<p>Metal toxicity depends on water chemistry characteristics such as hardness, pH, and dissolved organic matter. In this study, we investigated the effects of pH and hardness on the toxicity of Zn, Cu, Cd, and Ni to a freshwater diatom, <i>Navicula pelliculosa</i>, using a fluorescence microplate toxicity assay. The toxicity of Zn, Cu, and Cd to <i>N. pelliculosa</i> increased with increasing pH, but the relationship between pH and Ni toxicity was unclear. The toxicity of Zn, Cd, and Ni to <i>N. pelliculosa</i> decreased with increasing hardness, whereas Cu toxicity showed no clear relationship with hardness. These results were used to develop a bioavailability model to predict metal toxicities in natural water samples. We modelled the effect of pH by using a log-linear relationship between pH and toxicity with the biotic ligand model-type competition effects of Ca²⁺ and Mg²⁺. In addition, the metal bioavailability model was based on the free ion activity of metals considering the complexation of metals with dissolved organic matter. Model applicability was tested by comparing experimentally observed toxicities with predicted ones. The differences between predicted and observed EC₅₀ and EC₁₀ were within a factor of 2 for all metals in both synthetic and natural water samples except for Cu in natural water samples. Although the model was shown to be applicable for Zn, Cd, and Ni, it greatly underestimated the Cu toxicity in natural water samples, probably due to overestimation of the toxicity mitigation effect of dissolved organic matter.</p>