Fractionation with biological and chemical analysis has been proposed to characterize the risk associated with complex samples. Thus, we have employed hot pressurized water for fractionating a herbicide-contaminated soil. Genotoxicity was found in polar and midpolarity fractions extracted from a historically herbicide-contaminated soil. However, the parent herbicides as well as organic solvent extracts (consisting of herbicides) were not genotoxic. Thus, polar organics formed by natural degradation of herbicides may be responsible for the genotoxic response. For correct assessment of the toxicity of complex matrices such as soils, both contaminated and uncontaminated samples need to be studied for their toxicological impact.