1、BS EN ISO11348-1:2008ICS 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 1: Method using freshly preparedbacteria (
2、ISO 11348-1:2007)This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 December2008 BSI 2008ISBN 978 0 580 54630 3Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 11348-1:2008National forewordThis British Standard is the UK imple
3、mentation of EN ISO11348-1:2008. It is identical to ISO 11348-1:2007. It supersedes BS ENISO 11348-1:1999 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee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 on
4、request 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 113
5、48-1November 2008ICS 13.060.70 Supersedes EN ISO 11348-1:1998 English VersionWater quality - Determination of the inhibitory effect of watersamples on the light emission of Vibrio fischeri (Luminescentbacteria test) - Part 1: Method using freshly prepared bacteria(ISO 11348-1:2007)Qualit de leau - D
6、termination de leffet inhibiteurdchantillons deau sur la luminescence de Vibrio fischeri(Essai de bactries luminescentes) - Partie 1: Mthodeutilisant des bactries frachement prpares (ISO 11348-1:2007)Wasserbeschaffenheit - Bestimmung der Hemmwirkungvon Wasserproben auf die Lichtemission von Vibrio f
7、ischeri(Leuchtbakterientest) - Teil 1: Verfahren mit frischgezchteten Bakterien (ISO 11348-1:2007)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 th
8、e status of a 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
9、). A version in 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 Republ
10、ic, Denmark, Estonia, 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 NORMA
11、LISATIONEUROPISCHES 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 11348-1:2008: EBS EN ISO 11348-1:2008EN ISO 11348-1:2008 (E) 3 Foreword The text of
12、 ISO 11348-1:2007 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 11348-1:2008 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European S
13、tandard shall 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
14、document may be 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 11348-1:1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countri
15、es are bound to 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,
16、 Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 11348-1:2007 has been approved by CEN as a EN ISO 11348-1:2008 without any modification. BS EN ISO 11348-1:2008ISO 11348-1:2007(E) ISO 2007 All rights reserved vIntroduction The measurements specified in ISO 11348 can be
17、 carried out using 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, especial
18、ly in the presence 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
19、 that the origin 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
20、 cannot be interpreted by the user. For this reason, in addition to toxicity measurements with liquid-dried bacteria (ISO 11348-2) and freeze-dried bacteria (ISO 11348-3), a procedure with freshly prepared bacteria is described in this part of ISO 11348, the performance of which can be interpreted b
21、y the user in 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 11348-1:2008BS EN ISO 11348-1:2008INTERNATIONAL STANDARD ISO 11348-1:2007(E) IS
22、O 2007 All rights reserved 1Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 1: Method using freshly prepared bacteria WARNING Persons using this part of ISO 11348 should be familiar with normal laboratory
23、 practice. This standard 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 ess
24、ential that tests conducted in accordance with 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 specifie
25、s a method using freshly prepared bacteria. This method is applicable to: waste water; aqueous extracts and leachates; fresh water (surface and ground water); sea and brackish water; eluates of sediment (fresh water, brackish and sea water); pore water; single substances, diluted in water. 2 Normati
26、ve 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 ISO 7027, Water quality Determination of turbidity BS EN ISO 11348-1:2008ISO 11348-1:2007(E) 2 ISO 2007 All rights reserved3 Principle The inhibition of light emission b
28、y cultures of Vibrio fischeri is determined by means 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
29、or 30 min and optionally 5 min, taking into account a 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 s
30、eries. (EC is the effective concentration). 4 Interferences 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. Lo
31、sses of luminescence caused by light absorption or light 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).
32、Since oxygen is required for the bioluminescence6, samples 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 p
33、H outside the range of pH = 6,0 and pH = 8,5 affect the luminescence of the bacteria6, 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
34、to stimulation effects of bioluminescence, which may 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.
