1、BSI Standards PublicationBS ISO 18187:2016Soil quality Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformisBS ISO 18187:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 18187:2016.The UK participation in its preparatio
2、n was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The Brit
3、ish Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 79672 2ICS 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 April 2
4、016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 18187:2016 ISO 2016Soil quality Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformisQualit du sol Essai contact pour chantillons solides utilisant lactivit dshydrognase de Arth
5、robacter globiformisINTERNATIONAL STANDARDISO18187First edition2016-05-01Reference numberISO 18187:2016(E)BS ISO 18187:2016ISO 18187:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this p
6、ublication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the c
7、ountry of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 18187:2016ISO 18187:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Pri
8、nciple 45 Reagents and material 45.1 Test organisms 45.2 Control substrates 45.2.1 General 45.2.2 Control for soils . 45.2.3 Control for waste material . 55.3 Test substrates 55.4 Chemicals . 66 Apparatus . 87 Procedure. 97.1 Preparation of dilutions 97.2 Reference substance and positive control pre
9、paration . 97.3 Contact test procedure . 97.3.1 General 97.3.2 Aeration 107.3.3 Deactivation 107.3.4 Preparation of the inoculum . 117.3.5 Incubation and fluorescence measurement 117.4 Interferences 118 Calculation and expression of results 128.1 Calculation . 128.1.1 Relative fluorescence 128.1.2 D
10、etermining the percentage of inhibition 128.2 Expression of results . 129 Validity of the test .1310 Statistical analysis 1311 Test report 14Annex A (informative) Results on the ring test .15Annex B (informative) Preparation of test organisms 21Annex C (informative) Testing chemical substances .23Bi
11、bliography .25 ISO 2016 All rights reserved iiiContents PageBS ISO 18187:2016ISO 18187:2016(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
12、 out through ISO technical committees. Each member body interested 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
13、 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different a
14、pproval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may be th
15、e subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any
16、 trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in th
17、e Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.iv ISO 2016 All rights reservedBS ISO 18187:2016ISO 18187:2016(E)IntroductionThis Internationa
18、l Standard describes the miniaturized solid contact assay with Arthrobacter globiformis that allows the preliminary assessment of solid material (i.e. soil and waste materials) within 6 h. The principle of the assay relies on dehydrogenase activity inhibition of an added test organism, caused by bio
19、available toxic substances in soil and waste samples. This is an ecologically relevant assay as far as it uses a ubiquitous soil bacteria species with high affinity to surfaces166which dehydrogenases are involved in different biological mechanisms withstanding bacteria integrity (e.g. respiratory ch
20、ains). Moreover, it has been noticed that this parameter (dehydrogenase activity inhibition) is quite sensitive to different toxic substances.19101415Overall, this assay is non-labour-intensive, rapid, cost-effective and sensitive, providing results that improve the physical and chemical assessment
21、of natural samples while allowing a quick indication of their biological effects.The miniaturized solid contact assay is based on the solid contact assay established by Reference 7.This International Standard is also based on Reference 23.The results of an interlaboratory trial towards the evaluatio
22、n of test variability to assess different waste and soil samples, as well as chemicals, are presented in Annex A. ISO 2016 All rights reserved vBS ISO 18187:2016BS ISO 18187:2016Soil quality Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformis1 ScopeThis Intern
23、ational Standard specifies a rapid method for assessing solid samples in an aerobic suspension, by determining the inhibition of dehydrogenase activity of Arthrobacter globiformis using the redox dye resazurin.It is applicable for assessing the effect of water-soluble and solid matter bounded non-vo
24、latile contaminants of natural samples, such as soils and waste materials. The test yields a result within 6 h and can therefore be used for screening potentially contaminated material.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and
25、 are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 5667-15, Water quality Sampling Part 15: Guidance on the preservation and handling of sludge and
26、sediment samplesISO 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 laboratoryCEN/TR 15310-1, Characterization of waste Sampling of waste materials P
27、art 1: Guidance on selection and application of criteria for sampling under various conditionsEN 14735, Characterization of waste Preparation of waste samples for ecotoxicity tests3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1contact timeexpos
28、ure period of the bacteria to a suspension of solid matter3.2negative controlsample of a control substrate (3.6) with a mixture of known solutions distilled water, medium B or inoculum (3.12).Note 1 to entry: It is used to standardize the analysis.3.3positive controlsample of a control substrate (3.
