Ballast water is a likely cause for worldwide transfer of non-indigenous aquatic species because of the large volumes and frequency of possible inoculations. Ozone is one treatment option being considered for eliminating non-indigenous species in ballast water. When ozone is applied to seawater, secondary disinfectants are formed, commonly measured and expressed as total residual oxidant (TRO). The goal of this study was to determine those variables most likely to affect the rate of TRO increase during ozonation and the subsequent TRO decline that occurs over time. These parameters strongly influence the efficacy of ozone treatments aimed to eliminate organisms present in ballast water. Seawater was obtained from Puget Sound, Washington; Cape Fear, North Carolina; and San Francisco Bay. Results indicated that seawater characteristics, including the organic content and ammonia, affect the amount of ozone required to achieve a desired TRO level and rate of TRO decay, and therefore need to be considered in determining ozone requirements for ballast water treatment.