High hydrophobicity and volatility of chemicals often lead to substantial experimental challenges but were here utilized in headspace passive dosing (HS-PD) to establish and maintain exposure: the pure chemical served as a passive dosing donor for controlling exposure at saturation, whereas triglyceride oil containing the chemical was used to control lower exposure levels. These donor solutions were added to glass inserts placed in the closed test systems. Mass balance calculations confirmed a dominant donor capacity for all chemicals except isooctane. This HS-PD method was applied to algal growth inhibition and springtail lethality tests with terpenes, alkanes, and cyclic siloxanes. Headspace concentrations above the lipid donors were measured for three chemicals to determine their chemical activity, using saturated vapor as the analytical standard and thermodynamic reference. Toxicity was related to chemical activity and calculated concentrations in membranes at equilibrium with the lipid donor. For both tests and all chemicals, toxic effects were observed within or above the reported range for baseline toxicity, meaning that no excess toxicity was observed. The toxicity of siloxanes was markedly higher to the terrestrial springtail than the aquatic algae, which is consistent with a more efficient mass transfer of these volatile hydrophobic chemicals in air compared to water.