29、6) with a mixture of known solutions distilled water, medium B or inoculum (3.12) and a reference substanceNote 1 to entry: It is used to check the sensitivity of the test organism.INTERNATIONAL STANDARD ISO 18187:2016(E) ISO 2016 All rights reserved 1BS ISO 18187:2016ISO 18187:2016(E)3.4blank Ablan
30、k, which sets the own fluorescence of the substrate after being deactivatedNote 1 to entry: Blank is not added with bacteria.3.5blank Bblank, which sets the natural fluorescence of the substrate without being deactivatedNote 1 to entry: Blank is not added with bacteria.3.6control substratereference
31、or standard substrate used as a control and as medium (3.13) for preparing dilution/concentration series with test substrates (3.7) or a reference substanceEXAMPLE Quartz sand or LUFA standard soil type 2.2.3.7test substratenatural or artificial substrate that is naturally contaminated or spiked wit
32、h a test chemicalNote 1 to entry: The test substrate is the test material (3.8) after being prepared for testing (e.g. sieved) and/or diluted with a control substrate (3.6).3.8test materialoriginal sample of soil or waste material without any changes (e.g. sieving)3.9dehydrogenase activityactivity o
33、f hydrogen-abstracting enzymes which are involved in many energy and biosynthesis metabolic processes (e.g. the respiratory chain) and which require cell integrity to be produced Note 1 to entry: These enzymes can reduce resazurin into resorufin in the extracellular environment.6Note 2 to entry: See
34、 Reference 21.3.10effect concentration for x % effectECxconcentration (mass fraction) of a test substance or sample that causes x % of an effect on a given endpoint within a given exposure period when compared with a controlEXAMPLE An EC50 is a concentration estimated to cause an effect on a test en
35、d point in 50 % of an exposed population over a defined exposure period.Note 1 to entry: The ECx is expressed as a percentage of soil or waste tested per dry mass of soil mixture. When chemicals are tested, the ECx is expressed as mass of the test substance per dry mass of soil, in milligrams per ki
36、logram.3.11freeze-dried bacteriabacterial culture preserved through the water removing of a frozen cell suspension by sublimation under reduced vacuum pressureNote 1 to entry: The preserved cultures can be stored at (-20 2) C. The bacteria are active after being reconstituted with sterilized distill
37、ed water 20 min to 30 min at (6 2) C and ready to be used in the test, see 7.3.4 b).2 ISO 2016 All rights reservedBS ISO 18187:2016ISO 18187:2016(E)3.12inoculumsuspension of bacteria used to inoculate a nutrient solution3.13mediumaqueous nutritive solution required for bacterial growth3.14optical de
38、nsity of bacterial inoculummeasurement of the attenuation of a light beam passing through a bacterial suspension at 600 nm (used to determine the cell count indirectly)Note 1 to entry: In a bacterial test, the absorbance is usually measured as FAU (formazine attenuation units) at 600 nm (see Referen
39、ce 3).3.15test startmoment when the substrates, reagents and the bacterial inoculum (3.12) are prepared immediately before the incubation and reaction periodNote 1 to entry: Here is when preparing the test and control substrates (3.6) for incubation (i.e. Table 1, day 0).3.16reaction timetime it tak
40、es for the enzyme to react (from the addition of the resazurin solution until the end of the reaction)3.17slopequotient of the relative fluorescence (3.18) variation along the reaction time (3.16) between 15 min and 45 minNote 1 to entry: The slope (expressed as min-1) results from fitting a linear
41、regression model to the fluorescence readings over time.3.18relative fluorescencefluorescence measured for each treatment (control and test) after subtracting the fluorescence of the respective blank A (3.4)3.19stock culturebacterial culture obtained from a pure strain culture acquired from a certif
42、ied laboratoryNote 1 to entry: This stock culture provides an inoculum (3.12) for the pre-culture in the test procedure.3.20lowest ineffective dilutionLID-valuelowest value of the dilution factor (LID) for which the test does not give an ecotoxicological relevant reductionNote 1 to entry: The LID is
43、 expressed as the reciprocal value of dilution.EXAMPLE An often used dilution series is 1/2/4/8/16 = 100 %/50 %/25 %/12,5 %/6,25 % test substrate (3.7) to control substrate (3.6). A LID 8 corresponds to a dilution of soil or waste of 1 : 8. ISO 2016 All rights reserved 3BS ISO 18187:2016ISO 18187:20
44、16(E)4 PrincipleThe bacteria Arthrobacter globiformis is added to the solid material and incubated at (30 1) C for 2 h. After this contact time, the non-toxic redox dye resazurin is added. Due to the dehydrogenase activity, resazurin is transformed into resorufin, in the extracellular environment.6R
45、esorufin can be detected fluorometrically (excitation at 535 nm, emission at 590 nm) in the presence of solid matter. The increase of resorufin is determined by measuring the fluorescence every 15 min for a period of 1 h. In order to determine the inhibition of the dehydrogenase activity, the rate o
46、f resorufin increase in the sample is compared with the rate of resorufin increase in the control. In the presence of toxic substances, an inhibition of dehydrogenase activity is expected. This is reflected by the reduction of resorufin production and subsequent lowering of fluorescence emission.5 R
47、eagents and material5.1 Test organismsThe test organism is Arthrobacter globiformis (Conn 1982) Conn and Dimmick 1947 (strain number ATCC 8010), which is common in soils. Arthrobacter species belong to the Microccocaceae family. They are mostly obligate aerobic organisms, although some species may e
48、xhibit anaerobic metabolism under limiting oxygen conditions.9Arthrobacter spp. are chemoheterotrophic, and present pleomorphic characteristics, since they show a rod-to-coccus morphology change as they enter in the stationary phase. Although Arthrobacter is gram-positive, it can stain gram-negative
49、 during the log-phase. Variations in the cell wall thickness along the bacteria growth can lead to gram variability by differential staining of the granules.22However, this characteristic does not induce a differential sensitivity between assays, as far as an inoculum in exponential growth phase is used during the reaction time. Arthrobacter globiformis is classified in the risk group I non-pathogenic organism.The bacteria strain can be achieved from commercially available freeze-dried or
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