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1、BRITISH STANDARD BS 6920-2.5: 2000 Incorporating Amendment No. 1 Suitability of non-metallic products for use in contact with water intended for human consumption with regard to their effect on the quality of the water Part 2: Methods of test Section 2.5: The extraction of substances that may be of
2、concern to public health ICS 13.060.20 ? BS 6920-2.5:2000 This British Standard, having been prepared under the direction of the Health and Environment Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 May 2000 BSI 21 January 2004 The followin
3、g BSI references relate to the work on this British Standard: Committee reference EH/6 Draft for comment 99/560130 DC ISBN 0 580 33110 5 Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Committee EH/6, Effects of materials on water
4、quality, upon which the following bodies were represented: Automatic Vending Association of Britain British Cement Association BCF British Coatings Federation Ltd British Malleable Tube Fittings Association British Plastics Federation British Plumbing Fittings Manufacturers Association British Preca
5、st Concrete Federation Ltd British Rubber Manufacturers Association Ltd British Water DEFRA Water and Land Directorate Galvanizers Association Laboratory of the Government Chemist Pipeline Industries Guild UK Steel Association Water Regulations Advisory Scheme Water Research Centre plc Amendments is
6、sued since publication Amd. No.DateComments 1472121 January 2004Revision of 10.5 and 10.6 BS 6920-2.5:2000 BSI 21 January 2004 i Contents Page Committees responsibleInside front cover Forewordii Introduction1 1Scope1 2Normative references1 3Terms and definitions1 4Principle2 5Test premises2 6Safety2
7、 7Reagents3 8Apparatus4 9Samples4 10Test procedures5 11Expression of results6 12Test report7 Annex A (informative) Test sequence8 Bibliography9 Figure A.1 Test sequence8 BS 6920-2.5:2000 ii BSI 21 January 2004 Foreword This section of BS 6920 has been prepared by Technical Committee EH/6. It superse
8、des BS 6920-2.5:1996, which is withdrawn. This edition introduces technical changes but it does not reflect a full review or revision of the standard. BS 6920 is published in several parts, namely Part 1: Specification, Part 2: Methods of test, Part 3: High temperature tests and Part 4: Method for t
9、he GCMS identification of water leachable organic substances. Part 2 is further subdivided into a number of sections and subsections as follows. Section 2.1: Samples for testing; Section 2.2: Odour and flavour of water; Subsection 2.2.1: General method of test; Subsection 2.2.2: Method of testing od
10、ours and flavours imparted to water by hoses and composite pipes and tubes; Subsection 2.2.3: Method of testing odours and flavours imparted to water by hoses for conveying water for food and drink preparation; Section 2.3: Appearance of water; Section 2.4: Growth of aquatic microorganisms test; Sec
11、tion 2.5: The extraction of substances that may be of concern to public health; Section 2.6: The extraction of metals. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does n
12、ot of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages 1 to 9 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. BS 6920-2.5:2000 BSI 21
13、January 2004 1 Introduction WARNING. As well as observing safe working practices, take particular care in handling continuous cell lines since they may become infected with pathogenic viruses and bacteria during the course of their manipulation. The nutrient media used in this test are capable of su
14、pporting microbial growth, and the cell line is capable of being infected by human viruses. CAUTION. It is essential that this test procedure is carried out only by persons with experience of mammalian tissue culture techniques and cell line morphology. This section of BS 6920 describes a simple cyt
15、otoxicity technique to test leachates from materials and articles, used in customers premises in contact with water for human consumption, for biologically active compounds. Materials and chemicals used by water suppliers are subjected to a fuller assessment using an extraction procedure followed by
16、 sophisticated analytical methods. This method should be regarded as only an initial screening test for substances potentially hazardous to health. A satisfactory result indicates that the leachate probably does not contain significant amounts of acutely toxic substances, but it does not indicate th
17、e absence of small quantities of substances which may be harmful on prolonged exposure. 1 Scope This section of BS 6920 specifies a screening procedure (simple cytotoxicity test) using a mammalian cell line and a leachate from a product. The results of this procedure will assist in the toxicological
18、 assessment of the product for use in contact with water intended for human consumption. The procedure given in this section of BS 6920 is suitable for all non-metallic materials that may be used in contact with water intended for human consumption. NOTEUnder the requirements of the Water Supply (Wa
19、ter Quality) Regulations (Regulation 25) and the Water Supply (Water Fittings) Regulations (Clause 2 of Schedule 2), the National Regulator may specify additional provisions in some cases and will assess the significance of the results obtained. 2 Normative references The following normative documen
20、ts contain provisions which, through reference in this text, constitute provisions of this section of BS 6920. For dated references, subsequent amendments to or revisions of any of these publications do not apply. For undated references, the latest edition of the publication referred to applies. BS
21、748, Specification for haemacytometer and particle counting chambers. BS 6920-2.1:2000, Suitability of non-metallic products for use in contact with water intended for human consumption with regard to their effect on the quality of the water Part 2: Methods of test Section 2.1: Samples for testing.
