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Significance and Use
5.1 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 presence of cellular-ATP is an indicator of total metabolically active microbial contamination in fuels. ATP is not associated with matter of non-biological origin.
5.2 This test method is similar to Test Method except for the volumes sampled.
5.3 This test method differs from Test Method in that it utilizes filtration and wash steps designed to eliminate interferences that have historically rendered ATP testing unusable with complex organic fluids such as fuel and fuel-associated water.
5.4 This test method differs from Test Method in several regards:
5.4.1 Test Method reports relative light units (RLU). Consistent with Test Methods and , this test method 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 which small quantities of water do not separate readily (for example, ethanol blended gasoline containing ≥5 % v/v ethanol). Test Method cannot be used to recover ATP from fuels from which small quantities of water do not separate readily (for example, ethanol blended gasoline containing ≥5 % v/v ethanol).
5.4.3 This test method measures cellular-ATP in a single measurement (as pg ATP/mL). Test Method detects total ATP (as RLU) and extra-cellular ATP (as RLU) using two separate analyses and permits computation of cellular-ATP (as RLU) as the difference between total and extracellular ATP.
5.4.4 Test Method suggests a nominal 500 mL fuel sample volume. This test method suggests a nominal 20 mL fuel sample.
5.5 This test method can be used with all fuels specified in Specifications , , , , , , , and and other fuels with nominal viscosities ≤75 cSt at 20° ± 2°.
5.6 The ATP test provides rapid test results that reflect the total bioburden in the sample. It thereby reduces the delay between test initiation and data capture, from the 36 h to 48 h (or longer) required for culturable colonies to become visible, to approximately 5 min.
5.7 Although ATP data generally covary with culture data in fuel and fuel-associated water, different factors affect ATP concentration than those that affect culturability.
5.7.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample may be viable but not detected by any one culture test.
5.7.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see ).
22.214.171.124 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than bacteria.
126.96.36.199 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells, such as fungal spores. Because fungal spores are more hydrophobic than active fungal material (mycelium), spores may be the only indicator of fungal proliferation when fuel samples are taken from some fuel systems, but they will not be detected by a test for ATP.
188.8.131.52 The greater the total bioburden, the greater the ATP concentration in a sample.
5.7.3 The possibility exists that the rinse step ( ) may not eliminate all chemical substances that can interfere with the bioluminescence reaction ( ).
184.108.40.206 The presence of any such interferences can be evaluated by performing a standard addition test series or dilution series as described in . The precision statement in Section will not apply.
5.8 As explained in Test Method , there are inherent difficulties in assessing precision of microbiological procedures for fuels on account of the inherent variability of the determinant and various determinable and indeterminable sources of inaccuracy (see Guide ).
5.8.1 The precision of any microbiological analytical method will generally be considerably less than that of methods widely used in the petroleum industry for analysis of physical and chemical properties of fuels.
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 and fuel-associated water.
1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of cellular-ATP in the samples. The light is produced and measured quantitatively as relative light units (RLU) which are converted by comparison with an ATP standard, computation to pg ATP/mL and optional further transformation to Log10[pg ATP/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 ≤75 cSt at test temperature.
1.5 This test method detects ATP concentrations in the range of 5.0 pg ATP/mL (≈0.699 log10[pg ATP/mL]) to 100 000 pg ATP/mL (≈5.000 log10[pg ATP/mL]) for 20 mL samples of fuel and 20 pg ATP/mL (≈1.301 log10[pg ATP/mL]) to 400 000 pg ATP/mL (≈5.602 log10[pg ATP/mL]) for 5 mL samples of fuel-associated water.
Note 1: These ranges were calculated with the formula for calculating sample ATP in pg/mL provided in based on the minimum recommended RLU for a 1 ng/mL ATP standard when using the reagents specified in Section and the luminometer specified in and corrected with a reagent-method blank as determined in .
1.6 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP. This test method does not differentiate between ATP from different sources, for example: from different types of microorganisms, such as bacteria and fungi.
1.7 The 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 responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.
D396 Specification for Fuel Oils
D975 Specification for Diesel Fuel
D1129 Terminology Relating to Water
D1655 Specification for Aviation Turbine Fuels
D2069 Specification for Marine Fuels
D2880 Specification for Gas Turbine Fuel Oils
D3699 Specification for Kerosine
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration, and Reverse Osmosis Membrane Processes
D6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and Lubricants
D6751 Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels
D7463 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
D7464 Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
D7467 Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)
D7847 Guide for Interlaboratory Studies for Microbiological Test Methods
D7978 Test Method for Determination of the Viable Aerobic Microbial Content of Fuels and Associated WaterThixotropic Gel Culture Method
E2523 Terminology for Metalworking Fluids and Operations
E2694 Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids
F1671 Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using Phi-X174 Bacteriophage Penetration as a Test System
ICS Number Code 75.160.20 (Liquid fuels)
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ASTM D7687-21, Standard Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With Sample Concentration by Filtration, ASTM International, West Conshohocken, PA, 2021, www.astm.orgBack to Top