1、Designation: D8070 16Standard Test Method forScreening of Fuels and Fuel Associated AqueousSpecimens for Microbial Contamination by Lateral FlowImmunoassay1This standard is issued under the fixed designation D8070; the number immediately following the designation indicates the year oforiginal adopti
2、on or, in the case of revision, the year 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. Scope1.1 This test method describes a procedure that can be usedin the field or in
3、a laboratory to detect antigens indicative ofmicrobial contamination in liquid fuels, including thoseblended with synthesized hydrocarbons or biofuels, with kine-matic viscosities (at 40 C) of 24 mm2s1(for example,Specifications D396, D975, and D1655) and in fuel-associatedwater.1.1.1 This test meth
4、od has been validated by an ILS for arange of middle distillate fuels meeting Specification D1655,EN590, Specification D975, and ISO 8217:2012.1.2 This test method semi-quantitatively assesses the con-centration of specific antigens generated by aerobic microor-ganisms during active growth in fuels.
5、1.2.1 A proprietary formulation of antibodies and antibodymixtures is used to detect three types of microbial antigencontamination: antigens generally found in aerobic bacteria,antigens generally present in common fungi (yeast and molds),and an antigen that is characteristic of Hormoconis resinae (t
6、hefungus most commonly associated with fuel biodeterioration).1.2.2 Although the antibodies and antibody mixtures arecharacteristic of diverse types of bacteria and fungi, it isunlikely that they are universal. Recognizing that for everymicrobe that has been isolated and characterized, it is likelyt
7、hat there are a billion that have not. Consequently, as is thecase with all microbiological test methods, this test methoddoes not purport to detect 100 % of the microbes present in afuel or fuel-associated water sample.1.3 For each of the three sets of antigen detected (H.resinae, common fungi, and
8、 aerobic bacteria), the test detectswhether the antigen concentration present is within set rangesrepresenting negligible, moderate, or heavy microbial contami-nation.1.3.1 For fuel specimens, the antigen concentration rangesdetected are 750 g L (heavy).1.3.2 For specimens of water associated with f
9、uel, theantigen concentration ranges detected are 166 g mL (heavy).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 any, associated with its use. It
10、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. For a specifichazard statement, see Section 8.2. Referenced Documents2.1 ASTM Standards:2D396 Specification for Fuel OilsD9
11、75 Specification for Diesel Fuel OilsD1129 Terminology Relating to WaterD1655 Specification for Aviation Turbine FuelsD4175 Terminology Relating to Petroleum Products, LiquidFuels, and LubricantsD4176 Test Method for Free Water and Particulate Contami-nation in Distillate Fuels (Visual Inspection Pr
12、ocedures)D6469 Guide for Microbial Contamination in Fuels and FuelSystemsD6974 Practice for Enumeration of Viable Bacteria andFungi in Liquid FuelsFiltration and Culture ProceduresD7464 Practice for Manual Sampling of Liquid Fuels, As-sociated Materials and Fuel System Components forMicrobiological
13、TestingD7687 Test Method for Measurement of Cellular Adenosine1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition appro
14、ved Nov. 15, 2016. Published January 2017. DOI: 10.1520/D8070-16.2For 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 webs
15、ite.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of I
16、nternational Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1Triphosphate in Fuel, Fuel/Water Mixtures, and Fuel-Associated Water with Sample Concentration by FiltrationD7847 Guide for Interlaboratory Studies for Microbiologic
17、alTest MethodsD7978 Test Method for Determination of the Viable AerobicMicrobial Content of Fuels and Associated WaterThixotropic Gel Culture MethodE1326 Guide for Evaluating Non-culture MicrobiologicalTestsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 Other Standards:BS EN590 S
18、tandard for Diesel Fuel3ISO 8217:2012 Petroleum productsFuels (Class F)Specifications of marine fuels4NATO Logistics Handbook Chapter 15: Fuels, Oils, Lubri-cants and Petroleum Handling Equipment, Annex A: AideMemoire on Fuels in NATO5IATA Guidance Material on Microbiological Contaminationin Aircraf
19、t Fuel Tanks, Fifth Edition, 201563. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this test method, referto Terminologies D1129, D4175, E2756, and Guide E1326.3.2 Definitions:3.2.1 aerobic, adj(1) taking place in the presence ofoxygen, (2) living or active in the presence of oxy
