetrazoli
36、um chloride in tris buffer solution (4.3). The solution may be stored for one week at 4 C in the dark. Depending on the humus and clay content of the sample, use the following substrate concentrations: a) sandy soil, slightly humic and slightly clayey soil: 0,1 % to 0,5 % TTC; b) loam, humic and loa
37、my soil: 0,6 % to 1,0 % TTC; c) clayey and humic soil: 1,0 % to 2,0 % TTC. 4.5 Analytical grade acetone 4.6 Triphenylformazan (TPF) solutions 4.6.1 TPF stock solution, dissolve 1 000 mg of triphenylformazan (TPF) in acetone (4.5) to make up 100 ml. 4.6.2 TPF working solution, dilute 1,0 ml of stock
38、solution (4.6.1) to 100 ml with acetone (4.5). 4.6.3 TPF calibration solutions Pipette 0 ml, 1,0 ml, 2,0 ml, 5,0 ml and 10,0 ml respectively, of the solution specified in 4.6.2 into a series of five test tubes and make the volume up to 30 ml with acetone (4.5). The solutions obtained have TPF concen
39、trations of 0 g/ml, 3,33 g/ml, 6,67 g/ml, 16,7 g/ml and 33,3 g/ml respectively. Since TTC and TPF are light-sensitive, the solutions should be protected from exposure to light throughout the analysis. BS EN ISO 23753-1:2011ISO 23753-1:2005(E) ISO 2005 All rights reserved 35 Apparatus 5.1 Photometer
40、5.2 pH meter 5.3 Test tubes For example, 2 cm diameter and at least 30 ml capacity (test portion 5 g, see also Clause 6). 5.4 Incubator, capable of being set to (25 1) C. 5.5 Suitable conical flasks, volumetric flasks, pipettes and funnels 5.6 Test tube shaker 5.7 Fluted filter, with slow filtering
41、action (90 s to 100 s). 6 Procedure Weigh 5,00 g portions of naturally moist soil into each of four test tubes (5.3). Add 5 ml of substrate solution (4.4) to three samples (complete samples). Pipette 5 ml of tris buffer solution (4.3) instead of the substrate solution to the blank sample. Shake each
42、 tube, seal the tubes with rubber stoppers and incubate them at 25 C for 16 h. To extract the triphenylformazan formed, add 25 ml of acetone (4.5) to the samples and allow them to stand for 2 h in the dark. Shake the samples after adding the extractant, after 1 h, and again after 2 h. Then filter th
43、e samples in semidarkness using a fluted filter. Within 1 h, measure the absorbance of the filtrates against the calibration curve zero by photometry at a wavelength of 485 nm. According to Reference 1, optimum conditions are given at a soil/solution ratio of 1:1. There is no linear correlation betw
44、een test portion and TTC reduction if the same test tubes are used. If the test tube diameter is fitted to the test portion, a linear correlation between TTC reduction and the mass of test portion is obtained. Optimum test tube diameters are 1,5 cm for 2 g of soil, 2 cm for 5 g of soil and 2,5 cm fo
45、r 10 g of soil. For L, F, H horizons, soil portions of 0,5 g or 1 g and a test tube of 2 cm diameter are recommended 4. 7 Calculation Determine the dehydrogenase activity (based on dry soil) from the calibration curve and the following equation: cs bs( ) 100DMVamt=where: a is the dehydrogenase activ
46、ity in g/g of dry soil per hour; cs is the complete sample average TPF concentration, in g/ml; bs is the average TPF concentration of the blank sample, in g/ml; V is the solution volume (= volume of substrate or buffer solution + volume of extractant), in ml; BS EN ISO 23753-1:2011ISO 23753-1:2005(E
47、) 4 ISO 2005 All rights reservedm is the initial soil sample mass, in g; DM is the sample dry matter content in accordance with ISO 11465, as a percentage; t time of incubation (here 16 h), in h. 8 Test report The test report shall refer to this part of ISO 23753 and include the following details: a
48、) sample identification; b) details of storage temperature and duration (4.1); c) soil textural class; d) pH value (pHCaCl2) of soil sample; e) test results; f) any details that are optional or deviations from the specifications of this part of ISO 23753, and any effects which may have affected the
49、results. BS EN ISO 23753-1:2011ISO 23753-1:2005(E) ISO 2005 All rights reserved 5Bibliography 1 THALMANN, H.: Zur Methodik der Bestimmung der Dehydrogenaseaktivitt im Boden mittels Triphenyltetrazoliumchlorid (TTC). Landwirtsch. Forsch. 21, 1968, pp. 243258 2 GLATHE, H. and THALMANN, H.: Microbial activity and its relationship to fertility characteristics of some agricultural soils, with particular reference to dehydrogenase activity. Part 2. Zbl Bakt II, 124, 1970, pp. 2436
