The use of fertilizers in agriculture has a well-known effect on river, lake and coastal sea eutrophication. The effect of agricultural pesticides on aquatic ecosystems has been less investigated. Pesticides are transferred to the rivers via soil water runoff and spray drift. The toxicity of pesticides can affect non-target aquatic organisms and thus perturbate ecosystems. Herbicides may be toxic especially for aquatic photosynthetic organisms including algae. Periphytic diatoms are a major component of the biofilm growing at the light exposed surface of immerged substrates. As primary producers they are at the basis of the lotic trophic web. Diatoms have been extensively used as bioindicators of trophic and organic pollution. However, diatom standard indicators fail to properly detect toxic impact of molecules which do not target the photosystem II. The purpose of the study is to evaluate the effect of an herbicide, the metolachlor (a cell division inhibitor, inhibitor of Very-Long-Chain Fatty Acid) on a diatom species isolated from the field (Gomphonema gracile Ehrenberg), exposed to three concentrations of metolachlor (1, 10, 100 µg/L). Assessing metolachlor toxicity is of major concerns, because of its presence at high concentration in rivers. The effects of metolachlor on diatoms were evaluated according to physiological and behavioral parameters. These parameters were analyzed immediately after exposure, and 1, 2, 3, 4 and 7 days post-exposure. Metolachlor did not affect much population dynamics, photosynthesis and ATP content. Though metolachlor did not bioaccumulate in the biota, the mobility parameters provided early responses (on day 3) for all concentrations. These finding demonstrate the importance of use of various parameters and the potential of behavioral descriptors in toxicity assessment.