1、BRITISH STANDARDBS ISO 16869:2008Plastics Assessment of the effectiveness of fungistatic compounds in plastics formulationsICS 83.080.01g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g3
2、8g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 16869:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2008 BSI 2008ISBN 978 0 580 62557 2National forewordThis British Standard is the UK implementation of ISO 16869:2008. It supersedes B
3、S ISO 16869:2001 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee PRI/21, Testing of plastics.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 necessa
4、ry provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate CommentsReference numberISO 16869:2008(E)INTERNATIONAL STANDARD ISO16869Second edition200
5、8-06-01Plastics Assessment of the effectiveness of fungistatic compounds in plastics formulations Plastiques valuation de lefficacit des composs fongistatiques dans les formulations de plastiques BS ISO 16869:2008ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3
6、 Terms and definitions. 2 4 Principle. 2 5 Apparatus and materials 2 5.1 Apparatus 2 5.2 Culture media and reagents 3 5.3 Organisms and cultivation. 5 6 Test specimens . 5 6.1 Shape and dimensions. 5 6.2 Number of specimens 5 7 Preparation of specimens 5 7.1 Cleaning. 5 7.2 Labelling and storage. 5
7、8 Procedure 6 8.1 Test temperature. 6 8.2 Filling the Petri dishes 6 8.3 Arrangement of test specimens 6 8.4 Inoculation of the test specimens. 6 8.4.1 Preparation of the spore suspension . 6 8.4.2 Inoculation of the nutrient-salt overlay agar 6 8.4.3 Overlay of specimen. 6 8.4.4 Incubation 7 8.4.5
8、Viability control . 7 9 Assessment of fungal growth . 7 10 Expression of results . 7 11 Precision and bias 8 12 Test report . 8 Bibliography . 9 BS ISO 16869:2008iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bo
9、dies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental an
10、d non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives,
11、 Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies cas
12、ting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16869 was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 6, Ag
13、eing, chemical and environmental resistance. This second edition cancels and replaces the first edition (ISO 16869:2001), of which it constitutes a minor technical revision. The main changes are an increase in the maximum diameter of the test specimen to 4 cm (see 6.1) and the introduction of centri
14、fuging operations in the preparation of the spore suspension in 8.4.1. BS ISO 16869:2008vIntroduction It is a well known phenomenon that plasticizers as well as other ingredients in plastics formulations can be attacked by bacteria, yeasts and fungi, the latter being the most important deteriogens.
15、Microbial attack results in a reduction of the quality of the plastic, causing embrittlement as well as discoloration. This deterioration is of economic importance. The prevention of fungal attack can be achieved by the incorporation of a fungistatic compound into the formulation. The function of th
16、is fungistat is to inhibit the growth of any fungi present on the surface of the plastic product. The method described in this International Standard determines the effectiveness of fungistatic compounds incorporated into the plastic against the fungi used in the test. BS ISO 16869:2008blank1Plastic
17、s Assessment of the effectiveness of fungistatic compounds in plastics formulations WARNING Handling and manipulation of microorganisms that are potentially hazardous requires a high degree of technical competence and may be subject to current national legislation and regulations. Only personnel tra
18、ined in microbiological techniques should carry out such tests. Codes of practice for disinfection, sterilization and personal hygiene must be strictly observed. It is recommended that workers consult IEC 60068-2-10:2005, Annex A “Danger to personnel”, and ISO 7218, Microbiology of food and animal f
19、eeding stuffs General requirements and guidance for microbiological examinations. 1 Scope This International Standard specifies a method for determining the effectiveness of fungistatic compounds in protecting susceptible ingredients like plasticizers, stabilizers, etc., in plastics formulations. Th
20、e method demonstrates whether or not a plastic product is actively protected against fungal attack. The evaluation is by visual examination. The test is applicable to any articles made of plastic that are in the form of films or plates no thicker than 10 mm. In addition, porous materials such as pla
