Exposure to ZnO nanoparticles alters neuronal and vascular development in zebrafish: Acute and transgenerational effects mitigated with dissolved organic matter.

Exposure to ZnO-nanoparticles (NPs) in embryonic zebrafish reduces hatching rates which can be mitigated with dissolved organic material (DOM). Although hatching rate can be a reliable indicator of toxicity and DOM mitigation potential, a fish that has been exposed to ZnO-NPs or any other toxicant may also exhibit other abnormal phenotypes not readily detected by the unaided eye. In this study, we moved beyond hatching rate analysis to investigate the consequences of ZnO-NPs exposure on the nervous and vascular systems in developing zebrafish. Zebrafish exposed to ZnO-NPs (1-100 ppm) exhibited an array of cellular phenotypes including: abnormal secondary motoneuron (SMN) axonal projections, abnormal dorsal root ganglion development and abnormal blood vessel development. Dissolved Zn (<10 kDa) exposure also caused abnormal SMN axonal projections, but to a lesser extent than ZnO-NPs. The ZnO-NPs-induced abnormal phenotypes were reversed in embryos concurrently exposed with various types of DOM. In these acute mitigation exposure experiments, humic acid and carbohydrate, along with natural organic matter obtained from the Suwannee River in Georgia and Milwaukee River in Wisconsin, were the best mitigators of ZnO-NPs-induced motoneuron toxicity at 96 h post fertilization. Further experiments were performed to determine if the ZnO-NPs-induced, abnormal axonal phenotypes and the DOM mitigated axonal phenotypes could persist across generations. Abnormal SMN axon phenotypes caused by ZnO-NPs-exposure were detected in F1 and F2 generations. These are fish that have not been directly exposed to ZnO-NPs. Fish mitigated with DOM during the acute exposure (F0 generation) had a reduction in abnormal motoneuron axon errors in larvae of subsequent generations. Therefore, ZnO-NPs exposure results in neurotoxicity in developing zebrafish which can persist from one generation to the next. Mitigation with DOM can reverse the abnormal phenotypes in an acute embryonic exposure context, as well as across generations, resulting in healthy fish.

Zinc oxide nanoparticle toxicity in embryonic zebrafish: Mitigation with different natural organic matter.

Exposure experiments were conducted to evaluate the influence of dissolved organic matter (DOM) on the toxicity of ZnO-NPs (10-30 nm) and dissolved Zn at sub-lethal doses (50 and 5 ppm, respectively) to zebrafish (Danio rerio). Humic acid, alginic acid, bovine serum albumin and various natural DOM isolated from rivers as the Milwaukee River-WI (NOMW), Yukon River-AK (NOMA) and Suwannee River-GA DOM (NOMS) were used to represent humic substances (HA), carbohydrates (CHO), proteins (PTN), and natural organic matter (NOM), respectively. Initial experiments were carried out to confirm the toxic effect of ZnO-NPs at 50 ppm, followed by mitigation experiments with different types and concentrations of DOM (0.4-40 mg-C/L). Compared to 0% hatch of 50 ppm ZnO-NPs exposed embryos at 72 h post fertilization (hpf), NOMS, NOMW and HA had the best mitigative effects on hatching (53-65%), followed by NOMA, CHO and PTN (19-35%); demonstrating that the mitigation effects on ZnO-NPs toxicity were related to DOM's quantity and composition. At 96 hpf, 20% of embryos exposed to 50 ppm ZnO-NPs hatched, 100% of embryos reared in embryo medium hatched, and close to 100% of the embryos hatched upon mitigation, except for those mitigated with PTN which had less effect. Dissolved Zn (5 ppm) also exhibited the same toxicity on embryos as ZnO-NPs (50 ppm). However, in the presence of HA, NOM and CHO, the hatching rates at 72 and 96 hpf increased significantly compared to 5% hatch without DOM. The overall mitigation effects produced by DOM followed the order of HA ≥ NOMS > NOM (A&W) > CHO >> PTN, although specific mitigation effects varied with DOM concentration and functionalities. Our results also indicate that the toxicity of ZnO-NPs to embryos was mostly derived from NPs although dissolved Zn released from ZnO-NPs also interacted with embryos, affecting hatching, but to a less extent.