Tributaries are important for fish recruitment and diversity. Here, we examine the biological impact of inorganic and organic contaminants in Paint Branch stream (PBS), a tributary of the Anacostia river in Washington D.C. The Anacostia has suffered severe ecological damage because of decades of pollution and deposited wastewater runoff; however, PBS, which connects to the northern part of the river, is forested and less urbanized, suggesting higher water quality. However, the impact of PBS water on early fish development has not been studied. To address this question, we examined if chronic (28 day) exposure to water collected from PBS can support the proper early development of zebrafish (Danio rerio), a vertebrate model in toxicological studies. We assessed their overall growth and swimming behaviors and correlated these results with a water quality analysis. The water chemistry identified high levels of calcium, sodium, and nitrate in PBS water samples. A gas chromatography–mass spectroscopy analysis of extracted non-polar compounds in the water column revealed siloxanes (congeners D6–D10) were the only component identified with >90% certainty. In our fish experiments, we observed age-dependent increases in growth and eye development, consistent with normal development. In contrast, general swimming behaviors showed an early increase in angular velocity at 7 days postfertilization (dpf; p = 0.001) and a decreased total distance traveled at 14 dpf (p = 0.015) for PBS-treated larvae. Using the open field test, we observed that the PBS-treated fish made fewer visits to the edge at 7 (p = 0.01), 14 (p < 0.001), and 21 dpf (p = 0.038) and spent significantly more time at the edge at 21 dpf (p < 0.001). Fewer visits to the center were also noted at 14 and 21 dpf, suggesting reduced overall movement at these two ages in response to chronic PBS water exposure. Interestingly, by 28 dpf, no differences were noted in any parameter measured. Overall, these results indicate zebrafish larvae grew well in PBS water; however, their reduced movement and anxiogenic behavior suggested subtle behavioral abnormalities. The identified chemicals likely originated from runoff or sewage and have potentially deleterious consequences for fish living in PBS or migrating to/from upstream spawning/nursery locations.