ASTM E2694-21

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2694 − 16 E2694 − 21 An American National Standard
Standard Test Method for
Measurement of Adenosine Triphosphate in Water-Miscible
Metalworking Fluids
This standard is issued under the fixed designation E2694; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
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).(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 (RLU)(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 safety, health, and healthenvironmental 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.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
This test method is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Health
and Safety Standards for Metal Working Fluids.
Current edition approved Oct. 1, 2016Nov. 1, 2021. Published October 2016November 2021. Originally approved in 2009. Last previous edition approved in 20112016
as E2694 - 11.E2694 – 16. DOI:10.1520/E2694-16. DOI:10.1520/E2694-21.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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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
2.2 Government Standards:
29 CFR 1910.1000 Occupational Safety and Health Standards; Air contaminantsAir Contaminants
29 CFR 1910.1450 Occupational Exposure to Hazardous Chemicals in Laboratories
3. Terminology
3.1 Definitions: For definition of terms used in this method, refer to Terminology standards D1129, D6161, and E2523.
3.1 Definitions:
3.1.1 For definitions of terms used in this test method, refer to Terminologies D1129, D6161, and E2523.
3.1.2 adenosine monophosphate (AMP), n—the molecule formed by the removal of two molecules of phosphate (one
pyrophosphate molecule) from ATP.
3.1.3 adenosine triphosphate (ATP), n—a molecule comprised of a purine and three phosphate groups that serves as the primary
energy transport molecule in all biological cells.
3.1.4 aseptic, adj—sterile, free from viable microbial contamination.
3.1.5 bioluminescence, n—the production and emission of light by a living organism as the result of a chemical reaction during
which chemical energy is converted to light energy.
3.1.6 biomass, n—any matter which is or was a living organism or excreted from a microorganism (D6161).
3.1.7 culturable, adj—microorganisms that proliferate as indicated by the formation of colonies on solid growth media or the
development of turbidity in liquid growth media under specific growth conditions.
3.1.8 Luciferase, n—a general term for a class of enzymes that catalyze bioluminescent reactions.
3.1.9 Luciferin, n—a general term for a class of light-emitting biological pigments found in organisms capable of bioluminescence.
3.1.10 luminometer, n—an instrument capable of measuring light emitted as a result of non-thermal excitation.
3.1.11 relative light unit (RLU), n—an instrument-specific unit of measurement reflecting the number of photons emitted by the
Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.
3.1.11.1 Discussion—
RLU is not an SI unit, however, RLU is proportional to ATP concentration.
3.1.12 viable microbial biomass, n—metabolically active (living) microorganisms.
3.2 adenosine triphosphate (ATP), n—a molecule comprised of a purine and three phosphate groups that serves as the primary
energy transport molecule in all biological cells.
3.3 adenosine monophosphate (AMP), n—the molecule formed by the removal of two molecules of phosphate (one pyrophosphate
molecule) from ATP.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
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3.4 aseptic, adj—sterile, free from viable microbial contamination.
3.5 bioluminescence, n—the production and emission of light by a living organism as the result of a chemical reaction during
which chemical energy is converted to light energy.
3.6 biomass, n—any matter which is or was a living organism or excreted from a microorganism (D6161).
3.7 culturable, adj—microorganisms that proliferate as indicated by the formation of colonies on solid growth media or the
development of turbidity in liquid growth media under specific growth conditions.
3.8 Luciferase, n—a general term for a class of enzymes that catalyze bioluminescent reactions.
3.9 Luciferin, n—a general term for a class of light-emitting biological pigments found in organisms capable of bioluminescence.
3.10 luminometer, n—an instrument capable of measuring light emitted as a result of non-thermal excitation.
3.11 relative light unit (RLU), n—an instrument-specific unit of measurement reflecting the number of photons emitted by the
Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.
3.11.1 Discussion—
RLU is not an SI unit, however, RLU are proportional to ATP concentration.