22、BS EN ISO 369, Water for analytical laboratory use Specification and test methods. 3 Terms and definitions For the purposes of this section of BS 6920, the following definitions apply. 3.1 cytotoxic poisonous (toxic) to cells under the conditions of the test 3.2 monolayer resulting cell layer, only
23、one cell thick, formed when a cell culture grows in contact with a suitable solid surface, normally provided by the walls of the culture vessel. The cells are contact dependent and spread out onto the surface and multiply until adjacent cells are in contact whereupon the growth ceases NOTEOnly certa
24、in types of cell culture (including the cell line used in this test) form monolayers. BS 6920-2.5:2000 2 BSI 21 January 2004 3.3 tissue culture technique whereby cells are grown and maintained as a monolayer sheet on the inner surface of a container in a nutrient (culture) medium 3.4 cell line cells
25、 capable of growth under laboratory conditions using a tissue culture technique; a cell line capable of unlimited growth in vitro is described as a continuous cell line 3.5 biologically clean atmosphere atmosphere in which the numbers of microorganisms are sufficiently low to not have any adverse ef
26、fect on the test 3.6 morphology microscopic study of physical form; when applied to a cell line it is the overall appearance of both the cell monolayer and the individual appearance of the constituent cells 3.7 rounding off abnormal cells which are spherical or oval in shape and exhibit no visible i
27、nternal structures when using a low-power microscope 3.8 cell suspension suspension prepared from a monolayer by the application of digestive enzymes and/or chelating agents, thereby loosening the cells from the surface to which they are attached and dispersing them to give a suspension, which can b
28、e used to start fresh cultures or to perform tests 4 Principle The product is immersed in test water for 24 h. This water is used subsequently in the preparation of a nutrient medium. The morphology of the mammalian cell line grown in this medium is observed. A blank extract is assessed in parallel
29、with the material. NOTEA flow diagram showing the sequence of this test procedure is given in Annex A. 5 Test premises Manipulate the cells and media only in a laboratory with a biologically clean atmosphere that is dust free, and carry out the test in an environment which avoids infection of the ce
30、ll line. NOTEFor example, the test can be carried out in a laminar flow cabinet conforming to BS 5726-3. 6 Safety Observe the warning given in the Introduction. BS 6920-2.5:2000 BSI 21 January 2004 3 7 Reagents 7.1 Waters 7.1.1 Test water, obtained from a tap connected directly to a service pipe at
31、mains pressure. Before collection of the water, the tap shall be flushed until the temperature of the flowing water does not vary by more than 1 C over a period of 1 min and does not exceed 25 C. Alternatively, glass-distilled, demineralized or reverse osmosis treated water conforming to grade 3 of
32、BS EN ISO 3696 shall be used. The test water shall be free from substances that are toxic to or inhibit the growth of the cell line. NOTEFor example, this may be verified by growing at least three successive generations of the cell line in a nutrient medium made with the test water and comparing the
33、 morphology of the cells with that of cells from the same generations grown in the same medium made with distilled water (see 10.2). 7.1.2 Distilled water (for the preparation of media), glass-distilled water, or water produced by reverse osmosis and conforming to grade 3 of BS EN ISO 3696. NOTEDeio
34、nized water is not suitable. 7.2 Media 7.2.1 General All reagents shall be of analytical quality. NOTE 1Foetal calf serum may be used in place of newborn-calf serum. NOTE 2Other established growth and maintenance media may be used in place of those specified provided that healthy growth of the test
35、cell line can be maintained using them. The use of gentamicin shall be preferred to other antibiotics to control possible bacterial growth/contamination from the non-sterile test samples and test water. NOTE 3Commercially available sterile media constituents are available and should be used wherever
