1、December 2013Translation by DIN-Sprachendienst.English price group 11No 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).IC
2、S 13.080.30!%*c1“2076414www.din.deDDIN ISO 17155Soil quality Determination of abundance and activity of soil microflora usingrespiration curves (ISO 17155:2012),English translation of DIN ISO 17155:2013-12Bodenbeschaffenheit Bestimmung der Abundanz und Aktivitt der Bodenmikroflora mit Hilfe vonAtmun
3、gskurven (ISO 17155:2012),Englische bersetzung von DIN ISO 17155:2013-12Qualit du sol Dtermination de labondance et de lactivit de la microflore du sol laide de courbes derespiration (ISO 17155:2012),Traduction anglaise de DIN ISO 17155:2013-12SupersedesDIN ISO 17155:2003-06www.beuth.deIn case of do
4、ubt, the German-language original shall be considered authoritative.Document comprises 17 pages11.13 A comma is used as the decimal marker. Contents Page National foreword .3 National Annex NA (informative) Bibliography .4 1 Scope .6 2 Normative references .6 3 Terms and definitions 6 4 Principle .7
5、 5 Reagents 7 6 Apparatus 7 7 Sampling 8 7.1 Sample quantities .8 7.2 Sampling and storage 8 7.3 Soil sample characteristics .9 8 Procedure 9 8.1 Test .9 8.2 Toxicity testing 9 9 Calculation 9 9.1 Microbial parameters9 9.2 Interpretation of data . 10 10 Test report 13 Annex A (informative) Results o
6、f a laboratory ring test carried out in Germany . 14 Annex B (informative) Comparison of microbial biomass determination by respiration curve measurement (this International Standard) and substrate-induced respiration (ISO 14240-11) . 15 Bibliography . 18 2DIN ISO 17155:2013-12National foreword This
7、 standard has been prepared by Technical Committee ISO/TC 190 “Soil quality” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Subcommittee NA 119-01-02-04 UA Biologische Verfahren. Atten
8、tion is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN and/or DKE shall not be held responsible for identifying any or all such patent rights. Expert assistance and specialized laboratories will be required to perform the analyses describ
9、ed in this standard. Notes to Figure 2 and the key ISO 17155:2012 has been taken over as DIN ISO 17155 without any modifications. Editorial remarks concerning ISO 17155:2012: In Figure 2, RSband RSc are to be interchanged. The key has been corrected to read: X t h time Y R g gdm1 h1CO2respiration da
10、te CRcumulative CO2evolution or O2consumption RSsubstrate-induced respiration tpeakmaxtime to the peak maximum 1 unpolluted soil (control) 2 Cu-polluted soil (190 mg kgdm1) 3 TNT-polluted soil (50 mg kgdm1) 4 substrate addition a unpolluted soil (control) bCu-polluted soil (190 mg kgdm1) cTNT-pollut
11、ed soil (50 mg kgdm1) 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 10694 DIN ISO 10694 ISO 11277 DIN ISO 11277 ISO 11465 DIN ISO 11465 ISO 14238 DIN ISO 14238 ISO 14240-1 DIN EN ISO
12、 14240-1 ISO 16072 DIN EN ISO 16072 3DIN ISO 17155:2013-12Amendments This standard differs from DIN ISO 17155:2003-06 as follows: a) the standard has been editorially revised; b) Clause 3 “Terms and definitions” has been revised; c) the figures have been revised; d) Subclause 9.1 “Microbial paramete
13、rs” has been revised; e) Annex B has been added. Previous editions DIN ISO 17155: 2003-06 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 microbiologica
14、l processes, biomass and diversity in the laboratory DIN ISO 10390, Soil quality Determination of pH DIN ISO 10694, Soil quality Determination of organic and total carbon after dry combustion (elementary analysis) DIN ISO 11277, Soil quality Determination of particle size distribution in mineral soi
15、l material Method by sieving and sedimentation DIN ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method DIN ISO 14238, Soil quality Biological methods Determination of nitrogen mineralization and nitrification in soils and the influence of chemical
16、s on these processes DIN EN ISO 14240-1, Soil quality Determination of soil microbial biomass Part 1: Substrate-induced respiration method DIN EN ISO 16072, Soil quality Laboratory methods for determination of microbial soil respiration 4DIN ISO 17155:2013-12Soil quality Determination of abundance a
17、nd activity of soil microflora using respiration curves1 ScopeThis International Standard specifies a test method for determining the activity of active aerobic, heterotrophic microbial biomass in soils. This method is applicable to the monitoring of soil quality and to the evaluation of the ecotoxi