21、stic foams may be tested provided that they are in the above-mentioned form. A minimum diffusion of the fungicides that migrate out of the matrix is necessary with this procedure. In contrast to ISO 846, the test films are not sprayed with a fungal spore suspension but covered with a layer of test a
22、gar containing spores. It has been found that this leads to a better distribution of the spores as well as providing a good supply of water necessary for spore germination on plastic surfaces that are normally hydrophobic. 2 Normative references The following referenced documents are indispensable f
23、or 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 291:2008, Plastics Standard atmospheres for conditioning and testing BS ISO 16869:20082 3 Terms and
24、 definitions For the purposes of this document, the following terms and definitions apply. 3.1 plastic susceptible to fungal attack plastic material that contains in its formulation one or more nutrients that support fungal growth 3.2 fungistat compound that prevents fungal growth on a material that
25、 is normally susceptible to fungal attack 4 Principle Test specimens are exposed to a suspension of mixed fungal spores. The spores are applied to the surface of the test specimen in a thin layer of an agar medium without an added carbon source. In this way, uniform distribution of the spores is ach
26、ieved as well as an optimum supply of water. The absence of fungistatic agents in the plastic material will lead to germination of the fungal spores and initial growth. When the ingredients in the material are susceptible to fungal attack and no active fungistat is included in the formulation, furth
27、er growth and sporulation will occur over and around the test specimen. The presence of an active fungistat in the material will lead to suppression of spore germination and initial growth in the area over and around the test specimen. Fungistatic agents can migrate into the agar around the test spe
28、cimen, thereby suppressing germination and appearing to give an increased zone of inhibition. Although not relevant to the interpretation of the test results, the inhibition zone can be an indication of the behaviour of the fungistat under test. 5 Apparatus and materials 5.1 Apparatus Sterilize all
29、glassware and all parts of the rest of the apparatus that will come into contact with the culture media and/or reagents (except those which are supplied sterile) by one of the following methods: method A: autoclave (see 5.1.2) at 121 C for a minimum of 15 min; method B: use a dry-heat sterilizer (se
30、e 5.1.2) at 180 C for at least 30 min, at 170 C for at least 1 h, or at 160 C for at least 2 h; method C: use a membrane-filtration system of pore size 0,45 m. 5.1.1 Incubator, maintained at 24 C 1 C. 5.1.2 Sterilization apparatus: 5.1.2.1 For moist-heat sterilization, a suitable autoclave. 5.1.2.2
31、For dry-heat sterilization, a hot-air oven maintained at one of the temperatures specified in method B above. 5.1.2.3 For membrane-filtration sterilization, a membrane-filtration apparatus, of pore size as specified in method C above. 5.1.3 Analytical balance, accurate to 0,1 mg. 5.1.4 Laboratory ce
32、ntrifuge, speed 2 000 rpm to 5 000 rpm. 5.1.5 Counting chamber (for direct counting with the help of a microscope). BS ISO 16869:200835.1.6 Microscope, magnification 100. 5.1.7 pH-meter, having an accuracy of 0,1 pH-units, capable of temperature correction. 5.1.8 Vortex shaker, operating at 2 000 rp
33、m to 2 500 rpm. 5.1.9 Containers: test tubes, flasks or bottles of suitable capacity. 5.1.10 Petri dishes, 90 mm to 100 mm in diameter and at least 15 mm deep. 5.1.11 Graduated pipettes, with nominal capacities of 1,0 ml and 15,0 ml. Calibrated automatic pipettes may be used. 5.1.12 Graduated measur
34、ing cylinder, minimum capacity 30 ml. 5.1.13 Glass beads, diameter 3 mm to 5 mm. 5.2 Culture media and reagents All reagents shall be of analytical grade and/or of a grade appropriate for microbiological purposes. 5.2.1 Water Any water used shall be distilled or deionized and have a conductivity of
35、1 S/cm. 5.2.2 Malt-extract agar (MEA) Malt extract 30,0 g Soya peptone 3,0 g Agar-agar 15,0 g Water (5.2.1) to make up to 1 000 ml Sterilize in the autoclave (see 5.1.2). After sterilization, the pH of the medium shall be 7,0 0,2. 5.2.3 Chaetomium agar NaNO32,0 g MgSO47H2O 0,5 g KCl 0,5 Fe2(SO4)3H2O
