ASTM D7463-21

Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water

SIGNIFICANCE AND USE
5.1 This test 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 a reliable indicator of microbial contamination in fuel systems. ATP is not associated with matter of non-biological origin.  
5.2 This test method differs from Test Method D4012 as follows:  
5.2.1 By providing for the rapid determination of ATP present in a fuel (petroleum) sample, a fuel and water mixture sample, fuel-associated bottom water sample, and extracellular ATP freely available in the fuel or aqueous sample matrix;  
5.2.2 By providing for a method to capture, extract, and quantify ATP using self-contained test device and luminometer;  
5.2.3 By providing a method of quantifying ATP present in fuel or water matrices in generally less than 10 min; and  
5.2.4 By providing for the rapid separation of the ATP from chemical interferences that have previously prevented the use of ATP determinations in complex fluids containing hydrocarbons and other organic molecules.  
5.3 This test method does not require the use of hazardous materials and does not generate biohazard waste.  
5.4 This test method can be used to estimate viable microbial biomass, to evaluate the efficacy of antimicrobial pesticides, and to monitor microbial contamination in fuel storage and distribution systems.
SCOPE
1.1 This test method provides a protocol for capturing, concentrating, and testing the adenosine triphosphate (ATP) present in a fuel system sub-sample (that is, test specimen) associated with:  
1.1.1 Microorganisms and hydrophilic particles found in liquid fuels as described in Table X6.1, or  
1.1.2 Microorganisms and hydrophilic particles found in mixture of fuel and associated bottom water or just associated bottom water.  
1.1.3 ATP detected by this bioluminescence test can be derived from cellular ATP, extra-cellular ATP, or some combination of both.  
1.1.4 Cellular and extra-cellular ATP utilized to perform ATP bioluminescence are captured and concentrated from a fuel system sample into an aqueous test specimen (that is, sub-sample) for testing. For example, for a fuel system sample that does not contain any visible fuel associated bottom water, the aqueous test specimen is the capture solution itself described in 8.2.1.1. For fuel system samples that are a mixture of fuel and associated bottom water (that is, free water), the test specimen is an aliquant of the capture solution and associated bottom water.  
1.2 The ATP is measured using a patented bioluminescence enzyme assay, whereby light is generated in amounts proportional to the concentration of ATP in the sample. The light is produced and measured quantitatively using dedicated ATP test pens2 and a dedicated luminometer2 and reported in (instrument specific) Relative Light Units.  
1.3 This test method is equally suitable for use in the laboratory or field.  
1.4 Although bioluminescence is a reliable and proven technology, this method does not differentiate ATP from bacteria or fungi.  
1.5 For water or capture solution samples, the concentration range of ATP detectable by this test method is 1 × 10–11  M to 3 × 10–8  M which is equivalent to 1 × 10–14  moles/mL to 3 × 10–11  moles/mL for water samples or capture solution. Assuming testing on fuel phase is performed on a 500 mL volume of fuel the equivalent concentrations is fuel would be: 6 × 10–11 M to 2 × 10–14 M.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6.1 There is one exception—Relative Light Unit (RLU) as defined in 3.1.19.  
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 r...

General Information

• Status: Published
• Publication Date: 31-Mar-2021
• ICS: 07.100.99 - Other standards related to microbiology
• ICS: 75.160.20 - Liquid fuels
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.14 - Stability, Cleanliness and Compatibility of Liquid Fuels

Relations

• Refers: ASTM D1655-24 - Standard Specification for Aviation Turbine Fuels
• Effective Date: 15-Mar-2024
• Refers: ASTM D6300-24 - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Mar-2024
• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D975-23a - Standard Specification for Diesel Fuel
• Effective Date: 15-Dec-2023
• Refers: ASTM D6300-23a - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Dec-2023
• Refers: ASTM D4012-23a - Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
• Effective Date: 01-Nov-2023
• Refers: ASTM D1655-23a - Standard Specification for Aviation Turbine Fuels
• Effective Date: 01-Oct-2023
• Refers: ASTM D7467-23 - Standard Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)
• Effective Date: 01-Oct-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D7467-20a - Standard Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)
• Effective Date: 01-Jun-2020
• Refers: ASTM D7464-20 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-May-2020
• Refers: ASTM D7467-20 - Standard Specification for Diesel Fuel Oil, Biodiesel Blend (B6 to B20)
• Effective Date: 01-Jan-2020
• Refers: ASTM D396-19a - Standard Specification for Fuel Oils
• Effective Date: 15-Dec-2019
• Refers: ASTM D6300-19a - Standard Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products and Lubricants
• Effective Date: 01-Dec-2019
• Refers: ASTM D7464-19 - Standard Practice for Manual Sampling of Liquid Fuels, Associated Materials and Fuel System Components for Microbiological Testing
• Effective Date: 01-Nov-2019

Overview

ASTM D7463-21: Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water is a key protocol for detecting microbial contamination in fuels and associated water phases. By quantifying the ATP content-a universal biomarker of living organisms-this method provides a rapid, accurate indication of the presence of bacteria and fungi in liquid fuels, fuel/water mixtures, and related water samples. ATP testing is essential for maintaining fuel system quality, ensuring storage stability, and protecting critical equipment from biofouling and biodeterioration.

