1、September 2011 Translation by DIN-Sprachendienst.English price group 8No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).I
2、CS 13.080.30!$tY#“1815400www.din.deDDIN EN ISO 23753-2Soil quality Determination of dehydrogenase activity in soils Part 2: Method using iodotetrazolium chloride (INT) (ISO 23753-2:2005)English translation of DIN EN ISO 23753-2:2011-09Bodenbeschaffenheit Bestimmung der Dehydrogenaseaktivitt in Bden
3、Teil 2: Verfahren mit Iodotetrazoliumchlorid (INT) (ISO 23753-2:2005)Englische bersetzung von DIN EN ISO 23753-2:2011-09Qualit du sol Dtermination de lactivit des dshydrognases dans les sols Partie 2: Mthode au chlorure de iodottrazolium (CIT) (ISO 23753-2:2005)Traduction anglaise de DIN EN ISO 2375
4、3-2:2011-09SupersedesDIN ISO 23753-2:2006-04www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises pages0 .11813DIN EN ISO 23753-2:2011-09 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Co
5、mmittee ISO/TC 190 “Soil quality” in collaboration with Technical Committee CEN/TC 345 “Characterization of soils” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Working Committee NA 1
6、19-01-02-04 UA Biologische Verfahren of NA 119-01-02 AA Abfall- und Bodenuntersuchung. ISO 23753 consists of the following parts, under the general title Soil quality Determination of dehydro-genase activity in soils: Part 1: Method using triphenyltetrazolium chloride (TTC) Part 2: Method using iodo
7、tetrazolium chloride (INT) Expert assistance and specialized laboratories will be required to perform the procedures described in this standard. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 10381-6 DIN ISO 10381-6 ISO 10390 DIN ISO 1
8、0390 ISO 10693 DIN ISO 10693 ISO 11465 DIN ISO 11465 Amendments This standard differs from DIN ISO 23753-2:2006-04 as follows: a) the text of DIN ISO 23753-2:2006-04 has been adopted as European Standard DIN EN ISO 23753-2:2011-09. Previous editions DIN 19733-2: 1998-07 DIN ISO 23753-2: 2006-04 2 DI
9、N EN ISO 23753-2:2011-09 National Annex NA (informative) Bibliography DIN ISO 10381-6, Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory DIN ISO 1039
10、0, Soil quality Determination of pH DIN ISO 10693, Soil quality Determination of carbonate content Volumetric method DIN ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method 3 DIN EN ISO 23753-2:2011-09 4 This page is intentionally blank EUROPEAN S
11、TANDARD NORME EUROPENNE EUROPISCHE NORM 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) Qualit du sol - Dtermination de lactivit des dshydrognases dans les sols - Partie 2: Mt
12、hode au chlorure 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
13、CEN/CENELEC 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
14、Centre or to 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
15、 the official versions. 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, Rom
16、ania, Slovakia, Slovenia, 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
17、 worldwide for CEN national Members. Ref. No. EN ISO 23753-2:2011: E(ISO 23753-2:2005) Contents Page Foreword. 3 Introduction 4 1 Scope . 5 2 Normative references . 5 3 Principle. 5 4 Reagents and materials . 6 5 Apparatus 7 6 Procedure 7 7 Calculation. 7 8 Test report . 8 Bibliography . DIN EN ISO
18、23753-2:2011-09 EN ISO 23753-2:2011 (E) 29Foreword 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 been taken over as EN ISO 23753-2:2011 by Technical Committee CEN/TC 345 “Characteriz
19、ation 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 2012, and conflicting national standards shall be withdrawn at the latest by Januar
20、y 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 rights. According to the CEN/CENELEC Internal Regulations, the national standards orga
21、nizations 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, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Po
22、rtugal, 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. DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 3Introduction The soil microflora is
23、responsible for the decomposition and conversion 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
24、two hydrogen atoms. Dehydrogenases are essential 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. DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 41 Scope
25、 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 the INT reduction is less sensitive to O2, the method is more reproducible than the TTC-method described in ISO 23753-1. It is not ap
26、plicable 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 reducing properties (e.g. waterlogged soils). 2 Normative references The following referenced documents are indispensable for the a
27、pplication of this document. For dated references, only the edition cited applies. For undated references, 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 ass
28、essment of aerobic microbial processes in the laboratory ISO 10390, Soil quality Determination of pH 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 INT solution is added to a soi
29、l 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 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
30、 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), dehydrogenase activity should not be used as a measure of the biological activity in the soil 3. Abiotic components, such as iron(II) com
31、pounds or sulfides can reduce INT. DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 54 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 are not analysed immediately, they may be kept for up to three mont
32、hs 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 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
33、,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 carbonate 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
34、 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-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 ad
35、d 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 as possible and a suspension is obtained. Remove portions of the suspension later while stirring to keep it homogeneous. Determine the soil texture in a
36、ccordance 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 to 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
37、.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. 4.6.2 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 volumetri
38、c 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 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
39、light-sensitive, the solutions should be protected from exposure to light throughout the analysis. DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 65 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).
40、5.4 Incubator, capable of being set to (30 g114 1) C. 5.5 Suitable conical flasks, volumetric flasks, pipettes and funnels 5.6 Fluted 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
41、 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 (4.3) 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
42、 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 for 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
43、 filter. Within 1 h measure the absorbance of the filtrates against the calibration curve zero by photometry at a wavelength of 485 nm. 7 Calculation Determine the dehydrogenase activity (based on dry soil) from the calibration curve and the following equation: cs bs( ) 100DMVamtg85g85g16g117g117g32
44、g117g117where a is the dehydrogenase activity in g/g of dry soil per hour; csg85 is the complete sample average INTF concentration, in g/ml; bsg85 is the average INTF concentration of the blank sample, in g/ml; V is the solution volume = volume of substrate or buffer solution (5 ml) + volume of extr
45、actant (25 ml), in ml; m is the initial soil sample mass, in g; DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 7DM is the sample dry matter content 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
46、 23753 and include the following details: a) sample identification; b) details of storage temperature and duration (4.1); c) soil textural class; d) 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 IS
47、O 23753, and any effects which may have affected the results. DIN EN ISO 23753-2:2011-09 EN ISO 23753-2:2011 (E) 8Bibliography 1 SPOTHELFER-MAGANA, J., THALMANN, A. and SCHWEIKLE, V.: Methode zur Bestimmung der Dehydrogenaseaktivitt von Bden unter Einsatz von Iodotetrazoliumchlorid (INT): Chemische
48、Reduktion in autoklavierten sowie bestrahlten Bden sowie der Einflu der Inkubationstemperatur und zeit. Agribiol. Res., 46, 1993, pp. 250268 2 THALMANN, H.: Zur Methodik der Bestimmung der Dehydrogenaseaktivitt im Boden mittels Triphenyltetrazoliumchlorid (TTC). Landwirtsch. Forsch., 21, 1968, pp. 2
49、43258 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 determining the activity of soils with iodotetrazolium chloride. Bio. Fertil. Soils, 11, 1991, pp. 216220 5 HLINGER, R.: Determination of dehydrogenase activity using TTC. In: Me
