1、INTERNATIONAL STANDARD IS0 10708 First edition 1997-02-01 Water quality - Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds - Determination of biochemical oxygen demand in a two-phase closed bottle test Qualit de Ieau - Evaluation en milieu aqueux de la bi
2、odkgradabilitk akrobie Mime des compos a=400net; p=iso; o=isocs; s=central Printed in Switzerland ii 0 IS0 IS0 10708:1997(E) Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing Internation
3、al Standards is normally carried out through IS0 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,
4、 also take part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an Inter
5、national Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 10708 was prepared by Technical Committee lSO/TC 147, Water quality, Subcommittee SC 5, Biological methods. Annexes A to E of this International Standard are for information only. INT
6、ERNATIONAL STANDARD 0 IS0 IS0 10708: 1997(E) Water quality - Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds - Determination of biochemical oxygen demand in a two-phase closed bottle test WARNING - SAFETY PRECAUTIONS - Activated sludge and sewage may con
7、tain potentially pathogenic organisms. Therefore appropriate precautions should be taken when handling them. Toxic test compounds and those whose properties are unknown should be handled with care. 1 Scope This International Standard specifies a method for the evaluation of the ultimate biodegradabi
8、lity by aerobic microorganisms of organic compounds, in particular poorly water-soluble compounds, at a given concentration. NOTE 1 The conditions described in this International Standard do not necessarily always correspond to the optimal conditions allowing the maximum degree of biodegradation to
9、occur. The method applies to organic compounds which - are water soluble or poorly water soluble at the concentration used under the test conditions; - do not adsorb onto or have any effect on the oxygen electrode (see 8.1.2 and 8.3.4); - are not inhibitory to the test microorganisms at the concentr
10、ation chosen for the test. NOTE 2 For special measures to introduce poorly water-soluble test compounds into the test vessels, see IS0 10634. NOTE 3 inhibitory effects can be determined as described in 8.1.4 and 8.3.1, or by using any other method for determining the inhibitory effect on bacteria of
11、 a substance (e.g. IS0 8192). 2 Normative reference The following standard contains provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the edition indicated was valid. All standards are subject to revision, and parties
12、to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the standard indicated below. Members of IEC and IS0 maintain registers of currently valid International Standards. IS0 10634:1995, Water quality- Guidance for the
13、preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium. 3 Definitions For the purposes of this International Standard, the following definitions apply. IS0 10708: 1997(E) 0 IS0 3.1 ultimate biodegradation break
14、down of an organic chemical compound by microorganisms in the presence of oxygen to yield carbon dioxide, water and mineral salts of any other elements present (mineralization) and new biomass 3.2 primary biodegradation structural change (transformation) of an organic chemical compound by microorgan
15、isms resulting in the loss of a specific property 3.3 biochemical oxygen demand (BOD) mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological oxidation of a chemical compound or organic matter in water, expressed in this case as milligrams of oxygen uptak
16、e per milligram or gram of test compound 3.4 theoretical oxygen demand (ThOD) theoretical maximum amount of oxygen required to oxidize a chemical compound completely, calculated from the molecular formula, expressed in this case as milligrams of oxygen required per milligram or gram of test compound
17、 3.5 chemical oxygen demand (COD) mass concentration of oxygen equivalent to the amount of a specified oxidant consumed by a chemical compound or organic matter when a water sample is treated with that oxidant under defined conditions, expressed in this case as milligrams of oxygen consumed per mill
18、igram or gram of test compound 3.6 dissolved organic carbon (DOC) that part of the organic carbon in water which cannot be removed by specified phase separation, for example by centrifugation at 40 000 mes-* for 15 min or by membrane filtration using membranes with pores of 0,2 pm to 0,45 brn diamet
19、er 3.7 concentration of suspended solids of an activated sludge amount of solids obtained by filtration or centrifugation of a known volume of activated sludge after filtration or centrifugation and drying at about 105 “C to constant mass 3.8 lag phase time from the start of a test until adaptation