35、The resulting salt concentration in the test samples 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
36、strain of luminescence bacteria belonging to the species Vibrio fischeri NRRL B-11177. The bacteria strain can be taken from commercially available freeze-dried or liquid-dried reagents or from culture collections, e.g. Deutsche Sammlung fr Mikrorganismen und Zellkulturen GmbH, Mascheroder Weg 10, D
37、-38124 Braunschweig, Germany, or NRRL, ARS Culture collection NCAUR, 1815 N, University Street, Peoria, Illinois 61604, USA. The bacterial suspensions used for toxicity measurements shall be freshly prepared from cultures. 5.2 Sodium chloride solution, as diluent. Dissolve 20 g of sodium chloride (N
38、aCl) in water and make up to 1 l with water. BS EN ISO 11348-1:2008ISO 11348-1:2007(E) ISO 2007 All rights reserved 35.3 Sodium hydroxide solution, e.g. c(NaOH) = 1 mol/l. 5.4 Hydrochloric acid, e.g. c(HCl) = 1 mol/l. For the adjustment of the pH, it may be necessary to use acids or bases of lower o
39、r higher concentration. 5.5 Solution for freshly prepared bacteria. 8,0 g D(+)-Glucose monohydrate (C6H12O6H2O) 20,0 g Sodium chloride (NaCl) 2,035 g Magnesium chloride hexahydrate (MgCl26 H2O) 0,30 g Potassium chloride (KCl) 11,9 g N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) Diss
40、olve 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 stored in portions at 18 C to 20 C. 5.6 Reference substances. Prepare the following reference-substance stock solutions w
41、ith 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) (purity W 99 %) 22,6 mg/l Potassium dichromate (K2Cr2O7) These concentrations are approximately twice the expected EC50-value
42、s 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 preparations with defined concentrations of ZnSO4and K2Cr2O7(titrisol) for the preparation of the stock solutions of the ref
43、erence substances. 5.7 Liquid broth for pre- and main cultures. 30 g Sodium chloride (NaCl) 6,10 g Sodium dihydrogenphosphate monohydrate (NaH2PO4H2O) 2,75 g Dipotassium hydrogenphosphate trihydrate (K2HPO43 H2O) 0,204 g Magnesium sulfate heptahydrate (MgSO47 H2O) 0,500 g Diammonium hydrogenphosphat
44、e (NH4)2HPO4 3 ml Glycerol 5,00 g Caso-peptone 0,50 g Yeast extract Dissolve in water 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. Transfer 50 ml each to Erlenmeyer flasks (e.g. 250 ml) and sterilize in an autoclave at 121 C
45、 for 20 min. Caso-peptone and yeast extract offered by different suppliers can vary in quality. In case of problems (e.g. growth inhibition), it is recommended to purchase the product from another manufacturer. BS EN ISO 11348-1:2008ISO 11348-1:2007(E) 4 ISO 2007 All rights reserved5.8 Agar medium f
46、or stock cultures. Adjust the liquid broth (5.7) to pH 7,0 0,2. Add 12 g of agar per litre and dissolve by gentle warming; sterilize and transfer to sterile Petri dishes. 5.9 Protective medium. 66 g D(+)-Glucose monohydrate (C6H12O6H2O) 4 g Sodium chloride (NaCl) 2 g L-Histidine 0,5 g Bovine serum a
47、lbumin, BSA Dissolve completely in water at about 37 C and adjust the pH to 7,0 0,2 at room temperature with sodium hydroxide solution (5.3) or hydrochloric acid (5.4) as necessary. Make up to 100 ml with water. Damage of bacterial cells during the freezing procedure is prevented by the use of the p
48、rotective medium. BSA offered by different suppliers can vary in quality. If problems occur, it is recommended to purchase the product from another manufacturer. Prepare protective medium freshly before use. 6 Apparatus 6.1 Thermostatically controlled thermo-block, to maintain the test samples at a
49、temperature of 15 C 1 C. Within one test, the temperature deviation should be at most 0,3 C. 6.2 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. 6.3 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
