1、Designation: D7687 11D7687 17Standard Test Method forMeasurement of Cellular Adenosine Triphosphate in Fuel,Fuel/Water Mixtures, and Fuel-Associated Water withFueland Fuel-associated Water With Sample Concentration byFiltration1This standard is issued under the fixed designation D7687; the number im
2、mediately following the designation indicates the year oforiginal 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. Scope Scope
3、*1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate(cellular-ATP) content associated with microorganisms found in fuels, fuel/water mixtures fuels and fuel-associated water.1.2 The ATP is measured using a bioluminescence enzyme assay,
4、 whereby light is generated in amounts proportional to theconcentration of cellular-ATPin the samples. The light is produced and measured quantitatively as relative light units (RLU) whichare converted by comparison with an ATP standard, computation to pg ATP/mL and optional further transformation t
5、o Log10pgATP/mL.1.3 This test method is equally suitable for use as a laboratory or portable method.1.4 This test method is limited to fuels with a nominal viscosity 75cSt 75 cSt at test temperature.1.5 This test method detects ATP concentrations in the range of 5.0 pg ATP/mL (0.699(0.699 log10pg AT
6、P/mL) to100 000100 000 pg ATP/mL (5.000(5.000 log10pg ATP/mL) for 20 mL 20 mL samples of fuel or fuel/water mixtures, and 20pg ATP/mL (1.301and 20 pg ATP/mL (1.301 log10pg ATP/mL) to 400 000400 000 pg ATP/mL (5.602(5.602 log10pgATP/mL) for 5 mL samples of fuel-associated water.NOTE 1These ranges wer
7、e calculated with the formula for calculating sample ATP in pg/mL provided in 12.1 based on the minimum recommendedRLU for a 11 ng ng ATP/mL mLATP standard when using the reagents specified in Section 7 and the luminometer specified in 6.4 and corrected witha reagent-method blank as determined in Ap
8、pendix X5.1.6 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifyingATP. This test method does not differentiate between ATP from different sources, for example: from different types ofmicroorganisms, such as bacteria and fungi.1.7 The
9、 values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.8 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 appropri
10、ate safety and health practices and determine the applicability of regulatorylimitations prior to use.1.9 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of Internationa
11、l Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D396 Specification for Fuel OilsD975 Specification for Diesel Fuel OilsD1129 Terminology Relating to Water1 This test method is under t
12、he jurisdiction of ASTM Committee D02 on Petroleum Products Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.Current edition approved April 1, 2011May 1, 2017. Published April 2011June 2017. DOI:10.1520/D768711
13、.Originally approved in 2011. Last previous edition approvedin 2011 as D7687 11. DOI:10.1520 D7687-17.2 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 D
14、ocument Summary page on the ASTM website.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, AST
15、M recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published 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
16、Box C700, West Conshohocken, PA 19428-2959. United States1D1655 Specification for Aviation Turbine FuelsD2069 Specification for Marine Fuels (Withdrawn 2003)3D2880 Specification for Gas Turbine Fuel OilsD3699 Specification for KerosineD4012 Test Method for Adenosine Triphosphate (ATP) Content of Mic
17、roorganisms in WaterD4175 Terminology Relating to Petroleum Products, Liquid Fuels, and LubricantsD6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration and Reverse Osmosis Membrane ProcessesD6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for
18、Petroleum Products and LubricantsD6751 Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate FuelsD7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and FuelAssociated WaterD7464 Practice for Manual Sampling of Liquid Fuels,
19、 Associated Materials and Fuel System Components for MicrobiologicalTestingD7467 Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)D7847 Guide for Interlaboratory Studies for Microbiological Test MethodsD7978 Test Method for Determination of theViableAerobic Microbial Content of Fuels an
20、dAssociatedWaterThixotropic GelCulture MethodE2523 Terminology for Metalworking Fluids and OperationsE2694 Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking FluidsF1671 Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Born
