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 E2694 except for the volumes sampled.

5.3 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 and fuel-associated water.

5.4 This test method differs from Test Method D7463 in several regards:

5.4.1 Test Method D7463 reports relative light units (RLU). Consistent with Test Methods D4012 and E2694, 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 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).

5.4.3 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 the difference between total and extracellular ATP.

5.4.4 Test Method D7463 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 D396, D975, D1655, D2069, D2880, D3699, D6751, and D7467 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.4

5.7.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (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 than bacteria.

5.7.2.2 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.

5.7.2.3 The greater 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 the bioluminescence reaction (11.37).

5.7.3.1 The presence of any such interferences can be evaluated by performing a standard addition test series or dilution series as 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 for fuels 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 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 12.1 based on the minimum recommended RLU for a 1 ng/mL ATP 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.

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.