Di-(2-ethylhexyl) phthalate (DEHP) associated in vitro/vivo toxicity at current environmentally relevant concentration (ERC) with attendant ecological risks in the Three Gorges Reservoir Area (TGRA) is still elusive. Responding to this challenge, a novel integrated study based on analytical and biological assays was designed to elucidate the underlying mechanisms for toxicity of DEHP and its ecological risks at ERC. In this study, GC-MS analysis showed that the highest environmental concentration of DEHP in the TGRA surface water was nearly double that of WHO and USEPA standards. Both distribution and ecological risk decreased from the upper to middle and lower reaches of the TGRA. In vitro toxicity was assessed by cell viability and DNA damage assays: DEHP exposure at ERCs (100-800 mu g/L) caused significant reduction in cell viability and elevated DNA damage. Further, DEHP exposure above 400 mu g/L resulted in enhanced migration behavior of cancer cells. For in vivo toxicity assessment, short term acute exposure (7 d, 400 mu g/L) apparently activated the PI3K-AKT-mTOR pathway, and chronic low-level exposure (3 months, 10 -33 mu g/L) suppressed the hypothalamus pituitary thyroid (HPT) axis pathway in zebrafish. In addition, acute low-level exposure (5 d, 33-400 mu g/L) to DEHP increased aryl hydrocarbon receptor (AhR) activity in Tg(cyp1a:gfp) zebrafish in a concentration-dependent manner. In short, DEHP at ERC has extended potential to induce diverse in vitro and in vivo toxicity at concentrations that also cause impairment of biochemical function in aquatic species of the TGRA. (C) 2018 Elsevier Ltd. All rights reserved.