1、BSI Standards PublicationBS EN ISO 23753-2:2011Soil Quality Determination of dehydrogenase activity in soilsPart 2: Method using iodotetrazolium chloride (INT) (ISO 23753-2:2005)Incorporating corrigendum April 2014BS EN ISO 23753-2:2011National forewordThis British Standard is the UK implementation
2、of EN ISO 23753-2:2011. It is identical to ISO 23753-2:2005.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 obtained on request to its secretary.This publication does not purport to include a
3、ll the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014ISBN 978 0 580 86306 6ICS 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British
4、 Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2011.Amendments/corrigenda issued since publicationDate Text affected30 April 2014 CEN adoption sheet, Foreword and Endorsement insertedBRITISH STANDARDEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE N
5、ORM EN ISO 23753-2 July 2011 ICS 13.080.30 English Version Soil Quality - Determination of dehydrogenase activity in soils - Part 2: Method using iodotetrazolium chloride (INT) (ISO 23753-2:2005) Qualit du sol - Dtermination de lactivit des dshydrognases dans les sols - Partie 2: Mthode au chlorure
6、de iodottrazolium (CIT) (ISO 23753-2:2005) Bodenbeschaffenheit - Bestimmung der Dehydrogenaseaktivitt in Bden - Teil 2: Verfahren mit Iodotetrazoliumchlorid (INT) (ISO 23753-2:2005) This European Standard was approved by CEN on 17 June 2011. CEN members are bound to comply with the CEN/CENELEC Inter
7、nal 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 any
8、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 official ver
9、sions. CEN members are the national standards bodies of 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, Romania, Slovakia, S
10、lovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE 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 CE
11、N national Members. Ref. No. EN ISO 23753-2:2011: EBS EN ISO 23753-2:2011ISO 23753-2:2005(E) ii ISO 2005 All rights reservedForeword The text of ISO 23753-2:2005 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has bee
12、n taken over as EN ISO 23753-2:2011 by Technical Committee CEN/TC 345 “Characterization of soils” 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 2
13、012, and conflicting national standards shall be withdrawn at the latest by January 2012. 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 any or all such patent righ
14、ts. According to the CEN/CENELEC Internal Regulations, the national standards organizations of 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, Ire
15、land, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 23753-2:2005 has been approved by CEN as a EN ISO 23753-2:2011 without any modification. BS EN
16、ISO 23753-2:2011ISO 23753-2:2005(E) ISO 2005 All rights reserved iiiContents Page Foreword iv Introduction 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 23753-2:2011ISO 23
17、753-2:2005(E) iv ISO 2005 All rights reservedForeword ISO (the International Organization for Standardization) 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 memb
18、er body interested in a subject for which a technical committee has been established has the right 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 Ele
19、ctrotechnical Commission (IEC) on all matters of electrotechnical standardization. International 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 adop
20、ted by the technical committees are circulated to the member bodies for voting. Publication as 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 pate
21、nt rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 23753-2 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 o
22、f dehydrogenase activity in soils: Part 1: Method using triphenyltetrazolium chloride (TTC) Part 2: Method using iodotetrazolium chloride (INT) BS EN ISO 23753-2:2011ISO 23753-2:2005(E) ISO 2005 All rights reserved vIntroduction The soil microflora is responsible for the decomposition and conversion
23、 of organic substances, aggregation stability and the carbon, nitrogen, sulfur and phosphorus cycles. 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
24、 components of the enzyme system of microorganisms. Dehydrogenase activity can therefore be used as an indicator of biological redox systems and as a measure of microbial activity in the soil. BS EN ISO 23753-2:2011BS EN ISO 23753-2:2011INTERNATIONAL STANDARD ISO 23753-2:2005(E) ISO 2005 All rights
25、reserved 1Soil quality Determination of dehydrogenase activity in soils Part 2: Method using iodotetrazolium chloride (INT) 1 Scope This part of ISO 23753 specifies a method for determining soil dehydrogenase activity using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT). As th
26、e INT reduction is less sensitive to O2, the method is more reproducible than the TTC-method described in ISO 23753-1. It is not applicable for determining the dehydrogenase activity in upper layers (L, F, H horizons) of forest humus forms with low microbial activity (e.g. mor), or in soils showing
27、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 references, the latest edition of the referenced document (including any a
28、mendments) 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 ISO 11259, Soil quality Simplified soil description ISO 11465, Soil
29、quality Determination of dry matter and water content on a mass basis Gravimetric method 3 Principle INT solution is added to a soil sample and the mixture is incubated at 30 C for 18 h. The iodonitrotetrazolium formazan (INTF) released is extracted with acetone (in the case of humic soil) and deter
