High volume horizontal Hydraulic fracturing (HF) has emerged as a revolutionary method to stimulate the tight shale well, and dramatically increased the unconventional oil and gas production. Once the fracturing pressure released, the stimulated well starts to produce a complex saline mixture, referred as hydraulic fracturing flowback and produced water (HF-FPW). Increasing concerns have been raised regarding the environmental impact of releases of this wastewater to aquatic ecosystems. The toxicity of HF-FPW has not been previously reported, and is complicated by the combined complexity of organic and inorganic constituents in HF fluids and deep formation water. My research focuses on characterizing the chemical and toxicological profiles, and exploring the mechanisms of toxicity of HF-FPW. Targeted and untargeted organic analyses have revealed the HF-FPW contains numerous organics including natural hydrocarbons, additive organics, and potential secondary by-products. Acute and sub-chronic toxicity of HF-FPW was assessed on aquatic invertebrate and fish models. The adverse effects and toxic mechanism of HF-FPW on biotransformation, oxidative stress, endocrine disruption, and embryo development were also evaluated in fish models by using a variety of enzymatic, biochemical, and molecular tools. Overall, this work is the first study to demonstrate the chemical and toxicological complexity of real HF-FPW samples, and has improved our understanding on mechanisms of toxicity of this complex petroleum-associated wastewater and its potential impacts on aquatic ecosystem.