When an invasive wetland grass degrades a Ramsar wetland and Important Bird Area, decisive management action is called for. To limit the extent and spread of European Phragmites australis, the Ontario government began the first, large-scale application of glyphosate (Roundup Custom®) over standing water to control an invasive species in Canadian history. Between 2016 and 2018, over 1000 ha of marsh were treated. To assess the risk this herbicide presented to aquatic biota, we measured the concentration of glyphosate, its primary breakdown product aminomethylphosphonic acid (AMPA), and the alcohol ethoxylate-based adjuvant Aquasurf® in water and sediments in areas of the highest exposure risk and up to 150 m into adjacent bays. We never detected glyphosate or AMPA at concentrations exceeding thresholds of toxicological concern. The maximum observed concentration of glyphosate in water was 0.320 ppm, occurring within 24 hr of application. The maximum glyphosate concentration in sediment was 0.250 ppm, occurring within 30 days of application. AMPA was detectable in water and sediment, indicating microbial breakdown of glyphosate in the marsh, but at low concentrations (maxwater = 0.025 ppm, maxsed = 0.012 ppm). The maximum distance from the point of application at which glyphosate was detected in the water was 100 m, vs. 0 m for AMPA. Concentrations in water returned to pre-treatment levels (<DL) within 20-30 days of application. In sediment, glyphosate residue persisted above detection limits (>0.005 ppm) for over one year but less than two years. Concentrations of alcohol ethoxylates were variable in space and time, following a pattern that could not be attributed to Aquasurf® application. The direct, over-water application of Roundup Custom® with Aquasurf® to control invasive P. australis does not pose a toxicological risk to aquatic biota.HighlightsGlyphosate-based herbicide was applied directly to >1000 ha of marsh to control invasive P. australisGlyphosate and AMPA did not reach levels of toxicological concern for aquatic biotaAquasurf® exceedances were observed but could not be attributed to P. australis control activityGlyphosate, AMPA, and Aquasurf® dispersed no more than 100 m from the point of applicationGlyphosate, AMPA and Aquasurf® in water returned to baseline levels within 30 days of applicationGlyphosate, AMPA and Aquasurf® in sediment returned to baseline levels within 2 years