The development of standardized toxicity tests is critical for assessing pollutants in aquatic ecosystems. The mayfly Neocloeon triangulifer offers advantages as a test species due to its asexual reproduction, short life cycle, and high sensitivity to pollutants. However, we identified variability in response sensitivity to pollutants, which may stem from the nutritional composition and growth stage of its primary food source, Navicula spp. diatoms. This study aimed to evaluate the repeatability and reproducibility of 96 h toxicity tests to sodium chloride with N. triangulifer fed diatoms of varying ages (target concentrations: 329, 494, 741, 1111, 1666, and 2500 mg/L). We tracked Navicula spp. growth stages under controlled conditions and characterized fatty acid and lipid contents at each stage to assess how these factors influence N. triangulifer sensitivity to sodium chloride, a common reference toxicant. Sensitivity increased significantly as the diatom feed aged, highlighting the need for standardized culturing methods to improve test reliability. This study is the first to simultaneously characterize the growth stages and fatty acid profiles of Navicula spp. diatoms as feed species for N. triangulifer. This work contributes valuable insights to the optimization of standardized toxicity testing with this sensitive aquatic insect.