1、Designation: E1259 10Standard 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 last re
2、vision. 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 antimicrobialagents for the prevention of microbially influenced deteriora-tion of liqu
3、id fuels (as defined by Specification D396, D910,D975, D1655, D2069, D2880, D3699, D4814, D6227, D6751,and D7467), system deterioration, or both.1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 It is the responsibility of the investigator to determinewhether Good Labor
4、atory Practice (GLP) is required and tofollow them where appropriate (40 CFR, 160), or as revised.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if an
5、y, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D396 Specification for Fuel OilsD910 Specification
6、 for Aviation GasolinesD975 Specification for Diesel Fuel OilsD1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels3D2880 Specification for Gas Turbine Fuel OilsD3699 Specification for KerosineD4814 Specification for Automotive Spark-Ignition EngineFuelD5465 Practice for
7、 Determining Microbial Colony Countsfrom Waters Analyzed by Plating MethodsD6227 Specification for Grade 82 Unleaded Aviation Gaso-lineD6293 Test Method for Oxygenates and Paraffin, Olefin,Naphthene, Aromatic(O-PONA) Hydrocarbon Types inLow-Olefin Spark Ignition Engine Fuels by Gas Chroma-tography3D
8、6469 Guide for Microbial Contamination in Fuels andFuel SystemsD6729 Test Method for Determination of Individual Com-ponents in Spark Ignition Engine Fuels by 100 MetreCapillary High Resolution Gas ChromatographyD6733 Test Method for Determination of Individual Com-ponents in Spark Ignition Engine F
9、uels by 50-MetreCapillary High Resolution Gas ChromatographyD6751 Specification for Biodiesel Fuel Blend Stock (B100)for Middle Distillate FuelsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresD7463 Test Method for Adenosine Triphosphate (ATP
10、)Content of Microorganisms in Fuel, Fuel/Water Mixturesand Fuel Associated WaterD7464 Practice for Manual Sampling of Liquid Fuels,Associated Materials and Fuel System Components forMicrobiological TestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend(B6 to B20)E1326 Guide for Evaluating N
11、onconventional Microbio-logical Tests Used for Enumerating Bacteria2.2 NACE Standard:TM0172 Determining Corrosive Properties of Cargoes inPetroleum Product Pipelines42.3 Federal Standards:40 CFR Part 79 Fuels and Fuel Additives RegistrationRegulations540 CFR Part 152 Pesticide Registration and Class
12、ificationProcedures51This practice is under the jurisdiction of ASTM Committee E35 on Pesticidesand Alternative Control Agents and is the direct responsibility of SubcommitteeE35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2010. Published October 2010. Originallyapproved in 1988. Las
13、t previous edition approved in 2005 as E1259 05. DOI:10.1520/E1259-10.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM
14、 website.3Withdrawn. The last approved version of this historical standard is referencedon www.astm.org.4Item No. 21204, available from NACE International (NACE), 1440 SouthCreek Dr., Houston, TX 77084-4906, http:/www.nace.org.5Available from U.S. Government Printing Office Superintendent of Documen
15、ts,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 antimicrobial, nsee biocide.3.1.2 biocide, na phys
16、ical or chemical agent that killsliving organisms.3.1.2.1 DiscussionBiocides are further classified as bac-tericides (kill bacteria), fungicides (kill fungi), and microbi-cides (kill both bacterial and fungi). They are also referred toas antimicrobials.3.1.3 microbially-influenced deterioration,ndec
17、omposition /degradation of material (fuel) or makingunsuitable for use, as a result of metabolic activity or thepresence of microbes.3.1.4 microbicide, nsee biocide.3.1.5 microcosm, na miniature system used to modellarger systems.3.1.5.1 DiscussionIt is generally impractical to evaluatemicrobicide p
18、erformance in large fuel storage system capaci-ties ( 24 000 m3), consequently small volume (1.0 to 208 Lcapacity) microcosms are used as model systems.4. Summary of Practice4.1 This practice is conducted on a fuel representative of thegrade to be treated, and determines the antimicrobial efficacyun
19、der well-defined conditions that may include specific in-ocula: Pseudomonas aeruginosa, American Type Culture Col-lection, (ATCC) No. 33988, Hormoconis resinae, ATCC No.20495, and Yarrowia tropicalis (formerly Candida tropicalis,ATCC No. 18138; or an uncharacterized inoculum from amicrobially contam
20、inated fuel system. Additionally, water/fuelratios and containment time are also defined. This practiceallows for impact of fuel/water partitioning and time, on theantimicrobial agent, as well as the effect of continual rechal-lenge. At each sampling time interval, treated and untreatedaliquots are
21、checked for the three types of organisms in theinitial inoculum. These counts are coupled with gross obser-vations of each system for biofilm formation and interfacialgrowth. The size of the test system, total volume of fluid, fuelto bottom-water ratio and test duration may vary depending onthe spec
22、ific objectives of the test. Before beginning any testplan intended to meet performance testing compliance require-ments, confirm that the cognizant authority accepts the testprotocol.5. Significance and Use5.1 Guide D6469 details the types of problems associatedwith uncontrolled microbial growth in
23、 fuels and fuel systems.Treatment with effective antimicrobial agents is one element ofcontamination control strategy.5.2 The procedure should be used to evaluate the relativeefficacy of microbicides in liquid fuels boiling below 390C.The effect of environmental conditions, such as a variety offuel
24、additives, metal surfaces, and climatology, are variablesthat can be included in specific tests using this protocol.5.3 This practice addresses product performance issuesonly. Regulatory Agencies restrict and control the use of bothpesticides (in the U.S.: 40 CFR 152) and fuel additives (40CFR 79).
