1、BRITISH STANDARD BS EN ISO14240-2:2011Soil quality Determination of soil microbial biomass - Part 2: Fumigation-extraction methodICS 13.080National forewordThis British Standard is the UK implementation of EN ISO 14240-2:2011. It is identical to ISO 14240-2:1997. It supersedes BS 7755-4.4.2:1997, wh
2、ich is withdrawn.The UK participation in its preparation was entrusted to Technical Committee EH/4, Soil quality.A list of organizations represented on this committee can be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.Compliance with a British Standard cannot confer immunity from legal obligations.BS EN IS
3、O 14240-2:2011This British Standard, having been prepared under the direction of the Health and Environment Sector Board, was published under the authority of the Standards Board and comes into effect on15 May 1997 BSI 2011Amendments/corrigenda issued since publicationDate Comments 30 September 2011
4、 This corrigendum renumbers BS 7755-4.4.2:1997 as BS EN ISO 14240-2:2011ISBN 978 0 580 73149 5EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 14240-2 June 2011 ICS 13.080.30 English Version Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-extraction method (ISO 14
5、240-2:1997) Qualit du sol - Dtermination de la biomasse microbienne du sol - Partie 2 : Mthode par fumigation-extraction (ISO 14240-2:1997) Bodenbeschaffenheit - Bestimmung der mikrobiellen Biomasse von Bden - Teil 2: Fumigations-Extraktionsverfahren (ISO 14240-2:1997) This European Standard was app
6、roved by CEN on 3 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 bibliographical references concerning such national s
7、tandards 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 responsibility of a CEN member into its own language an
8、d 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, Hungary, Iceland, Ireland, Italy, Latvia, Li
9、thuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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 201
10、1 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 14240-2:2011: EForeword The text of ISO 14240-2:1997 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (IS
11、O) and has been taken over as EN ISO 14240-2:2011 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 lates
12、t by December 2011, and conflicting national standards shall be withdrawn at the latest by December 2011. 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
13、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, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hunga
14、ry, 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 14240-2:1997 has been approved by CEN as a EN ISO 14240-2:2011 without any modi
15、fication. BS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E)iiiContentPageIntroduction 11 Scope 12 Normative references 13 Definition 14 Principle 15 Materials and reagents 16 Apparatus 27 Fumigation and extraction 28 Determination of carbon in the extracts 39 Precision 5Annex A (normative) Measurement o
16、f water-holding capacity of soil 6Annex B (informative) Estimation of soil microbial biomass in soils containinglarge amounts of living roots Pre-extraction procedure 6Annex C (informative) Results of an interlaboratory trial 7Annex D (informative) Bibliography 7Table C.1 Result of German interlabor
17、atory ring test for the estimationofsoilmicrobial biomass by the fumigation-extraction method 7BS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E) ISO 2011blankBS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E)1IntroductionSoil consists of both living and nonliving components which exist in a complex and heter
18、ogeneous environment. Soil microflora is responsible for the degradation of organic matter, stability of aggregates and most nutrient cycling which occurs in soils. The purpose of determining the microbial biomass of soils is to allow assessment of the continued maintenance of soil fertility, the po
19、tential ability to degrade added organic materials, and the effects of added materials on the natural microbial population.1 ScopeThis part of ISO14240 specifies a method for the estimation of microbial biomass of soils by measurement of total extractable organic biomass material mainly from freshly
20、 killed microorganisms. The method is also applicable to the estimation of microbial nitrogen and microbial ninhydrin-reactive nitrogen in soil, but this part of ISO14240 describes only the measurement of extractable organic carbon. The fumigation-extraction (FE) method is applicable to aerobic and
