1、BS EN ISO11348-3:2008ICS 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 3: Method using freeze-driedbacteria (ISO
2、11348-3:2007)This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 December2008 BSI 2008ISBN 978 0 580 54632 7Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 11348-3:2008National forewordThis British Standard is the UK implement
3、ation of EN ISO11348-3:2008. It is identical to ISO 11348-3:2007. It supersedes BS ENISO 11348-3:1999 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee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 11348-3
5、November 2008ICS 13.060.70 Supersedes EN ISO 11348-3:1998 English VersionWater quality - Determination of the inhibitory effect of watersamples on the light emission of Vibrio fischeri (Luminescentbacteria test) - Part 3: Method using freeze-dried bacteria (ISO11348-3:2007)Qualit de leau - Dterminat
6、ion de leffet inhibiteurdchantillons deau sur la luminescence de Vibrio fischeri(Essai de bactries luminescentes) - Partie 3: Mthodeutilisant des bactries lyophilises (ISO 11348-3:2007)Wasserbeschaffenheit - Bestimmung der Hemmwirkungvon Wasserproben auf die Lichtemission von Vibrio fischeri(Leuchtb
7、akterientest) - Teil 3: Verfahren mitgefriergetrockneten Bakterien (ISO 11348-3:2007)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
9、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 Republic, Denmark,
10、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 NORMALISATIONEUROP
11、ISCHES 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 11348-3:2008: EBS EN ISO 11348-3:2008EN ISO 11348-3:2008 (E) 3 Foreword The text of ISO 11348-3:
12、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 11348-3:2008 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
14、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 11348-3:1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound
15、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, Sweden, Swit
16、zerland and the United Kingdom. Endorsement notice The text of ISO 11348-3:2007 has been approved by CEN as a EN ISO 11348-3:2008 without any modification. BS EN ISO 11348-3:2008ISO 11348-3:2007(E) ISO 2007 All rights reserved vIntroduction The measurements specified in ISO 11348 can be carried out
17、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 give different results, especially in the presen
18、ce 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 origin
19、 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 inter
20、preted by the user. For this reason, in addition to toxicity measurements with liquid-dried bacteria (ISO 11348-2) and freshly prepared bacteria (ISO 11348-1), a procedure with freeze-dried bacteria is described in this part of ISO 11348, the performance of which can be interpreted by the user in ev
21、ery 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 11348-3:2008BS EN ISO 11348-3:2008INTERNATIONAL STANDARD ISO 11348-3:2007(E) ISO 2007 All right
22、s reserved 1Water quality Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) Part 3: Method using freeze-dried bacteria WARNING Persons using this part of ISO 11348 should be familiar with normal laboratory practice. This stan
23、dard 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 co
24、nducted 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 specifies a method using fre
25、eze-dried 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 (freshwater, brackish and sea water); pore water; single substances, diluted in water. 2 Normative references The followi
26、ng 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 Part 16: Guidance on bio
27、testing of samples ISO 5814, Water quality Determination of dissolved oxygen Electrochemical probe method BS EN ISO 11348-3:2008ISO 11348-3:2007(E) 2 ISO 2007 All rights reserved3 Principle The inhibition of light emission by cultures of Vibrio fischeri is determined by means of a batch test. This i
28、s 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 and optionally 5 min, taking into account a correction factor (fkt
29、), 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 Interferences Insoluble, sligh
30、tly 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 light scattering may occu
31、r 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, samples with a high oxyge
32、n 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 bioluminescence 1. Samples with a pH outside the range of pH = 6,0 and pH = 8,5 affect the luminescence of the
33、 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 mask inhibition effec
34、ts (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 should not exceed the
35、 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 species Vibrio fischeri N
36、RRL B-11177. The bacterial suspensions used for toxicity measurements are prepared from commercially available freeze-dried reagents which can be stored in a freezer at 18 C to 20 C. The bacteria start glowing immediately after reconstitution and are ready to be used for the test. 5.2 Sodium chlorid
37、e 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, e.g. c(NaOH) = 1 mol/l. BS EN ISO 11348-3:2008ISO 11348-3:2007(E) ISO 2007 All rights reserved 35.4 Hydrochloric acid, e.g. c(HCl) = 1 mol/l. For the adjustment of t
38、he pH, it may be necessary to use acids or bases of lower or higher concentration. 5.5 Solution for freeze-dried bacteria. 20 g Sodium chloride (NaCl) 2,035 g Magnesium chloride hexahydrate (MgCl26 H2O) 0,30 g Potassium chloride (KCl) Dissolve in water and make up to 1 l with water. The solution may
39、 be stored in portions in a freezer 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: 19,34 mg/l Zinc sulfate heptahydrate (ZnSO47 H2O) 6,8 mg/l 3,5-Dichlorophe
40、nol (C6H4OCl2) (purity 99 %) 105,8 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 avai
41、lable chemical preparations with defined concentrations of ZnSO4 and 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 Incubator or refrigerator, to maintain the stock suspension (8.2) and, op
42、tionally, the “solution for freeze-dried bacteria” (5.5) (variant B) at a temperature of 4 C 3 C. 6.3 Thermostatically controlled thermo-block, to maintain the test samples at a temperature of 15 C 1 C. Within one test, the temperature deviation should be at most 0,3 C. 6.4 Luminometer, measuring ce
43、ll maintained at 15 C 1 C, equipped with suitable test tubes. 6.5 Test tubes, made of a chemically inert material, appropriate for the selected luminometer and having a capacity which facilitates the taking of a reading over the largest possible surface area and able to fit into the thermo-block (6.
44、3). 6.6 pH-meter. 6.7 Chronometer. 6.8 Piston pipettes or plastic syringes, 10 l, 500 l and 1 000 l. 6.9 Piston pipettes, with variable volume, 10 ml to 200 ml and 200 l to 5 000 l. 6.10 Conductometer. 6.11 Oxygen probe, in accordance with ISO 5814. BS EN ISO 11348-3:2008ISO 11348-3:2007(E) 4 ISO 20
45、07 All rights reserved7 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, store samples at 2 C to 5 C i
46、n 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 concentration in all samples
47、. 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 adjustment is usually not n
48、ecessary. 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
49、 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 purpose of the test, the pH may be adjusted to 7,0 0,2 or to the upper (8,5 0,2) and lower limits (6,0 0,2). Choose the concentration of the hydrochloric acid or the sodium hydroxide solution to restrict the volume added to not more than 5 % of total volume. Add 20 g of sodium chloride per litre to the water sample or to the neut
