Sequential supercritical fluid extraction together with a two-site desorption model were employed to estimate the bioaccessible fraction of polycyclic aromatic hydrocarbons (PAHs) in four historically contaminated soils. The ecotoxicity of the soils was assayed by four different contact tests. The same soils were exposed to exhaustive extraction and the extracts were returned to the soils to ensure total 100% bioaccessibility of the pollutants. Then the soils were assayed again. Statistical evaluation revealed that the predicted bioaccessible PAHs generally correlated with the ecotoxicity responses of the tests. The estimated bioaccessible fractions varied from 10 to 98%. This value increased for PAHs with higher lipophilicity and showed no correlation with the organic carbon content in the soils. The ecotoxicity tests in the study indicated different sensitivity toward PAHs and the tests employing Heterocypris incongruens and Eisenia fetida were found to be more suitable than Lemna minor and Vibrio fischeri. Mortality and growth inhibition of ostracods correlated with all the types of PAHs and earthworm growth inhibition and mortality were preferentially sensitive to PAHs with only 3-4 aromatic rings. Determination of the biota-soil accumulation factors indicated that the earthworm growth inhibition corresponded to increased accumulation of PAHs in the earthworm tissue. (C) 2013 Elsevier B.V. All rights reserved.