1、Designation: E1259 10E1259 16Standard Practice forEvaluation of Antimicrobials in Liquid Fuels Boiling Below390C1This 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 year of
2、 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. Scope1.1 This practice is designed to evaluate antimicrobial agents for the prevention of microbially influenced deterioration
3、 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 determine whether
4、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 the safety co
5、ncerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D396 Specification for Fuel OilsD910 Spe
6、cification 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Specification for Diesel Fuel OilsD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels (Withdrawn 2003)3D2880 Specification for Gas Turbine Fuel OilsD3699 Specification for KerosineD4814 Specification for Automotive Spark-Ign
7、ition Engine FuelD5465 Practice 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 Paraffin, Olefin, Naphthene, Aromatic(O-PONA) Hydrocarbon Types
8、 in Low-OlefinSpark Ignition Engine Fuels by Gas Chromatography (Withdrawn 2009)3D6469 Guide for Microbial Contamination in Fuels and Fuel SystemsD6729 Test Method for Determination of Individual Components in Spark Ignition Engine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 by 100 Metre Capillary HighResolution Gas ChromatographyD6733
9、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 FuelsD6974 Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuel
10、sFiltration and Culture ProceduresD7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and FuelAssociated Water1 This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is th
11、e direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2010April 1, 2016. Published October 2010May 2016. Originally approved in 1988. Last previous edition approved in 20052010 asE1259 05.E1259 10. DOI: 10.1520/E1259-10.10.1520/E1259-16.2 For referenc
12、edASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3 The last approved version of this historical standard is referenced on ww
13、w.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 version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consul
14、t prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1D7464 Practice for Manual Sampling of
15、 Liquid Fuels, Associated Materials and Fuel System Components for MicrobiologicalTestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-AssociatedWater with Sample Concent
16、ration by FiltrationD7978 Test Method for Determination of theViableAerobic Microbial Content of Fuels andAssociatedWaterThixotropic GelCulture MethodE1259 Practice for Evaluation of Antimicrobials in Liquid Fuels Boiling Below 390CE1326 Guide for Evaluating Non-culture Microbiological Tests2.2 NACE
17、 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 Pesticide Registration and Classification Procedures53. Terminology3.1 Definitions of Terms Specific to T
18、his 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 bacteria), fungicides (kill fungi), and microbicides (kill both bacterial and fungi).They are also referred
19、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 microbes.3.1.4 microbicide, nsee biocide.3.1.5 microcosm, na miniature system used to model larger systems
20、.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) microcosms are used as model systems.4. Summary of Practice4.1 This practice is conducted on a fuel representative
21、 of the grade to be treated, and determines the antimicrobial efficacy underwell-defined conditions that may include specific inocula:inocula Pseudomonas aeruginosa, American Type Culture Collection,(ATCC) No. 33988, Hormoconis resinae, ATCC No. 20495, and Yarrowia tropicalis (formerly Candida tropi
22、calis, ATCC No.18138; or an uncharacterized inoculum from a microbially contaminated fuel system. Additionally, water/fuel ratios andcontainment time are also defined. This practice allows for impact of fuel/water partitioning and time, on the antimicrobial agent,as well as the effect of continual r
23、echallenge. At each sampling time interval, treated and untreated aliquots are checked for thethree types of organisms in the initial inoculum. These counts are coupled with gross observations of each system for biofilmformation and interfacial growth. The size of the test system, total volume of fl
24、uid, fuel to bottom-water ratio and test duration mayvary depending on the specific objectives of the test. Before beginning any test plan intended to meet performance testingcompliance requirements, confirm that the cognizant authority accepts the test protocol.4.1.1 Water/fuel ratios and containme
25、nt 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 for the treated population survival.Microbiologica
26、l 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 objectives of the test.4.1.4 Before beginning any test pla
27、n intended to meet performance testing compliance requirements, confirm that the cognizantauthority accepts the test protocol.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
