Biological effects of selected metal nanoparticles in zebrafish (Danio rerio)
Osborne, Olivia Joanna
Date: 11 December 2013
University of Exeter
PhD in Biological Sciences
As the nanotechnology industry grows rapidly, the release of nanoparticles (NPs) will increase in the environment. The special functional properties of NPs bring uncertainty on how they will affect exposed organisms. Aquatic organisms may be particularly at risk because this environmental compartment acts as a sink for most contaminants. ...
As the nanotechnology industry grows rapidly, the release of nanoparticles (NPs) will increase in the environment. The special functional properties of NPs bring uncertainty on how they will affect exposed organisms. Aquatic organisms may be particularly at risk because this environmental compartment acts as a sink for most contaminants. Two major NPs used in the nano industry are silver (Ag) and titanium dioxide (TiO2). Ag is being exploited principally for its antimicrobial properties and TiO2 for its photocatalytic properties. There is evidence that both of these particles can induce harmful effects in exposed organisms but our understanding on the mechanisms for their effects, uptake and fate of these NPs is still very limited, especially for environmentally relevant exposure regimes. The studies conducted during this thesis investigated the potential for biological effects to Ag and TiO2 in the aquatic environment using the zebrafish (Danio rerio). Uptake and effects of a range of NPs of different sizes (Ag -10nm, 35nm and a bulk counterpart 600-1600nm, and TiO2 -7nm, 10nm, 35nm and a bulk counterpart 134nm) were assessed in zebrafish embryos exposed via the water column. It was established that TiO2NPs had no adverse effects on zebrafish embryos even at an exposure concentration of 25 000 µg/L. In contrast, AgNPs induced dose dependent lethality and the AgNP (35nm) was most toxic. At lower exposure concentrations Ag induced a number of morphological defects in embryos and apoptosis was seen to occur around 7 hours post fertilisation (hpf) in the extended yolk sac region of the embryo. Coating the AgNP materials with citrate and fulvic acid significantly reduced toxicity. Coherent Anti Raman Scattering (CARS) microscopy was applied to the exposed embryos and indicated that there was little, if any uptake of Ag/TiO2 NPs as particles into the embryo. CARS however showed very significant aggregation of both NPs at the chorion surface. It was concluded from this work that the toxicity observed was most likely mediated via silver ions dissociating from the AgNPs. In this work, the metallothionein (mt2) gene was activated (detected using whole mount in-situ hybridisation-WISH) at sublethal exposure concentrations (500 µg AgNP/L and 12 µg AgNO3/L) in the extended yolk sac region of the embryo (24hpf), which is known to be a site of metal detoxification. In a second series of studies to explore the mechanisms and target tissues for AgNPs, WISH was adopted in early life stages of zebrafish using various genes that were markers of stress responses. These genes were metallothionein (mt2), glutathionine S-Transferase Pi (gstp), glutathionine S-Transferase Mu 1 (gstm1), hemeoxygenase 1 (hmox1) and ferritin heavy chain 1 (fth1). In the first instance the ontogeny of expression of these genes was established for up to 12 days post fertilisation (dpf) to determine the optimal time point to test for responses of these genes for the particle exposures. Early life stages of the zebrafish were exposed to citrate coated AgNP (10nm, 500 µg/L), Ag bulk (160nm, 500 µg/L) and silver ions (20 µg/L). The Ag materials induced upregulation in mt2, gstp and gstm1 in various target tissues including the yolk sac, olfactory bulbs, lateral line neuromasts, ionocytes in the skin and in regions of the head. Silver ions affected the same target tissues and induced the same gene responses as AgNPs, albeit there were differences in the levels of these gene responses between these two treatments. In contrast, both hmox1 and fth1 were downregulated as a result of Ag exposure. To further explore the molecular mechanism by which AgNP toxicity occurs, an Nrf2 (a transcription pathway involved in oxidative stress) mutant zebrafish was included in exposures to Ag. These studies found that mt2 and gstp were both expressed at lower levels in the Nrf2 mutant zebrafish exposed to Ag materials compared with in the wild type zebrafish. This suggests that the Nrf2-Keap pathway plays a key role in controlling the expression of these genes that are responding to the AgNPs exposures. This work demonstrated that WISH provided a highly effective integrative approach for identifying target tissues exposure to the different silver materials and for exploring functional pathways of effects. In the final study of this thesis, adult breeding zebrafish were fed AgNP via the diet to investigate uptake and accumulation into target organs, the possibility of maternal transfer and the potential for subsequent effects in exposed offspring. Adult zebrafish were fed at a rate of 5µg Ag/g at 3% of their body weight to Ag materials (AgNP 10nm and AgB 600-1600nm) over a period of 26 days. No effects were seen in the adult fish on any of the measures taken of fitness (condition factor index, gonadsomatic index, hepatosomatic index, haematocrit index). Ag did not significantly affect fecundity (numbers of eggs spawned) or fertility (numbers of eggs fertilised). A detectable level of uptake of Ag in target organs occurred only in the AgNP treatments: liver (up to 2.1 µg/g in males) and gonads (up to 0.5 µg/g in males), suggesting particle size plays a role in the uptake and translocation through membranes into target organs and thus an enhanced bioavailability for AgNP compared with AgB. Maternal transfer was only detectable in the AgNP treatment (up to 0.43 ng Ag/per embryo). Gene responses of mt2 and gstp measured in the subsequent offspring via WISH analysis at 24 hpf indicated significant mt2 upregulation occurred after 2 weeks of adult exposure in both AgNP and AgB treatments. This demonstrates the potential for maternal exposure effects for exposure to AgNPs. Furthermore, challenging the subsequent embryos to the same Ag materials and measuring responses of mt2 via WISH indicated de-sensitisation to Ag in offspring where adults were treated with both nano and bulk forms of Ag. This finding has important considerations for risk assessments for silver base materials. Overall, the findings presented in this thesis have provided a body of evidence to show that silver material on a nanoscale may be of a greater hazard to fish and this warrants further investigation to consider more appropriately the potential risks associated with discharge of AgNPs into the aquatic environment.
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