3.12 viable microbial biomass, n—metabolically active (living) microorganisms
3.2 Acronyms:
3.2.1 AMP—adenosine monophosphate
3.2.2 ATP—adenosine triphosphate
3.2.3 HDPE—high density polyethylene
3.2.4 MWF—metalworking fluid
3.2.5 PP—polypropylene
3.2.6 RLU—relative light unit
4. Summary of Test Method
4.1 A control assay is performed using 100 μL of 1.0 ng ATP/mL standard.
4.2 A 5.0 mL 5.0 mL sample of MWF is placed into a syringe and then pressure-pressure filtered through a 0.7 μm, 0.7 μm,
glass-fiber, in-line depth filter.
4.3 The retentate is then washed with a reagent to remove extra-cellularextracellular ATP and other contaminants that might
otherwise interfere with the ATP assay.
4.4 The filter is air-dried.air dried.
4.5 A lysing reagent is used to release ATP from microbial cells that have been captured on the glass-fiber filter, and the filtrate
is dispensed into an unused culture tube.
4.6 The filtrate is diluted 1+9 with a buffer solution.
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4.7 A 100-μL100 μL volume of diluted filtrate is transferred to an unused culture tube into which 100 μL of Luciferin-Luciferase
reagent has previously been dispensed.
4.8 The culture tube is placed into a luminometer and the light intensity is read in RLU.
4.9 RLU areis converted to Log [pg ATP/mL] of sample by computation.
4.10 A procedure for differentiating between bacterial and fungal cATP-biomass cATP biomass is provided in Appendix X4.
4.11 A procedure for determining the total ATP (tATP) biomass on MWF system surfaces is provided in Appendix X5.
5. 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 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 MWF.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
five minutes.5 min.
5.5 Although ATP data generally covary with culture data in MWF, 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 havehas been estimated that less than 1 % of the species present in an environmental sample will
form colonies under any given set of growth conditions.
5.5.2 ATP concentration is affected by: the microbial species present, the physiological states of those species, and the total
bioburden (See(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.
Passman, et al. “Real-timeal., “Real-Time Testing of Bioburdens in Metalworking Fluids using Adenosine Triphosphate as a Biomass Indicator,” 2009 STLE Annual
Meeting, Orlando, FL.
Sloan, W. T., C. QuinceQuince, C., and Curtis, T. P., “The Uncountables,” Accessing Uncultivated Microorganisms, ASM Press, Washington, DC, 2008, p. 35.
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6. Apparatus
6.1 Culture tube,Tube, PP, 12 by 55 mm.
6.2 Culture tube,Tube, PP, 17 by 100 mm with caps.
6.3 Filter, 25 mm, sterile, disposable, in-line, 0.7 μm pore size, glass-fiber, depth-type with Luer-Lok inlet.
6.4 Luminometer, using photomultiplier tube, capable of detecting light emission at 420 nm and with a cuvette chamber that can
hold a 12 by 55-mm55 mm culture tube.
6.5 Macropipeter, adjustable, 1.0 to 5.0 mL.
6.6 Micropipeter, adjustable, 100 to 1000 μL.
6.7 Pipet tips,Tips, sterile, disposable, PP, 100 to 1000 μL.
6.8 Pipet tips,Tips, sterile, disposable, PP, 1.0 to 5.0 mL.
6.9 Sample collection container,Collection Container, sterile, wide-mouth bottle, 100 mL.
NOTE 1—ATP can adsorb onto glass surfaces. Consequently, PP or HDPE containers are strongly preferred.
6.10 Syringe, Luer-Lok, 20 mL, PP, sterile, disposable.
6.11 Syringe, Luer-Lok, 60 mL, PP, sterile disposable.
6.12 Test tube rack,Tube Rack, 12 mm.
6.13 Test tube rack,Tube Rack, 17 mm.
6.14 Waste receptacle, Receptacle—anyAny container suitable for receiving and retaining filtrate fluid for ultimate disposal.