Key Topics

• Microbial Contamination Detection: ATP is present in all living cells; its detection serves as a sensitive, non-specific marker of microbial presence in petroleum-based fuels and fuel-associated water.
• Bioluminescence Enzyme Assay: The method utilizes bioluminescent reactions catalyzed by enzymes (luciferase-luciferin system) measured with a luminometer, yielding results in Relative Light Units (RLU) proportional to ATP concentration.
• Versatile Sampling: Protocols are applicable to fuel-only, water-only, and fuel/water mixture samples. Both cellular and extracellular ATP can be measured to provide insight into total biomass and possible biofilm activity.
• Rapid and Field Deployable: Results are typically available in under 10 minutes, and the method is suitable for both laboratory and on-site field use without generating hazardous or biohazard waste.
• Matrix Interference Management: The method includes provisions for handling complex matrices containing hydrocarbons and other organics that previously interfered with ATP assays.
• Non-hazardous Procedure: No hazardous reagents are required, enhancing operational safety and environmental compliance.

Applications

• Fuel Quality Monitoring: Detect and quantify microbial activity in storage tanks, pipelines, and fuel distribution systems to prevent filter plugging, corrosion, and degradation.
• Biomass Estimation: Estimate viable microbial biomass, including bacteria and fungi, to monitor system cleanliness or diagnose suspected fuel contamination events.
• Efficacy Evaluation of Biocides: Assess the effectiveness of antimicrobial treatments (biocides) by monitoring changes in ATP levels before and after treatment.
• Maintenance in Critical Sectors: Widely used in the petroleum industry, aviation fuel management, diesel storage, and power generation sectors to ensure reliable operation and fuel lifespan.
• Trend Analysis: Generate data for historical trending and preventative maintenance, supporting proactive management decisions in fuel system integrity.

Related Standards

• ASTM D4012 - Test Method for ATP Content of Microorganisms in Water (less rapid, does not address hydrocarbon interference).
• ASTM D396 - Specification for Fuel Oils.
• ASTM D975 - Specification for Diesel Fuel.
• ASTM D1655 - Specification for Aviation Turbine Fuels.
• ASTM D2880 - Specification for Gas Turbine Fuel Oils.
• ASTM D4175 - Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants.
• ASTM D7464 - Practice for Manual Sampling of Liquid Fuels for Microbiological Testing.
• ASTM D7467 - Specification for Biodiesel Blend Diesel Fuel Oil (B6 to B20).

Summary

ASTM D7463-21 provides a fast, reliable, and practical method for monitoring microbial ATP in fuel systems. Its application supports improved risk management, fuel quality assurance, and operational efficiency in industries that rely on clean and stable fuels. The method’s compatibility with on-site testing and lack of hazardous waste makes it a valuable tool for routine surveillance as well as troubleshooting microbial contamination events. Integrating this standard into fuel management protocols helps protect assets and ensures compliance with industry best practices for fuel cleanliness and storage stability.

Standards Content (Sample)

