The prevalence of polycyclic aromatic hydrocarbons and their oxygenated and nitrogen-containing analogs in freshwater ecosystems are of concern due to their reported toxicity to several aquatic species including Daphnia magna. The aim of this study was to explore the mode of action of phenanthrene (PHEN), 9,10-phenanthrene quinone (PHQ), and phenanthridine (PN) as little is known about the molecular-level impairments, especially at low levels. For this purpose, D. magna was exposed to three sub-lethal levels of pollutants for 24 h. To assess biochemical responses, 52 polar metabolites were extracted from individual adult daphnids, and analyzed using a mass spectrometry-based targeted metabolomics approach. Changes in the normalized metabolite concentrations revealed up and down-regulation relative to the control group for all pollutants. PN exposure resulted in the most statistically significant changes to metabolite concentrations across all applied sub-lethal levels. Moreover, PN exposure responses were non-monotonic across exposure concentrations, whereas monotonic responses were observed for PHEN and PHQ. Biochemical pathway analysis for PN showed that all exposure concentrations had the same perturbed metabolic pathways. However, the number of perturbed pathways increased with increasing exposure concentrations for PHEN and PHQ. The results suggest that PN, and PHQ are more disruptive due to the presence of reactive functional groups when compared to PHEN. The findings of this study indicate that the sub-lethal mode of action of PN was equally disruptive across all applied levels; however, for PHEN and PHQ, the mode of action was concentration-dependent. Although the reported median effective concentration for PN is higher than PHEN and PHQ, our data shows that metabolomics captures molecular-level changes that may not be detected by traditional toxicity metrics.