7. Reagents and Materials
7.1 ATP standard,Standard, 1 ng ATP/mLATP/mL.
7.1.1 Commercially available; or
7.1.2 Dilute 1 mg ATP into 1000 mL ATP dilution buffer to get a 1000-ng1000 ng ATP/mL stock solution. Then, dilute 1.0 mL
of 1000 ng ATP/mL stock solution into 999.0 mL ATP dilution buffer to get a 1 ng ATP/mL ATP standard.
7.2 ATP extract dilution bufferExtract Dilution Buffer (proprietary) (proprietary).
7.3 ATP extraction reagentExtraction Reagent (proprietary) (proprietary).
7.4 Filter wash reagentWash Reagent (proprietary) (proprietary).
The sole source of supply of the proprietary ATP dilution buffer, ATP extraction reagent, filter wash reagent, and Luciferin-Luciferase reagent,reagent is LuminUltra
Technologies Ltd., Fredericton, New Brunswick, Canada, www.luminultra.com. If you are aware of alternative suppliers, please provide this information to ASTM
International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
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7.5 Luciferin-Luciferase reagentReagent (proprietary); store between -20°C–20 °C and 4°C;4 °C; allow to equilibrate to ambient
temperature before using.
8. Hazards
8.1 The analyst must know and observe good laboratory safety practice in accordance with 29 CFR1910.1450.
8.2 Inhalation or dermal exposure to MWF can pose health problems for personnel involved with MWF sampling. Provision of
personal protective equipment (PPE) in the form of respirators, protective clothing, or both may be indicated (see Practice E1497).
8.3 Review material safety data sheets for materials in use at the facility to identify potential hazards in order to determine
appropriate PPE (see 29 CFR 1910.1000).
9. Sampling and Test Specs and Units
9.1 Sampling Site:
9.1.1 Select sampling site that will yield a representative MWF sample.
9.1.2 For routine condition monitoring, select individual sump(s) or central systems that have actively circulating fluid.
9.1.3 For diagnostic testing, select zones of pooled or stagnant MWF.
9.2 Sampling:
9.2.1 If practical, draw sample from the mid-pointmidpoint of the fluid reservoir,reservoir; otherwise draw sample from below
surface of the MWF at an accessible location.
9.2.1.1 Microbial contamination will vary considerably within the fluid system and it is important to be consistent in selecting the
sampling location; this should be appropriate for the analysis objectives.
9.2.2 Collect sample by removing lid from sample container, immersing the open container (6.9), opening-down, below the fluid
surface and inverting the container to allow it to fill with the sampled fluid.
9.2.3 If the fluid depth is insufficient to permit 9.2.1, use a sterile pipet to draw sample from the fluid and dispense it into the
sample container;container, collecting at least 25 mL of sample.
9.3 Sample Storage/Shipment:
9.3.1 Label the sample container and follow accepted chain-of-custody procedures (Guide D4840).
9.3.2 Optimally samples should be tested on-siteonsite as soon as possible (<4 h) after testing.
9.3.3 If testing is to be delayed for longer than 4 h, or to be performed by an outside testing facility, samples may be stored on
ice or in a refrigerator for up to 24 h. Samples older than 24 h 24 h are unlikely to microbiologically representative of the MWF
at the time it was collected.
10. Calibration and Standardization
10.1 Turn on power to luminometer (6.4) and allow instrument to warm-up, warm up, in accordance with manufacturer’s
recommendations.
10.2 Ensure that all reagents have equilibrated to ambient temperature before running any tests.
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10.3 Use a micropipeter (6.6) with a new 100 to 1000-μL1000 μL tip (6.7) to dispense 100 μL Luciferin- Luciferase
Luciferin-Luciferase reagent (7.5) to an unused 12 by 55-mm55 mm culture tube (6.1).
10.4 Replace the micropipeter tip with a fresh tip.
10.5 Dispense 100 μL of 1 ng ATP/mL standard solution (7.1) into the culture tube.
10.6 Swirl gently for five times.
10.7 Place the culture tube into the luminometer.
10.8 Read and record RLU (RLU ).
ctrl
11. Procedure
11.1 Use aseptic procedure while performing this test method; ATP from analyst’s hands, sputum, etc. can contaminate the sample
with ATP from sources other than the sample itself.