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.
Designation: D7463 − 21
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
This standard is issued under the fixed designation D7463; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope* 1.3 This test method is equally suitable for use in the
laboratory or field.
1.1 This test method provides a protocol for capturing,
1.4 Although bioluminescence is a reliable and proven
concentrating, and testing the adenosine triphosphate (ATP)
technology, this method does not differentiate ATP from
present in a fuel system sub-sample (that is, test specimen)
bacteria or fungi.
associated with:
1.1.1 Microorganisms and hydrophilic particles found in
1.5 Forwaterorcapturesolutionsamples,theconcentration
–11
liquid fuels as described in Table X6.1,or
range ofATP detectable by this test method is1×10 Mto
–8 –14
1.1.2 Microorganisms and hydrophilic particles found in
3×10 M which is equivalent to1×10 moles/mL to 3 ×
–11
mixture of fuel and associated bottom water or just associated
10 moles/mLfor water samples or capture solution.Assum-
bottom water.
ing testing on fuel phase is performed on a 500mLvolume of
1.1.3 ATP detected by this bioluminescence test can be
fuel the equivalent concentrations is fuel would be: 6 ×
–11 –14
derived from cellularATP, extra-cellularATP, or some combi- 10 Mto2×10 M.
nation of both.
1.6 The values stated in SI units are to be regarded as
1.1.4 Cellular and extra-cellular ATP utilized to perform
standard. No other units of measurement are included in this
ATP bioluminescence are captured and concentrated from a
standard.
fuel system sample into an aqueous test specimen (that is,
1.6.1 Thereisoneexception—RelativeLightUnit(RLU)as
sub-sample) for testing. For example, for a fuel system sample
defined in 3.1.19.
that does not contain any visible fuel associated bottom water,
1.7 This standard does not purport to address all of the
the aqueous test specimen is the capture solution itself de-
safety concerns, if any, associated with its use. It is the
scribed in 8.2.1.1. For fuel system samples that are a mixture
responsibility of the user of this standard to establish appro-
offuelandassociatedbottomwater(thatis,freewater),thetest
priate safety, health, and environmental practices and deter-
specimen is an aliquant of the capture solution and associated
mine the applicability of regulatory limitations prior to use.
bottom water.
1.8 This international standard was developed in accor-
1.2 TheATPis measured using a patented bioluminescence
dance with internationally recognized principles on standard-
enzyme assay, whereby light is generated in amounts propor-
ization established in the Decision on Principles for the
tional to the concentration of ATP in the sample. The light is
Development of International Standards, Guides and Recom-
producedandmeasuredquantitativelyusingdedicatedATPtest
mendations issued by the World Trade Organization Technical
2 2
pens and a dedicated luminometer and reported in (instru-
Barriers to Trade (TBT) Committee.
ment specific) Relative Light Units.
2. Referenced Documents
2.1 ASTM Standards:
1 D396Specification for Fuel Oils
This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of D975Specification for Diesel Fuel
Subcommittee D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels.
D1655Specification for Aviation Turbine Fuels
Current edition approved April 1, 2021. Published April 2021. Originally
D2880Specification for Gas Turbine Fuel Oils
approved in 2008. Last previous edition approved in 2018 as D7463–18. DOI:
D4012TestMethodforAdenosineTriphosphate(ATP)Con-
10.1520/D7463-21.
The sole source of supply, repair, recertification, and technical support of the
tent of Microorganisms in Water
apparatus or test pen known to the committee at this time is Merck KGaA, 64271
Darmstadt,Germany(Worldwide)orFuelQualityServices,Inc.,4584CantrellRd.,
FloweryBranch,GA30542(USA).Ifyouareawareofalternativesuppliers,please For referenced ASTM standards, visit the ASTM website, www.astm.org, or
provide this information toASTM International Headquarters.Your comments will contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
receive careful consideration at a meeting of the responsible technical committee, Standards volume information, refer to the standard’s Document Summary page on
which you may attend. 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
D7463 − 21
D4175Terminology Relating to Petroleum Products, Liquid carbon diesel fuel that can be present as suspended haze,
Fuels, and Lubricants nonvisible suspended water droplets, as droplets on the walls
D6300Practice for Determination of Precision and Bias of the vessel, or as a separate layer on the bottom of the vessel
Data for Use in Test Methods for Petroleum Products, or sample container.
Liquid Fuels, and Lubricants
3.1.12 fungus, (pl. fungi), n—singlecell(yeasts)orfilamen-
D7464Practice for Manual Sampling of Liquid Fuels, As-
tous (molds) microorganisms that share the property of having
sociated Materials and Fuel System Components for
the true intracellular membranes (organelles) that characterize
