Abstract:The environmental quality standards based on total contents of heavy metals can’t meet the requirements of current soil management, studies on risk assessment and toxicity threshold that considering the bioavailability of heavy metals have great significance for the revision of corresponding standards. In this study, three farmland soils with different properties, i.e, black soil, meadow cinnamon soil, unsubmerged paddy soil, were selected and biological toxicity tests were conducted by simulating Cu pollution, four chemical extractants with different extraction capacities, including HNO3, EDTA-Na2, NH4OAc and CaCl2, were used to extract bioavailable Cu, the relationship between Cu extractability, bioavailability and toxicity were analyzed, and the toxicity thresholds of Cu were deduced for lettuce and earthworm Eisenia foetida. The results showed that HNO3 (41.4%) and EDTA-Na2 (56.8%) had strong Cu extraction ability, but NH4OAc (0.12%) and CaCl2 (8.70%) had weak ability. Cu extracted by CaCl2 significantly correlated with Cu accumulated in lettuce, the toxic effect, and the acute mortality of earthworms, while Cu extracted by HNO3 significantly correlated with the chronic toxicity and Cu accumulated in earthworms. Based on the effective concentration of different extractable Cu, the deduced EC20 for lettuce ranged from 90.5 mg/kg to 170 mg/kg based on HNO3, 103 mg/kg to 195 mg/kg based on EDTA-Na2, 3.97 mg/kg to 20.1 mg/kg based on NH4OAc and 0.21 mg/kg to 8.68 mg/kg based on CaCl2, while the deduced EC20 for earthworm Eisenia foetida ranged from 138 mg/kg to 193 mg/kg based on HNO3, 108 mg/kg to 226 mg/kg based on EDTA-Na2, 8.92 mg/kg to 11.6 mg/kg based on NH4OAc and 0.36 mg/kg to 10.6 mg/kg based on CaCl2. The deduced EC50 for lettuce ranged from 110 mg/kg to 188 mg/kg based on HNO3, 119 mg/kg to 230 mg/kg based on EDTA-Na2, 5.69 mg/kg to 32.2 mg/kg based on NH4OAc and 0.26 mg/kg to 9.62 mg/kg based on CaCl2, while the deduced EC50 for earthworm Eisenia foetida ranged from 183 mg/kg to 221 mg/kg based on HNO3, 180 mg/kg to 331 mg/kg based on EDTA-Na2, 13.1 mg/kg to 18.3 mg/kg based on NH4OAc and 0.54 mg/kg to 13.2 mg/kg based on CaCl2. The results have great significance for the selection and optimization of effective extraction methods of heavy metals in soils and the revision of environmental quality standards based on heavy metals bioavailability.