20、and selection of the degrading microorganisms are achieved and the biodegradation degree of a chemical compound or organic matter has increased to about 10 % of the theoretical maximum biodegradation; it is expressed in days 3.9 maximum level of biodegradation maximum biodegradation degree of a chem
21、ical compound or organic matter in a test, above which no further biodegradation takes place during the test; it is expressed as a percentage 3.10 degradation phase time from the end of the lag phase of a test until about 90 % of the maximum level of biodegradation has been reached; it is expressed
22、in days 3.11 plateau phase time from the end of the biodegradation phase when the maximum level of biodegradation is reached until the end of the test 3.12 pre-exposure pre-incubation of an inoculum in the presence of the test compound, with the aim of enhancing the ability of an inoculum to biodegr
23、ade the test compound by adaptation and selection of the microorganisms 3.13 pre-conditioning pre-incubation of an inoculum under the conditions of the test in the absence of the test compound, with the aim of improving the performance of the test by acclimatization of the microorganisms to the test
24、 conditions 2 0 IS0 Is0 10708:1997(E) 4 Principle Biodegradation of organic compounds by aerobic microorganisms is determined in an aquatic medium. The organic compound is the sole source of carbon and energy in the medium. The inoculated medium is shaken or stirred in closed bottles, containing kno
25、wn volumes of medium and air, to assure steady-state oxygen partitioning between liquid and gas phases. The degradation is followed by regular measurements of the dissolved oxygen concentration in the aqueous phase for up to 28 days. The total oxygen uptake in the test flasks is calculated from the
26、difference in the measured dissolved oxygen concentrations in the blank and test flasks divided by the oxygen saturation value under normal conditions and multiplied by the total oxygen content originally present in the liquid and gaseous phases. Biodegradability is calculated as the total oxygen up
27、take divided by the theoretical oxygen demand (ThOD) or chemical oxygen demand (COD), expressed as a percentage. 5 Test environment Incubation shall take place in the dark or in diffused light in an enclosure which is maintained at a constant temperature (2 0,5 “C) in a range between 20 “C and 25 “C
28、. 6 Reagents Use only reagents of recognized analytical grade. 6.1 Water Distilled or deionized water containing less than 2 mg/l of DOC and/or less than 10 % of the initial organic Garbon content introduced by the test compound. 6.2 Test medium 6.2.1 Composition 6.2.1 .l Solution A Anhydrous potass
29、ium dihydrogenphosphate (KH,PO,) 85 g Anhydrous dipotassium hydrogenphosphate (K,HPO,) 21,75 g Disodium hydrogenphosphate dihydrate (Na,HPO,-2H,O) 3394 g Ammonium chloride (NH&I) 095 g Water (6.1) in a quantity necessary to make up to 1 litre NOTE - The correct composition of the medium can be check
30、ed by measuring the pH value, which should be 7,4. 6.2.1.2 Solution B Dissolve 22,5 g of magnesium sulfate heptahydrate (MgS04.7H20) in 1 000 ml of water (6.1). 6.2.1.3 Solution C Dissolve 27,5 g of anhydrous calcium chloride (CaCl2) in 1 000 ml of water (6.1). 3 IS0 10708: 1997(E) 0 IS0 6.2.1.4 Sol
31、ution D Dissolve 0,25 g of iron(W) chloride hexahydrate (FeCI,-GH,O) in 1 000 ml of water (6.1). Prepare this solution freshly before use. NOTE - It is not necessary to prepare this solution just before use if a drop of concentrated hydrochloric acid (HCI) or 0,4 g/l ethylenediaminetetraacetic acid
32、(EDTA) is added. 6.2.2 Preparation For 1 litre of test medium, add to about 500 ml of water (6.1) - 10 ml of solution A: - 1 ml of each of the solutions B to D. Make up to 1 000 ml with water (6.1). 6.3 Sodium hydroxide solution Dissolve sodium hydroxide (NaOH) in water (6.1) to obtain a solution of
33、 0,i mol/l to 0,5 mol/l. 6.4 Hydrochloric acid solution Dissolve hydrochloric acid (HCI) in water (6.1) to obtain a solution of 0,l mol/l to 0,5 mol/l. 7 Apparatus Ensure that all glassware is thoroughly cleaned and, in particular, is free from organic or toxic matter. Use usual laboratory equipment
34、 and the following items. 7.1 Incubation bottles, gas-tight, e.g. narrow-neck flasks with volumes of 200 ml to 300 ml with suitable stoppers (e.g. ground-glass stoppers, screw-caps or butyl rubber stoppers), shielded from light (e.g. made of brown glass). It is recommended to use stopper clamps. Mar
