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Significance and Use
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.
This test method is similar to Test Method E2694 except for the volumes sampled.
This test method differs from Test Method D4012 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, fuel/water mixtures and fuel-associated water.
This test method differs from Test Method D7463 in several regards:
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 D7463 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).
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 the difference between total and extracellular ATP.
Test Method D7463 suggests a nominal 500 mL fuel sample volume. This test method suggests a nominal 20 mL fuel sample.
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 to 48 h (or longer) required for culturable colonies to become visible, to approximately 5 min.
Although ATP data generally covary with culture data in fuel, fuel/water mixtures, and fuel-associated water, different factors affect ATP concentration than those that affect culturability.
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.
ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see Appendix X1).
One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than bacteria.
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.
The greater the total bioburden, the greater the ATP concentration in a sample.
The possibility exists that the rinse step (11.15) may not eliminate all chemical substances that can interfere with the bioluminescence reaction (11.39).
The presence of any such interferences can be evaluated by performing a standard addition test series or dilution series as described in Appendix X4.
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 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 ≤ 75cSt 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 or fuel/water mixtures, 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 12.1 based on the minimum recommended RLU for a 1 ng ATP/mL standard when using the reagents specified in Section 7 and the luminometer specified in 6.4 and corrected with a reagent-method blank as determined in Appendix X5.
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 and health practices and determine the applicability of regulatory limitations prior to use.
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 Oils
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, Petroleum Products, 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 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)
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)