1、September 2011 Translation by DIN-Sprachendienst.English price group 9No 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!$th“1816991www.din.deDDIN EN ISO 23753-1Soil quality Determination of dehydrogenase activity in soil Part 1: Method using triphenyltetrazolium chloride (TTC)(ISO 23753-1:2005)English translation of DIN EN ISO 23753-1:2011-09Bodenbeschaffenheit Bestimmung der Dehydrogenaseaktivitt in Bde
3、n Teil 1: Verfahren mit Triphenyltetrazoliumchlorid (TTC) (ISO 23753-1:2005)Englische bersetzung von DIN EN ISO 23753-1:2011-09Qualit du sol Dtermination de lactivit des dshydrognases dans les sols Partie 1: Mthode au chlorure de triphnylttrazolium (CTT) (ISO 23753-1:2005)Traduction anglaise de DIN
4、EN ISO 23753-1:2011-09SupersedesDIN ISO 23753-1:2006-04www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises pages0 .11119 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee ISO/TC
5、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 119-01-02-04 UA
6、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 iodotetrazolium chl
7、oride (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 10390 ISO 11465
8、DIN ISO 11465 Amendments This standard differs from DIN ISO 23753-1:2006-04 as follows: a) DIN ISO 23753-1:2006-04 has been adopted as European Standard DIN EN ISO 23753-1:2011-09. Previous editions DIN 19733-1: 1998-07 DIN ISO 23753-1: 2006-04 National Annex NA (informative) Bibliography DIN ISO 10
9、381-6, 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 10390, Soil quality Determination of pH DIN ISO 11465, Soil quality Determination of
10、 dry matter and water content on a mass basis Gravimetric method 2 DIN EN ISO 23753-1:2011-09 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 23753-1 July 2011 ICS 13.080.30 English Version Soil Quality - Determination of dehydrogenase activity in soil - Part 1: Method using triphenyltetraz
11、olium chloride (TTC) 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 der Dehydrogenaseaktivitt in Bden - Teil 1: Verfahren mit Triphenyltetrazoliumchlorid (TTC) (ISO
12、 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 European Standard the status of a national standard without any alteration. Up-to-date lists and bibliog
13、raphical 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 (English, French, German). A version in any other language made by translation under the responsib
14、ility 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, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece
15、, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any
16、 means reserved worldwide for CEN national Members. Ref. No. EN ISO 23753-1:2011: E(ISO 23753-1:2005) EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG 1Contents Page Foreword. 3 Introduction 4 1 Scope . 5 2 Normative references . 5 3 Principle. 5 4
17、 Reagents and materials . 6 5 Apparatus 7 6 Procedure 7 7 Calculation. 7 8 Test report . Bibliography . 9 28DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) 3DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) Foreword The text of ISO 23753-1:2005 has been prepared by Technical Committee ISO/TC 190
18、 “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 soils” the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, e
19、ither 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. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN a
20、nd/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 the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Cz
21、ech 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 the United Kingdom. Endorsement notice The text of ISO 23753-1:
22、2005 has been approved by CEN as a EN ISO 23753-1:2011 without any modification. Introduction The soil microflora is responsible for the decomposition and conversion of organic substances, aggregation stability and the carbon, nitrogen, sulfur and phosphorus cycles. Dehydrogenases, as respiratory ch
23、ain 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 used as an indicator of biological redox sy
24、stems and as a measure of microbial activity in the soil. 4DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) 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
25、 dehydrogenase activity in the 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 application of this docum
26、ent. 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 assessment of aerobic micro
27、bial 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 TTC solution is added to a soil sample and the mixture
28、 is incubated at 25 C for 16 h. The triphenylformazan (TPF) released is extracted 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
29、. ethanol, acetone-CCl4mixture). According to Reference 1, methanol is not suitable 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 meas
30、ure of the biological activity in the soil 2. Abiotic components, such as iron(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 diffe
31、r from measured values (XVP) in soils with low microbial activity. 5DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) 4 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, the
32、y 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 dehydrogenase activity of samples with different storage times should not be compared. 4.2 Hydrochloric acid, c(HCl) = 1 mol/
33、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) 7,8 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 hydr
34、ochloric acid and make up to 1 000 ml with distilled water. 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 acco
35、rdance with ISO 11259. Dissolve 2,3,5-triphenyltetrazolium 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
36、 clayey soil: 0,1 % to 0,5 % TTC; b) loam, humic and loamy 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
37、 ml. 4.6.2 TPF working solution, dilute 1,0 ml of stock 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 wi
38、th acetone (4.5). The solutions obtained have TPF concentrations 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. 6DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:
39、2011 (E) 5 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 (25 g114 1) C. 5.5 Suitable conical flasks, volumetric flasks, pipettes and funnels 5.6 Test tube shake
40、r 5.7 Fluted filter, with slow filtering 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
41、 solution to the blank sample. Shake each 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
42、 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. According to Reference 1, optimum conditions are given at a soil/solution ratio of
43、 1:1. There is no linear correlation between 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
44、 soil, 2 cm for 5 g of soil and 2,5 cm for 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( ) 100D
45、MVamtg85g85g16g117g117g32g117g117where: a is the dehydrogenase activity in g/g of dry soil per hour; csg85 is the complete sample average TPF concentration, in g/ml; bsg85 is the average TPF concentration of the blank sample, in g/ml; V is the solution volume (= volume of substrate or buffer solutio
46、n + volume of extractant), in ml; 7DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) m 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
47、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) pH value (pHCaCl2) of soil sample; e) test results; f) any details that are optional or deviations from the specifications of this part of I
48、SO 23753, and any effects which may have affected the results. 8DIN EN ISO 23753-1:2011-09 EN ISO 23753-1:2011 (E) Bibliography 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.
