1、Designation: E1259 16E1259 18Standard Practice forEvaluation of Antimicrobials in Liquid Fuels Boiling Below390C390 C1This standard is issued under the fixed designation E1259; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the ye
2、ar of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope Scope*1.1 This practice is designed to evaluate antimicrobial agents for the prevention of microbially influenced d
3、eterioration of liquidfuels (as defined by Specification D396, D910, D975, D1655, D2069, D2880, D3699, D4814, D6227, D6751, and D7467), systemdeterioration, or both.1.2 Knowledge of microbiological techniques is required for these procedures.1.3 It is the responsibility of the investigator to determ
4、ine whether Good Laboratory Practice (GLP) is required and to followthem where appropriate (40 CFR, 160), or as revised.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all of t
5、he safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.6 This international standard was develo
6、ped in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Document
7、s2.1 ASTM Standards:2D396 Specification for Fuel OilsD910 Specification for Leaded Aviation GasolinesD975 Specification for Diesel Fuel OilsD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels (Withdrawn 2003)3D2880 Specification for Gas Turbine Fuel OilsD3699 Specifica
8、tion for KerosineD4814 Specification for Automotive Spark-Ignition Engine FuelD5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating MethodsD6227 Specification for Unleaded Aviation Gasoline Containing a Non-hydrocarbon ComponentD6293 Test Method for Oxygenates and P
9、araffin, Olefin, Naphthene, Aromatic(O-PONA) Hydrocarbon Types in Low-OlefinSpark Ignition Engine Fuels by Gas Chromatography (Withdrawn 2009)3D6469 Guide for Microbial Contamination in Fuels and Fuel SystemsD6729 Test Method for Determination of Individual Components in Spark Ignition Engine Fuels
10、by 100 Metre Capillary HighResolution Gas ChromatographyD6733 Test Method for Determination of Individual Components in Spark Ignition Engine Fuels by 50-Metre Capillary HighResolution Gas ChromatographyD6751 Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels1 This pract
11、ice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2016Oct. 1, 2018. Published May 2016October 2018. Originally approved in
12、1988. Last previous edition approved in 20102016 asE1259 10.E1259 16. DOI: 10.1520/E1259-16.10.1520/E1259-18.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the stan
13、dards Document Summary page on the ASTM website.3 The last approved version of this historical standard is referenced on www.astm.org.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous versio
14、n. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.*A Summary of Changes sectio
15、n appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1D6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid FuelsFiltration and Culture ProceduresD7463 Test Method for Adenosine Triphosph
16、ate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and FuelAssociated WaterD7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for MicrobiologicalTestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)D7687 Test Metho
17、d for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With SampleConcentration by FiltrationD7978 Test Method for Determination of theViableAerobic Microbial Content of Fuels andAssociatedWaterThixotropic GelCulture MethodE1259 Practice for Evaluation of Antimicrobia
18、ls in Liquid Fuels Boiling Below 390 CE1326 Guide for Evaluating Non-culture Microbiological Tests2.2 NACE Standard:TM0172 Determining Corrosive Properties of Cargoes in Petroleum Product Pipelines42.3 Federal Standards:40 CFR Part 79 Fuels and Fuel Additives Registration Regulations540 CFR Part 152
19、 Pesticide Registration and Classification Procedures53. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 antimicrobial, nsee biocide.3.1.2 biocide, na physical or chemical agent that kills living organisms.3.1.2.1 DiscussionBiocides are further classified as bactericides (kill ba
20、cteria), fungicides (kill fungi), and microbicides (kill both bacterial and fungi).They are also referred to as antimicrobials.3.1.3 microbially-influenced deterioration, ndecomposition /degradation of material (fuel) or making unsuitable for use, as aresult of metabolic activity or the presence of
