All in all, there remain several challenges to define the exposure concentration in the solvent spiking toxicity testing of BACs with a long alkyl chain. Using Cnom to calculate the effect concentrations of BACs with a long alkyl chain (#C ≥ 14) in solvent spiking tests may not provide reliable data to assess their toxicity, and even measured Cw may not represent the true bioavailable exposure concentration for C18-BAC.