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1、- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - w - - - - - - - - - - EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13623 September 2010 ICS 71.100.35 English Version Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bacteric
2、idal activity against Legionella of chemical disinfectants for aqueous systems - Test method and requirements (phase 2, step 1) Antiseptiques et dsinfectants chimiques - Essai quantitatif de suspension pour lvaluation de lactivit bactricide contre des lgionelles des dsinfectants chimiques pour les s
3、ystmes aqueux - Mthode dessai et prescriptions (phase 2, tape 1) Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung gegen Legionella von chemischen Desinfektionsmitteln fr wasserfhrende Systeme - Prfverfahren und Anforderungen (Ph
4、ase 2, Stufe 1) This European Standard was approved by CEN on 15 April 2010. 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 national standard without any alteration. Up-to-date lists and bibli
5、ographical 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 any other language made by translation under the responsibility
6、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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ice
7、land, 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 NORM ALISATION EUROPISCHES KOM ITEE FR NORMUNG Management Centre: Ave
8、nue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13623:2010: E EN 13623:2010 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Requirem
9、ents .5 5 Test methods 6 5.1 Principle 6 5.2 Materials and reagents 6 5.2.1 Test organism 6 5.2.2 Culture media and reagents .6 5.3 Apparatus and glassware 10 5.3.1 General . 10 5.3.2 Usual microbiological laboratory equipment 10 5.4 Preparation of test organism suspensions and test solutions 11 5.4
10、.1 Test organism suspension (test and validation suspension) 11 5.4.2 Product test solution 13 5.5 Procedure for assessing the bactericidal activity of the product . 13 5.5.1 General . 13 5.5.2 Dilution-neutralization method 15 5.5.3 Membrane filtration method 17 5.6 Experimental data and calculatio
11、ns 18 5.6.1 Explanation of terms and abbreviations 18 5.6.2 Calculation . 19 5.7 Verification of methodology 22 5.7.1 General . 22 5.7.2 Control of weighted mean counts . 23 5.7.3 Basic limits 23 5.8 Expression of results and precision . 23 5.8.1 Reduction 23 5.8.2 Control of active and non-active p
12、roduct test solution (5.4.2) 23 5.8.3 Bactericidal concentration . 23 5.8.4 Precision, repetition . 24 5.9 Interpretation of results conclusion 24 5.9.1 General . 24 5.9.2 Bactericidal activity for general purposes . 24 5.9.3 Bactericidal activity for specific purposes 24 5.10 Test report . 24 Annex
13、 A (informative) Determination of the bactericidal activity against Legionella pneumophila . 26 Annex B (informative) Neutralizer . 28 Annex C (informative) Graphical representation of test procedures 30 C.1 Dilution-neutralization method 30 C.2 Membrane filtration method 32 Bibliography . 34 EN 136
14、23:2010 (E) 3 Foreword This document (EN 13623:2010) has been prepared by Technical Committee CEN/TC 216 “Chemical disinfectants and antiseptics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical
15、 text or by endorsement, at the latest by March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. 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 responsi
16、ble for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland,
17、 France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. EN 13623:2010 (E) 4 Introduction This European Standard specifies a suspension test
18、 for establishing whether a chemical disinfectant has a bactericidal activity against Legionella pneumophila in the fields described in the scope. This standard is specifically prepared for water treatment products, but it may also be possible to use it for other products. Proliferation of Legionell
19、a only occurs in waters under certain conditions, and predominantly poses a risk when aerosolised. Many systems containing water do not require treatment. A decision to add chemical disinfectants to any water should be based on a risk assessment. If the product complies with the requirements of this
20、 standard, it can be considered bactericidal against Legionella pneumophila, but it should not necessarily be inferred that the product is acceptable for a specific site of application without consideration of other relevant factors such as the pH, water, chemistry, temperature and degree of biologi
21、cal fouling at that site of application. It does not take into account the protective effect conveyed by biofilm on the organisms. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each concentration of the chemical disinfectant foun
22、d by this test corresponds to defined experimental conditions. However, for some applications the recommendations of use of a product may differ and therefore additional test conditions need to be used. EN 13623:2010 (E) 5 1 Scope This European Standard specifies a test method and the minimum requir
23、ements for bactericidal activity of chemical disinfectant products intended to be used for treatment in aqueous systems against Legionella pneumophila that form a homogeneous, physically stable preparation when diluted with buffered ferrous hard water or hard water. Whenever Legionella pneumophila p