20、gen.3.2.2 antibody, nan immunoglobulin, a protein that isproduced as a part of the immune response which is capable ofspecifically combining with the antigen.3.2.2.1 DiscussionIn the context of this test method,antibodies created for this purpose are utilized in conjunctionwith visual indicators to
21、detect presence of microbial antigens.3.2.3 antigen, na substance that stimulates the host toproduce an immune response. In the context of this testmethod, specific antigens are detected as indicators of micro-bial contamination.3.2.4 buffer, na compound or mixture that, when con-tained in solution,
22、 causes the solution to resist change in pH.3.2.4.1 DiscussionEach buffer has a characteristic limitedrange of pH over which it is effective.3.2.5 colony, na discreet visible aggregate of microorgan-isms that develops when a viable microorganism, or particlecontaining viable microorganisms, is intro
23、duced into a gel-based nutritive culture medium and reproduces there.3.2.6 colony forming unit (CFU), na viable microorgan-ism or aggregate of viable microorganisms, which prolifer-ate(s) in a culture medium to produce a viable colony.3.2.7 culturable, adjin microbiology, a microorganismcapable of p
24、roliferating in or on a nutrient medium, under agiven set of growth conditions, to either form a colony (in or ongel media) or generate turbidity (in a liquid medium).3.2.8 lateral flow device, nin immunology, an antibody-impregnated, porous medium through which an antigen-containing buffer is permi
25、tted to wick in order to bring theantigen into contact with the antibody.3.2.8.1 DiscussionTypically, the antibody is linked to anindicator which produces a color reaction when antibody andantigen combine.3.2.9 metabolite, na chemical substance produced by anyof the many complex chemical and physica
26、l processes involvedin the maintenance of life.3.2.10 microorganisms, norganisms too small to be seenwith the naked eye, which generally include bacteria, protozoa,fungi, and microalgae (sometimes collectively called slime ormicrobial contamination).3.2.10.1 DiscussionIn the context of this test met
27、hod,microorganisms are bacteria and fungi (yeasts and molds) thatare capable of growth in fuels and associated aqueous-phasefluid.3.2.11 surfactant (surface active agent), na substance thataffects the interfacial or surface tension of solutions even whenpresent in very low concentrations.3.3 Definit
28、ions of Terms Specific to This Standard:3.3.1 extraction fluid, na mixture of buffer and surfactantsused to extract antigens from the specimens.3.4 Acronyms:3.4.1 CFUcolony forming unit3.4.2 FSIIfuel system icing inhibitor3.4.3 LFDlateral flow device4. Summary of Test Method4.1 Microbial contaminati
29、on is detected using a series ofantibodies immobilized onto a test tray in the form of threepairs of lateral flow devices (LFDs). These antibodies are usedto detect antigens from common bacteria and fungi thatproliferate in fuel tanks and systems.4.1.1 The LFDs contain broad spectrum antibodies rais
30、edagainst cell components and materials generated during micro-bial growth on hydrocarbons (the antigens). These antibodiesindicate the presence of H. resinae, other common fungi, andaerobic bacteria within concentration ranges described in 1.3.4.2 Fuel or aqueous specimens from fuel are mixed with
31、anaqueous extraction fluid in an extraction bottle. The extractionfluid captures antigenic material present in the specimen.4.3 Four drops of the separated extraction fluid are dis-pensed into the sample well of each of the six LFDs, which arearranged in three pairs.3Available from British Standards
32、 Institution (BSI), 389 Chiswick High Rd.,London W4 4AL, U.K., http:/.4Available from International Organization for Standardization (ISO), ISOCentral Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,Geneva, Switzerland, http:/www.iso.org.5Available from North Atlantic Treaty Organ
33、ization (NATO) at http:/www.nato.int/docu/logi-en/1997/lo-15a.htm.6Available from International Aviation Transport Association (IATA) at http:/store.iata.org.D8070 1624.4 For each of the three types of contamination (H. resinae,other common fungi, and aerobic bacteria), a pair of LFDsindicates the c
34、oncentration range of the detected antigenpresent in the specimen. For each LFD pair, the minimumdetection level is set so that one is at the lower boundary of themoderate contamination range and the other at the lowerboundary of the heavy contamination range of antigen concen-tration stated in 1.3.