18、c potential of soils and soil materials. It is also applicable for soils sampled along contamination gradients in the field and to soils that are contaminated experimentally in the field or in the laboratory.2 Normative referencesThe following referenced documents are indispensable for the applicati
19、on 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 under aerobic condit
20、ions for the assessment of microbiological processes, biomass and diversity in the laboratoryISO 10390, Soil quality Determination of pHISO 10694, Soil quality Determination of organic and total carbon after dry combustion (elementary analysis)ISO 11277, Soil quality Determination of particle size d
21、istribution in mineral soil material Method by sieving and sedimentationISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric methodISO 14238, Soil quality Biological methods Determination of nitrogen mineralization and nitrification in soils and the influ
22、ence of chemicals on these processes3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1basal respiration rateRBconstant mass of CO2released or mass of O2consumed per unit mass of soil per unit time without substrate additionNOTE See Figure 1 for a
23、typical basal respiration curve.3.2substrate-induced respiration rateRSconstant mass of CO2released or mass of O2consumed per unit mass of soil per unit time shortly after addition of a carbon substrateNOTE 1 See Figure 1 for a typical substrate-induced respiration curve.NOTE 2 If glucose is used as
24、 a carbon substrate, microbial biomass can be determined from the substrate-induced respiration rate (see ISO 14240-11).5DIN ISO 17155:2013-123.3respiratory activation quotientQRbasal respiration rate divided by substrate-induced respiration rateQRRRBS= (1)3.4specific growth rateexponent representin
25、g respiration rate per unit of time during the exponential phase of growthNOTE See Equation (3).3.5time to the peak maximumtpeakmaxtime from addition of substrate to the maximum respiration rateNOTE 1 See Figure 1.NOTE 2 The time to the peak maximum also reflects the viability of the growing organis
26、ms.3.6cumulative CO2evolution or O2consumptionCRtotal area bounded by the line of the soil respiration rate curve to the time axis from time of the addition of substrate to the time of peak maximum (tpeakmax)NOTE See Figure 1.3.7soil materialmaterial composed of excavated soil, dredged materials, ma
27、nufactured soils, treated soils or fill materials4 PrincipleThe CO2production or O2consumption (respiration rate) from unamended soils as well as the decomposition of an easily degraded substrate (glucose + ammonium + phosphate) is monitored regularly (e.g. every hour). From the CO2production or O2c
28、onsumption data, the different microbial parameters (basal respiration, substrate-induced respiration, respiratory activation quotient, tpeakmax, CR) can be calculated.5 Reagents5.1 Glucose, C6H12O6.5.2 Potassium dihydrogenphosphate, KH2PO4.5.3 Diammonium sulfate, (NH4)2SO4.5.4 Substrate, consisting
29、 of a mixture of 80 g of glucose (5.1), 13 g of diammonium sulfate (5.3), and 2 g of KH2PO4(5.2), which is thoroughly ground and mixed in a mortar.6 ApparatusOrdinary laboratory equipment and 6.1.6DIN ISO 17155:2013-126.1 Respirometer for continuous measurement of CO2evolution or O2consumption, main
30、tained at a constant temperature (preferably 20 C). Suitable examples of equipment are given in ISO 16072.2KeyX t h timeY R g g1dmh1CO2or O2 respiration ratesCRcumulative CO2evolution or O2consumptiondp/dt rate of product formation after substrate additionK respiration rate of K-strategist at the ti
31、me of substrate additionr respiration rate of r-strategist at the time of substrate additiontpeakmaxtime to the peak maximum specific growth rate1 RSsubstrate-induced respiration RS= K + r (at t = 0)2 RBbasal respiration 3 substrate additionFigure 1 Soil respiration rate before and after addition of
32、 an easily degraded substrate7 Sampling7.1 Sample quantitiesChoose the size of the soil samples taking into account the apparatus (6.1) used, the organic matter content of the samples (7.3) and the soil needed for sample characterization (7.3). It is recommended that at least three replicates per sa
33、mple be measured.7.2 Sampling and storageThe recommendations in ISO 10381-6 for collection, handling and storage of soil samples shall be followed.7DIN ISO 17155:2013-127.3 Soil sample characteristicsSoil samples generating soil respiration curves can be obtained from mineral, organic, polluted, and
34、 unpolluted soils. Determine the following characteristics for each soil sample: particle size distribution in accordance with ISO 11277; water content in accordance with ISO 11465; water-holding capacity in accordance with Annex A of ISO 14238:2012; pH in accordance with ISO 10390; organic matter c
