1、BSI Standards PublicationBS ISO 13641-1:2003Water quality Determinationof inhibition of gas productionof anaerobic bacteriaPart 1: General testCopyright British Standards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted with
2、out license from IHS-,-,-BS ISO 13641-1:2003 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 13641-1:2003.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committe
3、e 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 British Standards Institution 2014. Published by BSI StandardsLimited 2014ISBN 978 0 580 86078 2ICS 13.060.70Comp
4、liance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 March 2014.Amendments issued since publicationDate Text affectedCopyright British Standards Institution Provided by
5、IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:2003Reference numberISO 13641-1:2003(E)ISO 2003INTERNATIONAL STANDARD ISO13641-1First edition2003-05-15Water quality Determination of inhibition of gas pro
6、duction of anaerobic bacteria Part 1: General test Qualit de leau Dtermination de linhibition de la production de gaz des bactries anarobies Partie 1: Essai gnral Copyright British Standards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or netwo
7、rking permitted without license from IHS-,-,-BS ISO 13641-1:2003ISO 13641-1:2003(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed
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10、t is found, please inform the Central Secretariat at the address given below. ISO 2003 All 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 permi
11、ssion in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org ii ISO 2003 All rights reservedCopyright British Stan
12、dards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:2003ISO 13641-1:2003(E) ISO 2003 All rights reserved iiiContents Page Foreword iv 1 Scope 1 2 Normative references . 1 3 Prin
13、ciple . 2 4 Reagents and media . 2 5 Apparatus. 2 6 Test environment and interferences . 3 7 Procedure. 4 8 Calculation. 7 9 Validity criteria 8 10 Test report 8 Annex A (informative) Calibration of the pressure meter. 10 Annex B (informative) Testing of liquid samples and sludges 11 Annex C (inform
14、ative) Expression of results in tests with wastewater 13 Bibliography . 14 Copyright British Standards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:2003ISO 13641-1:2003(E) iv I
15、SO 2003 All rights reservedForeword ISO (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
16、 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
17、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 technic
18、al 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 sha
19、ll not be held responsible for identifying any or all such patent rights. ISO 13641-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. ISO 13641 consists of the following parts, under the general title Water quality Determination of inhibition of
20、gas production of anaerobic bacteria: Part 1: General test Part 2: Test for low biomass concentrations Copyright British Standards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:
21、2003INTERNATIONAL STANDARD ISO 13641-1:2003(E) ISO 2003 All rights reserved 1Water quality Determination of inhibition of gas production of anaerobic bacteria Part 1: General test WARNING Sludge samples may contain hazardous and inflammable substances. They contain pathogens and are liable to biolog
22、ical action. Consequently, it is recommended that samples be handled with special care. The gases that may be produced by microbiological activity are potentially inflammable and will pressurize sealed bottles. Exploding bottles are likely to result in infectious shrapnel and/or pathogenic aerosols.
23、 Glass bottles should be avoided wherever possible. Care is necessary when sampling, transporting and utilizing the sludge and when using microsyringes and pressure-meter syringe needles. National regulations should be followed with respect to microbiological hazards associated with this method. Tox
24、ic test materials and those with unknown properties should be handled with care. 1 Scope This part of ISO 13641 specifies a screening method for assessing the potential toxicity of substances, mixtures, surface waters, groundwaters and wastewaters, effluents, sludges or other environmental samples b
25、y determining the production of biogas (carbon dioxide and methane) from the anaerobic digestion of sewage sludge over periods of up to 3 days. The growth rate of anaerobic bacteria is much lower compared with that of aerobic microorganisms. For this reason, the test periods are longer for anaerobic
26、 methods than those used for aerobic bacteria. This method is applicable to substances, soluble or insoluble in water, including volatile chemicals (see Reference 1 in the Bibliography). NOTE Special care is necessary with compounds of low water-solubility, and in these cases, see for example, ISO 1
27、0634. For general information on biotesting see ISO 5667-162. This method can provide information that is useful in predicting the likely effect of a test material on biogas production in anaerobic digesters. For example, only longer tests, which simulate working digesters more closely can indicate
28、whether adaptation of the microorganisms to the test material can occur or whether compounds likely to be adsorbed onto sludge can build up to a toxic concentration over a longer period than allowed in this test. Information obtained by this part of ISO 13641 can also be helpful in choosing suitable
29、 initial mass concentrations for anaerobic biodegradability tests (e.g. ISO 11734). However, this part of ISO 13641 using undiluted sludge is less suitable for testing dilute digesting sludge than the method described in Part 2 of ISO 13641 where the mass concentrations are a hundredfold more dilute
30、. 2 Normative references The 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. Copyright British Standa
31、rds Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:2003ISO 13641-1:2003(E) 2 ISO 2003 All rights reservedISO 10634, Water quality Guidance for the preparation and treatment of po
32、orly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium 3 Principle Aliquots of a mixture of undiluted anaerobically digesting sludge (20 g/l to 40 g/l of total dry solids) and a degradable substrate are incubated alone and simultaneously wit
33、h a range of mass concentrations of the test material in sealed bottles for 2 days to 3 days. The amount of biogas (methane plus carbon dioxide) produced is measured by the increase in pressure in the bottles. The percentage inhibition of biogas production at the various mass concentrations of the t
34、est material is calculated from the amounts produced in the respective test and control bottles. The EC50and other effective mass concentrations are calculated from plots of percentage inhibition against the logarithm of mass concentration of the test material. 4 Reagents and media 4.1 Reagents 4.1.