35、4.5 Presence of antigen below the minimum level of detec-tion for each LFD is indicated by development of red controland test lines. If only the control line becomes visible, thenantigen concentration in the specimen is above the minimumlevel of detection for the LFD. When antigen is present at orab
36、ove threshold concentrations, test lines on the LFD fail toappear. By reading both LFDs in the pair, it can be establishedwhich range the antigen concentration falls within, that is,negligible, moderate, or heavy.4.6 The control lines appear within 10 min and the result isread at 15 min, although th
37、e lines are stable up to 30 min.4.7 If contaminating antigens are not present, test lines willalso appear within 10 min and the result is read within 30 min(see 4.6). If test lines do not appear, then this indicates thepresence of contamination at the concentration ranges de-scribed in 1.3.4.8 The r
38、esults and antigen concentration ranges are deter-mined and recorded.5. Significance and Use5.1 This test method is intended to provide a tool forassessing whether fuel storage and distribution facilities, or enduser fuel tanks, are subject to microbial growth, and to alertfuel suppliers or users to
39、 the potential for fuel quality oroperational problems or the requirement for preventative orremedial measures, or both.5.2 This test method allows assessment of whether antigensgenerated by microbial activity in the specimens are presentwithin specific defined ranges.5.3 This test method measures t
40、he presence of microbial andmetabolite antigens in a specimen. The antigens are generatedfrom the living cells and metabolites created by fungi andbacteria during growth on fuel. Consequently, the presence ofantigens is an indicator of microbial contamination in fuelsystems. Antigens are not associa
41、ted with matter of nonbio-logical origin.5.3.1 Some of the antigens detected by this test method canpersist after treatment with a biocide. See 11.4.5.4 This test method is semi-quantitative and can be used todetermine whether contamination in samples drawn from fueltanks and systems is negligible o
42、r present at moderate or heavylevels.5.4.1 Further information on using the test to assess biode-terioration risk is provided in Appendix X1.5.5 The significance of these levels to the operator willdepend on the fuel type, the sampling location, the equipmentor facility sampled, and the specific ope
43、rating circumstances.5.6 Further guidance on interpretation of test results can befound in Guide D6469, in Energy Institute guidelines for theinvestigation of the microbial content of petroleum fuels, andin the IATA Guidance Material on Microbial Contamination inAircraft Fuel Tanks.5.7 Further guida
44、nce on sampling can be found in PracticeD7464.5.8 Testing can be conducted on a routine basis or toinvestigate incidents.5.9 Microbiological tests are not intended to be used todetermine compliance with fuel specifications or limits. Theimplementation of specification limits for microbiologicalconta
45、mination in fuels is generally not appropriate, and micro-bial contamination levels cannot be used alone or directly tomake inferences about fuel quality or fitness for use.5.10 When interpreting results, it must be appreciated thatthe test result applies only to the specific sample and specimentest
46、ed and not necessarily to bulk fuel. Microbiological con-tamination usually shows a highly heterogeneous distributionin fuel systems, and therefore, analysis of a single sample willrarely provide a complete assessment of the overall levels ofcontamination present.5.11 Water phase will usually contai
47、n substantially higheramounts of microbial contamination than fuel phase and,consequently, a different interpretation of results is required.This is why this test method reports antigen concentration permL for water and per L for fuel.5.12 This test method differs from some other methods (forexample
48、 Test Methods D7687 and D7978) and practices (forexample Practice D6974) in that it detects microbial activity infuels or associated aqueous specimens in the field and does notneed to be performed in a laboratory or in an aseptic environ-ment. It may be used in a laboratory.5.13 This test method doe
49、s not require specialist microbio-logical experience or knowledge.5.14 This test method provides rapid results that reflect thetotal active microbial contamination in the specimen, andenables result to be obtained within 15 min.5.15 This test method differentiates among three ranges ofcontamination for H. resinae, other fungi, and aerobic bacteria(see 1.3).6. Interferences and Possible Test Method Errors6.1 Drops of the extraction fluid can fail to be expelled fromthe extraction bottle if particulate material in the sample blocksthe dr
