1、Designation: D7463 161D7463 18Standard Test Method forAdenosine Triphosphate (ATP) Content of Microorganismsin Fuel, Fuel/Water Mixtures, and Fuel Associated Water1This standard is issued under the fixed designation D7463; the number immediately following the designation indicates the year oforigina
2、l adoption 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 NOTESubsection 12.4 was corrected editorially in October 2016.1. Scope*1.1 T
3、his test method provides a protocol for capturing, concentrating, and testing the adenosine triphosphate (ATP) present ina fuel system sub-sample (that is, test specimen) associated with:1.1.1 Microorganisms and hydrophilic particles found in liquid fuels as described in Table X6.1, or1.1.2 Microorg
4、anisms and hydrophilic particles found in mixture of fuel and associated bottom water or just associated bottomwater.1.1.3 ATP detected by this bioluminescence test can be derived from cellular ATP, extra-cellular ATP, or some combination ofboth.1.1.4 Cellular and extra-cellularATPutilized to perfor
5、mATPbioluminescence are captured and concentrated from a fuel systemsample into an aqueous test specimen (that is, sub-sample) for testing. For example, for a fuel system sample that does not containany visible fuel associated bottom water, the aqueous test specimen is the capture solution itself de
6、scribed in 8.2.1.1. For fuelsystem samples that are a mixture of fuel and associated bottom water (that is, free water), the test specimen is an aliquant of thecapture solution and associated bottom water.1.2 The ATP is measured using a patented bioluminescence enzyme assay, whereby light is generat
7、ed in amounts proportionalto the concentration of ATP in the sample. The light is produced and measured quantitatively using dedicated ATP test pens2 anda dedicated luminometer2 and reported in (instrument specific) Relative Light Units.1.3 This test method is equally suitable for use in the laborat
8、ory or field.1.4 Although bioluminescence is a reliable and proven technology, this method does not differentiate ATP from bacteria orfungi.1.5 For water or capture solution samples, the concentration range of ATP detectable by this test method is 1 1011 M to 3 108 M which is equivalent to 1 1014 mo
9、les/mLto 3 1011 moles/mLfor water samples or capture solution.Assuming testingon fuel phase is performed on a 500 mL volume of fuel the equivalent concentrations is fuel would be: 6 1011 M to 2 1014M.1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are
10、included in this standard.1.6.1 There is one exceptionRelative Light Unit (RLU) as defined in 3.1.19.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety,
11、 health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.8 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of I
12、nternational Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.1 This test method is under the jurisdiction ofASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of SubcommitteeD
13、02.14 on Stability Stability, Cleanliness and CleanlinessCompatibility of Liquid Fuels.Current edition approved June 1, 2016Dec. 1, 2018. Published June 2016December 2018. Originally approved in 2008. Last previous edition approved in 20152016 asD7463 15.D7463 161. DOI: 10.1520/D7463-16E01.10.1520/D
14、7463-18.2 The sole source of supply, repair, recertification, and technical support of the apparatus or test pen known to the committee at this time is Merck KGaA, 64271 Darmstadt,Germany (Worldwide) or Fuel Quality Services, Inc., 4584 Cantrell Rd., Flowery Branch, GA 30542 (USA). If you are aware
15、of alternative suppliers, please provide thisinformation to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee,1 which you mayattend.This document is not an ASTM standard and is intended only to provide the user of an
16、 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 consult prior editions as appropriate. In all cases only the current versionof the standard as publis
17、hed by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12. Referenced Documents2.1 ASTM Standards:3D396 Specification for F
18、uel OilsD975 Specification for Diesel Fuel OilsD1655 Specification for Aviation Turbine FuelsD2880 Specification for Gas Turbine Fuel OilsD4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in WaterD4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricant
19、sD6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products and LubricantsD7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for MicrobiologicalTestingD7467 Specification for Diesel Fuel Oil, Biodiesel B
