Thus, according to the solvent-spiking tests, the toxicity of BACs increases with increasing alkyl chain length up to 14 carbon atoms, beyond which the trend apparently decreases. In contrast, the EC50 values are similar with the alkyl chain length ≥ 14 carbon atoms in the passive dosing tests (Figure ). It is commonly known that toxicity of chemicals increases with increasing hydrophobicity. However, a "cutoff effect" has often been observed in toxicity of amphiphile homologous series, including cationic surfactants; that is, toxicity increases with alkyl chain length up to a certain point, above which toxicity remains more or less constant or even decreases. 20, Sorption losses to glass beakers do not explain the cutoff in this study, because we used the measured Cw to calculate the EC50. There are several possible explanations for this behavior. For long-chain cationic surfactants in the spiking tests (e.g., C18-BAC), there may be difference between the freely dissolved and total concentrations due to association with dissolved organic components or formation of micelles, resulting in low bioavailability. As mentioned, this difference is considered negligible in passive dosing methods, thereby mitigating a cutoff effect. Another possible explanation for the cutoff point could be that the time required to reach organism/water equilibrium is longer for BACs with a long chain compared to those with a short chain, and the former do not reach equilibrium within the experimental time. . This could also explain why a slight cutoff effect was observed for C14-18-BACs in the 48 h toxicity test even using the passive dosing method.