Microbiological Testing
all higher life forms (Eukaryotes).
D7467Specification for Diesel Fuel Oil, Biodiesel Blend
3.1.13 hydrophilic particles, n—compounds such as ATP,
(B6 to B20)
+ +
NAD , NADP , NADH, NADPH, enzymes, free fatty acids,
preservatives, biocides, salts, as well as microorganisms or
3. Terminology
other articles are often dispersed or distributed in hydrophobic
3.1 Definitions:
liquid matrices such as crude oil, vegetable oil, petrol, and
3.1.1 For definition of terms used in this test method, refer
kerosine.
to Terminology D4175.
3.1.14 invert emulsion layer, n—interfacebetweenthewater
3.1.2 adenosine monophosphate (AMP), n—molecule
phase and fuel phase of a fuel water sample which consists of
formed by the removal of two molecules of phosphate (one
water micelles dispersed in the fuel.
pyrophosphate molecule) from ATP.
3.1.15 luciferase, n—general term for a class of enzymes
3.1.3 adenosine triphosphate (ATP), n—molecule com-
that catalyze bioluminescent reactions.
prised of a purine and three phosphate groups, that serves as
the primary energy transport molecule in all biological cells.
3.1.16 luciferin,n—generaltermforaclassoflight-emitting
biological pigments found in organisms capable of biolumi-
3.1.4 aseptic, adj—sterile, free from viable microbiological
nescence.
contamination.
3.1.17 luminometer, n—instrument capable of measuring
3.1.5 bioluminescence, n—production and emission of light
light emitted as a result of non-thermal excitation.
byalivingorganismastheresultofachemicalreactionduring
which chemical energy is converted to light energy.
3.1.18 pyrogen free, n—freeofsubstanceswhichcaninduce
fever.
3.1.6 biomass, n—biological material including any mate-
rial other than fossil fuels which is or was a living organism or
3.1.19 relative light unit (RLU), n—instrument and assay
component or product of a living organism.
specific unit of measurement reflecting the number of photons
3.1.7 capture solution, n—aqueous solution of proprietary
emitted by the Luciferin-Luciferase driven hydrolysis of ATP
composition used to capture and concentrate hydrophilic com- to AMP plus pyrophosphate.
pounds and particles from liquid fuels.
3.1.19.1 Discussion—RLU is not an SI unit, however, RLU
are proportional to ATP concentration.
3.1.8 cellular adenosine triphosphate (cellular-ATP),
n—ATPpresentinwholecells,whethertheyarelivingordead.
3.1.20 test specimen, n—a representative piece of a sample.
3.1.8.1 Discussion—Cellular-ATP is released upon inten-
3.1.20.1 Discussion—Forthistestmethod,thetestspecimen
tional lysis (rupturing) of microbial cells during the sample
is an aqueous sub-sample drawn from the fuel system sample
preparation process. Microbially infected fluids contain both
that is tested for the presence of cellular and/or extra-cellular
cellular (cell-associated/cell-bound) and extra-cellular ATP.
ATP.Inthecaseofafuelsystemsamplethatisfuelonlyinthe
3.1.9 culturable, adj—(microorganisms that are) able to absence of associated bottom water, the test specimen is the
proliferate as indicated by the formation of colonies on or in capture solution (3.1.7). For fuel system samples that contain
solid,semi-solid,orliquidgrowthmediaunderspecificgrowth associatedbottomwater,thetestspecimenisanaliquantofthe
conditions, as indicated by the formation of colonies, the capture solution and associated bottom water (3.1.11).
development of turbidity, or other indicators.
3.1.21 viable microbial biomass, n—metabolically active
3.1.10 extracellular ATP, n—ATP that is not contained
(living) micro-organisms
inside a cell.
3.2 Abbreviations:
3.1.10.1 Discussion—ATP is released into the environment
3.2.1 AMP—adenosine monophosphate
when cells die and break open (lyse), for example, as when
3.2.2 ATP—adenosine triphosphate
theyarekilledbyexposuretosomemicrobicides.ATPreleased
intotheenvironmentcanpersistforseveraldaysafteracellhas
3.2.3 HDPE—high density polyethylene
beenlysed.ConsequentlyextracellularATPmustbesubtracted
+
3.2.4 NAD —nicotinamide adenine dinucleotide, oxidized
from total ATP to determine the concentration of viable
form
cell-associated (biomass associated) ATP. However, extracel-
lular ATP can also be an indicator of “distant” biomass, for 3.2.5 NADH—nicotinamide adenine dinucleotide, reduced
form
example, biofilm in the system.
+
3.1.11 free water, n—water that exists as a separate phase.
3.2.6 NADP —nicotinamide adenine dinucleotide
3.1.11.1 Discussion—Water present in fuel such as hydro- phosphate, oxidized form
D7463 − 21
FIG. 1 Luminometer
3.2.7 NADPH—nicotinamide adenine dinucleotide 5.2.4 By providing for the rapid separation of theATPfrom
phosphate, reduced form chemical interferences that have previously prevented the use
ofATP determinations in complex fluids containing hydrocar-
3.2.8 PP—polypropylene
bons and other organic molecules.
3.2.9 RLU—relative light units
5.3 This test method does not require the use of hazardous
materials and does not generate biohazard waste.
4. Summary of Test Method
5.4 This test method can be used to estimate viable micro-
4.1 A fuel system sample is obtained either for condition
