1、BS EN ISO11348-2:2008ICS 13.060.70NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDWater quality Determination of theinhibitory effect ofwater samples on thelight emission of Vibriofischeri (Luminescentbacteria test)Part 2: Method using liquid-driedbacteria (ISO
2、11348-2:2007)This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 December2008 BSI 2008ISBN 978 0 580 54631 0Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 11348-2:2008National forewordThis British Standard is the UK implement
3、ation of EN ISO11348-2:2008. It is identical to ISO 11348-2:2007. It supersedes BS ENISO 11348-2:1999 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained onrequ
4、est to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 11348-2
5、November 2008ICS 13.060.70 Supersedes EN ISO 11348-2:1998 English VersionWater quality - Determination of the inhibitory effect of watersamples on the light emission of Vibrio fischeri (Luminescentbacteria test) - Part 2: Method using liquid-dried bacteria (ISO11348-2:2007)Qualit de leau - Dterminat
6、ion de leffet inhibiteurdchantillons deau sur la luminescence de Vibrio fischeri(Essai de bactries luminescentes) - Partie 2: Mthodeutilisant des bactries dshydrates (ISO 11348-2:2007)Wasserbeschaffenheit - Bestimmung der Hemmwirkungvon Wasserproben auf die Lichtemission von Vibrio fischeri(Leuchtba
7、kterientest) - Teil 2: Verfahren mit flssiggetrockneten Bakterien (ISO 11348-2:2007)This European Standard was approved by CEN on 29 October 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a
8、national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version i
9、n any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, E
10、stonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPI
11、SCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 11348-2:2008: EBS EN ISO 11348-2:2008EN ISO 11348-2:2008 (E) 3 Foreword The text of ISO 11348-2:2
12、007 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 11348-2:2008 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall
13、be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn at the latest by May 2009. Attention is drawn to the possibility that some of the elements of this document may b
14、e the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 11348-2:1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound t
15、o implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switz
16、erland and the United Kingdom. Endorsement notice The text of ISO 11348-2:2007 has been approved by CEN as a EN ISO 11348-2:2008 without any modification. BS EN ISO 11348-2:2008ISO 11348-2:2007(E) ISO 2007 All rights reserved vIntroduction The measurements specified in ISO 11348 can be carried out u
17、sing freshly prepared bacteria, as well as freeze-dried or liquid-dried bacterial preparations. Standardized work carried out by DIN Normenausschuss Wasserwesen and ISO/TC 147/SC 5/WG 1 has shown that, in special cases, these different techniques may deliver different results, especially in the pres
18、ence of heavy metals. Such varying sensitivity is caused by differences in media composition used in the preparation of freeze-dried or liquid-dried bacteria. These protective media influence the bioavailability of toxicants and/or the light emission of luminescent bacteria. This means that the orig
19、in and type of preparation need to be taken into account when interpreting the results. This may be difficult sometimes, as freeze-dried and liquid-dried bacteria may be obtained from different suppliers. This, in turn, can mean that the composition is not known in detail and therefore cannot be int
20、erpreted by the user. For this reason, in addition to toxicity measurements with freshly prepared bacteria (ISO 11348-1) and freeze-dried bacteria (ISO 11348-3), a procedure with liquid-dried bacteria is described in this part of ISO 11348, the performance of which can be interpreted by the user in
21、every detail. The laboratories responsible for the results have the opportunity to select the most suitable technique based on expert judgement and information about the water sample to be tested. BS EN ISO 11348-2:2008BS EN ISO 11348-2:2008INTERNATIONAL STANDARD ISO 11348-2:2007(E) ISO 2007 All rig
22、hts reserved 1Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 2: Method using liquid-dried bacteria WARNING Persons using this part of ISO 11348 should be familiar with normal laboratory practice. This st
23、andard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests
24、conducted in accordance with to this part of ISO 11348 be carried out by suitably trained staff. 1 Scope ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B-11177). This part of ISO 11348 specifies a method usin
25、g liquid-dried bacteria. This method is applicable to: waste water; aqueous extracts and leachates; fresh water (surface water and ground water); sea water and brackish water; eluates of sediment (fresh water, brackish water and sea water); pore water; single substances, diluted in water. 2 Normativ
26、e 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. ISO 5667-16, Water quality Sampling
27、Part 16: Guidance on biotesting of samples ISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe method BS EN ISO 11348-2:2008ISO 11348-2:2007(E) 2 ISO 2007 All rights reserved3 Principle The inhibition of light emission by cultures of Vibrio fischeri is determined by means
28、 of a batch test. This is accomplished by combining specified volumes of the test sample or the diluted sample with the luminescent bacteria suspension in a test tube. The test criterion is the luminescence, measured after a contact time of 15 min or 30 min or optionally 5 min, taking into account a
29、 correction factor (fkt), which is a measure of intensity changes of control samples during the exposure time. The inhibitory effect of the water sample can be determined as LID (see Annex B) or as EC20- and/or EC50-values by means of a dilution series. (EC is the effective concentration.) 4 Interfe
