Measuring Adenosine triphosphate (ATP) provides a proxy to check compliance with IMO's ballast water D-2 standard: <10 cells mL⁻¹ in the 10–50 μm size class. Measured with standard boiling techniques the ATPconcentration in aquatic eukaryotic microorganisms is 0.6 mol m ⁻³. Model calculations with 10–50 μm spherical organisms show their ATP content is 0.2 to 20 pg cell⁻¹, in line with several cell lysis data. However, at 10 cells mL⁻¹, these ATP contents lead to a maximum of only 2 to 200 pg ATP mL⁻¹, at least 7.5× below a D-2 test kit 1500 pg ATP mL⁻¹ upper limit. Differentcell shape and ATP extraction scenarios to reach 1500 pg ATP mL ⁻¹ are discussed but remain improbable. Because cell lysis data are inconclusive, and a novel phosphoric acid-benzalkonium chloride method indicates up to 3× higher ATP concentrations, an independent test kit validation and a comparison of all three techniques are recommended.