bial biomass, to evaluate the efficacy of antimicrobial
monitoring or for diagnostic testing, for example, fuel from a
pesticides, and to monitor microbial contamination in fuel
fuel system that is exhibiting problems such as sediment
storage and distribution systems.
formation or filter plugging where the presence of micro-
organisms is suspected.
6. Interferences
4.2 MicrobialATPis captured from the fuel system sample,
6.1 Sample containers and sampling devices shall be clean
concentrated into a test specimen, and tested using a biolumi-
and free of both ATP and microbial contamination.
nescence reaction. The light generated by the luminescence
reactionisproportionaltotheamountofATPpresentinthetest
6.2 EnsurethatthesamplingstickontheATPTestPendoes
specimen as measured in a luminometer.
not come into contact with any contaminating surfaces. Con-
tact with a surface or substance can cause contamination with
4.3 Test results should be documented for evaluation and
high levels of ATP, giving erroneous results.
trending.
6.3 Luciferase is an enzyme, which can be inhibited or
4.4 Specialized test methods for fuel samples, water
denatured by high temperatures, the presence of heavy metals,
samples, extracellular determination, or resolving potential
and high salt concentrations in the sample. These conditions
matrix interference in bottom water samples are described in
are unlikely to occur except in samples containing large
Appendix X4 and Appendix X5.
volumes of bottom-water samples from storage tanks and
similar systems.
5. Significance and Use
6.3.1 For samples in which inhibition is suspected or likely
5.1 This test method measures the concentration of ATP
to occur, testing of a dilution of the sample is described in
present in the sample. ATP is a constituent of all living cells
Appendix X4.
including bacteria and fungi. Consequently, the presence of
ATP is a reliable indicator of microbial contamination in fuel
7. Apparatus
systems. ATP is not associated with matter of non-biological
7.1 An example of the luminometer is shown as a diagram
origin.
in Fig. 1.
5.2 This test method differs from Test Method D4012 as
7.2 Warning—The apparatus is not explosion-proof. The
follows:
instrument should not be operated in explosive atmospheres or
5.2.1 By providing for the rapid determination of ATP
in locations where there may be explosive fumes, as it cannot
present in a fuel (petroleum) sample, a fuel and water mixture
be grounded.
sample,fuel-associatedbottomwatersample,andextracellular
ATP freely available in the fuel or aqueous sample matrix; 7.3 Sample bottle, round wide-mouth, nominal capacity
5.2.2 By providing for a method to capture, extract, and 500mL or 1000mL, HDPE (High Density Poly Ethylene) or
quantify ATP using self-contained test device and luminom- equivalent. There shall be sufficient excess volume in the
eter; sample bottle so that there is at least 10% head space in
5.2.3 By providing a method of quantifyingATP present in additiontothe500mLor1000mLsamplevolumetofacilitate
fuel or water matrices in generally less than 10 min; and the shearing and mixing of the capture solution.
D7463 − 21
FIG. 2 Fuel Test Pen and Free ATP Test Pen
7.3.1 Sample bottles may be reused provided they are container(s)witha70%alcohol(isopropylalcoholorethanol)
cleanedanddriedcorrectly.Refertotestsupplier’sinformation and water solution and let air dry. All devices (except factory
regarding recommended cleaning procedure. new, clean bottles) should be disinfected in this manner to
minimize the likelihood of cross-contamination. Use care to
7.4 Pipettors,fixedvolumeoradjustable,capableofprovid-
not touch the interior of the freshly decontaminated sample
ingdiscretevolumesofbottomwatertodeterminethepresence
devices or sample bottles. Remove the container lid immedi-
of matrix interference as described in Appendix X4. Example
ately before dispensing the sample into the container and
pipettor volumes include 10 µL, 50 µL, and 100 µL.
replace the lid on the container as soon as possible.
8. Reagents and Materials
9.5 Microbial contaminant populations are dynamic. Mi-
crobes within the sample can proliferate or die during the
8.1 Reagents:
8.1.1 ATP di-sodium salt. interval between collection and testing. Consequently, samples
shall be processed within 24 h after collection.
8.1.2 Water, Pyrogen free.
8.2 Materials: 9.6 Ifsamplesaretobetestedlaterthan4haftercollection,
8.2.1 ATP test pens: store the samples either on ice or refrigerated at >0°C to 5°C
8.2.1.1 HY-LiTE Fuel Test Pen, as shown in Fig. 2. until tested. Avoid freezing samples. Allow samples to equili-
brate to room temperature before testing.
8.2.1.2 HY-LiTE Free ATP Pen, as shown in Fig. 2.
8.2.2 Pasteur pipettes, sterile, disposable, polyethylene,
1.0mL. 10. Calibration and Standardization
8.2.3 Pasteur pipettes sterile, disposable, polyethylene,
10.1 The luminometer , which is specific to this test, is
10.0mL.
factorycalibratedandtemperaturecompensatedtogivealinear
response from 0 to 99000 RLU at temperatures between 5°C
9. Sampling, Test Specimens, and Test Units
and 35°C (41°F and 95°F). No calibration is nece
...