21、microbes.3.1.4 microbicide, nsee biocide.3.1.5 microcosm, na miniature system used to model larger systems.3.1.5.1 DiscussionIt is generally impractical to evaluate microbicide performance in large fuel storage system capacities ( 24 000 m3), consequentlysmall volume (1.0 to 208 L capacity) microcos
22、ms are used as model systems.4. Summary of Practice4.1 This practice is conducted on a fuel representative of the grade to be treated, and determines the antimicrobial efficacy underwell-defined conditions that may include specific inocula or an uncharacterized inoculum from a microbially contaminat
23、ed fuelsystem.4.1.1 Water/fuel ratios and containment time are also defined. This practice allows for impact of fuel/water partitioning andtime, on the antimicrobial agent, as well as the effect of continual rechallenge.4.1.2 At each sampling time interval, treated and untreated aliquots are checked
24、 for the treated population survival.Microbiological testing is coupled with gross observations of each system for biofilm formation and interfacial growth.4.1.3 The size of the test system, total volume of fluid, fuel to bottom-water ratio and test duration may vary depending on thespecific objecti
25、ves of the test.4.1.4 Before beginning any test plan intended to meet performance testing compliance requirements, confirm that the cognizantauthority accepts the test protocol.5. Significance and Use5.1 Guide D6469 details the types of problems associated with uncontrolled microbial growth in fuels
26、 and fuel systems.Treatment with effective antimicrobial agents is one element of contamination control strategy.4 Item No. 21204, available from NACE International (NACE), 1440 South Creek Dr., Houston, TX 77084-4906, http:/www.nace.org.5 Available from U.S. Government Printing Office Superintenden
27、t of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.E1259 1825.2 The procedure should be used to evaluate the relative efficacy of microbicides in liquid fuels boiling below 390C.390 C.The effect of environmental conditions, such as a variety of fuel additives, metal surface
28、s, and climatology, are variables that canbe included in specific tests using this protocol.5.3 This practice addresses product performance issues only. RegulatoryAgencies restrict and control the use of both pesticides(in the U.S.: 40 CFR 152) and fuel additives (40 CFR 79). Regardless of performan
29、ce in this method, antimicrobials must onlybe used in compliance with applicable regulations. Specific industries, for example, the aviation industry, may place furtherrestrictions on chemicals used for fuel treatment.6. Apparatus6.1 Colony CounterAny of several types, for example, a Quebec Colony C
30、ounter may be used.6.2 Drums; Steel208 L (55 gal) 16 ga. steel, open-head drum with removable 16 ga. lid fitted with 2.05 cm and 1.90 cmthreaded ports for venting and sampling.6.3 IncubatorAny incubator capable of maintaining temperature of 30 to 35C35 C may be used.6.4 Glass JarsFrench square or si
31、milar configuration.NOTE 1Jar capacity should be determined based on the test plan designed fuel to water ratio and the expected sample volume size needed for weeklytesting (9.5 and 9.9).6.5 Pails; Steel18.9 L (5 gal) steel, open-head pail with removable 16 ga. lid fitted with 2.05 cm and 1.90 cm th
32、readed portsfor venting and sampling.6.6 SterilizerAny suitable steam sterilizer capable of producing the conditions of sterility is acceptable. A pressurized filtersterilization apparatus of appropriate capacity to filter sterilize the test fuels and bottom-water used in the negative controlmicroco
33、sms. A 0.2 m pore-size methyl cellulose or cellulose acetate membrane should be used as the filtration medium.6.7 VortexMixer.7. Reagents and Materials67.1 Petri Dishes100 by 15 mm required for performing standard plate count.7.2 Bacteriological Pipets10.0 mL and 1.1, or 2.2 mL capacity.7.3 Water Di
34、lution BottlesAny sterilizable glass container having a 150 to 200 mL capacity and tight closure may be used.7.4 Fuel.NOTE 2Representative fuel samples from each product grade are available from all petroleum refiners.7.5 Synthetic Bottom Water.NOTE 3In order to promote microbial growth of the inocu
35、lum when using the fuel as the sole source of organic nutrients, synthetic bottom watermay contain various inorganic nutrients. An example, of a commonly used synthetic bottom water is Bushnell-Haas Mineral Salts medium (BHMSS).7with the concentration adjusted to simulate a particular type of bottom