21、e Pathogens UsingPhi-X174 Bacteriophage Penetration as a Test System3. Terminology3.1 Definitions:3.1.1 For definition of terms used in this test method, refer to Terminology D1129, D4175, D6161, and E2523.3.1.2 adenosine monophosphate (AMP), nmolecule formed by the removal of two molecules of phosp
22、hate (one pyrophos-phate molecule) from ATP.3.1.3 adenosine triphosphate (ATP), nmolecule comprised of a purine and three phosphate groups that serves as the primaryenergy transport molecule in all biological cells.3.1.4 aseptic, adjsterile, free from viable microbial contamination.3.1.5 background
23、RLU, nquantity of relative light units resulting from running the test method without incorporation of thesample.3.1.6 bioluminescence, nproduction and emission of light by a living organism as the result of a chemical reaction duringwhich chemical energy is converted to light energy.3.1.7 biomass,
24、nany matter which is or was a living organism or excreted from a microorganism. D61613.1.8 cellular adenosine triphosphate (cellular-ATP), nATP present in whole cells, whether they are living or dead.3.1.8.1 DiscussionCellular-ATPis released upon intentional lysis of microbial cells during the sampl
25、e preparation process. Microbially infected fluidscontain both cellular (cell-associated/ cell-bound) (cell-associated/cell-bound) and extra-cellular ATP.3.1.9 culturable, adjmicroorganisms that proliferate as indicated by the formation of colonies on solid growth media or thedevelopment of turbidit
26、y in liquid growth media under specific growth conditions.3.1.10 extra-cellular, adjmolecules or substances that are either excreted by living cells or released from microbial cells thathave lysed (see 3.1.14) in the sample.3.1.10.1 DiscussionExtra-cellular ATP is ATP that has been released from mic
27、robial cells that have either fully or partially lysed in the sample, theupstream fluid (fuel or water phase), or both.3.1.10.2 Discussion3 The last approved version of this historical standard is referenced on www.astm.org.D7687 172Lysis can occur due to natural life cycle process, antimicrobial tr
28、eatment or a combination of these factors. Extra-cellularATP canunder certain circumstances persist for periods greater than 24 h 24 h after cell lysis depending on physical/chemical conditions.3.1.11 luciferase, ngeneral term for a class of enzymes that catalyze bioluminescent reactions.3.1.12 luci
29、ferin, ngeneral term for a class of light-emitting biological pigments found in organisms capable of biolumines-cence.3.1.13 luminometer, ninstrument capable of measuring light emitted as a result of nonthermal excitation.3.1.14 lysis, ndisintegration or destruction of whole bacterial cells. F16713.
30、1.15 relative light unit (RLU), ninstrument and assay specific unit of measurement reflecting the number of photons emittedby the Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.3.1.15.1 DiscussionRLU is not an SI unit, however, RLU are proportional to ATP concentration.3.1.1
31、6 viable microbial biomass, nmetabolically active (living) microorganisms.3.2 Acronyms:3.2.1 AMPadenosine monophosphate.3.2.2 ATP adenosine triphosphate.3.2.3 HDPEhigh density polyethylene.3.2.4 PPpolypropylene.3.2.5 RLUrelative light unit.4. Summary of Test Method4.1 A control assay is performed us
32、ing 100 L 100 L of 1.01.0 ng 6 0.05 ng 0.05 ng ATP/mL standard to produce RLUctrl.4.2 A 20 mL 20 mL sample of fuel or fuel/water mixture or 5.0 mL 5.0 mL bottom-water is placed into a syringe and thenpressure-filtered through a 0.7 m, 0.7 m, glass-fiber, in-line, depth filter.4.3 The retentate is th
33、en washed with a reagent to remove extra-cellular ATP and non-ATP contaminants that might otherwiseinterfere with the cellular-ATP assay.4.4 The filter is air-dried.4.5 A lysing reagent is used to release cellular-ATP from microbial cells that have been captured on the glass-fiber filter, andthe fil
34、trate is dispensed into an unused culture tube.4.6 The filtrate is diluted 1 to 10 with a buffer solution.4.7 A 100 L 100 L volume of diluted filtrate is transferred to an unused culture tube into which 100 L 100 L ofLuciferin-Luciferase reagent has been previously dispensed.4.8 The culture tube is
