ASTM D4012-23a
(Test Method)Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Water
SIGNIFICANCE AND USE
5.1 A rapid and routine procedure for determining biomass of the living microorganisms in cultures, waters, wastewaters, and in plankton and periphyton samples taken from surface waters is frequently of vital importance. However, classical techniques such as direct microscope counts, turbidity, organic chemical analyses, cell tagging, and plate counts are expensive, time-consuming, or tend to underestimate total numbers. In addition, some of these methods do not distinguish between living and nonliving cells.
5.2 This test method measures the concentration of cellular-ATP present in the sample. ATP is a constituent of all living cells, including bacteria, algae, protozoa, and fungi. Consequently, the presence of cellular-ATP is an indicator of total metabolically active microbial contamination in water. ATP is not associated with matter of non-biological origin.
5.3 The ATP (luciferin-luciferase) method is a rapid, sensitive determination of viable microbial biomass. ATP is the primary energy donor for life processes, does not exist in association with nonliving detrital material, and the amount of ATP per unit of biomass (expressed in weight) is relatively constant. (ATP per cell varies with species and physiological state of the organism.)
5.4 This test method can be used to:
5.4.1 Estimate viable microbial biomass in cultures and waters.
5.4.2 Estimate the amount of total viable biomass in plankton and periphyton samples.
5.4.3 Estimate the number of viable cells in a unispecies culture if the cATP content (or if the average amount of cATP) per cell is known.
5.4.4 Estimate and differentiate between zooplanktonic, phytoplanktonic, bacterial, and fungal cATP through size fractionation of water samples.
5.4.5 Measure the mortality rate of microorganisms in toxicity tests in entrainment studies, and in other situations where populations or assemblages of microorganisms are placed under stress.
5.5 This test method is similar to Test Metho...
SCOPE
1.1 This test method covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content associated with microorganisms normally found in laboratory cultures and waters in plankton and periphyton samples from waters.
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 method does not remove all known chemical interferences, known to either luminesce in the 530 nm ± 20 nm range, or to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).
1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms (Appendix X1).
1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility.
1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily in the matrix being tested.
1.7 This test method is equally suitable for use in the laboratory or field.
1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log10 [pg cATP/mL]) to
4 000 000 pg cATP/mL (6.6 Log10 [pg cATP/mL]) in 50 mL water samples.
1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP, although the method does not differentiate between ATP from different sources, for example, from...
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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: D4012 − 23a
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
1
in Water
This standard is issued under the fixed designation D4012; 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 1.9 Providing interferences can be overcome, biolumines-
cence is a reliable and proven method for qualifying and
1.1 This test method covers a protocol for capturing, ex-
quantifying ATP, although the method does not differentiate
tracting and quantifying the cellular adenosine triphosphate
between ATP from different sources, for example, from differ-
(cATP) content associated with microorganisms normally
ent types of microorganisms, such as bacteria, fungi, algae and
found in laboratory cultures and waters in plankton and
protozoa.
periphyton samples from waters.
1.10 The values stated in SI units are to be regarded as
1.2 The ATP is measured using a bioluminescence enzyme
standard. No other units of measurement are included in this
assay, whereby light is generated in amounts proportional to
standard.
the concentration of ATP in the samples. The light is produced
1.11 This standard does not purport to address all of the
and measured quantitatively as relative light units (RLU)
safety concerns, if any, associated with its use. It is the
which are converted by comparison with an ATP standard and
responsibility of the user of this standard to establish appro-
computation to pg ATP/mL.
priate safety, health, and environmental practices and deter-
1.3 This method does not remove all known chemical
mine the applicability of regulatory limitations prior to use.
interferences, known to either luminesce in the 530 nm 6
1.12 This international standard was developed in accor-
20 nm range, or to quench light emitted in that range. It should
dance with internationally recognized principles on standard-
not be used to determine ATP concentrations in samples with
ization established in the Decision on Principles for the
dissolved organic compounds, heavy metals or >10 000 ppm
Development of International Standards, Guides and Recom-
total dissolved solids. Alternative methods have been devel-
mendations issued by the World Trade Organization Technical
oped for determining ATP concentrations in fluids samples
Barriers to Trade (TBT) Committee.
likely to contain such interferences (Test Methods D7687 and
E2694).
