Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.

A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses.

The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.

Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.

Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.

My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.

Theory and Practice in Sustainability Science: Influence of Urban Form on the Urban Heat Island and Implications for Urban Systems

As the world population continues to grow past seven billion people and global challenges continue to persist including resource availability, biodiversity loss, climate change and human well-being, a new science is required that can address the integrated nature of these challenges and the multiple scales on which they are manifest. Sustainability science has emerged to fill this role. In the fifteen years since it was first called for in the pages of Science, it has rapidly matured, however its place in the history of science and the way it is practiced today must be continually evaluated. In Part I, two chapters address this theoretical and practical grounding. Part II transitions to the applied practice of sustainability science in addressing the urban heat island (UHI) challenge wherein the climate of urban areas are warmer than their surrounding rural environs. The UHI has become increasingly important within the study of earth sciences given the increased focus on climate change and as the balance of humans now live in urban areas.

In Chapter 2 a novel contribution to the historical context of sustainability is argued. Sustainability as a concept characterizing the relationship between humans and nature emerged in the mid to late 20th century as a response to findings used to also characterize the Anthropocene. Emerging from the human-nature relationships that came before it, evidence is provided that suggests Sustainability was enabled by technology and a reorientation of world-view and is unique in its global boundary, systematic approach and ambition for both well being and the continued availability of resources and Earth system function. Sustainability is further an ambition that has wide appeal, making it one of the first normative concepts of the Anthropocene.

Despite its widespread emergence and adoption, sustainability science continues to suffer from definitional ambiguity within the academe. In Chapter 3, a review of efforts to provide direction and structure to the science reveals a continuum of approaches anchored at either end by differing visions of how the science interfaces with practice (solutions). At one end, basic science of societally defined problems informs decisions about possible solutions and their application. At the other end, applied research directly affects the options available to decision makers. While clear from the literature, survey data further suggests that the dichotomy does not appear to be as apparent in the minds of practitioners.

In Chapter 4, the UHI is first addressed at the synoptic, mesoscale. Urban climate is the most immediate manifestation of the warming global climate for the majority of people on earth. Nearly half of those people live in small to medium sized cities, an understudied scale in urban climate research. Widespread characterization would be useful to decision makers in planning and design. Using a multi-method approach, the mesoscale UHI in the study region is characterized and the secular trend over the last sixty years evaluated. Under isolated ideal conditions the findings indicate a UHI of 5.3 ± 0.97 °C to be present in the study area, the magnitude of which is growing over time.

Although urban heat islands (UHI) are well studied, there remain no panaceas for local scale mitigation and adaptation methods, therefore continued attention to characterization of the phenomenon in urban centers of different scales around the globe is required. In Chapter 5, a local scale analysis of the canopy layer and surface UHI in a medium sized city in North Carolina, USA is conducted using multiple methods including stationary urban sensors, mobile transects and remote sensing. Focusing on the ideal conditions for UHI development during an anticyclonic summer heat event, the study observes a range of UHI intensity depending on the method of observation: 8.7 °C from the stationary urban sensors; 6.9 °C from mobile transects; and, 2.2 °C from remote sensing. Additional attention is paid to the diurnal dynamics of the UHI and its correlation with vegetation indices, dewpoint and albedo. Evapotranspiration is shown to drive dynamics in the study region.

Finally, recognizing that a bridge must be established between the physical science community studying the Urban Heat Island (UHI) effect, and the planning community and decision makers implementing urban form and development policies, Chapter 6 evaluates multiple urban form characterization methods. Methods evaluated include local climate zones (LCZ), national land cover database (NCLD) classes and urban cluster analysis (UCA) to determine their utility in describing the distribution of the UHI based on three standard observation types 1) fixed urban temperature sensors, 2) mobile transects and, 3) remote sensing. Bivariate, regression and ANOVA tests are used to conduct the analyses. Findings indicate that the NLCD classes are best correlated to the UHI intensity and distribution in the study area. Further, while the UCA method is not useful directly, the variables included in the method are predictive based on regression analysis so the potential for better model design exists. Land cover variables including albedo, impervious surface fraction and pervious surface fraction are found to dominate the distribution of the UHI in the study area regardless of observation method.

Chapter 7 provides a summary of findings, and offers a brief analysis of their implications for both the scientific discourse generally, and the study area specifically. In general, the work undertaken does not achieve the full ambition of sustainability science, additional work is required to translate findings to practice and more fully evaluate adoption. The implications for planning and development in the local region are addressed in the context of a major light-rail infrastructure project including several systems level considerations like human health and development. Finally, several avenues for future work are outlined. Within the theoretical development of sustainability science, these pathways include more robust evaluations of the theoretical and actual practice. Within the UHI context, these include development of an integrated urban form characterization model, application of study methodology in other geographic areas and at different scales, and use of novel experimental methods including distributed sensor networks and citizen science.