1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 17155:2012Soil quality Determinationof abundance and activity ofsoil microflora using respirationcurvesBS ISO 17155:2012 BRITISH STANDARDNational forewordThis British Stan
2、dard is the UK implementation of ISO 17155:2012. Itsupersedes BS ISO 17155:2002 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can beobtained on request to its secretary.This publ
3、ication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 71396 5ICS 13.080.30Compliance with a British Standard cannot confer immunity
4、fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 October 2012.Amendments issued since publicationDate Text affectedBS ISO 17155:2012 ISO 2012Soil quality Determination of abundance and activity of soil microflora using
5、respiration curvesQualit du sol Dtermination de labondance et de lactivit de la microflore du sol laide de courbes de respirationINTERNATIONAL STANDARDISO17155Second edition2012-10-01Reference numberISO 17155:2012(E)BS ISO 17155:2012ISO 17155:2012(E)ii ISO 2012 All rights reservedCOPYRIGHT PROTECTED
6、 DOCUMENT ISO 2012All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member
7、body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 17155:2012ISO 17155:2012(E) ISO 2012 All rights reserved iiiContents PageForeword iv1 Scope 12 Nor
8、mative references . 13 Terms and definitions . 14 Principle . 25 Reagents 26 Apparatus 27 Sampling 37.1 Sample quantities 37.2 Sampling and storage 37.3 Soil sample characteristics 48 Procedure 48.1 Test 48.2 Toxicity testing . 49 Calculation 59.1 Microbial parameters 59.2 Interpretation of data 610
9、 Test report . 8Annex A (informative) Results of a laboratory ring test carried out in Germany 9Annex B (informative) Comparison of microbial biomass determination by respiration curve measurement (this International Standard) and substrate-induced respiration (ISO 14240-11) 11Bibliography .13BS ISO
10、 17155:2012ISO 17155:2012(E)ForewordISO (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 member body interested
11、 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 Electrotechnical Comm
12、ission (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 adopted by the technical
13、 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 patent rights. ISO shall
14、not be held responsible for identifying any or all such patent rights.ISO 17155 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.This second edition cancels and replaces the first edition (ISO 17155:2002), which has been technically revised.iv ISO 2
15、012 All rights reservedBS ISO 17155:2012INTERNATIONAL STANDARD ISO 17155:2012(E)Soil quality Determination of abundance and activity of soil microflora using respiration curves1 ScopeThis International Standard specifies a test method for determining the activity of active aerobic, heterotrophic mic
16、robial biomass in soils. This method is applicable to the monitoring of soil quality and to the evaluation of the ecotoxic 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 th
17、e field or in the laboratory.2 Normative referencesThe 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 amendments) applies
18、.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 laboratoryISO 10390, Soil quality Determination of pHISO 10694, Soil quality Determination of or
19、ganic and total carbon after dry combustion (elementary analysis)ISO 11277, Soil quality Determination of particle size distribution 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
20、 14238, Soil quality Biological methods Determination of nitrogen mineralization and nitrification in soils and the influence 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
21、 CO2released or mass of O2consumed per unit mass of soil per unit time without substrate additionNOTE See Figure 1 for a 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 additio
22、n of a carbon substrateNOTE 1 See Figure 1 for a typical substrate-induced respiration curve.NOTE 2 If glucose is used as a carbon substrate, microbial biomass can be determined from the substrate-induced respiration rate (see ISO 14240-11). ISO 2012 All rights reserved 1BS ISO 17155:2012ISO 17155:2
23、012(E)3.3respiratory activation quotientQRbasal respiration rate divided by substrate-induced respiration rateQRRRBS= (1)3.4specific growth rateexponent representing respiration rate per unit of time during the exponential phase of growthNOTE See Equation (3).3.5time to the peak maximumtpeakmaxtime
24、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 organisms.3.6cumulative CO2evolution or O2consumptionCRtotal area bounded by the line of the soil respiration rate curve to the time axis from
25、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, manufactured soils, treated soils or fill materials4 PrincipleThe CO2production or O2consumption (respiration rate) from unamended soils a
26、s well as the decomposition of an easily degraded substrate (glucose + ammonium + phosphate) is monitored regularly (e.g. every hour). From the CO2production or O2consumption data, the different microbial parameters (basal respiration, substrate-induced respiration, respiratory activation quotient,
27、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 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 i
28、n a mortar.6 ApparatusOrdinary laboratory equipment and 6.1.2 ISO 2012 All rights reservedBS ISO 17155:2012ISO 17155:2012(E)6.1 Respirometer for continuous measurement of CO2evolution or O2consumption, maintained at a constant temperature (preferably 20 C). Suitable examples of equipment are given i
29、n 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 time of substrate additionr respiration rate of r-strategist at the time of substrate additiontp
30、eakmaxtime 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 an easily degraded substrate7 Sampling7.1 Sample quantitiesChoose the size of the soil sample
31、s 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 sample be measured.7.2 Sampling and storageThe recommendations in ISO 10381-6 for collection, ha
32、ndling and storage of soil samples shall be followed. ISO 2012 All rights reserved 3BS ISO 17155:2012ISO 17155:2012(E)7.3 Soil sample characteristicsSoil samples generating soil respiration curves can be obtained from mineral, organic, polluted, and unpolluted soils. Determine the following characte
33、ristics 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 content in accordance with ISO 10694.8 Procedure8.1
34、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 first. Measure the respiration rates until constan
35、t 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 gram humus (see References 45).8.2 Toxicity testingIn
36、 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 of soil microorganisms, a soil with low content of
37、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 References 7 and 8).Moreover, polluted samples often do no
38、t 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 growing fungi with the marker 18:29,12 seem to be respon
39、sible 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. 6 ISO 2012 All rights reservedBS ISO 17155:2012ISO 17155:2012(E)KeyX t h timeY R g g
40、1dmh1CO2respiration 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 additionaReference 1.bRefer
41、ence 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 four differen
42、t 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 temporarily as high
43、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 less effic
44、ient and less biomass is formed (see Reference 11). The activity and vitality of the microorganisms might also be reduced. ISO 2012 All rights reserved 7BS ISO 17155:2012ISO 17155:2012(E) No observed effect: the toxicity of the test substance is small. If some species are affected they are replaced
45、by others that are as effective as the original flora (see Reference 12). CO2from the slow degradation of an organic test substance 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. Respira
46、tion is increased until the test substance is consumed (see Reference 13). The biomass and vitality of the growing organisms 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 info
47、rmation:a) general: soil collection, treatment, incubation, including date collected, length of storage, temperature of storage, test substance: chemical identification data (chemical testing), soil characteristics (see also 7.3): particle size distribution in accordance with ISO 11277 water content
48、 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 conditions: date and place of sampling, date of start and end of the test, temperature, incubation time with
49、the test substance before microbial measurements, concentrations tested or range of concentrations in test area compared to background levels, solvent used to add the test substance;c) results: list of the microbial parameters for each sub-sample, mean values for each sample, plot of log of concentration of test substance vs. microbial parameters, EC10and EC50, regression of these relationships.8 ISO 2012 All rights reservedBS ISO 17155:201
