Humic substances, which account for the majority of dissolved organic matter in aquatic and soil, have the various properties due to the differences of its origin such as water quality, soil property and differences of ecosystem. In addition, humic substances affect the mobility and bioavailability of heavy metals in aquatic and soil ecosystem. In order to estimate the ecotoxicity induced by the copper in aquatic and soil ecosystems, it is important to analyze which characteristics of humic substances have strong influences on the existing form of heavy metals and also it is important to examine if these interactions affect the toxicity of heavy metals. The objective of this study is to analyze the binding of proton and cupric ion to three types of binding sites in the humic acids by NICA (Non-Ideal Competitive Adsorption)-Donnan model and to examine the influences of each humic acid on the algal (P. subcapitata.) growth inhibition by copper ecotoxicity. In terms of behavior of proton and cupric ion binding analyzed by NICA-Donnan model, behavior of proton and cupric ion binding differ from source to source of humic acids. It is due to the differences of molecular weight of humic acids, abundance of functional groups that become binding sites of humic acids. And also it is due to the number and density of electron donor groups or electron attractive groups because the electrostatic potential, which changes depending on electron donor groups or electron attractive groups, affect the amount of binding sites. In terms of the influences of humic acid on copper growth inhibition to P. subcapitata, the toxicity reduction levels are almost same among sources like liver, and soil.