Technical Abstract: This study was performed to investigate the potential of vegetated drainage ditches for mitigating the impact of agricultural irrigation runoff on downstream aquatic ecosystems. Water column toxicity to larval fathead minnow (Pimephales promelas), and the amphipod, Hyalella azteca, was measured for =12 h at the ditch inflow and outflow (approx. 400 m downstream) using custom-built in situ exposure systems. In addition, water and sediment samples were subject to standard toxicity tests with Ceriodaphnia dubia and H. azteca, respectively. No acute toxicity to larval fathead minnow was observed, however, runoff was highly toxic to invertebrates. Passage through a 389- 402 m section of vegetated ditch had a mitigating effect and reduced toxicity to some degree. However, runoff from an alfalfa field treated with chlorpyrifos remained very highly toxic to both invertebrate species, and runoff from a tomato field treated with permethrin remained very highly toxic to H. azteca after passage through the ditch. Predicted TU based on insecticide concentrations in runoff and 96-h LC50 values generally agreed with C. dubia toxicity of grab samples, but significantly underestimated in situ toxicity to H. azteca. Sediments collected near the ditch outflow were toxic to H. azteca. Our results demonstrate that experimental field ditch lengths, alone, were unable to eliminate the risk of irrigation runoff to aquatic ecosystems. In addition, pyrethroid toxicity may commonly be underestimated when monitoring programs rely on laboratory test with C. dubia or analytical chemistry.