1、BRITISH STANDARD BS6068-5.25: 1999 ISO 15522: 1999 Water quality Part 5: Biological methods Section 5.25: Determination of the inhibitory effect of water constituents on the growth of activated sludge microorganisms ICS 13.060.70BS6068-5.25:1999 This British Standard, having been prepared under the
2、directionof the Health andEnvironment Sector Committee, was published underthe authority of the Standards Committee and comesinto effect on 15 August 1999 BSI 03-2000 ISBN 0 580 32866 X Amendments issued since publication Amd. No. Date CommentsBS6068-5.25:1999 BSI 03-2000 i Contents Page National fo
3、reword ii Foreword iii Text of ISO 15522 1BS6068-5.25:1999 ii BSI 03-2000 National foreword This British Standard reproduces verbatim ISO 15522:1999 and implements it as the UK national standard. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Sub
4、committee EH/3/5, Biological methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and
5、 European developments and promulgate them in the UK. A list of organizations represented on this subcommittee can be obtained on request to its secretary. This is one of a series of standards on water quality, others of which have been, or will be, published as Sections of BS 6068. The various Sect
6、ions of BS 6068 are comprised within Parts 1 to 7, which, together with Part 0, are listed below. Part 0: Introduction; Part 1: Glossary; Part 2: Physical, chemical and biological methods; Part 3: Radiological methods; Part 4: Microbiological methods; Part 5: Biological methods; Part 6: Sampling; Pa
7、rt 7: Precision and accuracy. NOTEThe tests described in this British Standard should only be carried out by suitably qualified persons with an appropriate level of biological expertise. Standard biological procedures should be followed throughout. Cross-references The British Standards which implem
8、ent international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purpor
9、t to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cov
10、er, pagesi andii, theISO title page, pagesii toiv, pages 1 to 10 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.BS6068-5.25:1999 ii BSI 03-2000 Contents Page Forew
11、ord iii Introduction 1 1 Scope 1 2 Normative references 1 3 Terms and definitions 1 4 Principle 2 5 Test environment 2 6 Reagents 2 7 Inoculum 3 8 Apparatus 3 9 Procedure 3 10 Expression of results 5 11 Interpretation of results 5 12 Reproducibility 5 13 Test report 6 Annex A (informative) Examples
12、of contents of test and control flasks 7 Annex B (informative) Typical growth curve using 3,5-dichlorophenol as test material 8 Annex C (informative) Determination of lowest ineffective dilution of wastewater 9 Bibliography 10 Figure B.1 Typical growth curve using 3,5-dichlorophenol as test material
13、 8 Table 1 Results of a ring test 6 Table C.1 9BS6068-5.25:1999 BSI 03-2000 iii Foreword 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
14、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 collaborates
15、 closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circul
16、ated to the member bodies for voting. Publication as an International Standard requires approval by at least 75% of the member bodies casting a vote. International Standard ISO 15522 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. Annex A, Annex
17、B and Annex C of this International Standard are for information only.iv blankBS6068-5.25:1999 BSI 03-2000 1 Introduction Information generated by this method may be helpful in estimating the effect of a test material on mixed bacterial communities in aerobic biological wastewater treatment systems
18、and in choosing suitable initial concentrations for aerobic biodegradability tests The results of this test should be considered only as a guide to the likely toxicity of the test material, since activated sludge from different sources, or even from the same source taken at different times, may diff
19、er in bacterial composition and concentration. Also, laboratory tests cannot truly simulate environmental conditions. For example, no account is taken of longer term adaptation of the microorganisms to the test material or of materials which may adsorb onto biofilm or activated sludge in subsequent
