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Excess rainfall events could lead to overflows and combined sewer overflows, which could threaten local mussel populations. This study sought to compare the long- term effects of combined sewer overflows and treated municipal effluents in caged Elliptio complanata mussels. Mussels were caged at 2 overflow sites, one downstream site of a major municipal effluent dispersion plume and a reference upstream site for 3 months during the summer. At the end of the exposure period, mussels were collected, analyzed for municipal contaminants (including pharmaceuticals), and effects biomarkers based on endocrine disruption (vitellogenin expression), xenobiotic detoxification (glutathione S-transferase and metallothioneins), oxidative stress/inflammation (cyclooxygenase and lipid peroxidation) and DNA damage. The data revealed that surface waters contained less pharmaceutical products than the downstream site but atrazine and its metabolite were at higher levels in overflow sites. Mussels contained elevated amounts of total heterotrophic bacteria, caffeine, acebutolol and venlafaxine at the downstream site relative to the upstream site where caffeine was higher at one of the overflow site. The levels of vitellogenin gene expression were significantly increased in both sexes of mussels caged at the downstream site only. Multivariate analysis revealed that the biomarker responses were completely separated between upstream, overflow and downstream sites. The site discrimination was based on vitellogenin, metallothioneins, DNA damage in gonad and digestive gland, gonad lipids/proteins reserves, lipid peroxidation, gonado-somatic index and condition factor. Adverse outcome pathways analysis using the power law approach revealed that most changes in the biomarkers identified by discriminant function analysis were significantly scaled to gonad energy reserves, tissue/ mussel size and loss of weight following air emersion stress. In conclusion, the toxic effects of mussels caged at overflow sites generally displayed lower responses than mussels caged downstream a treated municipal effluent suggesting that these overflows pose a lower risk than the continuous exposure to treated municipal effluent., Invertebrate Survival Journal, Vol 17 No 1 (2020)