35、ontent in accordance with ISO 10694.8 Procedure8.1 TestPre-incubate moist soil samples (preferably 40 % to 60 % of maximum water holding capacity or 0,01 MPa to 0,03 MPa suction pressure) at 20 C for 3 d to 4 d before the beginning of the measurement. Measure the basal respiration of the sub-samples
36、 first. Measure the respiration rates until constant rates are obtained.After measuring the basal respiration, add 10 mg of the substrate (5.4) per gram soil (dry mass) and mix homogeneously with a spatula into the soil samples. If the organic matter content is 5 %, add 0,2 g of the substrate per gr
37、am humus (see References 45).8.2 Toxicity testingIn principle, testing the influence of chemicals should also be possible with the method. Up to the time of publication, there is only scarce experience available in the literature.To determine the influence of chemicals on the abundance and activity
38、of soil microorganisms, a soil with low content of organic carbon (mass fraction between 0,5 % and 1,5 %). Particles of size 0,3 (mineral soils arable, grassland), QR 0,4 (mineral forest soils) and QR 0,6 (organic layers L, Of, Oh) and tpeakmax 50 h are indicative for polluted materials (see Referen
39、ces 7 and 8).Moreover, polluted samples often do not show any logarithmic increase of respiration rates after addition of substrate and/or formation of double peaks (see Figure 2). Double peak formation is caused by a short-term or selective toxic effect of a contaminant. In particular, slowly growi
40、ng fungi with the marker 18:29,12 seem to be responsible for the formation of a second respiration maximum (Reference 15).NOTE Double peaks can also occur in unpolluted samples. Reasons for this phenomenon are growth of fungi due to suboptimal (high) water contents. 10DIN ISO 17155:2013-12KeyX t h t
41、imeY R g g1dmh1CO2respiration rateCRcumulative CO2evolution or O2consumptionRSsubstrate-induced respirationtpeakmaxtime to the peak maximum1 unpolluted soil (control), Reference 42 Cu-polluted soil (190 mg kg1dm), Reference 53 TNT-polluted soil (50 mg kg1dm), Reference 64 substrate additionaReferenc
42、e 1.bReference 2.cReference 2.Figure 2 Soil respiration curves of an unpolluted and two polluted soils9.2.2 Additional criteria for the interpretation of the results from soils deliberately contaminated with chemicalsTheoretically, when a chemical is added to soil, the micro-flora can respond in fou
43、r different ways, of which the two intermediate are most likely to occur. Death: the substance is very toxic. The respiration decreases rapidly, but if the toxic substance is removed, either by degradation or evaporation, the survivors can decompose the dead biomass and the respiration is temporaril
44、y as high or higher than before the addition of the substance, but the biomass remains low for a considerable time (see Reference 10). Intolerance: the toxicity of the test substance is moderate. Sensitive species are replaced by more resistant ones. The decomposition of soil organic matter becomes
45、less efficient and less biomass is formed (see Reference 11). The activity and vitality of the microorganisms might also be reduced.11DIN ISO 17155:2013-12N1) National footnote: The errors contained in the key and Figure 2 of ISO 17155:2012 have been included, uncorrected, in the text of this docume
46、nt. The corrected version is given in the National foreword. N1) No observed effect: the toxicity of the test substance is small. If some species are affected they are replaced by others that are as effective as the original flora (see Reference 12). CO2from the slow degradation of an organic test s
47、ubstance can possibly mask a reduced degradation of soil organic matter. Enhancement: the test substance is a suitable substrate for at least some of the soil organisms. Respiration is increased until the test substance is consumed (see Reference 13). The biomass and vitality of the growing organism
48、s are also increased.The responses of intolerance or no observed effect to a chemical are the most likely to occur.10 Test reportThe test report shall include the following information:a) general: soil collection, treatment, incubation, including date collected, length of storage, temperature of sto
49、rage, test substance: chemical identification data (chemical testing), soil characteristics (see also 7.3): particle size distribution in accordance with ISO 11277 water content in accordance with ISO 11465 water-holding capacity in accordance with Annex A of ISO 14238:2012; pH in accordance with ISO 10390 organic matter content in accordance with ISO 10694;b) test condit