11.2 Remove plunger from a new 20-mL20 mL syringe (6.10) and place onto a 17-mm17 mm test tube rack so that plunger tip
does not contact any surfaces.
11.3 Affix filter (6.3) onto the 20-mL20 mL syringe.
11.4 Place a fresh 1.0 to 5.0-mL5.0 mL tip (6.8) onto the macropipeter (6.5).
11.5 Shake sample for 15 secondss to ensure homogeneity.
11.6 With minimal delay, remove lid from sample container and, using the macropipeter, transfer 5.0 mL of sample to the
20-mL20 mL syringe barrel.
11.7 While holding the barrel over the waste receptacle (6.14), replace the plunger into the 20-mL20 mL syringe.
11.8 Apply even pressure to the 20-mL20 mL syringe plunger to pressure filter MWF sample, having filtrate discharge into the
waste receptacle.
11.9 Remove filter from the 20-mL20 mL syringe and place onto a 17-mm17 mm test tube rack so that filter outlet does not contact
any surfaces.
11.10 Remove plunger from the 20-mL20 mL syringe (6.10) and place onto a 17-mm17 mm test tube rack so that the plunger tip
does not contact any surfaces.
11.11 Replace filter onto the end of the syringe barrel.
11.12 Place a fresh 1.0 to 5.0 mL tip onto the macropipeter.
11.13 Transfer 5 mL of filter wash reagent (7.4) into the syringe barrel.
11.14 While holding the barrel over the waste receptacle (6.14), replace the 20-mL20 mL syringe plunger.
11.15 Apply even pressure to syringe plunger to pressure filter MWF sample;sample, having filtrate discharge into the waste
receptacle.
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11.16 Remove filter from the 20-mL20 mL syringe. Place the 20-mL20 mL syringe to the side for later use (11.25).
11.17 Remove plunger from a 60-mL60 mL syringe (6.11) and place onto a 17-mm17 mm test tube rack so that barrel tip does
not contact any surfaces.
NOTE 2—The 60 mL 60 mL syringe used for the air-drying step may be used for multiple samples, however,samples. However, used syringes should not
be stored overnight for re-use.reuse.
11.18 Attach the filter onto the 60-mL60 mL syringe.
11.19 While holding the barrel over the waste receptacle (6.14), replace the 60-mL60 mL syringe plunger.
11.20 Apply even pressure to the 60-mL60 mL syringe plunger to air dry the filter.
11.21 Repeat steps 11.1711.17 – 11.20 through 11.20one more time, first separating the filter before removing the plunger from
the 60-mL60 mL syringe.
11.22 Remove the filter from the 60-mL60 mL syringe and place onto a 17-mm17 mm test tube rack so that the filter outlet does
not contact any surfaces. Place the 60-mL60 mL syringe to the side for later use (see Note 2).
11.23 Place an unused 17 by 100-mm100 mm culture tube (6.2) into the 17-mm17 mm test tube rack.
11.24 Remove the barrel from the 20-mL20 mL syringe (11.16) and place onto the 17-mm17 mm test tube rack so that the barrel
tip does not contact any surfaces.
11.25 Attach filter from step 11.22 onto end of the 20-mL20 mL syringe.
11.26 Place a fresh 100 to 1000-μL1000 μL pipet tip onto micropipeter.
11.27 Use micropipeter to dispense 1.0 mL of ATP Extraction Reagentextraction reagent (7.3) into the 20-mL20 mL syringe barrel.
11.28 While holding the barrel over the 17 by 100-mm100 mm culture tube (11.23), replace the 20-mL20 mL syringe plunger.
11.29 Apply even pressure to the 20-mL20 mL syringe plunger,plunger to dispense ATP Extraction Reagentextraction reagent and
extracted ATP into the 17 by 100-mm100 mm culture tube.