36、s-water (marine, brackish, fresh, etc.).7.6 Soy Peptone Casein Digest Agar.7.7 Sabouraud Dextrose Agar.7.8 Agar, Bacteriological Grade.7.9 Potassium Tellurite Solutionsterile 1 %.7.10 Gentamicin Sulfate50 g/mL.7.11 Plate Count Agar.7.12 Potato Dextrose Agar.NOTE 4Items 7.5 7.12 are available from a
37、variety of media manufacturers and chemical supply companies.8. Inoculum8.1 Inoculum Selection:8.1.1 Depending on the objectives of a test plan, one or more characterized cultures (for example: bacterium, yeast and mold)can be selected or microbially contaminated bottoms-water collected from a fuel
38、system can be used.6 Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents not listed bythe American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the Un
39、ited States Pharmacopeia and NationalFormulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.7 Bushnell, L.D. and Haas, H.F. 1941. The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41: 653- 673.E1259 1838.1.2 Contaminated fuel system microbial communities can be q
40、uite diverse and contain 50 different taxa. Consequently, whenPractice E1259 is to be used in order to assess a products general antimicrobial performance properties in fuel systems, multi-taxainocula provide a more realistic challenge population than either single or commonly used, three taxa inocu
41、la.8.1.3 The use of standardized cultures to prepare microcosm inocula facilitates corroborative testing.8.1.4 Inoculum taxa should be selected from cultures known to grow using fuel as their sole carbon source.8.1.5 Depending on microcosm design, it can be appropriate to include aerobic and anaerob
42、ic taxa. If inhibition ofmicrobiologically influence corrosion is to be assessed, the challenge population should include iron related bacteria, acidproducing bacteria and sulfate reducing bacteria as part of the inoculum mixture.8.1.6 Uncharacterized, bottoms-water, contaminant populations are most
43、 appropriate when Practice E1259 is to be used toevaluate microbicide performance efficacy in a single system or family of systems (for example, bulk storage tanks for a specificfuel grade at a specific facility).8.2 Inoculum Preparation and Maintenance:8.2.1 Inoculum RevitalizationCommonly used cul
44、tures are Pseudomonas aeruginosa,ATCC No. 33988, Hormoconis resinae,ATCC No. 20495, and Yarrowia tropicalisCandida viswanathii (formerly Candida tropicalis and Yarrowia tropicalis), ATCC No.48138. However, in accordance with 8.1, additional cultures can be used.8.2.1.1 Obtain cultures fromATCC. Befo
45、re initiating fuel antimicrobial tests, revitalize each of the three cultures in accordancewith the instructions contained with each culture.8.2.2 Maintenance and Preparation of Pre-InoculaAll cultures are transferred from slants of a specified agar, (for example,a) Pseudomonas aeruginosa (Plate Cou
46、ntAgar), (b) Hormoconis resinae Potato DextroseAgar), and (c) Yarrowia tropicali (PotatoDextrose Agar) to synthetic bottom water medium in a suitable size screw-cap glass bottle (6.4).8.2.2.1 Overlay inoculated bottom water with fuel to give a final fuel to water ratio of 10.8.2.2.2 Keep this two-ph
47、ase system at room temperature (20 to 30C)30 C) for seven days.8.2.2.3 Weekly, transfer the interface, along with half the bottom water to a similar system until the inoculum used.8.2.2.4 During this inoculum preparation period the bacterial levels should be maintained at approximately 107 CFU/mL or
48、non-culture test bioburden equivalent, the yeast levels at approximately 106 CFU/mL, and mold levels at approximately 104spores/mL.8.2.2.5 Freshly collected, microbially contaminated bottoms-water can be maintained per 8.2.2.1 8.2.2.4.8.2.3 Preparation of Challenge (Test) Inoculum:8.2.3.1 To prepare
49、 the test inoculum, dilute bacterial pre-inocula 1:100 to achieve a population equivalent to approximately 105CFU/mL. Dilute yeast and molds 1:10 to achieve a population equivalent to approximately 103 CFU/mL.8.2.3.2 At time zero, just prior to adding inoculum to each setup, and at each subsequent time point, determine the microbialpopulation density (9.9).8.2.3.3 If test systems larger than 1.0 L will be used, the challenge inoculum should first be acclimated to growth in systemsthat contain the same volume and fuel to bottom-water
