Abstract The overarching goal of the Duke Superfund Research Center (DUSRC) is to determine later life consequences of early life exposure to polycyclic aromatic hydrocarbons (PAHs) and heavy metals. In accordance with the Superfund Mandate on understanding epidemiological and ecological impacts of exposure to hazardous chemicals, DUSRC-Project 4 addresses ecological outcomes of PAH and metal exposure in an evolutionary toxicology context. Evolved resistance to hazardous contaminants has far reaching implications for environmental health, ecological risk assessment, and management. However, key knowledge gaps exist in our understanding of the mechanisms of pollution adaptation and fitness consequences, especially when animals are exposed to multiple chemicals (simultaneous or sequential) and other abiotic stressors in their natural environment. Addressing these gaps, we extend our extensive previous research on evolved resistance to PAHs in Atlantic killifish Fundulus heteroclitus in the Elizabeth River (ER), VA, in a new direction. Specifically, we will elucidate the role of mitochondria as an important target of PAHs and metals during early development in mediating adverse behavioral and bioenergetic outcomes later in life and in subsequent generations under optimal and suboptimal thermal and dissolved oxygen conditions. Our specific aims are: Aim 1 – to determine later life fitness consequences of early life simultaneous exposures to PAHs and Cd and Pb; Aim 2 – to elucidate later life and cross generational fitness consequences of developmental exposures to PAHs and subsequent Cd or Pb exposure and determine links to epigenetic modifications; Aim 3 – to compare the gut microbiome differences in PAH-resistant and sensitive fish, and elucidate the potential contributions of the gut microbiome on PAH resistance using germ-free killifish studies; Aim 4 – to develop an ecological-effect directed analysis based on medium-throughput mitochondrial and behavioral assays to assess toxicity of environmental samples including remediated samples derived in DUSRC-Project 5. Moreover, we will bi-directionally collaborate with Project 2 (behavioral studies) and Project 3 (mitochondrial studies), focusing on aspects of experimental design and toxicity mechanisms. We will also work closely with the community engagement core for dissemination of our findings to the relevant communities, the data management core for experimental design, data management and analyses, and the analytical chemistry core for chemical analyses. The project will be heavily involved in the training of graduate students and post-doctoral researchers and will coordinate activities with the training core as well as the admin core for overall support and research translation. Overall, studies will elucidate ecologically relevant fitness outcomes of PAH and metal exposures and selected aspects of underlying molecular mechanisms in killifish populations, while rendering the toxicity assays for...