NOTE 3—At this point in the protocol, this ATP extract may be stored for up to seven days at 2-8°C 2 to 8 °C prior to completing the test.
11.30 If not already performed (10.1), turn on power onto luminometer (6.4) and allow instrument to warm up, in accordance with
the manufacturer’s recommendations.
11.31 Place a fresh 1.0 to 5.0-mL5.0 mL pipet tip onto the macropipeter.
11.32 Use macropipeter to dispense two 4.5 mL portions (9 mL 4.5 mL portions (9 mL total) of ATP Extract Dilution Bufferextract
dilution buffer (7.2) into the culture tube to prepare the diluted ATP extract.
11.33 Place cap on culture tube and invert three times to mix well.
NOTE 4—Diluted ATP extract is stable for at least 4h4 h at room temperature (20 6 2°C).2 °C).
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11.34 Place one 12 by 55-mm55 mm culture tube into the 12-mm12 mm test tube rack (6.12).
11.35 As in 10.3, use a micropipeter with a fresh 100 to 1000-μL1000 μL tip to dispense 100 μL of Luciferin-Luciferase reagent
into the 12 by 55-mm55 mm culture tube.
11.36 Using a fresh 100 to 1000-μL1000 μL pipet tip, use micropipeter to transfer 100 μL of diluted sample (11.33) to the 12 by
55-mm55 mm culture tube containing 100-μL100 μL of Luciferin- Luciferase Luciferin-Luciferase reagent (11.35).
11.37 Remove the culture tube formfrom the test tube rack and swirl gently five times.
11.38 Place culture tube into luminometer chamber.
11.39 Read and record RLU .
obs
NOTE 5—If RLU areis outside of the luminometer’s range (that is, below the background level or greater than the maximum read-out),readout), see
Appendix X2 for guidance on steps to prepare sample so that RLU reading is within the luminometer’s measurement range.
11.40 When testing multiple samples, perform steps 11.111.1 – 11.29 through 11.29in sequence for each sample. After turning on
the luminometer (11.30), perform steps 11.3111.31 – 11.39 through 11.39for each prepared ATP extract.
12. Calculation or Interpretation of Results
12.1 Compute ATP in pg ATP/mL:
Sample
RLU 10,000 pgATP
~ !
obs
ATP pgATP/mL 5 3 (1)
~ !
Sample
RLU V mL
~ !
ctrl Sample
Where:
RLU is the sample RLU reading (11.39),
obs
RLU is the RLU for the 1 ng ATP/mL control (10.8),
ctrl
V is the sample volume in mL (5 mL per 11.611.6), and
Sample
10,000 pg ATP is derived from:
where:
RLU = the sample RLU reading (11.39),
obs
RLU = the RLU for the 1 ng ATP/mL control (10.8),
ctrl
V = the sample volume in mL (5 mL per 11.6), and 10 000 pg ATP is derived from:
Sample
1000 pg ATP
10,000 pg ATP 5 31 ng ATP 3dilution factor (2)
S D
ng ATP
Where:
1000 pg ATP/ng ATP is a unit conversion factor,
1 ng ATP is the concentration of the ATP standard used to acquire RLU (10.8), and
ctrl
the dilution factor is 10 (1.0 mL ATP extract (11.29) in 9.0 mL ATP extract dilution buffer.
where:
1000 pg ATP/ng ATP = a unit conversion factor,
1 ng ATP = the concentration of the ATP standard used to acquire RLU (10.8), and
ctrl
dilution factor = 10 (1.0 mL ATP extract (11.29) in 9.0 mL ATP extract dilution buffer.
12.2 Transform and report results as Log [pg ATP/mL].(pg ATP/mL).
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13. Precision and Bias
13.1 The precision of this test method is based on an interlaboratory study of ASTM E2694 conducted in 2011. Ten laboratories
tested 22 different metalworking fluids for ATP content. Every “test result” represents an individual determination. All labs were
asked to submit triplicate test results for each material tested (see Table 1). Practice E691 was followed for the overall design and
analysis of the data; the details are given in ASTM Research Report No. E34-1002.
