5.1 This method measures the concentration of ATP present in the sample. ATP is a constituent of all living cells, including bacteria and fungi. Consequently, the presence of ATP is an indicator of total microbial contamination in metalworking fluids. ATP is not associated with matter of non-biological origin.

5.2 Test Method D4012 validated ATP as a surrogate for culturable bacterial data (Guide E1326).

5.3 This method differs from Test Method D4012 in that it eliminates interferences that have historically rendered ATP testing unusable with complex organic fluids such as MWFs.

5.4 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.

5.5 Although ATP data generally covary with culture data in MWF,4 different factors affect ATP concentration than those that affect culturability.

5.5.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. It has been estimated that less than 1 % of the species present in an environmental sample will form colonies under any given set of growth conditions.5

5.5.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total bioburden (see Appendix X1).

5.5.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for fungi than bacteria.

5.5.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells.

5.5.2.3 The greater the total bioburden, the greater the ATP concentration in a sample.

5.5.3 The possibility exists that the rinse step (11.15) may not eliminate all chemical substances that can interfere with the bioluminescence reaction (11.39).

5.5.3.1 The presence of any such interferences can be evaluated by performing a standard addition test series as described in Appendix X3.

5.5.3.2 Any impact of interfering chemicals will be reflected as bias relative to data obtained from fluid that does not contain interfering chemicals.

1.1 This test method provides a protocol for capturing, extracting, and quantifying the adenosine triphosphate (ATP) content associated with microorganisms found in water-miscible metalworking fluids (MWFs).

1.2 The ATP is measured using a bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of ATP in the samples. The light is produced and measured quantitatively as relative light units (RLUs) which are converted by comparison with an ATP standard and computation to pg ATP/mL.

1.3 This test method is equally suitable for use in the laboratory or field.

1.4 The test method detects ATP concentrations in the range of 4.0 pg ATP/mL to 400 000 pg ATP/mL.

1.5 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP. The method does not differentiate between ATP from different sources, for example, from different types of microorganisms, such as bacteria and fungi.

1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.7 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.

1.8 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.