There is substantial demand for safe chemical treatments for diseases such as Ichthyophthiriasis (ICH) which is caused by the ectoparasite Ichthyophthirius multifiliis. In this study, we aimed to determine the minimum effective concentrations of copper sulfate and chloramine-T to kill the theronts of ectoparasite I. multifiliis at all environmentally relevant alkalinity and hardness levels in vitro. Using Fibonacci dose-escalation schemes, we exposed theronts cultured from the trophonts of infected rainbow trout (Oncorhynchus mykiss) to copper sulfate (1/34, 1/21, 1/13, 1/8, 1/5, 1/2, 1 and 2 ppm) or chloramine-T (1/2, 1, 2, 3, 5, 8, 13, 21,34 and 55 ppm). The theronts were submitted to various levels of alkalinity (50, 100, 175, 281 and 400 mgCaCO(3)/L), hardness (35, 83, 110, 170, 276 and 415 mgCaCO(3)/L) and the effectiveness of each chemical was monitored at 13, 21, 34, 55, 89, 144, and 233 min post exposure. Alkalinity and hardness of water altered both copper sulfate and chloramine-T toxicity to theronts. A linear relationship between alkalinity and copper sulfate toxicity was found, with five times more copper sulfate required to eliminate all theronts at 281 mg CaCO3/L compared with 50 mg CaCO3/L alkalinity. Relationship between hardness levels and the toxicity of copper sulfate and chloramine-T was logarithmic. Fibonacci dose escalation scale helped determine the minimum effective doses of each chemical tested, which may decrease costs and environmental impact of chemical treatment of fish with ICH.