36、 possible. 7.2.2 Growth medium, prepared from the following. Sterilized ingredients shall be used. The medium shall be prepared aseptically and stored in the absence of light at (4 1) C. NOTE199 concentrate ( 10) with Earles salts but without glutamine and sodium hydrogen carbonate is available comm
37、ercially. Before use, 0.34 ml of a 200 mmol/l solution of L-glutamine should be added to each 100 ml of concentrate. Newborn-calf serum, mycoplasma screened, is available commercially. 7.2.3 Maintenance medium, prepared from the following. Sterilized ingredients shall be used. The medium shall be pr
38、epared aseptically and stored in the absence of light at (4 1) C. 7.2.4 Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide, prepared from the following. Sterile distilled water 90 ml 199 concentrate ( 10) with Earles salts but without sodium hydrogen carbonate buffer 10
39、 ml Newborn-calf serum 7 ml Gentamicin solution (4 000 i.u./ml) 1 ml Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide (7.2.4) 2 ml Sterile distilled water 90 ml 199 concentrate ( 10) with Earles salts but without sodium hydrogen carbonate buffer 10 ml Newborn-calf ser
40、um 2 ml Gentamicin solution (4 000 i.u./ml) 1 ml Sodium hydrogen carbonate buffer solution, 44 g/l, saturated with carbon dioxide (7.2.4) 3 ml Sodium hydrogen carbonate 8.8 g Phenol red (2 g/l aqueous solution) 10 ml Distilled water to 200 ml BS 6920-2.5:2000 4 BSI 21 January 2004 The ingredients sh
41、all be mixed and the solution saturated with carbon dioxide either by bubbling the gas through it, or by the addition of pieces of solid carbon dioxide. Immediately after preparation, the buffer shall be placed into 10 ml glass bottles, filled to the rim, closed tightly and sterilized at a temperatu
42、re of 115 C and at a gauge pressure of 69 kPa for 20 min in a small autoclave. Store at (4 1) C. Discard bottles showing a bright magenta colouration. 7.2.5 Phosphate-buffered saline solution, prepared from the following. The ingredients shall be mixed and the solution shall be dispensed into glass
43、bottles in 20 ml amounts and sterilized at a temperature of 115 C and a gauge pressure of 69 kPa for 10 min. 7.2.6 Trypsin-EDTA solution, as commercially available trypsin-EDTA solution or equivalent. NOTEThis should be stored at a temperature lower than p18 C. 7.2.7 Concentrated growth medium, prep
44、ared as described in 7.2.2, but omitting the distilled water. The medium shall be stored at (4 1) C in the absence of light. 7.3 Cell line The established VERO cell line of African green monkey kidney cells (ATCC number CCL 81) shall be used. Establish that the cell line is healthy upon receipt (see
45、 10.2). NOTEThe BGM monkey kidney cell line may be used in place of the VERO cell line for this test, but the VERO cell line is preferred. 8 Apparatus 8.1 Tissue-culture ware, prepared in accordance with the requirements given below. All glassware used for this test shall be cleaned using an aqueous
46、 solution of a proprietary detergent specifically designed for use in tissue culture techniques. Glassware shall be thoroughly rinsed after cleaning, and given two final rinses in distilled water (7.1.2). Glassware shall be sterilized at a temperature of 121 C and at a gauge pressure of 103 kPa for
47、15 min. NOTEAn alternative to the above requirements is to use presterilized plastics containers supplied specifically for tissue culture work. 8.2 Haemocytometer counting chamber, conforming to BS 748. 8.3 Extraction containers, consisting of borosilicate glass beakers calibrated for a capacity in
48、accordance with BS 6920-2.1:2000, 4.1.2. The beakers shall be cleaned using an aqueous solution of a biodegradable laboratory detergent. The beakers shall be rinsed in test water (7.1.1) and then once in distilled water (7.1.2). Following this, the beakers shall be drained and dried in a hot air cab
49、inet. Before use, the beakers shall be rinsed in the test water (7.1.1). 9 Samples 9.1 General Samples shall conform to the pertinent requirements given in BS 6920-2.1, except in the following respects. 9.2 Reference materials At present there is no safe and proven reference material or substance that can be used in this test. Sodium chloride 8.0 g Potassium chloride 0.2 g Disodium hydrogen orthophosphate 1.15 g Potassium dihydrogen orthophosphate 0.2 g Distilled w
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