Critical methodological challenges in the microbial biosensor approach to assessing Hg(II) bioavailability were evaluated from the perspective of analytical chemists. The main challenge stems from the fact that the chemical speciation of Hg(II) in natural waters exerts a major control on its bioavailability, yet its natural complexation equilibria are extensively altered during conventional bioassays. New data, obtained using a bioluminescent Hg(II)-biosensor, that illustrate these challenges are presented and potential solutions proposed.