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1、BS EN ISO 11348-1:2008 ICS 13.060.70 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BRITISH STANDARD Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 1: Method using freshly prepare
2、d bacteria (ISO 11348-1:2007) Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 December 2008 BSI 2008 ISBN 978 0 580 54630 3 Am
3、endments/corrigenda issued since publication DateComments BS EN ISO 11348-1:2008 National foreword This British Standard is the UK implementation of EN ISO 11348-1:2008. It is identical to ISO 11348-1:2007. It supersedes BS EN ISO 11348-1:1999 which is withdrawn. The UK participation in its preparat
4、ion was entrusted to Technical Committee EH/3/5, Biological Methods. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct appli
5、cation. Compliance with a British Standard cannot confer immunity from legal obligations. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11348-1 November 2008 ICS 13.060.70Super
6、sedes EN ISO 11348-1:1998 English Version Water 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 (ISO 11348-1:2007) Qualit de leau - Dtermination de leffet inhibiteu
7、r dchantillons deau sur la luminescence de Vibrio fischeri (Essai de bactries luminescentes) - Partie 1: Mthode utilisant des bactries frachement prpares (ISO 11348- 1:2007) Wasserbeschaffenheit - Bestimmung der Hemmwirkung von Wasserproben auf die Lichtemission von Vibrio fischeri (Leuchtbakterient
8、est) - Teil 1: Verfahren mit frisch gezchteten 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 European Standard the status of a nation
9、al standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards 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 in an
10、y other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Es
11、tonia, 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 STANDARDIZATION COMIT EUROPEN DE NORMALISATION EU
12、ROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2008 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11348-1:2008: E Licensed Copy: London South Bank University, South Bank University, 31/01/2
13、009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008 EN ISO 11348-1:2008 (E) 3 Foreword The text of 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
14、by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard 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 wi
15、thdrawn at the latest by May 2009. Attention is drawn to the possibility that some of the elements of this 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. Accor
16、ding to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvi
17、a, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, 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. Licensed Copy: London So
18、uth Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO 11348-1:2008 ISO 11348-1:2007(E) ISO 2007 All rights reserved v Introduction The measurements specified in ISO 11348 can be carried out using freshly prepared bacteria, as well as
19、 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 presence of heavy metals. Such varying sensiti
20、vity 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 origin and type of preparation need to be take
21、n 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 interpreted by the user. For this reason, in
22、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 by the user in every detail. The laboratories responsible
23、 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. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO
24、11348-1:2008 Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO 11348-1:2008 INTERNATIONAL STANDARD ISO 11348-1:2007(E) ISO 2007 All rights reserved 1 Water quality Determination of the inhibitory effect of
25、 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 practice. This standard does not purport to address all of the safety problems, if
26、 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 conducted in accordance with this part of ISO 11348 be carried ou
27、t 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 using freshly prepared bacteria. This method is applicable to: waste wat
28、er; 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 Normative references The following referenced documents are indispensable for the applicat
29、ion 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 Part 16: Guidance on biotesting of samples ISO 5814, Water quality Determination o
30、f dissolved oxygen Electrochemical probe method ISO 7027, Water quality Determination of turbidity Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO 11348-1:2008 ISO 11348-1:2007(E) 2 ISO 2007 All rights r
31、eserved 3 Principle The inhibition of light emission by 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
32、luminescence, measured after a contact time of 15 min 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) o
33、r as EC20- and/or EC50-values by means of a dilution series. (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
34、 or impair the reproducibility of the test results. Losses 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-cha
35、mbered absorption correction test tube (see Annex A). Since oxygen is required for the bioluminescence 6, 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-inde
36、pendent reduction in bioluminescence 1. Samples with a pH outside the range of pH = 6,0 and pH = 8,5 affect the 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 sa
37、lt-water samples with the standard procedure often leads 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 sal
38、t spiking required by the test, to hyperosmotic effects. 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
39、 or water of equivalent purity. 5.1 Test bacteria. Use a 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 Mi
40、krorganismen und Zellkulturen GmbH, Mascheroder Weg 10, D-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
41、solution, as diluent. Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 l with water. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO 11348-1:2008 ISO 11348-1:2007(E) ISO 2007 All rights
42、reserved 3 5.3 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 or higher concentration. 5.5 Solution for freshly prepared bacteria. 8,0 g D(+)-Glucose monohydrate (C6H12O
43、6H2O) 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) Dissolve in water, stir for about 30 min and adjust the pH to 7,0 0,2 with sodium hydroxide solution (5.3) or
44、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 with sodium chloride solution (5.2) as diluent separately, without adjustment of the pH: 219,8 mg/l Zinc su
45、lfate 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-values for the respective reference substances in this part of ISO 11348. The volumes required depend on the te
46、st set-up. NOTE It is possible to use commercially available chemical preparations with defined concentrations of ZnSO4 and K2Cr2O7 (titrisol) for the preparation of the stock solutions of the reference substances. 5.7 Liquid broth for pre- and main cultures. 30 g Sodium chloride (NaCl) 6,10 g Sodiu
47、m dihydrogenphosphate monohydrate (NaH2PO4H2O) 2,75 g Dipotassium hydrogenphosphate trihydrate (K2HPO43 H2O) 0,204 g Magnesium sulfate heptahydrate (MgSO47 H2O) 0,500 g Diammonium hydrogenphosphate (NH4)2HPO4 3 ml Glycerol 5,00 g Caso-peptone 0,50 g Yeast extract Dissolve in water and adjust the pH
48、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 for 20 min. Caso-peptone and yeast extract offered by different suppliers can vary in quality. In case
49、of problems (e.g. growth inhibition), it is recommended to purchase the product from another manufacturer. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 02:23, Uncontrolled Copy, (c) BSI BS EN ISO 11348-1:2008BS EN ISO 11348-1:2008 ISO 11348-1:2007(E) 4 ISO 2007 All rights reserved 5.8 Agar medium for 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 steril