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    ASTM E2694 - 16

    Standard Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids

    Active Standard ASTM E2694 | Developed by Subcommittee: E34.50

    Book of Standards Volume: 11.03

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    Significance and Use

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

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

    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 five minutes.

    5.5 Although ATP data generally covary with culture data in MWF4, 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 have 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). One example of the species effect is that the amount of ATP per cell is substantially greater for fungi than bacteria. Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells. 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). The presence of any such interferences can be evaluated by performing a standard addition test series as described in Appendix X3. Any impact of interfering chemicals will be reflected as bias relative to data obtained from fluid that does not contain interfering chemicals.

    1. Scope

    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 (MWF).

    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 (RLU) 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 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.

    ASTM Standards

    D1129 Terminology Relating to Water

    D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water

    D4840 Guide for Sample Chain-of-Custody Procedures

    D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration and Reverse Osmosis Membrane Processes

    E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods

    E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method

    E1326 Guide for Evaluating Non-culture Microbiological Tests

    E1497 Practice for Selection and Safe Use of Water-Miscible and Straight Oil Metal Removal Fluids

    E2523 Terminology for Metalworking Fluids and Operations

    ICS Code

    ICS Number Code 75.100 (Lubricants, industrial oils and related products)

    UNSPSC Code

    UNSPSC Code

    Referencing This Standard
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    Link to Active (This link will always route to the current Active version of the standard.)

    DOI: 10.1520/E2694-16

    Citation Format

    ASTM E2694-16, Standard Test Method for Measurement of Adenosine Triphosphate in Water-Miscible Metalworking Fluids, ASTM International, West Conshohocken, PA, 2016, www.astm.org

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