20、wastewater treatment and build up to a toxic concentration over a longer period of time. WARNING Take appropriate precautions when handling sewage, as it may contain potentially pathogenic organisms. Handle with care all toxic test materials or those whose properties are unknown. 1 Scope This Intern
21、ational Standard specifies a method for assessing the potential toxicity of a test material to the growth of aerobic bacteria present in activated sludge. The inhibitory effect is restricted to those microorganisms capable of growth on the chosen organic test medium. This method gives information on
22、 inhibitory effects on the microorganisms over incubation periods up to6 h. This method is applicable to water, wastewater and chemical substances which are soluble under the conditions of the test. Special care is needed with volatile or coloured materials and materials which form turbid suspension
23、s or dispersions. NOTE 1Results with volatile test material should be interpreted with caution and are likely to underestimate any inhibitory effect because of the difficulties in maintaining the initial concentration in the test flasks. NOTE 2Coloured materials and materials of low water-solubility
24、 which form turbid suspensions or dispersions can be tested in many cases using colour/turbidity controls as blanks (see 9.4). NOTE 3Inhibitory effects on activated sludge microorganisms of test materials for which this test is not applicable can be determined by using an inhibition test based on re
25、spirometric measurements (see ISO8192). 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications
26、do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. M
27、embers of ISO and IEC maintain registers of currently valid International Standards. ISO 5667-16, Water quality Sampling Part16:General guidance for the biotesting of water, waste water, and water ingredients Sampling, pretreatment, performance and evaluation. ISO 8192, Water quality Test for inhibi
28、tion of oxygen consumption of activated sludge. ISO 11733, Water quality Evaluation of the elimination and biodegradability of organic compounds in an aqueous medium Activated sludge simulation test. 3 Terms and definitions For the purposes of this International Standard, the following terms and def
29、initions apply. 3.1 growth increase in the number of microbial cells during the test period, assessed by measurement of the biomass of a culture NOTEBiomass can be determined by any suitable method, for example as turbidity optical density (OD) or absorbance with a spectrophotometer at 530 nm and ex
30、pressed in relative turbidity units (OD 530 ). 3.2 growth curve measured biomass growth graphically plotted against the incubation time 3.3 specific growth rate doubling of biomass (x) per time unit (t) = 1/x dx/dt NOTEGrowth rate is usually expressed in reciprocal hours(h 1 ).BS6068-5.25:1999 2 BSI
31、 03-2000 3.4 growth inhibition difference in the growth at the end of the incubation time in the presence of organic test medium and test material, compared with that in a similar mixture without test material NOTEGrowth inhibition is expressed as a percentage. 3.5 inhibition curve growth inhibition
32、, in percent, plotted against the logarithm of the test material concentration 3.6 effective concentration EC value concentration of the test material giving a calculated growth inhibition, or interpolated from the inhibition curve, of 50% (EC 50 ), 20% (EC 20 ) or80% (EC 80 ) compared with that of
33、a similar mixture without test material 4 Principle Flasks containing organic test medium and test material are inoculated with an overnight culture of activated sludge microorganisms and incubated on a shaking device at 22 C 2 C. The total test duration is normally 6 h, including an exposure time o
34、f about 4,5 h. The biomass of these cultures and of blank controls without test material is determined using an appropriate method. Measurement of turbidity in a spectrophotometer at a wavelength of530 nm and expression in relative units (OD 530 ) is recommended. The growth inhibition, in percent, a
35、t the end of incubation is calculated by comparison with blank controls and is plotted, for example in a semilogarithmic curve against the test material concentration, to derive the EC values. The sensitivity of the activated sludge microorganisms can be checked against a reference substance (see6.7