30、rences Insoluble, slightly soluble or volatile substances or substances which react with the dilution water or the suspension, or alter their state during the test period, may affect the result or impair the reproducibility of the test results. Losses of luminescence caused by light absorption or li
31、ght scattering may occur in the case of strongly coloured or turbid waters. This interference can be compensated by a sample treatment for turbidity (7.2) or, for example, by using a double-chambered absorption correction test tube (see Annex A). Since oxygen is required for the bioluminescence6, sa
32、mples with a high oxygen demand (and/or a low oxygen concentration) may cause a deficiency of oxygen and be inhibitory. Readily biodegradable nutrients in the sample may cause a pollutant-independent reduction in bioluminescence1. Samples with a pH outside the range of pH = 6,0 and pH = 8,5 affect t
33、he luminescence of the bacteria 6, 7. An adjustment of the sample is required when the toxic effect of pH is not wanted. As the test organism Vibrio fischeri is a marine bacterium, testing salt-water samples with the standard procedure often leads to stimulation effects of bioluminescence, which may
34、 mask inhibition effects (see Annex D). Salt concentrations in the initial sample exceeding 30 g/l NaCl, or contents of other compounds giving equal osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. The resulting salt concentration in the test samples
35、 should not exceed the osmolarity of a 35 g/l NaCl solution in order to avoid these effects. 5 Reagents and materials Use chemicals of recognized analytical grade quality. Use distilled water or water of equivalent purity. 5.1 Test bacteria. Use a strain of luminescence bacteria belonging to the spe
36、cies Vibrio fischeri NRRL B-11177. The bacterial suspensions used for toxicity measurements are prepared from commercially available liquid-dried reagents Store the liquid-dried bacteria at u 18 C and consider the recommendations of the supplier. The bacteria start glowing immediately after reconsti
37、tution and are ready to be used for the test. 5.2 Sodium chloride solution, as diluent. Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 l with water. 5.3 Sodium hydroxide solution, c(NaOH) = e.g. 1 mol/l. BS EN ISO 11348-2:2008ISO 11348-2:2007(E) ISO 2007 All rights reserved 35.4 H
38、ydrochloric acid, c(HCl) = e.g. 1 mol/l. For the adjustment of the pH, it may be necessary to use acids or bases of lower or higher concentration. 5.5 Solution for liquid-dried bacteria. 8,0 g D(+)-Glucose monohydrate (C6H12O6H2O) 20,0 g Sodium chloride (NaCl) 2,035 g Magnesium chloride hexahydrate
39、(MgCl26 H2O) 0,30 g Potassium chloride (KCl) 11,9 g N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) Dissolve in water, stir for about 30 min and adjust the pH to 7,0 0,2 with sodium hydroxide solution (5.3) or hydrochloric acid (5.4). Make up to 1 l with water. This solution may be st
40、ored in portions at 18 C to 20 C. 5.6 Reference substances. Prepare the following reference-substance stock solutions with sodium chloride solution (5.2) as diluent separately, without adjustment of the pH: 219,8 mg/l Zinc sulfate heptahydrate (ZnSO47 H2O) 9 mg/l 3,5-Dichlorophenol (C6H4OCl2) (purit
41、y W 99 %) 22,6 mg/l Potassium dichromate (K2Cr2O7) These concentrations are approximately twice the expected EC50-values for the respective reference substances in this part of ISO 11348. The volumes required depend on the test set-up. NOTE It is possible to use commercially available chemical prepa
42、rations with defined concentrations of ZnSO4and K2Cr2O7(titrisol) for the preparation of the stock solutions of the reference substances. 6 Apparatus 6.1 Freezer, for the storage of preserved bacteria. 6.2 Thermostatically controlled thermo-block, to maintain the test samples at a temperature of 15
43、C 1 C. Within one test, the temperature deviation should be at most 0,3 C. 6.3 Luminometer, measuring cell maintained at 15 C 1 C, equipped with suitable test tubes. 6.4 Test tubes, made of a chemically inert material, appropriate for the selected luminometer, with a capacity which facilitates the t
44、aking of a reading over the largest possible surface area and able to fit into the thermo-block (6.2). 6.5 pH-meter. 6.6 Chronometer. 6.7 Piston pipettes or plastic syringes, 100 l, 500 l and 1 000 l. 6.8 Piston pipettes, with variable volume, 10 ml to 200 ml and 200 l to 5 000 l. 6.9 Water bath, ca
45、pable of maintaining a temperature of 20 C 2 C. BS EN ISO 11348-2:2008ISO 11348-2:2007(E) 4 ISO 2007 All rights reserved6.10 Water bath or thermostatically controlled thermo-block, to maintain at least 12 ml volume (e.g. reagent vessel) of the solution prepared in 5.5 at 15 C 1 C. 6.11 Conductometer
46、. 6.12 Oxygen probe, in accordance with ISO 5814. 7 Sampling and sample pretreatment 7.1 Sampling Collect samples in chemically inert, clean containers as specified in ISO 5667-16. Fill the containers completely and seal them. Test the samples as soon as possible after collection. Where necessary, s
47、tore samples at 2 C to 5 C in the dark in the containers for not longer than 48 h. For periods up to two months, store at u 18 C. Do not use chemicals to preserve the samples. Perform the necessary pH-adjustment and salt addition immediately before testing. 7.2 Sample preparation Measure the oxygen
48、concentration in all samples. An oxygen concentration 3 mg/l is required for the test. If the oxygen concentration of the undiluted sample is less than 3 mg/l, use adequate methods to oxygenate the sample, e.g. aeration or stirring. Measure the pH of all samples. If the pH is between 6,0 and 8,5, an
49、 adjustment is usually not necessary. Adjustment of the pH-value, however, may alter the nature of the sample. On the other hand, the pH of the sample and the pH of the test batch may differ because of the buffer capacity of the test medium. It may be necessary to carry out tests on both the pH-adjusted and the non-pH-adjusted samples. If necessary, adjust the pH of the sample by adding either hydrochloric acid (5.4) or sodium hydroxide solution (5.3). Depending on the pu
