This study aimed to evaluate the development of Canavalia ensiformis seedlings under high concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in different substrates. In the first analysis, seeds from different origins were disinfected and introduced into a modified Leonard vessel (PET vessel) containing washed sand or washed sand plus vermiculite substrates and under high concentrations of 2,4-D (0 - control, 250 and 500 mg/L). For a second analysis, seed germination, callogenesis and contamination in different concentrations of 2,4-D (0 - control, 20, 40, 250 and 500 mg/L) were evaluated with sterile sand as substrate. Results showed that higher concentrations of 2,4-D could induce larger areas of callus formation. At a concentration of 40 mg/L of 2,4-D, germination in riparian forest substrates washed sand resulted in root growth and callus formation without contamination. Although C. ensiformis seeds showed tolerance to regions with high concentrations of 2,4-D, they do not demonstrate phytoremediation capacity for this herbicide since it caused damage to the plant’s initial development.