20、lend (B6 to B20)3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this test method, refer to Terminology D4175.3.1.2 adenosine triphosphate, nmolecule comprised of a purine and three phosphate groups, that serves as the primary energytransport molecule in all biological cells.3.1.3
21、 adenosine monophosphate, nmolecule formed by the removal of two (2) molecules of phosphate (one pyrophosphatemolecule) from ATP.3.1.4 aseptic, adjsterile, free from viable microbiological contamination.3.1.5 bioluminescence, nproduction and emission of light by a living organism as the result of a
22、chemical reaction duringwhich chemical energy is converted to light energy.3.1.6 biomass, nbiological material including any material other than fossil fuels which is or was a living organism orcomponent or product of a living organism.3.1.7 capture solution, naqueous solution of proprietary composi
23、tion used to capture and concentrate hydrophilic compoundsand particles from liquid fuels.3.1.8 cellular adenosine triphosphate (cellular-ATP), nATP present in whole cells, whether they are living or dead.3.1.8.1 DiscussionCellular-ATP is released upon intentional lysis (rupturing) of microbial cell
24、s during the sample preparation process. Microbiallyinfected fluids contain both cellular (cell-associated/cell-bound) and extra-cellular ATP.3.1.9 culturable, adjmicroorganisms that proliferate as indicated by the formation of colonies in or on solid growth media,or the development of turbidity in
25、liquid growth media under specified growth conditions.3.1.10 extracellular ATP, nATP that is not contained inside a cell.3.1.10.1 DiscussionATP is released into the environment when cells die and break open (lyse), for example, as when they are killed by exposure tosome microbicides. ATP released in
26、to the environment can persist for several days after a cell has been lysed. Consequentlyextracellular ATP must be subtracted from total ATP to determine the concentration of viable cell-associated (biomass associated)ATP. However, extracellular ATP can also be an indicator of “distant” biomass, for
27、 example, biofilm in the system.3.1.11 free water, nundissolved water present in a hydrophobic material.water that exists as a separate phase.3.1.11.1 DiscussionFree waterWater present in fuel such as hydrocarbon diesel fuel that can be present as a suspended haze, nonvisible suspendedwater droplets
28、, as droplets on the walls of the vessel, or as a separate layer on the bottom of the vessel.vessel or sample container.3.1.12 fungus, (pl. fungi), nsingle cell (yeasts) or filamentous (molds) microorganisms that share the property of having thetrue intracellular membranes (organelles) that characte
29、rize all higher life forms (Eukaryotes).3 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 standards Document Summary page on the ASTM website.D7463 1823.1.13 hydro
30、philic particles, ncompounds such as ATP, NAD+, NADP+, NADH, NADPH, enzymes, free fatty acids,preservatives, biocides, salts, as well as microorganisms or other articles are often dispersed or distributed in hydrophobic liquidmatrices such as crude oil, vegetable oil, petrol, and kerosine.3.1.14 inv
31、ert emulsion layer, ninterface between the water phase and fuel phase of a fuel water sample which consists of watermicelles dispersed in the fuel.3.1.15 luciferase, ngeneral term for a class of enzymes that catalyze bioluminescent reactions.3.1.16 luciferin, ngeneral term for a class of light-emitt
32、ing biological pigments found in organisms capable of biolumines-cence.3.1.17 luminometer, ninstrument capable of measuring light emitted as a result of non-thermal excitation.3.1.18 pyrogen free, nfree of substances which can induce fever.3.1.19 relative light unit (RLU), ninstrument-specific unit
33、of measurement reflecting the number of photons emitted by theLuciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.3.1.19.1 DiscussionRLU is not an SI unit, however, RLU are proportional to ATP concentration.3.1.20 test specimen, na representative piece of a sample.3.1.20.1 Discus
34、sionFor this test method, the test specimen is an aqueous sub-sample drawn from the fuel system sample that is tested for the presenceof cellular and/or extra-cellularATP. In the case of a fuel system sample that is fuel only in the absence of associated bottom water,the test specimen is the capture
35、 solution (3.1.7). For fuel system samples that contain associated bottom water, the test specimenis an aliquant of the capture solution and associated bottom water (3.1.11).3.1.21 viable microbial biomass, nmetabolically active (living) micro-organisms3.2 Abbreviations:3.2.1 AMPadenosine monophosph
