- Incorporation of nanoparticles (NPs) into consumer products is on the rise and human exposure to NPs is unavoidable. Currently, there is insufficient data to assess the safety of nanoparticles. I conducted a series of five studies using the zebrafish model to determine which NP components (i.e., core material or surface functionalization) contribute to biological responses and how ionic strength influences these results. The first study employed a systematic, rapid embryonic zebrafish assay to identify specific responses to precisely engineered lead sulfide (PbS-NPs) and gold nanoparticles (AuNPs) functionalized with different surface ligands. Lead sulfide nanoparticles functionalized with either 3-mercaptopropanesulfane (MT) or sodium 2,3-dimercaptopropanesulfonate (DT) ligands with nearly identical core sizes caused differential responses at the same concentration. I determined that the different responses were because MT-functionalized NPs released more soluble lead ions than DT-functionalized NPs due to different decomposition and oxidation rates. The second study investigated the different biological responses of three NPs identified during toxicity screening of a gold nanoparticle library. AuNPs functionalized with 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE), induced differential biological responses in embryonic zebrafish at the same concentration. Exposure to MES-AuNPs induced sublethal effects, while TMAT-AuNPs were embryo-lethal and MEEE-AuNPs were benign. Gold tissue concentration was confirmed to be similar in exposed embryos using inductively coupled-mass spectrometry. Microarrays were used to gain insight to the causes of the different responses. This approach identified that MES- and TMAT-AuNPs perturbed inflammatory and immune responses. These differential biological responses may be due to misregulated transport mechanisms causing numerous downstream defects unique to each surface functional group‟s property. In the next study, I tested the long-term consequences of developmental exposure to TMAT-, MES, and MEEE-AuNPs, and showed that MES- and TMAT-AuNPs affected larval behavior that persisted into adulthood. During the course of these investigations, I found that high ion concentration in exposure solutions results in NP agglomeration, presenting a problem for NP testing in the zebrafish model. For the fourth study, I focused on solving this by determining that zebrafish can be raised in nearly ion-free media without adverse consequences. When 3-MPA-AuNPs were dispersed in this new low ionic media, I observed adverse responses in the embryonic zebrafish toxicity assay, but not when the NPs were suspended in high ionic media. Thus, I demonstrated that the media greatly influences both agglomeration rates and biological responses, but most importantly, that the zebrafish is insensitive to external ions. The fifth study focused on the adverse response observed when embryonic zebrafish were exposed to 3-MPA-AuNPs. Exposed larvae failed to respond to a touch in the caudal fin at 120 hours post fertilization (hpf). Addition of a neuromuscular stimulus, nicotine, revealed the exposed embryos were not paralyzed, but experienced a reduction in axonal projections. A global genomic analysis (RNA-seq) using embryos exposed to 3-MPA-AuNP and MEEE-AuNPs (non-toxic control) from 6 to 120 hpf suggested that neurophysiological and signal transduction processes were perturbed. Functional analysis of the data led to the hypothesis that the most elevated gene, early growth response 1 (EGR-1), impacts axonogenesis in the caudal fin, interfering with glutaminergic synapses and preventing the connection of sensory neurons and touch perception. Although MEEE-AuNPs did not cause morphological defects, the RNA-seq analysis identified that these NPs perturbed immune and inflammatory system processes. Collectively, these results suggest that surface functional groups drive the differential responses to nanomaterials. The five studies summarized here confirm that a systems toxicological approach using the zebrafish model enables the rapid identification of structure-activity relationships, which will facilitate the design of safer nano-containing products.