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 Methods D7687 and E2694 except for the volumes sampled, and omission of wash and drying steps used in Test Methods D7687 and E2694 to remove interferences (1.3).

5.6 Although ATP data generally covary with culture data in water samples, different factors affect cATP concentration than those that affect culturability.

5.6.1 Culturability is affected primarily by the ability of captured microbes to proliferate on the growth medium provided, under specific growth conditions. Consequently, a proportion of the active or inactive microbial population present in a sample may be viable but not detected by any one culture test.3

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

5.6.2.1 One example of the species effect is that the amount of ATP per cell is substantially greater for active fungal cells than bacteria (Appendix X1).

5.6.2.2 Within a species, cells that are more metabolically active will have more ATP per cell than dormant cells, such as fungal spores.

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

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 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.

1.11 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.