This study examined the biological response of aquatic organisms, collected in situ and from laboratory toxicity testing of water and soil to the herbicide Arsenal® primarily used to decrease the invasive species Arundo donax in the Rio Grande River through Big Bend National Park in Texas (USA). Biological, toxicological, physicochemical, and flow data were collected before, during, and after treatment from 4 reference and 3 treatment sites. Significant differences existed in scoring, and in toxicity testing of water and sediment, between treatment and reference sites. Greater toxicity was observed in sediment samples compared to water samples. This could be due to the surfactant nonylphenol ethoxylate (NPE), added to the Arsenal® formulation rather than the active ingredient imazapyr. Care must be implemented with the results of this research due to many confounding variables that occurred during the study period. Flooding could impact the sediment toxicity results as sediment-bound contaminants could be spread some distance from their origin. Due to treatment timing this represented an extreme condition as far as concentration of potential toxicants in the water are concerned and these relatively high concentrations would be expected to have decreased within a relatively short period of time, especially in the water. We believe the findings from this study provide indication of potential toxicity of some magnitude as well as provide direction for management. Decisions of whether the toxic effects of herbicide use to aquatic macroinvertebrates outweigh the beneficial effects of Arundo donax control should be carefully considered.