35、k each bottle with waterproof markings. If the oxygen electrode used has no mounted stirrer, provide each bottle with a magnetic stirrer-bar coated with polytetrafluorethylene. Either pre- select bottles of standard volume such that the standard deviation about the mean volume of the batch of bottle
36、s is less than 1 ml, or measure and record the volumes of individual, numbered bottles to an accuracy of 1 ml. Carefully grease the stoppers of the bottles with inert silicone grease to assure proper closing and easy removal. 7.2 Oxygen electrode to measure over a range of 0 mg/l to 10 mg/l with a p
37、recision of 1 %. Steady-state shall be reached within about 2 min. Mount the electrode for example in an inert stopper which makes a leak-proof fit in the ground glass neck of the incubation bottle. Preferably use electrodes with mounted stirrers. 7.3 Magnetic stirrer with speed regulation, if requi
38、red. Stirrer-rods attached to the oxygen electrode shall be of such a material that no ingredients of any plastic coatings will contaminate the test medium and no adsorption of test compounds will occur. Heating the test vessels by stirring and raising the test temperature shall be avoided. 7.4 Shak
39、ing device, if required. 7.5 Water bath, or other device to guarantee an accurate temperature control of rt 0,5 “C in a range of 20 “C to 25 “C. 7.6 pH-meter. Q IS0 IS0 10708:1997(E) 7.7 Dissolved organic carbon (DOC) analyser (only in special cases, see 8.3.4, note 3). 8 Procedure 8.1 Preparation o
40、f test and reference compounds 8.1.1 Water-soluble test compounds Prepare a stock solution of water-soluble test compounds in water (6.1) or test medium (6.2). Dilute a suitable amount of this solution in the test medium (6.2) to obtain a final test concentration which corresponds to 100 mg/l ThOD (
41、see 8.3.3). For calculation of ThOD, see annex A. If ThOD cannot be calculated from the formula, use elemental analyses or determine COD (see annex B). Be aware that COD determination of poorly water-soluble substances may be difficult. If DOC removal is to be determined, measure the equivalent DOC
42、test concentration (in milligrams per litre) or calculate it from the measured stock solution. 8.1.2 Water-insoluble test compounds Grind dry solids in a mortar, weigh them on a piece of glass and place directly into the test bottles. Smear oily and waxy substances onto a piece of glass and, after r
43、eweighing, place directly into the test bottles. If dispersions or emulsions are used, disperse the test compound in water (6.1) by ultrasonic treatment or emulsify with a non-biodegradable emulsifier, or use both techniques in combination. A suitable emulsifier is nonylphenol ethoxylate (about 10 E
44、O) and propoxylate (about 3 to 7 PO). Ensure that the dispersion or emulsion is homogeneous when an aliquot is used to obtain the desired test concentration. Ensure that the final amount of test compound in the test vessel is at about 100 mg/l of ThOD (see annex A). It is not important to adjust exa
45、ctly to 100 mg/l, but record the exact amount for each numbered bottle. For further information on handling of poorly water-soluble test compounds, see IS0 10634. NOTE - If the test compound (e.g. an oily or fatty substance) adsorbs on the oxygen electrode, the oxygen measurement may be reduced and
46、the test results could be influenced. Adsorption can also occur on the bottle walls or the stoppers. If this occurs, use another test method e.g. the respirometer test (IS0 9408) or the C02-evolution test (IS0 9439). 8.1.3 Solution of the reference compound Prepare a stock solution of the reference
47、compound (an organic compound of known biodegradability such as sodium acetate, sodium benzoate or aniline) in the same way as in 8.1 .I in order to obtain a final test concentration which corresponds to a 100 mg/l ThOD. 8.1.4 Solution to check inhibition If information on a possible inhibition of t
48、he inoculum by the test compound is desired, prepare in the test medium (6.2) a solution containing the test compound and the reference compound in the respective concentrations indicated in 8.1.1, 8.1.2 and 8.1.3. 8.2 Preparation of the inoculum Prepare the inoculum using the sources as described i
49、n 8.2.1 to 8.2.3 or use a mixture of these sources to obtain a microbial population that offers sufficient biodegradative activity. Stabilize the inoculum as described in 8.3.2 before use in the test. 8.2.1 lnoculum from a secondary effluent Take a sample of a secondary effluent collected from a treatment plant or a laboratory plant dealing with predominantly domestic sewage. If the density of microorganisms in the inoculum is too low so that the requirements for suitable votume see b) cannot be fulfilled, concentrate the sample by filtration or centrifugation. Mix well, keep the sample u
