Honors Theses

Date of Award

Spring 5-7-2022

Document Type

Undergraduate Thesis



First Advisor

Kristine Willett

Second Advisor

Thomas Werfel

Third Advisor

Teresa Lefmann

Relational Format



Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is a known carcinogen leading to adverse effects in the development of both humans and animals. BaP is also continuously present in the environment leading to regular exposure via inhalation or ingestion. Because organisms’ early life stages can be more susceptible to contaminant exposure, our focus was on BaP’s adverse impacts on survival, length, weight, behavior, bioenergetic state, and fecundity following developmental exposures. To study BaP’s impacts, zebrafish (Danio rerio) were used as a model organism. BaP is a ligand for the aryl hydrocarbon receptor (AHR in humans; Ahr in fish). This receptor mediates some of BaP’s adverse effects (i.e., metabolic activation of reactive oxygen species). To understand the role of Ahr in behavior and bioenergetics, wild-type (5D) and a presumptive Ahr2-/- line were used. Embryos were exposed to waterborne BaP exposure at confirmed concentrations of 0, 4.02, or 53.9 µg/L from 6 – 120 hours post fertilization (hpf). Fish were then raised in clean water until 4 months postfertilization (mpf). Subsequently, survival, hatch, size, and behavioral effects (locomotion and anxiety-like behaviors) were recorded at various life stages: larval photomotor response at 120 hpf (larval); and open field test at 1 mpf (juvenile); 3 mpf (adolescent); and 4 mpf (adult). In addition, at 4 mpf, fecundity was assessed by breeding the F0 adults who were developmentally exposed to BaP to produce the F1 generation. The Seahorse XFe96 Flux Analyzer was used to assess the relative mitochondrial bioenergetic state at 4 mpf in the F0 fish. Furthermore, F1 survival and hatch was recorded, and behavior was assessed using a larval photomotor response assay at 120 hpf. In the wildtype (5D) F0 generation, while there were no significant differences in survival between control and 53.9 µg/L BaP through adulthood, larvae from the 4.02 µg/L BaP treatment group did not survive past 2 weeks post fertilization (wpf). The 53.9 µg/L BaP exposed males weighed significantly more than controls at 3 mpf and length was significantly increased at 4 mpf. With respect to behavior, BaP exposed larvae displayed increased activity in the dark phase compared to controls. No behavioral differences were observed in the open field assessment at 1 mpf. However, at 3 mpf, BaP exposed fish had a significant decrease in total distance traveled and a significant increase in freezing duration. At 4 mpf, adult females, regardless of treatment, spent significantly more time in the periphery than males indicating anxiety-related behavior, but there were no significant treatment effects. There were no significant differences in the bioenergetic state between treatments. No differences were observed in F0 fecundity or F1 survival and hatch. In the F1 generation, larvae whose parents were exposed to BaP were significantly less active in the dark phase compared to the F1 controls, which is the opposite effect of what was observed in the F0 larvae. The Ahr2-/- fish when genotyped represented a mix of Ahrgenotypes so no conclusions were drawn from null animals. Overall, our wild-type results suggest that BaP-related behavioral impacts are present and can cause multigenerational effects and is potentially harmful in terms of cognition and development.



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