28、Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.E1259 1625. Significance and Use5.1 Guide D6469 details the types of problems associated with uncontrolled microbial growth in fuels and fuel systems.Treatment with effective antimicrobial agents is one element
29、 of contamination control strategy.5.2 The procedure should be used to evaluate the relative efficacy of microbicides in liquid fuels boiling below 390C.The effectof environmental conditions, such as a variety of fuel additives, metal surfaces, and climatology, are variables that can be includedin s
30、pecific 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 performance in this method, antimicrobials must onlybe used in comp
31、liance 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 Counter may be used.6.2 Drums; Steel208 L (55 gal) 16 ga. s
32、teel, 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 35C may be used.6.4 Glass Jars1 L capacity, French square or similar configuration.NOTE 1Jar capacity should be
33、 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 threaded portsfor venting and sampling.6.6 Sterili
34、zerAny 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 controlmicrocosms. A 0.2 m pore-size methyl cellulose or cellu
35、lose acetate membrane should be used as the filtration medium.6.7 VortexMixer.7. Reagents and Materials7.1 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 Dilution BottlesAny sterilizable glass container ha
36、ving 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 inoculum when using the fuel as the sole source of org
37、anic nutrients, synthetic bottom watermay contain various inorganic nutrients. An example, of a commonly used synthetic bottom water is Bushnell-Haas Mineral Salts medium (BHMSS).6with the concentration adjusted to simulate a particular type of bottoms-water (marine, brackish, fresh, etc.).7.6 Soy P
38、eptone 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 variety of media manufacturers and chemical suppl
39、y 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 system can be used.6 Bushnell, L.D. and Haas, H.F
40、. 1941. The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41: 653- 673.E1259 1638.1.2 Contaminated fuel system microbial communities can be quite diverse and contain 50 different taxa. Consequently, whenPractice E1259 is to be used in order to assess a products general antimic
41、robial performance properties in fuel systems, multi-taxainocula provide a more realistic challenge population than either single or commonly used, three taxa inocula.8.1.3 The use of standardized cultures to prepare microcosm inocula facilitates corroborative testing.8.1.4 Inoculum taxa should be s
42、elected 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 anaerobic taxa. If inhibition ofmicrobiologically influence corrosion is to be assessed, the challenge population should include iron related b
43、acteria, acidproducing bacteria and sulfate reducing bacteria as part of the inoculum mixture.8.1.6 Uncharacterized, bottoms-water, contaminant populations are most appropriate when Practice E1259 is to be used toevaluate microbicide performance efficacy in a single system or family of systems (for
44、example, bulk storage tanks for a specificfuel grade at a specific facility).8.2 Inoculum Preparation and Maintenance:8.2.1 Inoculum RevitalizationCultures Commonly used cultures are Pseudomonas aeruginosa,ATCC No. 33988, Hormoconisresinae, ATCC No. 20495, and Yarrowia tropicalis (formerly Candida t
45、ropicalis), ATCC No. 18138. Obtain cultures from ATCC.Before initiating fuel antimicrobial tests, revitalize each of the three cultures 48138. However, in accordance with 8.1theinstructions contained with each culture. , additional cultures can be used.8.2.1.1 Obtain cultures fromATCC. Before initia
46、ting fuel antimicrobial tests, revitalize each of the three cultures in accordancewith the instructions contained with each culture.8.2.2 Maintenance and Preparation of InoculaPre-InoculaAll three cultures are transferred from slants of a specified agar,(for example, a) Pseudomonas aeruginosa (Plate
47、 Count Agar), (b) Hormoconis resinae Potato Dextrose Agar), and (c) Yarrowiatropicali (Potato Dextrose Agar)Agar) to synthetic bottom water medium in a suitable size screw-cap glass bottle (French(6.4square), and then overlaid with 10 times the volume of fuel. This two-phase system is kept at room t
48、emperature (20 to 30C)for seven days, and the interface with half the bottom water is transferred weekly to a similar system weekly until used. Thebacterial levels expected are about 10).7 CFU/mL, the yeast levels 106 CFU/mL, and mold levels 104 spores/mL. For the testinoculum, the bacteria are dilu
49、ted 1:100 while yeast and molds are diluted 1:10.The counting of the inoculum is done directly fromthe prepared synthetic bottom water mixture at time zero, just prior to adding inoculum to each setup, and at each subsequent timepoint. This procedure may also be followed to maintain and prepare uncharacterized inocula. If test systems lager than 1.0 L willbe used, the challenge inoculum should first be acclimated to growth in systems that contain the same volume and fuel tobottom-water ratio as the test systems.NOTE 3In the distillate
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