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: D7463 − 18 D7463 − 21
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
in Fuel, Fuel/Water Mixtures, and Fuel Associated Water
This standard is issued under the fixed designation D7463; 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, concentrating, and testing the adenosine triphosphate (ATP) present in a
fuel system sub-sample (that is, test specimen) associated with:
1.1.1 Microorganisms and hydrophilic particles found in liquid fuels as described in Table X6.1, or
1.1.2 Microorganisms and hydrophilic particles found in mixture of fuel and associated bottom water or just associated bottom
water.
1.1.3 ATP detected by this bioluminescence test can be derived from cellular ATP, extra-cellular ATP, or some combination of
both.
1.1.4 Cellular and extra-cellular ATP utilized to perform ATP bioluminescence are captured and concentrated from a fuel system
sample into an aqueous test specimen (that is, sub-sample) for testing. For example, for a fuel system sample that does not contain
any visible fuel associated bottom water, the aqueous test specimen is the capture solution itself described in 8.2.1.1. For fuel
system samples that are a mixture of fuel and associated bottom water (that is, free water), the test specimen is an aliquant of the
capture solution and associated bottom water.
1.2 The ATP is measured using a patented bioluminescence enzyme assay, whereby light is generated in amounts proportional to
the concentration of ATP in the sample. The light is produced and measured quantitatively using dedicated ATP test pens and a
dedicated luminometer and reported in (instrument specific) Relative Light Units.
1.3 This test method is equally suitable for use in the laboratory or field.
1.4 Although bioluminescence is a reliable and proven technology, this method does not differentiate ATP from bacteria or fungi.
–11
1.5 For water or capture solution samples, the concentration range of ATP detectable by this test method is 1 × 10 M to 3 ×
This test method is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcommittee
D02.14 on Stability, Cleanliness and Compatibility of Liquid Fuels.
Current edition approved Dec. 1, 2018April 1, 2021. Published December 2018April 2021. Originally approved in 2008. Last previous edition approved in 20162018 as
ɛ1
D7463 – 16D7463 – 18. . DOI: 10.1520/D7463-18. 10.1520/D7463-21.
The sole source of supply, repair, recertification, and technical support of the apparatus or test pen known to the committee at this time is Merck KGaA, 64271 Darmstadt,
Germany (Worldwide) or Fuel Quality Services, Inc., 4584 Cantrell Rd., Flowery Branch, GA 30542 (USA). 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.
*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
D7463 − 21
–8
–14 –11
10 M which is equivalent to 1 × 10 moles/mL to 3 × 10 moles/mL for water samples or capture solution. Assuming testing
–11 –14
on fuel phase is performed on a 500 mL volume of fuel the equivalent concentrations is fuel would be: 6 × 10 M M to 2 × 10
M.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6.1 There is one exception—Relative Light Unit (RLU) as defined in 3.1.19.
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.
2. Referenced Documents
2.1 ASTM Standards:
D396 Specification for Fuel Oils
D975 Specification for Diesel Fuel
D1655 Specification for Aviation Turbine Fuels
D2880 Specification for Gas Turbine Fuel Oils
D4012 Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and
Lubricants
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)
3. Terminology
3.1 Definitions:
3.1.1 For definition of terms used in this test method, refer to Terminology D4175.
3.1.2 adenosine monophosphate (AMP), n—molecule formed by the removal of two molecules of phosphate (one pyrophosphate
molecule) from ATP.
3.1.3 adenosine triphosphate, triphosphate (ATP), n—molecule comprised of a purine and three phosphate groups, that serves as
the primary energy transport molecule in all biological cells.
3.1.3 adenosine monophosphate, n—molecule formed by the removal of two (2) molecules of phosphate (one pyrophosphate
molecule) from ATP.
3.1.4 aseptic, adj—sterile, free from viable microbiological contamination.
3.1.5 bioluminescence, n—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—biological material including any material other than fossil fuels which is or was a living organism or
component or product of a living organism.
3.1.7 capture solution, n—aqueous solution of proprietary composition used to capture and concentrate hydrophilic compounds
and particles from liquid fuels.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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3.1.8 cellular adenosine triphosphate (cellular-ATP), n—ATP present in whole cells, whether they are living or dead.
3.1.8.1 Discussion—