36、). 5 Test environment Incubation shall take place in the dark or in diffuse light in an enclosure which is maintained at 22 C 2 C and which is free from vapours toxic to microorganisms. 6 Reagents Use only reagents of recognized analytical grade. 6.1 Distilled or deionized water 6.2 Organic test med
37、ium 6.2.1 Composition 6.2.1.1 Solution A In order to check this buffer solution, measurement of the pH, which should be about 7,4, is recommended. If this is not the case, prepare a new solution. 6.2.1.2 Solution B Dissolve 22,5 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) in 1000 ml of water
38、 (6.1). 6.2.1.3 Solution C Dissolve 36,4 g of calcium chloride dihydrate (CaCl 2 H 2 O) in 1000 ml of water (6.1). 6.2.1.4 Solution D Dissolve 0,25 g of iron(III) chloride hexahydrate (FeCl 3 6H 2 O) in 1000 ml of water (6.1). Prepare this solution freshly before use. NOTEIt is not necessary to prep
39、are this solution just before use if a drop of concentrated hydrochloric acid (HCl) is added. 6.2.1.5 Solution E The addition of the following trace elements to improve aerobic growth is recommended. Dissolve reagents in 1000 ml of water (6.1) 6.2.1.6 Solution F (organic substrate) Dissolve 80 g of
40、dry extract of nutrient broth (commercially available mixture of beef extract and peptone) and 60 g of sodium acetate in 1000 ml of water (6.1), or use the organic components of the synthetic sewage in accordance with ISO 11733. Anhydrous potassium dihydrogenphosphate (KH 2 PO 4 ) 8,5g Anhydrous dip
41、otassium hydrogenphosphate (K 2 HPO 4 ) 21,75g Disodium hydrogenphosphate dihydrate (Na 2 HPO 4 2H 2 O) 33,4 g Ammonium chloride (NH 4 Cl) 0,5g Water (6.1) quantity necessary to make up to 1000 ml Boric acid, H 3 BO 3 50mg Cobalt chloride hexahydrate, CoCl 2 6H 2 O 50 mg Manganese sulfate monohydrat
42、e, MnSO 4 H 2 O 15 mg Disodium molybdate dihydrate, Na 2 MoO 4 2H 2 O 15 mg Nickel chloride hexahydrate, NiCl 2 6H 2 O 10 mg Zinc sulfate heptahydrate, ZnSO 4 7H 2 O 50 mgBS6068-5.25:1999 BSI 03-2000 3 6.2.2 Preparation For 1000 ml of test medium, add to about 800 ml of the water (6.1): 10 ml of sol
43、ution A; 1ml of each of solutions B to E (solution E is optional, but recommended); 25 ml of solution F. Make up to 1000 ml with the water (6.1). 6.3 Sodium azide solution (optional) Dissolve 100 g of sodium azide (NaN 3 ) in 1000 ml of water (6.1); this solution is used as a preservative. 6.4 Sodiu
44、m hydroxide solution Dissolve 40 g sodium hydroxide (NaOH) in 1000 ml of water (6.1); this solution is used for pH adjustment. 6.5 Sulfuric acid solution Dissolve 98 g of sulfuric acid (H 2 SO 4 ) in 1000 ml of water (6.1); this solution is used for pH adjustment. 6.6 Solution of test material Use w
45、ater and wastewater samples directly or, if necessary, appropriately diluted. Dissolve a suitable amount of a water-soluble test material, e.g. 1000 mg in 1000 ml of water (6.1) as a stock solution. Determine the pH of the stock solution and the wastewater and adjust to pH 7,0 0,2 with solutions 6.4
46、 or 6.5 if necessary. Do not adjust the pH if the acid or alkaline effect is to be determined. For preparation see also ISO 5667-16. Samples with significant turbidity may influence the determination of turbidity. In this case use ISO8192. 6.7 Stock solution of reference substance Dissolve 1000 mg o
47、f 3,5-dichlorophenol in 1000 ml of water (6.1) and adjust to pH 7,0 0,2 with solutions 6.4 or 6.5 if necessary. 7 Inoculum Collect activated sludge from the aeration basin of a wastewater treatment plant. Normally, wastewater treatment plants treating predominantly domestic sewage are sampled. If po
48、ssible inhibitory effects shall not be determined for general purpose, but rather for a specific plant, using sludge from this plant. Let the sludge flocs settle for about 15 min, or longer if required, and use the supernatant for inoculation. Use the supernatant fresh or if necessary store up to 24
49、 h at about 4 C. 8 Apparatus Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter. Usual laboratory equipment is required and the following apparatus. 8.1 Test flasks, such as 1000 ml and 100 ml Erlenmeyer flasks, preferably with one baffle, closed e.g. with cotton plugs. 8.2 Shaking device for Erlenmeyer flasks, with a shaking speed of about 150r/min. 8.3 Room with constant temperature or incubator at22 C 2 C. 8.4 UV/visible spectrophotometer an