21、anaerobic (water-logged, paddy) soils over the whole range of soil pH. Biomass can be also measured in soils containing actively decomposing substrates and soils supersaturated with potassium sulfate solution.NOTEChloroform fumigation also affects soil fauna. The contribution of carbon from these or
22、ganisms is generally small ( 5%) and can usually be neglected.2 Normative referencesThe following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO14240. At the time of publication, the editions indicated were valid. All standards are subje
23、ct to revision, and parties to agreements based on this part of ISO14240 are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.ISO 10381-6:1993, Soil qu
24、ality Sampling Part.6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory. ISO 10694:1995, Soil quality Determination of organic and total carbon after dry combustion (elementary analysis). ISO 11465:1993, Soil quality Determi
25、nation of dry matter and water content on a mass basis Gravimetric method. 3 DefinitionFor the purposes of this part of ISO14240, the following definition applies.3.1 soil microbial biomassmass of intact microbial cells in a given soilNOTEThis parameter can be estimated from the measurement of the c
26、arbon or nitrogen content of these cells or by the measurement of their ability to mineralize an added carbon source. Dead cells and cell fragments may be detected when carbon or nitrogen analysis is used but only intact cells will be detected when respiration is measured.4 PrincipleThrough fumigati
27、on of the soil sample, intact microbial cells are lysed and the microbial organic matter released. Nonliving soil organic matter is not seriously affected by such fumigation. Soil samples are fumigated with chloroform for24h. The organic carbon extracted using0,5mol/l potassium sulfate is determined
28、 in fumigated and unfumigated samples, and the difference in extracted organic carbon is used to determine microbial biomass carbon. The method is usually termed fumigation-extraction (FE).5 Materials and reagents5.1 SoilsGuidance for the collection, handling and storage of soil (ISO10381-6) shall b
29、e followed, as far as applicable. Sieve the soil samples at approximately40% water-holding capacity (WHC) if homogeneous samples are required.Determine the soil WHC in accordance withAnnex A.BS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E) ISO 20112The water content of samples shall be higher than30% W
30、HC to ensure uniform chloroform distribution and effective fumigation. Take care to avoid smearing and compaction of wet soil in this method. Samples of waterlogged soils do not have to be dried prior to analysis.5.2 ReagentsReagents of recognized analytical grade shall be used, including:5.2.1 Sili
31、cone grease (medium viscosity).5.2.2 Ethanol-free chloroformWARNING In the presence of light, ethanol-free chloroform degrades rapidly to form phosgene gas (COCl2) which is odourless and highly toxic.5.2.3 Potassium sulfate solution, c(K2SO4) = 0,5 mol/l ( = 87,135 g/l).5.2.4 Soda lime6 Apparatus6.1
32、 Room, or incubator, capable of being maintained at (25 2)C.6.2 Implosion-protected desiccator6.3 Filter paper1)6.4 Glass beakers6.5 Petri dishes6.6 Polyethylene bottles, of capacity250ml6.7 Vacuum line (water-pump or electric pump).6.8 Horizontal or overhead shaker6.9 Freezer, operation at (15 to 2
33、0)C.6.10 Antibumping granules7 Fumigation and extraction7.1 FumigationLine the desiccator(6.2) with moist filter paper(6.3) for fumigation of the soil samples.Weigh at least three samples of moist soil(5.1), each containing a mass equivalent to25 to50g of oven-dry soil, into glass beakers(6.4) or Pe
34、tri dishes(6.5) and place them in the desiccator with a beaker containing25ml of ethanol-free chloroform(5.2.2) and a few antibumping granules(6.10), and a beaker with soda lime(5.2.4). Evacuate the desiccator until the chloroform has boiled vigorously for approximately2min. Close the vacuum tap on
35、the desiccator and incubate in the dark(6.1) at(25 2)C for22h to24h.If there is insufficient soil available, use smaller samples provided the ratio of soil mass to volume of extractant is kept the same(1 : 4). In soils containing more than20% organic material (as determined according to ISO10694), i
