2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Cluronomus riparius after long-term exposure to 2,4-D The larvae were treated with 0 1, 1 or, 10 mu g L-1 of 2.4-D for short- and long-term exposure periods. The sex ratio was changed in C riparius exposed to only 10 mu g L-1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C riparius exposed to a 10 mu g L-1 of 2,4-D Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), terrains and glutathione S-transferases (GSTs) in C riparius exposed to 2,4-D for 24 h The expressions of HSP70, HSP40, HSP90 and GST levels in C riparius were significantly increased after exposure to a 10 mu g L-1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C riparius Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment (C) 2010 Elsevier Inc All rights reserved.