Glyphosate is the most used herbicide globally, but little is known of its prevalence in the Australian environment or its behaviour during wastewater treatment. This study examined the concentration of glyphosate and its primary biodegradation product, aminomethylphosphonic acid (AMPA), in influent and effluent from 22 Australian wastewater treatment plants (WWTPs) to inform exposure risks. Glyphosate was detected in all 22 WWTP influent samples, with concentrations ranging from 0.37 to 370 μg/L (mean: 22 ± 76 μg/L). In treated effluent, glyphosate was found in 82 % of samples with concentrations ranging from <
LOQ (0.2 μg/L) to 11 μg/L (mean: 2.1 ± 3.1 μg/L). AMPA was detected in the influent of four of the 22 WWTP, with concentrations ranging from <
LOQ to 32 μg/L (mean: 1.4 ± 5.2 μg/L). AMPA was detected in the effluent samples at eleven sites, with concentrations ranging from <
LOQ to 19 μg/L. Glyphosate removal efficiency varied by treatment process. Plants with tertiary treatment (n = 10) removed on average 76 % of the glyphosate, while primary treatment sites (n = 4) removed the lowest (mean: -102 %
higher in the effluent). One tertiary treatment site had glyphosate concentrations 160 % higher in the treated effluent, suggesting additional sources post treatment. The mean population weighted mass load of glyphosate in treated effluent was 572 ± 856 mg per day per 1000 inhabitants, with an estimated 5000 kg/year of glyphosate reaching the Australian aquatic environment. A temporal study at two influent sites identified increasing glyphosate use over the past decade. Despite the widespread presence of glyphosate in effluent, all concentrations were all below the current Australian freshwater species protection guideline of 180 μg/L.