25、Regardless of performance in this method, antimi-crobials must only be used in compliance with applicableregulations. Specific industries, for example, the aviationindustry, may place further restrictions on chemicals used forfuel treatment.6. Apparatus6.1 Colony CounterAny of several types, for exa
26、mple, aQuebec Colony Counter may be used.6.2 Drums; Steel208 L (55 gal) 16 ga. steel, open-headdrum with removable 16 ga. lid fitted with 2.05 cm and 1.90cm threaded ports for venting and sampling.6.3 IncubatorAny incubator capable of maintaining tem-perature of 30 to 35C may be used.6.4 Glass Jars1
27、 L capacity, French square or similarconfiguration.6.5 Pails; Steel18.9 L (5 gal) steel, open-head pail withremovable 16 ga. lid fitted with 2.05 cm and 1.90 cm threadedports for venting and sampling.6.6 SterilizerAny suitable steam sterilizer capable ofproducing the conditions of sterility is accep
28、table. A pressur-ized filter sterilization apparatus of appropriate capacity tofilter sterilize the test fuels and bottom-water used in thenegative control microcosms. A 0.2 m pore-size methylcellulose or cellulose acetate membrane should be used as thefiltration medium.6.7 VortexMixer.7. Reagents a
29、nd Materials7.1 Petri Dishes100 by 15 mm required for performingstandard plate count.7.2 Bacteriological Pipets10.0 mL and 1.1, or 2.2 mLcapacity.7.3 Water Dilution BottlesAny sterilizable glass containerhaving a 150 to 200 mLcapacity and tight closure may be used.7.4 Fuel.NOTE 1Representative fuel
30、samples from each product grade areavailable from all petroleum refiners.7.5 Synthetic Bottom Water.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 Pot
31、ato Dextrose Agar.NOTE 2Items 7.5-7.12 are available from a variety of media manu-facturers and chemical supply companies.8. Inoculum8.1 Inoculum Preparation and Maintenance:8.1.1 Inoculum RevitalizationCultures are Pseudomonasaeruginosa,ATCC No. 33988, Hormoconis resinae,ATCC No.20495, and Yarrowia
32、 tropicalis (formerly Candida tropicalis),ATCC No. 18138. Obtain cultures from ATCC. Before initiat-ing fuel antimicrobial tests, revitalize each of the three culturesin accordance with the instructions contained with each cul-ture.E1259 1028.1.2 Maintenance and Preparation of InoculaAll threecultur
33、es are transferred from slants of a specified agar, (a)Pseudomonas aeruginosa (Plate Count Agar), (b) Hormoconisresinae Potato Dextrose Agar), and (c) Yarrowia tropicali(Potato Dextrose Agar) to synthetic bottom water medium in asuitable size screw-cap glass bottle (French square), and thenoverlaid
34、with 10 times the volume of fuel. This two-phasesystem is kept at room temperature (20 to 30C) for sevendays, and the interface with half the bottom water is transferredweekly to a similar system weekly until used. The bacteriallevels expected are about 107CFU/mL, the yeast levels 106CFU/mL, and mol
35、d levels 104spores/mL. For the test inocu-lum, the bacteria are diluted 1:100 while yeast and molds arediluted 1:10. The counting of the inoculum is done directlyfrom the prepared synthetic bottom water mixture at time zero,just prior to adding inoculum to each setup, and at eachsubsequent time poin
36、t. This procedure may also be followed tomaintain and prepare uncharacterized inocula. If test systemslager than 1.0 L will be used, the challenge inoculum shouldfirst be acclimated to growth in systems that contain the samevolume and fuel to bottom-water ratio as the test systems.NOTE 3In the disti
37、llate fuel industry, additives, including biocides,are calculated on a weight per weight basis so that the specific gravity ofboth the fuel and the biocide (if a liquid formulation) must be taken intoaccount.9. Procedure9.1 Test Array DeterminationThe test plan determines thenumber and capacities of