Cellular-ATP is released upon intentional lysis (rupturing) of microbial cells during the sample preparation process. Microbially
infected fluids contain both cellular (cell-associated/cell-bound) and extra-cellular ATP.
3.1.9 culturable, adj—microorganisms that (microorganisms that are) able to proliferate as indicated by the formation of colonies
in or on solid growth media, or the development of turbidity in on or in solid, semi-solid, or liquid growth media under specified
growth conditions.specific growth conditions, as indicated by the formation of colonies, the development of turbidity, or other
indicators.
3.1.10 extracellular ATP, n—ATP that is not contained inside a cell.
3.1.10.1 Discussion—
ATP is released into the environment when cells die and break open (lyse), for example, as when they are killed by exposure to
some microbicides. ATP released into the environment can persist for several days after a cell has been lysed. Consequently
extracellular ATP must be subtracted from total ATP to determine the concentration of viable cell-associated (biomass associated)
ATP. However, extracellular ATP can also be an indicator of “distant” biomass, for example, biofilm in the system.
3.1.11 free water, n—water that exists as a separate phase.
3.1.11.1 Discussion—
Water present in fuel such as hydrocarbon diesel fuel that can be present as suspended haze, nonvisible suspended water droplets,
as droplets on the walls of the vessel, or as a separate layer on the bottom of the vessel or sample container.
3.1.12 fungus, (pl. fungi), n—single cell (yeasts) or filamentous (molds) microorganisms that share the property of having the true
intracellular membranes (organelles) that characterize all higher life forms (Eukaryotes).
+ +
3.1.13 hydrophilic particles, n—compounds such as ATP, NAD , NADP , NADH, NADPH, enzymes, free fatty acids,
preservatives, biocides, salts, as well as microorganisms or other articles are often dispersed or distributed in hydrophobic liquid
matrices such as crude oil, vegetable oil, petrol, and kerosine.
3.1.14 invert emulsion layer, n—interface between the water phase and fuel phase of a fuel water sample which consists of water
micelles dispersed in the fuel.
3.1.15 luciferase, n—general term for a class of enzymes that catalyze bioluminescent reactions.
3.1.16 luciferin, n—general term for a class of light-emitting biological pigments found in organisms capable of bioluminescence.
3.1.17 luminometer, n—instrument capable of measuring light emitted as a result of non-thermal excitation.
3.1.18 pyrogen free, n—free of substances which can induce fever.
3.1.19 relative light unit (RLU), n—instrument-specific instrument and assay specific unit of measurement reflecting the number
of photons emitted by the Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate.
3.1.19.1 Discussion—
RLU is not an SI unit, however, RLU are proportional to ATP concentration.
3.1.20 test specimen, n—a representative piece of a sample.
3.1.20.1 Discussion—
For this test method, the test specimen is an aqueous sub-sample drawn from the fuel system sample that is tested for the presence
of cellular and/or extra-cellular ATP. In the case of a fuel system sample that is fuel only in the absence of associated bottom water,
the test specimen is the capture solution (3.1.7). For fuel system samples that contain associated bottom water, the test specimen
is an aliquant of the capture solution and associated bottom water (3.1.11).
3.1.21 viable microbial biomass, n—metabolically active (living) micro-organisms
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3.2 Abbreviations:
3.2.1 AMP—adenosine monophosphate
3.2.2 ATP—adenosine triphosphate
3.2.3 HDPE—high density polyethylene
+
3.2.4 NAD —nicotinamide adenine dinucleotide, oxidized form
3.2.5 NADH—nicotinamide adenine dinucleotide, reduced form
+
3.2.6 NADP —nicotinamide adenine dinucleotide phosphate, oxidized form
3.2.7 NADPH—nicotinamide adenine dinucleotide phosphate, reduced form
3.2.8 PP—polypropylene
3.2.9 RLU—relative light units
4. Summary of Test Method
4.1 A fuel system sample is obtained either for condition monitoring or for diagnostic testing, for example, fuel from a fuel system
that is exhibiting problems such as sediment formation or filter plugging where the presence of micro-organisms is suspected.
4.2 Microbial ATP is captured from the fuel system sample, concentrated into a test specimen, and tested using a bioluminescence
reaction. The light generated by the luminescence reaction is proportional to the amount of ATP present in the test specimen as
measured in a luminometer.
4.3 Test results should be documented for evaluation and trending.
4.4 Specialized test methods for fuel samples, water samples, extracellular determination, or resolving potential matrix
interference in bottom water samples are described in Appendix X4 and Appendix X5.