36、ncrease the ratio soil : extract above1 : 4 (to a maximum of1 : 30 for soils containing95% organic matter, e.g.L-layer) in order to obtain sufficient extracted matter. Record the mass of soil used.After fumigation is completed, remove the beaker containing the chloroform and the filter paper from th
37、e desiccators. Remove the chloroform vapour from the soil by repeated evacuation(6 times for2min each) in the desiccator. The samples are ready for extraction.Place three unfumigated, moist control samples(50g dry mass) into polyethylene bottles(6.6) and immediately extract with200ml of the potassiu
38、m sulfate(5.2.3), as indicated in7.2.1) Whatman No. 42, Schleicher VH is the volume titrant(8.1.2.4) consumed by the refluxed blank, in millilitres;VC is the volume titrant(8.1.2.4) consumed by the unrefluxed blank, in millilitres;M is c(K2Cr2O7), in moles per litre;PD is the added volume of K2Cr2O7
39、 solution, in millilitres;PS is the added volume of sample, in millilitres;E is 3conversion of organic CC to CO2C(+IV).C (4g/g dry soil) = C (4g/ml) (PK/DW + SW) . (2)wherePK is the mass of extractant, in grams;DW is the dry mass of sample (determined according to ISO11465), in grams;SW is the soil
40、water (grams of water/grams of dry soil) (determined according to ISO11465).BC = EC/kEC . (3)whereEC = (mass of organic C extracted from fumigated soils) (mass of organic C extracted from unfumigated soils);kEC = 0,38.BS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E) ISO 201158.2.3 Additional apparatus8
41、.2.3.1 Automatic carbon analyzer with IR detection2) or continuous-flow system with colorimetric detection3)For the purposes of this part of ISO14240, the determination of microbial biomass carbon is based on UV-activated persulfate oxidation of organic carbon.8.2.4 ProcedureFor the automated UV-per
42、sulfate oxidation method, mix5ml of the potassium sulfate soil extract(7.1) with5ml of sodium polyphosphate reagent(8.2.2.5). Any precipitate of CaSO4 in the soil extracts is dissolved by this procedure. The potassium persulfate reagent(8.2.2.4) is automatically fed into the UV oxidation chamber, wh
43、ere the oxidation to CO2 is activated by UV light. The resulting CO2 is measured by IR absorption or UV spectrophotometric detection.8.2.5 Calculation of resultsCalculate the extractable organic carbon (C) using equation(4).Calculate the biomass BC using equation(5).NOTE 1The factor KECwas calculate
44、d by relating the results of the fumigation-incubation method to those of fumigation-extraction method(23 soils).NOTE 2It is possible to use the fumigation-extraction method in conjunction with decomposition studies of 14C-labelled organic substrates.9 PrecisionDetails of an interlaboratory test on
45、the precision of the method are summarized inAnnex C. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given.2) Dohrman DC80, Maihak Tocor4, are examples of suitable equipment available commercially. This information is giv
46、en for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products.3) Skalar, Perstorp Alpkem, are examples of suitable equipment available commercially. This information is given for the convenience of users of this International Standard
47、and does not constitute an endorsement by ISO of these products.C (4g/g dry soil) = (V DV) (B DB) (Pk/DW + SW) . (4)whereV is the C (4g/ml) of sample;B is the C (4g/ml) of blank;DV is the dilution of sample with sodium phosphate, in millilitres;DB is the dilution of blank with sodium phosphate, in m
48、illilitres;Pk see equation(2);DW see equation(2);SW see equation(2).BC = EC/kEC . (5)whereEC = (organic C extracted from fumigated soils) (organic C extracted from unfumigated soils);kEC = 0,45.BS EN ISO 14240-2:2011EN ISO 14240-2:2011 (E) ISO 20116Annex A (normative) Measurement of water-holding ca
49、pacity of soilA.1 ScopeThe method is applicable for obtaining a value for the water-holding capacity of a soil for applications in which the exact value is not critical.A.2 PrincipleA cylinder with perforated base is partially filled with soil, capped and immersed in water and drained. The quantity of water taken up by the soil is determined by weighing, drying to constant mass at105C and reweighing.A.3 ApparatusA.3.1 Cylinder, of known volume, of approximate dimensions50mm to150mm in length and50mm to100mm in diameter, with a p