36、 0,01 g KH2PO40,14 K2HPO41,20 g Agar-agar 15,0 g Yeast extract 0,02 g Microcellulose 20,0 g or Carboxymethyl-cellulose (Na salt) 10,0 g Water (5.2.1) to make up to 1 000 ml Sterilize in the autoclave (see 5.1.2). After sterilization, the pH of the medium shall be 7,2 0,2. BS ISO 16869:20084 5.2.4 We
37、tting agent Prepare a 5 % (m/V) stock solution of polysorbate 80 (polyoxyethylenesorbitane monooleate) in water. To harvest the spores, dilute the stock solution with water to 0,05 % (m/V). 5.2.5 Stock solution for nutrient-salt solution and agar NaCl 0,5 g FeSO47H2O 0,2 g ZnSO47H2O 0,2 g MnSO41H2O
38、0,06 g Water (5.2.1) to make up to 1 000 ml Before storage for a lengthy period, the stock solution shall be sterilized by membrane filtration. 5.2.6 Nutrient-salt solution KH2PO42,62 g Na2HPO42H2O 0,2 g MgSO47H2O 0,7 g NH4NO31,0 g Stock solution (5.2.5) 10 ml Water (5.2.1) to make up to 1 000 ml St
39、erilize in the autoclave (see 5.1.2). After sterilization, the pH of the medium shall be 5,5 0,2. 5.2.7 Nutrient-salt agar KH2PO42,62 g Na2HPO42H2O 0,20 g MgSO47H2O 0,70 g NH4NO31,0 g Agar-agar 15,0 g Stock solution (5.2.5) 10 ml Water (5.2.1) to make up to 1 000 ml Heat the prepared medium till the
40、 agar-agar is molten, then measure the temperature and adjust the pH to 5,5 0,1. Sterilize in an autoclave (see 5.1.2). Agar plates shall be freshly prepared when no validated method for long-term storage (longer than 3 days) is available. The sterilized solutions may be stored for up to 3 months. T
41、he storage conditions shall preclude any evaporation occurring. BS ISO 16869:200855.3 Organisms and cultivation 5.3.1 Test organisms 5.3.1.1 Aspergillus niger ATCC 6275 5.3.1.2 Chaetomium globosum ATCC 6205 5.3.1.3 Paecilomyces variotii CBS 628.66 5.3.1.4 Penicillium funiculosum ATCC 9644 5.3.1.5 Tr
42、ichoderma longibrachiatum ATCC 13631 The test fungi shall be obtained from a national culture collection (e.g. ATCC = American Type Culture Collection, USA; CBS = Centraalbureau voor Schimmelcultures, NL). If there are particular reasons for doing so, and by agreement between the interested parties,
43、 other fungi (for example, Aspergillus terreus, Aureobasidium pullulans) may be used. In any case, all strains used shall be listed in the test report. 5.3.2 Culture conditions Cultivation of strains 5.3.1.1 and 5.3.1.3 to 5.3.1.5 shall be on a malt-extract agar (5.2.2) slant in a test tube at 24 C
44、1 C for 14 to 21 days. Cultivation of strain 5.3.1.2 shall be on Chaetomium agar (5.2.3) at 24 C 1 C for 14 to 21 days. Stock cultures may be kept on agar slants or, preferably, freeze-dried or frozen. 6 Test specimens 6.1 Shape and dimensions Sterilize a suitable punch, and punch out round specimen
45、s from each test film to obtain discs with a diameter of 1 cm to 4 cm as required. The thickness of the specimens shall not exceed 10 mm. 6.2 Number of specimens Prepare at least three replicate specimens of each material to be evaluated. 7 Preparation of specimens 7.1 Cleaning Handling them with tw
46、eezers, clean the specimens mechanically (if necessary with a brush) and store in a clean container. Carry out all subsequent handling of the specimens using tweezers to avoid contamination. 7.2 Labelling and storage Labelling or marking may result in interactions with the plastic during the test. T
47、herefore store the specimens separately in closed containers (e.g. Petri dishes) at ambient temperature. Mark the containers and not the specimens. BS ISO 16869:20086 8 Procedure 8.1 Test temperature Prepare and condition the specimens in an atmosphere complying with ISO 291 Class 2 23 C 2 C and (50
48、 10) % rh. 8.2 Filling the Petri dishes After sterilization, pour 20 ml of the nutrient-salt agar (5.2.7) into each Petri dish, allow to solidify and dry until no water is visible on the surface. 8.3 Arrangement of test specimens Place the test specimen discs separately, as flat as possible, in the
49、middle of the solidified medium. If the test specimens are thicker than 5 mm, punch holes in the agar using a punch of the same size as that used when preparing the specimens. The punch shall be sterilized, e.g. by flame treatment. Fit the specimens into the holes in the agar. 8.4 Inoculation of the test specimens 8.4.1 Preparation of the spore suspension Produce a mixed spore suspension from well sporulated cultures as follows: Introduce into each culture tube (see 5.3.2) 5 ml of wetting-agent solution (5.2.4). Gently scrape