5. Significance and Use
5.1 This test 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 a reliable indicator of microbial contamination in fuel systems. ATP is
not associated with matter of non-biological origin.
5.2 This test method differs from Test Method D4012 as follows:
5.2.1 By providing for the rapid determination of ATP present in a fuel (petroleum) sample, a fuel and water mixture sample,
fuel-associated bottom water sample, and extracellular ATP freely available in the fuel or aqueous sample matrix;
5.2.2 By providing for a method to capture, extract, and quantify ATP using self-contained test device and luminometer;
5.2.3 By providing a method of quantifying ATP present in fuel or water matrices in generally less than 10 min; and
5.2.4 By providing for the rapid separation of the ATP from chemical interferences that have previously prevented the use of ATP
determinations in complex fluids containing hydrocarbons and other organic molecules.
5.3 This test method does not require the use of hazardous materials and does not generate biohazard waste.
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FIG. 1 Luminometer
5.4 This test method can be used to estimate viable microbial biomass, to evaluate the efficacy of antimicrobial pesticides, and
to monitor microbial contamination in fuel storage and distribution systems.
6. Interferences
6.1 Sample containers and sampling devices shall be clean and free of both ATP and microbial contamination.
6.2 Ensure that the sampling stick on the ATP Test Pen does not come into contact with any contaminating surfaces. Contact with
a surface or substance can cause contamination with high levels of ATP, giving erroneous results.
6.3 Luciferase is an enzyme, which can be inhibited or denatured by high temperatures, the presence of heavy metals, and high
salt concentrations in the sample. These conditions are unlikely to occur except in samples containing large volumes of
bottom-water samples from storage tanks and similar systems.
6.3.1 For samples in which inhibition is suspected or likely to occur, testing of a dilution of the sample is described in Appendix
X4.
7. Apparatus
7.1 An example of the luminometer is shown as a diagram in Fig. 1.
7.2 Warning—The apparatus is not explosion-proof. The instrument should not be operated in explosive atmospheres or in
locations where there may be explosive fumes, as it cannot be grounded.
7.3 Sample bottle, round wide-mouth, nominal capacity 500 mL or 1000 mL, HDPE (High Density Poly Ethylene) or equivalent.
There shall be sufficient excess volume in the sample bottle so that there is at least 10 % head space in addition to the 500 mL or
1000 mL sample volume to facilitate the shearing and mixing of the capture solution.
7.3.1 Sample bottles may be reused provided they are cleaned and dried correctly. Refer to test supplier’s information regarding
recommended cleaning procedure.
7.4 Pipettors, fixed volume or adjustable, capable of providing discrete volumes of bottom water to determine the presence of
matrix interference as described in Appendix X4. Example pipettor volumes include 10 μL, 50 μL, and 100 μL.
8. Reagents and Materials
8.1 Reagents:
8.1.1 ATP di-sodium salt.
8.1.2 Water, Pyrogen free.
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FIG. 2 Fuel Test Pen and Free ATP Test Pen
8.2 Materials:
8.2.1 ATP test pens:
8.2.1.1 HY-LiTE Fuel Test Pen, as shown in Fig. 2.
8.2.1.2 HY-LiTE Free ATP Pen, as shown in Fig. 2.
8.2.2 Pasteur pipettes, sterile, disposable, polyethylene, 1.0 mL.
8.2.3 Pasteur pipettes sterile, disposable, polyethylene, 10.0 mL.
9. Sampling, Test Specimens, and Test Units
9.1 Samples shall be drawn in accordance with Practice D7464 and dispensed into a clean 500 mL sample bottle (7.3).
9.2 Aircraft fuel systems shall be drawn in accordance with the applicable Aircraft Maintenance Manual and dispensed into a clean
1000 mL sample bottle (7.3).
9.3 To reduce the risk of accidental contamination, samples intended for microbiological testing shall not be used for other tests
until after they are no longer needed for microbiological testing.
9.4 It may be possible to accidentally cross contaminate the sample under field conditions. To reduce risk of potential
cross-contamination, rinse the sample device(s) and sample container(s) with a 70 % alcohol (isopropyl alcohol or ethanol) and
water solution and let air dry. All devices (except factory new, clean bottles) should be disinfected in this manner to minimize the
likelihood of cross-contamination. Use care to not touch the interior of the freshly decontaminated sample devices or sample
bottles. Remove the container lid immediately before dispensing the sample into the container and replace the lid on the container
as soon
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