30、mined by photometry at a wavelength of 485 nm. NOTE 1 The method is based on a modified version of the method reported in Reference 1. NOTE 2 Acetone is used as extractant and samples are not extracted to completion. In the case of soil having reducing characteristics (e.g. waterlogged soil), dehydr
31、ogenase activity should not be used as a measure of the biological activity in the soil 3. Abiotic components, such as iron(II) compounds or sulfides can reduce INT. BS EN ISO 23753-2:2011ISO 23753-2:2005(E) 2 ISO 2005 All rights reserved4 Reagents and materials 4.1 Soil Take and prepare soil sample
32、s as specified in ISO 10381-6. If samples which have been sieved in the fresh state are not analysed immediately, they may be kept for up to three months at 4 C. Determine the dry matter content of the sample in accordance with ISO 11465. The storage can affect the enzyme activity and hence dehydrog
33、enase 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 12,11 g of tris(hydroxymethyl)aminomethane in 600 ml of distilled water and adjust the pH value to a) 7,7 for soil having a carbon
34、ate content exceeding 2 %, or b) 7,9 for soil having a carbonate content not exceeding 2 % using 1 mol/l hydrochloric acid and make up to 1 000 ml with distilled water. Determine pH value in accordance with ISO 10390. 4.4 Substrate solution (INT) Weigh 900 mg of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
35、phenyltetrazolium chloride into a 100 ml conical flask and wet it on a magnetic stirrer with a portion of 100 ml of tris buffer solution (4.3). Then add the remainder of the tris buffer solution and treat the mixture with an ultrasonic probe for 1 min to 2 min, so that the INT is dissolved as much a
36、s possible and a suspension is obtained. Remove portions of the suspension later while stirring to keep it homogeneous. Determine the soil texture in accordance with ISO 11259. The solubility of INT in pH 7,7 tris buffer solution is at most 5 mg/ml. During incubation there is a slight increase due t
37、o dissolution of INT from the suspension to a maximum of 7 mg/ml. Therefore 9 mg/ml should be used. 4.5 Analytical grade acetone 4.6 INTF solutions 4.6.1 INTF stock solution Weigh 50 mg of iodonitrotetrazolium formazan into a 100 ml-volumetric-flask; dissolve it in acetone; and make up to 100 ml. 4.
38、6.2 INTF calibration solutions Pipette 0 ml, 0,5 ml, 1,0 ml, 2,0 ml, 3,0 ml and 4,0 ml respectively, of stock solution into a series of 50 ml volumetric flasks, add a little acetone (use the solvent which is also being used as extractant in the samples), add 10 ml of tris buffer solution and make up
39、 to the mark with acetone to give concentrations of 0 g, 5 g, 10 g, 20 g, 30 g and 40 g INTF per ml of acetone buffer solution. Since INT and INTF are light-sensitive, the solutions should be protected from exposure to light throughout the analysis. BS EN ISO 23753-2:2011ISO 23753-2:2005(E) ISO 2005
40、 All rights reserved 35 Apparatus 5.1 Photometer 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 (30 1) C. 5.5 Suitable conical flasks, volumetric flasks, pipettes and funnels 5.6 Fluted
41、filter, with slow filtering action (90 s to 100 s). 5.7 Ultrasonic probe 5.8 Test tube shaker 6 Procedure Weigh 2,00 g to 5,00 g portions of naturally moist soil into each four test tubes. Add 2 ml to 5 ml of substrate solution (4.4) to three samples. Pipette 2 ml to 5 ml of tris buffer solution (4.
42、3) instead of the substrate solution (4.4) to the blank sample. Homogenize the samples on the test tube shaker; seal the tubes with rubber stoppers; and incubate them at 30 C in the dark for 18 h. In order to extract the formazan formed, add 20 ml of acetone to the samples and allow them to stand fo
43、r 2 h in the dark. Shake the samples on the shaker after adding the extractant, after 1 h, and again after 2 h. Then filter the 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
44、 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 activity in g/g of dry soil per hour; cs is the complete sample average INTF concentration, in g/ml; bs is the average INTF
45、concentration of the blank sample, in g/ml; V is the solution volume = volume of substrate or buffer solution (5 ml) + volume of extractant (25 ml), in ml; m is the initial soil sample mass, in g; BS EN ISO 23753-2:2011ISO 23753-2:2005(E) 4 ISO 2005 All rights reservedDM is the sample dry matter con
46、tent in accordance with ISO 11465, as a percentage; t time of incubation (here 18 h), in h. 8 Test report The test report shall refer to this part of ISO 23753 and include the following details: a) sample identification; b) details of storage temperature and duration (4.1); c) soil textural class; d
47、) carbonate content of soil (see Reference 7); 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 results. BS EN ISO 23753-2:2011ISO 23753-2:2005(E) ISO 2005 All rights reserved 5Bibliography
48、1 SPOTHELFER-MAGANA, J., THALMANN, A. and SCHWEIKLE, V.: Methode zur Bestimmung der Dehydrogenaseaktivitt von Bden unter Einsatz von Iodotetrazoliumchlorid (INT): Chemische Reduktion in autoklavierten sowie bestrahlten Bden sowie der Einflu der Inkubationstemperatur und zeit. Agribiol. Res., 46, 199
49、3, pp. 250268 2 THALMANN, H.: Zur Methodik der Bestimmung der Dehydrogenaseaktivitt im Boden mittels Triphenyltetrazoliumchlorid (TTC). Landwirtsch. Forsch., 21, 1968, pp. 243258 3 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 4 MERSI, W. VON and SCHINNER, F.: An improved and accurate method for
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