35、1 Dilution water, previously de-aerated and de-ionized. Analytical controls of this water are not necessary, but make sure that the deionizing apparatus is regularly maintained. Prior to addition of the anaerobic inoculum to any solution or dilution of test material, make sure that these are oxygen-
36、free. Therefore, either bubble nitrogen gas (4.1.2) through the dilution water or through the dilutions for 1 h before adding the inoculum, or alternatively heat the dilution water to boiling then cool it to room temperature in an oxygen-free atmosphere. 4.1.2 Nitrogen gas, of high purity with a con
37、tent of less than 5 l/l oxygen. 4.2 Media 4.2.1 Inoculum (digesting sludge). Collect active digesting anaerobic sludge from the digester at a sewage treatment plant or, alternatively, from a laboratory digester, treating sludge from primarily domestic sewage into collection bottles (5.1). Add sludge
38、 to these bottles to about 1 cm from the top, seal them tightly and place in insulated containers (5.2) to minimize temperature shock, until being transferred to an incubator (5.12) maintained at 35 C 2 C. When opening the bottles, take care to release excess gas pressure either by periodically loos
39、ening the seal, or by fitting a three-way pressure-release valve (5.3) to the bottle cap. It is preferable to use the sludge within a few hours of collection, otherwise store the bottles at 35 C 2 C under a headspace of nitrogen for up to 3 days when little loss of activity normally occurs. Immediat
40、ely prior to use, mix the sludge by gentle stirring and pass it through a mesh sieve (5.5) into a suitable reservoir (5.6) through the headspace of which a stream of nitrogen is passed. Set aside a sample for determination of the mass concentration of total dry solids (e.g. see ISO 11923). In genera
41、l, use the sludge without dilution. The solids mass concentration should be between 20 g/l and 40 g/l dry material. Check the pH value of the sludge and, if necessary, adjust it to 7 0,5. 4.2.2 Substrate. Dissolve 10 g of nutrient broth, 10 g of yeast extract and 10 g of D-glucose in deionized water
42、 (4.1.1) and dilute to 100 ml. Filter-sterilize (0,2 m) (5.7) and use immediately or store at 4 C for no longer than 1 day. 5 Apparatus 5.1 Inoculum collection bottles, equipped with gas-tight seals and made of high density polyethylene or a similar material, which can expand. Glass is not recommend
43、ed since the bottle may explode. Copyright British Standards Institution Provided by IHS under license with BSI - Uncontrolled Copy Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS ISO 13641-1:2003ISO 13641-1:2003(E) ISO 2003 All rights reserved 35.2 Insulated co
44、ntainers, for transport of sludge. 5.3 Three-way pressure-release valves, capable of being fitted to the caps of inoculum collection bottles. 5.4 Centrifuge, for determination of the mass concentration of inoculum solids. 5.5 Sieve, having a mesh size of 1 mm2. 5.6 Reservoir for digesting sludge, co
45、nsisting of a glass or plastic bottle (capacity about 5 l) fitted with a stirrer and facilities for passing a stream of nitrogen gas through the headspace. 5.7 Membrane filters, with a pore size of 0,2 m for sterilizing the substrate. 5.8 Microsyringes, for the gas-tight connection of the pressure m
46、eter to the headspace in the bottles; also for adding insoluble liquid test materials into the bottles. 5.9 Pressure-resistant gas-tight closed glass test bottles, with an appropriate nominal size (0,1 l to 1 l). Use, for example, nominal 125-ml serum bottles with an actual total volume of 160 ml, g
47、as-tight sealed with septa and crimped aluminium rings. Use septa preferably made of silicone or polytetrafluoroethene-coated butyl rubber capable of withstanding a pressure of about 2 105Pa. Gas-tightness of the caps used, especially butyl rubber septa, should be tested in advance because several c
48、ommercially available septa are not sufficiently gas-tight against methane, and some septa do not stay tight when they are pierced with a needle as required under the conditions of this test. 5.10 Precision pressure meter for measuring total biogas production (methane plus carbon dioxide). A needle attachment is adapted to enable measurement and venting of the biogas produced. An example of a suitable instrument is a hand-held precision pressure meter connected to a suitable syringe needle; a 3-way gas-tight valve facilitates the release of excess pressure. Calibrate the mete