2. Referenced Documents
1.4 Knowledge of the concentration of ATP can be related to 2
2.1 ASTM Standards:
viable biomass or metabolic activity of microorganisms (Ap-
D1129 Terminology Relating to Water
pendix X1).
D1193 Specification for Reagent Water
1.5 This test method offers a high degree of sensitivity,
D1601 Test Method for Dilute Solution Viscosity of Ethyl-
rapidity, accuracy, and reproducibility. ene Polymers
D4175 Terminology Relating to Petroleum Products, Liquid
1.6 The analyst should be aware that the precision statement
Fuels, and Lubricants
pertains only to determinations in reagent water and not
D5847 Practice for Writing Quality Control Specifications
necessarily in the matrix being tested.
for Standard Test Methods for Water Analysis
1.7 This test method is equally suitable for use in the
D6161 Terminology Used for Microfiltration, Ultrafiltration,
laboratory or field.
Nanofiltration, and Reverse Osmosis Membrane Processes
1.8 The method normally detects cATP concentrations in the D6300 Practice for Determination of Precision and Bias
range of 0.1 pg cATP/mL (–1.0Log [pg cATP/mL]) to Data for Use in Test Methods for Petroleum Products,
10
4 000 000 pg cATP/mL (6.6 Log [pg cATP/mL]) in 50 mL Liquid Fuels, and Lubricants
10
water samples. D7687 Test Method for Measurement of Cellular Adenosine
1
This test method is under the jurisdiction of ASTM Committee D19 on Water
2
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Nov. 1, 2023. Published November 2023. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1981. Last previous edition approved in 2023 as D4012 – 23. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D4012-23A. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ---------
...
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: D4012 − 23 D4012 − 23a
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
1
in Water
This standard is issued under the fixed designation D4012; 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 covers a protocol for capturing, extracting and quantifying the cellular adenosine triphosphate (cATP) content
associated with microorganisms normally found in laboratory cultures and waters in plankton and periphyton samples from waters.
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 method does not remove all known chemical interferences, known to either luminesce in the 530 nm 6 20 nm range, or
to quench light emitted in that range. It should not be used to determine ATP concentrations in samples with dissolved organic
compounds, heavy metals or >10 000 ppm total dissolved solids. Alternative methods have been developed for determining ATP
concentrations in fluids samples likely to contain such interferences (Test Methods D7687 and E2694).
1.4 Knowledge of the concentration of ATP can be related to viable biomass or metabolic activity of microorganisms (Appendix
X1).
1.5 This test method offers a high degree of sensitivity, rapidity, accuracy, and reproducibility.
1.6 The analyst should be aware that the precision statement pertains only to determinations in reagent water and not necessarily
in the matrix being tested.
1.7 This test method is equally suitable for use in the laboratory or field.
1.8 The method normally detects cATP concentrations in the range of 0.1 pg cATP/mL (–1.0Log [pg cATP/mL]) to
10
4 000 000 pg cATP/mL (6.6 Log [pg cATP/mL]) in 50 mL water samples.
10
1.9 Providing interferences can be overcome, bioluminescence is a reliable and proven method for qualifying and quantifying ATP,
although the method does not differentiate between ATP from different sources, for example, from different types of
microorganisms, such as bacteria, fungi, algae and protozoa.
1.10 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved July 1, 2023Nov. 1, 2023. Published August 2023November 2023. Originally approved in 1981. Last previous edition approved in 20152023
as D4012 – 15.D4012 – 23. DOI: 10.1520/D4012-23.10.1520/D4012-23A.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
D4012 − 23a
1.11 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.
1.12 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
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1601 Test Method for Dilute Solution Viscosity of Ethylene Polymers
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
D6161 Terminology Used for Microfiltration, Ultrafiltration, Nanofiltration, and Reverse Osmosis Membrane Processes
D6300 Practice for Determination of Precision and Bias Data for Use in Test Methods for Petroleum Products, Liquid Fuels, and
Lubricants
D7687 Test Method for Measurement of Cellular Adenosine Triphosphate in Fuel and Fuel-associated Water With S
...
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