1 Title: Toxicity in aquatic model species exposed to a temporal series of three different flowback and 1 produced water samples collected from a horizontal hydraulically fractured well. 2 3 Authors: Erik J. Folkerts 1 , Tamzin A. Blewett 1 , Perrine Delompré 1 , W. Tyler Mehler 1 , Shannon L. 4 Flynn 3 , Chenxing Sun 4 , Yifeng Zhang 4 , Jonathan W. Martin 4 , Daniel S. Alessi 2 , Greg G. Goss 1,5 . 5 1 Department of Biological Sciences, University of Alberta, Edmonton, T6G 2E9 Alberta, Canada 6 2 Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, T6G 2E3 Alberta, 7 Canada 8 3 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU, 9 UK 10 4 Division of Analytical and Environmental Toxicology, University of Alberta, Edmonton, T6G 2E9 11 Alberta, Canada 12 5 National Institute for Nanotechnology, Edmonton, Alberta, T6G 2M9, Canada 13 14 Abstract 15 In the present study, we compared the toxicity and associated chemical characterizations of 16 flowback and produced water (FPW) collected from a single horizontal hydraulically fractured well at 17 different time points during FPW production. Since few studies on whole mixture toxicity related to FPW 18 exist, our aims were to determine both overall toxicity of the FPW mixture in a suite of organisms 19 (Daphnia magna, Lumbriculus variegatus, Danio rerio, and Oncorhynchus mykiss) and also determine if 20 toxicity changes depending on variation in FPW chemical properties as a function of time sampled (1.33, 21 72, and 228 hr FPW samples collected immediately post-well production onset were analyzed in current 22