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Numerous state and federal agencies are increasingly concerned with the rapid expansion of invasive, noxious weeds across the United States. Herbicides are frequently applied as weed control measures in forest and rangeland ecosystems that frequently overlap with critical habitats of threatened and endangered fish species. However, there is little published chronic toxicity data for herbicides and fish that can be used to assess ecological risk of herbicides in aquatic environments. We conducted 96-h flowthrough acute and 30-day chronic toxicity studies with swim-up larvae and juvenile rainbow trout (Onchorhyncus mykiss) exposed to the free acid form of 2,4-D. Juvenile rainbow trout were acutely sensitive to 2,4-D acid equivalent at 494 mg/L (95% confidence interval [CI] 334-668 mg/L; 96-h ALC(50)). Accelerated life-testing procedures, used to estimate chronic mortality from acute data, predicted that a 30-day exposure of juvenile rainbow trout to 2,4-D would result in 1% and 10% mortality at 260 and 343 mg/L, respectively. Swim-up larvae were chronically more sensitive than juveniles using growth as the measurement end point. The 30-day lowest observable effect concentration (LOEC) of 2,4-D on growth of swim-up larvae was 108 mg/L, whereas the 30-day no observable effect concentration (NOEC) was 54 mg/L. The 30-day maximum acceptable toxicant concentration (MATC) of 2,4-D for rainbow trout, determined as the geometric mean of the NOEC and the LOEC, was 76 mg/L. The acute:chronic ratio was 6.5 (i.e., 494/76). We observed no chronic effects on growth of juvenile rainbow trout at the highest concentration tested (108 mg/L). Worst-case aquatic exposures to 2,4-D (4 mg/L) occur when the herbicide is directly applied to aquatic ecosystems for aquatic weed control and resulted in a 30-day safety factor of 19 based on the MATC for growth (i.e., 76/4). Highest nontarget aquatic exposures to 2,4-D applied following terrestrial use is calculated at 0.136 mg/L and resulted in a 30-day safety factor of 559 (e.g., 76/0.163). Assessment of the exposure and response data presented herein indicates that use of 2,4-D acid for invasive weed control in aquatic and terrestrial habitats poses no substantial risk to growth or survival of rainbow trout or other salmonids, including the threatened bull trout (Salvelinus confluentus).