24、oses a risk to human health, this method is suitable for water used in cooling towers and water for general purposes, like spas, pools, showers and other uses. The method is not suitable for electro-chemical disinfection. The European Standard applies to products used to treat water in order to kill
25、 Legionella pneumophila. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test . NOTE 3 This method does not take into account the fact tha
26、t Legionella pneumophila is often found in cells of amoebae and/or biofilms and that thereby a products activity against the bacteria may be reduced. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendation“. 2 Normative references The following re
27、ferenced 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. EN 14885, Chemical disinfectants and antiseptics Application of E
28、uropean Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 and the following apply. 3.1 cooling water water used to remove heat from a process or environment 3.2 water for general purposes water
29、used in premises other than water used as cooling water 4 Requirements The product shall demonstrate al least a four decimal log (lg) reduction, when diluted with buffered ferrous hard water (5.2.2.10) or hard water (5.2.2.7), and tested in accordance with Clause 5 under the obligatory test conditio
30、ns (one selected test organism, at either 20 C or 30 C) within 60 min for rapid acting products or 15 h for slower acting products. The bactericidal activity shall be evaluated using the following test organism: Legionella pneumophila. Where indicated, additional specific bactericidal activity shall
31、 be determined applying other contact times and test organisms (in accordance with 5.2.1 and 5.5.1.1) in order to take into account intended specific use conditions. EN 13623:2010 (E) 6 NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained und
32、er the obligatory test conditions. 5 Test methods 5.1 Principle 5.1.1 A sample of the product diluted with hard water (5.2.2.7 or 5.2.2.10) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is maintained at either (20 1) C or (30 1) C for 60 min 10 s or
33、 (15 1) h (obligatory test conditions). At the end of the chosen contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable n
34、eutralizer cannot be found membrane filtration is used. The numbers of surviving bacteria in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using Legionella pneumophila as test organism (obligatory test conditions). 5.1.3 Additional and optional contact times
35、 are specified. Additional test organisms may be used. 5.2 Materials and reagents 5.2.1 Test organism The bactericidal activity shall be evaluated using the following strain as test organism1 ): Legionella pneumophila: serogroup 1, Philadelphia (NCTC 11192; ATCC 33152). If required for specific appl
36、ications, additional test organisms may be used, e.g. Legionella pneumophila serogroup 1 Benidorm (NCTC 12006, ATCC 43108). The required incubation temperature for this test organism is (36 1) C or (37 1) C (5.3.2.3). The same temperature (either 36 C or 37 C) shall be used for all incubations perfo
37、rmed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, the
38、ir suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a ref
39、erence for five years. 5.2.2 Culture media and reagents 5.2.2.1 General Unless specifically stated, all weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent
40、 molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. 1) The NCTC and ATCC numbers are the collection numbers of strains supplied by the National
41、 Type Culture Collection (NCTC) and American Type Culture Collection (ATCC). This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named. EN 13623:2010 (E) 7 NOTE 1 To improve reproducibility, it is recommended that comme
42、rcially available dehydrated material is used for the preparation of culture media. The manufacturers instructions relating to the preparation of these products should be rigorously followed. NOTE 2 For each culture medium and reagent, a limitation for use should be fixed. 5.2.2.2 Water The water sh
43、all be freshly glass-distilled water and not demineralized water. If distilled water of adequate quality is not available, water for injections (bibliographic reference 1) can be used. Sterilize in the autoclave (5.3.2.1, a). Sterilization is not necessary if the water is used e.g. for preparation o
44、f culture media and subsequently sterilized. NOTE See 5.2.2.10 for the procedure to prepare buffered ferrous hard water. 5.2.2.3 Buffered Charcoal Yeast Extract (BCYE) Agar BCYE agar, consisting of yeast extract (bacteriological grade) 10,0 g; agar 12,0 g; activated charcoal 2,0 g; alpha-ketoglutara
45、te, monopotassium salt 1,0 g; ACES buffer (N-2-acetamido-2-aminoethanesulfonic acid) 10,0 g; potassium hydroxide (KOH) (pellets) 2,8 g; L-cysteine hydrochloride monohydrate 0,4 g; iron(III) pyrophosphate Fe4(P207)3 0,25 g; distilled water to 1 000,0 ml. Preparation a) Cysteine and iron solutions Prepare fresh solutions of L-cysteine hydrochloride and iron(III) pyrophosphate by adding 0,4 g and 0,25 g respectively to 10-ml-volumes of water (5.2.2.2). Sterilize each solution by membrane filtration (5.3.2.7). Stor