36、ate3.2.2 ATPadenosine triphosphate3.2.3 HDPEhigh density polyethylene3.2.4 NAD+nicotinamide adenine dinucleotide, oxidized form3.2.5 NADHnicotinamide adenine dinucleotide, reduced form3.2.6 NADP+nicotinamide adenine dinucleotide phosphate, oxidized form3.2.7 NADPHnicotinamide adenine dinucleotide ph
37、osphate, reduced form3.2.8 PPpolypropylene3.2.9 RLUrelative light units4. Summary of Test Method4.1 A fuel system sample is obtained either for condition monitoring or for diagnostic testing, for example, fuel from a fuelsystem that is exhibiting problems such as sediment formation or filter pluggin
38、g where the presence of micro-organisms issuspected.4.2 Microbial ATP is captured from the fuel system sample, concentrated into a test specimen, and tested using abioluminescence reaction. The light generated by the luminescence reaction is proportional to the amount of ATP present in thetest speci
39、men as measured in a luminometer.24.3 Test results should be documented for evaluation and trending.4.4 Specialized test methods for fuel samples, water samples, extracellular determination, or resolving potential matrixinterference in bottom water samples are described in Appendix X4 and Appendix X
40、5.5. Significance and Use5.1 This test method measures the concentration of ATP present in the sample. ATP is a constituent of all living cells includingbacteria and fungi. Consequently, the presence of ATP is a reliable indicator of microbial contamination in fuel systems. ATP isnot associated with
41、 matter of non-biological origin.5.2 This test method differs from Test Method D4012 as follows:D7463 1835.2.1 By providing for the rapid determination of ATP present in a fuel (petroleum) sample, a fuel and water mixture sample,fuel-associated bottom water sample, and extracellular ATP freely avail
42、able in the fuel or aqueous sample matrix;5.2.2 By providing for a method to capture, extract, and quantify ATP using self-contained test device and luminometer;5.2.3 By providing a method of quantifying ATP present in fuel or water matrices in generally less than 10 min; and5.2.4 By providing for t
43、he rapid separation of the ATP from chemical interferences that have previously prevented the use ofATP determinations in complex fluids containing hydrocarbons and other organic molecules.5.3 This test method does not require the use of hazardous materials and does not generate biohazard waste.5.4
44、This test method can be used to estimate viable microbial biomass, to evaluate the efficacy of antimicrobial pesticides, andto monitor microbial contamination in fuel storage and distribution systems.6. Interferences6.1 Sample containers and sampling devices shall be clean and free of both ATP and m
45、icrobial contamination.6.2 Ensure that the sampling stick on the ATP Test Pen does not come into contact with any contaminating surfaces. Contactwith a surface or substance can cause contamination with high levels of ATP, giving erroneous results.6.3 Luciferase is an enzyme, which can be inhibited o
46、r denatured by high temperatures, the presence of heavy metals, and highsalt concentrations in the sample. These conditions are unlikely to occur except in samples containing large volumes ofbottom-water samples from storage tanks and similar systems.6.3.1 For samples in which inhibition is suspecte
47、d or likely to occur, testing of a dilution of the sample is described in AppendixX4.7. Apparatus7.1 An example of the luminometer2 is shown as a diagram in Fig. 1.7.2 WarningThe apparatus is not explosion-proof. The instrument should not be operated in explosive atmospheres or inlocations where the
48、re may be explosive fumes, as it cannot be grounded.7.3 Sample bottle, round wide-mouth, nominal capacity 500 mL or 1000 mL, HDPE (High Density Poly Ethylene) orequivalent. There shall be sufficient excess volume in the sample bottle so that there is at least 10 % head space in addition to the500 mL
49、 or 1000 mL sample volume to facilitate the shearing and mixing of the capture solution.7.3.1 Sample bottles may be reused provided they are cleaned and dried correctly. Refer to test suppliers information regardingrecommended cleaning procedure.7.4 Pipettors, fixed volume or adjustable, capable of providing discrete volumes of bottom water to determine the presence ofmatrix interference as described in Appendix X4. Example pipettor volumes include 10 L, 50 L, and 100 L.8. Reagents and Materials8.1 Reagents:8.1.1 ATP