13.2 Repeatability Limit (r)—Two test results obtained within one laboratory shall be judged not equivalent if they differ by more
than the “r” value for that material; “r” is the interval representing the critical difference between two test results for the same
material, obtained by the same operator using the same equipment on the same day in the same laboratory.
13.2.1 Repeatability limits are listed in Table 1 below.
13.3 Reproducibility Limit (R)—Two test results shall be judged not equivalent if they differ by more than the “R” value for that
material; “R” is the interval representing the critical difference between two test results for the same material, obtained by different
operators using different equipment in different laboratories.
13.3.1 Reproducibility limits are listed in Table 1 below.
13.4 The above terms (repeatability limit and reproducibility limit) are used as specified in Practice E177.
13.5 Any judgment in accordance with statements 9.1.113.2 and 9.1.213.3 would have an approximate 95 % probability of being
correct.
14. Keywords
14.1 adenosine triphosphate; ATP; bacteria; bioburden; biodeterioration; biomass; fungi; metalworking; microbial contamination;
microbiology; microorganisms
APPENDIXES
TABLE 1 Precision Data for Log pg ATP/mL
Repeatability Reproducibility
Repeatability Reproducibility
Average Standard Standard
Material Limit Limit
x¯ Deviation Deviation
r R
s s
r R
x¯ s s r R
r R
EO0101 4.38 0.07 0.14 0.18 0.40
EO0102 3.35 0.07 0.12 0.18 0.35
E00103 1.85 0.30 0.32 0.83 0.91
E00104 0.93 0.13 0.33 0.37 0.92
SS0101 4.85 0.06 0.14 0.17 0.39
SS0102 4.26 0.67 0.95 1.89 2.66
SS0103 3.16 0.40 0.42 1.11 1.18
SS0104 1.03 0.26 0.46 0.74 1.28
SO0101 4.59 0.07 0.10 0.21 0.28
SO0102 3.90 0.06 0.21 0.17 0.58
SO0103 1.66 0.09 0.29 0.25 0.82
SO0104 0.89 0.25 0.53 0.70 1.48
EO0201 4.21 0.06 0.67 0.16 1.87
EO0202 2.46 0.15 0.56 0.42 1.58
EO0203 1.06 0.25 0.39 0.70 1.09
EO0204 0.80 0.26 0.55 0.74 1.53
SS0201 1.74 0.44 0.49 1.22 1.36
SS0204 0.74 0.29 0.59 0.82 1.66
SO0201 3.31 0.27 0.27 0.75 0.75
SO0202 2.12 0.16 0.25 0.45 0.69
SO0203 1.26 0.14 0.39 0.39 1.09
Supporting data have been filed at ASTM International Headquarters and may be obtained by requesting Research Report RR:E34-1002. Contact ASTM Customer
Service at service@astm.org.
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(Nonmandatory Information)
X1. RELATIONSHIP BETWEEN ATP CONCENTRATION AND POPULATION DENSITY
-15–15
X1.1 Bacterial cells typically contain 0.5 to 5 fg ATP/cell (1 fg = 10(1 fg = 10 g). Fungal cells can have 10 to 100 times as
much ATP/cell as bacteria. Consequently, although ATP concentration tends to covary with culturability (CFU/mL) data, it is
inappropriate to attempt to convert ATP data into CFU/mL data mathematically.
X1.2 Based on the information provided in X1.1X1.1,, the 4.0 pg ATP/mL lower detection limit for this method ranges from 800
to 8000 bacteria/mL and 8 to 800 fungal cells/mL. Without first determining the actual cell count (cells/mL), it is impossible to
correlate ATP concentration to cell counts of CFU/mL. However Passman,However, Passman et al. have demonstrated strong
correlations between ATP data and other commonly used MWF condition monitoring parameters, including: CFU bacteria/mL,
biocide concentration, and pH.
X1.3 As for all condition monitoring parameters, ATP data are best used based on data trends. Upper control limits (UC
...