1、Designation: E 1259 05Standard Practice forEvaluation of Antimicrobials in Liquid Fuels Boiling Below390C1This standard is issued under the fixed designation E 1259; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 l
3、iquid fuels (as defined by Specification D 396, D 910,D 975, D 1655, D 2069, D 2880, D 3699, D 4814, D 6227, andD 6751), 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
4、 Laboratory Practice (GLP) is required and tofollow them where appropriate (40 CFR, 160), or as revised.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and
5、health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 396 Specification for Fuel OilsD 910 Specification for Aviation GasolinesD 975 Specification for Diesel Fuel OilsD 1655 Specification for Aviation Turbine FuelsD 2069
6、 Specification for Marine FuelsD 2880 Specification for Gas Turbine FuelsD 3699 Specification for KerosineD 4057 Practice for Manual Sampling of Petroleum andPetroleum ProductsD 4814 Specification forAutomotive Spark-Ignition EngineFuelD 5465 Practice for Determining Microbial Colony Countsfrom Wate
7、rs Analyzed by Plating MethodsD 6227 Specification for Grade 82 Unleaded AviationGasolineD 6293 Test Method for Oxygenates and Paraffin, Olefin,Napththene, Aromatic (O-PNOA) Types in Low-OlefinSpark Ignition Engine Fuels by Gas ChromatographyD 6469 Guide to Microbial Contamination in Fuels andFuel S
8、ystemsD 6729 Test Method for Determination of Individual Com-ponents in Spark ignition Engine Fuels by 100-MeterCapillary High Resolution Gas ChromatographyD 6733 Test Method for Determination of Individual Com-ponents in Spark ignition Engine Fuels by 50-MeterCapillary High Resolution Gas Chromatog
9、raphyD 6751 Specification for Biodiesel (B100) Blend Stock forDistillate FuelsD 6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresE 1326 Guide for Evaluating Nonconventional Microbio-logical Tests Used for Enumerating Bacteria2.2 NACE Standard:
10、TM0172 Determining Corrosive Properties of Cargoes inPetroleum Product Pipelines32.3 Federal Standards:40 CFR, Part 79, Fuels and Fuel Additives RegistrationRegulations440 CFR, Part 152, Pesticide Registration and ClassificationProcedures43. Terminology3.1 Definitions of Terms Specific to This Stand
11、ard:3.1.1 antimicrobial, nsee biocide.3.1.2 biocide, na physical 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 an
12、timicrobials.3.1.3 microbially-influenced deterioration,ndecomposition/degradation of material (fuel) or makingunsuitable for use, as a result of metabolic activity or thepresence of microbes.3.1.4 microbicide, nsee biocide.1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides
13、and is the direct responsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 1, 2005. Published December 2005. Originallyapproved in 1988. Last previous edition approved in 2001 as E 1259 01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcont
14、act ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Item No. 21204, available from NACE International, Houston TX.4Available from U.S. Government Printing Office Superintendent of Document
15、s,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.3.1.5 microcosm, na miniature system used to modellarger systems.3.1.5.1 DiscussionIt is generally impractical to evaluate
16、microbicide performance 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 antimicrobi
17、al efficacyunder 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 amicro
18、bially contaminated 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 untreated
19、aliquots are 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 dependi
20、ng onthe specific 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 D 6469 details the types of problems associatedwith uncontrolled micro
21、bial growth in 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 v
22、ariety offuel 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 additive
23、s (40CFR 79). 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
24、types, for example, 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
25、.4 Glass Jars1 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 ster
26、ility is acceptable. 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
27、.7. Reagents and 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.57.5 Synthet
28、ic Bottom Water.67.6 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.8. Inoculum8.1 Inoculum Preparation and Maintenance:8.1.1 Inoculu
29、m RevitalizationCultures are Pseudomonasaeruginosa,ATCC No. 33988, Hormoconis resinae,ATCC No.20495, and Yarrowia 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 t
30、he instructions contained with each cul-ture.8.1.2 Maintenance and Preparation of InoculaAll threecultures 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 s
31、ynthetic bottom water medium in asuitable size screw-cap glass bottle (French square), and thenoverlaid 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
32、weekly until used. The bacteriallevels expected are about 107CFU/mL, the yeast levels 106CFU/mL, and mold 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
33、 water mixture at time zero,5Representative fuel samples from each product grade are available from allpetroleum refiners.6Items 7.5-7.12 are available from a variety of media manufacturers andchemical supply companies.E1259052just prior to adding inoculum to each setup, and at eachsubsequent time p
34、oint. 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 1Caution:
35、In the distillate fuel industry, additives, includingbiocides, are calculated on a weight per weight basis so that the specificgravity of both the fuel and the biocide (if a liquid formulation) must betaken into account.9. Procedure9.1 Test Array DeterminationThe test plan determines thenumber and c
36、apacities of 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 2Som
37、e commercially 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
38、DoseTesting 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 t
39、reatment costs 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 Microcosms VolumeMicroc
40、osm volumewill 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 rations range form 50:1 to 500:1.NO
41、TE 3All fuel-grades 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 w
42、ith a vapor trapping 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 (simulati
43、ng condensate-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 singe, To challenge or repe
44、ated challenges. 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
45、 of Effect TestingSample at monthlyintervals until microcosm with highest microbicide dose fails(see 10.2.3).NOTE 4To 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 volu
46、mes andre-challenge 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 viab
47、le counts (see Practice D 6469 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 D 6469 andsection 9.9).NOTE 5Viable count data may be replaced by or augmented withnon-conv
48、entional data (see Guide E 1326).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 conditions (in particular: temperature,relative humidity, and dew
49、point) will effect microcosm ecol-ogy.10.2.3.3 Fuel ChemistryFuels are manufactured to speci-fications. Fuels with substantially different chemical profiles(for example, as determined by Test Method D 6293) mayyield identical specification test results. Moreover, fuels arechemically unstable, consequently, their chemistry changeswith time. Production lot variability and aging contribute tovariability in a fuels tendency to support microbial growth infuel associated water.10.2.3.4 SamplingFor larger microcosm systems, 100 %system capture fo