35、placed into a luminometer and the light intensity is read as RLUobs.4.9 RLUobs is normalized to an actual pg ATP/mL concentration through an equation that accounts for the result of the controlassay (RLUctrl), the volume of the sample processed, and the method dilution factor.NOTE 2Optionally, for c
36、ondition monitoring purposes, pg ATP/mL are converted to Log10pg ATP/mL of sample by computation.5. Significance and Use5.1 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells,including bacteria and fungi. Consequently, the pres
37、ence of cellular-ATP is an indicator of total metabolically active microbialcontamination in fuels. ATP is not associated with matter of non-biological origin.5.2 This test method is similar to Test Method E2694 except for the volumes sampled.5.3 This test method differs from Test Method D4012 in th
38、at it utilizes filtration and wash steps designed to eliminateinterferences that have historically rendered ATP testing unusable with complex organic fluids such as fuel, fuel/water mixturesfuel and fuel-associated water.5.4 This test method differs from Test Method D7463 in several regards:5.4.1 Te
39、st Method D7463 reports relative light units (RLU). Consistent with Test MethodMethods D4012 and E2694, this testmethod reports ATP concentration.5.4.2 This test method detects only cellular-ATP and it can be used to detect cellular-ATP in fuels and fuel stocks from whichsmall quantities of water do
40、 not separate readily (for example, ethanol blended gasoline containing 5 % v 5% v/v v ethanol).D7687 173Test Method D7463 cannot be used to recover ATP from fuels from which small quantities of water do not separate readily (forexample, ethanol blended gasoline containing 5%5 % v v/v v ethanol).5.4
41、.3 This test method measures cellular-ATP in a single measurement (as pg ATP/mL). Test Method D7463 detects total ATP(as RLU) and extra-cellular ATP (as RLU) using two separate analyses and permits computation of cellular-ATP (as RLU) as thedifference between total and extracellular ATP.5.4.4 Test M
42、ethod D7463 suggests a nominal 500 mL 500 mL fuel sample volume. This test method suggests a nominal 20 mL20 mL fuel sample.5.5 This test method can be used with all fuels specified in Specifications D396, D975, D1655, D2069, D2880, D3699, D6751,and D7467 and other fuels with nominal viscosities 75
43、cSt at 2075 cSt at 20 6 2.5.6 The ATP test provides rapid test results that reflect the total bioburden in the sample. It thereby reduces the delay betweentest initiation and data capture, from the 3636 h to 48 h 48 h (or longer) required for culturable colonies to become visible, toapproximately 5
44、min.5 min.5.7 Although ATP data generally covary with culture data in fuel, fuel/water mixtures, fuel and fuel-associated water, differentfactors affect ATP concentration than those that affect culturability.5.7.1 Culturability is affected primarily by the ability of captured microbes to proliferate
45、 on the growth medium provided, underspecific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample may beviable but not detected by any one culture test.45.7.2 ATP concentration is affected by: the microbial species present, the physiologic
46、al states of those species, and the totalbioburden (see Appendix X1).5.7.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells thanbacteria.5.7.2.2 Within a species, cells that are more metabolically active will have moreATP per cel
47、l than dormant cells, such as fungalspores. Because fungal spores are more hydrophobic than active fungal material (mycelium), spores may be the only indicator offungal proliferation when fuel samples are taken from some fuel systems, but they will not be detected by a test for ATP.5.7.2.3 The great
48、er the total bioburden, the greater the ATP concentration in a sample.5.7.3 The possibility exists that the rinse step (11.15) may not eliminate all chemical substances that can interfere with thebioluminescence reaction (11.3911.37).5.7.3.1 The presence of any such interferences can be evaluated by
49、 performing a standard addition test series or dilution seriesas described in Appendix X4. The precision statement in Section 13 will not apply.5.8 As explained in Test Method D7978, there are inherent difficulties in assessing precision of microbiological procedures forfuels on account of the inherent variability of the determinant and various determinable and indeterminable sources of inaccuracy(see Guide D7847).5.8.1 The precision of any microbiological analytical method will generally be considerably less t
