An in vitro bioaccessibility test was applied for assessing the transfer of polycyclic aromatic hydrocarbons (PAHs) present in road dust, into benthic organisms living in a receiving water body. The road dust is supposed to be urban runoff particles under wet weather conditions. Sodium dodecyl sulfate (SDS) solution was used as a hypothetical gut fluid. Pyrene, fluoranthene and phenanthrene were the main PAH species in the SIDS extractable fraction of road dust, as well as the whole extract. Benzo(ghi)perylene showed relatively low concentrations in the SIDS extract in spite of a high concentration in the original dust. The PAH composition in benthic organisms (polychaetes) did not correspond with that of the surrounding sediment and the PAHs detected were also detected in high concentrations in the SDS extract of road dust. When testing the toxicity of the extracted contaminants by a standardised algal toxicity test, SIDS extracts of a detention pond sediment showed higher toxicity than the pore water of the corresponding sediment. Sediment suspension showed a comparative toxicity with 0.1% SIDS extract. From the results, the in vitro bioaccessibility test seems more suitable to evaluate the exposed contaminants than the traditional organic solvent extraction method and the SDS extracted fraction is applicable to toxicity tests reflecting the digestive process.