38、 microcosms needed for the test plan.Preferably, duplicate microcosms will be set up for eachcontrol and test treatment.9.1.1 Controls may include any combination of:9.1.1.1 Filter sterilized fuel over filter sterilized water.9.1.1.2 Challenged, microbicide-free fuel over water.NOTE 4Some commercial
39、ly available fuels contain additives withantimicrobial properties. It may be necessary to filter such fuels throughactivated carbon filters before using them for microbicide performancetesting.9.1.1.3 Reference ControlMicrobicide treated fuel overbottom-water.9.1.2 Microbicide Treatment DoseTesting
40、may be per-formed using a single dose or a range of doses. Typically theminimum and maximum doses permitted under the microbi-cides FIFRA registration are used. One or intermediate con-centrations may also be used. For cost-effectiveness compari-sons, dose selection may be based on the treatment cos
41、ts of themicrobicide against which the test product is being evaluated.9.1.3 To determine the number of microcosms needed forthe test array, add the total number of control and testtreatments and multiply by the number of replicate microcosmsrequired.9.2 Determine Microcosm VolumeMicrocosm volumewil
42、l depend on test objectives. Preliminary microbicidal prod-uct screening may be performed in 1 L microcosms. Achievingthe desired fuel to water ratio, to simulate tank storageconditions, may require drum-size (208 L) microcosms. Typi-cal fuel to water ratios range from 50:1 to 500:1.NOTE 5All fuel-g
43、rades covered by this practice have sufficiently highvapor pressures to permit off-gassing of noxious, potentially toxic volatileorganic carbon (VOC) molecules. Small microcosms should be set upinside a fume hood. Microcosms too large to be stored inside a fume hoodshould be equipped with a vapor tr
44、apping system. A simple system can bedesigned from polyvinylchloride (PVC) piping and buckets filled withactivated carbon (see Fig. 1).9.3 Determine Bottom-Water CompositionDepending onthe anticipated end-use application, bottom-water compositionmay range from distilled water (simulating condensate-
45、wateraccumulation) to sea-water. Recognizing that bottom-waterchemistry varies substantially amongst fuel tanks, site-specifictesting should be performed using filter-sterilized water fromfuel tanks.9.4 Determine Challenge FrequencyThe test plan mayinclude a single challenge or repeated challenges.
46、Typically,when repeated challenges are used, they are scheduled forimmediately after each sample collection time.9.5 Determine Sampling Schedule:9.5.1 Kill-Rate TestingFor speed of kill or kill-rate test-ing, collect samples after 30 min; 4, 8, 16, 24, 48, and 72 h.9.5.2 Persistence of Effect Testin
47、gSample at monthlyintervals until microcosm with highest microbicide dose fails(see 10.2.3).NOTE 6To simulate long-term storage, replace fuel and bottom-watervolumes removed after sampling, but do not re-challenge. To simulatehigh turnover systems, replace fuel and bottom-water volumes andre-challen
48、ge after each sampling.9.6 Set Up Microcosms:9.6.1 If test will include corrosion testing (NACETMO172), prepare corrosion coupons and place them inmicrocosms.9.6.2 Dispense bottom-water then fuel into each microcosm.9.6.3 Draw pre-test samples and enumerate fuel andbottom-water viable counts (see Pr
49、actice D6469 and section9.9).9.7 Add Challenge InoculumInoculate test and positivecontrol microcosms with challenge population. Draw time zero(T0) fuel and bottom-water samples (see Practice D6469 andsection 9.9).NOTE 7Viable count data may be replaced by or augmented withnon-conventional data (see Guide E1326 and Test Method D7463).9.8 SamplingPredetermined intervals, the following pro-tocol is observed.9.8.1 Small (100 L) microcosm arrays. Con-sequently, for test arrays not set up in an a climate controlledenvironment, climatic condit
